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Sample records for a2 strain replicates

  1. A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor.

    PubMed

    Fenard, D; Lambeau, G; Maurin, T; Lefebvre, J C; Doglio, A

    2001-08-01

    We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.

  2. Phospholipase A2 activity during the replication cycle of the flavivirus West Nile virus.

    PubMed

    Liebscher, Susann; Ambrose, Rebecca L; Aktepe, Turgut E; Mikulasova, Andrea; Prier, Julia E; Gillespie, Leah K; Lopez-Denman, Adam J; Rupasinghe, Thusitha W T; Tull, Dedreia; McConville, Malcolm J; Mackenzie, Jason M

    2018-04-30

    Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.

  3. Variable Inhibition of Zika Virus Replication by Different Wolbachia Strains in Mosquito Cell Cultures

    PubMed Central

    Schultz, Michaela J.; Isern, Sharon; Michael, Scott F.; Corley, Ronald B.

    2017-01-01

    ABSTRACT Mosquito-borne arboviruses are a major source of human disease. One strategy to reduce arbovirus disease is to reduce the mosquito's ability to transmit virus. Mosquito infection with the bacterial endosymbiont Wolbachia pipientis wMel is a novel strategy to reduce Aedes mosquito competency for flavivirus infection. However, experiments investigating cyclic environmental temperatures have shown a reduction in maternal transmission of wMel, potentially weakening the integration of this strain into a mosquito population relative to that of other Wolbachia strains. Consequently, it is important to investigate additional Wolbachia strains. All Zika virus (ZIKV) suppression studies are limited to the wMel Wolbachia strain. Here we show ZIKV inhibition by two different Wolbachia strains: wAlbB (isolated from Aedes albopictus mosquitoes) and wStri (isolated from the planthopper Laodelphax striatellus) in mosquito cells. Wolbachia strain wStri inhibited ZIKV most effectively. Single-cycle infection experiments showed that ZIKV RNA replication and nonstructural protein 5 translation were reduced below the limits of detection in wStri-containing cells, demonstrating early inhibition of virus replication. ZIKV replication was rescued when Wolbachia was inhibited with a bacteriostatic antibiotic. We observed a partial rescue of ZIKV growth when Wolbachia-infected cells were supplemented with cholesterol-lipid concentrate, suggesting competition for nutrients as one of the possible mechanisms of Wolbachia inhibition of ZIKV. Our data show that wAlbB and wStri infection causes inhibition of ZIKV, making them attractive candidates for further in vitro mechanistic and in vivo studies and future vector-centered approaches to limit ZIKV infection and spread. IMPORTANCE Zika virus (ZIKV) has swiftly spread throughout most of the Western Hemisphere. This is due in large part to its replication in and spread by a mosquito vector host. There is an urgent need for approaches

  4. Role of Prion Replication in the Strain-dependent Brain Regional Distribution of Prions.

    PubMed

    Hu, Ping Ping; Morales, Rodrigo; Duran-Aniotz, Claudia; Moreno-Gonzalez, Ines; Khan, Uffaf; Soto, Claudio

    2016-06-10

    One intriguing feature of prion diseases is their strain variation. Prion strains are differentiated by the clinical consequences they generate in the host, their biochemical properties, and their potential to infect other animal species. The selective targeting of these agents to specific brain structures have been extensively used to characterize prion strains. However, the molecular basis dictating strain-specific neurotropism are still elusive. In this study, isolated brain structures from animals infected with four hamster prion strains (HY, DY, 139H, and SSLOW) were analyzed for their content of protease-resistant PrP(Sc) Our data show that these strains have different profiles of PrP deposition along the brain. These patterns of accumulation, which were independent of regional PrP(C) production, were not reproduced by in vitro replication when different brain regions were used as substrate for the misfolding-amplification reaction. On the contrary, our results show that in vitro replication efficiency depended exclusively on the amount of PrP(C) present in each part of the brain. Our results suggest that the variable regional distribution of PrP(Sc) in distinct strains is not determined by differences on prion formation, but on other factors or cellular pathways. Our findings may contribute to understand the molecular mechanisms of prion pathogenesis and strain diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair.

    PubMed

    Mestre, Olga; Luo, Tao; Dos Vultos, Tiago; Kremer, Kristin; Murray, Alan; Namouchi, Amine; Jackson, Céline; Rauzier, Jean; Bifani, Pablo; Warren, Rob; Rasolofo, Voahangy; Mei, Jian; Gao, Qian; Gicquel, Brigitte

    2011-01-20

    The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes. A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs). A recent Beijing genotype (Bmyc10), which included 60% of strains from distinct parts of the world, appeared to be predominant. We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family.

  6. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts.

    PubMed

    Al Dahouk, Sascha; Köhler, Stephan; Occhialini, Alessandra; Jiménez de Bagüés, María Pilar; Hammerl, Jens Andre; Eisenberg, Tobias; Vergnaud, Gilles; Cloeckaert, Axel; Zygmunt, Michel S; Whatmore, Adrian M; Melzer, Falk; Drees, Kevin P; Foster, Jeffrey T; Wattam, Alice R; Scholz, Holger C

    2017-03-16

    Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species.

  7. Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts

    PubMed Central

    Al Dahouk, Sascha; Köhler, Stephan; Occhialini, Alessandra; Jiménez de Bagüés, María Pilar; Hammerl, Jens Andre; Eisenberg, Tobias; Vergnaud, Gilles; Cloeckaert, Axel; Zygmunt, Michel S.; Whatmore, Adrian M.; Melzer, Falk; Drees, Kevin P.; Foster, Jeffrey T.; Wattam, Alice R.; Scholz, Holger C.

    2017-01-01

    Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species. PMID:28300153

  8. The route of inoculation dictates the replication patterns of the infectious laryngotracheitis virus (ILTV) pathogenic strain and chicken embryo origin (CEO) vaccine.

    PubMed

    Beltrán, Gabriela; Williams, Susan M; Zavala, Guillermo; Guy, James S; García, Maricarmen

    2017-12-01

    Infectious laryngotracheitis virus (ILTV) has a high proclivity to replicate in the larynx and trachea of chickens causing severe lesions. There is a lack of knowledge on the ability of ILTV to replicate in other respiratory associated tissues apart from in the trachea. The objective of this study was to investigate how tissues that first encounter the virus dictate further sites of viral replication during the lytic stage of infection. Replication patterns of the pathogenic strain 63140 and the chicken embryo origin (CEO) vaccine in the conjunctiva, the Harderian gland, nasal cavity and trachea were evaluated after ocular, oral, intranasal or intratracheal inoculation of specific pathogen-free chickens. Viral replication was assessed by detection of microscopic cytolytic lesions, detection of viral antigen and viral genome load. The route of viral entry greatly influenced virus replication of both strain 63140 and CEO vaccine in the conjunctiva and trachea, while replication in the nasal cavity was not affected. In the Harderian gland, independently of the route of viral entry, microscopic lesions characteristic of lytic replication were absent, whereas viral antigen and viral genomes for either virus were detected, suggesting that the Harderian gland may be a key site of antigen uptake. Findings from this study suggest that interactions of the virus with the epithelial-lymphoid tissues of the nasal cavity, conjunctiva and the Harderian gland dictate patterns of ILTV lytic replication.

  9. Ellagitannins as synergists of ACV on the replication of ACV-resistant strains of HSV 1 and 2.

    PubMed

    Vilhelmova-Ilieva, N; Jacquet, R; Quideau, S; Galabov, A S

    2014-10-01

    The plant-derived polyphenolic compounds castalagin, vescalagin and grandinin (C-glucosidic ellagitannins containing nonahydroxyterphenoyl) manifested a strong inhibitory effect on the replication of acyclovir-resistant strains of herpes simplex viruses (HSV) type 1 and 2 in MDBK cells in focus forming units (i.e., microscopically registered microplaques) reduction assay and in two variants of cytopathic effect inhibition test. The effect on the acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) strain was markedly higher compared to that on the ACV-resistant herpes simplex virus type 2 (HSV-2). The three compounds showed comparable levels of antiviral activity against ACV-resistant HSV strains, in contrast with previous results where castalagin exerted the highest degree of activity against wild type HSV strains (Vilhelmova et al., 2011). Combinations of ellagitannins and ACV were tested on the ACV-resistant strains of both HSV-1 and 2 and produced synergistic effects that were revealed by applying the three-dimensional approach of Prichard and Shipman (1990). The ellagitannin(s)-ACV combination applied against ACV-resistant HSV-1 produced a much stronger synergistic effect compared to the effect observed against ACV-resistant HSV-2. The study of the effects of the combination ellagitannin(s)-ACF on intact cell monolayers did not show any toxicity resulting from interaction between the two substances. Altogether, the results obtained in this study demonstrate the highly promising potential of these plant polyphenols as antiherpetic agents. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Protective efficacy of multivalent replication-abortive vaccine strains in horses against African horse sickness virus challenge.

    PubMed

    Lulla, Valeria; Losada, Andres; Lecollinet, Sylvie; Kerviel, Adeline; Lilin, Thomas; Sailleau, Corinne; Beck, Cecile; Zientara, Stephan; Roy, Polly

    2017-07-24

    African horse sickness virus (AHSV) is an orbivirus, a member of the Reoviridae family. Nine different serotypes have been described so far. AHSV is vectored by Culicoides spp. to equids, causing high mortality, particularly in horses, with considerable economic impacts. For development of a safe attenuated vaccine, we previously established an efficient reverse genetics (RG) system to generate Entry Competent Replication-Abortive (ECRA) virus strains, for all nine serotypes and demonstrated the vaccine potential of these strains in type I interferon receptor (IFNAR)-knockout mice. Here, we evaluated the protective efficacies of these ECRA viruses in AHSV natural hosts. One monoserotype (ECRA.A4) vaccine and one multivalent cocktail (ECRA.A1/4/6/8) vaccine were tested in ponies and subsequently challenged with a virulent AHSV4. In contrast to control animals, all vaccinated ponies were protected and did not develop severe clinical symptoms of AHS. Furthermore, the multivalent cocktail vaccinated ponies produced neutralizing antibodies against all serotypes present in the cocktail, and a foal born during the trial was healthy and had no viremia. These results validate the suitability of these ECRA strains as a new generation of vaccines for AHSV. Copyright © 2017. Published by Elsevier Ltd.

  11. Genetically diverse CC-founder mouse strains replicate the human influenza gene expression signature.

    PubMed

    Elbahesh, Husni; Schughart, Klaus

    2016-05-19

    Influenza A viruses (IAV) are zoonotic pathogens that pose a major threat to human and animal health. Influenza virus disease severity is influenced by viral virulence factors as well as individual differences in host response. We analyzed gene expression changes in the blood of infected mice using a previously defined set of signature genes that was derived from changes in the blood transcriptome of IAV-infected human volunteers. We found that the human signature was reproduced well in the founder strains of the Collaborative Cross (CC) mice, thus demonstrating the relevance and importance of mouse experimental model systems for studying human influenza disease.

  12. Immunity raised by recent European subtype 1 PRRSV strains allows better replication of East European subtype 3 PRRSV strain Lena than that raised by an older strain.

    PubMed

    Trus, Ivan; Frydas, Ilias S; Reddy, Vishwanatha R A P; Bonckaert, Caroline; Li, Yewei; Kvisgaard, Lise K; Larsen, Lars E; Nauwynck, Hans J

    2016-01-08

    Stable spatial distribution of porcine reproductive and respiratory syndrome (PRRSV)-1 subtypes in Europe is accompanied by a strong population immunity induced by local PRRSV strains. In the present study, it was examined if the immunity induced by three West European subtype 1 PRRSV strains (2007 isolate 07V063 and 2013 isolates 13V091 and 13V117) offers protection against the highly virulent East European subtype 3 PRRSV strain Lena. The number of fever days was greater (p < 0.05) in the control group (7.6 ± 1.7 days) compared to the immune groups (07V063-immune: 4.0 ± 1.2 days, 13V091-immune: 4.6 ± 1.1 days, 13V117-immune: 4.0 ± 2.9 days). In all groups, protection was characterized by reduction (p < 0.05) of AUC values of nasal shedding (control: 14.6, 07V063-immune: 3.4, 13V091-immune: 8.9, 13V117-immune: 8.0) and viremia (control: 28.1, 07V063-immune: 5.4, 13V091-immune: 9.0, 13V117-immune: 8.3). Reduction of respiratory disease, nasal shedding (mean AUC and mean peak values) and viremia (mean AUC and mean peak values) was more pronounced in 07V063-immune (p < 0.05) than in 13V091-immune and 13V117-immune animals. Inoculation with subtype 1 PRRSV strains caused priming of the Lena-specific virus neutralization antibody response. Upon challenge with Lena, we observed a very strong serological booster effect for neutralizing antibodies against strains used for the first inoculation. Our results indicate that inoculation with subtype 1 PRRSV strains can partially protect against antigenically divergent subtype 3 strains. The lower protection level elicited by recently isolated subtype 1 PRRSV strains may impair the outcome of the spatial expansion of subtype 3 strains from East Europe to West Europe.

  13. DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair.

    PubMed

    Patrick, Steve M; Oakley, Greg G; Dixon, Kathleen; Turchi, John J

    2005-06-14

    Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro, we have addressed the effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in a decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on) and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form, we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.

  14. A comprehensive analysis of replicative lifespan in 4,698 single-gene deletion strains uncovers conserved mechanisms of aging

    PubMed Central

    McCormick, Mark A.; Delaney, Joe R.; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; Shemorry, Anna; Sim, Sylvia; Chou, Annie Chia-Zong; Ahmed, Umema; Carr, Daniel; Murakami, Christopher J.; Schleit, Jennifer; Sutphin, George L.; Wasko, Brian M.; Bennett, Christopher F.; Wang, Adrienne M.; Olsen, Brady; Beyer, Richard P.; Bammler, Theodor K.; Prunkard, Donna; Johnson, Simon C.; Pennypacker, Juniper K.; An, Elroy; Anies, Arieanna; Castanza, Anthony S.; Choi, Eunice; Dang, Nick; Enerio, Shiena; Fletcher, Marissa; Fox, Lindsay; Goswami, Sarani; Higgins, Sean A.; Holmberg, Molly A.; Hu, Di; Hui, Jessica; Jelic, Monika; Jeong, Ki-Soo; Johnston, Elijah; Kerr, Emily O.; Kim, Jin; Kim, Diana; Kirkland, Katie; Klum, Shannon; Kotireddy, Soumya; Liao, Eric; Lim, Michael; Lin, Michael S.; Lo, Winston C.; Lockshon, Dan; Miller, Hillary A.; Moller, Richard M.; Muller, Brian; Oakes, Jonathan; Pak, Diana N.; Peng, Zhao Jun; Pham, Kim M.; Pollard, Tom G.; Pradeep, Prarthana; Pruett, Dillon; Rai, Dilreet; Robison, Brett; Rodriguez, Ariana A.; Ros, Bopharoth; Sage, Michael; Singh, Manpreet K.; Smith, Erica D.; Snead, Katie; Solanky, Amrita; Spector, Benjamin L.; Steffen, Kristan K.; Tchao, Bie Nga; Ting, Marc K.; Wende, Helen Vander; Wang, Dennis; Welton, K. Linnea; Westman, Eric A.; Brem, Rachel B.; Liu, Xin-guang; Suh, Yousin; Zhou, Zhongjun; Kaeberlein, Matt; Kennedy, Brian K.

    2015-01-01

    SUMMARY Many genes that affect replicative lifespan (RLS) in the budding yeast Saccharomyces cerevisiae also affect aging in other organisms such as C. elegans and M. musculus. We performed a systematic analysis of yeast RLS in a set of 4,698 viable single-gene deletion strains. Multiple functional gene clusters were identified, and full genome-to-genome comparison demonstrated a significant conservation in longevity pathways between yeast and C. elegans. Among the mechanisms of aging identified, deletion of tRNA exporter LOS1 robustly extended lifespan. Dietary restriction (DR) and inhibition of mechanistic Target of Rapamycin (mTOR) exclude Los1 from the nucleus in a Rad53-dependent manner. Moreover, lifespan extension from deletion of LOS1 is non-additive with DR or mTOR inhibition, and results in Gcn4 transcription factor activation. Thus, the DNA damage response and mTOR converge on Los1-mediated nuclear tRNA export to regulate Gcn4 activity and aging. PMID:26456335

  15. Infection with equine infectious anemia virus vaccine strain EIAVDLV121 causes no visible histopathological lesions in target organs in association with restricted viral replication and unique cytokine response.

    PubMed

    Liu, Qiang; Ma, Jian; Wang, Xue-Feng; Xiao, Fei; Li, Li-Jia; Zhang, Jiao-Er; Lin, Yue-Zhi; Du, Cheng; He, Xi-Jun; Wang, Xiaojun; Zhou, Jian-Hua

    2016-02-01

    The live equine infectious anemia virus (EIAV) vaccine strain EIAVDLV121 was developed by in vitro attenuation of a virulent strain, EIAVLN40, in the 1970s, and it has been demonstrated to induce protective immunity under laboratory and natural EIAV infection conditions. The detailed biological features of this attenuated virus remain to be further investigated. Experimental inoculation with EIAVDLV121 did not result in clinical symptoms even with immunosuppressive treatment in our previous studies. Here, we further investigated whether the replication of the vaccine strain EIAVDLV121 in experimentally infected horses causes histopathological lesions to develop in the targeted organs. Both the lungs and the spleen have been demonstrated to support EIAV replication. By evaluating the gross macroscopic and histological changes, we found that EIAVDLV121 did not cause detectable histopathological lesions and that it replicated several hundred times more slowly than its parental virulent strain, EIAVLN40, in tissues. Immunochemical assays of these tissues indicated that the primary target cells of EIAVDLV121 were monocytes/macrophages, but that EIAVLN40 also infected alveolar epithelial cells and vascular endothelial cells. In addition, both of these viral strains promoted the up- and down-regulation of the expression of various cytokines and chemokines, implicating the potential involvement of these cellular factors in the pathological outcomes of EIAV infection and host immune responses. Taken together, these results demonstrate that the EIAV vaccine strain does not cause obvious histopathological lesions or clinical symptoms and that it induces a unique cytokine response profile. These features are considered essential for EIAVDLV121 to function as an effective live vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance.

    PubMed

    Luan, Guodong; Bao, Guanhui; Lin, Zhao; Li, Yang; Chen, Zugen; Li, Yin; Cai, Zhen

    2015-12-25

    Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Influence of Foot-and-Mouth Disease Virus O/CHN/Mya98/33-P Strain Leader Protein on Viral Replication and Host Innate Immunity.

    PubMed

    Jiang, Shaodong; Bai, Xingwen; Li, Pinghua; Zhang, Meng; Bao, Huifang; Sun, Pu; Lu, Zengjun; Cao, Yimei; Chen, Yingli; Li, Dong; Fu, Yuanfang; Liu, Zaixin

    2015-09-01

    Foot-and-mouth disease virus (FMDV) O/CHN/Mya98/33-P strain was isolated from the esophageal-pharyngeal fluid sample of cattle, and was shown to cause persistent infection. Its leader protein contains 200 amino acids with one amino acid deletion, which is upstream and next to the second initiation codon compared with the majority of FMDV Mya98 strains. The FMDV genome includes two initiation codons that can produce two different leader proteins, Lab (from the first AUG) and Lb (from the second AUG). For convenience, the inter-AUG region was named as La. Previously, it was found that a recombinant virus with Lab of FMDV O/CHN/Mya98/33-P strain had higher proliferation efficiency, and better ability to inhibit the host innate immune response. Three full-length infectious cDNA clones-rHN33-Lb, rHN33-La, and rHNGSLX-Lb-containing the FMDV O/CHN/Mya98/33-P strain leader proteins Lb, La, or the FMDV O/GSLX/2010 strain leader protein Lb, respectively, were constructed based on an established infectious clone r-HN rescued from FMDV O/HN/CHN/93 strain. After infecting pig kidney primary cells, rHN33-La showed higher replication efficiency than r-HN, and rHN33-Lb displayed better ability to resist host innate immunity than rHNGSLX-Lb. These results demonstrated that the inter-AUG region of FMDV strain O/CHN/Mya98/33-P leader protein must be involved in increasing viral replication efficiency. Additionally, the Lb of FMDV O/CHN/Mya98/33-P must be involve in increasing its ability to inhibit host innate immune response, and the distinctive amino acids G56 and/or R118 of FMDV leader protein may play essential roles in it.

  18. Simian rhesus rotavirus is a unique heterologous (non-lapine) rotavirus strain capable of productive replication and horizontal transmission in rabbits.

    PubMed

    Ciarlet, M; Estes, M K; Conner, M E

    2000-05-01

    Simian rhesus rotavirus (RRV) is the only identified heterologous (non-lapine) rotavirus strain capable of productive replication at a high inoculum dose of virus (>10(8) p.f.u.) in rabbits. To evaluate whether lower doses of RRV would productively infect rabbits and to obtain an estimate of the 50% infectious dose, rotavirus antibody-free rabbits were inoculated orally with RRV at inoculum doses of 10(3), 10(5) or 10(7) p.f.u. Based on faecal virus antigen or infectious virus shedding, RRV replication was observed with inoculum doses of 10(7) and 10(5) p.f.u., but not 10(3) p.f.u. Horizontal transmission of RRV to one of three mock-inoculated rabbits occurred 4-5 days after onset of virus antigen shedding in RRV-infected rabbits. Rabbits infected at 10(7) and 10(5), but not 10(3), p.f.u. of RRV developed rotavirus-specific immune responses and were completely (100%) protected from lapine ALA rotavirus challenge. These data confirm that RRV can replicate productively and spread horizontally in rabbits. In attempts to elucidate the genetic basis of the unusual replication efficacy of RRV in rabbits, the sequence of the gene encoding the lapine non-structural protein NSP1 was determined. Sequence analysis of the NSP1 of three lapine rotaviruses revealed a high degree of amino acid identity (85-88%) with RRV. Since RRV and lapine strains also share similar VP7s (96-97%) and VP4s (69-70%), RRV might replicate efficiently in rabbits because of the high relatedness of these three gene products, each implicated in host range restriction.

  19. Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

    PubMed Central

    Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Pérez-Jiménez, Eva; Oliveros, Juan Carlos

    2013-01-01

    Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4+ and CD8+ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4+ whereas DNA-LACK/M101-LACK preferentially induced CD8+ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors. PMID

  20. 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.

    PubMed

    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.

  1. Host-cell-dependent role of actin cytoskeleton during the replication of a human strain of influenza A virus.

    PubMed

    Arcangeletti, M C; De Conto, F; Ferraglia, F; Pinardi, F; Gatti, R; Orlandini, G; Covan, S; Motta, F; Rodighiero, I; Dettori, G; Chezzi, C

    2008-01-01

    This study was aimed at investigating the possible involvement of the actin cytoskeleton in the modulation of host permissiveness to A/NWS/33 human influenza virus infection in two mammalian (MDCK and LLC-MK2) cell lines in vitro. During the early stages of infection, no appreciable association between incoming NWS/33 virions and cortical actin was detectable in the permissive MDCK model by confocal microscopy, while extensive colocalization and a slower infection progression were observed in LLC-MK2 cells. In the latter model, we also demonstrated the inability of the virus to carry out multiple replication cycles, irrespective of the presence of cleaved HA subunits in the released virions. Treatment with the actin-depolymerizing agent cytochalasin D significantly increased the infection efficiency in LLC-MK2 cells, while a detrimental effect was observed in the MDCK cell line. Our data suggest a selective role of the actin network in inducing a restriction to influenza virus replication, mostly depending on its molecular organization, the host cell type and virus replication phase.

  2. Replication potential and different modes of transmission of West Nile virus in an Indian strain of Culex gelidus Theobald (Diptera: Culicidae) mosquitoes.

    PubMed

    Sudeep, A B; Ghodke, Y S; Gokhale, M D; George, R P; Dhaigude, S D; Bondre, V P

    2014-12-01

    Culex gelidus mosquito, an important vector of Japanese encephalitis virus, has shown to transmit West Nile virus (WNV), Kunjin and Murray Valley encephalitis viruses experimentally. An attempt was, therefore, made to study the replication kinetics and vector competence of an Indian strain of Cx. gelidus to WNV. Mosquitoes were infected by both intrathoracic inoculation and oral feeding and studied the growth kinetics by determining the virus titre on different days post-infection (PI). Vector competence was studied by determining the presence of WNV in saliva on subsequent days PI. Horizontal transmission was determined by demonstrating infection in infant mice by bite of mosquitoes that were fed on viraemic mice previously. Vertical transmission was studied by screening progeny derived from infected mosquitoes. Trans-stadial transmission was determined by screening adult mosquitoes emerged from parenterally inoculated IV instar larvae. The mosquito replicated WNV to 7log10 TCID50/ml on Day 8 PI and maintained the titre for 14 days. Virus dissemination to legs and salivary glands could be detected, but not to ovaries up to Day 10 PI. The mosquitoes picked up infection from viraemic blood and transmitted successfully to infant mice on subsequent feeding. Trans-stadial transmission also could be demonstrated. However, vertical transmission could not be demonstrated. The replication potential, maintenance of WNV for prolonged periods and ability to transmit WNV experimentally makes the mosquito a serious threat to public health especially in the wake of active WNV activity in certain parts of India.

  3. Increased replication of T-cell-tropic HIV strains and CXC-chemokine receptor-4 induction in T cells treated with macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES beta-chemokines.

    PubMed

    Dolei, A; Biolchini, A; Serra, C; Curreli, S; Gomes, E; Dianzani, F

    1998-01-22

    To study, in T-lymphoid cells, the effects of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and RANTES beta-chemokines on the replication of T-cell-tropic HIV-1 strains, since it has been reported that beta-chemokines interfere with the replication of macrophage-tropic HIV-1 strains, but not T-cell-tropic strains. Freshly phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL) and cultured PHA-activated T cells from healthy volunteers, as well as the C8166 T-cell line, were treated overnight with beta-chemokines before infection with T-cell-tropic HIV-1 isolates, or human T-lymphotropic virus type IIIB. HIV replication was followed by detecting the production of infectious particles, p24 antigen, and viral sequences. CXC-chemokine receptor (CXCR)-4 expression was followed by detection and quantification of specific transcripts. Pretreatment of T cells with MIP-1alpha, MIP-1beta and RANTES affected T-cell-tropic strains, increased the replication of HIV-1beta and HIV-1RPdT strains dose-dependently, as well as virus absorption and provirus DNA accumulation. These findings were associated with increased accumulation of CXCR-4 transcripts, and mediated by the protein tyrosine kinase signalling. Moreover, beta-chemokines stimulated PBL proliferation. Beta-chemokines increase the adsorption and replication of at least some T-cell-tropic HIV-1 strains, and this is related to stimulated expression of the CXCR-4 coreceptor.

  4. Pathogenesis of chronic Epstein-Barr virus infection: detection of a virus strain with a high rate of lytic replication.

    PubMed

    Jäger, M; Prang, N; Mitterer, M; Larcher, C; Huemer, H P; Reischl, U; Wolf, H; Schwarzmann, F

    1996-12-01

    In rare cases Epstein-Barr virus (EBV) leads to chronic active infection (CAEBV) which is characterized by persistant symptoms of infectious mononucleosis. Previously we described a case of persisting polyclonal B-cell lymphocytosis (PPBL) that was associated with CAEBV. Using reverse transcription and polymerase chain reaction we showed that in late passages of a spontaneous cell line, SM, latent EB viral genes such as EBNA1, EBNA2, EBNA3A/3B/ 3C, LMP1 and LMP2A were active. The master gene of the lytic cycle, BZLF1, was silent. This indicated that there was no general defect in immortalization and establishing latency by this CAEBV isolate SM. We obtained virus from the standard immortalizing strain B95-8 and the CAEBV strain SM from latently infected LCL quantified the number of virus particles by competitive PCR and demonstrated that the impaired capacity to immortalize umbilical cord blood lymphocytes was a virus strain-specific property, and was not due to an incapability to infect purified CD19+ B lymphocytes. Transcription of latency- and immortalization-associated genes such as EBNA1, EBNA2 and LMP2A was reduced, in contrast to a strongly enhanced activity of the master gene of the lytic cycle, BZLF1. A scenario for an antagonistic regulation of lytic and latent cycle genes is presented and a role for the pathogenesis of CAEBV is discussed.

  5. Control regions for chromosome replication are conserved with respect to sequence and location among Escherichia coli strains

    PubMed Central

    Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A.; Løbner-Olesen, Anders

    2015-01-01

    In Escherichia coli, chromosome replication is initiated from oriC by the DnaA initiator protein associated with ATP. Three non-coding regions contribute to the activity of DnaA. The datA locus is instrumental in conversion of DnaAATP to DnaAADP (datA dependent DnaAATP hydrolysis) whereas DnaA rejuvenation sequences 1 and 2 (DARS1 and DARS2) reactivate DnaAADP to DnaAATP. The structural organization of oriC, datA, DARS1, and DARS2 were found conserved among 59 fully sequenced E. coli genomes, with differences primarily in the non-functional spacer regions between key protein binding sites. The relative distances from oriC to datA, DARS1, and DARS2, respectively, was also conserved despite of large variations in genome size, suggesting that the gene dosage of either region is important for bacterial growth. Yet all three regions could be deleted alone or in combination without loss of viability. Competition experiments during balanced growth in rich medium and during mouse colonization indicated roles of datA, DARS1, and DARS2 for bacterial fitness although the relative contribution of each region differed between growth conditions. We suggest that this fitness advantage has contributed to conservation of both sequence and chromosomal location for datA, DARS1, and DARS2. PMID:26441936

  6. An attenuated EIAV vaccine strain induces significantly different immune responses from its pathogenic parental strain although with similar in vivo replication pattern.

    PubMed

    Lin, Yue-Zhi; Shen, Rong-Xian; Zhu, Zhen-Ying; Deng, Xi-Lin; Cao, Xue-Zhi; Wang, Xue-Feng; Ma, Jian; Jiang, Cheng-Gang; Zhao, Li-Ping; Lv, Xiao-Ling; Shao, Yi-Ming; Zhou, Jian-Hua

    2011-11-01

    The EIAV (equine infectious anemia virus) multi-species attenuated vaccine EIAV(DLV121) successfully prevented the spread of equine infectious anemia (EIA) in China in the 1970s and provided an excellent model for the study of protective immunity to lentiviruses. In this study, we compared immune responses induced by EIAV(DLV121) to immunity elicited by the virulent EIAV(LN40) strain and correlated immune responses to protection from infection. Horses were randomly grouped and inoculated with either EIAV(DLV121) (Vaccinees, Vac) or a sublethal dose of EIAV(LN40) (asymptomatic carriers, Car). Car horses became EIAV(LN40) carriers without disease symptoms. Two of the four Vac horses were protected against infection and the other two had delayed onset or reduced severity of EIA with a lethal EIAV(LN40) challenge 5.5 months post initial inoculation. In contrast, all three Car animals developed acute EIA and two succumbed to death. Specific humoral and cellular immune responses in both Vac and Car groups were evaluated for potential correlations with protection. These analyses revealed that although plasma viral loads remained between 10(3) and 10(5)copies/ml for both groups before EIAV(LN40) challenge, Vac-treated animals developed significantly higher levels of conformational dependent, Env-specific antibody, neutralizing antibody as well as significantly elevated CD4(+) T cell proliferation and IFN-γ-secreting CD8(+) T cells than those observed in EIAV(LN40) asymptomatic carriers. Further analysis of protected and unprotected cases in vaccinated horses identified that cellular response parameters and the reciprocal anti-p26-specific antibody titers closely correlated with protection against infection with the pathogenic EIAV(LN40). These data provide a better understanding of protective immunity to lentiviruses. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

    PubMed

    Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

    2017-03-01

    The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10 5 ) were infected with T. gondii tachyzoites (5×10 5 ) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10 5 ) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

  8. Complete genome sequence of Spiroplasma citri strain R8-A2T, causal agent of stubborn disease in Citrus spp

    USDA-ARS?s Scientific Manuscript database

    Spiroplasma citri is the causal agent of stubborn disease in Citrus spp., as well as the cause of diseases in numerous other plant genera. Here we report the nucleotide sequence of the 1,599,709 bp circular chromosome and two plasmids of strain R8-A2T. This information will facilitate comparative ...

  9. Complete Genome Sequence of Spiroplasma citri Strain R8-A2T, Causal Agent of Stubborn Disease in Citrus Species.

    PubMed

    Davis, Robert E; Shao, Jonathan; Zhao, Yan; Gasparich, Gail E; Gaynor, Brady J; Donofrio, Nicole

    2017-04-20

    Spiroplasma citri causes stubborn disease in Citrus spp. and diseases in other plants. Here, we report the nucleotide sequence of the 1,599,709-bp circular chromosome and two plasmids of S. citri strain R8-A2 T This information will facilitate analyses to understand spiroplasmal pathogenicity and evolutionary adaptations to lifestyles in plants and arthropod hosts. Copyright © 2017 Davis et al.

  10. Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

    PubMed

    Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

    2014-04-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

  11. Typing of Clinical Mycobacterium avium Complex Strains Cultured during a 2-Year Period in Denmark by Using IS1245

    PubMed Central

    Bauer, Jeanett; Andersen, Åse B.; Askgaard, Dorthe; Giese, Sten B.; Larsen, Birger

    1999-01-01

    In the present study restriction fragment length polymorphism analyses with the recently described insertion sequence IS1245 as a probe was performed with clinical Mycobacterium avium complex strains cultured in Denmark during a 2-year period. The overall aim of the study was to disclose potential routes of transmission of these microorganisms. As a first step, the genetic diversity among isolates from AIDS patients and non-human immunodeficiency virus (HIV)-infected patients was described. In addition, a number of isolates from nonhuman sources cultured during the same period were analyzed and compared to the human isolates. A total of 203 isolates from AIDS patients (n = 90), non-HIV-infected patients (n = 91), and nonhuman sources (n = 22) were analyzed. The presence of IS1245 was restricted to Mycobacterium avium subsp. avium isolates. The majority of human isolates had large numbers of IS1245 copies, while nonhuman isolates could be divided into a high-copy-number group and a low-copy-number group. Groups of identical strains were found to be geographically widespread, comprising strains from AIDS patients as well as strains from non-HIV-infected patients. Samples of peat (to be used as potting soil) and veterinary samples were found to contain viable M. avium isolates belonging to genotypes also found in humans. PMID:9986819

  12. hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

    SciTech Connect

    Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

    2014-01-20

    The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to themore » cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.« less

  13. Infection of human islets of Langerhans with two strains of Coxsackie B virus serotype 1: assessment of virus replication, degree of cell death and induction of genes involved in the innate immunity pathway.

    PubMed

    Anagandula, Mahesh; Richardson, Sarah J; Oberste, M Steven; Sioofy-Khojine, Amir-Babak; Hyöty, Heikki; Morgan, Noel G; Korsgren, Olle; Frisk, Gun

    2014-08-01

    Type 1 diabetes mellitus is believed to be triggered, in part, by one or more environmental factors and human enteroviruses (HEVs) are among the candidates. Therefore, this study has examined whether two strains of HEV may differentially affect the induction of genes involved in pathways leading to the synthesis of islet hormones, chemokines and cytokines in isolated, highly purified, human islets. Isolated, purified human pancreatic islets were infected with strains of Coxsackievirus B1.Viral replication and the degree of CPE/islet dissociation were monitored. The expression of insulin, glucagon, CXCL10, TLR3, IF1H1, CCL5, OAS-1, IFNβ, and DDX58 was analyzed. Both strains replicated in islets but only one of strain caused rapid islet dissociation/CPE. Expression of the insulin gene was reduced during infection of islets with either viral strain but the gene encoding glucagon was unaffected. All genes analyzed which are involved in viral sensing and the development of innate immunity were induced by Coxsackie B viruses, with the notable exception of TLR3. There was no qualitative difference in the expression pattern between each strain but the magnitude of the response varied between donors. The lack of virus induced expression of TLR3, together with the differential regulation of IF1H1, OAS1 and IFNβ, (each of which has polymorphic variants influence the predisposition to type 1 diabetes), that might result in defective clearance of virus from islet cells. The reduced expression of the insulin gene and the unaffected expression of the gene encoding glucagon by Coxsackie B1 infection is consistent with the preferential β-cell tropism of the virus. © 2013 Wiley Periodicals, Inc.

  14. Whole-Genome Sequence of Clostridium botulinum A2B3 87, a Highly Virulent Strain Involved in a Fatal Case of Foodborne Botulism in Italy

    PubMed Central

    Giordani, Francesco; Fillo, Silvia; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Gentile, Bernardina; Pittiglio, Valentina; Spagnolo, Ferdinando; Anniballi, Fabrizio; Fiore, Alfonsina; Auricchio, Bruna; De Medici, Dario

    2015-01-01

    Here, we report the genome sequence of a rare bivalent strain of Clostridium botulinum, A2B3 87. The strain was isolated from a foodborne botulism case that occurred in Italy in 1995. The case was characterized by rapid evolution of the illness and failure of conventional treatments. PMID:25814616

  15. Replicating vaccines

    USDA-ARS?s Scientific Manuscript database

    Early work on fish immunology and disease resistance demonstrated fish (like animals and humans) that survived infection were typically resistant to re-infection with the same pathogen. The concepts of resistance upon reinfection lead to the research and development of replicating (live) vaccines in...

  16. Draft genome sequence of Bacillus velezensis 2A-2B strain: a rhizospheric inhabitant of Sporobolus airoides (Torr.) Torr., with antifungal activity against root rot causing phytopathogens.

    PubMed

    Martínez-Raudales, Inés; De La Cruz-Rodríguez, Yumiko; Alvarado-Gutiérrez, Alejandro; Vega-Arreguín, Julio; Fraire-Mayorga, Ahuitz; Alvarado-Rodríguez, Miguel; Balderas-Hernández, Victor; Fraire-Velázquez, Saúl

    2017-01-01

    A Bacillus velezensis strain from the rhizosphere of Sporobolus airoides (Torr.) Torr . , a grass in central-north México, was isolated during a biocontrol of phytopathogens scrutiny study. The 2A-2B strain exhibited at least 60% of growth inhibition of virulent isolates of phytopathogens causing root rot. These phytopathogens include Phytophthora capsici , Fusarium solani , Fusarium oxysporum and Rhizoctonia solani . Furthermore, the 2A-2B strain is an indolacetic acid producer, and a plant inducer of PR1, which is an induced systemic resistance related gene in chili pepper plantlets. Whole genome sequencing was performed to generate a draft genome assembly of 3.953 MB with 46.36% of GC content, and a N50 of 294,737. The genome contains 3713 protein coding genes and 89 RNA genes. Moreover, comparative genome analysis revealed that the 2A-2B strain had the greatest identity (98.4%) with Bacillus velezensis.

  17. Virulence difference between the prototypic Schu S4 strain (A1a) and Francisella tularensis A1a, A1b, A2 and type B strains in a murine model of infection.

    PubMed

    Molins, Claudia R; Delorey, Mark J; Yockey, Brook M; Young, John W; Belisle, John T; Schriefer, Martin E; Petersen, Jeannine M

    2014-02-06

    The use of prototypic strains is common among laboratories studying infectious agents as it promotes consistency for data comparability among and between laboratories. Schu S4 is the prototypic virulent strain of Francisella tularensis and has been used extensively as such over the past six decades. Studies have demonstrated virulence differences among the two clinically relevant subspecies of F. tularensis, tularensis (type A) and holarctica (type B) and more recently between type A subpopulations (A1a, A1b and A2). Schu S4 belongs to the most virulent subspecies of F. tularensis, subspecies tularensis. In this study, we investigated the relative virulence of Schu S4 in comparison to A1a, A1b, A2 and type B strains using a temperature-based murine model of infection. Mice were inoculated intradermally and a hypothermic drop point was used as a surrogate for death. Survival curves and the length of temperature phases were compared for all infections. Bacterial burdens were also compared between the most virulent type A subpopulation, A1b, and Schu S4 at drop point. Survival curve comparisons demonstrate that the Schu S4 strain used in this study resembles the virulence of type B strains, and is significantly less virulent than all other type A (A1a, A1b and A2) strains tested. Additionally, when bacterial burdens were compared between mice infected with Schu S4 or MA00-2987 (A1b) significantly higher burdens were present in the blood and spleen of mice infected with MA00-2987. The knowledge gained from using Schu S4 as a prototypic virulent strain has unquestionably advanced the field of tularemia research. The findings of this study, however, indicate that careful consideration of F. tularensis strain selection must occur when the overall virulence of the strain used could impact the outcome and interpretation of results.

  18. Factors influencing the attenuation of serotype 1 Marek's disease virus by serial cell culture passage, and evaluation of attenuated strains for protection and replication

    USDA-ARS?s Scientific Manuscript database

    This study was to better understand factors that influenced the process of attenuation of Marek’s disease (MD) virus by serial passage in cell cultures. Three virulent (v) pathotype and 3 very virulent plus (vv+) pathotype strains were passed by 3 techniques up to 131 times and the passage level at...

  19. Role of trypsin in the replication of Avian metapneumovirus subtype C (strain MN-2a) and its entry into the Vero cells.

    PubMed

    Paudel, Sarita; Shin, Hyun-Jin

    2015-12-01

    To understand the molecular mechanisms of Avian metapneumovirus (aMPV) and the requirements involved in the infection and fusion, trypsin treatment was done in the different stages of virus; before infection, during entry and after virus infection followed by aMPV infection. The growth kinetics of aMPV was compared in time dependent manner. The effect of trypsin was found in the later stage of aMPV infection increasing the numbers of infected cells with the significant higher titer of infectious virions to that of trypsin treated before infection, during entry and aMPV. A serine protease inhibitor reduced aMPV replication in a significant way, whereas cysteine peptidase (E-64), aspartic protease (pepstatin A), and metalloprotease (phosphoramidon) inhibitors had no effect on aMPV replication. Inoculation of aMPV on Vero cells expressing the membrane-associated protease TMPRSS2 resulted in higher virus titers than that inoculated on normal Vero cells and is statistically significant (p < 0.05). Also, an inhibitor of clathrin/caveolae-mediated endocytosis had no effect on virus progeny, indicating that aMPV does not use the endocytic pathway for entry but undergoes direct fusion. The effect of lysosomotropic agents was not significant, suggesting that aMPV does not require low-pH environment in endosomes to fuse its envelope with the plasma membrane. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

    PubMed Central

    Kenney, Scott P.

    2015-01-01

    ABSTRACT Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that contains an insertion of a 171-nucleotide sequence encoding amino acids 21 to 76 of the human ribosomal protein S17 (RPS17) within the hypervariable region (HVR) of the HEV ORF1 protein. When the RPS17 insertion was placed into a strain of genotype 1 HEV which infects only humans, it expanded the host range of the virus, allowing it to infect cell lines from multiple animal species, including cow, dog, cat, chicken, and hamster. In this study, we utilized forward and reverse genetics to attempt to define which aspects of the RPS17 insertion allow for the ability of the Kernow C-1 P6 HEV to adapt in cell culture and allow for expanded host tropism. We demonstrate that the RPS17 sequence insertion in HEV bestows novel nuclear/nucleolar trafficking capabilities to the ORF1 protein of Kernow P6 HEV and that lysine residues within the RPS17 insertion, but not nuclear localization of the ORF1 protein, correlate with the enhanced replication of the HEV Kernow C-1 P6 strain. The results from this study have important implications for understanding the mechanism of cross-species infection and replication of HEV. IMPORTANCE HEV is an important pathogen worldwide. The virus causes high mortality (up to 30%) in pregnant women and has been recognized to cause chronic hepatitis in immunocompromised populations. The life cycle of HEV has been understudied due to a lack of sufficient cell culture systems in which to propagate the virus. Recently, insertions and rearrangements of the

  1. Failure of Sterne- and Pasteur-like strains of Bacillus anthracis to replicate and survive in the urban bluebottle blow fly Calliphora vicina under laboratory conditions.

    PubMed

    von Terzi, Britta; Turnbull, Peter C B; Bellan, Steve E; Beyer, Wolfgang

    2014-01-01

    This study aimed to elucidate the bacteriological events occurring within the gut of Calliphora vicina, selected as the European representative of blow flies held responsible for the spread of anthrax during epidemics in certain parts of the world. Green-fluorescent-protein-carrying derivatives of Bacillus anthracis were used. These lacked either one of the virulence plasmids pXO1 and pXO2 and were infected, or not infected, with a worm intestine phage (Wip4) known to influence the phenotype and survival of the pathogen. Blood meals were prepared for the flies by inoculation of sheep blood with germinated and, in case of pXO2+ strains, encapsulated cells of the four B. anthracis strains. After being fed for 4 h an initial 10 flies were externally disinfected with peracetic acid to ensure subsequent quantitation representing ingested B. anthracis only. Following neutralization, they were crushed in sterile saline. Over each of the ensuing 7 to 10 days, 10 flies were removed and processed the same way. In the absence of Wip4, strains showed steady declines to undetectable in the total B. anthracis counts, within 7-9 days. With the phage infected strains, the falls in viable counts were significantly more rapid than in their uninfected counterparts. Spores were detectable in flies for longer periods than vegetative bacteria. In line with the findings in both biting and non-biting flies of early workers our results indicate that B. anthracis does not multiply in the guts of blow flies and survival is limited to a matter of days.

  2. Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus.

    PubMed

    Stewart, Hazel; Adema, Karen W; McMonagle, Elizabeth L; Hosie, Margaret J; Willett, Brian J

    2012-05-31

    The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C. Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C. Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent

  3. Construction and immunogenicity of replication-competent adenovirus 5 host range mutant recombinants expressing HIV-1 gp160 of SF162 and TV1 strains.

    PubMed

    Hidajat, Rachmat; Kuate, Seraphin; Venzon, David; Kalyanaraman, Vaniambadi; Kalisz, Irene; Treece, James; Lian, Ying; Barnett, Susan W; Robert-Guroff, Marjorie

    2010-05-21

    An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIV(SF162) gp160 (subtype B) and HIV(TV1) gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIV(TV1) gp160 induced better cellular immunity than Ad5hr-HIV(SF162) gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIV(TV1) gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector. Published by Elsevier Ltd.

  4. Construction and immunogenicity of replication-competent adenovirus 5 host range mutant recombinants expressing HIV-1 gp160 of SF162 and TV1 strains

    PubMed Central

    Hidajat, Rachmat; Kuate, Seraphin; Venzon, David; Kalyanaraman, Vaniambadi; Kalisz, Irene; Treece, James; Lian, Ying; Barnett, Susan W.; Robert-Guroff, Marjorie

    2010-01-01

    An HIV Env immunogen capable of eliciting broad immunity is critical for a successful vaccine. We constructed and characterized Adenovirus 5 host range mutant (Ad5hr) recombinants encoding HIVSF162 gp160 (subtype B) and HIVTV1 gp160 (subtype C). Immunization of mice with one or both induced cellular immunity to subtype B and C peptides by ELISpot, and antibody responses with high binding titers to HIV Env of subtypes A, B, C, and E. Notably, Ad5hr-HIVTV1 gp160 induced better cellular immunity than Ad5hr-HIVSF162 gp160, either alone or following co-administration. Thus, the TV1 Env recombinant alone may be sufficient for eliciting immune responses against both subtype B and C envelopes. Further studies of Ad5hr-HIVTV1 gp160 in rhesus macaques will evaluate the suitability of this insert for a future phase I clinical trial using a replication-competent Ad4 vector. PMID:20382241

  5. CENTRIOLE REPLICATION

    PubMed Central

    Mizukami, Ikuko; Gall, Joseph

    1966-01-01

    Sperm formation was studied in the fern, Marsilea, and the cycad, Zamia, with particular emphasis on the centrioles. In Marsilea, the mature sperm possesses over 100 flagella, the basal bodies of which have the typical cylindrical structure of centrioles. Earlier observations by light microscopy suggested that these centrioles arise by fragmentation of a body known as the blepharoplast. In the youngest spermatids the blepharoplast is a hollow sphere approximately 0.8 µ in diameter. Its wall consists of closely packed immature centrioles, or procentrioles. The procentrioles are short cylinders which progressively lengthen during differentiation of the spermatid. At the same time they migrate to the surface of the cell, where each of them puts out a flagellum. A blepharoplast is found at each pole of the spindle during the last antheridial mitosis, and two blepharoplasts are found in the cytoplasm before this mitosis. Blepharoplasts are also found in the preceding cell generation, but their ultimate origin is obscure. Before the last mitosis the blepharoplasts are solid, consisting of a cluster of radially arranged tubules which bear some structural similarity to centrioles. In Zamia, similar stages are found during sperm formation, although here the number of flagella on each sperm is close to 20,000 and the blepharoplast measures about 10 µ in diameter. These observations are discussed in relation to theories of centriole replication. PMID:5950730

  6. The effect of mismatch strain induced by the a1-a2 domain pattern on dislocation generation of epitaxial ferroelectric films

    NASA Astrophysics Data System (ADS)

    He, J. M.; Xie, C.; Ma, T. F.

    2018-01-01

    This study theoretically models both mismatch strain and lattice dislocation generation induced by the a1-a2 domain pattern in epitaxial ferroelectric films. For the ferroelectric film/substrate structure, the precise complex potential functions for the Somigliana screw dislocation quadrupole that represents the lattice mismatch strain are presented, the free boundary condition of the film is approximately processed by an equivalent method, and the effect of mismatch strain on lattice dislocation generation is discussed. The results show that: the higher mismatch degree and domain wall energy evidently lead to interfacial dislocation multiplication; the density of the dislocations decreases with decreasing film thickness; the generated dislocations are mainly concentrated at the sites far away from domain walls.

  7. COMPARISON OF OVERALL METABOLISM OF 1, 2, 7, 8-PECDD IN CYP1A2(-L-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    COMPARISON OF OVERALL METABOLISM OF 1,2,3,7,8-PeCDD
    IN CYP1A2 (-/-) KNOCKOUT AND C57BL/6N PARENTAL
    STRAINS OF MICE

    Heldur Hakk1 and Janet J. Diliberto2

    1 USDA-ARS, Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    2 US EPA, ORD, National Heal...

  8. Acute-phase CD4+ T-cell proliferation and CD152 upregulation predict set-point virus replication in vaccinated simian-human immunodeficiency virus strain 89.6p-infected macaques.

    PubMed

    Koopman, Gerrit; Mortier, Daniella; Hofman, Sam; Koutsoukos, Marguerite; Bogers, Willy M J M; Wahren, Britta; Voss, Gerald; Heeney, Jonathan L

    2009-04-01

    Human immunodeficiency virus (HIV) infection in humans and simian immunodeficiency virus (SIV) infection in macaques are accompanied by a combined early loss of CCR5 (CD195)-expressing CD4(+) memory T cells, loss of T-helper function and T-cell hyperactivation, which have all been associated with development of high virus load and disease progression. Here, a cohort of vaccinated simian-human immunodeficiency virus strain 89.6p (SHIV(89.6p))-infected rhesus macaques, where preferential depletion of these memory T-cell subsets does not take place and CD4(+) T cells are relatively well maintained, was used to study the role of hyperactivation as an independent factor in the establishment of set-point virus load. In the acute phase of the infection, a transient loss of CD4(+) T cells, as well as strong increases in expression of proliferation and activation markers on CD4(+) and CD8(+) T cells, together with CD152 expression on CD4(+) T cells, were observed. Peak expression levels of these markers on CD4(+) T cells, but not on CD8(+) T cells, were correlated with high virus replication in the chronic phase of the infection. In addition, the peak expression level of these markers was correlated inversely with acute-phase, but not chronic-phase, HIV/SIV-specific gamma interferon responses. These data highlight a central role for an acute but transient CD4 decrease, as well as CD4(+) T-cell activation, as independent factors for prediction of set-point levels of virus replication.

  9. Clostridium botulinum strain Af84 produces three enzymatically active neurotoxins: BoNT/A2, BoNT/F4, and BoNT/F5

    PubMed Central

    Kalb, Suzanne R.; Baudys, Jakub; Smith, Theresa J.; Smith, Leonard A.; Barr, John R.

    2015-01-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F; designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MSE quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates which mimic the toxins’ natural targets and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum. PMID:24605815

  10. The FhaB/FhaC two-partner secretion system is involved in adhesion of Acinetobacter baumannii AbH12O-A2 strain

    PubMed Central

    Pérez, A.; Merino, M.; Rumbo-Feal, S.; Álvarez-Fraga, L.; Vallejo, J. A.; Beceiro, A.; Ohneck, E. J.; Mateos, J.; Fernández-Puente, P.; Actis, L. A.; Poza, M.; Bou, G.

    2017-01-01

    ABSTRACT Acinetobacter baumannii is a hospital-acquired pathogen that shows an extraordinary capacity to stay in the hospital environment. Adherence of the bacteria to eukaryotic cells or to abiotic surfaces is the first step for establishing an infection. The A. baumannii strain AbH12O-A2 showed an exceptional ability to adhere to A549 epithelial cells. The AbFhaB/FhaC 2-partner secretion (TPS) system involved in adhesion was discovered after the screening of the recently determined A. baumannii AbH12O-A2 strain genome (CP009534.1). The AbFhaB is a large exoprotein which transport to the bacterial surface is mediated by the AbFhaC protein. In the present study, the role of this TPS system in the AbH12O-A2 adherence phenotype was investigated. The functional inactivation of this 2-partner secretion system was addressed by analyzing the outer membrane vesicles (OMV) proteomic profile from the wild-type strain and its derivative mutant AbH12O-A2ΔfhaC demonstrating that AbFhaB is no longer detected in the absence of AbFhaC. Scanning electron microscopy (SEM) and adhesion experiments demonstrated that inactivation of the AbFhaB/FhaC system significantly decreases bacterial attachment to A549 alveolar epithelial cells. Moreover, it has been demonstrated that this 2-partner secretion system is involved in fibronectin-mediated adherence of the A. baumannii AbH12O-A2 isolate. Finally, we report that the AbFhaB/FhaC system is involved in virulence when tested using invertebrate and vertebrate hosts. These data suggest the potential role that this AbFhaB/FhaC secretion system could play in the pathobiology of A. baumannii. PMID:27858524

  11. RNA segment 9 exists as a duplex concatemer in an Australian strain of epizootic haemorrhagic disease virus (EHDV): Genetic analysis and evidence for the presence of concatemers as a normal feature of orbivirus replication.

    PubMed

    Anthony, S J; Darpel, K E; Belaganahalli, M N; Maan, N; Nomikou, K; Sutton, G; Attoui, H; Maan, S; Mertens, P P C

    2011-11-25

    This paper reports a concatemeric RNA in a strain of epizootic haemorrhagic disease virus (EHDV) serotype 5. Sequencing showed that the concatemeric RNA contains two identical full-length copies of genome segment 9, arranged in series, which has apparently replaced the monomeric form of the segment. In vitro translation demonstrated that the concatemeric RNA can act as a viable template for VP6 translation, but that no double-sized protein is produced. Studies were also performed to assess whether mutations might be easily introduced into the second copy (which might indicate some potential evolutionary significance of a concatemeric RNA segment), however multiple (n=40) passages generated no changes in the sequence of either the upstream or downstream segments. Further, we present results that demonstrate the presence of concatemers or partial gene duplications in multiple segments of different orbiviruses (in tissue culture and purified virus), suggesting their generation is likely to be a normal feature of orbivirus replication. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  13. The evolution of replicators.

    PubMed

    Szathmáry, E

    2000-11-29

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators.

  14. The evolution of replicators.

    PubMed Central

    Szathmáry, E

    2000-01-01

    Replicators of interest in chemistry, biology and culture are briefly surveyed from a conceptual point of view. Systems with limited heredity have only a limited evolutionary potential because the number of available types is too low. Chemical cycles, such as the formose reaction, are holistic replicators since replication is not based on the successive addition of modules. Replicator networks consisting of catalytic molecules (such as reflexively autocatalytic sets of proteins, or reproducing lipid vesicles) are hypothetical ensemble replicators, and their functioning rests on attractors of their dynamics. Ensemble replicators suffer from the paradox of specificity: while their abstract feasibility seems to require a high number of molecular types, the harmful effect of side reactions calls for a small system size. No satisfactory solution to this problem is known. Phenotypic replicators do not pass on their genotypes, only some aspects of the phenotype are transmitted. Phenotypic replicators with limited heredity include genetic membranes, prions and simple memetic systems. Memes in human culture are unlimited hereditary, phenotypic replicators, based on language. The typical path of evolution goes from limited to unlimited heredity, and from attractor-based to modular (digital) replicators. PMID:11127914

  15. Prelife catalysts and replicators

    PubMed Central

    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 activity, thereby enhancing the rates of certain prelife reactions. We study the selection criteria for these prelife catalysts. Their catalytic efficiency must be above certain critical values. We find a maintenance threshold and an initiation threshold. The former is a linear function of sequence length, and the latter is an exponential function of sequence length. Therefore, it is extremely hard to select for prelife catalysts that have long sequences. We compare prelife catalysis with a simple model for replication. Assuming fast template-based elongation reactions, we can show that replicators have selection thresholds that are independent of their sequence length. Our calculation demonstrates the efficiency of replication and provides an explanation of why replication was selected over other forms of prelife catalysis. PMID:19692408

  16. Who Needs Replication?

    ERIC Educational Resources Information Center

    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…

  17. CHARACTERIZATION AND NUCLEOTIDE SEQUENCE DETERMINATION OF A REPEAT ELEMENT ISOLATED FROM A 2,4,5,-T DEGRADING STRAIN OF PSEUDOMONAS CEPACIA

    EPA Science Inventory

    Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T− strain PT88 by a ColE1 :: Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) wa...

  18. Adenovirus DNA Replication

    PubMed Central

    Hoeben, Rob C.; Uil, Taco G.

    2013-01-01

    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease. PMID:23388625

  19. Eukaryotic DNA Replication Fork.

    PubMed

    Burgers, Peter M J; Kunkel, Thomas A

    2017-06-20

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

  20. HIV-1 replicative fitness in elite controllers

    PubMed Central

    Lobritz, Michael A.; Lassen, Kara G.; Arts, Eric J.

    2017-01-01

    Purpose of review Differential rates of disease progression are obviously multifactorial, but the virulence of the actual infecting virus is most frequently ignored as potential source of slow or rapid disease progression. In this review, the argument will be made that nearly all elite suppressors are infected by weak HIV-1 strain (in terms of replicative capacity). Whether this poor virus replication is the cause of elite suppression or the consequence of a strong immune response remains a leading question in the field. Recent findings Although numerous research studies have related HIV-1 replicative capacity/fitness in tissue culture to virulence within patients, this review will focus on several recent and key discoveries on the important role of HIV-1 fitness in elite suppression. First, elite suppressors appear to harbor HIV-1 variants that encode Gag, Pol, and Env proteins that are less efficient than their counterparts of HIV-1 in typical/chronic progressors. Second, the actual HIV-1 clone(s) that establish acute infection may be less fit in patients who become elite controllers as compared with typical progressors. Finally, the fitness costs of cytotoxic T lymphocyte escape in HIV-1 may be easily compensated by secondary mutations if the infecting strain is capable of high replication kinetics and rapid evolution. A strain with weak replicative capacity might not compensate for fitness loss or even generate the initial escape mutations. Summary A combination of good, anti-HIV-1 host genetics (e.g. HLA-B*57) along with infection by a ‘whimpy’ HIV-1 strain may be necessary for elite suppression, whereas only one of these may lead to slow progression and viremia. PMID:21430530

  1. DNA Replication Origins

    PubMed Central

    Leonard, Alan C.; Méchali, Marcel

    2013-01-01

    The onset of genomic DNA synthesis requires precise interactions of specialized initiator proteins with DNA at sites where the replication machinery can be loaded. These sites, defined as replication origins, are found at a few unique locations in all of the prokaryotic chromosomes examined so far. However, replication origins are dispersed among tens of thousands of loci in metazoan chromosomes, thereby raising questions regarding the role of specific nucleotide sequences and chromatin environment in origin selection and the mechanisms used by initiators to recognize replication origins. Close examination of bacterial and archaeal replication origins reveals an array of DNA sequence motifs that position individual initiator protein molecules and promote initiator oligomerization on origin DNA. Conversely, the need for specific recognition sequences in eukaryotic replication origins is relaxed. In fact, the primary rule for origin selection appears to be flexibility, a feature that is modulated either by structural elements or by epigenetic mechanisms at least partly linked to the organization of the genome for gene expression. PMID:23838439

  2. Organization of DNA Replication

    PubMed Central

    Chagin, Vadim O.; Stear, Jeffrey H.; Cardoso, M. Cristina

    2010-01-01

    The discovery of the DNA double helix structure half a century ago immediately suggested a mechanism for its duplication by semi-conservative copying of the nucleotide sequence into two DNA daughter strands. Shortly after, a second fundamental step toward the elucidation of the mechanism of DNA replication was taken with the isolation of the first enzyme able to polymerize DNA from a template. In the subsequent years, the basic mechanism of DNA replication and its enzymatic machinery components were elucidated, mostly through genetic approaches and in vitro biochemistry. Most recently, the spatial and temporal organization of the DNA replication process in vivo within the context of chromatin and inside the intact cell are finally beginning to be elucidated. On the one hand, recent advances in genome-wide high throughput techniques are providing a new wave of information on the progression of genome replication at high spatial resolution. On the other hand, novel super-resolution microscopy techniques are just starting to give us the first glimpses of how DNA replication is organized within the context of single intact cells with high spatial resolution. The integration of these data with time lapse microscopy analysis will give us the ability to film and dissect the replication of the genome in situ and in real time. PMID:20452942

  3. Replication-Competent Controlled Herpes Simplex Virus.

    PubMed

    Bloom, David C; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria; Voellmy, Richard

    2015-10-01

    We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of

  4. COMPARISON OF OVERALL METABOLISM OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN IN CYP1A2(-/-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    Comparison of Overall Metabolism of 2,3,7,8-TCDD
    in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    Heldur Hakk* and Janet J. Diliberto**

    * USDA-ARS Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    ** US-EPA ORD, National Health Eff...

  5. Continuous Passaging of a Recombinant C-Strain Virus in PK-15 Cells Selects Culture-Adapted Variants that Showed Enhanced Replication but Failed to Induce Fever in Rabbits.

    PubMed

    Tong, Chao; Chen, Ning; Liao, Xun; Yuan, Xuemei; Sun, Mengjiao; Li, Xiaoliang; Fang, Weihuan

    2017-09-28

    Classical swine fever virus (CSFV) is the etiologic agent of classical swine fever, a highly contagious disease that causes significant economic losses to the swine industry. The lapinized C-strain, a widely used vaccine strain against CSFV, has low growth efficiency in cell culture, which limits the productivity in the vaccine industry. In this study, a recombinant virus derived from C-strain was constructed and subjected to continuous passaging in PK-15 cells with the goal of acquiring a high progeny virus yield. A cell-adapted virus variant, RecCpp80, had nearly 1,000-fold higher titer than its parent C-strain but lost the ability to induce fever in rabbits. Sequence analysis of cell-adapted RecC variants indicated that at least six nucleotide changes were fixed in RecCpp80. Further adaption of RecCpp80 variant in swine testicle cells led to a higher virus yield without additional mutations. Introduction of each of these residues into the wild-type RecC backbone showed that one mutation, M979R (T3310G), located in the C-terminal region of E2 might be closely related to the cell-adapted phenotype. Rabbit inoculation revealed that RecCpp80 +10 failed to induce fever in rabbits, whereas RecCpp40 +10 caused a fever response similar to the commercial C-strain vaccine. In conclusion, the C-strain can be adapted to cell culture by introducing specific mutations in its E2 protein. The mutations in RecCpp80 that led to the loss of fever response in rabbits require further investigation. Continuous passaging of the C-strain-based recombinant viruses in PK-15 cells could enhance its in vitro adaption. The non-synonymous mutations at 3310 and 3531 might play major roles in the enhanced capacity of general virus reproduction. Such findings may help design a modified C-strain for improved productivity of commercial vaccines at reduced production cost.

  6. Glycoprotein 5 of porcine reproductive and respiratory syndrome virus strain SD16 inhibits viral replication and causes G2/M cell cycle arrest, but does not induce cellular apoptosis in Marc-145 cells

    SciTech Connect

    Mu, Yang, E-mail: muyang@nwsuaf.edu.cn; Experimental Station of Veterinary Pharmacology and Veterinary Biotechnology, Ministry of Agriculture of the People's Republic of China, No. 22 Xinong Road, Yangling, Shaanxi 712100; Li, Liangliang, E-mail: lifeiyang2007@126.com

    2015-10-15

    Cell apoptosis is common after infection with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV GP5 has been reported to induce cell apoptosis. To further understand the role of GP5 in PRRSV induced cell apoptosis, we established Marc-145 cell lines stably expressing full-length GP5, GP5{sup Δ84-96} (aa 84-96 deletion), and GP5{sup Δ97-119} (aa 97-119 deletion). Cell proliferation, cell cycle progression, cell apoptosis and virus replication in these cell lines were evaluated. Neither truncated nor full-length GP5 induced cell apoptosis in Marc-145 cells. However, GP5{sup Δ97-119}, but not full-length or GP5{sup Δ84-96}, induced a cell cycle arrest at the G2/M phasemore » resulting in a reduction in the growth of Marc-145 cells. Additionally, GP5{sup Δ84-96} inhibited the replication of PRRSV in Marc-145 cells through induction of IFN-β. These findings suggest that PRRSV GP5 is not responsible for inducing cell apoptosis in Marc-145 cells under these experimental conditions; however it has other important roles in virus/host cell biology. - Highlights: • Marc-145 cell lines stable expression PRRSV GP5 or truncated GP5 were constructed. • GP5{sup Δ97-119} expression in Marc-145 cell induced cell cycle arrest at G2/M phase. • Expression of GP5 and truncated GP5 could not induce Marc-145 cells apoptosis. • PRRSV replication in Marc-145-GP5{sup Δ84-96} was significantly inhibited.« less

  7. DNA Replication Timing

    PubMed Central

    Rhind, Nicholas; Gilbert, David M.

    2013-01-01

    Patterns of replication within eukaryotic genomes correlate with gene expression, chromatin structure, and genome evolution. Recent advances in genome-scale mapping of replication kinetics have allowed these correlations to be explored in many species, cell types, and growth conditions, and these large data sets have allowed quantitative and computational analyses. One striking new correlation to emerge from these analyses is between replication timing and the three-dimensional structure of chromosomes. This correlation, which is significantly stronger than with any single histone modification or chromosome-binding protein, suggests that replication timing is controlled at the level of chromosomal domains. This conclusion dovetails with parallel work on the heterogeneity of origin firing and the competition between origins for limiting activators to suggest a model in which the stochastic probability of individual origin firing is modulated by chromosomal domain structure to produce patterns of replication. Whether these patterns have inherent biological functions or simply reflect higher-order genome structure is an open question. PMID:23838440

  8. Hepatitis B virus replication

    PubMed Central

    Beck, Juergen; Nassal, Michael

    2007-01-01

    Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ε RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development. PMID:17206754

  9. Replication of chaos

    NASA Astrophysics Data System (ADS)

    Akhmet, M. U.; Fen, M. O.

    2013-10-01

    We propose a rigorous method for replication of chaos from a prior one to systems with large dimensions. Extension of the formal properties and features of a complex motion can be observed such that ingredients of chaos united as known types of chaos, Devaney's, Li-Yorke and obtained through period-doubling cascade. This is true for other appearances of chaos: intermittency, structure of the chaotic attractor, its fractal dimension, form of the bifurcation diagram, the spectra of Lyapunov exponents, etc. That is why we identify the extension of chaos through the replication as morphogenesis. To provide rigorous study of the subject, we introduce new definitions such as chaotic sets of functions, the generator and replicator of chaos, and precise description of ingredients for Devaney and Li-Yorke chaos in continuous dynamics. Appropriate simulations which illustrate the chaos replication phenomenon are provided. Moreover, in discussion form we consider inheritance of intermittency, replication of Shil'nikov orbits and quasiperiodical motions as a possible skeleton of a chaotic attractor. Chaos extension in an open chain of Chua circuits is also demonstrated.

  10. The right ventricular response to high afterload: comparison between healthy persons and patients with transposition of the great arteries: a 2D strain study.

    PubMed

    Becker, Michael; Hümpel, Carmen; Ocklenburg, Christina; Muehler, Eberhard; Schroeder, Joerg; Eickholt, Christian; Hoffmann, Rainer; Marx, Nikolaus; Franke, Andreas

    2010-11-01

    To evaluate the contraction pattern of the systemic right ventricle (RV) in comparison to healthy controls. 31 patients (14 female, mean age 21.5 ± 3.3 years) with congenital corrected (ccTGA) and surgical corrected (D-TGA) TGA and 31 age-matched healthy controls (17 female, mean age 23.1 ± 2.6 years) were included in the study. We compared the contraction pattern of the systemic RV with findings in the right and left ventricle (LV) of healthy controls. Echocardiograms were performed for all patients (19.3 ± 2.5 years after the operation) and all healthy controls. Using a novel computer software (GE Ultrasound, Horton, Norway) that allows automatic frame-by-frame tracking of accoustic markers during the heart cycle longitudinal and circumferential strain and strain rate were defined as parameters of myocardial deformation. In the systemic free RV wall, circumferential strain was greater than longitudinal strain (-25.6 ± 4.2% vs. -15.2 ± 3.9%, P < 0.001), opposite to the contraction pattern of the subpulmonary free RV wall (-17.7 ± 4.1% vs. -28.3 ± 3.3%, P < 0.001). Compared with controls, segmental analysis of the free RV wall resulted in lower systolic longitudinal and higher systolic circumferential strain and strain rate values. In the free LV wall, circumferential strain was lower than longitudinal strain (-19.1 ± 2.7% vs. -26.8 ± 4.5%, P < 0.001) in contrast to the data in the systemic free LV wall (-24.5 ± 3.1% vs. -16.7 ± 3.5%, P < 0.001). Myocardial deformation parameters were significantly different in systemic and normal RV indicating a dependency of myocardial deformation parameters on right ventricular afterload. The use of myocardial deformation imaging identified a shift from longitudinal to circumferential contraction pattern in the systemic RV in comparison to the LV. This might demonstrate a response to the systemic load creating a myocardial hypertrophy. © 2010, Wiley Periodicals, Inc.

  11. Psychology, replication & beyond.

    PubMed

    Laws, Keith R

    2016-06-01

    Modern psychology is apparently in crisis and the prevailing view is that this partly reflects an inability to replicate past findings. If a crisis does exists, then it is some kind of 'chronic' crisis, as psychologists have been censuring themselves over replicability for decades. While the debate in psychology is not new, the lack of progress across the decades is disappointing. Recently though, we have seen a veritable surfeit of debate alongside multiple orchestrated and well-publicised replication initiatives. The spotlight is being shone on certain areas and although not everyone agrees on how we should interpret the outcomes, the debate is happening and impassioned. The issue of reproducibility occupies a central place in our whig history of psychology.

  12. Replicated Composite Optics Development

    NASA Technical Reports Server (NTRS)

    Engelhaupt, Darell

    1997-01-01

    Advanced optical systems for applications such as grazing incidence Wolter I x-ray mirror assemblies require extraordinary mirror surfaces in ten-ns of fine surface finish and figure. The impeccable mirror surface is on the inside of the rotational mirror form. One practical method of producing devices with these requirements is to first fabricate an exterior surface for the optical device then replicate that surface to have the inverse component with lightweight characteristics. The replicate optic is not better than the master or mandrel from which it is made. This task is a continuance of previous studies to identify methods and materials for forming these extremely low roughness optical components.

  13. Quantitative analysis of echocardiographic cine-loops using a 2D-strain algorithm in correlation with thallium-201 SPECT imaging.

    PubMed

    Miller, Aaron; Chouraqui, Pierre; Zwirn, Gil; Feinberg, Micha S; Akselrod, Solange

    2008-11-01

    The aim of this study was to investigate the correlation between the regional left ventricular (LV) contractility, assessed by two-dimensional (2D)-strain echocardiographic images, and regional LV perfusion, determined by thallium-201 single photon emission computed tomography (SPECT) imaging. Forty-five hospitalized patients with suspected myocardial ischemia (32 males, mean age 69 +/- 12 years) have undergone both echocardiography and SPECT imaging on the same day. The echocardiographic data has been obtained in three apical views. The data for each view has been processed by the 2D-strain algorithm, to produce six peak systolic longitudinal strain measurements per view, one per segment. The resulting measurements have been compared with the corresponding regions within the SPECT bull's-eye map (at rest). In order to handle inaccuracies in the positioning of the echocardiographic transducer, the two datasets have undergone registration based on local correlation. The mean correlation coefficients between SPECT perfusion and peak systolic longitudinal strain for the apical long-axis, apical two-chamber, and apical four-chamber views were 0.55 +/- 0.36, 0.47 +/- 0.35, and 0.64 +/- 0.30 respectively (negative correlation coefficients have been considered as zero). The overall mean correlation coefficient was 0.56 +/- 0.34. The scatter graphs of the average values of perfusion and the strain values showed a nonlinear relation between the two parameters. The average correlation coefficient is comparable to the values reported for the correlation between the average LV function, based on visual analysis of echocardiographic imaging, and the average LV perfusion, based on SPECT imaging.

  14. Evolution of Replication Machines

    PubMed Central

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

    2016-01-01

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

  15. Replicating viruses for gynecologic cancer therapy.

    PubMed

    Park, J W; Kim, M

    2016-01-01

    Despite advanced therapeutic treatments, gynecologic malignancies such as cervical and ovarian cancers are still the top ten leading cause of cancer death among women in South Korea. Thus a novel and innovative approach is urgently needed. Naturally occurring viruses are live, replication-proficient viruses that specifically infect human cancer cells while sparing normal cell counterparts. Since the serendipitous discovery of the naturally oncotropic virus targeting gynecologic cancer in 1920s, various replicating viruses have shown various degrees of safety and efficacy in preclinical or clinical applications for gynecologic cancer therapy. Cellular oncogenes and tumor suppressor genes, which are frequently dysregulated in gynecologic malignancies, play an important role in determining viral oncotropism. Published articles describing replicating, oncolytic viruses for gynecologic cancers are thoroughly reviewed. This review outlines the discovery of replication-proficient virus strains for targeting gynecologic malignancies, recent progresses elucidating molecular connections between oncogene/tumor suppressor gene abnormalities and viral oncotropism, and the associated preclinical/clinical implications. The authors would also like to propose future directions in the utility of the replicating viruses for gynecologic cancer therapy.

  16. A Survey of Genomic Maps in Strains of Staphylococcus Aureus

    DTIC Science & Technology

    chromosomal replication procedure. Strains possessing multiple antibiotic resistance yielded a genomic map with all genes shifted toward significantly later...replication times than in strains not bearing multiple antibiotic resistance . It appears that there is a unique site on the S. aureus chromosome for the initiation of replication.

  17. Replication of plasmids from Staphylococcus aureus in Escherichia coli.

    PubMed Central

    Goze, A; Ehrlich, S D

    1980-01-01

    Plasmid pBR322 derives from plasmid ColE1 and does not replicate in Escherichia coli strains lacking DNA polymerase I. Hybrids between pBR322 and a plasmid isolated from Staphylococcus aureus, pC194, replicate in such E. coli strains, provided that the pC194 replication region is intact. Inactivation of the pBR322 replication region does not interfere with the replication of hybrids in E. coli. Hybrids between pBR322 and two other plasmids from S. aureus, pT127 and pUB112, and replicate at the restrictive temperature in E. coli having thermosensitive DNA polymerase I. Similar hybrids involving pC221 and pHV400, plasmids from S. aureus and Bacillus subtilis, respectively, do not replicate under such conditions. These results show that some plasmids from a Gram-positive bacterium, S. aureus, can replicate in a Gram-negative one, E. coli. Images PMID:7012836

  18. Coronavirus Attachment and Replication

    DTIC Science & Technology

    1988-03-28

    dis~ertation I will discuss two aspects of coronaviruses: 1) the RNA polymerase of the A59 strain of MHV; and 2) the role of coronavirus receptors ...proteins. Therefore, species-specific host restriction for MHV may occur at the level of viral attachment or penetration. MHV receptors in mouse...except the fully resistant SJL/J strain expressed a 100-120 kilodalton MHV receptor , but C57BL/ 6 mice expressed a larger receptor on the intestine

  19. Neonatal neosporosis in a 2-week-old Bernese mountain dog infected with multiple Neospora caninum strains based on MS10 microsatellite analysis.

    PubMed

    Prandini da Costa Reis, Rodrigo; Crisman, Robin; Roser, Margie; Malik, Richard; Šlapeta, Jan

    2016-05-15

    Neonatal neosporosis is a challenging disease to diagnose in neonatal and young puppies because the first signs of this condition may not be strongly suggestive of an infectious aetiology. Within two weeks of birth, three of four pups died with a subacute clinical course, some with dyspnea, some with diarrhoea and some with neurologic signs. Neosporosis was diagnosed post-mortem, but only after microscopic examination of tissues collected at necropsy. Histological findings consisted of (i) necrotizing, diffuse interstitial pneumonia associated with intralesional protozoa and (ii) necrotizing multifocal myocarditis with mineralization and intralesional protozoa. No significant alterations were found in the cerebrum or cerebellum (spinal cord was not examined). Immunohistochemistry confirmed protozoal stages and cysts were Neospora caninum. Immunohistochemistry for Toxoplasma gondii was negative. Lung and heart were the most severely affected tissues with large numbers of free zoites, BAG5 positive bradyzoites and tissue cysts of N. caninum further confirmed by N. caninum-specific quantitative real-time PCR. One affected pup which displayed knuckling, ataxia and diarrhoea were treated with trimethoprim sulfadiazine and clindamycin, and made a complete recovery. This surviving pup (at 8 weeks-of-age) and dam were both positive for N. caninum antibody (reciprocal titres 4096 and 256, respectively). Three other intact bitches on the same property were seropositive for N. caninum, suggesting horizontal transmission and a common source of infection, possibly due to consumption of infected meat. Analysis using microsatellite-10 (MS10) demonstrated that multiple strains of N. caninum were present. It was likely that all MS10 N. caninum strains were transplacentally transmitted from dam to pups. This is the first time that multiple N. caninum strains have been demonstrated to be vertically transmitted in dogs. N. caninum should be considered in the differential diagnosis for

  20. Ultrastructural Characterization of Zika Virus Replication Factories.

    PubMed

    Cortese, Mirko; Goellner, Sarah; Acosta, Eliana Gisela; Neufeldt, Christopher John; Oleksiuk, Olga; Lampe, Marko; Haselmann, Uta; Funaya, Charlotta; Schieber, Nicole; Ronchi, Paolo; Schorb, Martin; Pruunsild, Priit; Schwab, Yannick; Chatel-Chaix, Laurent; Ruggieri, Alessia; Bartenschlager, Ralf

    2017-02-28

    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. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A2A Receptor in Hippocampal Glutamate Synapses.

    PubMed

    Machado, Nuno J; Simões, Ana Patrícia; Silva, Henrique B; Ardais, Ana Paula; Kaster, Manuella P; Garção, Pedro; Rodrigues, Diana I; Pochmann, Daniela; Santos, Ana Isabel; Araújo, Inês M; Porciúncula, Lisiane O; Tomé, Ângelo R; Köfalvi, Attila; Vaugeois, Jean-Marie; Agostinho, Paula; El Yacoubi, Malika; Cunha, Rodrigo A; Gomes, Catarina A

    2017-03-01

    Caffeine prophylactically prevents mood and memory impairments through adenosine A 2A receptor (A 2A R) antagonism. A 2A R antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher A 2A R density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the A 2A R agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the A 2A R antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal A 2A R controlling synaptic glutamatergic function.

  2. Taxonomy by Carbon Replication

    PubMed Central

    Dietz, Alma; Mathews, John

    1962-01-01

    Pre-shadowed carbon replication of spore surfaces (carbon repligraphy) provides a new technique for the characterization of streptomycetes. Carbon repligraphs of five members of the Streptomyces hygroscopicus complex show two distinct types. Type I shows a nonsegmented spore structure with an extremely wrinkled surface. Type II has a segmented spore chain with detailed surface structure resembling a basket weave. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 FIG. 11 FIG. 12 FIG. 13 FIG. 14 FIG. 15 FIG. 16 FIG. 17 PMID:13886326

  3. Replication Research and Special Education

    ERIC Educational Resources Information Center

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

    2016-01-01

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

  4. Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations.

    PubMed

    Rodríguez-Cousiño, Nieves; Esteban, Rosa

    2017-02-15

    Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed. Killer strains of S. cerevisiae secrete protein toxins that kill nonkiller yeasts. The "killer phenomenon" depends on two dsRNA viruses: L-A and M. M encodes the toxin, and L-A, the helper virus, provides the capsids for both viruses. Different killer toxins exist: K1, K2, K28, and Klus, encoded on different M viruses. Our data indicate that each M dsRNA depends on a specific helper virus; these helper viruses have

  5. Relationships and Evolution of Double-Stranded RNA Totiviruses of Yeasts Inferred from Analysis of L-A-2 and L-BC Variants in Wine Yeast Strain Populations

    PubMed Central

    Rodríguez-Cousiño, Nieves

    2016-01-01

    ABSTRACT Saccharomyces cerevisiae killer strains secrete a protein toxin active on nonkiller strains of the same (or other) yeast species. Different killer toxins, K1, K2, K28, and Klus, have been described. Each toxin is encoded by a medium-size (1.5- to 2.3-kb) M double-stranded RNA (dsRNA) located in the cytoplasm. M dsRNAs require L-A helper virus for maintenance. L-A belongs to the Totiviridae family, and its dsRNA genome of 4.6 kb codes for the major capsid protein Gag and a minor Gag-Pol protein, which form the virions that separately encapsidate L-A or the M satellites. Different L-A variants exist in nature; on average, 24% of their nucleotides are different. Previously, we reported that L-A-lus was specifically associated with Mlus, suggesting coevolution, and proposed a role of the toxin-encoding M dsRNAs in the appearance of new L-A variants. Here we confirm this by analyzing the helper virus in K2 killer wine strains, which we named L-A-2. L-A-2 is required for M2 maintenance, and neither L-A nor L-A-lus shows helper activity for M2 in the same genetic background. This requirement is overcome when coat proteins are provided in large amounts by a vector or in ski mutants. The genome of another totivirus, L-BC, frequently accompanying L-A in the same cells shows a lower degree of variation than does L-A (about 10% of nucleotides are different). Although L-BC has no helper activity for M dsRNAs, distinct L-BC variants are associated with a particular killer strain. The so-called L-BC-lus (in Klus strains) and L-BC-2 (in K2 strains) are analyzed. IMPORTANCE Killer strains of S. cerevisiae secrete protein toxins that kill nonkiller yeasts. The “killer phenomenon” depends on two dsRNA viruses: L-A and M. M encodes the toxin, and L-A, the helper virus, provides the capsids for both viruses. Different killer toxins exist: K1, K2, K28, and Klus, encoded on different M viruses. Our data indicate that each M dsRNA depends on a specific helper virus; these

  6. Enhanced Deletion Formation by Aberrant DNA Replication in Escherichia Coli

    PubMed Central

    Saveson, C. J.; Lovett, S. T.

    1997-01-01

    Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Escherichia coli strains mutant for various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E. coli chromosome. Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ, the ε editing subunit of Pol III, and dnaB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the α polymerase (dnaE), the γ clamp loader complex (holC, dnaX), and the β clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent. Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways. PMID:9177997

  7. Multiaxial yield behaviour of Al replicated foam

    NASA Astrophysics Data System (ADS)

    Combaz, E.; Bacciarini, C.; Charvet, R.; Dufour, W.; Mortensen, A.

    2011-09-01

    Multiaxial experiments are performed on replicated aluminium foam using a custom-built apparatus. The foam structure is isotropic, and features open monomodal pores 75 μm in average diameter. Plane stress ( σ1, σ2, σ3=0) and axisymmetric ( σ1, σ2=σ3) yield envelopes are measured using cubical specimens, supplemented by tests on hollow cylindrical and uniaxial samples. In addition to the three stress components at 0.2% offset strain, the computer-controlled testing apparatus also measures the three instantaneous displacement vectors. Results show that the shape of the yield surface is independent of the relative density of the foam in the explored range (13-28%). Strain increment vectors lie, within error, roughly normal to the line traced through data points in stress space. Replicated foams feature asymmetric yield behaviour between tension and compression. The data additionally show an influence on the yield surface of the third stress tensor invariant (i.e., of the Lode angle). Simple general expressions for the yield surface are fitted to the data, leading to conclude that their behaviour is slightly better captured by parabolic rather than elliptic expressions dependent on all three stress invariants.

  8. Inhibition of Zika Virus Replication by Silvestrol.

    PubMed

    Elgner, Fabian; Sabino, Catarina; Basic, Michael; Ploen, Daniela; Grünweller, Arnold; Hildt, Eberhard

    2018-03-27

    The Zika virus (ZIKV) outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the plant Aglaia foveolata was found to have very potent antiviral effects against the (-)-strand RNA-virus Ebola virus as well as against Corona- and Picornaviruses with a (+)-strand RNA-genome. This antiviral activity is based on the impaired translation of viral RNA by the inhibition of the DEAD-box RNA helicase eukaryotic initiation factor-4A (eIF4A) which is required to unwind structured 5´-untranslated regions (5'-UTRs) of several proto-oncogenes and thereby facilitate their translation. Zika virus is a flavivirus with a positive-stranded RNA-genome harboring a 5'-capped UTR with distinct secondary structure elements. Therefore, we investigated the effects of silvestrol on ZIKV replication in A549 cells and primary human hepatocytes. Two different ZIKV strains were used. In both infected A549 cells and primary human hepatocytes, silvestrol has the potential to exert a significant inhibition of ZIKV replication for both analyzed strains, even though the ancestor strain from Uganda is less sensitive to silvestrol. Our data might contribute to identify host factors involved in the control of ZIKV infection and help to develop antiviral concepts that can be used to treat a variety of viral infections without the risk of resistances because a host protein is targeted.

  9. Replication of Venezuelan Equine Encephalomyelitis Virus In Vitro

    PubMed Central

    Hearn, H. J.; Tribble, H. R.; Nagle, S. C.; Bowersox, O. C.

    1971-01-01

    An attempt was made to identify nutritional additives that influence the replication of Venezuelan equine encephalomyelitis virus in suspension cultures grown in a defined serum-free medium. Proline, serine, and choline enhanced titers of the virulent PES strain; the progeny population, however, possessed a virulence character that was somewhat different from that of the PES inocula. These nutritional supplements did not appreciably influence the titers of the attenuated 9t and 20t viral strains. When both the PES and 20t strains were employed as a mixed inoculum in culture, the presence of the latter strain appeared to interfere with the growth of the PES strain and reduced its response to the medium supplements. PMID:5544296

  10. Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

    PubMed Central

    Rousseau, Geneviève M.; Capra, María L.; Quiberoni, Andrea; Tremblay, Denise M.; Labrie, Simon J.

    2015-01-01

    Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: CL1, CL2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (CL1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages CL1, CL2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages CL1, CL2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts. PMID:26475105

  11. Checkpoint independence of most DNA replication origins in fission yeast

    PubMed Central

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

    2007-01-01

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

  12. Self-replication: Nanostructure evolution

    NASA Astrophysics Data System (ADS)

    Simmel, Friedrich C.

    2017-10-01

    DNA origami nanostructures were utilized to replicate a seed pattern that resulted in the growth of populations of nanostructures. Exponential growth could be controlled by environmental conditions depending on the preferential requirements of each population.

  13. Natural Strain

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.

    1997-01-01

    Logarithmic strain is the preferred measure of strain used by materials scientists, who typically refer to it as the "true strain." It was Nadai who gave it the name "natural strain," which seems more appropriate. This strain measure was proposed by Ludwik for the one-dimensional extension of a rod with length l. It was defined via the integral of dl/l to which Ludwik gave the name "effective specific strain." Today, it is after Hencky, who extended Ludwik's measure to three-dimensional analysis by defining logarithmic strains for the three principal directions.

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

    PubMed

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

    2018-04-04

    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.

  15. Natural Strain

    NASA Technical Reports Server (NTRS)

    Freed, Alan D.

    1995-01-01

    The purpose of this paper is to present a consistent and thorough development of the strain and strain-rate measures affiliated with Hencky. Natural measures for strain and strain-rate, as I refer to them, are first expressed in terms of of the fundamental body-metric tensors of Lodge. These strain and strain-rate measures are mixed tensor fields. They are mapped from the body to space in both the Eulerian and Lagrangian configurations, and then transformed from general to Cartesian fields. There they are compared with the various strain and strain-rate measures found in the literature. A simple Cartesian description for Hencky strain-rate in the Lagrangian state is obtained.

  16. Personality and Academic Motivation: Replication, Extension, and Replication

    ERIC Educational Resources Information Center

    Jones, Martin H.; McMichael, Stephanie N.

    2015-01-01

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

  17. Replicating systems concepts: Self-replicating lunar factory and demonstration

    NASA Technical Reports Server (NTRS)

    1982-01-01

    Automation of lunar mining and manufacturing facility maintenance and repair is addressed. Designing the factory as an automated, multiproduct, remotely controlled, reprogrammable Lunar Manufacturing Facility capable of constructing duplicates of itself which would themselves be capable of further replication is proposed.

  18. Exploiting replication in distributed systems

    NASA Technical Reports Server (NTRS)

    Birman, Kenneth P.; Joseph, T. A.

    1989-01-01

    Techniques are examined for replicating data and execution in directly distributed systems: systems in which multiple processes interact directly with one another while continuously respecting constraints on their joint behavior. Directly distributed systems are often required to solve difficult problems, ranging from management of replicated data to dynamic reconfiguration in response to failures. It is shown that these problems reduce to more primitive, order-based consistency problems, which can be solved using primitives such as the reliable broadcast protocols. Moreover, given a system that implements reliable broadcast primitives, a flexible set of high-level tools can be provided for building a wide variety of directly distributed application programs.

  19. A novel benzonitrile analogue inhibits rhinovirus replication.

    PubMed

    Lacroix, Céline; Querol-Audí, Jordi; Roche, Manon; Franco, David; Froeyen, Mathy; Guerra, Pablo; Terme, Thierry; Vanelle, Patrice; Verdaguer, Núria; Neyts, Johan; Leyssen, Pieter

    2014-10-01

    To study the characteristics and the mode of action of the anti-rhinovirus compound 4-[1-hydroxy-2-(4,5-dimethoxy-2-nitrophenyl)ethyl]benzonitrile (LPCRW_0005). The antiviral activity of LPCRW_0005 was evaluated in a cytopathic effect reduction assay against a panel of human rhinovirus (HRV) strains. To unravel its precise molecular mechanism of action, a time-of-drug-addition study, resistance selection and thermostability assays were performed. The crystal structure of the HRV14/LPCRW_0005 complex was elucidated as well. LPCRW_0005 proved to be a selective inhibitor of the replication of HRV14 (EC(50) of 2 ± 1 μM). Time-of-drug-addition studies revealed that LPCRW_0005 interferes with the earliest stages of virus replication. Phenotypic drug-resistant virus variants were obtained (≥30-fold decrease in susceptibility to the inhibitory effect of LPCRW_0005), which carried either an A150T or A150V amino acid substitution in the VP1 capsid protein. The link between the mutant genotype and drug-resistant phenotype was confirmed by reverse genetics. Cross-resistance studies and thermostability assays revealed that LPCRW_0005 has a similar mechanism of action to the capsid binder pleconaril. Elucidation of the crystal structure of the HRV14/LPCRW_0005 complex revealed the existence of multiple hydrophobic and polar interactions between the VP1 pocket and LPCRW_0005. LPCRW_0005 is a novel inhibitor of HRV14 replication that acts as a capsid binder. The compound has a chemical structure that is markedly smaller than that of other capsid binders. Structural studies show that LPCRW_0005, in contrast to pleconaril, leaves the toe end of the pocket in VP1 empty. This suggests that extended analogues of LPCRW_0005 that fill the full cavity could be more potent inhibitors of rhinovirus replication. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e

  20. Replication and Reporting: A Commentary.

    ERIC Educational Resources Information Center

    Polio, Charlene; Gass, Susan

    1997-01-01

    Addresses the need for replication studies in the field of second-language acquisition and discusses the problems surrounding standards of reporting research. Notes a lack of uniform standards in reporting second-language learners' proficiency levels and proposes ways to achieve more thorough reporting of research that will allow others to engage…

  1. Chameleon Chasing II: A Replication.

    ERIC Educational Resources Information Center

    Newsom, Doug A.; And Others

    1993-01-01

    Replicates a 1972 survey of students, educators, and Public Relations Society of America members regarding who the public relations counselor really serves. Finds that, in 1992, most respondents thought primary responsibility was to the client, then to the client's relevant publics, then to self, then to society, and finally to media. Compares…

  2. Replication of kinetoplast minicircle DNA

    SciTech Connect

    Sheline, C.T.

    1989-01-01

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

  3. Discovery and Optimization of Benzotriazine Di-N-Oxides Targeting Replicating and Non-replicating Mycobacterium tuberculosis

    PubMed Central

    Chopra, Sidharth; Koolpe, Gary A.; Tambo-ong, Arlyn A.; Matsuyama, Karen N.; Ryan, Kenneth J.; Tran, Tran B.; Doppalapudi, Rupa S.; Riccio, Edward S.; Iyer, Lalitha V.; Green, Carol E.; Wan, Baojie; Franzblau, Scott G.; Madrid, Peter B.

    2012-01-01

    Compounds bactericidal against both replicating and non-replicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and non-replicating Mtb. Medicinal chemistry optimization, guided by semi-empirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 μg/mL against H37Rv and a cytotoxicity (CC50) against Vero cells of 25 μg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs. PMID:22691154

  4. Phenotype Switching Is a Natural Consequence of Staphylococcus aureus Replication

    PubMed Central

    2012-01-01

    The pathogen Staphylococcus aureus undergoes phenotype switching in vivo from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small-colony-variant (SCV) phenotype that is associated with persistence in host cells and tissues. However, it is not clear whether phenotype switching is the result of a constitutive process that is selected for under certain conditions or is triggered by particular environmental stimuli. Examination of cultures of diverse S. aureus strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV subpopulation that emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was reestablished by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure, however, phenotype switching was bidirectional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that S. aureus phenotype switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant subpopulation of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the S. aureus life cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population. PMID:22865841

  5. Replication of nanoscale DNA patterns

    NASA Astrophysics Data System (ADS)

    Maass, Corinna; Wang, Tong; Sha, Ruojie; Leunissen, Mirjam; Dreyfus, Remi; Seeman, Nadrian; Chaikin, Paul

    2011-03-01

    We present an artificial supramolecular system mimicking self- replication and information transmission strategies in nature, but without the aid of enzymes or equivalent biological mechanisms. Using DNA nanotechnology techniques, we can make DNA tiles with selective interactions based on complementary single-strand connections. A linear tile pattern distinguished by their connector sequences is transmitted to a subsequent generation of copies by connector hybridisation. Longitudinal pattern formation and transverse copy attachment are well separated by different melting temperatures. We have achieved a faithful transmission of the pattern information to the second replication generation. We use AFM imaging to test for pattern fidelity and gel electrophoresis for quantitative yield analysis. supported by a DAAD postdoc grant.

  6. Replication Clamps and Clamp Loaders

    PubMed Central

    Hedglin, Mark; Kumar, Ravindra; Benkovic, Stephen J.

    2013-01-01

    To achieve the high degree of processivity required for DNA replication, DNA polymerases associate with ring-shaped sliding clamps that encircle the template DNA and slide freely along it. The closed circular structure of sliding clamps necessitates an enzyme-catalyzed mechanism, which not only opens them for assembly and closes them around DNA, but specifically targets them to sites where DNA synthesis is initiated and orients them correctly for replication. Such a feat is performed by multisubunit complexes known as clamp loaders, which use ATP to open sliding clamp rings and place them around the 3′ end of primer–template (PT) junctions. Here we discuss the structure and composition of sliding clamps and clamp loaders from the three domains of life as well as T4 bacteriophage, and provide our current understanding of the clamp-loading process. PMID:23545418

  7. Replication of Japanese Encephalitis Virus.

    DTIC Science & Technology

    1980-12-10

    replicative intermediate in minor amounts, were the only intracellular viral RNA molecules present (Boulton and Westaway, 1977; Wengler et al, 1978). We had...for (Svitkin et al., 1978) and against ( Wengler et al., 1979) this hypothesis has been presented and the question of number and sizes of flavivirus...West Nile virus ( Wengler , Wengler , and Gross, 1978) intracellular 40S RNAs are reported to have the type I cap structure m7GpppAm. Labeling with 3H

  8. Strain Gage

    NASA Technical Reports Server (NTRS)

    1995-01-01

    HITEC Corporation developed a strain gage application for DanteII, a mobile robot developed for NASA. The gage measured bending forces on the robot's legs and warned human controllers when acceptable forces were exceeded. HITEC further developed the technology for strain gage services in creating transducers out of "Indy" racing car suspension pushrods, NASCAR suspension components and components used in motion control.

  9. Bunyaviridae and Their Replication. Part 2. Replication of Bunyaviridae

    DTIC Science & Technology

    1990-01-01

    Virol 1984:137:227-240. 1984;49:717-723. 45. Eshita Y. Ericson B, Romanowski V, Bishop DHL. Analyses 23. Broadwell R, Oliver C. Golgi apparatus, GERL, and...McPhee DA. Bunyavirus pathogenesis. Adv 136. Romanowski V, Bishop DHL. Conserved sequences and cod- Viru.s Res 1985:30:279-316. ing of two strains of

  10. Saikosaponin A inhibits influenza A virus replication and lung immunopathology

    PubMed Central

    Zhao, Yaqin; Ling, Fangfang; Xiao, Kun; Li, Qian; Li, Bin; Lu, Chunni; Qi, Wenbao; Zeng, Zhenling; Liao, Ming; Liu, Yahong; Chen, Weisan

    2015-01-01

    Fatal influenza outcomes result from a combination of rapid virus replication and collateral lung tissue damage caused by exaggerated pro-inflammatory host immune cell responses. There are few therapeutic agents that target both biological processes for the attenuation of influenza-induced lung pathology. We show that Saikosaponin A, a bioactive triterpene saponin with previouslyestablished anti-inflammatory effects, demonstrates both in vitro and in vivo anti-viral activity against influenza A virus infections. Saikosaponin A attenuated the replication of three different influenza A virus strains, including a highly pathogenic H5N1 strain, in human alveolar epithelial A549 cells. This anti-viral activity occurred through both downregulation of NF-κB signaling and caspase 3-dependent virus ribonucleoprotein nuclear export as demonstrated by NF-κB subunit p65 and influenza virus nucleoprotein nuclear translocation studies in influenza virus infected A549 cells. Critically, Saikosaponin A also attenuated viral replication, aberrant pro-inflammatory cytokine production and lung histopathology in the widely established H1N1 PR8 model of influenza A virus lethality in C57BL/6 mice. Flow cytometry studies of mouse bronchoalveolar lavage cells revealed that SSa exerted immunomodulatory effects through a selective attenuation of lung neutrophil and monocyte recruitment during the early peak of the innate immune response to PR8 infection. Altogether, our results indicate that Saikosaponin A possesses novel therapeutic potential for the treatment of pathological influenza virus infections. PMID:26637810

  11. DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

    DTIC Science & Technology

    2006-04-01

    detecting gene duplications; (2) demonstrate our ability to perform high throughput microarray CGH on hundreds of yeast isolates ; (3) suggest that...detailed description of plasmid and strain construction. Yeast media , growth and arrest Cells were grown in YEP, synthetic complete (SC), or...replication in yeast cells. In the prior reporting period we demonstrated that re-replication induces a rapid and significant decrease in cell viability

  12. DNA Replication in Mycobacterium tuberculosis

    PubMed Central

    DITSE, ZANELE; LAMERS, MEINDERT H.; WARNER, DIGBY F.

    2017-01-01

    Faithful replication and maintenance of the genome are essential to the ability of any organism to survive and propagate. For an obligate pathogen such as Mycobacterium tuberculosis that has to complete successive cycles of transmission, infection, and disease in order to retain a foothold in the human population, this requires that genome replication and maintenance must be accomplished under the metabolic, immune, and antibiotic stresses encountered during passage through variable host environments. Comparative genomic analyses have established that chromosomal mutations enable M. tuberculosis to adapt to these stresses: the emergence of drug-resistant isolates provides direct evidence of this capacity, so too the well-documented genetic diversity among M. tuberculosis lineages across geographic loci, as well as the microvariation within individual patients that is increasingly observed as whole-genome sequencing methodologies are applied to clinical samples and tuberculosis (TB) disease models. However, the precise mutagenic mechanisms responsible for M. tuberculosis evolution and adaptation are poorly understood. Here, we summarize current knowledge of the machinery responsible for DNA replication in M. tuberculosis, and discuss the potential contribution of the expanded complement of mycobacterial DNA polymerases to mutagenesis. We also consider briefly the possible role of DNA replication—in particular, its regulation and coordination with cell division—in the ability of M. tuberculosis to withstand antibacterial stresses, including host immune effectors and antibiotics, through the generation at the population level of a tolerant state, or through the formation of a subpopulation of persister bacilli—both of which might be relevant to the emergence and fixation of genetic drug resistance. PMID:28361736

  13. Multiple replication origins with diverse control mechanisms in Haloarcula hispanica

    PubMed Central

    Wu, Zhenfang; Liu, Jingfang; Yang, Haibo; Liu, Hailong; Xiang, Hua

    2014-01-01

    The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is essential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompatible with the wild-type (but not the ΔoriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea. PMID:24271389

  14. How to securely replicate services

    NASA Technical Reports Server (NTRS)

    Reiter, Michael; Birman, Kenneth

    1992-01-01

    A method is presented for constructing replicated services that retain their availability and integrity despite several servers and clients corrupted by an intruder, in addition to others failing benignly. More precisely, a service is replicated by n servers in such a way that a correct client will accept a correct server's response if, for some prespecified parameter k, at least k servers are correct and fewer than k servers are corrupt. The issue of maintaining causality among client requests is also addressed. A security breach resulting from an intruder's ability to effect a violation of causality in the sequence of requests processed by the service is illustrated. An approach to counter this problem is proposed that requires fewer than k servers to be corrupt and that is live if at least k+b servers are correct, where b is the assumed maximum total number of corrupt servers in any system run. An important and novel feature of these schemes is that the client need not be able to identify or authenticate even a single server. Instead, the client is required only to possess at most two public keys for the service. The practicality of these schemes is illustrated through a discussion of several issues pertinent to their implementation and use, and their intended role in a secure version of the Isis system is also described.

  15. Bunyavirales ribonucleoproteins: the viral replication and transcription machinery.

    PubMed

    Sun, Yeping; Li, Jing; Gao, George F; Tien, Po; Liu, Wenjun

    2018-03-08

    The Bunyavirales order is one of the largest groups of segmented negative-sense single-stranded RNA viruses, which includes many pathogenic strains that cause severe human diseases. The RNA segments of the bunyavirus genome are separately encapsidated by multiple copies of nucleoprotein (N), and both termini of each N-encapsidated genomic RNA segment bind to one copy of the viral L polymerase protein. The viral genomic RNA, N and L protein together form the ribonucleoprotein (RNP) complex that constitutes the molecular machinery for viral genome replication and transcription. Recently, breakthroughs have been achieved in understanding the architecture of bunyavirus RNPs with the determination of the atomic structures of the N and L proteins from various members of this order. In this review, we discuss the structures and functions of these bunyavirus RNP components, as well as viral genome replication and transcription mechanisms.

  16. Minireview: DNA Replication in Plant Mitochondria

    PubMed Central

    Cupp, John D.; Nielsen, Brent L.

    2014-01-01

    Higher plant mitochondrial genomes exhibit much greater structural complexity as compared to most other organisms. Unlike well-characterized metazoan mitochondrial DNA (mtDNA) replication, an understanding of the mechanism(s) and proteins involved in plant mtDNA replication remains unclear. Several plant mtDNA replication proteins, including DNA polymerases, DNA primase/helicase, and accessory proteins have been identified. Mitochondrial dynamics, genome structure, and the complexity of dual-targeted and dual-function proteins that provide at least partial redundancy suggest that plants have a unique model for maintaining and replicating mtDNA when compared to the replication mechanism utilized by most metazoan organisms. PMID:24681310

  17. Optimal Placement of Origins for DNA Replication

    NASA Astrophysics Data System (ADS)

    Karschau, Jens; Blow, J. Julian; de Moura, Alessandro P. S.

    2012-02-01

    DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyze these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to the grouping of origins, and relate this to experimental data in a number of species showing origin grouping.

  18. Replication of prions in differentiated muscle cells.

    PubMed

    Herbst, Allen; Aiken, Judd M; McKenzie, Debbie

    2014-01-01

    We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication. (1) We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrP(Sc), differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions.

  19. Replication of prions in differentiated muscle cells

    PubMed Central

    Herbst, Allen; Aiken, Judd M; McKenzie, Debbie

    2014-01-01

    We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication.1 We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrPSc, differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions. PMID:24518642

  20. Cytosolic Replication of Group A Streptococcus in Human Macrophages.

    PubMed

    O'Neill, Alan M; Thurston, Teresa L M; Holden, David W

    2016-04-12

    As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus(GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria. Classically regarded as an extracellular pathogen, GAS can persist within human epithelial cells, as well as neutrophils and macrophages. Some studies suggest that GAS can modulate its intracellular vacuole to promote survival and perhaps replicate in macrophages. However, an in-depth single-cell analysis of the dynamics of survival and replication is lacking. We used macrophage-like cell lines and primary macrophages to measure the intracellular growth of GAS at both the population and single-cell levels. While CFU counts revealed no increase in overall bacterial growth, quantitative fluorescence microscopy

  1. Cell Cycle Regulation of DNA Replication

    PubMed Central

    Sclafani, R. A.; Holzen, T. M.

    2008-01-01

    Eukaryotic DNA replication is regulated to ensure all chromosomes replicate once and only once per cell cycle. Replication begins at many origins scattered along each chromosome. Except for budding yeast, origins are not defined DNA sequences and probably are inherited by epigenetic mechanisms. Initiation at origins occurs throughout the S phase according to a temporal program that is important in regulating gene expression during development. Most replication proteins are conserved in evolution in eukaryotes and archaea, but not in bacteria. However, the mechanism of initiation is conserved and consists of origin recognition, assembly of pre-replication (pre-RC) initiative complexes, helicase activation, and replisome loading. Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can only occur in G1 phase, whereas helicase activation and loading can only occur in S phase. Checkpoint regulation maintains high fidelity by stabilizing replication forks and preventing cell cycle progression during replication stress or damage. PMID:17630848

  2. Therapeutic targeting of replicative immortality

    PubMed Central

    Yaswen, Paul; MacKenzie, Karen L.; Keith, W. Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L.; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S.; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I.; Azmi, Asfar S.; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S. Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-01-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed “senescence,” can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells’ heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. PMID:25869441

  3. Herpesvirus saimiri terminal membrane proteins modulate HIV-1 replication by altering Nef and Tat functions.

    PubMed

    Raymond, Andrea D; Hasham, Muneer; Tsygankov, Alexander Y; Henderson, Earl E

    2007-01-01

    Herpesvirus saimiri (HVS)-transformed human T cells expressing terminal membrane proteins (TMPs) tyrosine kinase interacting protein (Tip) and saimiri transformation associated protein strain C (StpC) are highly permissive for R5 and X4 strains of HIV-1. StpC expression enhances replication of R5 and X4 strains of HIV-1 and induces latent reservoirs of replication competent HIV-1 in cell lines derived from T cells or monocytes. Paradoxically Tip expression restricts replication and cytopathic effects of R5 and X4 strains of HIV-1 in T cells and monocytes post-retrotransposition. Understanding the canonical pathways whereby Tip and StpC alter HIV-1 replication may uncover novel therapeutic approaches to HIV-1 infection. Here we show Tip inhibits Tat-mediated transcriptional activation of the long terminal repeat (LTR). Tip mediated inhibition of Tat transactivation is reversed by Nef. Tip also mediates restriction of late-stage replication of HIV-1 by disrupting Nef interaction with lymphocyte-specific protein-tyrosine kinase (Lck) in lipid rafts. Specifically, in the presence of Tip, Lck does not localize to lipid rafts reducing Nef interaction with Lck within the lipid rafts. Finally, the permissive phenotype conferred by StpC is the result of synergy with Tat during transcriptional activation of the HIV-1 LTR. This transcriptional synergy between StpC and Tat requires Lck and NF-kappaB consensus binding sequences. These findings demonstrate that the HVS TMPs influence transcriptional and post-transcriptional stages in HIV-1 replication. We propose that HVS-encoded TMPs associated with T cell transformation have evolved ability to modulate the replication of competing retroviruses. Gene based approaches utilizing Tip and StpC may provide therapeutic models for treating acute and latent HIV-1 infections, respectively.

  4. Overcoming natural replication barriers: differential helicase requirements.

    PubMed

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

    2012-02-01

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

  5. Self-replicating devices with dipolar colloids

    NASA Astrophysics Data System (ADS)

    Dempster, Joshua; Zhang, Rui; Olvera de La Cruz, Monica

    2014-03-01

    Ubiquitous in nature, self-replication on the nano-scale has been challenging to produce in the laboratory. Recent efforts with DNA tiles have shown great success in correctly replicating tile-sequence templates but require frequent manipulation by the experimenter. We propose a scheme for achieving self-replication with dipolar colloids. Dimers in these systems replicate exponentially over millisecond time scales with no intervention other than periodic energy pulses supplied by external fields. We develop a general formalism governing the rate of self-replication as a function of the interval between pulses. Results from kinetic Monte Carlo simulations show good agreement with the growth rates predicted by simple models of the replication process. We thank the Office of the Director of Defense Research and Engineering (DDR&E) and Air Force Office of Scientific Research (AFOSR) for their support under Award FA9550-10-1-0167.

  6. Targeting DNA Replication and Repair for the Development of Novel Therapeutics against Tuberculosis.

    PubMed

    Reiche, Michael A; Warner, Digby F; Mizrahi, Valerie

    2017-01-01

    Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), an infectious disease which results in approximately 10 million incident cases and 1.4 million deaths globally each year, making it the leading cause of mortality from infection. An effective frontline combination chemotherapy exists for TB; however, this regimen requires the administration of four drugs in a 2 month long intensive phase followed by a continuation phase of a further 4 months with two of the original drugs, and is only effective for the treatment of drug-sensitive TB. The emergence and global spread of multidrug-resistant (MDR) as well as extensively drug-resistant (XDR) strains of M. tuberculosis , and the complications posed by co-infection with the human immunodeficiency virus (HIV) and other co-morbidities such as diabetes, have prompted urgent efforts to develop shorter regimens comprising new compounds with novel mechanisms of action. This demands that researchers re-visit cellular pathways and functions that are essential to M. tuberculosis survival and replication in the host but which are inadequately represented amongst the targets of current anti-mycobacterial agents. Here, we consider the DNA replication and repair machinery as a source of new targets for anti-TB drug development. Like most bacteria, M. tuberculosis encodes a complex array of proteins which ensure faithful and accurate replication and repair of the chromosomal DNA. Many of these are essential; so, too, are enzymes in the ancillary pathways of nucleotide biosynthesis, salvage, and re-cycling, suggesting the potential to inhibit replication and repair functions at multiple stages. To this end, we provide an update on the state of chemotherapeutic inhibition of DNA synthesis and related pathways in M. tuberculosis . Given the established links between genotoxicity and mutagenesis, we also consider the potential implications of targeting DNA metabolic pathways implicated in the development of drug

  7. Targeting DNA Replication and Repair for the Development of Novel Therapeutics against Tuberculosis

    PubMed Central

    Reiche, Michael A.; Warner, Digby F.; Mizrahi, Valerie

    2017-01-01

    Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB), an infectious disease which results in approximately 10 million incident cases and 1.4 million deaths globally each year, making it the leading cause of mortality from infection. An effective frontline combination chemotherapy exists for TB; however, this regimen requires the administration of four drugs in a 2 month long intensive phase followed by a continuation phase of a further 4 months with two of the original drugs, and is only effective for the treatment of drug-sensitive TB. The emergence and global spread of multidrug-resistant (MDR) as well as extensively drug-resistant (XDR) strains of M. tuberculosis, and the complications posed by co-infection with the human immunodeficiency virus (HIV) and other co-morbidities such as diabetes, have prompted urgent efforts to develop shorter regimens comprising new compounds with novel mechanisms of action. This demands that researchers re-visit cellular pathways and functions that are essential to M. tuberculosis survival and replication in the host but which are inadequately represented amongst the targets of current anti-mycobacterial agents. Here, we consider the DNA replication and repair machinery as a source of new targets for anti-TB drug development. Like most bacteria, M. tuberculosis encodes a complex array of proteins which ensure faithful and accurate replication and repair of the chromosomal DNA. Many of these are essential; so, too, are enzymes in the ancillary pathways of nucleotide biosynthesis, salvage, and re-cycling, suggesting the potential to inhibit replication and repair functions at multiple stages. To this end, we provide an update on the state of chemotherapeutic inhibition of DNA synthesis and related pathways in M. tuberculosis. Given the established links between genotoxicity and mutagenesis, we also consider the potential implications of targeting DNA metabolic pathways implicated in the development of drug

  8. Mechanisms and strategies of papillomavirus replication.

    PubMed

    McBride, Alison A

    2017-07-26

    Animal and human papillomaviruses (HPVs) replicate persistently in specific types of stratified epithelia of their host. After the initial infection, the viral genome replicates at low levels in the dividing cells of the epithelium, and these cells form a reservoir of infection that can last for decades. When the infected cells differentiate, viral genomes replicate to high levels to form progeny virus that is released from the surface of the epithelium. This complex life cycle requires several different modes of viral DNA replication, but papillomaviruses are masters at hijacking key cellular processes to facilitate their own reproduction.

  9. Screening of the Pan-African Natural Product Library Identifies Ixoratannin A-2 and Boldine as Novel HIV-1 Inhibitors

    PubMed Central

    Tietjen, Ian; Ntie-Kang, Fidele; Mwimanzi, Philip; Onguéné, Pascal Amoa; Scull, Margaret A.; Idowu, Thomas Oyebode; Ogundaini, Abiodun Oguntuga; Meva’a, Luc Mbaze; Abegaz, Berhanu M.; Rice, Charles M.; Andrae-Marobela, Kerstin; Brockman, Mark A.; Brumme, Zabrina L.; Fedida, David

    2015-01-01

    The continued burden of HIV in resource-limited regions such as parts of sub-Saharan Africa, combined with adverse effects and potential risks of resistance to existing antiretroviral therapies, emphasize the need to identify new HIV inhibitors. Here we performed a virtual screen of molecules from the pan-African Natural Product Library, the largest collection of medicinal plant-derived pure compounds on the African continent. We identified eight molecules with structural similarity to reported interactors of Vpu, an HIV-1 accessory protein with reported ion channel activity. Using in vitro HIV-1 replication assays with a CD4+ T cell line and peripheral blood mononuclear cells, we confirmed antiviral activity and minimal cytotoxicity for two compounds, ixoratannin A-2 and boldine. Notably, ixoratannin A-2 retained inhibitory activity against recombinant HIV-1 strains encoding patient-derived mutations that confer resistance to protease, non-nucleoside reverse transcriptase, or integrase inhibitors. Moreover, ixoratannin A-2 was less effective at inhibiting replication of HIV-1 lacking Vpu, supporting this protein as a possible direct or indirect target. In contrast, boldine was less effective against a protease inhibitor-resistant HIV-1 strain. Both ixoratannin A-2 and boldine also inhibited in vitro replication of hepatitis C virus (HCV). However, BIT-225, a previously-reported Vpu inhibitor, demonstrated antiviral activity but also cytotoxicity in HIV-1 and HCV replication assays. Our work identifies pure compounds derived from African plants with potential novel activities against viruses that disproportionately afflict resource-limited regions of the world. PMID:25830320

  10. Character relations and replication identities in 2d Conformal Field Theory

    NASA Astrophysics Data System (ADS)

    Bantay, P.

    2016-10-01

    We study replication identities satisfied by conformal characters of a 2D CFT, providing a natural framework for a physics interpretation of the famous Hauptmodul property of Monstrous Moonshine, and illustrate the underlying ideas in simple cases.

  11. Quantifying the Antiviral Effect of IFN on HIV-1 Replication in Cell Culture

    PubMed Central

    Ikeda, Hiroki; Godinho-Santos, Ana; Rato, Sylvie; Vanwalscappel, Bénédicte; Clavel, François; Aihara, Kazuyuki; Iwami, Shingo; Mammano, Fabrizio

    2015-01-01

    Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses. PMID:26119462

  12. Quantifying the Antiviral Effect of IFN on HIV-1 Replication in Cell Culture

    NASA Astrophysics Data System (ADS)

    Ikeda, Hiroki; Godinho-Santos, Ana; Rato, Sylvie; Vanwalscappel, Bénédicte; Clavel, François; Aihara, Kazuyuki; Iwami, Shingo; Mammano, Fabrizio

    2015-06-01

    Type-I interferons (IFNs) induce the expression of hundreds of cellular genes, some of which have direct antiviral activities. Although IFNs restrict different steps of HIV replication cycle, their dominant antiviral effect remains unclear. We first quantified the inhibition of HIV replication by IFN in tissue culture, using viruses with different tropism and growth kinetics. By combining experimental and mathematical analyses, we determined quantitative estimates for key parameters of HIV replication and inhibition, and demonstrate that IFN mainly inhibits de novo infection (33% and 47% for a X4- and a R5-strain, respectively), rather than virus production (15% and 6% for the X4 and R5 strains, respectively). This finding is in agreement with patient-derived data analyses.

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

    NASA Astrophysics Data System (ADS)

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

    1994-07-01

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

  14. Directionality of replication fork movement determined by two-dimensional native-native DNA agarose gel electrophoresis.

    PubMed

    Ivessa, Andreas S

    2013-01-01

    The analysis of replication intermediates by the neutral-neutral two-dimensional agarose gel technique allows determining the chromosomal positions where DNA replication initiates, whether replication forks pause or stall at specific sites, or whether two DNA molecules undergo DNA recombination events. This technique does not, however, immediately tell in which direction replication forks migrate through the DNA region under investigation. Here, we describe the procedure to determine the direction of replication fork progression by carrying out a restriction enzyme digest of DNA imbedded in agarose after the completion of the first dimension of a 2D gel.

  15. Evaluation of porcine reproductive and respiratory syndrome virus replication in laboratory rodents

    PubMed Central

    Rosenfeld, Paul; Turner, Patricia V.; MacInnes, Janet I.; Nagy, Éva; Yoo, Dongwan

    2009-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major cause of economic losses in the swine industry. The disease is widespread worldwide, and so PRRSV-negative pigs are often difficult to find for the study of PRRSV in vivo. To determine if a small animal model could be developed for PRRSV, 3 strains of laboratory rodent were examined for their susceptibility to the virus. No virus replication was detected in BALB/c or SCID (severe combined immunodeficiency) mice after intraperitoneal inoculation. Moderate replication of PRRSV was detected in primary cotton rat lung cell cultures, but no viral replication was detected following intranasal or intraperitoneal inoculation. Following intratracheal inoculation, viral transcripts were detected in the lungs of cotton rats, but only for 1 day. This study indicates that PRRSV replication in common laboratory rodent species is inefficient, and suggests that a rodent model for this virus is not appropriate. PMID:20046635

  16. Arrest of Viral Proliferation by Ectopic Copies of Its Cognate Replication Origin

    PubMed Central

    Valenzuela, Manuel S.; Sharan, Chakradhari

    2015-01-01

    The initiation step of DNA replication is the crucial determinant of proliferation in all organisms. This step depends on the specific interaction of DNA sequences present at origins of DNA replication and their cognate activators. We wished to explore the hypothesis that the presence of ectopic origin copies may interfere with proper genome duplication. Bacteriophage λ was used as a model system. To this end, the outcome of an infection of an E. coli strain harboring ectopic copies of the λ origin region was analyzed. By measuring the effect on the host growth, viral production, and electro-microscopic visualization of the resulting λ replicative intermediates, we concluded that the ectopic copies had prevented the normal initiation step of λ DNA replication. These results suggest that DNA decoys encoding viral origins could constitute effective tools to specifically arrest viral proliferation. PMID:26110319

  17. Replicating Milgram Would People Still Obey Today?

    ERIC Educational Resources Information Center

    Burger, Jerry M.

    2009-01-01

    The author conducted a partial replication of Stanley Milgram's (1963, 1965, 1974) obedience studies that allowed for useful comparisons with the original investigations while protecting the well-being of participants. Seventy adults participated in a replication of Milgram's Experiment 5 up to the point at which they first heard the learner's…

  18. Replication and Robustness in Developmental Research

    ERIC Educational Resources Information Center

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

    2014-01-01

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

  19. How frog embryos replicate their DNA reliably

    NASA Astrophysics Data System (ADS)

    Bechhoefer, John; Marshall, Brandon

    2007-03-01

    Frog embryos contain three billion base pairs of DNA. In early embryos (cycles 2-12), DNA replication is extremely rapid, about 20 min., and the entire cell cycle lasts only 25 min., meaning that mitosis (cell division) takes place in about 5 min. In this stripped-down cell cycle, there are no efficient checkpoints to prevent the cell from dividing before its DNA has finished replication - a disastrous scenario. Even worse, the many origins of replication are laid down stochastically and are also initiated stochastically throughout the replication process. Despite the very tight time constraints and despite the randomness introduced by origin stochasticity, replication is extremely reliable, with cell division failing no more than once in 10,000 tries. We discuss a recent model of DNA replication that is drawn from condensed-matter theories of 1d nucleation and growth. Using our model, we discuss different strategies of replication: should one initiate all origins as early as possible, or is it better to hold back and initiate some later on? Using concepts from extreme-value statistics, we derive the distribution of replication times given a particular scenario for the initiation of origins. We show that the experimentally observed initiation strategy for frog embryos meets the reliability constraint and is close to the one that requires the fewest resources of a cell.

  20. Three Conceptual Replication Studies in Group Theory

    ERIC Educational Resources Information Center

    Melhuish, Kathleen

    2018-01-01

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

  1. Using Replication Projects in Teaching Research Methods

    ERIC Educational Resources Information Center

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

    2014-01-01

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

  2. Outreach: Replicating Services for Young Handicapped Children.

    ERIC Educational Resources Information Center

    Gunn, Lynn, Ed.

    Presented are eight author contributed chapters dealing with the outreach and replication of federally funded early education programs for handicapped children. M. Karnes and R. Zehrbach consider decisions regarding identification and assessment of replicable products (such as curricula and audiovisual presentations). Discussed by D. Stedman are…

  3. Chromatin signatures of the Drosophila replication program

    PubMed Central

    Eaton, Matthew L.; Prinz, Joseph A.; MacAlpine, Heather K.; Tretyakov, George; Kharchenko, Peter V.; MacAlpine, David M.

    2011-01-01

    DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events. PMID:21177973

  4. Characteristics of the poliovirus replication complex.

    PubMed

    Bienz, K; Egger, D; Pfister, T

    1994-01-01

    In the infected cell, the poliovirus replication complex (RC) is found in the center of a rosette formed by many virus-induced vesicles. The RC is attached to the vesicular membranes and contains a compact central part which encloses the replication forks of the replicative intermediate and all proteins necessary for strand elongation. The growing plus strands of the replicative intermediate protrude from the central part of the RC, but are still enclosed by membraneous structures of the rosette. After completion, progeny 36S RNA is set free at the surface of the rosette. In an in vitro transcription system, isolated replication complex-containing rosettes are active in initiation, elongation and maturation (release) of plus strand progeny RNA. Full functionality of the RC depends on an intact structural framework of all membraneous components of the rosette.

  5. Rescue from replication stress during mitosis.

    PubMed

    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.

  6. Rescue from replication stress during mitosis

    PubMed Central

    Naim, Valeria

    2017-01-01

    ABSTRACT 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. PMID:28166452

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

    ERIC Educational Resources Information Center

    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…

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

    ERIC Educational Resources Information Center

    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…

  9. Ribosome biogenesis in replicating cells: Integration of experiment and theory.

    PubMed

    Earnest, Tyler M; Cole, John A; Peterson, Joseph R; Hallock, Michael J; Kuhlman, Thomas E; Luthey-Schulten, Zaida

    2016-10-01

    Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016. © 2016 Wiley Periodicals, Inc.

  10. Replication efficiency of oncolytic vaccinia virus in cell cultures prognosticates the virulence and antitumor efficacy in mice

    PubMed Central

    2011-01-01

    Background We have shown that insertion of the three vaccinia virus (VACV) promoter-driven foreign gene expression cassettes encoding Renilla luciferase-Aequorea GFP fusion protein, β-galactosidase, and β-glucuronidase into the F14.5L, J2R, and A56R loci of the VACV LIVP genome, respectively, results in a highly attenuated mutant strain GLV-1h68. This strain shows tumor-specific replication and is capable of eradicating tumors with little or no virulence in mice. This study aimed to distinguish the contribution of added VACV promoter-driven transcriptional units as inserts from the effects of insertional inactivation of three viral genes, and to determine the correlation between replication efficiency of oncolytic vaccinia virus in cell cultures and the virulence and antitumor efficacy in mice Methods A series of recombinant VACV strains was generated by replacing one, two, or all three of the expression cassettes in GLV-1h68 with short non-coding DNA sequences. The replication efficiency and tumor cell killing capacity of these newly generated VACV strains were compared with those of the parent virus GLV-1h68 in cell cultures. The virus replication efficiency in tumors and antitumor efficacy as well as the virulence were evaluated in nu/nu (nude) mice bearing human breast tumor xenografts. Results we found that virus replication efficiency increased with removal of each of the expression cassettes. The increase in virus replication efficiency was proportionate to the strength of removed VACV promoters linked to foreign genes. The replication efficiency of the new VACV strains paralleled their cytotoxicity in cell cultures. The increased replication efficiency in tumor xenografts resulted in enhanced antitumor efficacy in nude mice. Similarly, the enhanced virus replication efficiency was indicative of increased virulence in nude mice. Conclusions These data demonstrated that insertion of VACV promoter-driven transcriptional units into the viral genome for the

  11. Factors influencing microinjection molding replication quality

    NASA Astrophysics Data System (ADS)

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

    2018-01-01

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

  12. The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2(-/_) knockout and C57BL/6N parental strains of mice

    EPA Science Inventory

    Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ra...

  13. The Effect of Dose on 2,3,7,8-TCDD Tissue Distribution, Metabolism and Elimination in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    USDA-ARS?s Scientific Manuscript database

    Numerous metabolism studies have demonstrated that the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver to fat concentration ratios. This study was in...

  14. Top 10 Replicated Findings from Behavioral Genetics

    PubMed Central

    Plomin, Robert; DeFries, John C.; Knopik, Valerie S.; Neiderhiser, Jenae M.

    2015-01-01

    In the context of current concerns about replication in psychological science, we describe 10 findings from behavioral genetic research that have robustly replicated. These are ‘big’ findings, both in terms of effect size and potential impact on psychological science, such as linearly increasing heritability of intelligence from infancy (20%) through adulthood (60%). Four of our top-10 findings involve the environment, discoveries that could only have been found using genetically sensitive research designs. We also consider reasons specific to behavioral genetics that might explain why these findings replicate. PMID:26817721

  15. Replication and Analysis of Ebbinghaus' Forgetting Curve.

    PubMed

    Murre, Jaap M J; Dros, Joeri

    2015-01-01

    We present a successful replication of Ebbinghaus' classic forgetting curve from 1880 based on the method of savings. One subject spent 70 hours learning lists and relearning them after 20 min, 1 hour, 9 hours, 1 day, 2 days, or 31 days. The results are similar to Ebbinghaus' original data. We analyze the effects of serial position on forgetting and investigate what mathematical equations present a good fit to the Ebbinghaus forgetting curve and its replications. We conclude that the Ebbinghaus forgetting curve has indeed been replicated and that it is not completely smooth but most probably shows a jump upwards starting at the 24 hour data point.

  16. Replication and Analysis of Ebbinghaus’ Forgetting Curve

    PubMed Central

    Murre, Jaap M. J.; Dros, Joeri

    2015-01-01

    We present a successful replication of Ebbinghaus’ classic forgetting curve from 1880 based on the method of savings. One subject spent 70 hours learning lists and relearning them after 20 min, 1 hour, 9 hours, 1 day, 2 days, or 31 days. The results are similar to Ebbinghaus' original data. We analyze the effects of serial position on forgetting and investigate what mathematical equations present a good fit to the Ebbinghaus forgetting curve and its replications. We conclude that the Ebbinghaus forgetting curve has indeed been replicated and that it is not completely smooth but most probably shows a jump upwards starting at the 24 hour data point. PMID:26148023

  17. Segregation of Induced Frameshift Mutations and the Sequence of Gene Replication in ESCHERICHIA COLI K12

    PubMed Central

    Hoess, Ronald H.; Herman, Robert K.

    1973-01-01

    We have constructed a strain of E. coli K12 carrying six mutations induced by the acridine half-mustard ICR-191. The mutations are widely spaced on the E. coli linkage map and are all easily reverted by ICR-191. Mapping of ten independent revertants for each of five markers indicated that the reversions induced by ICR-191 occurred near the original mutations. Exponentially and nonsynchronously growing cultures of this strain were exposed to ICR-191 for 0.85 generation, quickly washed free of mutagen, and resuspended in the original medium minus mutagen. Total viable cell number maintained its exponential increase both during and immediately after exposure to mutagen, whereas the number of revertants of any particular type remained constant for a characteristic period after removal of mutagen before finally assuming an exponential increase. Theoretically, the length of such a segregation lag should depend on the position of the particular reverted gene in the sequence of gene replication: the earlier a gene is replicated in the chromosome replication cycle, the longer its segregation lag should be. Our results are consistent with this prediction and fit a unidirectional, clockwise replication scheme with an origin between 55 and 74 min on the E. coli linkage map. The results also fit a very asymmetric bidirectional replication scheme. PMID:17248614

  18. Is psychology suffering from a replication crisis? What does "failure to replicate" really mean?

    PubMed

    Maxwell, Scott E; Lau, Michael Y; Howard, George S

    2015-09-01

    Psychology has recently been viewed as facing a replication crisis because efforts to replicate past study findings frequently do not show the same result. Often, the first study showed a statistically significant result but the replication does not. Questions then arise about whether the first study results were false positives, and whether the replication study correctly indicates that there is truly no effect after all. This article suggests these so-called failures to replicate may not be failures at all, but rather are the result of low statistical power in single replication studies, and the result of failure to appreciate the need for multiple replications in order to have enough power to identify true effects. We provide examples of these power problems and suggest some solutions using Bayesian statistics and meta-analysis. Although the need for multiple replication studies may frustrate those who would prefer quick answers to psychology's alleged crisis, the large sample sizes typically needed to provide firm evidence will almost always require concerted efforts from multiple investigators. As a result, it remains to be seen how many of the recently claimed failures to replicate will be supported or instead may turn out to be artifacts of inadequate sample sizes and single study replications. (PsycINFO Database Record (c) 2015 APA, all rights reserved).

  19. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing.

    PubMed

    Hansen, R Scott; Thomas, Sean; Sandstrom, Richard; Canfield, Theresa K; Thurman, Robert E; Weaver, Molly; Dorschner, Michael O; Gartler, Stanley M; Stamatoyannopoulos, John A

    2010-01-05

    Faithful transmission of genetic material to daughter cells involves a characteristic temporal order of DNA replication, which may play a significant role in the inheritance of epigenetic states. We developed a genome-scale approach--Repli Seq--to map temporally ordered replicating DNA using massively parallel sequencing and applied it to study regional variation in human DNA replication time across multiple human cell types. The method requires as few as 8,000 cytometry-fractionated cells for a single analysis, and provides high-resolution DNA replication patterns with respect to both cell-cycle time and genomic position. We find that different cell types exhibit characteristic replication signatures that reveal striking plasticity in regional replication time patterns covering at least 50% of the human genome. We also identified autosomal regions with marked biphasic replication timing that include known regions of monoallelic expression as well as many previously uncharacterized domains. Comparison with high-resolution genome-wide profiles of DNaseI sensitivity revealed that DNA replication typically initiates within foci of accessible chromatin comprising clustered DNaseI hypersensitive sites, and that replication time is better correlated with chromatin accessibility than with gene expression. The data collectively provide a unique, genome-wide picture of the epigenetic compartmentalization of the human genome and suggest that cell-lineage specification involves extensive reprogramming of replication timing patterns.

  20. DNA replication origins, ORC/DNA interaction, and assembly of pre-replication complex in eukaryotes.

    PubMed

    Sun, Jingya; Kong, Daochun

    2010-07-01

    Chromosomal DNA replication in eukaryotic cells is highly complicated and sophisticatedly regulated. Owing to its large size, a typical eukaryotic genome contains hundreds to tens of thousands of initiation sites called DNA replication origins where DNA synthesis takes place. Multiple initiation sites remove the constraint of a genome size because only a certain amount of DNA can be replicated from a single origin in a limited time. The activation of these multiple origins must be coordinated so that each segment of chromosomal DNA is precisely duplicated only once per cell cycle. Although DNA replication is a vital process for cell growth and its mechanism is highly conserved, recent studies also reveal significant diversity in origin structure, assembly of pre-replication complex (pre-RC) and regulation of replication initiation along evolutionary lines. The DNA replication origins in the fission yeast Schizosaccharomyces pombe are found to contain a second essential element that is bound by Sap1 protein besides the essential origin recognition complex-binding site. Sap1 is recently demonstrated to be a novel replication initiation protein that plays an essential role in loading the initiation protein Cdc18 to origins and thus directly participates in pre-RC formation. In this review, we summarize the recent advance in understanding how DNA replication origins are organized, how pre-RC is assembled and how DNA replication is initiated and regulated in yeast and metazoans.

  1. A novel system for identification of inhibitors of rift valley Fever virus replication.

    PubMed

    Piper, Mary E; Gerrard, Sonja R

    2010-03-01

    Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication.

  2. Commercial Building Partnerships Replication and Diffusion

    SciTech Connect

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

    2013-09-16

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

  3. Replication and robustness in developmental research.

    PubMed

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

    2014-11-01

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

  4. Nucleosomes influence multiple steps during replication initiation

    PubMed Central

    Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F; Kang, Sukhyun; Belsky, Jason A; MacAlpine, David M; Peterson, Craig L; Bell, Stephen P

    2017-01-01

    Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin. DOI: http://dx.doi.org/10.7554/eLife.22512.001 PMID:28322723

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

    PubMed

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

    2018-03-01

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

  6. In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies

    PubMed Central

    Marzio, Giuseppe; Verhoef, Koen; Vink, Monique; Berkhout, Ben

    2001-01-01

    A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tet-controlled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine. PMID:11353837

  7. Cryptoporic acid E from Cryptoporus volvatus inhibits influenza virus replication in vitro.

    PubMed

    Gao, Li; Han, Jiayuan; Si, Jianyong; Wang, Junchi; Wang, Hexiang; Sun, Yipeng; Bi, Yuhai; Liu, Jinhua; Cao, Li

    2017-07-01

    Influenza virus infection is a global public health issue. The efficacy of antiviral agents for influenza virus has been limited by the emergence of drug-resistant virus strains. Thus, there is an urgent need to identify novel antiviral therapies. Our previous studies have found that Cryptoporus volvatus extract can potently inhibit influenza virus replication in vitro and in vivo. However, the effective component of Cryptoporus volvatus, which mediates the antiviral activity, hasn't been identified. Here, we identified a novel anti-influenza virus molecule, Cryptoporic acid E (CAE), from Cryptoporus volvatus. Our results showed that CAE had broad-spectrum anti-influenza activity against 2009 pandemic strain A/Beijing/07/2009 (H1N1/09pdm), seasonal strain A/Beijing/CAS0001/2007(H3N2), mouse adapted strains A/WSN/33 (H1N1), and A/PR8/34 (H1N1). We further investigated the mode of CAE action. Time-course-analysis indicated that CAE exerted its inhibition mainly at the middle stages of the replication cycle of influenza virus. Subsequently, we confirmed that CAE inhibited influenza virus RNA polymerase activity and blocked virus RNA replication and transcription in MDCK cells. In addition, we found that CAE also impaired influenza virus infectivity by directly targeting virus particles. Our data suggest that CAE is a major effective component of Cryptoporus volvatus. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The structure and function of replication protein A in DNA replication.

    PubMed

    Prakash, Aishwarya; Borgstahl, Gloria E O

    2012-01-01

    In all organisms from bacteria and archaea to eukarya, single-stranded DNA binding proteins play an essential role in most, if not all, nuclear metabolism involving single-stranded DNA (ssDNA). Replication protein A (RPA), the major eukaryotic ssDNA binding protein, has two important roles in DNA metabolism: (1) in binding ssDNA to protect it and to keep it unfolded, and (2) in coordinating the assembly and disassembly of numerous proteins and protein complexes during processes such as DNA replication. Since its discovery as a vital player in the process of replication, RPAs roles in recombination and DNA repair quickly became evident. This chapter summarizes the current understanding of RPA's roles in replication by reviewing the available structural data, DNA-binding properties, interactions with various replication proteins, and interactions with DNA repair proteins when DNA replication is stalled.

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

    PubMed

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

    2014-09-02

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

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

    PubMed Central

    Xu, Kai

    2017-01-01

    ABSTRACT 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

  11. Species-barrier-independent prion replication in apparently resistant species

    NASA Astrophysics Data System (ADS)

    Hill, Andrew F.; Joiner, Susan; Linehan, Jackie; Desbruslais, Melanie; Lantos, Peter L.; Collinge, John

    2000-08-01

    Transmission of prions between mammalian species is thought to be limited by a "species barrier," which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.

  12. Digoxin Suppresses HIV-1 Replication by Altering Viral RNA Processing

    PubMed Central

    Wong, Raymond W.; Balachandran, Ahalya; Ostrowski, Mario A.; Cochrane, Alan

    2013-01-01

    To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2β, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection. PMID:23555254

  13. Poliovirus replication and spread in primary neuron cultures.

    PubMed

    Daley, John K; Gechman, Lisa A; Skipworth, Jason; Rall, Glenn F

    2005-09-15

    While some neurotropic viruses cause rapid central nervous system (CNS) disease upon entry into the brain parenchyma, other viruses that are cytolytic in the periphery either result in little neuropathology or are associated with a protracted course of CNS disease consistent with persistent infection. One such virus, poliovirus (PV), is an extremely lytic RNA virus that requires the expression of CD155, the poliovirus receptor (PVR), for infection. To compare the kinetics of PV infection in neuronal and non-neuronal cell types, primary hippocampal neurons and fibroblasts were isolated from CD155+ transgenic embryos and infected with the Mahoney and Sabin strains of PV. Despite similar levels of infection in these ex vivo cultures, PV-infected neurons produced 100-fold fewer infectious particles as compared to fibroblasts throughout infection, and death of PV-infected neurons was delayed approximately 48 h. Spread in neurons occurred primarily by trans-synaptic transmission and was CD155-dependent. Together, these results demonstrate that the magnitude and speed with which PV replication, spread, and subsequent cell death occur in neurons is decreased as compared to non-neuronal cells, implicating cell-specific effects on replication that may then influence viral pathogenesis.

  14. AN ORGANOTYPIC UNIAXIAL STRAIN MODEL USING MICROFLUIDICS

    PubMed Central

    Dollé, Jean-Pierre; Morrison, Barclay; Schloss, Rene R.; Yarmush, Martin L.

    2012-01-01

    Traumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. The injury effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strains to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This uniaxial strain model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The device has the ability to strain either individual axons or bundles of axons through the control of microchannel dimensions. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects and breakdown in microtubules. Microtubule breakdown was shown to be dependent on the degree of the applied strain field, where maximal breakdown was observed at peak strain and minimal breakdown is observed at low strain. This strain injury model could be a powerful tool in assessing strain injury effects on functional axonal connections. PMID:23233120

  15. [Genetic and biochemical studies on the mechanism of mammalian chromosome replication].

    PubMed

    Hanaoka, F

    1989-11-01

    It has been clarified that basic mechanism of deoxyribonucleic acid (DNA) replication is conserved from bacteria to higher cells. What distinguishes prokaryotic and eukaryotic modes of DNA replication most clearly is that bacterial chromosomes form a single replicon copied from a single initiation point and eukaryotic chromosomes consist of multiple replicons that initiate at multiple points. Thus, eukaryotes have to coordinate orderly replication of the genome. In order to understand this complex problem as a whole, three approaches were chosen. First approach is a genetic one. Certain number of temperature-sensitive (ts) mutants were isolated from mouse FM3A cells. One of the ts mutants, designated as tsFT20, was shown to contain heat-labile DNA polymerase alpha (pol. alpha). By the use of this mutant strain, it was proved that pol. alpha is essential for mammalian DNA replication. In addition, the human gene for pol. alpha on the X chromosome was assigned. Second approach is an enzymological one. FM3A cells were used for the identification and characterization of enzymes and proteins supposed to be involved in DNA replication. Four DNA-dependent ATPases, three pol. alpha stimulation factors, DNA topoisomerases I and II have been identified, as well as a stimulation factor for the assembly of nucleosome. DNA helicase activity was detected in two of the DNA-dependent ATPase (B and C1). Third approach is the reconstitution of DNA replication in cell-free system. By use of polyoma virus DNA as a template, cell-free extract from FM3A cells supported DNA replication in the presence of polyoma virus large T-antigen. This cell-free system will be useful for the analysis of the function of replication enzymes and proteins as well as the characterization of ts mutants.

  16. Optical tweezers reveal how proteins alter replication

    NASA Astrophysics Data System (ADS)

    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

  17. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication

    PubMed Central

    Guo, Longjun; Yu, Haidong; Gu, Weihong; Luo, Xiaolei; Li, Ren; Zhang, Jian; Xu, Yunfei; Yang, Lijun; Shen, Nan; Feng, Li; Wang, Yue

    2016-01-01

    Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication. PMID:27029407

  18. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

    PubMed

    Guo, Longjun; Yu, Haidong; Gu, Weihong; Luo, Xiaolei; Li, Ren; Zhang, Jian; Xu, Yunfei; Yang, Lijun; Shen, Nan; Feng, Li; Wang, Yue

    2016-03-31

    Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

  19. The evolutionary ecology of molecular replicators.

    PubMed

    Nee, Sean

    2016-08-01

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

  20. The evolutionary ecology of molecular replicators

    PubMed Central

    2016-01-01

    By reasonable criteria, life on the Earth consists mainly of molecular replicators. These include viruses, transposons, transpovirons, coviruses and many more, with continuous new discoveries like Sputnik Virophage. Their study is inherently multidisciplinary, spanning microbiology, genetics, immunology and evolutionary theory, and the current view is that taking a unified approach has great power and promise. We support this with a new, unified, model of their evolutionary ecology, using contemporary evolutionary theory coupling the Price equation with game theory, studying the consequences of the molecular replicators' promiscuous use of each others' gene products for their natural history and evolutionary ecology. Even at this simple expository level, we can make a firm prediction of a new class of replicators exploiting viruses such as lentiviruses like SIVs, a family which includes HIV: these have been explicitly stated in the primary literature to be non-existent. Closely connected to this departure is the view that multicellular organism immunology is more about the management of chronic infections rather than the elimination of acute ones and new understandings emerging are changing our view of the kind of theatre we ourselves provide for the evolutionary play of molecular replicators. This study adds molecular replicators to bacteria in the emerging field of sociomicrobiology. PMID:27853598

  1. Methadone enhances human influenza A virus replication.

    PubMed

    Chen, Yun-Hsiang; Wu, Kuang-Lun; Tsai, Ming-Ta; Chien, Wei-Hsien; Chen, Mao-Liang; Wang, Yun

    2017-01-01

    Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs. © 2015 Society for the Study of Addiction.

  2. A new method for culturing Plasmodium falciparum shows replication at the highest erythrocyte densities

    NASA Technical Reports Server (NTRS)

    Li, Tao; Glushakova, Svetlana; Zimmerberg, Joshua

    2003-01-01

    Plasmodium falciparum replicates poorly in erythrocyte densities greater than a hematocrit of 20%. A new method to culture the major malaria parasite was developed by using a hollow fiber bioreactor that preserves healthy erythrocytes at hematocrit up to 100%. P. falciparum replicated equally well at all densities studied. This method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof), because high yields ( approximately 10(10) in 4 days) could be prepared with less cost and labor. Concomitantly, secreted proteins were concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8%-12% with the 3D7 strain. The finding that P. falciparum can replicate at packed erythrocyte densities suggests that this system may be useful for study of the pathogenesis of fatal cerebral malaria, of which one feature is densely packed blood cells in brain microvasculature.

  3. Lagomorphs (rabbits, pikas and hares) do not use telomere-directed replicative aging in vitro.

    PubMed

    Forsyth, Nicholas R; Elder, Frederick F B; Shay, Jerry W; Wright, Woodring E

    2005-01-01

    Telomere shortening is used for replicative aging in primates and ungulates but not rodents. We examined telomere biology in rabbits to expand the comparative biology of telomere-directed replicative senescence within mammals. The order Lagomorpha consists of two families; Leporidae and Ochotonidae. We examined telomere biology in species representing three leporid genera (European White Rabbit, Black-tailed Jack Rabbit, and Swamp Rabbit) and the monotypic ochotonid genus (North American Pika). Of the leporids one species was a laboratory strain and the others were wild caught. The leporids neither exhibited cellular senescence after sustained periods in culture nor displayed detectable telomerase activity. Continued culture was possible because of their extremely long telomeric arrays. Immunofluorescence showed robust telomere signals at chromosome ends and significant internal chromosomal staining in some instances. Pika was unique in displaying endogenous telomerase activity throughout time in culture. These results show that it is unlikely that lagomorphs use telomere shortening and replicative senescence as a tumor protective mechanism.

  4. The role of equine herpesvirus type 4 glycoprotein k in virus replication.

    PubMed

    Azab, Walid; El-Sheikh, Abuelyazeed

    2012-08-01

    Equine herpesvirus 4 (EHV-4) is an important equine pathogen that causes respiratory tract disease among horses worldwide. Glycoprotein K (gK) homologues have been identified in several alphaherpesviruses as a major player in virus entry, replication, and spread. In the present study, EHV-4 gK-deletion mutant has been generated by using bacterial artificial chromosome technology and Red mutagenesis to investigate the role of gK in EHV-4 replication. Our findings reported here show that gK is essential for virus replication in vitro and that the gK-negative strain was not able to be reconstituted in equine cells. It is noteworthy that these findings agree with the previously published study describing gK deletion in other alphaherpesviruses.

  5. SLFN11 Blocks Stressed Replication Forks Independently of ATR.

    PubMed

    Murai, Junko; Tang, Sai-Wen; Leo, Elisabetta; Baechler, Simone A; Redon, Christophe E; Zhang, Hongliang; Al Abo, Muthana; Rajapakse, Vinodh N; Nakamura, Eijiro; Jenkins, Lisa M Miller; Aladjem, Mirit I; Pommier, Yves

    2018-02-01

    SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites. Published by Elsevier Inc.

  6. PTEN Regulates DNA Replication Progression and Stalled Fork Recovery

    PubMed Central

    He, Jinxue; Kang, Xi; Yin, Yuxin; Chao, K.S. Clifford; Shen, Wen H.

    2015-01-01

    Faithful DNA replication is a cornerstone of genomic integrity. PTEN plays multiple roles in genome protection and tumor suppression. Here we report on the importance of PTEN in DNA replication. PTEN depletion leads to impairment of replication progression and stalled fork recovery, indicating an elevation of endogenous replication stress. Exogenous replication inhibition aggravates replication-originated DNA lesions without inducing S-phase arrest in cells lacking PTEN, representing replication stress tolerance. Our analysis reveals the physical association of PTEN with DNA replication forks and PTEN-dependent recruitment of Rad51. PTEN deletion results in Rad51 dissociation from replication forks. Stalled replication forks in Pten null cells can be reactivated by ectopic Rad51 or PTEN, the latter facilitating chromatin loading of Rad51. These data highlight the interplay of PTEN with Rad51 in promoting stalled fork restart. We propose that loss of PTEN may initiate a replication stress cascade that progressively deteriorates through the cell cycle. PMID:26158445

  7. Replicating Large Genomes: Divide and Conquer

    PubMed Central

    Rivera-Mulia, Juan Carlos; Gilbert, David M.

    2016-01-01

    Complete duplication of large metazoan chromosomes requires thousands of potential initiation sites, only a small fraction of which are selected in each cell. Assembly of the replication machinery is highly conserved and tightly regulated during cell cycle, but the sites of initiation are highly flexible their temporal order of firing is regulated at the level of large-scale multi-replicon domains. Additionally, the number of replication forks needs to be quickly adjusted in response to stress to ensure complete replication and prevent genome instability. Here we argue that large genomes are divided into domains for exactly this reason. Domain structure abrogates the need for precise initiation sites and creates a scaffold for the evolution of other chromosome functions. PMID:27259206

  8. Replication factory activation can be decoupled from the replication timing program by modulating Cdk levels

    PubMed Central

    Thomson, Alexander M.; Gillespie, Peter J.

    2010-01-01

    In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase. PMID:20083602

  9. Invasive Salmonella Typhimurium ST313 with naturally attenuated flagellin elicits reduced inflammation and replicates within macrophages.

    PubMed

    Ramachandran, Girish; Perkins, Darren J; Schmidlein, Patrick J; Tulapurkar, Mohan E; Tennant, Sharon M

    2015-01-01

    Invasive non-typhoidal Salmonella (iNTS) are an important cause of septicemia in children under the age of five years in sub-Saharan Africa. A novel genotype of Salmonella enterica subsp. enterica serovar Typhimurium (multi-locus sequence type [ST] 313) circulating in this geographic region is genetically different to from S. Typhimurium ST19 strains that are common throughout the rest of the world. S. Typhimurium ST313 strains have acquired pseudogenes and genetic deletions and appear to be evolving to become more like the typhoidal serovars S. Typhi and S. Paratyphi A. Epidemiological and clinical data show that S. Typhimurium ST313 strains are clinically associated with invasive systemic disease (bacteremia, septicemia, meningitis) rather than with gastroenteritis. The current work summarizes investigations of the broad hypothesis that S. Typhimurium ST313 isolates from Mali, West Africa, will behave differently from ST19 isolates in various in vitro assays. Here, we show that strains of the ST313 genotype are phagocytosed more efficiently and are highly resistant to killing by macrophage cell lines and primary mouse and human macrophages compared to ST19 strains. S. Typhimurium ST313 strains survived and replicated within different macrophages. Infection of macrophages with S. Typhimurium ST19 strains resulted in increased apoptosis and higher production of proinflammatory cytokines, as measured by gene expression and protein production, compared to S. Typhimurium ST313 strains. This difference in proinflammatory cytokine production and cell death between S. Typhimurium ST19 and ST313 strains could be explained, in part, by an increased production of flagellin by ST19 strains. These observations provide further evidence that S. Typhimurium ST313 strains are phenotypically different to ST19 strains and instead share similar pathogenic characteristics with typhoidal Salmonella serovars.

  10. Mycobacterial DNA Replication as a Target for Antituberculosis Drug Discovery.

    PubMed

    Płocinska, Renata; Korycka-Machala, Malgorzata; Plocinski, Przemyslaw; Dziadek, Jaroslaw

    2017-06-16

    Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is a leading infectious disease organism, causing millions of deaths each year. This serious pathogen has been greatly spread worldwide and recent years have observed an increase in the number of multi-drug resistant and totally drug resistant M. tuberculosis strains (WHO report, 2014). The danger of tuberculosis becoming an incurable disease has emphasized the need for the discovery of a new generation of antimicrobial agents. The development of novel alternative medical strategies, new drugs and the search for optimal drug targets are top priority areas of tuberculosis research. Key characteristics of mycobacteria include: slow growth, the ability to transform into a metabolically silent - latent state, intrinsic drug resistance and the relatively rapid development of acquired drug resistance. These factors make finding an ideal antituberculosis drug enormously challenging, even if it is designed to treat drug sensitive tuberculosis strains. A vast majority of canonical antibiotics including antituberculosis agents target bacterial cell wall biosynthesis or DNA/RNA processing. Novel therapeutic approaches are being tested to target mycobacterial cell division, twocomponent regulatory factors, lipid synthesis and the transition between the latent and actively growing states. This review discusses the choice of cellular targets for an antituberculosis therapy, describes putative drug targets evaluated in the recent literature and summarizes potential candidates under clinical and pre-clinical development. We focus on the key cellular process of DNA replication, as a prominent target for future antituberculosis therapy. We describe two main pathways: the biosynthesis of nucleic acids precursors - the nucleotides, and the synthesis of DNA molecules. We summarize data regarding replication associated proteins that are critical for nucleotide synthesis, initiation, unwinding and

  11. African swine fever virus replication and genomics.

    PubMed

    Dixon, Linda K; Chapman, David A G; Netherton, Christopher L; Upton, Chris

    2013-04-01

    African swine fever virus (ASFV) is a large icosahedral DNA virus which replicates predominantly in the cytoplasm of infected cells. The ASFV double-stranded DNA genome varies in length from about 170 to 193 kbp depending on the isolate and contains between 150 and 167 open reading frames. These are closely spaced and read from both DNA strands. The virus genome termini are covalently closed by imperfectly base-paired hairpin loops that are present in two forms that are complimentary and inverted with respect to each other. Adjacent to the termini are inverted arrays of different tandem repeats. Head to head concatemeric genome replication intermediates have been described. A similar mechanism of replication to Poxviruses has been proposed for ASFV. Virus genome transcription occurs independently of the host RNA polymerase II and virus particles contain all of the enzymes and factors required for early gene transcription. DNA replication begins in perinuclear factory areas about 6h post-infection although an earlier stage of nuclear DNA synthesis has been reported. The virus genome encodes enzymes required for transcription and replication of the virus genome and virion structural proteins. Enzymes that are involved in a base excision repair pathway may be an adaptation to enable virus replication in the oxidative environment of the macrophage cytoplasm. Other ASFV genes encode factors involved in evading host defence systems and modulating host cell function. Variation between the genomes of different ASFV isolates is most commonly due to gain or loss of members of multigene families, MGFs 100, 110, 300, 360, 505/530 and family p22. These are located within the left terminal 40kbp and right terminal 20kbp. ASFV is the only member of the Asfarviridae, which is one of the families within the nucleocytoplasmic large DNA virus superfamily. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Strain and sex based characterization of behavioral expressions in non-induced compulsive-like mice

    PubMed Central

    Mitra, Swarup; Bastos, Cristiane P.; Chesworth, Savanna; Frye, Cheryl; Bult-Ito, Abel

    2016-01-01

    There is currently a lack of understanding how genetic background and sex differences attribute to the heterogeneity of obsessive-compulsive disorder (OCD). An animal model of compulsive-like behaviors has been developed through bidirectional selection of house mice (Mus musculus) for high (big cotton nests; BIG mice) and low levels (small nests; SMALL mice) of nest-building behavior. The BIG male strains have predictive and face validity as a spontaneous animal model of OCD. Here, we evaluated compulsive-, anxiety-, cognitive-, and depression-like behaviors among male and proestrus female replicate strains each of BIG (BIG1, BIG2) and SMALL (SML1, SML2) nest-builders, and randomly-bred Controls (C1, C2). BIG1 and BIG2 males and females had higher nesting scores when compared to SMALL and Control strains. Male BIG1 and BIG2 strains showed more compulsive-like nesting than BIG1 and BIG2 proestrus females, which was not observed among the other strains. Nesting scores were also different between BIG replicate male strains. A similar pattern was observed in the compulsive-like marble burying behavior with BIG strains burying more marbles than SMALL and Control strains. Significant replicate and sex differences were also observed in marble burying among the BIG strains. The open field test revealed replicate effects while the BIG strains showed less anxiety-like behavior in the elevated plus maze test compared to the SMALL strains. For novel object recognition only the Control strains showed replicate and sex differences. In the depression-like forced swim test proestrus females demonstrated less depression-like behavior than males. BIG and SMALL nest-building strains had a higher corticosterone stress response than the Control strains. Together these results indicate a strong interplay of genetic background and sex in influencing expression of behaviors in our compulsive-like mouse model. These results are in congruence with the clinical heterogeneity of OCD. PMID

  13. Replication, Neurotropism, and Pathogenicity of Avian Paramyxovirus Serotypes 1–9 in Chickens and Ducks

    PubMed Central

    Kim, Shin-Hee; Xiao, Sa; Shive, Heather; Collins, Peter L.; Samal, Siba K.

    2012-01-01

    Avian paramyxovirus (APMV) serotypes 1–9 have been isolated from many different avian species. APMV-1 (Newcastle disease virus) is the only well-characterized serotype, because of the high morbidity, mortality, and economic loss caused by highly virulent strains. Very little is known about the pathogenesis, replication, virulence, and tropism of the other APMV serotypes. Here, this was evaluated for prototypes strains of APMV serotypes 2–9 in cell culture and in chickens and ducks. In cell culture, only APMV-1, -3 and -5 induced syncytium formation. In chicken DF1 cells, APMV-3 replicated with an efficiency approaching that of APMV-1, while APMV-2 and -5 replicated to lower, intermediate titers and the others were much lower. Mean death time (MDT) assay in chicken eggs and intracerebral pathogenicity index (ICPI) test in 1-day-old SPF chicks demonstrated that APMV types 2–9 were avirulent. Evaluation of replication in primary neuronal cells in vitro as well as in the brains of 1-day-old chicks showed that, among types 2–9, only APMV-3 was neurotropic, although this virus was not neurovirulent. Following intranasal infection of 1-day-old and 2-week-old chickens, replication of APMV types 2–9 was mostly restricted to the respiratory tract, although APMV-3 was neuroinvasive and neurotropic (but not neurovirulent) and also was found in the spleen. Experimental intranasal infection of 3-week-old mallard ducks with the APMVs did not produce any clinical signs (even for APMV-1) and exhibited restricted viral replication of the APMVs (including APMV-1) to the upper respiratory tract regardless of their isolation source, indicating avirulence of APMV types 1–9 in mallard ducks. The link between the presence of a furin cleavage site in the F protein, syncytium formation, systemic spread, and virulence that has been well-established with APMV-1 pathotypes was not evident with the other APMV serotypes. PMID:22558104

  14. Iterated function systems for DNA replication

    NASA Astrophysics Data System (ADS)

    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.

  15. Temporal organization of cellular self-replication

    NASA Astrophysics Data System (ADS)

    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.

  16. Signal replication in a DNA nanostructure

    NASA Astrophysics Data System (ADS)

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

    2017-01-01

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

  17. Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA

    PubMed Central

    2017-01-01

    In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage. PMID:28806726

  18. Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

    PubMed

    Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

    2004-05-15

    Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

  19. Pertussis toxin B-oligomer inhibits HIV infection and replication in hu-PBL-SCID mice.

    PubMed

    Lapenta, Caterina; Spada, Massimo; Santini, Stefano M; Racca, Sara; Dorigatti, Fernanda; Poli, Guido; Belardelli, Filippo; Alfano, Massimo

    2005-04-01

    Bordetella pertussis toxin B-oligomer (PTX-B) has been shown to inhibit HIV infection and replication in vitro. The potential anti-viral effect of PTX-B was tested here in an in vivo surrogate model of HIV infection, i.e. SCID mice reconstituted with human peripheral blood leukocytes (PBL) (hu-PBL-SCID) and infected with a CCR5-dependent (R5) HIV-1 strain. SCID mice inoculated intra-peritoneal (i.p.) with PTX-B and then infected with the R5 strain SF-162 were sacrificed 7 days later and analyzed for human PBL (hu-PBL) lymphoid tissue reconstitution, infection of hu-PBL, plasma viremia and viral rescue from ex vivo-cultivated i.p. hu-PBL. Unlike mice treated with 500 ng per animal of PTX-B showing no evidence of viral inhibition, daily administration of PTX-B (50 ng per mouse) strongly inhibited virus infection and replication, as determined by undetectable viremia, absence of infected hu-PBL and lack of rescue of infectious HIV in most animals. Furthermore, PTX-B injection 2 h before and twice after infection prevented HIV-1 infection and replication in all (10/10) tested animals. Thus, PTX-B potently inhibited virus infection and replication in hu-PBL-SCID mice, supporting the hypothesis that it may represent a new pharmacological agent against HIV-1 infection.

  20. Genetic-genomic replication to identify candidate mouse atherosclerosis modifier genes.

    PubMed

    Hsu, Jeffrey; Smith, Jonathan D

    2013-01-23

    Genetics plays a large role in atherosclerosis susceptibility in humans and mice. We attempted to confirm previously determined mouse atherosclerosis-associated loci and use bioinformatics and transcriptomics to create a catalog of candidate atherosclerosis modifier genes at these loci. A strain intercross was performed between AKR and DBA/2 mice on the apoE(-/-) background generating 166 F2 progeny. Using the phenotype log10 of the aortic root lesion area, we identified 3 suggestive atherosclerosis quantitative trait loci (Ath QTLs). When combined with our prior strain intercross, we confirmed 3 significant Ath QTLs on chromosomes 2, 15, and 17, with combined logarithm of odds scores of 5.9, 5.3, and 5.6, respectively, which each met the genome-wide 5% false discovery rate threshold. We identified all of the protein coding differences between these 2 mouse strains within the Ath QTL intervals. Microarray gene expression profiling was performed on macrophages and endothelial cells from this intercross to identify expression QTLs (eQTLs), the loci that are associated with variation in the expression levels of specific transcripts. Cross tissue eQTLs and macrophage eQTLs that replicated from a prior strain intercross were identified. These bioinformatic and eQTL analyses produced a comprehensive list of candidate genes that may be responsible for the Ath QTLs. Replication studies for clinical traits as well as gene expression traits are worthwhile in identifying true versus false genetic associations. We have replicated 3 loci on mouse chromosomes 2, 15, and 17 that are associated with atherosclerosis. We have also identified protein coding differences and multiple replicated eQTLs, which may be useful in the identification of atherosclerosis modifier genes.

  1. Geobacteraceae strains and methods

    DOEpatents

    Lovley, Derek R.; Nevin, Kelly P.; Yi, Hana

    2015-07-07

    Embodiments of the present invention provide a method of producing genetically modified strains of electricigenic microbes that are specifically adapted for the production of electrical current in microbial fuel cells, as well as strains produced by such methods and fuel cells using such strains. In preferred embodiments, the present invention provides genetically modified strains of Geobacter sulfurreducens and methods of using such strains.

  2. The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid.

    PubMed

    Michelsen, Ole; Hansen, Flemming G; Albrechtsen, Bjarne; Jensen, Peter Ruhdal

    2010-02-01

    Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division.

  3. The MG1363 and IL1403 Laboratory Strains of Lactococcus lactis and Several Dairy Strains Are Diploid▿ †

    PubMed Central

    Michelsen, Ole; Hansen, Flemming G.; Albrechtsen, Bjarne; Jensen, Peter Ruhdal

    2010-01-01

    Bacteria are normally haploid, maintaining one copy of their genome in one circular chromosome. We have examined the cell cycle of laboratory strains of Lactococcus lactis, and, to our surprise, we found that some of these strains were born with two complete nonreplicating chromosomes. We determined the cellular content of DNA by flow cytometry and by radioactive labeling of the DNA. These strains thus fulfill the criterion of being diploid. Several dairy strains were also found to be diploid while a nondairy strain and several other dairy strains were haploid in slow-growing culture. The diploid and haploid strains differed in their sensitivity toward UV light, in their cell size, and in their D period, the period between termination of DNA replication and cell division. PMID:20023021

  4. Two Replicable Suppressor Situations in Personality Research

    ERIC Educational Resources Information Center

    Paulhus, Delroy L.; Robins, Richard W.; Trzesniewski, Kali H.; Tracy, Jessica L.

    2004-01-01

    Suppressor situations occur when the simultaneous inclusion of two predictors improves one or both validities. A common allegation is that suppressor effects rarely replicate and have little substantive import. We present substantive examples from two established research domains to counter this skepticism. In the first domain, we show how…

  5. Multiseason occupancy models for correlated replicate surveys

    USGS Publications Warehouse

    Hines, James; Nichols, James D.; Collazo, Jaime

    2014-01-01

    Occupancy surveys collecting data from adjacent (sometimes correlated) spatial replicates have become relatively popular for logistical reasons. Hines et al. (2010) presented one approach to modelling such data for single-season occupancy surveys. Here, we present a multiseason analogue of this model (with corresponding software) for inferences about occupancy dynamics. We include a new parameter to deal with the uncertainty associated with the first spatial replicate for both single-season and multiseason models. We use a case study, based on the brown-headed nuthatch, to assess the need for these models when analysing data from the North American Breeding Bird Survey (BBS), and we test various hypotheses about occupancy dynamics for this species in the south-eastern United States. The new model permits inference about local probabilities of extinction, colonization and occupancy for sampling conducted over multiple seasons. The model performs adequately, based on a small simulation study and on results of the case study analysis. The new model incorporating correlated replicates was strongly favoured by model selection for the BBS data for brown-headed nuthatch (Sitta pusilla). Latitude was found to be an important source of variation in local colonization and occupancy probabilities for brown-headed nuthatch, with both probabilities being higher near the centre of the species range, as opposed to more northern and southern areas. We recommend this new occupancy model for detection–nondetection studies that use potentially correlated replicates.

  6. Reflections on "Replicating Milgram" (Burger, 2009)

    ERIC Educational Resources Information Center

    Miller, Arthur G.

    2009-01-01

    In "Replicating Milgram: Would People Still Obey Today?" Jerry M. Burger (see record 2008-19206-001) reported a high base rate of obedience, comparable to that observed by Stanley Milgram (1974). Another condition, involving a defiant confederate, failed to significantly reduce obedience. This commentary discusses the primary contributions of…

  7. Projector Center: Replication, Transcription, and Translation.

    ERIC Educational Resources Information Center

    Ruth, Edward B.

    1984-01-01

    Describes the use of a chart that systematically summarizes three basic steps that involve DNA and its decoding in both eukaryotic and prokaryotic cells: replication; transcription, and translation. Indicates that the chart (mounted on a tranparency) does an adequate job of conveying basic information about nucleic acids to students. (DH)

  8. The Intergenerational Caregiving Program: A Replication Manual.

    ERIC Educational Resources Information Center

    Cohen, Marlene Cresci; And Others

    The Intergenerational Caregiving Program (ICP), a year long educational experience in human development and child care for older adults, is described in this replication manual. In its first year of operation, the ICP recruited older, usually retired adults from agencies and organizations in San Francisco that serve older people. The 22 older…

  9. 36 CFR 910.64 - Replication.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Replication. 910.64 Section 910.64 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT CORPORATION GENERAL GUIDELINES AND UNIFORM STANDARDS FOR URBAN PLANNING AND DESIGN OF DEVELOPMENT WITHIN THE PENNSYLVANIA AVENUE...

  10. 36 CFR 910.64 - Replication.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 3 2011-07-01 2011-07-01 false Replication. 910.64 Section 910.64 Parks, Forests, and Public Property PENNSYLVANIA AVENUE DEVELOPMENT CORPORATION GENERAL GUIDELINES AND UNIFORM STANDARDS FOR URBAN PLANNING AND DESIGN OF DEVELOPMENT WITHIN THE PENNSYLVANIA AVENUE...

  11. Flow Liner Slot Edge Replication Feasibility Study

    NASA Technical Reports Server (NTRS)

    Newman, John A.; Willard, Scott A.; Smith, Stephen W.; Piascik, Robert S.

    2006-01-01

    Surface replication has been proposed as a method for crack detection in space shuttle main engine flowliner slots. The results of a feasibility study show that examination of surface replicas with a scanning electron microscope can result in the detection of cracks as small as 0.005 inch, and surface flaws as small as 0.001 inch, for the flowliner material.

  12. Suppression of Adenovirus Replication by Cardiotonic Steroids

    PubMed Central

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha

    2016-01-01

    ABSTRACT The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent

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

    PubMed

    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.

  14. Attenuated oncolytic Measles Virus strains as cancer therapeutics

    PubMed Central

    Msaouel, P.; Iankov, I.D.; Dispenzieri, A.; Galanis, E.

    2011-01-01

    Attenuated measles virus vaccine strains have emerged as a promising oncolytic vector platform, having shown significant anti-tumor activity against a broad range of malignant neoplasms. Measles virus strains derived from the attenuated Edmonston-B (MV-Edm) vaccine lineage have been shown to selectively infect, replicate in and lyse cancer cells while causing minimal cytopathic effect on normal tissues. This review summarizes the preclinical data that led to the rapid clinical translation of oncolytic measles vaccine strains and provides an overview of early clinical data using this oncolytic platform. Furthermore, novel approaches currently under development to further enhance the oncolytic efficacy of MV-Edm strains, including strategies to circumvent immunity or modulate immune system responses, combinatorial approaches with standard treatment modalities, virus retargeting as well as strategies for in vivo monitoring of viral replication are discussed. PMID:21740361

  15. Enhanced replication of herpes simplex virus type 1 in human cells

    SciTech Connect

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

    1991-02-01

    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 themore » 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.« less

  16. Muscle strain treatment

    MedlinePlus

    Treatment - muscle strain ... Question: How do you treat a muscle strain ? Answer: Rest the strained muscle and apply ice for the first few days after the injury. Anti-inflammatory medicines or acetaminophen ( ...

  17. Muscle strain (image)

    MedlinePlus

    A muscle strain is the stretching or tearing of muscle fibers. A muscle strain can be caused by sports, exercise, a ... something that is too heavy. Symptoms of a muscle strain include pain, tightness, swelling, tenderness, and the ...

  18. Strains and Sprains

    MedlinePlus

    ... Strains and Sprains Print Strains are injuries to muscle due to overstretching, while sprains involve a stretch or a partial tear of ligaments (which connect two bones) or tendons (which connect muscle to bone). Sprains and strains happen more often ...

  19. West Nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion.

    PubMed

    Ambrose, Rebecca L; Mackenzie, Jason M

    2011-03-01

    For intracellular survival it is imperative that viruses have the capacity to manipulate various cellular responses, including metabolic and biosynthetic pathways. The unfolded protein response (UPR) is induced by various external and internal stimuli, including the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Our previous studies have indicated that the replication and assembly of the flavivirus West Nile virus strain Kunjin virus (WNV(KUN)) is intimately associated with the ER. Thus, we sought to determine whether the UPR was induced during WNV(KUN) infection. WNV(KUN) induces UPR signaling during replication, which is coordinated with peak replication. Interestingly, signaling is biased toward the ATF6/IRE-1 arm of the response, with high levels of Xbp-1 activation but negligible eukaryotic translation initiation factor 2α phosphorylation and downstream transcription. We show that the PERK-mediated response may partially regulate replication, since external UPR stimulation had a limiting effect on early replication events and cells deficient for PERK demonstrated increased replication and virus release. Significantly, we show that the WNV(KUN) hydrophobic nonstructural proteins NS4A and NS4B are potent inducers of the UPR, which displayed a high correlation in inhibiting Jak-STAT signaling in response to alpha interferon (IFN-α). Sequential removal of the transmembrane domains of NS4A showed that reducing hydrophobicity decreased UPR signaling and restored IFN-α-mediated activation. Overall, these results suggest that WNV(KUN) can stimulate the UPR to facilitate replication and that the induction of a general ER stress response, regulated by hydrophobic WNV(KUN) proteins, can potentiate the inhibition of the antiviral signaling pathway.

  20. RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

    PubMed Central

    Date, Tomoko; Akazawa, Daisuke; Tian, Xiao; Suzuki, Tetsuro; Kato, Takanobu; Tanaka, Yasuhito; Mizokami, Masashi; Wakita, Takaji; Toyoda, Tetsuya

    2010-01-01

    We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells. PMID:20442786

  1. Effect of Temperature on Replication of Epizootic Hemorrhagic Disease Viruses in Culicoides sonorensis (Diptera: Ceratopogonidae).

    PubMed

    Ruder, Mark G; Stallknecht, David E; Howerth, Elizabeth W; Carter, Deborah L; Pfannenstiel, Robert S; Allison, Andrew B; Mead, Daniel G

    2015-09-01

    Replication of arboviruses, including orbiviruses, within the vector has been shown to be temperature dependent. Cooler ambient temperatures slow virus replication in arthropod vectors, whereas viruses replicate faster and to higher titers at warmer ambient temperatures. Previous research with epizootic hemorrhagic disease virus (EHDV) serotype 1 demonstrated that higher temperatures were associated with shorter extrinsic incubation periods in Culicoides sonorensis Wirth & Jones, a confirmed vector of EHDV in North America. To further our understanding of the effect of temperature on replication of EHDV within the vector, C. sonorensis were experimentally infected with one of three EHDV strains representing three serotypes (1, 2, and 7). Midges were fed defibrinated white-tailed deer (Odocoileus virginianus) blood spiked with EHDV (≥10(6.5) TCID(50)/ml) through a parafilm membrane using an artificial feeding device and were then held at 20, 25, or 30°C. In addition to this in vitro method, a white-tailed deer experimentally infected with EHDV-7 was used to provide an infectious bloodmeal to determine if the results were comparable with those from the in vitro feeding method. Whole midges were processed for virus isolation and titration at regular intervals following feeding; midges with ≥10(2.7) TCID(50) were considered potentially competent to transmit virus. The virus recovery rates were high throughout the study and all three viruses replicated within C. sonorensis to high titer (≥ 10(2.7) TCID(50)/midge). Across all virus strains, the time to detection of potentially competent midges decreased with increasing temperature: 12-16 d postfeeding (dpf) at 20°C, 4-6 dpf at 25°C, and 2-4 dpf at 30°C. Significant differences in replication of the three viruses in C. sonorensis were observed, with EHDV-2 replicating to a high titer in a smaller proportion of midges and with lower peak titers. The findings are consistent with previous studies of related

  2. Superior infectivity for mosquito vectors contributes to competitive displacement among strains of dengue virus

    PubMed Central

    Hanley, Kathryn A; Nelson, Jacob T; Schirtzinger, Erin E; Whitehead, Stephen S; Hanson, Christopher T

    2008-01-01

    Background Competitive displacement of a weakly virulent pathogen strain by a more virulent strain is one route to disease emergence. However the mechanisms by which pathogens compete for access to hosts are poorly understood. Among vector-borne pathogens, variation in the ability to infect vectors may effect displacement. The current study focused on competitive displacement in dengue virus serotype 3 (DENV3), a mosquito-borne pathogen of humans. In Sri Lanka in the 1980's, a native DENV3 strain associated with relatively mild dengue disease was displaced by an invasive DENV3 strain associated with the most severe disease manifestations, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), resulting in an outbreak of DHF/DSS. Here we tested the hypothesis that differences between the invasive and native strain in their infectivity for Aedes aegypti mosquitoes, the primary vector of DENV, contributed to the competitive success of the invasive strain Results To be transmitted by a mosquito, DENV must infect and replicate in the midgut, disseminate into the hemocoel, infect the salivary glands, and be released into the saliva. The ability of the native and invasive DENV3 strains to complete the first three steps of this process in Aedes aegypti mosquitoes was measured in vivo. The invasive strain infected a similar proportion of mosquitoes as the native strain but replicated to significantly higher titers in the midgut and disseminated with significantly greater efficiency than the native strain. In contrast, the native and invasive strain showed no significant difference in replication in cultured mosquito, monkey or human cells. Conclusion The invasive DENV3 strain infects and disseminates in Ae. aegypti more efficiently than the displaced native DENV3 strain, suggesting that the invasive strain is transmitted more efficiently. Replication in cultured cells did not adequately characterize the known phenotypic differences between native and invasive DENV3

  3. Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2

    SciTech Connect

    Gorman, Nadia; Department of Biochemistry, Dartmouth Medical School, Hanover, NH; Trask, Heidi S.

    2007-06-01

    Hexachlorobenzene (HCB), a weak ligand of the aryl hydrocarbon receptor (AHR), causes hepatic uroporphyrin (URO) accumulation (uroporphyria) in humans and animals. CYP1A2 has been shown to be necessary in the development of uroporphyria in mice. Using mice expressing the low affinity form of the AH receptor (AHRd), we investigated whether the enhancement of uroporphyria by HCB involves an obligatory increase in CYP1A2 as measured by specific enzyme assays and immunoblotting. We compared the ability of HCB, in combination with iron dextran and the porphyrin precursor, 5-aminolevulinate (ALA), to cause uroporphyria in a strain of mice (C57BL/6) which expresses the highmore » affinity form of the receptor (AHRb{sub 1}), with three strains of mice (SWR and two 129 sublines) expressing the low affinity AHRd. In C57BL/6 mice, HCB-enhanced uroporphyria was associated with a doubling of CYP1A2. HCB treatment produced uroporphyria in iron-loaded mice expressing AHRd, even though there was little or no increase in CYP1A2. Cyp1a2(-/-) mice in a 129 background were completely resistant to HCB-induced uroporphyria, and female Hfe(-/-) 129 mice, in which the levels of hepatic CYP1A2 were half of those of the male levels, responded poorly. The effect of exogenous iron, administered in the form of iron dextran, on HCB enhancement of uroporphryia could be replicated utilizing the endogenous hepatic iron accumulated in 129 Hfe(-/-) mice. In conclusion, some minimal basal expression of CYP1A2 is essential for HCB-mediated enhancement of uroporphyria, but increases in CYP1A2 above that level are not essential.« less

  4. Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response.

    PubMed

    Qu, Lin; Murakami, Kosuke; Broughman, James R; Lay, Margarita K; Guix, Susana; Tenge, Victoria R; Atmar, Robert L; Estes, Mary K

    2016-10-01

    Human noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (α/β) and III (interleukin-29 [IL-29]) IFN treatment. However, in cells capable of a strong IFN response to Sendai virus (SeV) and poly(I·C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is enhanced neither by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R nor by short hairpin RNA (shRNA) knockdown of mitochondrial antiviral signaling protein (MAVS) or interferon regulatory factor 3 (IRF3) in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells, and an SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce an IFN response, suggesting that the epithelial IFN response may play a limited role in host restriction of HuNoV replication. Human noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small-animal model, little is known about the innate host defense to these viruses. Studies on

  5. Replication of the Escherichia coli chromosome in RNase HI-deficient cells: Multiple initiation regions and fork dynamics

    PubMed Central

    Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

    2013-01-01

    DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. PMID:24164596

  6. Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

    PubMed

    Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

    2014-01-01

    DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. © 2013 John Wiley & Sons Ltd.

  7. ATP-dependent chromatin remodeling shapes the DNA replication landscape

    PubMed Central

    Vincent, Jack A.; Kwong, Tracey J.; Tsukiyama, Toshio

    2009-01-01

    Summary The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 play especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes promoting DNA replication, and define a specific stage of replication that requires remodeling for normal function. PMID:18408730

  8. Replicative Homeostasis: A fundamental mechanism mediating selective viral replication and escape mutation

    PubMed Central

    Sallie, Richard

    2005-01-01

    Hepatitis C (HCV), hepatitis B (HBV), the human immunodeficiency viruses (HIV), and other viruses that replicate via RNA intermediaries, cause an enormous burden of disease and premature death worldwide. These viruses circulate within infected hosts as vast populations of closely related, but genetically diverse, molecules known as "quasispecies". The mechanism(s) by which this extreme genetic and antigenic diversity is stably maintained are unclear, but are fundamental to understanding viral persistence and pathobiology. The persistence of HCV, an RNA virus, is especially problematic and HCV stability, maintained despite rapid genomic mutation, is highly paradoxical. This paper presents the hypothesis, and evidence, that viruses capable of persistent infection autoregulate replication and the likely mechanism mediating autoregulation – Replicative Homeostasis – is described. Replicative homeostasis causes formation of stable, but highly reactive, equilibria that drive quasispecies expansion and generates escape mutation. Replicative homeostasis explains both viral kinetics and the enigma of RNA quasispecies stability and provides a rational, mechanistic basis for all observed viral behaviours and host responses. More importantly, this paradigm has specific therapeutic implication and defines, precisely, new approaches to antiviral therapy. Replicative homeostasis may also modulate cellular gene expression. PMID:15707489

  9. Regulation of Flavivirus RNA synthesis and replication

    PubMed Central

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-01-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  10. Organization of the Influenza Virus Replication Machinery

    PubMed Central

    Moeller, Arne; Kirchdoerfer, Robert N.; Potter, Clinton S.; Carragher, Bridget; Wilson, Ian A.

    2013-01-01

    Influenza virus ribonucleoprotein complexes (RNPs) are central to the viral life cycle and in adaptation to new host species. RNPs are composed of the viral genome, viral polymerase and many copies of the viral nucleoprotein. In vitro cell expression of all RNP protein components with four of the eight influenza virus gene segments enabled structural determination of native influenza virus RNPs by cryo-electron microscopy. The cryo-EM structure reveals the architecture and organization of the native RNP, thereby defining the attributes of its largely helical structure and how polymerase interacts with NP and the viral genome. Observations of branched-RNP structures in negative stain EM and their putative identification as replication intermediates suggest a mechanism for viral replication by a second polymerase on the RNP template. PMID:23180774

  11. Experimental Replication of an Aeroengine Combustion Instability

    NASA Technical Reports Server (NTRS)

    Cohen, J. M.; Hibshman, J. R.; Proscia, W.; Rosfjord, T. J.; Wake, B. E.; McVey, J. B.; Lovett, J.; Ondas, M.; DeLaat, J.; Breisacher, K.

    2000-01-01

    Combustion instabilities in gas turbine engines are most frequently encountered during the late phases of engine development, at which point they are difficult and expensive to fix. The ability to replicate an engine-traceable combustion instability in a laboratory-scale experiment offers the opportunity to economically diagnose the problem (to determine the root cause), and to investigate solutions to the problem, such as active control. The development and validation of active combustion instability control requires that the causal dynamic processes be reproduced in experimental test facilities which can be used as a test bed for control system evaluation. This paper discusses the process through which a laboratory-scale experiment was designed to replicate an instability observed in a developmental engine. The scaling process used physically-based analyses to preserve the relevant geometric, acoustic and thermo-fluid features. The process increases the probability that results achieved in the single-nozzle experiment will be scalable to the engine.

  12. Self-Replication Without Life (yet)

    NASA Astrophysics Data System (ADS)

    Chaikin, Paul

    2010-03-01

    We want to make a non-``biological'' system which can self-replicate. The idea is to design particles with specific and reversible and irreversible interactions, introduce seed motifs, and cycle the system in such a way that a copy is made. Repeating the cycle would double the number of offspring in each generation leading to exponential growth. Using the chemistry of DNA either on colloids or on DNA Tiles makes the specific recognition part easy. In the case of DNA Tiles we have in fact replicated the seed at least to the third generation. The DNA linkers can also be self-protected so that particles don't interact unless they are held together for sufficient time - a nano-contact glue. We have also designed and produced colloidal particles that use novel geometries to get specific and reversible physical interactions. **With M. Leunissen, R. Dreyfus, R. Sha, T. Wang, S. Sacanna, D. Pine and N. Seeman

  13. The Replication Stress Response in Pancreatic Cancer

    DTIC Science & Technology

    2014-12-01

    AWARD NUMBER: W81XWH-12-1-0407 TITLE: The Replication Stress Response in Pancreatic Cancer PRINCIPAL INVESTIGATOR: DavidS. Yu CONTRACTING...will become reliant on backup pathways that can be targeted to cause cell death through the principal of synthetic lethality (inactivation of one gene...calculated and normalized to that of nontargeting siRNA. Principal compo nents analysis (PCA) was used to account for possible vari ability between the cell

  14. Replication of Functional MRI Detection of Deception

    PubMed Central

    Kozel, F. Andrew; Laken, Steven J.; Johnson, Kevin A.; Boren, Bryant; Mapes, Kimberly S.; Morgan, Paul S.; George, Mark S.

    2009-01-01

    Background Several studies support the use of fMRI for detecting deception. There have been, however, no reported replications on different scanners or at different locations. In a previous study, deception was accurately detected in at least 90% of the participants in two independent cohorts. This study attempted to replicate those findings using a different scanner and location. Methods Healthy participants 18–50 years of age were recruited from the local community. After providing written informed consent, participants were screened to ensure that they were healthy, not taking any medications, and safe to have an MRI. For the testing paradigm, subjects chose one of two objects (ring or watch) to “steal” and placed it in their locker. Participants were then scanned while being visually presented with a series of questions. Functional MRI analysis was performed in the same manner as described in Kozel et al. 2005. A Chi-Squared test was used to test for a significant difference between the results in the previous study and in this replication study. Results Thirty subjects (20 women, mean age 29.0±6.5 years) were scanned with one subject being noncompliant with the protocol. Twenty-five of twenty-nine (86%) participants were correctly identified when being deceptive. There was no statistical difference between the accuracy rate obtained in this study (25/29) versus the previous study (28/31) (Chi-Squared, χ2=0.246, p=0.6197). Conclusions Our methodology for detecting deception was successfully replicated at a different site suggesting that this methodology is robust and independent of both scanner and location. PMID:19844599

  15. DNA ligase I, the replicative DNA ligase

    PubMed Central

    Howes, Timothy R.L.; Tomkinson, Alan E.

    2013-01-01

    Multiple DNA ligation events are required to join the Okazaki fragments generated during lagging strand DNA synthesis. In eukaryotes, this is primarily carried out by members of the DNA ligase I family. The C-terminal catalytic region of these enzymes is composed of three domains: a DNA binding domain, an adenylation domain and an OB-fold domain. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. This proline-rich unstructured region contains the nuclear localization signal and a PCNA interaction motif that is critical for localization to replication foci and efficient joining of Okazaki fragments. DNA ligase I initially engages the PCNA trimer via this interaction motif which is located at the extreme N-terminus of this flexible region. It is likely that this facilitates an additional interaction between the DNA binding domain and the PCNA ring. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. DNA ligase I also interacts with replication factor C, the factor that loads the PCNA trimeric ring onto DNA. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. PMID:22918593

  16. Dipyridamole Reversibly Inhibits Mengovirus RNA Replication

    PubMed Central

    Fata-Hartley, Cori L.; Palmenberg, Ann C.

    2005-01-01

    Dipyridamole is an effective inhibitor of cardiovirus growth in cell culture. The effects of dipyridamole on mengovirus replication in vivo and in vitro were examined in the hope the drug could be used as an experimental analog of the poliovirus inhibitor guanidine. Guanidine selectively inhibits poliovirus RNA synthesis but not RNA translation, and as such, has been a valuable research tool. Although guanidine does not inhibit cardiovirus infection, a compound with similar discriminatory characteristics would be experimentally useful for parallel work with these viruses. We found that mengovirus plaque formation in HeLa or L cells was inhibited nearly 100% by the presence of 80 μM dipyridamole. The inhibitory effect was reversible and targeted an early step in the replication cycle. Studies with luciferase-expressing mengovirus replicons showed that viral protein synthesis was unaffected by dipyridamole, and rather, RNA synthesis was the step targeted by the drug. This assessment was confirmed by direct analyses of viral translation and RNA synthesis activities in a Krebs-2-derived in vitro system that supported complete, infectious cardiovirus replication. In Krebs extracts, dipyridamole specifically inhibited viral RNA synthesis to more than 95%, with no concomitant effect on viral protein translation or polyprotein processing. The observed inhibition reversibly affected an early step in both minus-strand and plus-strand RNA synthesis, although inhibition of plus-strand synthesis was more profound than that of minus-strand synthesis. We conclude that dipyridamole is a potent experimental tool that readily distinguishes between cardiovirus translation and RNA replication functions. PMID:16103157

  17. Replication of architectural antiquities using polymeric materials

    SciTech Connect

    Frye, E.R.

    1983-03-01

    In support of the University of New Mexico's Maxwell Museum of Anthropology, a number of volunteers undertook the replication of Chaco Canyon masonry. Composite rubber latex and glass fabric molds were developed and a series of molds fabricated directly on the Chaco structures. From these, a set of fiberglass epoxy models was constructed depicting about 200 ft/sup 2/ of various masonry styles, and doorway and kiva niche features.

  18. Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; McGrath, M. S.; Hanks, D.; Erickson, S.; Pulliam, L.

    1992-01-01

    We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

  19. Molecular Biology of Rotavirus Entry and Replication

    PubMed Central

    Ruiz, Marie Christine; Leon, Theresa; Diaz, Yuleima; Michelangeli, Fabian

    2009-01-01

    Rotavirus is a nonenveloped, double-stranded, RNA virus belonging to the Reoviridae family and is the major etiological agent of viral gastroenteritis in young children and young animals. Remarkable progress in the understanding of the rotavirus cycle has been made in the last 10 years. The knowledge of viral replication thus far acquired is based on structural studies, the expression and coexpression of individual viral proteins, silencing of individual genes by siRNAs, and the effects that these manipulations have on the physiology of the infected cell. The functions of the individual rotavirus proteins have been largely dissected; however, the interactions between them and with cell proteins, and the molecular mechanisms of virus replication, are just beginning to be understood. These advancements represent the basis for the development of effective vaccination and rational therapeutic strategies to combat rotavirus infection and diarrhea syndromes. In this paper, we review and try to integrate the new knowledge about rotavirus entry, replication, and assembly, and pose some of the questions that remain to be solved. PMID:20024520

  20. The molecular biology of Bluetongue virus replication.

    PubMed

    Patel, Avnish; Roy, Polly

    2014-03-01

    The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Registered Replication Report: Strack, Martin, & Stepper (1988).

    PubMed

    Acosta, Alberto; Adams, Reginald B; Albohn, Daniel N; Allard, Eric S; Beek, Titia; Benning, Stephen D; Blouin- Hudon, Eve-Marie; Bulnes, Luis Carlo; Caldwell, Tracy L; Calin-Jageman, Robert J; Capaldi, Colin A; Carfagno, Nicholas S; Chasten, Kelsie T; Cleeremans, Axel; Connell, Louise; DeCicco, Jennifer M.; Dijkhoff, Laura; Dijkstra, Katinka; Fischer, Agneta H; Foroni, Francesco; Gronau, Quentin F; Hess, Ursula; Holmes, Kevin J; Jones, Jacob L H; Klein, Olivier; Koch, Christopher; Korb, Sebastian; Lewinski, Peter; Liao, Julia D; Lund, Sophie; Lupiáñez, Juan; Lynott, Dermot; Nance, Christin N; Oosterwijk, Suzanne; Özdog˘ru, Asil Ali; Pacheco-Unguetti, Antonia Pilar; Pearson, Bethany; Powis, Christina; Riding, Sarah; Roberts, Tomi-Ann; Rumiati, Raffaella I; Senden, Morgane; Shea-Shumsky, Noah B; Sobocko, Karin; Soto, Jose A; Steiner, Troy G; Talarico, Jennifer M; vanAllen, Zack M; Wagenmakers, E-J; Vandekerckhove, Marie; Wainwright, Bethany; Wayand, Joseph F; Zeelenberg, Rene; Zetzer, Emily E; Zwaan, Rolf A

    2016-11-01

    According to the facial feedback hypothesis, people's affective responses can be influenced by their own facial expression (e.g., smiling, pouting), even when their expression did not result from their emotional experiences. For example, Strack, Martin, and Stepper (1988) instructed participants to rate the funniness of cartoons using a pen that they held in their mouth. In line with the facial feedback hypothesis, when participants held the pen with their teeth (inducing a "smile"), they rated the cartoons as funnier than when they held the pen with their lips (inducing a "pout"). This seminal study of the facial feedback hypothesis has not been replicated directly. This Registered Replication Report describes the results of 17 independent direct replications of Study 1 from Strack et al. (1988), all of which followed the same vetted protocol. A meta-analysis of these studies examined the difference in funniness ratings between the "smile" and "pout" conditions. The original Strack et al. (1988) study reported a rating difference of 0.82 units on a 10-point Likert scale. Our meta-analysis revealed a rating difference of 0.03 units with a 95% confidence interval ranging from -0.11 to 0.16. © The Author(s) 2016.

  2. Superlattice strain gage

    DOEpatents

    Noel, Bruce W.; Smith, Darryl L.; Sinha, Dipen N.

    1990-01-01

    A strain gage comprising a strained-layer superlattice crystal exhibiting piezoelectric properties is described. A substrate upon which such a strained-layer superlattice crystal has been deposited is attached to an element to be monitored for strain. A light source is focused on the superlattice crystal and the light reflected from, passed through, or emitted from the crystal is gathered and compared with previously obtained optical property data to determine the strain in the element.

  3. Superlattice strain gage

    DOEpatents

    Noel, B.W.; Smith, D.L.; Sinha, D.N.

    1988-06-28

    A strain gage comprising a strained-layer superlattice crystal exhibiting piezoelectric properties is described. A substrate upon which such a strained-layer superlattice crystal has been deposited is attached to an element to be monitored for strain. A light source is focused on the superlattice crystal and the light reflected from, passed through, or emitted from the crystal is gathered and compared with previously obtained optical property data to determine the strain in the element. 8 figs.

  4. Loss of Caenorhabditis elegans BRCA1 Promotes Genome Stability During Replication in smc-5 Mutants

    PubMed Central

    Wolters, Stefanie; Ermolaeva, Maria A.; Bickel, Jeremy S.; Fingerhut, Jaclyn M.; Khanikar, Jayshree; Chan, Raymond C.; Schumacher, Björn

    2014-01-01

    DNA damage by ultraviolet (UV) light poses a risk for mutagenesis and a potential hindrance for cell cycle progression. Cells cope with UV-induced DNA damage through two general strategies to repair the damaged nucleotides and to promote cell cycle progression in the presence of UV-damaged DNA. Defining the genetic pathways and understanding how they function together to enable effective tolerance to UV remains an important area of research. The structural maintenance of chromosomes (SMC) proteins form distinct complexes that maintain genome stability during chromosome segregation, homologous recombination, and DNA replication. Using a forward genetic screen, we identified two alleles of smc-5 that exacerbate UV sensitivity in Caenorhabditis elegans. Germ cells of smc-5-defective animals show reduced proliferation, sensitivity to perturbed replication, chromatin bridge formation, and accumulation of RAD-51 foci that indicate the activation of homologous recombination at DNA double-strand breaks. Mutations in the translesion synthesis polymerase polh-1 act synergistically with smc-5 mutations in provoking genome instability after UV-induced DNA damage. In contrast, the DNA damage accumulation and sensitivity of smc-5 mutant strains to replication impediments are suppressed by mutations in the C. elegans BRCA1/BARD1 homologs, brc-1 and brd-1. We propose that SMC-5/6 promotes replication fork stability and facilitates recombination-dependent repair when the BRC-1/BRD-1 complex initiates homologous recombination at stalled replication forks. Our data suggest that BRC-1/BRD-1 can both promote and antagonize genome stability depending on whether homologous recombination is initiated during DNA double-strand break repair or during replication stalling. PMID:24424777

  5. Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa.

    PubMed

    Lee, Min Woo; Rogers, Elizabeth E; Stenger, Drake C

    2010-12-01

    Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

  6. Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells.

    PubMed

    Yi, MinKyung; Lemon, Stanley M

    2004-08-01

    Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.

  7. Darwinian Evolution of Mutualistic RNA Replicators with Different Genes

    NASA Astrophysics Data System (ADS)

    Mizuuchi, R.; Ichihashi, N.

    2017-07-01

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

  8. A new model to produce infectious hepatitis C virus without the replication requirement.

    PubMed

    Triyatni, Miriam; Berger, Edward A; Saunier, Bertrand

    2011-04-01

    Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity

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

    PubMed

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

    2016-07-27

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

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

    PubMed

    Anderson, Samantha F; Maxwell, Scott E

    2017-01-01

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

  11. Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

    PubMed Central

    Tsaalbi-Shtylik, Anastasia; Moser, Jill; Mullenders, Leon H. F.; Jansen, Jacob G.; de Wind, Niels

    2014-01-01

    Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3−/− and Rev1−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold. PMID:24464993

  12. Zika Virus Replicates in Proliferating Cells in Explants From First-Trimester Human Placentas, Potential Sites for Dissemination of Infection.

    PubMed

    Tabata, Takako; Petitt, Matthew; Puerta-Guardo, Henry; Michlmayr, Daniela; Harris, Eva; Pereira, Lenore

    2018-03-28

    Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

  13. Dissecting host-virus interaction in lytic replication of a model herpesvirus.

    PubMed

    Dong, Xiaonan; Feng, Pinghui

    2011-10-07

    In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection. Murine γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus. γHV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in γHV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during γHV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during γHV68 lytic replication ex vivo. Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo. Recently, we have discovered that γHV68 usurps

  14. USF Binding Sequences from the HS4 Insulator Element Impose Early Replication Timing on a Vertebrate Replicator

    PubMed Central

    Cadoret, Jean-Charles; Ma, Meiji Kit-Wan; Boggetto, Nicole; West, Adam G.; Prioleau, Marie-Noëlle

    2012-01-01

    The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the β-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication. PMID:22412349

  15. A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment

    NASA Astrophysics Data System (ADS)

    Khademolhosseini, F.; Liu, C.-C.; Lim, C. J.; Chiao, M.

    2016-08-01

    Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

  16. Replication characteristics of equine herpesvirus 1 and equine herpesvirus 3: comparative analysis using ex vivo tissue cultures.

    PubMed

    Negussie, Haileleul; Li, Yewei; Tessema, Tesfaye Sisay; Nauwynck, Hans J

    2016-01-15

    Replication kinetics and invasion characteristics of equine herpesvirus-1 and -3 (EHV-1/-3) in nasal and vaginal mucosae were compared using explants. The explants were cultured during 96 h with little change in viability. The tissues were inoculated with EHV-1 03P37 (neuropathogenic), 97P70 (abortigenic) and EHV-3 04P57, collected at 0, 24, 48 and 72 h post inoculation (pi) and stained for viral antigens. Both EHV-1 and EHV-3 replicated in a plaquewise manner. The plaques were already observed at 24 h pi, their size increased over time and did not directly cross the basement membrane (BM). However, EHV-1 infected the monocytic cells (MC) and hijacked these cells to invade the lamina propria. In contrast, EHV-3 replication was fully restricted to epithelial cells; the virus did not breach the BM via a direct cell-to-cell spread nor used infected MC. EHV-1-induced plaques were larger in nasal mucosa compared to vaginal mucosa. The opposite was found for EHV-3-induced plaques. Both EHV-1 strains replicated with comparable kinetics in nasal mucosa. However, the extent of replication of the abortigenic strain in vaginal mucosa was significantly higher than that of the neuropathogenic strain. Two-to-five-fold lower numbers of EHV-1-infected MC underneath the BM were found in vaginal mucosa than in nasal mucosa. Our study has shown that (i) EHV-1 has developed in evolution a predisposition for respiratory mucosa and EHV-3 for vaginal mucosa, (ii) abortigenic EHV-1 replicates better in vaginal mucosa than neuropathogenic EHV-1 and (iii) EHV-3 demonstrated a strict epithelial tropism whereas EHV-1 in addition hijacked MC to invade the lamina propria.

  17. Investigate zero-stress replicated optics

    NASA Technical Reports Server (NTRS)

    Engelhaupt, Darell; Rood, Robert

    1993-01-01

    The contracted activities for the procurement of 'Investigate Zero-Stress Replicated Optics' to support the AXAF-S x-ray spectrometer mirrors has been completed. To date four large Wolter I grazing incidence x-ray optical shells have been electroformed from nickel. The mirrors were fabricated utilizing each of two nickel alloy plated aluminum substrates twice. A wide variety of testing has been completed by NASA MSFC and UAH. This testing includes heat treatment control tests, subscale plating and fixture testing, alloy control of the electroless nickel, adhesion and release testing of the gold to electroless nickel, electroforming instrumentation and software and fabrication of subscale models. The full scale shells are one millimeter thick nickel electrodeposited over a thin gold layer which in turn has the optical surface on the inside. The optical surface is the replicate of the surface prepared on the substrate. Appendix I briefly outlines the fabrication process. Major objectives which were shared by UAH and MSFC include the design of facilities, equipment and tooling and procurement of materials and equipment. Process development followed with the fabrication of small scale pilot units. Procurement commenced immediately and equipment and materials were ordered to implement the fabrication of first surface full scale substrates (mandrels) and the second surface electroformed optical components. All principal objectives have been achieved. Inspection of the mirrors in visible and x-ray modes validates that the required performance and the quality can be achieved by an electroforming replication process. A very distinct progressive improvement has been achieved with each of the four mirrors produced. The final mirror exceeded the original goals and set an improved standard for flight hardware. The future goal of a 30 arc second resolution at 8 KEV x-ray appears to be achievable by this process when proper cleanliness and process control is utilized.

  18. The IFITMs Inhibit Zika Virus Replication.

    PubMed

    Savidis, George; Perreira, Jill M; Portmann, Jocelyn M; Meraner, Paul; Guo, Zhiru; Green, Sharone; Brass, Abraham L

    2016-06-14

    Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses. Copyright © 2016. Published by Elsevier Inc.

  19. Replicative and Chronological Aging in Saccharomyces cerevisiae

    PubMed Central

    Longo, Valter D.; Shadel, Gerald S.; Kaeberlein, Matt; Kennedy, Brian

    2012-01-01

    Saccharomyces cerevisiae has directly or indirectly contributed to the identification of arguably more mammalian genes that affect aging than any other model organism. Aging in yeast is assayed primarily by measuring replicative or chronological lifespan. Here, we review the genes and mechanisms implicated in these two aging model systems and key remaining issues that need to be addressed to optimize these assays. Because of its well-characterized genome and proteome that is remarkably amenable to genetic manipulation and high-throughput screening procedures, S. cerevisiae will continue to serve as a leading model organism to study pathways relevant to human aging and disease. PMID:22768836

  20. Diphenylpyrazoles as Replication Protein A inhibitors

    DOE PAGES

    Waterson, Alex G.; Kennedy, J. Phillip; Patrone, James D.; ...

    2014-11-11

    Replication Protein A is the primary eukaryotic ssDNA binding protein that has a central role in initiating the cellular response to DNA damage. RPA recruits multiple proteins to sites of DNA damage via the N-terminal domain of the 70 kDa subunit (RPA70N). Here we describe the optimization of a diphenylpyrazole carboxylic acid series of inhibitors of these RPA–protein interactions. Lastly, we evaluated substituents on the aromatic rings as well as the type and geometry of the linkers used to combine fragments, ultimately leading to submicromolar inhibitors of RPA70N protein–protein interactions.

  1. The replicator equation and other game dynamics.

    PubMed

    Cressman, Ross; Tao, Yi

    2014-07-22

    The replicator equation is the first and most important game dynamics studied in connection with evolutionary game theory. It was originally developed for symmetric games with finitely many strategies. Properties of these dynamics are briefly summarized for this case, including the convergence to and stability of the Nash equilibria and evolutionarily stable strategies. The theory is then extended to other game dynamics for symmetric games (e.g., the best response dynamics and adaptive dynamics) and illustrated by examples taken from the literature. It is also extended to multiplayer, population, and asymmetric games.

  2. Termination sites for adenovirus type 2 DNA replication.

    PubMed Central

    Tolun, A; Pettersson, U

    1975-01-01

    Termination sites for replication of adenovirus type 2 DNA have been demonstrated at both ends of the viral chromosome by the procedure of Danna and Nathans (1972). Single-stranded DNA from replicating intermediates was also characterized by hybridization with separated strands of viral DNA. The results indicate that both strands are exposed during replication. PMID:1165592

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

    PubMed

    Marois, Isabelle; Cloutier, Alexandre; Meunier, Isabelle; Weingartl, Hana M; Cantin, André M; Richter, Martin V

    2014-01-01

    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.

  4. Replication of Toxoplasma gondii in chicken erythrocytes and thrombocytes compared to macrophages.

    PubMed

    Malkwitz, Irene; Berndt, Angela; Zhang, Runhui; Daugschies, Arwid; Bangoura, Berit

    2017-01-01

    Toxoplasma (T.) gondii is able to infect various cell types in different hosts. The replication of this parasite within different peripheral mononuclear blood cell populations in chicken has not yet been fully understood. Aim of the present study was to investigate the impact of chicken erythrocytes and thrombocytes as potential host cells for T. gondii. Cultures of primary avian erythrocytes and thrombocytes were inoculated with tachyzoites of T. gondii type II strain ME49. Parasite replication was detected by a quantitative real-time PCR at different times postinoculation until 24 or 48 h, respectively, displaying long-term investigations for the chosen cultures. The parasite replication curve showed a continuous decrease of parasite stages in erythrocytes and thrombocytes. Observations by light microscopy showed massive destruction for both cell populations. Few macrophages in between the infected thrombocytes were viable during the investigation period and showed internalised tachyzoites by confocal laser scanning microscopy. These findings show that T. gondii is not capable of replication in chicken erythrocytes and thrombocytes; therefore, both cannot be considered as potential host cells. In further consequence, monocyte-derived macrophages seem to be the key to the dissemination mechanisms for T. gondii in chicken.

  5. Inhibition of Influenza Virus Replication by Targeting Broad Host Cell Pathways

    PubMed Central

    Marois, Isabelle; Cloutier, Alexandre; Meunier, Isabelle; Weingartl, Hana M.; Cantin, André M.; Richter, Martin V.

    2014-01-01

    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. PMID:25333287

  6. The Rep20 replication initiator from the pAG20 plasmid of Acetobacter aceti.

    PubMed

    Babič, Martin; Rešková, Zuzana; Bugala, Juraj; Cimová, Viera; Grones, Peter; Grones, Jozef

    2014-01-01

    In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5'-TCCAAATTTGGAT'-3') and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.

  7. Inactivation of the Escherichia coli priA DNA replication protein induces the SOS response.

    PubMed Central

    Nurse, P; Zavitz, K H; Marians, K J

    1991-01-01

    Many of the proteins that operate at the replication fork in Escherichia coli have been defined genetically. These include some of the subunits of the DNA polymerase III holoenzyme, the DnaB replication fork helicase, and the DnaG primase. The multiprotein primosome (which includes the DnaB and DnaG proteins), defined biochemically on the basis of its requirement during bacteriophage phi X174 complementary-strand synthesis, could serve as the helicase-primase replication machine on the lagging-strand template. In order to determine if this is the case, we have begun an investigation of the phenotypes of mutants with mutations priA, priB, and priC, which encode the primosomal proteins factor Y (protein n'), n, and n", respectively. Inactivation of priA by insertional mutagenesis resulted in the induction of the SOS response, as evinced by induction of a resident lambda prophage, extreme filamentation, and derepression of an indicator operon in which beta-galactosidase production was controlled by the dinD1 promoter. In addition, the copy numbers of resident pBR322 plasmids were reduced four- to fivefold in these strains, and production of phi X174 phage was delayed considerably. These results are discussed in the context of existing models for SOS induction and possible roles for the PriA protein at the replication fork in vivo. Images FIG. 2 FIG. 4 FIG. 6 PMID:1938875

  8. Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

    PubMed

    Maura, Damien; Morello, Eric; du Merle, Laurence; Bomme, Perrine; Le Bouguénec, Chantal; Debarbieux, Laurent

    2012-08-01

    Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

  9. Human stem cell-derived astrocytes replicate human prions in aPRNPgenotype-dependent manner.

    PubMed

    Krejciova, Zuzana; Alibhai, James; Zhao, Chen; Krencik, Robert; Rzechorzek, Nina M; Ullian, Erik M; Manson, Jean; Ironside, James W; Head, Mark W; Chandran, Siddharthan

    2017-12-04

    Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype-dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery. © 2017 Krejciova et al.

  10. In vitro infection and replication of hepatitis E virus in primary cynomolgus macaque hepatocytes.

    PubMed

    Tam, A W; White, R; Yarbough, P O; Murphy, B J; McAtee, C P; Lanford, R E; Fuerst, T R

    1997-11-10

    An in vitro model was developed to replicate hepatitis E virus (HEV) in normal primary cynomolgus macaque hepatocytes using a hormonally defined, serum-free medium formulation. Primary hepatocytes were infected in tissue culture following isolation by collagenase treatment of liver wedge biopsy material. Viral replication was monitored by a highly strand-specific reverse transcription-polymerase chain reaction (RT-PCR) assay, which could detect the positive- and negative-strands of HEV RNA independently in a sensitive and specific manner. Several infectious HEV (Burma strain) inocula were titered by this RT-PCR assay, and a minimum effective infectious dose was determined. Appearance of newly replicated virus was demonstrated by detection of both strands of HEV RNA in experimentally infected hepatocytes as well as the genomic positive-strand viral RNA in the culture medium. Infectivity of the virus particles present in the media was confirmed by serial passage and replication of the virus in culture. Using this in vitro infection system, a neutralization assay was developed to assess the ability of anti-HEV antibodies to block virus infection of liver cells. Results presented in this report represent the first in vitro demonstration of a neutralizing anti-HEV antibody directed against the ORF2-encoded putative capsid protein.

  11. Different activities of the largest subunit of Replication protein A cooperate during SV40 DNA replication

    PubMed Central

    Taneja, Poonam; Boche, Irene; Hartmann, Hella; Nasheuer, Heinz-Peter; Grosse, Frank; Fanning, Ellen

    2007-01-01

    Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase α-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein-protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end. PMID:17673209

  12. Evolutionary factor analysis of replicated time series.

    PubMed

    Motta, Giovanni; Ombao, Hernando

    2012-09-01

    In this article, we develop a novel method that explains the dynamic structure of multi-channel electroencephalograms (EEGs) recorded from several trials in a motor-visual task experiment. Preliminary analyses of our data suggest two statistical challenges. First, the variance at each channel and cross-covariance between each pair of channels evolve over time. Moreover, the cross-covariance profiles display a common structure across all pairs, and these features consistently appear across all trials. In the light of these features, we develop a novel evolutionary factor model (EFM) for multi-channel EEG data that systematically integrates information across replicated trials and allows for smoothly time-varying factor loadings. The individual EEGs series share common features across trials, thus, suggesting the need to pool information across trials, which motivates the use of the EFM for replicated time series. We explain the common co-movements of EEG signals through the existence of a small number of common factors. These latent factors are primarily responsible for processing the visual-motor task which, through the loadings, drive the behavior of the signals observed at different channels. The estimation of the time-varying loadings is based on the spectral decomposition of the estimated time-varying covariance matrix. © 2012, The International Biometric Society.

  13. Conditionally Replicating Adenoviruses for Cancer Treatment

    PubMed Central

    Jounaidi, Youssef; Doloff, Joshua C.; Waxman, David J.

    2012-01-01

    Replication-conditional, oncolytic adenoviruses are emerging as powerful tools in the warfare on cancer. The ability to modify cell-specific infectivity or tissue-specific replication machinery, as well as the possibility of modifying viral-cellular protein interactions with cellular checkpoint regulators are emerging as new trends in the design of safer and more effective adenoviruses. The integration of oncolytic adenoviruses with mainstream cancer therapies, such as chemotherapy and radiotherapy, continues to yield significant therapeutic benefits. Adenoviruses can be armed with prodrug-activating enzymes as well as tumor suppressor genes or anti-angiogenic factors, thus providing for enhanced anti-tumor therapy and reduced host toxicity. Thus far, encouraging results have been obtained from extensive preclinical and human clinical studies. However, there is a need to improve adenoviral vectors to overcome unresolved problems facing this promising anti-cancer agent, chief among these issues is the adenovirus-triggered immune response threatening its efficacy. The continued expansion of the knowledge base of adenovirus biology will likely lead to further improvements in the design of the ideal oncolytic adenoviruses for cancer treatment. PMID:17504125

  14. Bioaerosol sampling system with replicated optics

    NASA Astrophysics Data System (ADS)

    Gross, Mike; Cunningham, Nicholas; Erickson, Joshua; Manning, Christopher J.; Samuels, Alan C.

    2004-09-01

    There is a critical need throughout DoD, the U.S. government, and the commercial sector for cost-effective monitoring systems to detect airborne biological warfare (BW) agents. At present, solutions for this sensor need are relatively expensive and have a high false alarm rate. Manning Applied Technology is developing a compact, portable bioaerosol sampling system for continuous monitoring of air quality, both at field locations and fixed installations. The instrument is premised on optical interrogation via a multi-step process. The first step is electrostatic concentration, to improve detection limits. An advantage of electrostatic particle concentration is the power efficiency, relative to impactors, cyclones and filter-based systems. The second step is presentation for particle analysis, which would employ one of several unique FT spectrometer designs. The advantage of spectroscopic interrogation of bioaerosol particles is the very low cost of each analysis, with no consumables required. It is thought that mid-IR and THz frequency ranges offer the best potential for accurate discrimination. The third, optional step, is archiving the collected particles for further analysis. To reduce component costs in the Fourier transform spectrometer, an optical replication process has been developed and tested, with promising results. The replication and optical testing methods are described in detail.

  15. The alphaviruses: gene expression, replication, and evolution.

    PubMed Central

    Strauss, J H; Strauss, E G

    1994-01-01

    The alphaviruses are a genus of 26 enveloped viruses that cause disease in humans and domestic animals. Mosquitoes or other hematophagous arthropods serve as vectors for these viruses. The complete sequences of the +/- 11.7-kb plus-strand RNA genomes of eight alphaviruses have been determined, and partial sequences are known for several others; this has made possible evolutionary comparisons between different alphaviruses as well as comparisons of this group of viruses with other animal and plant viruses. Full-length cDNA clones from which infectious RNA can be recovered have been constructed for four alphaviruses; these clones have facilitated many molecular genetic studies as well as the development of these viruses as expression vectors. From these and studies involving biochemical approaches, many details of the replication cycle of the alphaviruses are known. The interactions of the viruses with host cells and host organisms have been exclusively studied, and the molecular basis of virulence and recovery from viral infection have been addressed in a large number of recent papers. The structure of the viruses has been determined to about 2.5 nm, making them the best-characterized enveloped virus to date. Because of the wealth of data that has appeared, these viruses represent a well-characterized system that tell us much about the evolution of RNA viruses, their replication, and their interactions with their hosts. This review summarizes our current knowledge of this group of viruses. Images PMID:7968923

  16. Endoplasmic reticulum stress causes EBV lytic replication

    PubMed Central

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K.; Rowe, David T.; Wadowsky, Robert M.

    2011-01-01

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)–specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress–dependent and –independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)–treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress. PMID:21849482

  17. Endoplasmic reticulum stress causes EBV lytic replication.

    PubMed

    Taylor, Gwen Marie; Raghuwanshi, Sandeep K; Rowe, David T; Wadowsky, Robert M; Rosendorff, Adam

    2011-11-17

    Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

  18. How to securely replicate services (preliminary version)

    NASA Technical Reports Server (NTRS)

    Reiter, Michael; Birman, Kenneth

    1992-01-01

    A method is presented for constructing replicated services that retain their availability and integrity despite several servers and clients being corrupted by an intruder, in addition to others failing benignly. More precisely, a service is replicated by 'n' servers in such a way that a correct client will accept a correct server's response if, for some prespecified parameter, k, at least k servers are correct and fewer than k servers are correct. The issue of maintaining causality among client requests is also addressed. A security breach resulting from an intruder's ability to effect a violation of causality in the sequence of requests processed by the service is illustrated. An approach to counter this problem is proposed that requires that fewer than k servers are corrupt and, to ensure liveness, that k is less than or = n - 2t, where t is the assumed maximum total number of both corruptions and benign failures suffered by servers in any system run. An important and novel feature of these schemes is that the client need not be able to identify or authenticate even a single server. Instead, the client is required only to possess at most two public keys for the service.

  19. Le Chatelier's principle in replicator dynamics.

    PubMed

    Allahverdyan, Armen E; Galstyan, Aram

    2011-10-01

    The Le Chatelier principle states that physical equilibria are not only stable, but they also resist external perturbations via short-time negative-feedback mechanisms: a perturbation induces processes tending to diminish its results. The principle has deep roots, e.g., in thermodynamics it is closely related to the second law and the positivity of the entropy production. Here we study the applicability of the Le Chatelier principle to evolutionary game theory, i.e., to perturbations of a Nash equilibrium within the replicator dynamics. We show that the principle can be reformulated as a majorization relation. This defines a stability notion that generalizes the concept of evolutionary stability. We determine criteria for a Nash equilibrium to satisfy the Le Chatelier principle and relate them to mutualistic interactions (game-theoretical anticoordination) showing in which sense mutualistic replicators can be more stable than (say) competing ones. There are globally stable Nash equilibria, where the Le Chatelier principle is violated even locally: in contrast to the thermodynamic equilibrium a Nash equilibrium can amplify small perturbations, though both types of equilibria satisfy the detailed balance condition.

  20. Le Chatelier's principle in replicator dynamics

    NASA Astrophysics Data System (ADS)

    Allahverdyan, Armen E.; Galstyan, Aram

    2011-10-01

    The Le Chatelier principle states that physical equilibria are not only stable, but they also resist external perturbations via short-time negative-feedback mechanisms: a perturbation induces processes tending to diminish its results. The principle has deep roots, e.g., in thermodynamics it is closely related to the second law and the positivity of the entropy production. Here we study the applicability of the Le Chatelier principle to evolutionary game theory, i.e., to perturbations of a Nash equilibrium within the replicator dynamics. We show that the principle can be reformulated as a majorization relation. This defines a stability notion that generalizes the concept of evolutionary stability. We determine criteria for a Nash equilibrium to satisfy the Le Chatelier principle and relate them to mutualistic interactions (game-theoretical anticoordination) showing in which sense mutualistic replicators can be more stable than (say) competing ones. There are globally stable Nash equilibria, where the Le Chatelier principle is violated even locally: in contrast to the thermodynamic equilibrium a Nash equilibrium can amplify small perturbations, though both types of equilibria satisfy the detailed balance condition.

  1. Rubella Virus Replication and Links to Teratogenicity

    PubMed Central

    Lee, Jia-Yee; Bowden, D. Scott

    2000-01-01

    Rubella virus (RV) is the causative agent of the disease known more popularly as German measles. Rubella is predominantly a childhood disease and is endemic throughout the world. Natural infections of rubella occur only in humans and are generally mild. Complications of rubella infection, most commonly polyarthralgia in adult women, do exist; occasionally more serious sequelae occur. However, the primary public health concern of RV infection is its teratogenicity. RV infection of women during the first trimester of pregnancy can induce a spectrum of congenital defects in the newborn, known as congenital rubella syndrome (CRS). The development of vaccines and implementation of vaccination strategies have substantially reduced the incidence of disease and in turn of CRS in developed countries. The pathway whereby RV infection leads to teratogenesis has not been elucidated, but the cytopathology in infected fetal tissues suggests necrosis and/or apoptosis as well as inhibition of cell division of critical precursor cells involved in organogenesis. In cell culture, a number of unusual features of RV replication have been observed, including mitochondrial abnormalities, and disruption of the cytoskeleton; these manifestations are most probably linked and play some role in RV teratogenesis. Further understanding of the mechanism of RV teratogenesis will be brought about by the investigation of RV replication and virus-host interactions. PMID:11023958

  2. Field replication of classwide peer tutoring.

    PubMed

    Greenwood, C R; Dinwiddie, G; Bailey, V; Carta, J J; Dorsey, D; Kohler, F W; Nelson, C; Rotholz, D; Schulte, D

    1987-01-01

    We conducted a large-scale field replication study of classwide peer tutoring applied to spelling instruction (Greenwood, Delquadri, & Hall, 1984). Two hundred and eleven inner-city students in four schools participated during their first- and second-grade school years. The effects of classwide peer tutoring were compared to teacher instructional procedures and pretest probes using a group replication design (Barlow, Hayes, & Nelson, 1984). Analysis of group and individual results indicated that (a) both teacher instructional procedures and classwide peer tutoring were effective in increasing spelling performance above pretest levels, (b) peer tutoring produced statistically greater gains relative to the teachers' procedures for both low and high student groups formed on pretest levels, (c) these outcomes were representative of groups, classes, individuals, and years during the project, and (d) participant satisfaction with the program was generally high. A separate analysis of the social importance of treatment outcome revealed differential findings for low and high groups related to pretest levels. Implications of these findings are discussed.

  3. Field replication of classwide peer tutoring.

    PubMed Central

    Greenwood, C R; Dinwiddie, G; Bailey, V; Carta, J J; Dorsey, D; Kohler, F W; Nelson, C; Rotholz, D; Schulte, D

    1987-01-01

    We conducted a large-scale field replication study of classwide peer tutoring applied to spelling instruction (Greenwood, Delquadri, & Hall, 1984). Two hundred and eleven inner-city students in four schools participated during their first- and second-grade school years. The effects of classwide peer tutoring were compared to teacher instructional procedures and pretest probes using a group replication design (Barlow, Hayes, & Nelson, 1984). Analysis of group and individual results indicated that (a) both teacher instructional procedures and classwide peer tutoring were effective in increasing spelling performance above pretest levels, (b) peer tutoring produced statistically greater gains relative to the teachers' procedures for both low and high student groups formed on pretest levels, (c) these outcomes were representative of groups, classes, individuals, and years during the project, and (d) participant satisfaction with the program was generally high. A separate analysis of the social importance of treatment outcome revealed differential findings for low and high groups related to pretest levels. Implications of these findings are discussed. PMID:3610894

  4. Replicative DNA polymerase mutations in cancer.

    PubMed

    Heitzer, Ellen; Tomlinson, Ian

    2014-02-01

    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. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Replicated polymer optical waveguides and the application

    NASA Astrophysics Data System (ADS)

    Terakawa, Yukari; Hosokawa, Hayami

    2006-10-01

    Recently, optical communication networks, such as FTTH (Fiber-To-The-Home), are spreading rapidly to the end-user. And the expectation of replacing electrical signal lines in the body of mobile devices by optical lines is growing based on the recent increase of communication density and the need of smaller mobile devices, also. But, the business fields are facing directly to the consumers, therefore, the serious cost reduction and higher productivity for large-scale production are expected toward the optical communication devices. In order to achieve these expectations, many efforts are given to develop the replicated polymer optical waveguide, SPICA (Stacked Polymer optical IC/Advanced). SPICA is based our unique replication technology which realizes both low cost and large-scale production. And we already realized the single mode polymer optical waveguide by adopting this original technology. And, we evolved this technology to fabricate the V-groove integrated optical waveguide and the film optical waveguide. The V-groove integrated optical waveguide realizes cost reduction at the optical fiber alignment and assembling processes by introducing the optical fiber self-alignment for further cost reduction as a optical link module. The film optical waveguide which is as flexible as electrical cables and is durable of many bending cycle test are realized. In this paper, the technology of SPICA and the applications are discussed.

  6. Replicative DNA polymerase mutations in cancer☆

    PubMed Central

    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

  7. Replication origin of mitochondrial DNA in insects.

    PubMed

    Saito, Shigeru; Tamura, Koichiro; Aotsuka, Tadashi

    2005-12-01

    The precise position of the replication origin (O(R)) of mtDNA was determined for insect species belonging to four different orders (four species of Drosophila, Bombyx mori, Triborium castaneum, and Locusta migratoria, which belong to Diptera, Lepidoptera, Coleoptera, and Orthoptera, respectively). Since the free 5' ends of the DNA strands of mtDNA are interpreted as the O(R), their positions were mapped at 1-nucleotide resolution within the A + T-rich region by using the ligation-mediated PCR method. In all species examined, the free 5' ends were found within a very narrow range of several nucleotides in the A + T-rich region. For four species of Drosophila, B. mori, and T. castaneum, which belong to holometabolous insects, although the O(R)'s were located at different positions, they were located immediately downstream of a series of thymine nucleotides, the so-called T-stretch. These results strongly indicate that the T-stretch is involved in the recognition of the O(R) of mtDNA at least among holometabolous insects. For L. migratoria (hemimetabolous insect), on the other hand, none of the long stretches of T's was found in the upstream portion of the O(R), suggesting that the regulatory sequences involved in the replication initiation process have changed through insect evolution.

  8. Inhibition of vaccinia virus replication by nitazoxanide.

    PubMed

    Hickson, Sarah E; Margineantu, Daciana; Hockenbery, David M; Simon, Julian A; Geballe, Adam P

    2018-04-03

    Nitazoxanide (NTZ) is an FDA-approved anti-protozoal drug that inhibits several bacteria and viruses as well. However, its effect on poxviruses is unknown. Therefore, we investigated the impact of NTZ on vaccinia virus (VACV). We found that NTZ inhibits VACV production with an EC 50 of ~2 μM, a potency comparable to that reported for several other viruses. The inhibitory block occurs early during the viral life cycle, prior to viral DNA replication. The mechanism of viral inhibition is likely not due to activation of intracellular innate immune pathways, such as protein kinase R (PKR) or interferon signaling, contrary to what has been suggested to mediate the effects of NTZ against some other viruses. Rather, our finding that addition of exogenous palmitate partially rescues VACV production from the inhibitory effect of NTZ suggests that NTZ impedes adaptations in cellular metabolism that are needed for efficient completion of the VACV replication cycle. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Inhibitory effects on HAV IRES-mediated translation and replication by a combination of amantadine and interferon-alpha

    PubMed Central

    2010-01-01

    Hepatitis A virus (HAV) causes acute hepatitis and sometimes leads to fulminant hepatitis. Amantadine is a tricyclic symmetric amine that inhibits the replication of many DNA and RNA viruses. Amantadine was reported to suppress HAV replication, and the efficacy of amantadine was exhibited in its inhibition of the internal ribosomal entry site (IRES) activities of HAV. Interferon (IFN) also has an antiviral effect through the induction of IFN stimulated genes (ISG) and the degradation of viral RNA. To explore the mechanism of the suppression of HAV replication, we examined the effects of the combination of amantadine and IFN-alpha on HAV IRES-mediated translation, HAV replicon replication in human hepatoma cell lines, and HAV KRM003 genotype IIIB strain replication in African green monkey kidney cell GL37. IFN-alpha seems to have no additive effect on HAV IRES-mediated translation inhibition by amantadine. However, suppressions of HAV replicon and HAV replication were stronger with the combination than with amantadine alone. In conclusion, amantadine, in combination of IFN-alpha, might have a beneficial effect in some patients with acute hepatitis A. PMID:20815893

  10. Estimation of different coal compressibilities of coalbed methane reservoirs under replicated in situ condition

    NASA Astrophysics Data System (ADS)

    Liu, Shimin

    Studies completed recently have shown that desorption of methane results in a change in the matrix volume of coal thus altering the permeability of, and production rates from, coalbed methane (CBM) reservoirs. An accurate estimation of different coal compressibilities is, therefore, critical in CBM operations in order to model and project gas production rates. Furthermore, a comprehensive knowledge of the dynamic permeability helps in understanding the unique feature of CBM production, an initial negative gas decline rate. In this study, different coal compressibility models were developed based on the assumption that the deformation of a depleting coalbed is limited to the vertical direction, that is, the reservoir is under uniaxial strain conditions. Simultaneously, experimental work was carried out replicating these conditions. The results showed that the matrix volumetric strain typically follows the Langmuir-type relationship. The agreement between the experimental results and those obtained using the proposed model was good. The proposed volumetric strain model successfully isolated the sorption-induced strain from the strain resulting from mechanical compression. It, therefore, provides a technique to integrate the sorption-induced strain alone into different analytical permeability models. The permeability variation of coal with a decrease in pore pressure under replicated in situ stress/strain conditions was measured. The results showed that decreasing pore pressure resulted in a significant decrease in horizontal stress and increased permeability. The permeability increased non-linearly with decreasing pore pressure, with a small increase in the high pressure range, increasing progressively as the pressure dropped below a certain value. The experimental results were also used to test the proposed coupled sorption-induced strain model and several analytical permeability models. One of the commonly used models overestimated the permeability increase between

  11. Cryptoporus volvatus Extract Inhibits Influenza Virus Replication In Vitro and In Vivo

    PubMed Central

    Si, Jianyong; Liu, Jinhua; Sun, Guibo; Sun, Xiaobo; Cao, Li

    2014-01-01

    Influenza virus is the cause of significant morbidity and mortality, posing a serious health threat worldwide. Here, we evaluated the antiviral activities of Cryptoporus volvatus extract on influenza virus infection. Our results demonstrated that the Cryptoporus volvatus extract inhibited different influenza virus strain replication in MDCK cells. Time course analysis indicated that the extract exerted its inhibition at earlier and late stages in the replication cycle of influenza virus. Subsequently, we confirmed that the extract suppressed virus internalization into and released from cells. Moreover, the extract significantly reduced H1N1/09 influenza virus load in lungs and dramatically decreased lung lesions in mice. And most importantly, the extract protected mice from lethal challenge with H1N1/09 influenza virus. Our results suggest that the Cryptoporus volvatus extract could be a potential candidate for the development of a new anti-influenza virus therapy. PMID:25437846

  12. Materials Chemistry and Performance of Silicone-Based Replicating Compounds.

    SciTech Connect

    Brumbach, Michael T.; Mirabal, Alex James; Kalan, Michael

    2014-11-01

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

  13. Sprains and Strains

    MedlinePlus

    ... and strains Overview Sprains and strains are common injuries that share similar signs and symptoms, but involve different parts of your body. A sprain is a stretching or tearing of ligaments — the tough bands of ...

  14. Quantifying Limits on Replication, Death, and Quiescence of Mycobacterium tuberculosis in Mice

    PubMed Central

    McDaniel, Margaret M.; Krishna, Nitin; Handagama, Winode G.; Eda, Shigetoshi; Ganusov, Vitaly V.

    2016-01-01

    When an individual is exposed to Mycobacterium tuberculosis (Mtb) three outcomes are possible: bacterial clearance, active disease, or latent infection. It is generally believed that most individuals exposed to Mtb become latently infected and carry the mycobacteria for life. How Mtb is maintained during this latent infection remains largely unknown. During an Mtb infection in mice, there is a phase of rapid increase in bacterial numbers in the murine lungs within the first 3 weeks, and then bacterial numbers either stabilize or increase slowly over the period of many months. It has been debated whether the relatively constant numbers of bacteria in the chronic infection result from latent (dormant, quiescent), non-replicating bacteria, or whether the observed Mtb cell numbers are due to balance between rapid replication and death. A recent study of mice, infected with a Mtb strain carrying an unstable plasmid, showed that during the chronic phase, Mtb was replicating at significant rates. Using experimental data from this study and mathematical modeling we investigated the limits of the rates of bacterial replication, death, and quiescence during Mtb infection of mice. First, we found that to explain the data the rates of bacterial replication and death could not be constant and had to decrease with time since infection unless there were large changes in plasmid segregation probability over time. While a decrease in the rate of Mtb replication with time since infection was expected due to depletion of host's resources, a decrease in the Mtb death rate was counterintuitive since Mtb-specific immune response, appearing in the lungs 3–4 weeks after infection, should increase removal of bacteria. Interestingly, we found no significant correlation between estimated rates of Mtb replication and death suggesting the decline in these rates was driven by independent mechanisms. Second, we found that the data could not be explained by assuming that bacteria do not die

  15. Elevated temperature strain gages

    NASA Technical Reports Server (NTRS)

    Brittain, J. O.; Geslin, D.; Lei, J. F.

    1985-01-01

    Materials were evaluated that could be used in manufacturing electrical resistance strain gages for static strain measurements at temperatures at or above 1273 K. Strain gage materials must have a characteristic response to strain, temperature and time that is reproducible or that varies in a predictable manner within specified limits. Several metallic alloys were evaluated, as well as a series of transition metal carbides, nitrides and silicides.

  16. Chromosome I Controls Chromosome II Replication in Vibrio cholerae

    PubMed Central

    Baek, Jong Hwan; Chattoraj, Dhruba K.

    2014-01-01

    Control of chromosome replication involves a common set of regulators in eukaryotes, whereas bacteria with divided genomes use chromosome-specific regulators. How bacterial chromosomes might communicate for replication is not known. In Vibrio cholerae, which has two chromosomes (chrI and chrII), replication initiation is controlled by DnaA in chrI and by RctB in chrII. DnaA has binding sites at the chrI origin of replication as well as outside the origin. RctB likewise binds at the chrII origin and, as shown here, to external sites. The binding to the external sites in chrII inhibits chrII replication. A new kind of site was found in chrI that enhances chrII replication. Consistent with its enhancing activity, the chrI site increased RctB binding to those chrII origin sites that stimulate replication and decreased binding to other sites that inhibit replication. The differential effect on binding suggests that the new site remodels RctB. The chaperone-like activity of the site is supported by the finding that it could relieve the dependence of chrII replication on chaperone proteins DnaJ and DnaK. The presence of a site in chrI that specifically controls chrII replication suggests a mechanism for communication between the two chromosomes for replication. PMID:24586205

  17. Eukaryotic Mismatch Repair in Relation to DNA Replication

    PubMed Central

    Erie, Dorothy A.

    2017-01-01

    Three processes act in series to accurately replicate the eukaryotic nuclear genome. The major replicative DNA polymerases strongly prevent mismatch formation, occasional mismatches that do form are proofread during replication, and rare mismatches that escape proofreading are corrected by mismatch repair (MMR). This review focuses on MMR in light of increasing knowledge about nuclear DNA replication enzymology and the rate and specificity with which mismatches are generated during leading- and lagging-strand replication. We consider differences in MMR efficiency in relation to mismatch recognition, signaling to direct MMR to the nascent strand, mismatch removal, and the timing of MMR. These studies are refining our understanding of relationships between generating and repairing replication errors to achieve accurate replication of both DNA strands of the nuclear genome. PMID:26436461

  18. Prevention of DNA re-replication in eukaryotic cells

    PubMed Central

    Truong, Lan N.; Wu, Xiaohua

    2011-01-01

    DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints. Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and present possible mechanisms to prevent re-replication in order to maintain genome stability. PMID:21278447

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

    PubMed

    Earp, Brian D; Trafimow, David

    2015-01-01

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

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

    PubMed Central

    Earp, Brian D.; Trafimow, David

    2015-01-01

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

  1. Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces

    PubMed Central

    Tsai, Hsiu-Hui; Shu, Hung-Wei; Yang, Chien-Chin; Chen, Carton W.

    2012-01-01

    Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5′-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3′-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor. PMID:22006845

  2. Sprains and Strains

    MedlinePlus

    ... happens. A strain is a stretched or torn muscle or tendon. Tendons are tissues that connect muscle to bone. Twisting or pulling these tissues can ... suddenly or develop over time. Back and hamstring muscle strains are common. Many people get strains playing ...

  3. Do Neuroscience Journals Accept Replications? A Survey of Literature

    PubMed Central

    Yeung, Andy W. K.

    2017-01-01

    Background: Recent reports in neuroscience, especially those concerning brain-injury and neuroimaging, have revealed low reproducibility of results within the field and urged for more replication studies. However, it is unclear if the neuroscience journals welcome or discourage the submission of reports on replication studies. Therefore, the current study assessed the explicit position of neuroscience journals on replications. Methods: A list of active neuroscience journals publishing in English was compiled from Scopus database. These journal websites were accessed to read their aims and scope and instructions to authors, and to assess if they: (1) explicitly stated that they accept replications; (2) did not state their position on replications; (3) implicitly discouraged replications by emphasizing on the novelty of the manuscripts; or (4) explicitly stated that they reject replications. For journals that explicitly stated they accept or reject replications, their subcategory within neuroscience and their 5-year impact factor were recorded. The distribution of neuroscience replication studies published was also recorded by searching and extracting data from Scopus. Results: Of the 465 journals reviewed, 28 (6.0%) explicitly stated that they accept replications, 394 (84.7%) did not state their position on replications, 40 (8.6%) implicitly discouraged replications by emphasizing on the novelty of the manuscripts, and 3 (0.6%) explicitly stated that they reject replications. For the 28 journals that explicitly welcomed replications, three (10.7%) stated their position in the aims and scope, whereas 25 (89.3%) stated in within the detailed instructions to authors. The five-year impact factor (2015) of these journals ranged from 1.655 to 10.799, and nine of them (32.1%) did not receive a 5-year or annual impact factor in 2015. There was no significant difference in the proportions of journals explicitly welcomed replications (journals with vs. without impact factors

  4. Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle.

    PubMed

    Qian, Jie; Chen, Yueyue; Hu, Ying; Deng, Yingtian; Liu, Yang; Li, Gang; Zou, Wenxuan; Zhao, Jie

    2018-04-01

    Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two- to four-cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4-1/+ with AtRFC4 expression driven through the embryo-specific DD45pro and ABI3pro or the endosperm-specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4-1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4-1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S-phase and M-phase cells in the rfc4-1/- FIS 2; DD 45; ABI 3pro::At RFC 4 seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

  5. Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells.

    PubMed

    Spuul, Pirjo; Balistreri, Giuseppe; Hellström, Kirsi; Golubtsov, Andrey V; Jokitalo, Eija; Ahola, Tero

    2011-05-01

    For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.

  6. Bidirectional Replication of Simian Virus 40 DNA*

    PubMed Central

    Danna, Kathleen J.; Nathans, Daniel

    1972-01-01

    SV40 (Simian Virus 40) DNA was pulse-labeled with [3H]thymidine in infected monkey cells, and the distribution of label within newly completed molecules was determined by analysis of specific fragments produced by restriction endonuclease from Hemophilus influenzae. From these data, an order of synthesis or temporal order of the fragments was deduced. Comparison of the temporal order with the physical order of the fragments in the SV40 DNA molecule indicates a correspondence in these orders for two separate groups of fragments. From an analysis of the results, we conclude that SV40 DNA replication begins at a specific site and proceeds bidirectionally, terminating about halfway around the circular molecule from the initiation point. Images PMID:4343955

  7. Star Trek replicators and diatom nanotechnology.

    PubMed

    Drum, Ryan W; Gordon, Richard

    2003-08-01

    Diatoms are single celled algae, the 10(5)-10(6) species of which create a wide variety of three-dimensional amorphous silica shells. If we could get them to produce useful structures, perhaps by compustat selection experiments (i.e. forced evolution of development or evodevo), their exponential growth in suspension cultures could compete with the lithography techniques of present day nanotechnology, which have limited 3D capabilities. Alternatively, their fine detail could be used for templates for MEMS (micro electro mechanical systems), or their silica deposition systems isolated for guiding silica deposition. A recent paper has demonstrated that silica can be replaced atom for atom without change of shape--a step towards the Star Trek replicator.

  8. Population control of self-replicating systems

    NASA Technical Reports Server (NTRS)

    Mccord, R. L.

    1982-01-01

    The literature concerning fibonacci sequence and the mathematics of self replication are reviewed. One option allows each primary to generate n-replicas, one in each sequential time frame after its own generation with no restrictions on the number of ancestors per replica. The state vector of the replicas in an efficient manner is determined. Option-B has a fixed number of replicas per primary and no restrictions on the number of ancestors for a replica. Any element fij represents the number of elements of type-j in time frame k+1 generated from type-i in time frame k. Option-D is a diagonal matrix whose eigenvalues are precisely those of f.

  9. Experimenter gender and replicability in science

    PubMed Central

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

    2018-01-01

    There is a replication crisis spreading through the annals of scientific inquiry. Although some work has been carried out to uncover the roots of this issue, much remains unanswered. With this in mind, this paper investigates how the gender of the experimenter may affect experimental findings. Clinical trials are regularly carried out without any report of the experimenter’s gender and with dubious knowledge of its influence. Consequently, significant biases caused by the experimenter’s gender may lead researchers to conclude that therapeutics or other interventions are either overtreating or undertreating a variety of conditions. Bearing this in mind, this policy paper emphasizes the importance of reporting and controlling for experimenter gender in future research. As backdrop, it explores what we know about the role of experimenter gender in influencing laboratory results, suggests possible mechanisms, and suggests future areas of inquiry. PMID:29349293

  10. Best practice not being sufficiently replicated.

    PubMed

    Baillie, Jonathan

    2016-06-01

    'Efficient productivity through innovation' was the theme of the first ever Hospital Innovations conference and exhibition at Olympia, London in late April. At the two-day event--supported by organisations including IHEEM, the Legionella Control Association, the Water Management Society, the BRE, and a sizeable number of English NHS Trusts--the Day Two keynote address by Lord Carter very much reflected this theme. Following his address at Healthcare Estates 2015, the Labour Peer focused further in London on his team's recent review of the 'productivity and efficiency' of English NHS Trusts, and explained how the initiative would progress in coming months. One of his key conclusions was that while the NHS consistently rates as one of the world's most efficient public health systems, innovation and good practice are rarely sufficiently shared or widely replicated service-wide, resulting in a considerable 'gap' between the best and worst-performing Trusts. HEJ editor, Jonathan Baillie, reports.

  11. Replicator dynamics with turnover of players

    NASA Astrophysics Data System (ADS)

    Juul, Jeppe; Kianercy, Ardeshir; Bernhardsson, Sebastian; Pigolotti, Simone

    2013-08-01

    We study adaptive dynamics in games where players abandon the population at a given rate and are replaced by naive players characterized by a prior distribution over the admitted strategies. We demonstrate how such a process leads macroscopically to a variant of the replicator equation, with an additional term accounting for player turnover. We study how Nash equilibria and the dynamics of the system are modified by this additional term for prototypical examples such as the rock-paper-scissors game and different classes of two-action games played between two distinct populations. We conclude by showing how player turnover can account for nontrivial departures from Nash equilibria observed in data from lowest unique bid auctions.

  12. Functional characterization of a replication initiator protein.

    PubMed Central

    Gammie, A E; Tolmasky, M E; Crosa, J H

    1993-01-01

    Functional domains in the RepI replication initiator protein have been identified by classical and site-directed mutagenesis techniques. Mutations conferring an increase in plasmid copy number contained alterations in a key position of a putative helix-turn-helix DNA binding motif. The mutations did not appear to affect autorepressing functions. Regions of RepI important for autorepression were localized as well. Two classes of mutations resulting in diminished autorepression functions were identified. One class was distinguished by an elevated copy number, while the other class remained at the wild-type copy number level. Analysis of the various mutations leading to changes in copy number or autorepressing functions suggest that in some cases the autorepression and initiating functions of the RepI protein are separable. Finally, analysis with deletion clones suggests that the trans-acting autorepressing functions of RepI might depend on intermolecular coupling control. Images PMID:8501060

  13. Replication of Muscle Cell Using Bioimprint

    NASA Astrophysics Data System (ADS)

    Samsuri, Fahmi; Mitchell, John S.; Alkaisi, Maan M.; Evans, John J.

    2009-07-01

    In our earlier study a heat-curable PDMS or a UV curable elastomer, was used as the replicating material to introduce Bioimprint methodology to facilitate cell imaging [1-2] But, replicating conditions for thermal polymerization is known to cause cell dehydration during curing. In this study, a new type of polymer was developed for use in living cell replica formation, and it was tested on human muscle cells. The cells were incubated and cultured according to standard biological culturing procedures, and they were grown for about 10 days. The replicas were then separated from the muscle cells and taken for analysis under an Atomic Force Microscope (AFM). The new polymer was designed to be biocompatible with higher resolution and fast curing process compared to other types of silicon-based organic polymers such as polydimethylsiloxane (PDMS). Muscle cell imprints were achieved and higher resolution images were able to show the micro structures of the muscle cells, including the cellular fibers and cell membranes. The AFM is able to image features at nanoscale resolution. This capacity enables a number of characteristics of biological cells to be visualized in a unique manner. Polymer and muscle cells preparations were developed at Hamilton, in collaboration between Plant and Food Research and the Department of Electrical and Computer Engineering, University of Canterbury. Tapping mode was used for the AFM image analysis as it has low tip-sample forces and non-destructive imaging capability. We will be presenting the bioimprinting processes of muscle cells, their AFM imaging and characterization of the newly developed polymer.

  14. Biogenesis and architecture of arterivirus replication organelles.

    PubMed

    van der Hoeven, Barbara; Oudshoorn, Diede; Koster, Abraham J; Snijder, Eric J; Kikkert, Marjolein; Bárcena, Montserrat

    2016-07-15

    All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double-membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation

  15. Simian Virus 40 Deoxyribonucleic Acid Replication

    PubMed Central

    Kit, Saul; Kurimura, Takashi; de Torres, Ramon A.; Dubbs, Del R.

    1969-01-01

    Infectious deoxyribonucleic acid (DNA) was extracted from green monkey kidney (CV-1) cultures at various times after the cultures were infected with simian virus 40 (SV40) at input multiplicities of 0.01 and 0.1 plaque-forming unit (PFU) per cell. A pronounced decrease in infectious DNA was observed from 3 to 16 hr after virus infection, suggesting that structurally altered intracellular forms may have been generated early in infection. Evidence is also presented that SV40 DNA synthesis requires concurrent protein synthesis. DNA replication was studied in the presence and absence of cycloheximide in: (i) SV40-infected and uninfected cultures of CV-1 cells; (ii) cultures synchronized with 1-β-d-arabinofuranosylcytosine (ara-C) for 24 to 30 hr prior to the addition of cycloheximide; and (iii) in heterokaryons of SV40-transformed hamster and susceptible monkey kidney cells. DNA synthesis was determined by pulse-labeling the cultures with 3H-thymidine at various times from 24 to 46 hr after infection. In addition, the total infectious SV40 DNA was measured. Addition of cycloheximide, even after early proteins had been induced, grossly inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNA polymerase, deoxycytidylate deaminase, and thymidylate kinase were measured; these enzyme activities remained high for at least 9 hr in the presence of cycloheximide. SV40 DNA prelabeled with 3H-thymidine before the addition of cycloheximide was also relatively stable during the time required for cycloheximide to inhibit further DNA replication. PMID:4306301

  16. Multiple genetic pathways regulate replicative senescence in telomerase-deficient yeast

    PubMed Central

    Ballew, Bari J.; Lundblad, Victoria

    2013-01-01

    Summary Most human tissues express low levels of telomerase and undergo telomere shortening and eventual senescence; the resulting limitation on tissue renewal can lead to a wide range of age-dependent pathophysiologies. Increasing evidence indicates that the decline in cell division capacity in cells that lack telomerase can be influenced by numerous genetic factors. Here, we use telomerase-defective strains of budding yeast to probe whether replicative senescence can be attenuated or accelerated by defects in factors previously implicated in handling of DNA termini. We show that the MRX (Mre11-Rad50-Xrs2) complex, as well as negative (Rif2) and positive (Tel1) regulators of this complex, comprise a single pathway that promotes replicative senescence, in a manner that recapitulates how these proteins modulate resection of DNA ends. In contrast, the Rad51 recombinase, which acts downstream of the MRX complex in double-strand break (DSB) repair, regulates replicative senescence through a separate pathway operating in opposition to the MRX-Tel1-Rif2 pathway. Moreover, defects in several additional proteins implicated in DSB repair (Rif1 and Sae2) confer only transient effects during early or late stages of replicative senescence, respectively, further suggesting that a simple analogy between DSBs and eroding telomeres is incomplete. These results indicate that the replicative capacity of telomerase-defective yeast is controlled by a network comprised of multiple pathways. It is likely that telomere shortening in telomerase-depleted human cells is similarly under a complex pattern of genetic control; mechanistic understanding of this process should provide crucial information regarding how human tissues age in response to telomere erosion. PMID:23672410

  17. Replication of swine and human influenza viruses in juvenile and layer turkey hens.

    PubMed

    Ali, Ahmed; Yassine, Hadi; Awe, Olusegun O; Ibrahim, Mahmoud; Saif, Yehia M; Lee, Chang-Won

    2013-04-12

    Since the first reported isolation of swine influenza viruses (SIVs) in turkeys in the 1980s, transmission of SIVs to turkeys was frequently documented. Recently, the 2009 pandemic H1N1 virus, that was thought to be of swine origin, was detected in turkeys with a severe drop in egg production. In this study, we assessed the infectivity of different mammalian influenza viruses including swine, pandemic H1N1 and seasonal human influenza viruses in both juvenile and layer turkeys. In addition, we investigated the potential influenza virus dissemination in the semen of experimentally infected turkey toms. Results showed that all mammalian origin influenza viruses tested can infect turkeys. SIVs were detected in respiratory and digestive tracts of both juvenile and layer turkeys. Variations in replication efficiencies among SIVs were observed especially in the reproductive tract of layer turkeys. Compared to SIVs, limited replication of seasonal human H1N1 and no detectable replication of recent human-like swine H1N2, pandemic H1N1 and seasonal human H3N2 viruses was noticed. All birds seroconverted to all tested viruses regardless of their replication level. In turkey toms, we were able to detect swine H3N2 virus in semen and reproductive tract of infected toms by real-time RT-PCR although virus isolation was not successful. These data suggest that turkey hens could be affected by diverse influenza strains especially SIVs. Moreover, the differences in the replication efficiency we demonstrated among SIVs and between SIV and human influenza viruses in layer turkeys suggest a possible use of turkeys as an animal model to study host tropism and pathogenesis of influenza viruses. Our results also indicate a potential risk of venereal transmission of influenza viruses in turkeys. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Modulation of Replicative Lifespan in Cryptococcus neoformans: Implications for Virulence

    PubMed Central

    Bouklas, Tejas; Jain, Neena; Fries, Bettina C.

    2017-01-01

    The fungal pathogen, Cryptococcus neoformans, has been shown to undergo replicative aging. Old cells are characterized by advanced generational age and phenotypic changes that appear to mediate enhanced resistance to host and antifungal-based killing. As a consequence of this age-associated resilience, old cells accumulate during chronic infection. Based on these findings, we hypothesized that shifting the generational age of a pathogenic yeast population would alter its vulnerability to the host and affect its virulence. SIR2 is a well-conserved histone deacetylase, and a pivotal target for the development of anti-aging drugs. We tested its effect on C. neoformans’ replicative lifespan (RLS). First, a mutant C. neoformans strain (sir2Δ) was generated, and confirmed a predicted shortened RLS in sir2Δ cells consistent with its known role in aging. Next, RLS analysis showed that treatment of C. neoformans with Sir2p-agonists resulted in a significantly prolonged RLS, whereas treatment with a Sir2p-antagonist shortened RLS. RLS modulating effects were dependent on SIR2 and not observed in sir2Δ cells. Because SIR2 loss resulted in a slightly impaired fitness, effects of genetic RLS modulation on virulence could not be compared with wild type cells. Instead we chose to chemically modulate RLS, and investigated the effect of Sir2p modulating drugs on C. neoformans cells in a Galleria mellonella infection model. Consistent with our hypothesis that shifts in the generational age of the infecting yeast population alters its vulnerability to host cells, we observed decreased virulence of C. neoformans in the Galleria host when RLS was prolonged by treatment with Sir2p agonists. In contrast, treatment with a Sir2p antagonist, which shortens RLS enhanced virulence in Galleria. In addition, combination of Sir2p agonists with antifungal therapy enhanced the antifungal’s effect. Importantly, no difference in virulence was observed with drug treatment when sir2Δ cells

  19. Inhomogeneous DNA replication kinetics is associated with immune system response

    NASA Astrophysics Data System (ADS)

    Bechhoefer, John; Gauthier, Michel G.; Norio, Paolo

    2013-03-01

    In eukaryotic organisms, DNA replication is initiated at ``origins,'' launching ``forks'' that spread bidirectionally to replicate the genome. The distribution and firing rate of these origins and the fork progression velocity form the ``replication program.'' Previous models of DNA replication in eukaryotes have assumed firing rates and replication fork velocities to be homogeneous across the genome. But large variations in origin activity and fork velocity do occur. Here, we generalize our replication model to allow for arbitrary spatial variation of initiation rates and fork velocities in a given region of the genome. We derive and solve rate equations for the forks and replication probability, to obtain the mean-field replication program. After testing the model on simulations, we analyze the changes in replication program that occur during B cell development in the mouse. B cells play a major role in the adaptive immune system by producing the antibodies. We show that the process of cell differentiation is associated with a change in replication program, where the zones of high origin initiation rates located in the immunoglobulin heavy-chain locus shift their position as the locus prepares to undergo the recombination events responsible for generating antibody specificity. This work was funded by HSFP and NSERC-Canada (MGG and JB) and by NIH-NIGMS grant R01GM080606 (PN).

  20. Software for Replicating Data Between X.500 and LDAP Directories

    NASA Technical Reports Server (NTRS)

    Wolfe, Thomas

    2003-01-01

    X500/LDAP Directory Replication Utility is a computer program for replicating information between X.500 and LDAP directories. [X.500 is an international standard for on-line directory services. LDAP (Lightweight Directory Access Protocol) is a simple directory access protocol.] The utility can be used to replicate an object of any type from X.500 to LDAP or from LDAP to X.500. The program uses the LDAP version 2 protocol, which is capable of working with both X.500 and LDAP directories. The program can provide any or all of the following services: (1) replicate only modified objects; (2) force replication of all objects; (3) replicate individual objects, one level of objects, or a subtree of objects; (4) filter sets of objects to select ones to be replicated; (5) remove and/or modify object classes from objects that are replicated; and (6) select and/or limit attributes that are replicated. The program includes a separate program that is used to remove objects that are no longer required to be replicated.

  1. Exploring and exploiting the systemic effects of deregulated replication licensing.

    PubMed

    Petrakis, Theodoros G; Komseli, Eirini-Stavroula; Papaioannou, Marilena; Vougas, Kostas; Polyzos, Alexandros; Myrianthopoulos, Vassilios; Mikros, Emmanuel; Trougakos, Ioannis P; Thanos, Dimitris; Branzei, Dana; Townsend, Paul; Gorgoulis, Vassilis G

    2016-06-01

    Maintenance and accurate propagation of the genetic material are key features for physiological development and wellbeing. The replication licensing machinery is crucial for replication precision as it ensures that replication takes place once per cell cycle. Thus, the expression status of the components comprising the replication licensing apparatus is tightly regulated to avoid re-replication; a form of replication stress that leads to genomic instability, a hallmark of cancer. In the present review we discuss the mechanistic basis of replication licensing deregulation, which leads to systemic effects, exemplified by its role in carcinogenesis and a variety of genetic syndromes. In addition, new insights demonstrate that above a particular threshold, the replication licensing factor Cdc6 acts as global transcriptional regulator, outlining new lines of exploration. The role of the putative replication licensing factor ChlR1/DDX11, mutated in the Warsaw Breakage Syndrome, in cancer is also considered. Finally, future perspectives focused on the potential therapeutic advantage by targeting replication licensing factors, and particularly Cdc6, are discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production.

    PubMed

    Murayama, Asako; Sugiyama, Nao; Suzuki, Ryosuke; Moriyama, Masaki; Nakamura, Noriko; Mochizuki, Hidenori; Wakita, Takaji; Kato, Takanobu

    2017-02-15

    Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3'X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3'X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3'X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3'X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3'X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells. The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell

  3. Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production

    PubMed Central

    Murayama, Asako; Sugiyama, Nao; Suzuki, Ryosuke; Moriyama, Masaki; Nakamura, Noriko; Mochizuki, Hidenori; Wakita, Takaji

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3′X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3′X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3′X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3′X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3′X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells. IMPORTANCE The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life

  4. Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22.

    PubMed

    Cheng, Fan; Ramos da Silva, Suzane; Huang, I-Chueh; Jung, Jae U; Gao, Shou-Jiang

    2018-02-15

    The recent outbreak of Zika virus (ZIKV), a reemerging flavivirus, and its associated neurological disorders, such as Guillain-Barré (GB) syndrome and microcephaly, have generated an urgent need to develop effective ZIKV vaccines and therapeutic agents. Here, we used human endothelial cells and astrocytes, both of which represent key cell types for ZIKV infection, to identify potential inhibitors of ZIKV replication. Because several pathways, including the AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) signaling pathways, have been reported to play important roles in flavivirus replication, we tested inhibitors and agonists of these pathways for their effects on ZIKV replication. We identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. PKI effectively suppressed the replication of ZIKV from both the African and Asian/American lineages with a high efficiency and minimal cytotoxicity. While ZIKV infection does not induce PKA activation, endogenous PKA activity is essential for supporting ZIKV replication. Interestingly, in addition to PKA, PKI also inhibited another unknown target(s) to block ZIKV replication. PKI inhibited ZIKV replication at the postentry stage by preferentially affecting negative-sense RNA synthesis as well as viral protein translation. Together, these results have identified a potential inhibitor of ZIKV replication which could be further explored for future therapeutic application. IMPORTANCE There is an urgent need to develop effective vaccines and therapeutic agents against Zika virus (ZIKV) infection, a reemerging flavivirus associated with neurological disorders, including Guillain-Barré (GB) syndrome and microcephaly. By screening for inhibitors of several cellular pathways, we have identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. We show that PKI effectively suppresses the replication of all ZIKV

  5. Three-Dimensional Architecture of Tick-Borne Encephalitis Virus Replication Sites and Trafficking of the Replicated RNA

    PubMed Central

    Romero-Brey, Inés; Maiuri, Paolo; Hoppe, Simone; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

    2013-01-01

    Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle. PMID:23552408

  6. Replication of Avocado Sunblotch Viroid in the Yeast Saccharomyces cerevisiae▿

    PubMed Central

    Delan-Forino, Clémentine; Maurel, Marie-Christine; Torchet, Claire

    2011-01-01

    Viroids are the smallest known pathogenic agents. They are noncoding, single-stranded, closed-circular, “naked” RNAs, which replicate through RNA-RNA transcription. Viroids of the Avsunviroidae family possess a hammerhead ribozyme in their sequence, allowing self-cleavage during their replication. To date, viroids have only been detected in plant cells. Here, we investigate the replication of Avocado sunblotch viroid (ASBVd) of the Avsunviroidae family in a nonconventional host, the yeast Saccharomyces cerevisiae. We demonstrate that ASBVd RNA strands of both polarities are able to self-cleave and to replicate in a unicellular eukaryote cell. We show that the viroid monomeric RNA is destabilized by the nuclear 3′ and the cytoplasmic 5′ RNA degradation pathways. For the first time, our results provide evidence that viroids can replicate in other organisms than plants and that yeast contains all of the essential cellular elements for the replication of ASBVd. PMID:21270165

  7. Capitalizing on disaster: Establishing chromatin specificity behind the replication fork.

    PubMed

    Ramachandran, Srinivas; Ahmad, Kami; Henikoff, Steven

    2017-04-01

    Eukaryotic genomes are packaged into nucleosomal chromatin, and genomic activity requires the precise localization of transcription factors, histone modifications and nucleosomes. Classic work described the progressive reassembly and maturation of bulk chromatin behind replication forks. More recent proteomics has detailed the molecular machines that accompany the replicative polymerase to promote rapid histone deposition onto the newly replicated DNA. However, localized chromatin features are transiently obliterated by DNA replication every S phase of the cell cycle. Genomic strategies now observe the rebuilding of locus-specific chromatin features, and reveal surprising delays in transcription factor binding behind replication forks. This implies that transient chromatin disorganization during replication is a central juncture for targeted transcription factor binding within genomes. We propose that transient occlusion of regulatory elements by disorganized nucleosomes during chromatin maturation enforces specificity of factor binding. © 2017 WILEY Periodicals, Inc.

  8. Small telescopes: detectability and the evaluation of replication results.

    PubMed

    Simonsohn, Uri

    2015-05-01

    This article introduces a new approach for evaluating replication results. It combines effect-size estimation with hypothesis testing, assessing the extent to which the replication results are consistent with an effect size big enough to have been detectable in the original study. The approach is demonstrated by examining replications of three well-known findings. Its benefits include the following: (a) differentiating "unsuccessful" replication attempts (i.e., studies yielding p > .05) that are too noisy from those that actively indicate the effect is undetectably different from zero, (b) "protecting" true findings from underpowered replications, and (c) arriving at intuitively compelling inferences in general and for the revisited replications in particular. © The Author(s) 2015.

  9. DnaA and ORC: more than DNA replication initiators.

    PubMed

    Scholefield, Graham; Veening, Jan-Willem; Murray, Heath

    2011-03-01

    Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. Prestressed F-actin networks cross-linked by hinged filamins replicate mechanical properties of cells

    NASA Astrophysics Data System (ADS)

    Gardel, M. L.; Nakamura, F.; Hartwig, J. H.; Crocker, J. C.; Stossel, T. P.; Weitz, D. A.

    2006-02-01

    We show that actin filaments, shortened to physiological lengths by gelsolin and cross-linked with recombinant human filamins (FLNs), exhibit dynamic elastic properties similar to those reported for live cells. To achieve elasticity values of comparable magnitude to those of cells, the in vitro network must be subjected to external prestress, which directly controls network elasticity. A molecular requirement for the strain-related behavior at physiological conditionsis a flexible hinge found in FLNa and some FLNb molecules. Basic physical properties of the in vitro filamin-F-actin network replicate the essential mechanical properties of living cells. This physical behavior could accommodate passive deformation and internal organelle trafficking at low strains yet resist externally or internally generated high shear forces. cytoskeleton | cell mechanics | nonlinear rheology

  11. Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

    PubMed

    Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Chen, Chun-Long; Arneodo, Alain; Goldar, Arach; d'Aubenton-Carafa, Yves; Thermes, Claude; Audit, Benjamin; Hyrien, Olivier

    2011-12-01

    Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general

  12. Apoptosis was promoted at a nonpermissive temperature in DNA replication-defective temperature-sensitive mutants of mouse FM3A cells.

    PubMed

    Yamauchi, Y; Tanaka, A; Yamaguchi, K; Kobayashi, M; Shimamura, S; Hanaoka, F

    1998-02-01

    Apoptosis was promoted at the nonpermissive temperature in some temperature-sensitive (ts) mutant strains of mouse FM3A cells deficient in initiation of DNA replication. We examined expression of cell cycle regulation genes in the four ts mutant strains and found that two strains, tsFT107 and tsFT111, exhibited marked accumulation of p53 protein by a posttranscriptional mechanism at 16 h after temperature up-shift. These two strains also exhibited high levels of p21 mRNA expression, repression of cyclin A and D1 mRNAs, and obvious accumulation of underphosphorylated retinoblastoma protein. Only these two strains died by apoptosis at day 3 after up-shift, although no change was observed in the level of bax mRNA. These results suggest the existence of two types of responses after temperature up-shift in the four temperature-sensitive cell strains of the initiation of DNA replication: one type directs inappropriate DNA replication that then may produce endogenous DNA damage, p53-mediated cell cycle arrest, and subsequent apoptosis, while the other type exhibits only the p53-independent cell cycle arrest. Copyright 1998 Academic Press.

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

    PubMed

    Briner, Wayne; Kirwan, Jeral

    2017-01-01

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

  14. Antimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis.

    PubMed

    Segretti, Natanael Dante; Simões, Cristina Kortstee; Corrêa, Michelle Fidelis; Felli, Veni Maria Andres; Miyata, Marcelo; Cho, Sang Hyun; Franzblau, Scott Gary; Fernandes, João Paulo Dos Santos

    2016-07-01

    Tuberculosis (TB) is an important infectious disease caused by Mycobacterium tuberculosis (Mtb) and responsible for thousands of deaths every year. Although there are antimycobacterial drugs available in therapeutics, just few new chemical entities have reached clinical trials, and in fact, since introduction of rifampin only two important drugs had reached the market. Pyrazinoic acid (POA), the active agent of pyrazinamide, has been explored through prodrug approach to achieve novel molecules with anti-Mtb activity, however, there is no activity evaluation of these molecules against non-replicating Mtb until the present. Additionally, pharmacokinetic must be preliminary evaluated to avoid future problems during clinical trials. In this paper, we have presented six POA esters as prodrugs in order to evaluate their anti-Mtb activity in replicating and non-replicating Mtb, and these showed activity highly influenced by medium composition (especially by albumin). Lipophilicity seems to play the main role in the activity, possibly due to controlling membrane passage. Novel duplicated prodrugs of POA were also described, presenting interesting activity. Cytotoxicity of these prodrugs set was also evaluated, and these showed no important cytotoxic profile. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. RuvAB is essential for replication forks reversal in certain replication mutants

    PubMed Central

    Baharoglu, Zeynep; Petranovic, Mirjana; Flores, Maria-Jose; Michel, Bénédicte

    2006-01-01

    Inactivated replication forks may be reversed by the annealing of leading- and lagging-strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double-strand end. In Escherichia coli mutants deficient for double-strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ-specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR. PMID:16424908

  16. Three dimensional strained semiconductors

    SciTech Connect

    Voss, Lars; Conway, Adam; Nikolic, Rebecca J.

    2016-11-08

    In one embodiment, an apparatus includes a three dimensional structure comprising a semiconductor material, and at least one thin film in contact with at least one exterior surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the three dimensional structure. In another embodiment, a method includes forming a three dimensional structure comprising a semiconductor material, and depositing at least one thin film on at least onemore » surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the structure.« less

  17. Three dimensional strained semiconductors

    DOEpatents

    Voss, Lars; Conway, Adam; Nikolic, Rebecca J.; Leao, Cedric Rocha; Shao, Qinghui

    2016-11-08

    In one embodiment, an apparatus includes a three dimensional structure comprising a semiconductor material, and at least one thin film in contact with at least one exterior surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the three dimensional structure. In another embodiment, a method includes forming a three dimensional structure comprising a semiconductor material, and depositing at least one thin film on at least one surface of the three dimensional structure for inducing a strain in the structure, the thin film being characterized as providing at least one of: an induced strain of at least 0.05%, and an induced strain in at least 5% of a volume of the structure.

  18. Prion replication without host adaptation during interspecies transmissions

    PubMed Central

    Bian, Jifeng; Khaychuk, Vadim; Angers, Rachel C.; Fernández-Borges, Natalia; Meyerett-Reid, Crystal; Kim, Sehun; Calvi, Carla L.; Bartz, Jason C.; Hoover, Edward A.; Agrimi, Umberto; Richt, Jürgen A.; Castilla, Joaquín; Telling, Glenn C.

    2017-01-01

    Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC. Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders. PMID:28096357

  19. Prion replication without host adaptation during interspecies transmissions.

    PubMed

    Bian, Jifeng; Khaychuk, Vadim; Angers, Rachel C; Fernández-Borges, Natalia; Vidal, Enric; Meyerett-Reid, Crystal; Kim, Sehun; Calvi, Carla L; Bartz, Jason C; Hoover, Edward A; Agrimi, Umberto; Richt, Jürgen A; Castilla, Joaquín; Telling, Glenn C

    2017-01-31

    Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrP C ) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrP C Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrP C conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrP C was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrP C from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrP C also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.

  20. Integrative and replicative genetic transformation of Aureobasidium pullulans.

    PubMed

    Thornewell, S J; Peery, R B; Skatrud, P L

    1995-12-01

    A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1-alpha (TEF1) adjacent to the 5' end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmBR) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41bp of homologous DNA has also been shown to transform A. pullulans to HmBR. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmBR transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7-8kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmBR A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4kb) was unable to transform A. pullulans to HmBR.

  1. Functional Coupling between Replication and Packaging of Poliovirus Replicon RNA

    PubMed Central

    Nugent, Constance I.; Johnson, Kyle L.; Sarnow, Peter; Kirkegaard, Karla

    1999-01-01

    Poliovirus RNA genomes that contained deletions in the capsid-coding regions were synthesized in monkey kidney cells that constitutively expressed T7 RNA polymerase. These replication-competent subgenomic RNAs, or replicons (G. Kaplan and V. R. Racaniello, J. Virol. 62:1687–1696, 1988), were encapsidated in trans by superinfecting polioviruses. When superinfecting poliovirus resistant to the antiviral compound guanidine was used, the RNA replication of the replicon RNAs could be inhibited independently of the RNA replication of the guanidine-resistant helper virus. Inhibiting the replication of the replicon RNA also profoundly inhibited its trans-encapsidation, even though the capsid proteins present in the cells could efficiently encapsidate the helper virus. The observed coupling between RNA replication and RNA packaging could account for the specificity of poliovirus RNA packaging in infected cells and the observed effects of mutations in the coding regions of nonstructural proteins on virion morphogenesis. It is suggested that this coupling results from direct interactions between the RNA replication machinery and the capsid proteins. The coupling of RNA packaging to RNA replication and of RNA replication to translation (J. E. Novak and K. Kirkegaard, Genes Dev. 8:1726–1737, 1994) could serve as mechanisms for late proofreading of poliovirus RNA, allowing only those RNA genomes capable of translating a full complement of functional RNA replication proteins to be propagated. PMID:9847348

  2. Problem solving during artificial selection of self-replicating loops

    NASA Astrophysics Data System (ADS)

    Chou, Hui-Hsien; Reggia, James A.

    1998-05-01

    Past cellular automata models of self-replication have generally done only one thing: replicate themselves. However, it has recently been demonstrated that such self-replicating structures can be programmed to also carry out a task during the replication process. Past models of this sort have been limited in that the “program” involved is copied unchanged from parent to child, so that each generation of replicants is executing exactly the same program on exactly the same data. Here we take a different approach in which each replicant receives a distinct partial solution that is modified during replication. Under artificial selection, replicants with promising solutions proliferate while those with failed solutions are lost. We show that this approach can be applied successfully to solve an NP-complete problem, the satisfiability problem. Bounds are given on the cellular space size and time needed to solve a given problem, and simulations demonstrate that this approach works effectively. These and other recent results raise the possibility of evolving self-replicating structures that have a simulated metabolism or that carry out useful tasks.

  3. The architecture and function of the chromatin replication machinery.

    PubMed

    Miller, Thomas Cr; Costa, Alessandro

    2017-12-01

    Genomic DNA in eukaryotic cells is packaged into nucleosome arrays. During replication, nucleosomes need to be dismantled ahead of the advancing replication fork and reassembled on duplicated DNA. The architecture and function of the core replisome machinery is now beginning to be elucidated, with recent insights shaping our view on DNA replication processes. Simultaneously, breakthroughs in our mechanistic understanding of epigenetic inheritance allow us to build new models of how histone chaperones integrate with the replisome to reshuffle nucleosomes. The emerging picture indicates that the core eukaryotic DNA replication machinery has evolved elements that handle nucleosomes to facilitate chromatin duplication. Copyright © 2017. Published by Elsevier Ltd.

  4. FANCD2 Facilitates Replication through Common Fragile Sites.

    PubMed

    Madireddy, Advaitha; Kosiyatrakul, Settapong T; Boisvert, Rebecca A; Herrera-Moyano, Emilia; García-Rubio, María L; Gerhardt, Jeannine; Vuono, Elizabeth A; Owen, Nichole; Yan, Zi; Olson, Susan; Aguilera, Andrés; Howlett, Niall G; Schildkraut, Carl L

    2016-10-20

    Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress. In the absence of FANCD2, replication forks stall within the AT-rich fragility core of CFS, leading to dormant origin activation. Furthermore, FANCD2 deficiency is associated with DNA:RNA hybrid formation at CFS-FRA16D, and inhibition of DNA:RNA hybrid formation suppresses replication perturbation. In addition, we also found that FANCD2 reduces the number of potential sites of replication initiation. Our data demonstrate that FANCD2 protein is required to ensure efficient CFS replication and provide mechanistic insight into how FANCD2 regulates CFS stability. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena

    PubMed Central

    Sandoval, Pamela Y.; Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M.

    2015-01-01

    The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress. PMID:26218270

  6. PTEN regulates RPA1 and protects DNA replication forks

    PubMed Central

    Wang, Guangxi; Li, Yang; Wang, Pan; Liang, Hui; Cui, Ming; Zhu, Minglu; Guo, Limei; Su, Qian; Sun, Yujie; McNutt, Michael A; Yin, Yuxin

    2015-01-01

    Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication. PMID:26403191

  7. Human Cytomegalovirus Induces JC Virus DNA Replication in Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Heilbronn, Regine; Albrecht, Ingrid; Stephan, Sonja; Burkle, Alexander; Zur Hausen, Harald

    1993-12-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML.

  8. Mechanisms and regulation of DNA replication initiation in eukaryotes

    PubMed Central

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

    2017-01-01

    Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

  9. A new radial strain and strain rate estimation method using autocorrelation for carotid artery

    NASA Astrophysics Data System (ADS)

    Ye, Jihui; Kim, Hoonmin; Park, Jongho; Yeo, Sunmi; Shim, Hwan; Lim, Hyungjoon; Yoo, Yangmo

    2014-03-01

    Atherosclerosis is a leading cause of cardiovascular disease. The early diagnosis of atherosclerosis is of clinical interest since it can prevent any adverse effects of atherosclerotic vascular diseases. In this paper, a new carotid artery radial strain estimation method based on autocorrelation is presented. In the proposed method, the strain is first estimated by the autocorrelation of two complex signals from the consecutive frames. Then, the angular phase from autocorrelation is converted to strain and strain rate and they are analyzed over time. In addition, a 2D strain image over region of interest in a carotid artery can be displayed. To evaluate the feasibility of the proposed radial strain estimation method, radiofrequency (RF) data of 408 frames in the carotid artery of a volunteer were acquired by a commercial ultrasound system equipped with a research package (V10, Samsung Medison, Korea) by using a L5-13IS linear array transducer. From in vivo carotid artery data, the mean strain estimate was -0.1372 while its minimum and maximum values were -2.961 and 0.909, respectively. Moreover, the overall strain estimates are highly correlated with the reconstructed M-mode trace. Similar results were obtained from the estimation of the strain rate change over time. These results indicate that the proposed carotid artery radial strain estimation method is useful for assessing the arterial wall's stiffness noninvasively without increasing the computational complexity.

  10. Epitaxially strained strontium titanate

    NASA Astrophysics Data System (ADS)

    Biegalski, Michael David

    Although SrTiO3 is normally not ferroelectric at any temperature, predictions that predate this thesis based on thermodynamic analysis concluded that a biaxial tensile strain of order 1% would shift the paraelectric-to-ferroelectric transition temperature (Tc) of SrTiO3 to the vicinity of room temperature. In practice, uniformly straining SrTiO3 or related perovskite ferroelectrics to such strain levels is challenging and hitherto unheard of Nonetheless, using epitaxy and the misfit strain imposed by an underlying substrate, I have strained SrTiO 3 thin films to percent levels---far beyond where they would crack in bulk. Epitaxial ferroelectric films are often grown to thicknesses greatly exceeding their critical values, resulting in undesirable relaxation toward a zero-strain state by the introduction of dislocations. Dislocation densities of ˜1011 cm-2 are common in epitaxial ferroelectric films grown on lattice-mismatched substrates, and the resulting inhomogeneous strain smears out the ferroelectric phase transition. My approach to achieving the desired high strain levels in SrTiO3 films to assess strain predictions made use of new substrates (DyScO3 and GdScO3 ) that enabled the growth of uniformly strained SrTiO3 films below, or at least far closer to, the critical thickness for relaxation. The resulting strained SrTiO3 films have better structural perfection (narrower rocking curve widths) than the best bulk SrTiO3 single crystals. These films have the narrowest rocking curves ever reported for any heteroepeitaxial oxide thin film (6.5 arcsec). Modeling of ferroelectrics under these strain levels predicts dramatic shifts in the transition temperature and enhancement of the polarization. Indeed, in our strained SrTiO3, a ferroelectric state was induced with a Tc near room temperature. These films also exhibit a peak dielectric constant near room temperature of ˜20,000, comparable to that seen at very low temperatures (˜4K) in bulk SrTiO3. Unexpectedly, the

  11. Thin film strain transducer

    NASA Astrophysics Data System (ADS)

    Rand, J. L.

    1985-02-01

    A strain transducer system and process for making same is disclosed wherein a beryllium-copper ring having four strain gages disposed thereon is electrically connected in Wheatstone bridge fashion to output instrumentation. Tabs are bonded to a balloon or like surface with strain on the surface causing bending of the ring and providing an electrical signal through the gages proportional to the surface strain. A figure is provided which illustrates a pattern of a one-half ring segment as placed on a sheet of beryllium-copper for chem-mill etch formation, prior to bending and welding of a pair of the segments to form a ring structure.

  12. Strained Silicon Photonics

    PubMed Central

    Schriever, Clemens; Bohley, Christian; Schilling, Jörg; Wehrspohn, Ralf B.

    2012-01-01

    A review of recent progress in the field of strained silicon photonics is presented. The application of strain to waveguide and photonic crystal structures can be used to alter the linear and nonlinear optical properties of these devices. Here, methods for the fabrication of strained devices are summarized and recent examples of linear and nonlinear optical devices are discussed. Furthermore, the relation between strain and the enhancement of the second order nonlinear susceptibility is investigated, which may enable the construction of optically active photonic devices made of silicon. PMID:28817015

  13. Elevated temperature strain gages

    NASA Technical Reports Server (NTRS)

    Brittain, J. O.; Geslin, D.; Lei, J. F.

    1986-01-01

    One of the goals of the HOST Program is the development of electrical resistance strain gages for static strain measurements at temperatures equal to or greater than 1273 K. Strain gage materials must have a reproducible or predictable response to temperature, time and strain. It is the objective of this research to investigate criteria for the selection of materials for such applications through electrical properties studies. The results of the investigation of two groups of materials, refractory compounds and binary alloy solid solutions are presented.

  14. Thin film strain transducer

    NASA Technical Reports Server (NTRS)

    Rand, J. L. (Inventor)

    1984-01-01

    A strain transducer system and process for making the same is disclosed. A beryllium copper ring having four strain gages is electrically connected in Wheatstone bridge fashion to the output instrumentation. Tabs are bonded to a balloon or like surface with strain on the surface causing bending of a ring which provides an electrical signal through the gages proportional to the surface strain. A photographic pattern of a one half ring segment as placed on a sheet of beryllium copper for chem-mill etch formation is illustrated.

  15. Replication Experiments in Microgravity Liquid Phase Sintering

    NASA Astrophysics Data System (ADS)

    German, Randall M.; Johnson, John L.

    2016-05-01

    Although considerable experience exists with sintering on Earth, the behavior under reduced gravity conditions is poorly understood. This study analyzes replica microgravity liquid phase sintering data for seven tungsten alloys (35 to 88 wt pct tungsten) sintered for three hold times (1, 180, or 600 minutes) at 1773 K (1500 °C) using 0.002 pct of standard gravity. Equivalent sintering is performed on Earth using the same heating cycles. Microgravity sintering results in a lower density and more shape distortion. For Earth-based sintering, minimized distortion is associated with low liquid contents to avoid solid settling and slumping. Distortion in microgravity sintering involves viscous spreading of the component at points of contact with the containment crucible. Distortion in microgravity is minimized by short hold times; long hold times allow progressive component reshaping toward a spherical shape. Microgravity sintering also exhibits pore coalescence into large, stable voids that cause component swelling. The microgravity sintering results show good replication in terms of mass change and sintered density. Distortion is scattered but statistically similar between the replica microgravity runs. However, subtle factors, not typically of concern on Earth, emerge to influence microgravity sintering, such that ground experiments do not provide a basis to predict microgravity behavior.

  16. Macroevolution simulated with autonomously replicating computer programs.

    PubMed

    Yedid, Gabriel; Bell, Graham

    The process of adaptation occurs on two timescales. In the short term, natural selection merely sorts the variation already present in a population, whereas in the longer term genotypes quite different from any that were initially present evolve through the cumulation of new mutations. The first process is described by the mathematical theory of population genetics. However, this theory begins by defining a fixed set of genotypes and cannot provide a satisfactory analysis of the second process because it does not permit any genuinely new type to arise. The evolutionary outcome of selection acting on novel variation arising over long periods is therefore difficult to predict. The classical problem of this kind is whether 'replaying the tape of life' would invariably lead to the familiar organisms of the modern biota. Here we study the long-term behaviour of populations of autonomously replicating computer programs and find that the same type, introduced into the same simple environment, evolves on any given occasion along a unique trajectory towards one of many well-adapted end points.

  17. Inference by replication in densely connected systems.

    PubMed

    Neirotti, Juan P; Saad, David

    2007-10-01

    An efficient Bayesian inference method for problems that can be mapped onto dense graphs is presented. The approach is based on message passing where messages are averaged over a large number of replicated variable systems exposed to the same evidential nodes. An assumption about the symmetry of the solutions is required for carrying out the averages; here we extend the previous derivation based on a replica-symmetric- (RS)-like structure to include a more complex one-step replica-symmetry-breaking-like (1RSB-like) ansatz. To demonstrate the potential of the approach it is employed for studying critical properties of the Ising linear perceptron and for multiuser detection in code division multiple access (CDMA) under different noise models. Results obtained under the RS assumption in the noncritical regime give rise to a highly efficient signal detection algorithm in the context of CDMA; while in the critical regime one observes a first-order transition line that ends in a continuous phase transition point. Finite size effects are also observed. While the 1RSB ansatz is not required for the original problems, it was applied to the CDMA signal detection problem with a more complex noise model that exhibits RSB behavior, resulting in an improvement in performance.

  18. Implementation of the GARF replicated objects platform

    NASA Astrophysics Data System (ADS)

    Garbinato, B.; Guerraoui, R.; Mazouni, K. R.

    1995-03-01

    This paper presents the design and implementation of the GARF system, an object-oriented platform that helps programming fault-tolerant distributed applications in a modular way. The originality of GARF is to separate a distributed object into several objects, the complexity of distribution and fault-tolerance being encapsulated in reusable classes. The use of those classes by the GARF system is based on a run-time mechanism of invocation redirection, where most other systems use inheritance, a compile-time mechanism. Our runtime, which supports the GARF object model, is written in Smalltalk. It is presented in detail, as well as the reusable classes that support fault-tolerance. Fault-tolerant objects are implemented using groups of replicated objects. Our Dependable Object Toolkit provides group management facilities at the object level. Object groups are built on top of the Isis toolkit, which provides group management facilities at the Unix process level. Our mapping of object groups on process groups and our interfacing of Smalltalk and Isis are detailed. Performance analysis and a first evaluation of our prototype are also presented.

  19. Minority games, evolving capitals and replicator dynamics

    NASA Astrophysics Data System (ADS)

    Galla, Tobias; Zhang, Yi-Cheng

    2009-11-01

    We discuss a simple version of the minority game (MG) in which agents hold only one strategy each, but in which their capitals evolve dynamically according to their success and in which the total trading volume varies in time accordingly. This feature is known to be crucial for MGs to reproduce stylized facts of real market data. The stationary states and phase diagram of the model can be computed, and we show that the ergodicity breaking phase transition common for MGs, and marked by a divergence of the integrated response, is present also in this simplified model. An analogous majority game turns out to be relatively void of interesting features, and the total capital is found to diverge in time. Introducing a restraining force leads to a model akin to the replicator dynamics of evolutionary game theory, and we demonstrate that here a different type of phase transition is observed. Finally we briefly discuss the relation of this model with one strategy per player to more sophisticated minority games with dynamical capitals and several trading strategies per agent.

  20. Energy Proportionality for Disk Storage Using Replication

    SciTech Connect

    Kim, Jinoh; Rotem, Doron

    2010-09-09

    Energy saving has become a crucial concern in datacenters as several reports predict that the anticipated energy costs over a three year period will exceed hardware acquisition. In particular, saving energy for storage is of major importance as storage devices (and cooling them off) may contribute over 25 percent of the total energy consumed in a datacenter. Recent work introduced the concept of energy proportionality and argued that it is a more relevant metric than just energy saving as it takes into account the tradeoff between energy consumption and performance. In this paper, we present a novel approach, called FREPmore » (Fractional Replication for Energy Proportionality), for energy management in large datacenters. FREP includes areplication strategy and basic functions to enable flexible energy management. Specifically, our method provides performance guarantees by adaptively controlling the power states of a group of disks based on observed and predicted workloads. Our experiments, using a set of real and synthetic traces, show that FREP dramatically reduces energy requirements with a minimal response time penalty.« less

  1. Genetic networks controlled by the bacterial replication initiator and transcription factor DnaA in Bacillus subtilis.

    PubMed

    Washington, Tracy A; Smith, Janet L; Grossman, Alan D

    2017-10-01

    DnaA is the widely conserved bacterial AAA+ ATPase that functions as both the replication initiator and a transcription factor. In many organisms, DnaA controls expression of its own gene and likely several others during growth and in response to replication stress. To evaluate the effects of DnaA on gene expression, separate from its role in replication initiation, we analyzed changes in mRNA levels in Bacillus subtilis cells with and without dnaA, using engineered strains in which dnaA is not essential. We found that dnaA was required for many of the changes in gene expression in response to replication stress. We also found that dnaA indirectly affected expression of several regulons during growth, including those controlled by the transcription factors Spo0A, AbrB, PhoP, SinR, RemA, Rok and YvrH. These effects were largely mediated by the effects of DnaA on expression of sda. DnaA activates transcription of sda, and Sda inhibits histidine protein kinases required for activation of the transcription factor Spo0A. We also found that loss of dnaA caused a decrease in the development of genetic competence. Together, our results indicate that DnaA plays an important role in modulating cell physiology, separate from its role in replication initiation. © 2017 John Wiley & Sons Ltd.

  2. Unprecedented large inverted repeats at the replication terminus of circular bacterial chromosomes suggest a novel mode of chromosome rescue

    PubMed Central

    El Kafsi, Hela; Loux, Valentin; Mariadassou, Mahendra; Blin, Camille; Chiapello, Hélène; Abraham, Anne-Laure; Maguin, Emmanuelle; van de Guchte, Maarten

    2017-01-01

    The first Lactobacillus delbrueckii ssp. bulgaricus genome sequence revealed the presence of a very large inverted repeat (IR), a DNA sequence arrangement which thus far seemed inconceivable in a non-manipulated circular bacterial chromosome, at the replication terminus. This intriguing observation prompted us to investigate if similar IRs could be found in other bacteria. IRs with sizes varying from 38 to 76 kbp were found at the replication terminus of all 5 L. delbrueckii ssp. bulgaricus chromosomes analysed, but in none of 1373 other chromosomes. They represent the first naturally occurring very large IRs detected in circular bacterial genomes. A comparison of the L. bulgaricus replication terminus regions and the corresponding regions without IR in 5 L. delbrueckii ssp. lactis genomes leads us to propose a model for the formation and evolution of the IRs. The DNA sequence data are consistent with a novel model of chromosome rescue after premature replication termination or irreversible chromosome damage near the replication terminus, involving mechanisms analogous to those proposed in the formation of very large IRs in human cancer cells. We postulate that the L. delbrueckii ssp. bulgaricus-specific IRs in different strains derive from a single ancestral IR of at least 93 kbp. PMID:28281695

  3. Mutation Q95K enhances N155H-mediated integrase inhibitor resistance and improves viral replication capacity.

    PubMed

    Fun, Axel; Van Baelen, Kurt; van Lelyveld, Steven F L; Schipper, Pauline J; Stuyver, Lieven J; Wensing, Annemarie M J; Nijhuis, Monique

    2010-11-01

    The genetic barrier to development of raltegravir resistance is considered to be low, requiring at least one primary integrase mutation: Y143C, Q148H/K/R or N155H to confer raltegravir therapy failure. However, during continued raltegravir treatment failure, additional mutations may be selected. In a patient failing raltegravir therapy, we investigated the impact of multiple integrase mutations on resistance and viral replication. Furthermore, in vivo fitness was investigated during failure of raltegravir-containing highly active antiretroviral therapy and after raltegravir was discontinued from the regimen. Patient-derived viral integrase genes were cloned into a reference strain. These recombinant viruses were used to determine the contribution of individual integrase mutations to raltegravir resistance and replication capacity in vitro. To determine in vivo fitness, the relative proportion of specific integrase mutations was monitored over time by in-depth clonal analysis of the viral integrase at baseline, during and after raltegravir treatment. Raltegravir therapy failure was associated with the initial selection of primary resistance mutation N155H. This mutation conferred a 3.8-fold reduction in raltegravir susceptibility and a severe reduction in viral replication. Acquisition of integrase mutation Q95K increased resistance (6.2-fold) and partly restored viral replication. Selection of a third mutation, V151I, further increased raltegravir resistance (20-fold), but decreased viral replication. After prolonged raltegravir interruption, raltegravir resistance mutations were lost, demonstrating the reduced replication capacity of the resistant virus. We describe selection of Q95K as a secondary resistance mutation during raltegravir therapy failure. In the background of N155H, Q95K enhances raltegravir and elvitegravir resistance and improves the impaired replication of the virus.

  4. Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle, sheep, and deer.

    PubMed

    McLaughlin, Bridget E; DeMaula, Christopher D; Wilson, William C; Boyce, Walter M; MacLachlan, N James

    2003-07-01

    To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.

  5. Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

    PubMed

    Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

    2017-11-01

    The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

  6. Random mutagenesis by error-prone Pol I plasmid replication in Escherichia coli

    PubMed Central

    Alexander, David L.; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J.; Camps, Manel

    2014-01-01

    Summary Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available. PMID:25055769

  7. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication

    PubMed Central

    Jiang, Zhiwu; Gu, Liming; Chen, Yanxia

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy. PMID:27525278

  8. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication.

    PubMed

    Wang, Gefei; Li, Rui; Jiang, Zhiwu; Gu, Liming; Chen, Yanxia; Dai, Jianping; Li, Kangsheng

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy.

  9. Geodetic Strain Analysis Tool

    NASA Technical Reports Server (NTRS)

    Kedar, Sharon; Baxter, Sean C.; Parker, Jay W.; Webb, Frank H.; Owen, Susan E.; Sibthorpe, Anthony J.; Dong, Danan

    2011-01-01

    A geodetic software analysis tool enables the user to analyze 2D crustal strain from geodetic ground motion, and create models of crustal deformation using a graphical interface. Users can use any geodetic measurements of ground motion and derive the 2D crustal strain interactively. This software also provides a forward-modeling tool that calculates a geodetic velocity and strain field for a given fault model, and lets the user compare the modeled strain field with the strain field obtained from the user s data. Users may change parameters on-the-fly and obtain a real-time recalculation of the resulting strain field. Four data products are computed: maximum shear, dilatation, shear angle, and principal components. The current view and data dependencies are processed first. The remaining data products and views are then computed in a round-robin fashion to anticipate view changes. When an analysis or display parameter is changed, the affected data products and views are invalidated and progressively re-displayed as available. This software is designed to facilitate the derivation of the strain fields from the GPS and strain meter data that sample it to facilitate the understanding of the strengths and weaknesses of the strain field derivation from continuous GPS (CGPS) and other geodetic data from a variety of tectonic settings, to converge on the "best practices" strain derivation strategy for the Solid Earth Science ESDR System (SESES) project given the CGPS station distribution in the western U.S., and to provide SESES users with a scientific and educational tool to explore the strain field on their own with user-defined parameters.

  10. Replication, tissue tropisms and transmission of yellow fever virus in Aedes albopictus.

    PubMed

    Miller, B R; Mitchell, C J; Ballinger, M E

    1989-01-01

    Experimental studies undertaken to ascertain the dynamics of yellow fever virus replication in an introduced strain (Houston) of the Asian mosquito, Aedes albopictus (Skuse), indicate that this species is an efficient vector of yellow fever virus. Replication of virus in Ae. albopictus could be detected 3 d after feeding on a suspension containing 7.2 log10 Vero cell plaque forming units (PFU) per ml of virus; peak titres (3.5 log10 PFU/mosquito) occurred 7 d after exposure. Viral antigen, visualized by immunofluorescence, was first detected in midgut cells 4 d after exposure and appeared in fat cells 7 d after exposure. The only other mosquito tissues revealing viral antigen were the salivary glands, brain, and occasionally cells of the suboesophageal ganglion. Viral antigen was not detected in any of the tissues of the reproductive tract, nor could viral genomic ribonucleic acid (RNA) be detected in these tissues by RNA-RNA molecular hybridization in situ. We detected no vertical transmission of yellow fever virus in 6180 F1 adult progeny produced from infected females. The extrinsic incubation period at 26.7 degrees C was 9 d. We conclude that the Houston strain of Ae. albopictus is a competent vector of yellow fever virus and can serve as bridging vector between the jungle yellow fever cycle and the urban cycle in New World ecosystems.

  11. Statistical Analysis of Microarray Data with Replicated Spots: A Case Study with Synechococcus WH8102

    DOE PAGES

    Thomas, E. V.; Phillippy, K. H.; Brahamsha, B.; ...

    2009-01-01

    Until recently microarray experiments often involved relatively few arrays with only a single representation of each gene on each array. A complete genome microarray with multiple spots per gene (spread out spatially across the array) was developed in order to compare the gene expression of a marine cyanobacterium and a knockout mutant strain in a defined artificial seawater medium. Statistical methods were developed for analysis in the special situation of this case study where there is gene replication within an array and where relatively few arrays are used, which can be the case with current array technology. Due in partmore » to the replication within an array, it was possible to detect very small changes in the levels of expression between the wild type and mutant strains. One interesting biological outcome of this experiment is the indication of the extent to which the phosphorus regulatory system of this cyanobacterium affects the expression of multiple genes beyond those strictly involved in phosphorus acquisition.« less

  12. The effects of Mycoplasma hyopneumoniae on porcine circovirus type 2 replication in vitro PK-15 cells.

    PubMed

    Wang, Haiyan; Feng, Zhixin; Wu, Yuzi; Wei, Yanna; Gan, Yuan; Hua, Lizhong; Li, Bin; Wang, Xiaomin; Liu, Maojun; Xiong, Qiyan; Shao, Guoqing

    2016-04-01

    Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). Mycoplasma hyopneumoniae (Mhp) is a very well-known co-factor that potentially enhances PCV2 replication and thus the development of PMWS. However, co-infection with Mhp and PCV2 in vivo under different conditions can produce divergent clinical signs and lesions. In this study, PCV2 replication could be enhanced by subsequent co-inoculation with Mhp (PCV2+Mhp) in a time and dose dependent method, but not by prior (Mhp+PCV2) or simultaneous (Mhp/PCV2) co-inoculation. Furthermore, different magnitudes of PCV2-infected cells, varying from 150% ± 14% to 351% ± 28%, were detected when co-infected with different Mhp strains. The relative percentage of PCV2-infected cells greatly decreased from 351% ± 28 to 141% ± 18 when the Mhp strain was treated with UV light for 12 h. These results offer the evidences to better understand the complex clinical syndromes in Mhp/PCV2 co-infection cases, and the occurrence of PMWS. Copyright © 2016. Published by Elsevier Ltd.

  13. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    SciTech Connect

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be themore » potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.« less

  14. Interrelationship of Primary Virus Replication, Level of Latency, and Time to Reactivation in the Trigeminal Ganglia of Latently Infected Mice

    PubMed Central

    Matundan, Harry H.; Mott, Kevin R.; Allen, Sariah J.; Wang, Shaohui; Bresee, Catherine J.; Ghiasi, Yasamin N.; Town, Terrence

    2016-01-01

    ABSTRACT We sought to determine the possibility of an interrelationship between primary virus replication in the eye, the level of viral DNA in the trigeminal ganglia (TG) during latency, and the amount of virus reactivation following ocular herpes simplex virus type 1 (HSV-1) infection. Mice were infected with virulent (McKrae) or avirulent (KOS and RE) strains of HSV-1, and virus titers in the eyes and TG during primary infection, level of viral gB DNA in TG on day 28 postinfection (p.i.), and virus reactivation on day 28 p.i. as measured by explant reactivation were calculated. Our results suggest that the avirulent strains of HSV-1, even after corneal scarification, had lower virus titers in the eye, had less latency in the TG, and took a longer time to reactivate than virulent strains of HSV-1. The time to explant reactivation of avirulent strains of HSV-1 was similar to that of the virulent LAT(−) McKrae-derived mutant. The viral dose with the McKrae strain of HSV-1 affected the level of viral DNA and time to explant reactivation. Overall, our results suggest that there is no absolute correlation between primary virus titer in the eye and TG and the level of viral DNA in latent TG and time to reactivation. IMPORTANCE Very little is known regarding the interrelationship between primary virus replication in the eye, the level of latency in TG, and the time to reactivate in the mouse model. This study was designed to answer these questions. Our results point to the absence of any correlation between the level of primary virus replication and the level of viral DNA during latency, and neither was an indicator of how rapidly the virus reactivated following explant TG-induced reactivation. PMID:27512072

  15. Polygonum cuspidatum and Its Active Components Inhibit Replication of the Influenza Virus through Toll-Like Receptor 9-Induced Interferon Beta Expression

    PubMed Central

    Lin, Chao-jen; Lin, Hui-Ju; Chen, Ter-Hsin; Hsu, Yu-An; Liu, Chin-San; Hwang, Guang-Yuh; Wan, Lei

    2015-01-01

    Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9–induced IFN-β production. PMID:25658356

  16. Light intensity strain analysis

    NASA Technical Reports Server (NTRS)

    Williams, J. G. (Inventor)

    1973-01-01

    A process is described for the analysis of the strain field of structures subjected to large deformations involving a low modulus substrate having a high modulus, relatively thin coating. The optical properties of transmittance and reflectance are measured for the coated substrate while stressed and unstressed to indicate the strain field for the coated substrate.

  17. Chitinase producing Bt strains

    Treesearch

    Haim B. Gunner; Matthew Zimet; Sarah Berger

    1985-01-01

    Screening of 402 strains of more than 18 varieties of Bacillus thuringiensis showed chitinase to be inducible in virtually every serovar tested. Though the chitinase titre varied among strains, there was a strong correlation between enhanced lethality to spruce budworm, Choristoneura fumiferana (Clemens), and an increase in...

  18. Chromosomal DNA replication in higher plants

    SciTech Connect

    Van't Hof, J.; Bjerknes, C.A.

    1979-01-01

    Replicon-size estimations from DNA fiber autoradiograms must always be considered with the limits of resolution in mind. However, data from yeast obtained by autoradiography and electron-microscopy gave similar average sizes in the 20 to 30 ..mu..m range. These sizes are in agreement with those of C. capillaris observed in the present work, with those of Pisum sativum and Helianthus annuus, and with those of four other unrelated plant species. The curious fact that higher plants and yeast have replicons of about the same size raises the question of whether or not all members of the plant kingdom share this commonmore » statistic. Higher plants appear to have a common replicon size, and they also have a slower fork rate than either bacteria or mammalian cells when grown at optimal temperatures. Even at 38/sup 0/ sunflower (Helianthus annuus) root meristem cells have a fork rate a little less than 12 ..mu..m per hour. On the other hand, at about the same temperature, the rate is approximately 800 ..mu..m per hour in bacteria, and in mammalian cells it ranges from 30 to 60 ..mu..m per hour. Current data from higher plants show that they have a range in fork rate from 6 to 12 ..mu..m per hour. The lower rates observed among higher plants are similar to and more often less than those reported for the amphibians Triturus and Xenopus and that of fatheat minnow cells. Therefore, higher plants and cold-blooded animals commonly share the characteristic of a relatively low replication fork rate.« less

  19. Hypotension and environmental noise: a replication study.

    PubMed

    Lercher, Peter; Widmann, Ulrich; Thudium, Jürg

    2014-08-26

    Up to now, traffic noise effect studies focused on hypertension as health outcome. Hypotension has not been considered as a potential health outcome although in experiments some people also responded to noise with decreases of blood pressure. Currently, the characteristics of these persons are not known and whether this down regulation of blood pressure is an experimental artifact, selection, or can also be observed in population studies is unanswered. In a cross-sectional replication study, we randomly sampled participants (age 20-75, N = 807) from circular areas (radius = 500 m) around 31 noise measurement sites from four noise exposure strata (35-44, 45-54, 55-64, >64 Leq, dBA). Repeated blood pressure measurements were available for a smaller sample (N = 570). Standardized information on socio-demographics, housing, life style and health was obtained by door to door visits including anthropometric measurements. Noise and air pollution exposure was assigned by GIS based on both calculation and measurements. Reported hypotension or hypotension medication past year was the main outcome studied. Exposure-effect relationships were modeled with multiple non-linear logistic regression techniques using separate noise estimations for total, highway and rail exposure. Reported hypotension was significantly associated with rail and total noise exposure and strongly modified by weather sensitivity. Reported hypotension medication showed associations of similar size with rail and total noise exposure without effect modification by weather sensitivity. The size of the associations in the smaller sample with BMI as additional covariate was similar. Other important cofactors (sex, age, BMI, health) and moderators (weather sensitivity, adjacent main roads and associated annoyance) need to be considered as indispensible part of the observed relationship. This study confirms a potential new noise effect pathway and discusses potential patho-physiological routes of actions.

  20. Hypotension and Environmental Noise: A Replication Study

    PubMed Central

    Lercher, Peter; Widmann, Ulrich; Thudium, Jürg

    2014-01-01

    Up to now, traffic noise effect studies focused on hypertension as health outcome. Hypotension has not been considered as a potential health outcome although in experiments some people also responded to noise with decreases of blood pressure. Currently, the characteristics of these persons are not known and whether this down regulation of blood pressure is an experimental artifact, selection, or can also be observed in population studies is unanswered. In a cross-sectional replication study, we randomly sampled participants (age 20–75, N = 807) from circular areas (radius = 500 m) around 31 noise measurement sites from four noise exposure strata (35–44, 45–54, 55–64, >64 Leq, dBA). Repeated blood pressure measurements were available for a smaller sample (N = 570). Standardized information on socio-demographics, housing, life style and health was obtained by door to door visits including anthropometric measurements. Noise and air pollution exposure was assigned by GIS based on both calculation and measurements. Reported hypotension or hypotension medication past year was the main outcome studied. Exposure-effect relationships were modeled with multiple non-linear logistic regression techniques using separate noise estimations for total, highway and rail exposure. Reported hypotension was significantly associated with rail and total noise exposure and strongly modified by weather sensitivity. Reported hypotension medication showed associations of similar size with rail and total noise exposure without effect modification by weather sensitivity. The size of the associations in the smaller sample with BMI as additional covariate was similar. Other important cofactors (sex, age, BMI, health) and moderators (weather sensitivity, adjacent main roads and associated annoyance) need to be considered as indispensible part of the observed relationship. This study confirms a potential new noise effect pathway and discusses potential patho-physiological routes of actions

  1. Method for replicating an array of nucleic acid probes

    DOEpatents

    Cantor, Charles R.; Przetakiewicz, Marek; Smith, Cassandra L.; Sano, Takeshi

    1998-01-01

    The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5'- and/or 3'-overhangs.

  2. Method for replicating an array of nucleic acid probes

    DOEpatents

    Cantor, C.R.; Przetakiewicz, M.; Smith, C.L.; Sano, T.

    1998-08-18

    The invention relates to the replication of probe arrays and methods for replicating arrays of probes which are useful for the large scale manufacture of diagnostic aids used to screen biological samples for specific target sequences. Arrays created using PCR technology may comprise probes with 5{prime}- and/or 3{prime}-overhangs. 16 figs.

  3. Focus Article: Replication in Second Language Writing Research

    ERIC Educational Resources Information Center

    Porte, Graeme; Richards, Keith

    2012-01-01

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

  4. Evaluating Replicability of Regression Results Using the Jackknife Statistic.

    ERIC Educational Resources Information Center

    Yoon, Jina

    Contrary to popular opinion, significance testing does not inform the researcher of the likelihood of the replication of results from current research findings. Result replicability has been ignored by researchers because of an overreliance on significance testing. Several alternatives have been offered to provide the researcher with more…

  5. Perfectionism in Gifted Adolescents: A Replication and Extension

    ERIC Educational Resources Information Center

    Margot, Kelly C.; Rinn, Anne N.

    2016-01-01

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

  6. Reconsidering Replication: New Perspectives on Large-Scale School Improvement

    ERIC Educational Resources Information Center

    Peurach, Donald J.; Glazer, Joshua L.

    2012-01-01

    The purpose of this analysis is to reconsider organizational replication as a strategy for large-scale school improvement: a strategy that features a "hub" organization collaborating with "outlet" schools to enact school-wide designs for improvement. To do so, we synthesize a leading line of research on commercial replication to construct a…

  7. Replication of Psycholinguistic Experiments and the Resolution of Inconsistencies

    ERIC Educational Resources Information Center

    Rákosi, Csilla

    2017-01-01

    Non-exact replications are regarded as effective tools of problem solving in psycholinguistic research because they lead to more plausible experimental results; however, they are also ineffective tools of problem solving because they trigger cumulative contradictions among different replications of an experiment. This paper intends to resolve this…

  8. Can Successful Schools Replicate? Evidence from Boston Charter Schools

    ERIC Educational Resources Information Center

    Cohodes, Sarah; Setren, Elizabeth; Walters, Christopher

    2016-01-01

    Can successful schools replicate? In a climate of school turnarounds, charter conversions, and new school openings, an important question is whether schools that have demonstrated success with in one or several schools can replicate their success with additional schools. The federal government's Investing in Innovation (i3) grant program has been…

  9. Murine leukemia virus (MLV) replication monitored with fluorescent proteins.

    PubMed

    Sliva, Katja; Erlwein, Otto; Bittner, Alexandra; Schnierle, Barbara S

    2004-12-20

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

  10. Trajectory of Externalizing Child Behaviors in a KEEP Replication

    ERIC Educational Resources Information Center

    Uretsky, Mathew C.; Lee, Bethany R.; Greeno, Elizabeth J.; Barth, Richard P.

    2017-01-01

    Objective: The purpose of this study is to examine the correlates of child behavior change over time in a replication of the KEEP intervention. Method: The study sample was drawn from the treatment group of the Maryland replication of KEEP (n=65). Change over time was analyzed using multilevel linear mixed modeling. Results: Parents' use of…

  11. The Causes and Consequences of Topological Stress during DNA Replication.

    PubMed

    Keszthelyi, Andrea; Minchell, Nicola E; Baxter, Jonathan

    2016-12-21

    The faithful replication of sister chromatids is essential for genomic integrity in every cell division. The replication machinery must overcome numerous difficulties in every round of replication, including DNA topological stress. Topological stress arises due to the double-stranded helical nature of DNA. When the strands are pulled apart for replication to occur, the intertwining of the double helix must also be resolved or topological stress will arise. This intrinsic problem is exacerbated by specific chromosomal contexts encountered during DNA replication. The convergence of two replicons during termination, the presence of stable protein-DNA complexes and active transcription can all lead to topological stresses being imposed upon DNA replication. Here we describe how replication forks respond to topological stress by replication fork rotation and fork reversal. We also discuss the genomic contexts where topological stress is likely to occur in eukaryotes, focusing on the contribution of transcription. Finally, we describe how topological stress, and the ways forks respond to it, may contribute to genomic instability in cells.

  12. Replication stress in Mammalian cells and its consequences for mitosis.

    PubMed

    Gelot, Camille; Magdalou, Indiana; Lopez, Bernard S

    2015-05-22

    The faithful transmission of genetic information to daughter cells is central to maintaining genomic stability and relies on the accurate and complete duplication of genetic material during each cell cycle. However, the genome is routinely exposed to endogenous and exogenous stresses that can impede the progression of replication. Such replication stress can be an early cause of cancer or initiate senescence. Replication stress, which primarily occurs during S phase, results in consequences during mitosis, jeopardizing chromosome segregation and, in turn, genomic stability. The traces of replication stress can be detected in the daughter cells during G1 phase. Alterations in mitosis occur in two types: 1) local alterations that correspond to breaks, rearrangements, intertwined DNA molecules or non-separated sister chromatids that are confined to the region of the replication dysfunction; 2) genome-wide chromosome segregation resulting from centrosome amplification (although centrosomes do not contain DNA), which amplifies the local replication stress to the entire genome. Here, we discuss the endogenous causes of replication perturbations, the mechanisms of replication fork restart and the consequences for mitosis, chromosome segregation and genomic stability.

  13. Replication of Special Education Research: Necessary but Far Too Rare

    ERIC Educational Resources Information Center

    Makel, Matthew C.; Plucker, Jonathan A.; Freeman, Jennifer; Lombardi, Allison; Simonsen, Brandi; Coyne, Michael

    2016-01-01

    Increased calls for rigor in special education have often revolved around the use of experimental research design. However, the replicability of research results is also a central tenet to the scientific research process. To assess the prevalence, success rate, and authorship history of replications in special education, we investigated the…

  14. Template role of double-stranded RNA in tombusvirus replication.

    PubMed

    Kovalev, Nikolay; Pogany, Judit; Nagy, Peter D

    2014-05-01

    Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV

  15. Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

    PubMed

    Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

    2018-01-01

    Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

  16. Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair.

    PubMed

    Alexander, Jessica L; Barrasa, M Inmaculada; Orr-Weaver, Terry L

    2015-06-15

    Replication origins are under tight regulation to ensure activation occurs only once per cell cycle [1, 2]. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint [2-7]. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions [5]. These types of chromosomal aberrations are common in numerous human cancers, suggesting that re-replication events contribute to cancer progression. It was proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks [3]. However, previously studied systems lack the resolution to determine whether the observed DSBs are generated at sites of fork collisions. Here, we utilize the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control [8-10], to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs) [10-12]. Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation [13, 14]. Here, we show that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker γH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Evaluating genome-scale approaches to eukaryotic DNA replication

    PubMed Central

    Gilbert, David M.

    2010-01-01

    Mechanisms regulating where and when eukaryotic DNA replication initiates remain a mystery. Recently, genome-scale methods have been brought to bear on this problem. The identification of replication origins and their associated proteins in yeasts is a well-integrated investigative tool, but corresponding data sets from multicellular organisms are scarce. By contrast, standardized protocols for evaluating replication timing have generated informative data sets for most eukaryotic systems. Here, I summarize the genome-scale methods that are most frequently used to analyse replication in eukaryotes, the kinds of questions each method can address and the technical hurdles that must be overcome to gain a complete understanding of the nature of eukaryotic replication origins. PMID:20811343

  18. Persistent HIV-1 replication maintains the tissue reservoir during therapy

    PubMed Central

    Bedford, Trevor; Kim, Eun-Young; Archer, John; Pond, Sergei L. Kosakovsky; Chung, Yoon-Seok; Penugonda, Sudhir; Chipman, Jeffrey; Fletcher, Courtney V.; Schacker, Timothy W.; Malim, Michael H.; Rambaut, Andrew; Haase, Ashley T.; McLean, Angela R.; Wolinsky, Steven M.

    2015-01-01

    Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site of viral production, storage of viral particles in immune complexes, and viral persistence. Whilst combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. A spatial dynamic model of persistent viral replication and spread explains why the development of drug resistance is not a foregone conclusion under conditions where drug concentrations are insufficient to completely block virus replication. These data provide fresh insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and refill the viral reservoir despite potent antiretroviral therapy. PMID:26814962

  19. DNA replication stress and cancer: cause or cure?

    PubMed

    Taylor, Elaine M; Lindsay, Howard D

    2016-01-01

    There is an extensive and growing body of evidence that DNA replication stress is a major driver in the development and progression of many cancers, and that these cancers rely heavily on replication stress response pathways for their continued proliferation. This raises the possibility that the pathways that ordinarily protect cells from the accumulation of cancer-causing mutations may actually prove to be effective therapeutic targets for a wide range of malignancies. In this review, we explore the mechanisms by which sustained proliferation can lead to replication stress and genome instability, and discuss how the pattern of mutations observed in human cancers is supportive of this oncogene-induced replication stress model. Finally, we go on to consider the implications of replication stress both as a prognostic indicator and, more encouragingly, as a potential target in cancer treatment.

  20. Diversity of the DNA Replication System in the Archaea Domain

    PubMed Central

    Long, Feng; Cann, Isaac; Whitman, William B.

    2014-01-01

    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. PMID:24790526

  1. Autophagy Facilitates Salmonella Replication in HeLa Cells

    PubMed Central

    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

  2. Replication and transmission of influenza viruses in Japanese quail

    PubMed Central

    Makarova, Natalia V.; Ozaki, Hiroishi; Kida, Hiroshi; Webster, Robert G.; Perez, Daniel R.

    2015-01-01

    Quail have emerged as a potential intermediate host in the spread of avian influenza A viruses in poultry in Hong Kong. To better understand this possible role, we tested the replication and transmission in quail of influenza A viruses of all 15 HA subtypes. Quail supported the replication of at least 14 subtypes. Influenza A viruses replicated predominantly in the respiratory tract. Transmission experiments suggested that perpetuation of avian influenza viruses in quail requires adaptation. Swine influenza viruses were isolated from the respiratory tract of quail at low levels. There was no evidence of human influenza A or B virus replication. Interestingly, a human–avian recombinant containing the surface glycoprotein genes of a quail virus and the internal genes of a human virus replicated and transmitted readily in quail; therefore, quail could function as amplifiers of influenza virus reassortants that have the potential to infect humans and/or other mammalian species. PMID:12788625

  3. Diversity of the DNA replication system in the Archaea domain.

    PubMed

    Sarmiento, Felipe; Long, Feng; Cann, Isaac; Whitman, William B

    2014-01-01

    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.

  4. Characterization of the theta replication plasmid pGR7 from Acetobacter aceti CCM 3610.

    PubMed

    Grones, Peter; Grones, Jozef

    2012-07-01

    A cryptic plasmid of Acetobacter aceti CCM 3610, designated pGR7, was sequenced and characterized. It is a 2446-bp circular molecule with a G + C content of 30%, which is unusual when compared to the already known plasmids isolated from Acetobacter genera. Sequence analysis of pGR7 revealed three putative open reading frames (ORFs). ORF1 displays low similarity with other Acetobacter plasmid replication proteins. The other two ORFs show similarities only to hypothetical proteins and do not encode any important protein. The replication module comprises a DnaA box-like sequence, indirect repeats, a potential prokaryotic promoter and the rep gene. The rep module organization is similar to that found in other theta-replicating plasmids from acetic acid bacteria that stably maintain in both Acetobacter and Escherichia coli, with two repeated sequences containing modules. Nevertheless, the pGR7 plasmid could replicate and be stably maintained only in Acetobacter strains and not in E. coli, another uncommon feature of this plasmid. The Rep protein was cloned into the pET30a + expression vector and purified by high-performance liquid chromatography. The helicase activity was determined and the ability of the protein to bind to the plasmid regulation region was confirmed by an electrophoretic mobility shift assay. The plasmid was stable in the Acetobacter cells after cultivation under nonselective conditions. By real-time polymerase chain reaction, the relative copy number of pGR7 was estimated to be seven copies per host chromosome equivalent. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Effect of small interfering RNAs on in vitro replication and gene expression of feline coronavirus.

    PubMed

    Anis, Eman A; Wilkes, Rebecca P; Kania, Stephen A; Legendre, Alfred M; Kennedy, Melissa A

    2014-09-01

    To evaluate the ability of small interfering RNAs (siRNAs) to inhibit in vitro viral replication and gene expression of feline coronavirus (FCoV). Cell cultures of Crandell-Rees feline kidney cells. 5 synthetic siRNAs that each targeted a different region of the FCoV genome were tested individually and in various combinations for their antiviral effects against 2 strains of FCoV (feline infectious peritonitis virus WSU 79-1146 and feline enteric coronavirus WSU 79-1683) in cell cultures. Tested combinations targeted the FCoV leader and 3' untranslated region, FCoV leader region and nucleocapsid gene, and FCoV leader region, 3' untranslated region, and nucleocapsid gene. For each test condition, assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, reverse-transcription PCR analysis; flow cytometric evaluation of the reduction of viral protein expression in infected cells; and assessment of virus replication inhibition via titration of extracellular virus with a TCID₅₀ infectivity assay. The 5 siRNAs had variable inhibitory effects on FCoV when used singly. Combinations of siRNAs that targeted different regions of the viral genome resulted in more effective viral inhibition than did individual siRNAs that targeted a single gene. The tested siRNA combinations resulted in approximately 95% reduction in viral replication (based on virus titration results), compared with findings in negative control, nontargeting siRNA-treated, FCoV-infected cells. In vitro replication of FCoV was specifically inhibited by siRNAs that targeted coding and noncoding regions of the viral genome, suggesting a potential therapeutic application of RNA interference in treatment of feline infectious peritonitis.

  6. Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

    PubMed Central

    2011-01-01

    Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects. PMID:22078030

  7. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

    PubMed Central

    Kamgoué, Alain; Murray, Heath; Pasta, Franck

    2016-01-01

    Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of

  8. HIV-1 replication in CD4+ T cell lines: the effects of adaptation on co-receptor use, tropism, and accessory gene function.

    PubMed

    Dejucq, N

    2000-09-01

    We studied the replication of HIV-1 macrophage-tropic CCR5-using strains (R5) in CD4+ T cell lines to better understand the switch in co-receptor use of such strains during disease progression and to assess resulting changes in cell tropism. We found that the majority of R5 strains cannot replicate in CD4+ T cell lines without adaptation by serial passage. A small minority of primary R5 isolates, however, were able to infect two T cell lines, Molt4 and SupT1. This expanded tropism was due to the use of undetectable levels of CCR5 rather than CXCR4 or alternative receptors. In contrast, HIV-1sF162 adaptation for replication in the C8166 T cell line was due to the emergence of variant strains that could use CXCR4. Of two variants, one was dual-tropic and one T-tropic, although both could use CCR5 as well as CXCR4. A single mutation in the start codon of the accessory gene vpu accounted for the T-tropic phenotype of the second variant, indicating that a non-functional vpu impairs macrophage tropism. Thus, in vitro and in the absence of an immune response, R5 strains naturally adapt to infect CXCR4+ T cell lines. Such adaptation resembles the rare R5 to X4 switch that occurs in vivo. Mutations in accessory genes (e.g., vpu) not required for replication in rapidly dividing cell lines may also occur in vitro, abrogating replication in primary cell types such as macrophages. Such mutations, however, are normally selected against in vivo.

  9. Survival of the fittest: how Brucella strains adapt to their intracellular niche in the host.

    PubMed

    Roop, R Martin; Gaines, Jennifer M; Anderson, Eric S; Caswell, Clayton C; Martin, Daniel W

    2009-11-01

    Brucella strains produce abortion and infertility in their natural hosts and a zoonotic disease in humans known as undulant fever. These bacteria do not produce classical virulence factors, and their capacity to successfully survive and replicate within a variety of host cells underlies their pathogenicity. Extensive replication of the brucellae in placental trophoblasts is associated with reproductive tract pathology in natural hosts, and prolonged persistence in macrophages leads to the chronic infections that are a hallmark of brucellosis in both natural hosts and humans. This review describes how Brucella strains have efficiently adapted to their intracellular lifestyle in the host.

  10. Coxsackievirus B3 mutator strains are attenuated in vivo.

    PubMed

    Gnädig, Nina F; Beaucourt, Stéphanie; Campagnola, Grace; Bordería, Antonio V; Sanz-Ramos, Marta; Gong, Peng; Blanc, Hervé; Peersen, Olve B; Vignuzzi, Marco

    2012-08-21

    Based on structural data of the RNA-dependent RNA polymerase, rational targeting of key residues, and screens for Coxsackievirus B3 fidelity variants, we isolated nine polymerase variants with mutator phenotypes, which allowed us to probe the effects of lowering fidelity on virus replication, mutability, and in vivo fitness. These mutator strains generate higher mutation frequencies than WT virus and are more sensitive to mutagenic treatments, and their purified polymerases present lower-fidelity profiles in an in vitro incorporation assay. Whereas these strains replicate with WT-like kinetics in tissue culture, in vivo infections reveal a strong correlation between mutation frequency and fitness. Variants with the highest mutation frequencies are less fit in vivo and fail to productively infect important target organs, such as the heart or pancreas. Furthermore, whereas WT virus is readily detectable in target organs 30 d after infection, some variants fail to successfully establish persistent infections. Our results show that, although mutator strains are sufficiently fit when grown in large population size, their fitness is greatly impacted when subjected to severe bottlenecking, which would occur during in vivo infection. The data indicate that, although RNA viruses have extreme mutation frequencies to maximize adaptability, nature has fine-tuned replication fidelity. Our work forges ground in showing that the mutability of RNA viruses does have an upper limit, where larger than natural genetic diversity is deleterious to virus survival.

  11. Tight Chk1 Levels Control Replication Cluster Activation in Xenopus

    PubMed Central

    Wiggins, Jennifer M.; Barbosa, Pedro; Libeau, Pierre; Priam, Pierre; Narassimprakash, Hemalatha; Grodzenski, Xenia; Marheineke, Kathrin

    2015-01-01

    DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data. PMID:26046346

  12. Deletion of the M2-2 gene from avian metapneumovirus subgroup C impairs virus replication and immunogenicity in Turkeys.

    PubMed

    Yu, Qingzhong; Estevez, Carlos N; Roth, Jason P; Hu, Haixia; Zsak, Laszlo

    2011-06-01

    The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in viral RNA transcription or replication. To further characterize the function of the M2-2 protein in virus replication, the non-overlapping region of the M2-2 ORF was deleted from an infectious cDNA clone of the aMPV-C strain, and a viable virus was rescued by using reverse genetics technology. The recombinant virus, raMPV-C ΔM2-2, was characterized in vitro and in vivo. In Vero cells, raMPV-C ΔM2-2 replicated slightly less efficiently than the parental virus, 10-fold reduction at 48-h post-infection. The raMPV-C ΔM2-2 virus induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental virus infection. In specific-pathogen-free (SPF) turkeys, raMPV-C ΔM2-2 was attenuated and caused no clinical signs of disease. Less than 20% of the inoculated birds shed detectable virus in tracheal tissue during the first 5 days post-infection, and no virus shedding was detected afterward. Forty percent of infected birds produced a weak antibody response at 14 days post-infection. Upon challenge with a virulent aMPV-C strain, more than 80% of the raMPV-C ΔM2-2-inoculated birds showed typical disease signs and virus shedding in tracheal tissue. These results suggest that the M2-2 protein of aMPV-C virus is not essential for virus replication in vitro, but is required for sufficient virus replication to maintain pathogenicity and immunogenicity in the natural host.

  13. Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

    PubMed Central

    Ow, D W; Ausubel, F M

    1983-01-01

    P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration. Images PMID:6307977

  14. Comparison of the Transcription and Replication Strategies of Marburg Virus and Ebola Virus by Using Artificial Replication Systems

    PubMed Central

    Mühlberger, Elke; Weik, Michael; Volchkov, Viktor E.; Klenk, Hans-Dieter; Becker, Stephan

    1999-01-01

    The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses. PMID:9971816

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

    SciTech Connect

    Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au; Centre for Cancer Biology, SA Pathology, Adelaide; Hampton-Smith, Rachel J.

    2016-04-15

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

  16. Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication

    USDA-ARS?s Scientific Manuscript database

    Porcine circovirus (PCV) has been assumed to replicate its genome via the rolling-circle replication (RCR) mechanism because it encodes a Rep protein that contains several amino acid motifs commonly found in other RCR biological systems. Two proteins, Rep and Rep', are essential for PCV DNA replicat...

  17. High levels of virus replication and an intense inflammatory response contribute to the severe pathology in lymphoid tissues caused by Newcastle disease virus genotype VIId.

    PubMed

    Hu, Zenglei; Hu, Jiao; Hu, Shunlin; Song, Qingqing; Ding, Pingyun; Zhu, Jie; Liu, Xiaowen; Wang, Xiaoquan; Liu, Xiufan

    2015-03-01

    Some strains of Newcastle disease virus (NDV) genotype VIId cause more-severe tissue damage in lymphoid organs compared to other virulent strains. In this study, we aim to define the mechanism of this distinct pathological manifestation of genotype VII viruses. Pathology, virus replication, and the innate immune response in lymphoid tissues of chickens infected with two genotype VIId NDV strains (JS5/05 and JS3/05), genotype IX NDV F48E8 and genotype IV NDV Herts/33, were compared. Histopathologic examination showed that JS5/05 and JS3/05 produced more-severe lesions in the spleen and thymus, but these four virulent strains caused comparable mild lesions in the bursa. In addition, JS3/05 and JS5/05 replicated at significantly higher levels in the lymphatic organs than F48E8 and Herts/33. A microarray assay performed on the spleens of chickens infected with JS5/05 or Herts/33 revealed that JS5/05 elicited a more potent inflammatory response by increasing the number and expression levels of activated genes. Moreover, cytokine gene expression profiling showed that JS5/05 and JS3/05 induced a stronger cytokine response in lymphoid tissues compared to F48E8 and Herts/33. Taken together, our results indicate that the severe pathology in immune organs caused by genotype VIId NDV strains is associated with high levels of virus replication and an intense inflammatory response.

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Heat strain in cold.

    PubMed

    Rintamäki, Hannu; Rissanen, Sirkka

    2006-07-01

    In spite of increased environmental cold stress, heat strain is possible also in a cold environment. The body heat balance depends on three factors: environmental thermal conditions, metabolic heat production and thermal insulation of clothing and other protective garments. As physical exercise may increase metabolic heat production from rest values by ten times or even more, the required thermal insulation of clothing may vary accordingly. However, in most outdoor work, and often in indoor cold work, too, the thermal insulation of clothing is impractical, difficult or impossible to adjust according to the changes in physical activity. This is especially true with whole body covering garments like chemical protective clothing. As a result of this imbalance, heat strain may develop. In cold all the signs of heat strain (core temperature above 38 degrees C, warm or hot thermal sensations, increased cutaneous circulation and sweating) may not be present at the same time. Heat strain in cold may be whole body heat strain or related only to torso or core temperature. Together with heat strain in torso or body core, there can be at the same time even cold strain in peripheral parts and/or superficial layers of the body. In cold environment both the preservation of insulation and facilitation of heat loss are important. Development of clothing design is still needed to allow easy adjustments of thermal insulation.

  20. Candida albicans strain delineation.

    PubMed Central

    Merz, W G

    1990-01-01

    Candida albicans is a major opportunistic pathogen causing a wide spectrum of disease in human beings. Methods for strain delineation of this species to assess or predict virulence or to conduct epidemiologic or pathogenetic investigations have been developed. Although factors associated with virulence have been identified, there is no rapid system to quantitate them in a clinical laboratory. Therefore, many typing methods are based on variable phenotypic characteristics within this species including morphotyping, serotyping, antibiogram, resistogram typing, biotyping, biotyping based on commercial carbon assimilation patterns, enzyme profiles, sensitivity to yeast killer toxins, and typing based on protein variability. Phenotypically defined strains generally do not correlate with the pathogenic potential of a strain with the exception of morphotyping. However, these methods can be useful in epidemiologic investigations; for example, they have revealed that most individuals harbor one strain and that infections are frequently due to an endogenous strain. Problems with these methods usually relate to their discriminatory power. When this is maximized, reproducibility (especially between laboratories) suffers. Recently, methods based on differences in DNA structure (genotyping) for strain delineation have been developed, including electrophoretic karyotyping and restriction enzyme fragment length polymorphisms. The development of a computer-assisted data bank and analysis for these genotypic strain delineators will open investigations into the pathogenesis of this infection and permit epidemiologic studies previously not possible with this important human pathogen. PMID:1977511

  1. Strain gauge installation tool

    DOEpatents

    Conard, Lisa Marie

    1998-01-01

    A tool and a method for attaching a strain gauge to a test specimen by maaining alignment of, and applying pressure to, the strain gauge during the bonding of the gauge to the specimen. The tool comprises rigid and compliant pads attached to a spring-loaded clamp. The pads are shaped to conform to the specimen surface to which the gauge is to be bonded. The shape of the pads permits the tool to align itself to the specimen and to maintain alignment of the gauge to the specimen during the bond curing process. A simplified method of attaching a strain gauge is provided by use of the tool.

  2. Activation of Silent Replication Origins at Autonomously Replicating Sequence Elements near the HML Locus in Budding Yeast

    PubMed Central

    Vujcic, Marija; Miller, Charles A.; Kowalski, David

    1999-01-01

    In the budding yeast, Saccharomyces cerevisiae, replicators can function outside the chromosome as autonomously replicating sequence (ARS) elements; however, within chromosome III, certain ARSs near the transcriptionally silent HML locus show no replication origin activity. Two of these ARSs comprise the transcriptional silencers E (ARS301) and I (ARS302). Another, ARS303, resides between HML and the CHA1 gene, and its function is not known. Here we further localized and characterized ARS303 and in the process discovered a new ARS, ARS320. Both ARS303 and ARS320 are competent as chromosomal replication origins since origin activity was seen when they were inserted at a different position in chromosome III. However, at their native locations, where the two ARSs are in a cluster with ARS302, the I silencer, no replication origin activity was detected regardless of yeast mating type, special growth conditions that induce the transcriptionally repressed CHA1 gene, trans-acting mutations that abrogate transcriptional silencing at HML (sir3, orc5), or cis-acting mutations that delete the E and I silencers containing ARS elements. These results suggest that, for the HML ARS cluster (ARS303, ARS320, and ARS302), inactivity of origins is independent of local transcriptional silencing, even though origins and silencers share key cis- and trans-acting components. Surprisingly, deletion of active replication origins located 25 kb (ORI305) and 59 kb (ORI306) away led to detection of replication origin function at the HML ARS cluster, as well as at ARS301, the E silencer. Thus, replication origin silencing at HML ARSs is mediated by active replication origins residing at long distances from HML in the chromosome. The distal active origins are known to fire early in S phase, and we propose that their inactivation delays replication fork arrival at HML, providing additional time for HML ARSs to fire as origins. PMID:10454557

  3. Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

    PubMed Central

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek

    2012-01-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532

  4. Pyrimidine dimers block simian virus 40 replication forks

    SciTech Connect

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

    1986-10-01

    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 themore » 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.« less

  5. Inhibitors of Nucleotidyltransferase Superfamily Enzymes Suppress Herpes Simplex Virus Replication

    PubMed Central

    Wang, Hong; Tollefson, Ann E.; Ying, Baoling; Korom, Maria; Cheng, Xiaohong; Cao, Feng; Davis, Katie L.; Wold, William S. M.

    2014-01-01

    Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 μM, with suppression at 50 μM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 μM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family. PMID:25267681

  6. CRISPR-mediated control of the bacterial initiation of replication.

    PubMed

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

    2016-05-05

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

  7. EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

    PubMed Central

    Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F.; Rudel, Thomas

    2015-01-01

    The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and

  8. Distinct epigenetic features of differentiation-regulated replication origins.

    PubMed

    Smith, Owen K; Kim, RyanGuk; Fu, Haiqing; Martin, Melvenia M; Lin, Chii Mei; Utani, Koichi; Zhang, Ya; Marks, Anna B; Lalande, Marc; Chamberlain, Stormy; Libbrecht, Maxwell W; Bouhassira, Eric E; Ryan, Michael C; Noble, William S; Aladjem, Mirit I

    2016-01-01

    Eukaryotic genome duplication starts at discrete sequences (replication origins) that coordinate cell cycle progression, ensure genomic stability and modulate gene expression. Origins share some sequence features, but their activity also responds to changes in transcription and cellular differentiation status. To identify chromatin states and histone modifications that locally mark replication origins, we profiled origin distributions in eight human cell lines representing embryonic and differentiated cell types. Consistent with a role of chromatin structure in determining origin activity, we found that cancer and non-cancer cells of similar lineages exhibited highly similar replication origin distributions. Surprisingly, our study revealed that DNase hypersensitivity, which often correlates with early replication at large-scale chromatin domains, did not emerge as a strong local determinant of origin activity. Instead, we found that two distinct sets of chromatin modifications exhibited strong local associations with two discrete groups of replication origins. The first origin group consisted of about 40,000 regions that actively initiated replication in all cell types and preferentially colocalized with unmethylated CpGs and with the euchromatin markers, H3K4me3 and H3K9Ac. The second group included origins that were consistently active in cells of a single type or lineage and preferentially colocalized with the heterochromatin marker, H3K9me3. Shared origins replicated throughout the S-phase of the cell cycle, whereas cell-type-specific origins preferentially replicated during late S-phase. These observations are in line with the hypothesis that differentiation-associated changes in chromatin and gene expression affect the activation of specific replication origins.

  9. Carbon monoxide and biliverdin suppress bovine viral diarrhoea virus replication.

    PubMed

    Ma, Zhiqian; Pu, Fengxing; Zhang, Xiaobin; Yan, Yunhuan; Zhao, Lijuan; Zhang, Angke; Li, Na; Zhou, En-Min; Xiao, Shuqi

    2017-12-01

    Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

  10. Strain gage calibration

    NASA Technical Reports Server (NTRS)

    Moore, T. C. (Inventor)

    1984-01-01

    A temporary bonding system for accurately predetermining the individual apparent strain curve characteristics of the gages is used, and subsequently employs a computer to watch the apparent strain curves of the individual gages to determine which gages should be used together on transducers. The temporary bonding system requires a test block on which the gages are temporarily bonded, several thermocouples for monitoring temperature, and a data acquisition system for recording apparent strain data. Initially, a group of strain gages are attached to the test block using a bonding agent that disintegrates at high temperatures. The gages are then wired to an appropriate data acquisition and data, collected throughout a predetermined temperature excursion. Once the data is obtained, the test block is heated until the bonding agent disintegrates freeing the gages from the test block. The gages are then disconnected from the data acquisition system and cleaned, thereby ready for use on transducers.

  11. Strains and Sprains

    MedlinePlus

    ... Sprains? Muscles contract and relax (almost like rubber bands) to help your body move. So a strain ... where your body bends and rotates. Strong, elastic bands of tissue called ligaments hold bones together in ...

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  13. Evolution of complexity in RNA-like replicator systems

    PubMed Central

    Takeuchi, Nobuto; Hogeweg, Paulien

    2008-01-01

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

  14. Mutation at Expanding Front of Self-Replicating Colloidal Clusters

    NASA Astrophysics Data System (ADS)

    Tanaka, Hidenori; Zeravcic, Zorana; Brenner, Michael P.

    2016-12-01

    We construct a scheme for self-replicating square clusters of particles in two spatial dimensions, and validate it with computer simulations in a finite-temperature heat bath. We find that the self-replication reactions propagate through the bath in the form of Fisher waves. Our model reflects existing colloidal systems, but is simple enough to allow simulation of many generations and thereby the first study of evolutionary dynamics in an artificial system. By introducing spatially localized mutations in the replication rules, we show that the mutated cluster population can survive and spread with the expanding front in circular sectors of the colony.

  15. Strain Measurement - Unidirectional.

    DTIC Science & Technology

    1983-04-20

    pressure is used for positioning and clamping. 3.4 Gage Wiring and Protection. Electrical conductors used to transmit signals from the strain gage to the...wires do not introduce significant resistance, cause resistance changes, or generate electrical signals that are not related to the strain gage...reduction, it is often desirable and es- sential for precision work with analog recording systems, to provide for an electrical simulation of known

  16. Suppressor of Cytokine Signaling 3 Suppresses Hepatitis C Virus Replication in an mTOR-Dependent Manner▿

    PubMed Central

    Shao, Run-Xuan; Zhang, Leiliang; Peng, Lee F.; Sun, Eileen; Chung, Woo Jin; Jang, Jae Yong; Tsai, Wei-Lun; Hyppolite, Guibenson; Chung, Raymond T.

    2010-01-01

    We and others have observed that hepatic levels of suppressor of cytokine signaling 3 (SOCS3) are significantly higher in persons with chronic hepatitis C, particularly those who are nonresponders to interferon (IFN) treatment, than in healthy individuals. However, the relationship between SOCS3 and hepatitis C virus (HCV) replication remains unclear. Given its putative role, we hypothesized that SOCS3 is permissive for viral replication. We therefore used the OR6 cell line, which harbors a genotype 1b full-length HCV replicon, and the genotype 2a full-length HCV strain JFH1 infection system to analyze the effects of SOCS3 overexpression and short hairpin RNA (shRNA)-mediated knockdown on HCV replication. We further analyzed the role of mTOR in the effects of SOCS3 by treating selected cells with rapamycin. OR6 cells and JFH1-infected Huh7.5.1 cells expressed significantly less SOCS3 than control cells. Furthermore, inhibition of HCV replication with the HCV protease inhibitor BILN 2061 restored SOCS3 protein levels. SOCS3 overexpression in OR6 cells and JFH1-infected Huh7.5.1 cells resulted in significantly lower HCV replication than that in the control cells, despite SOCS3-related inhibition of STAT1 phosphorylation and type I IFN signaling. In contrast, JFH1-infected cells with stable SOCS3 knockdown expressed higher levels of HCV proteins and RNA than did control cells. SOCS3-targeting shRNA also knocked down mTOR and phospho-mTOR. The mTOR inhibitor rapamycin reversed the inhibitory effects of SOCS3. In independent investigations, SOCS3 unexpectedly suppressed HCV replication in an mTOR-dependent manner. These findings suggest that increased SOCS3 levels consistently observed in chronic IFN nonresponders may reflect a compensatory host antiviral response to persistent infection and that manipulation of SOCS3/mTOR may offer benefit against HCV infection. PMID:20375166

  17. Competitive replication kinetics and pathogenicity in pigs co-infected with historical and newly invading classical swine fever viruses.

    PubMed

    Huang, Yu-Liang; Deng, Ming-Chung; Tsai, Kuo-Jung; Liu, Hsin-Meng; Huang, Chin-Cheng; Wang, Fun-In; Chang, Chia-Yi

    2017-01-15

    Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Photothermal strain imaging

    NASA Astrophysics Data System (ADS)

    Choi, Changhoon; Ahn, Joongho; Jeon, Seungwan; Kim, Chulhong

    2017-07-01

    Vulnerable plaques are the major cause of cardiovascular disease, but they are difficult to detect with conventional intravascular imaging techniques. Techniques are needed to identify plaque vulnerability based on the presence of lipids in plaque. Thermal strain imaging (TSI) is an imaging technique based on ultrasound (US) wave propagation speed, which varies with the medium temperature. In TSI, the strain that occurs during tissue temperature change can be used for lipid detection because it has a different tendency depending on the type of tissue. Here, we demonstrate photothermal strain imaging (pTSI) using an intravascular ultrasound catheter. pTSI is performed by slightly and selectively heating lipid using a relatively inexpensive continuous laser source. We applied a speckle-tracking algorithm to US B-mode images for strain calculations. As a result, the strain produced in porcine fat was different from the strain produced in water-bearing gelatin phantom, which made it possible to distinguish the two. This suggests that pTSI could potentially be a way of differentiating lipids in coronary artery.

  19. The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.

    PubMed

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.

  20. The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

    PubMed Central

    de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent

    2013-01-01

    Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution. PMID:23853584

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

    SciTech Connect

    Wörmann, Xenia; Lesch, Markus; Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee

    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 themore » 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.« less

  2. The Bimodal Lifestyle of Intracellular Salmonella in Epithelial Cells: Replication in the Cytosol Obscures Defects in Vacuolar Replication

    PubMed Central

    Steele-Mortimer, Olivia

    2012-01-01

    Salmonella enterica serovar Typhimurium invades and proliferates within epithelial cells. Intracellular bacteria replicate within a membrane bound vacuole known as the Salmonella containing vacuole. However, this bacterium can also replicate efficiently in the cytosol of epithelial cells and net intracellular growth is a product of both vacuolar and cytosolic replication. Here we have used semi-quantitative single-cell analyses to investigate the contribution of each of these replicative niches to intracellular proliferation in cultured epithelial cells. We show that cytosolic replication can account for the majority of net replication even though it occurs in less than 20% of infected cells. Consequently, assays for net growth in a population of infected cells, for example by recovery of colony forming units, are not good indicators of vacuolar proliferation. We also show that the Salmonella Type III Secretion System 2, which is required for SCV biogenesis, is not required for cytosolic replication. Altogether this study illustrates the value of single cell analyses when studying intracellular pathogens. PMID:22719929

  3. Identification of Bacillus Strains for Biological Control of Catfish Pathogens

    PubMed Central

    Ran, Chao; Carrias, Abel; Williams, Malachi A.; Capps, Nancy; Dan, Bui C. T.; Newton, Joseph C.; Kloepper, Joseph W.; Ooi, Ei L.; Browdy, Craig L.; Terhune, Jeffery S.; Liles, Mark R.

    2012-01-01

    Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×107 CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture. PMID:23029244

  4. Identification of Bacillus strains for biological control of catfish pathogens.

    PubMed

    Ran, Chao; Carrias, Abel; Williams, Malachi A; Capps, Nancy; Dan, Bui C T; Newton, Joseph C; Kloepper, Joseph W; Ooi, Ei L; Browdy, Craig L; Terhune, Jeffery S; Liles, Mark R

    2012-01-01

    Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.

  5. Strain: Fact or Fiction?

    NASA Astrophysics Data System (ADS)

    Heilbronner, Renée

    2017-04-01

    2017 marks the 50th anniversary of the publication of John Ramsay's well known textbook "Folding and Fracturing of Rocks" - ... and the 30th anniversary of the rejection of a rather less well known paper entitled "Strain: Fact or Fiction?" submitted by Renée Panozzo to the Journal of Structural Geology. The gist of the paper was simple and straight forward: it was argued that not every fabric that can be observed in deformed rocks is necessarily a measure of the amount of strain the rock incurred. A distinction was made between a general "fabric", i.e., the traceable geometry of grain boundaries, for example, and a so-called "strain fabric", i.e., the model geometry that would result from homogeneously straining an initially isotropic fabric and that would exhibit at least orthorhombic symmetry. To verify if a given fabric was indeed a strain fabric it was therefore suggested to use the SURFOR method (published by Panozzo) and to carry out a so-called strain test, i.e., a check of symmetry, before interpreting the results of a fabric analysis in terms of strain. The problem with the paper was that it was very obviously written out of frustration. The frustration came form having reviewed a number of manuscripts which tried to use the then novel SURFOR method for strain analysis without first checking if the the fabric was a indeed a "strain fabric" or not, and then blaming the SURFOR method for producing ambiguous results. As a result, the paper was not exactly well balanced and carefully thought out. It was considered "interesting but not scholarly" by one of the reviewers and down-right offensive by the second. To tell the truth, however, the paper was not formally rejected. The editor Sue Treagus strongly encouraged Panozzo to revise the paper, ... and 30 years later, I will follow her advise and offer a revised paper as a tribute to John Ramsay. To quote from the original manuscript: "We should be a little more impressed that strain works so well, and less

  6. EPA Lean Government Initiative: How to Replicate Lean Successes

    EPA Pesticide Factsheets

    This Lean Replication Primer describes how EPA Offices and Regions can identify and adapt successful practices from previous Lean projects to “replicate” their successes and generate further improvements.

  7. Revisiting Mental Simulation in Language Comprehension: Six Replication Attempts

    PubMed Central

    Zwaan, Rolf A.; Pecher, Diane

    2012-01-01

    The notion of language comprehension as mental simulation has become popular in cognitive science. We revisit some of the original empirical evidence for this. Specifically, we attempted to replicate the findings from earlier studies that examined the mental simulation of object orientation, shape, and color, respectively, in sentence-picture verification. For each of these sets of findings, we conducted two web-based replication attempts using Amazon's Mechanical Turk. Our results are mixed. Participants responded faster to pictures that matched the orientation or shape implied by the sentence, replicating the original findings. The effect was larger and stronger for shape than orientation. Participants also responded faster to pictures that matched the color implied by the sentence, whereas the original studies obtained mismatch advantages. We argue that these results support mental simulation theory, show the importance of replication studies, and show the viability of web-based data collection. PMID:23300547

  8. Bayesian probabilistic network modeling from multiple independent replicates

    PubMed Central

    2012-01-01

    Often protein (or gene) time-course data are collected for multiple replicates. Each replicate generally has sparse data with the number of time points being less than the number of proteins. Usually each replicate is modeled separately. However, here all the information in each of the replicates is used to make a composite inference about signal networks. The composite inference comes from combining well structured Bayesian probabilistic modeling with a multi-faceted Markov Chain Monte Carlo algorithm. Based on simulations which investigate many different types of network interactions and experimental variabilities, the composite examination uncovers many important relationships within the networks. In particular, when the edge's partial correlation between two proteins is at least moderate, then the composite's posterior probability is large. PMID:22901091

  9. Varicella-Zoster Virus Glycoproteins: Entry, Replication, and Pathogenesis

    PubMed Central

    Yang, Edward; Arvin, Ann M.

    2016-01-01

    Varicella-zoster virus (VZV), an alphaherpesvirus that causes chicken pox (varicella) and shingles (herpes zoster), is a medically important pathogen that causes considerable morbidity and, on occasion, mortality in immunocompromised patients. Herpes zoster can afflict the elderly with a debilitating condition, postherpetic neuralgia, triggering severe, untreatable pain for months or years. The lipid envelope of VZV, similar to all herpesviruses, contains numerous glycoproteins required for replication and pathogenesis. Purpose of Review To summarize the current knowledge about VZV glycoproteins and their roles in cell entry, replication and pathogenesis. Recent Findings The functions for some VZV glycoproteins are known, such as gB, gH and gL in membrane fusion, cell-cell fusion regulation, and receptor binding properties. However, the molecular mechanisms that trigger or mediate VZV glycoproteins remains poorly understood. Summary VZV glycoproteins are central to successful replication but their modus operandi during replication and pathogenesis remain elusive requiring further mechanistic based studies. PMID:28367398

  10. Varicella-Zoster Virus Glycoproteins: Entry, Replication, and Pathogenesis.

    PubMed

    Oliver, Stefan L; Yang, Edward; Arvin, Ann M

    2016-12-01

    Varicella-zoster virus (VZV), an alphaherpesvirus that causes chicken pox (varicella) and shingles (herpes zoster), is a medically important pathogen that causes considerable morbidity and, on occasion, mortality in immunocompromised patients. Herpes zoster can afflict the elderly with a debilitating condition, postherpetic neuralgia, triggering severe, untreatable pain for months or years. The lipid envelope of VZV, similar to all herpesviruses, contains numerous glycoproteins required for replication and pathogenesis. To summarize the current knowledge about VZV glycoproteins and their roles in cell entry, replication and pathogenesis. The functions for some VZV glycoproteins are known, such as gB, gH and gL in membrane fusion, cell-cell fusion regulation, and receptor binding properties. However, the molecular mechanisms that trigger or mediate VZV glycoproteins remains poorly understood. VZV glycoproteins are central to successful replication but their modus operandi during replication and pathogenesis remain elusive requiring further mechanistic based studies.

  11. Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

    PubMed Central

    Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

    2015-01-01

    Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

  12. Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins.

    PubMed

    Samson, Rachel Y; Abeyrathne, Priyanka D; Bell, Stephen D

    2016-01-21

    Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Replication-Competent Influenza A Viruses Expressing Reporter Genes

    PubMed Central

    Breen, Michael; Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. PMID:27347991

  14. GABA metabolism pathway genes, UGA1 and GAD1, regulate replicative lifespan in Saccharomycescerevisiae

    SciTech Connect

    Kamei, Yuka; Tamura, Takayuki; Yoshida, Ryo

    2011-04-01

    Highlights: {yields}We demonstrate that two genes in the yeast GABA metabolism pathway affect aging. {yields} Deletion of the UGA1 or GAD1 genes extends replicative lifespan. {yields} Addition of GABA to wild-type cultures has no effect on lifespan. {yields} Intracellular GABA levels do not differ in longevity mutants and wild-type cells. {yields} Levels of tricarboxylic acid cycle intermediates positively correlate with lifespan. -- Abstract: Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for {gamma}-aminobutyric acid (GABA)-dependentmore » induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomycescerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The {Delta}uga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased lifespan. Therefore, two genes in the GABA metabolism pathway, UGA1 and GAD1, were identified as aging genes. Unexpectedly, intracellular GABA levels in mutant cells (except for {Delta}uga2 cells) did not differ from those in wild-type cells. Addition of GABA to culture media, which induces transcription of the UGA structural genes, had no effect on replicative lifespan of wild-type cells. Multivariate analysis of {sup 1}H nuclear magnetic resonance spectra for the whole-cell metabolite levels demonstrated a separation between long-lived and normal-lived strains. Gas chromatography-mass spectrometry analysis of identified metabolites showed that levels of tricarboxylic acid cycle intermediates positively correlated with

  15. Net replication of Salmonella enterica serovars Typhimurium and Choleraesuis in porcine intestinal mucosa and nodes is associated with their differential virulence.

    PubMed

    Paulin, Susan M; Jagannathan, Aparna; Campbell, June; Wallis, Timothy S; Stevens, Mark P

    2007-08-01

    Salmonella enterica is a facultative intracellular pathogen of worldwide importance and causes a spectrum of diseases depending on serovar- and host-specific factors. Oral infection of pigs with S. enterica serovar Typhimurium strain 4/74 produces acute enteritis but is rarely fatal, whereas serovar Choleraesuis strain A50 causes systemic disease with a high mortality rate. With a porcine ligated ileal loop model, we observed that systemic virulence of serovar Choleraesuis A50 is not associated with enhanced intestinal invasion, secretory responses, or neutrophil recruitment compared to serovar Typhimurium 4/74. The net growth in vivo of serovar Choleraesuis A50 and serovar Typhimurium 4/74 was monitored following oral inoculation of pigs with strains harboring pHSG422, which exhibits temperature-sensitive replication. Analysis of plasmid partitioning revealed that the enteric virulence of serovar Typhimurium 4/74 relative to that of serovar Choleraesuis A50 is associated with rapid replication in the intestinal wall, whereas systemic virulence of serovar Choleraesuis A50 is associated with enhanced persistence in intestinal mesenteric lymph nodes. Faster replication of serovar Typhimurium, compared to that of serovar Choleraesuis, in the intestinal mucosa was associated with greater induction of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-8 (IL-8), and IL-18 as detected by reverse transcriptase PCR analysis of transcripts from infected mucosa. During replication in batch culture and porcine alveolar macrophages, transcription of genes encoding components of type III secretion systems 1 (sipC) and 2 (sseC) was observed to be significantly higher in serovar Typhimurium 4/74 than in serovar Choleraesuis A50, and this may contribute to the differences in epithelial invasion and intracellular proliferation. The rapid induction of proinflammatory responses by strain 4/74 may explain why pigs confine serovar Typhimurium infection to the

  16. Verdinexor, a Novel Selective Inhibitor of Nuclear Export, Reduces Influenza A Virus Replication In Vitro and In Vivo

    PubMed Central

    Perwitasari, Olivia; Johnson, Scott; Yan, Xiuzhen; Howerth, Elizabeth; Shacham, Sharon; Landesman, Yosef; Baloglu, Erkan; McCauley, Dilara; Tamir, Sharon; Tompkins, S. Mark

    2014-01-01

    ABSTRACT Influenza is a global health concern, causing death, morbidity, and economic losses. Chemotherapeutics that target influenza virus are available; however, rapid emergence of drug-resistant strains is common. Therapeutic targeting of host proteins hijacked by influenza virus to facilitate replication is an antiviral strategy to reduce the development of drug resistance. Nuclear export of influenza virus ribonucleoprotein (vRNP) from infected cells has been shown to be mediated by exportin 1 (XPO1) interaction with viral nuclear export protein tethered to vRNP. RNA interference screening has identified XPO1 as a host proinfluenza factor where XPO1 silencing results in reduced influenza virus replication. The Streptomyces metabolite XPO1 inhibitor leptomycin B (LMB) has been shown to limit influenza virus replication in vitro; however, LMB is toxic in vivo, which makes it unsuitable for therapeutic use. In this study, we tested the anti-influenza virus activity of a new class of orally available small-molecule selective inhibitors of nuclear export, specifically, the XPO1 antagonist KPT-335 (verdinexor). Verdinexor was shown to potently and selectively inhibit vRNP export and effectively inhibited the replication of various influenza virus A and B strains in vitro, including pandemic H1N1 virus, highly pathogenic H5N1 avian influenza virus, and the recently emerged H7N9 strain. In vivo, prophylactic and therapeutic administration of verdinexor protected mice against disease pathology following a challenge with influenza virus A/California/04/09 or A/Philippines/2/82-X79, as well as reduced lung viral loads and proinflammatory cytokine expression, while having minimal toxicity. These studies show that verdinexor acts as a novel anti-influenza virus therapeutic agent. IMPORTANCE Antiviral drugs represent important means of influenza virus control. However, substantial resistance to currently approved influenza therapeutic drugs has developed. New antiviral

  17. Performance analysis of static locking in replicated distributed database systems

    NASA Technical Reports Server (NTRS)

    Kuang, Yinghong; Mukkamala, Ravi

    1991-01-01

    Data replications and transaction deadlocks can severely affect the performance of distributed database systems. Many current evaluation techniques ignore these aspects, because it is difficult to evaluate through analysis and time consuming to evaluate through simulation. Here, a technique is discussed that combines simulation and analysis to closely illustrate the impact of deadlock and evaluate performance of replicated distributed databases with both shared and exclusive locks.

  18. The hunt for origins of DNA replication in multicellular eukaryotes

    PubMed Central

    Urban, John M.; Foulk, Michael S.; Casella, Cinzia

    2015-01-01

    Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed. PMID:25926981

  19. Performance analysis of static locking in replicated distributed database systems

    NASA Technical Reports Server (NTRS)

    Kuang, Yinghong; Mukkamala, Ravi

    1991-01-01

    Data replication and transaction deadlocks can severely affect the performance of distributed database systems. Many current evaluation techniques ignore these aspects, because it is difficult to evaluate through analysis and time consuming to evaluate through simulation. A technique is used that combines simulation and analysis to closely illustrate the impact of deadlock and evaluate performance of replicated distributed database with both shared and exclusive locks.

  20. Intracellular Detection of Viral Transcription and Replication Using RNA FISH

    DTIC Science & Technology

    2016-05-26

    only allow entirely new investigations into the replication of these viruses , but also how this method can be applied to any virus with a known...resulted in the development of a variety of useful assays for virus detection. For hemorrhagic fever viruses with segmented genomes such as Crimean Congo...Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses

  1. Studies in Historical Replication in Psychology I: Introduction

    NASA Astrophysics Data System (ADS)

    Tweney, Ryan D.

    2008-05-01

    This special issue reports a project in which the replication of historically meaningful studies was carried out by graduate students in a history of psychology course. In this introduction, I outline the nature of the project and its rationale, and briefly sketch the results. The subsequent five papers represent scholarly presentations of five selected replications written by students in the course. These are followed by a commentary on the project by an educational psychologist.

  2. Did template-directed nucleation precede molecular replication?

    NASA Technical Reports Server (NTRS)

    Orgel, Leslie E.

    1986-01-01

    It is proposed that mononucleotides incorporated into the surfaces of microcrystals of inorganic phosphates such as hydroxyapatite can act as templates to assemble complementary mononucleotides from solution, and that the phosphate groups of the assembled nucleotides can facilitate nucleation of a second hydroxyapatite crystal. This would provide a mechanism of replication that is subject to natural selection. The possible role of a replicating system of this kind in the origins of life on the earth is discussed.

  3. Effects of strain and light intensity on growth performance and carcass characteristics of broilers grown to heavy weights.

    PubMed

    Olanrewaju, H A; Miller, W W; Maslin, W R; Collier, S D; Purswell, J L; Branton, S L

    2014-08-01

    Effects of genetic strain and light intensity on growth performance and carcass characteristics of broilers grown to heavy weights were investigated. The experimental design was a randomized complete block design. Treatment structure was a 2 × 5 factorial arrangement with the main factors being strain (Ross × Ross 308, Ross × Ross 708) and light intensity (25, 10, 5, 2.5, and 0.2 lx) with trial as replicates. In each of the 5 trials, chicks of 2 different strains of the same commercial hatchery were equally and randomly distributed into 10 environmentally controlled rooms (5 rooms/strain) at 1 d of age at 50% RH. Each room was randomly assigned 1 of 5 light intensities from 22 to 56 d of age. Feed and water were provided ad libitum. Birds and feed were weighed on 0, 14, 28, 42, and 56 d of age for growth performance. Humoral immune response was determined on d 28, whereas ocular and blood samples were performed on d 42 and 55, respectively. On d 56, 20 (10 males and 10 females) birds/strain from each room were processed to determine weights and yields. Genetic strain was significant (P ≤ 0.05) for most of the examined variables, where Ross × Ross 308 had better growth performance and meat yield in comparison with Ross × Ross 708. Although, there was no main effect of light intensity on growth performance and meat yield, results indicated that birds under 10 and 5 lx intensities showed slightly better growth performance and meat yield compared with birds under 25, 2.5, and 0.2 lx in both strains. There was no effect of strain and light intensity on ocular indices, immune response, plasma corticosterone levels, and mortality. This study shows the positive influence on profits to commercial poultry facilities that are using a low lighting environment to reduce energy cost, optimize feed conversion, and maximize production without compromising the welfare of the broilers. © Poultry Science Association Inc.

  4. Simultaneous regulation of cell size and chromosome replication in bacteria

    PubMed Central

    Ho, Po-Yi; Amir, Ariel

    2015-01-01

    Bacteria are able to maintain a narrow distribution of cell sizes by regulating the timing of cell divisions. In rich nutrient conditions, cells divide much faster than their chromosomes replicate. This implies that cells maintain multiple rounds of chromosome replication per cell division by regulating the timing of chromosome replications. Here, we show that both cell size and chromosome replication may be simultaneously regulated by the long-standing initiator accumulation strategy. The strategy proposes that initiators are produced in proportion to the volume increase and is accumulated at each origin of replication, and chromosome replication is initiated when a critical amount per origin has accumulated. We show that this model maps to the incremental model of size control, which was previously shown to reproduce experimentally observed correlations between various events in the cell cycle and explains the exponential dependence of cell size on the growth rate of the cell. Furthermore, we show that this model also leads to the efficient regulation of the timing of initiation and the number of origins consistent with existing experimental results. PMID:26217311

  5. Sustainable proliferation of liposomes compatible with inner RNA replication

    PubMed Central

    Tsuji, Gakushi; Fujii, Satoshi; Sunami, Takeshi; Yomo, Tetsuya

    2016-01-01

    Although challenging, the construction of a life-like compartment via a bottom–up approach can increase our understanding of life and protocells. The sustainable replication of genome information and the proliferation of phospholipid vesicles are requisites for reconstituting cell growth. However, although the replication of DNA or RNA has been developed in phospholipid vesicles, the sustainable proliferation of phospholipid vesicles has remained difficult to achieve. Here, we demonstrate the sustainable proliferation of liposomes that replicate RNA within them. Nutrients for RNA replication and membranes for liposome proliferation were combined by using a modified freeze–thaw technique. These liposomes showed fusion and fission compatible with RNA replication and distribution to daughter liposomes. The RNAs in daughter liposomes were repeatedly used as templates in the next RNA replication and were distributed to granddaughter liposomes. Liposome proliferation was achieved by 10 cycles of iterative culture operation. Therefore, we propose the use of culturable liposomes as an advanced protocell model with the implication that the concurrent supplement of both the membrane material and the nutrients of inner reactions might have enabled protocells to grow sustainably. PMID:26711996

  6. Dynamics of intracellular bacterial replication at the single cell level.

    PubMed

    Helaine, Sophie; Thompson, Jessica A; Watson, Kathryn G; Liu, Mei; Boyle, Cliona; Holden, David W

    2010-02-23

    Several important pathogens cause disease by surviving and replicating within host cells. Bacterial proliferation is the product of both replication and killing undergone by the population. However, these processes are difficult to distinguish, and are usually assessed together by determination of net bacterial load. In addition, measurement of net load does not reveal heterogeneity within pathogen populations. This is particularly important in persistent infections in which slow or nongrowing bacteria are thought to have a major impact. Here we report the development of a reporter system based on fluorescence dilution that enables direct quantification of the replication dynamics of Salmonella enterica serovar Typhimurium (S. Typhimurium) in murine macrophages at both the population and single-cell level. We used this technique to demonstrate that a major S. Typhimurium virulence determinant, the Salmonella pathogenicity island 2 type III secretion system, is required for bacterial replication but does not have a major influence on resistance to killing. Furthermore, we found that, upon entry into macrophages, many bacteria do not replicate, but appear to enter a dormant-like state. These could represent an important reservoir of persistent bacteria. The approach could be extended to other pathogens to study the contribution of virulence and host resistance factors to replication and killing, and to identify and characterize nonreplicating bacteria associated with chronic or latent infections.

  7. Relicensing of Transcriptionally Inactivated Replication Origins in Budding Yeast*

    PubMed Central

    Lõoke, Marko; Reimand, Jüri; Sedman, Tiina; Sedman, Juhan; Järvinen, Lari; Värv, Signe; Peil, Kadri; Kristjuhan, Kersti; Vilo, Jaak; Kristjuhan, Arnold

    2010-01-01

    DNA replication origins are licensed in early G1 phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G1 to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G1 might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase. PMID:20962350

  8. Visualization of replication initiation and elongation in Drosophila

    PubMed Central

    Claycomb, Julie M.; MacAlpine, David M.; Evans, James G.; Bell, Stephen P.; Orr-Weaver, Terry L.

    2002-01-01

    Chorion gene amplification in the ovaries of Drosophila melanogaster is a powerful system for the study of metazoan DNA replication in vivo. Using a combination of high-resolution confocal and deconvolution microscopy and quantitative realtime PCR, we found that initiation and elongation occur during separate developmental stages, thus permitting analysis of these two phases of replication in vivo. Bromodeoxyuridine, origin recognition complex, and the elongation factors minichromosome maintenance proteins (MCM)2–7 and proliferating cell nuclear antigen were precisely localized, and the DNA copy number along the third chromosome chorion amplicon was quantified during multiple developmental stages. These studies revealed that initiation takes place during stages 10B and 11 of egg chamber development, whereas only elongation of existing replication forks occurs during egg chamber stages 12 and 13. The ability to distinguish initiation from elongation makes this an outstanding model to decipher the roles of various replication factors during metazoan DNA replication. We utilized this system to demonstrate that the pre–replication complex component, double-parked protein/cell division cycle 10–dependent transcript 1, is not only necessary for proper MCM2–7 localization, but, unexpectedly, is present during elongation. PMID:12403810

  9. CTCF driven TERRA transcription facilitates completion of telomere DNA replication.

    PubMed

    Beishline, Kate; Vladimirova, Olga; Tutton, Stephen; Wang, Zhuo; Deng, Zhong; Lieberman, Paul M

    2017-12-13

    Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.

  10. Late replication domains are evolutionary conserved in the Drosophila genome.

    PubMed

    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.

  11. Mutations in DNA replication genes reduce yeast life span.

    PubMed

    Hoopes, Laura L Mays; Budd, Martin; Choe, Wonchae; Weitao, Tao; Campbell, Judith L

    2002-06-01

    Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated. We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span. All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type. In addition, the life span of dna2 mutants is extended by expression of an additional copy of SIR2 or by deletion of FOB1, which also increase wild-type life span. The ribosomal DNA locus and the nucleolus seem to be particularly sensitive to defects in dna2 mutants, although in dna2 mutants extrachromosomal ribosomal circles do not accumulate during the aging of a mother cell. Several other replication mutations, such as rad27 Delta, encoding the FEN-1 nuclease involved in several aspects of genomic stability, also show premature aging. We propose that replication fork failure due to spontaneous, endogenous DNA damage and attendant genomic instability may contribute to replicative senescence. This may imply that the genomic instability, segmental premature aging symptoms, and cancer predisposition associated with the human RecQ helicase diseases, such as Werner, Bloom, and Rothmund-Thomson syndromes, are also related to replicative stress.

  12. OriDB: a DNA replication origin database.

    PubMed

    Nieduszynski, Conrad A; Hiraga, Shin-ichiro; Ak, Prashanth; Benham, Craig J; Donaldson, Anne D

    2007-01-01

    Replication of eukaryotic chromosomes initiates at multiple sites called replication origins. Replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where several complementary studies have mapped their locations genome-wide. We have collated these datasets, taking account of the resolution of each study, to generate a single list of distinct origin sites. OriDB provides a web-based catalogue of these confirmed and predicted S.cerevisiae DNA replication origin sites. Each proposed or confirmed origin site appears as a record in OriDB, with each record comprising seven pages. These pages provide, in text and graphical formats, the following information: genomic location and chromosome context of the origin site; time of origin replication; DNA sequence of proposed or experimentally confirmed origin elements; free energy required to open the DNA duplex (stress-induced DNA duplex destabilization or SIDD); and phylogenetic conservation of sequence elements. In addition, OriDB encourages community submission of additional information for each origin site through a User Notes facility. Origin sites are linked to several external resources, including the Saccharomyces Genome Database (SGD) and relevant publications at PubMed. Finally, a Chromosome Viewer utility allows users to interactively generate graphical representations of DNA replication data genome-wide. OriDB is available at www.oridb.org.

  13. A Blm-Recql5 partnership in replication stress response.

    PubMed

    Lu, Xincheng; Lou, Hua; Luo, Guangbin

    2011-02-01

    Deficiencies in DNA damage response and repair not only can result in genome instability and cancer predisposition, but also can render the cancer cells intrinsically more vulnerable to certain types of DNA damage insults. Particularly, replication stress is both a hallmark of human cancers and a common instigator for genome instability and cell death. Here, we review our work based on the genetic knockout studies on Blm and Recql5, two members of the mammalian RecQ helicase family. These studies have uncovered a unique partnership between these two helicases in the implementation of proper mitigation strategies under different circumstances to promote DNA replication and cell survival and suppress genome instability and cancer. In particular, current studies have revealed the presence of a novel Recql5/RECQL5-dependent mechanism for suppressing replication fork collapse in response to global replication fork stalling following exposure to camptothecin (CPT), a topoisomerase I inhibitor, and a potent inhibitor of DNA replication. The unique partnership between Blm and Recql5 in coping with the challenge imposed by replication stress is discussed. In addition, given that irinotecan and topotecan, two CPT derivatives, are currently used in clinic for treating human cancer patients with very promising results, the potential implication of the new findings from these studies in anticancer treatments is also discussed.

  14. Direct Evidence for the Formation of Precatenanes during DNA Replication*

    PubMed Central

    Cebrián, Jorge; Castán, Alicia; Martínez, Víctor; Kadomatsu-Hermosa, Maridian J.; Parra, Cristina; Fernández-Nestosa, María José; Schaerer, Christian; Hernández, Pablo; Krimer, Dora B.; Schvartzman, Jorge B.

    2015-01-01

    The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination. PMID:25829493

  15. Break induced replication in eukaryotes: mechanisms, functions, and consequences.

    PubMed

    Sakofsky, Cynthia J; Malkova, Anna

    2017-08-01

    Break-induced replication (BIR) is an important pathway specializing in repair of one-ended double-strand DNA breaks (DSBs). This type of DSB break typically arises at collapsed replication forks or at eroded telomeres. BIR initiates by invasion of a broken DNA end into a homologous template followed by initiation of DNA synthesis that can proceed for hundreds of kilobases. This synthesis is drastically different from S-phase replication in that instead of a replication fork, BIR proceeds via a migrating bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual mode of DNA replication is responsible for frequent genetic instabilities associated with BIR, including hyper-mutagenesis, which can lead to the formation of mutation clusters, extensive loss of heterozygosity, chromosomal translocations, copy-number variations and complex genomic rearrangements. In addition to budding yeast experimental systems that were initially employed to investigate eukaryotic BIR, recent studies in different organisms including humans, have provided multiple examples of BIR initiated within different cellular contexts, including collapsed replication fork and telomere maintenance in the absence of telomerase. In addition, significant progress has been made towards understanding microhomology-mediated BIR (MMBIR) that can promote complex chromosomal rearrangements, including those associated with cancer and those leading to a number of neurological disorders in humans.

  16. Physical determinants of the self-replication of protein fibrils

    NASA Astrophysics Data System (ADS)

    Šarić, Anđela; Buell, Alexander K.; Meisl, Georg; Michaels, Thomas C. T.; Dobson, Christopher M.; Linse, Sara; Knowles, Tuomas P. J.; Frenkel, Daan

    2016-09-01

    The ability of biological molecules to replicate themselves is the foundation of life, requiring a complex cellular machinery. However, a range of aberrant processes involve the self-replication of pathological protein structures without any additional assistance. One example is the autocatalytic generation of pathological protein aggregates, including amyloid fibrils, involved in neurodegenerative disorders. Here, we use computer simulations to identify the necessary requirements for the self-replication of fibrillar assemblies of proteins. We establish that a key physical determinant for this process is the affinity of proteins for the surfaces of fibrils. We find that self-replication can take place only in a very narrow regime of inter-protein interactions, implying a high level of sensitivity to system parameters and experimental conditions. We then compare our theoretical predictions with kinetic and biosensor measurements of fibrils formed from the Aβ peptide associated with Alzheimer's disease. Our results show a quantitative connection between the kinetics of self-replication and the surface coverage of fibrils by monomeric proteins. These findings reveal the fundamental physical requirements for the formation of supra-molecular structures able to replicate themselves, and shed light on mechanisms in play in the proliferation of protein aggregates in nature.

  17. uvsF RFC1, the large subunit of replication factor C in Aspergillus nidulans, is essential for DNA replication, functions in UV repair and is upregulated in response to MMS-induced DNA damage.

    PubMed

    Kafer, Etta; Chae, Suhn-Kee

    2008-09-01

    uvsF201 was the first highly UV-sensitive repair-defective mutation isolated in Aspergillus nidulans. It showed epistasis only with postreplication repair mutations, but caused lethal interactions with many other repair-defective strains. Unexpectedly, closest homology of uvsF was found to the large subunit of human DNA replication factor RFC that is essential for DNA replication. Sequencing of the uvsF201 region identified changes at two close base pairs and the corresponding amino acids in the 5'-region of uvsF(RFC1). This viable mutant represents a novel and possibly important type. Additional sequencing of the uvsF region confirmed a mitochondrial ribosomal protein gene, mrpA(L16), closely adjacent, head-to-head with a 0.2kb joint promoter region. MMS-induced transcription of both the genes, but especially uvsF(RFC1), providing evidence for a function in DNA damage response.

  18. Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation

    SciTech Connect

    Ambrose, R.L.; Mackenzie, J.M., E-mail: jason.mackenzie@unimelb.edu.au

    2015-07-15

    The West Nile virus strain Kunjin virus (WNV{sub KUN}) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV{sub KUN} replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV{sub KUN} replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion andmore » membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein.« less

  19. A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase*

    PubMed Central

    Bruck, Irina; Dhingra, Nalini

    2017-01-01

    The assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2–7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2–7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ϵ, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. We show here that Dpb11 stimulates DDK phosphorylation of the minichromosome maintenance complex protein Mcm4 alone and also of the Mcm2–7 complex and the dsDNA-loaded Mcm2–7 complex. We further demonstrate that Dpb11 can directly recruit DDK to Mcm4. A DDK phosphomimetic mutant of Mcm4 bound Dpb11 with substantially higher affinity than wild-type Mcm4, suggesting a mechanism to recruit Dpb11 to DDK-phosphorylated Mcm2–7. Furthermore, dsDNA-loaded Mcm2–7 harboring the DDK phosphomimetic Mcm4 mutant bound GINS in the presence of Dpb11, suggesting a mechanism for how GINS is recruited to Mcm2–7. We isolated a mutant of Dpb11 that is specifically defective for binding to Mcm4. This mutant, when expressed in budding yeast, diminished cell growth and DNA replication, substantially decreased Mcm4 phosphorylation, and decreased association of GINS with replication origins. We conclude that Dpb11 functions with DDK and Mcm4 in a positive amplification mechanism to trigger the assembly of the replication fork helicase. PMID:28082681

  20. Strain superlattices in graphene

    NASA Astrophysics Data System (ADS)

    Zhang, Yingjie; Kim, Youngseok; Lyding, Joseph; Gilbert, Matthew; Mason, Nadya

    Superlattices have been widely explored to tailor the electronic properties of two-dimensional electron systems. Previous approaches to create superlattices have been limited to periodic potential modulations, either in the form of electrostatic gating or moiré heterostructures. Here we present a new strategy to generate superlattices in 2D materials. We deposit these 2D membranes on a periodic array of dielectric nanospheres, and achieve superlattices with periodic strain modulations. We studied the electronic and magneto-transport properties of strained graphene superlattices, and observed salient features of Dirac point cloning and Hofstadter's butterfly. Furthermore, we were able to tune the transport properties by changing the magnitude of strain in the graphene superlattice. This new degree of freedom provides a novel platform both for fundamental studies of 2D electron correlations and for prospective applications in 2D electronic devices. Y.Z. and N.M. acknowledge support from the National Science Foundation under Grant No. ENG-1434147.

  1. Strain gauge installation tool

    DOEpatents

    Conard, L.M.

    1998-06-16

    A tool and a method are disclosed for attaching a strain gauge to a test specimen by maintaining alignment of, and applying pressure to, the strain gauge during the bonding of the gauge to the specimen. The tool comprises rigid and compliant pads attached to a spring-loaded clamp. The pads are shaped to conform to the specimen surface to which the gauge is to be bonded. The shape of the pads permits the tool to align itself to the specimen and to maintain alignment of the gauge to the specimen during the bond curing process. A simplified method of attaching a strain gauge is provided by use of the tool. 6 figs.

  2. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

    PubMed

    Celma, Cristina C; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A; Beer, Martin; Roy, Polly

    2017-01-01

    Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce

  3. Differential virulence mechanisms of infectious hematopoietic necrosis virus in rainbow trout (Oncorhynchus mykiss) include host entry and virus replication kinetics

    USGS Publications Warehouse

    Penaranda, M.M.D.; Purcell, M.K.; Kurath, G.

    2009-01-01

    Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response. ?? 2009 SGM.

  4. A strain of Serratia marcescens pathogenic for larvae of Lymantria dispar: Infectivity and mechanisms of pathogenicity

    Treesearch

    J.D. Podgwaite; B.J. Cosenza

    1976-01-01

    The ED50 of a strain of Serratia marcescens for microinjected instar III and IV gypsy moth larvae was 7.5 and 14.5 viable cells, respectively. Percentage and rate of mortality were found to be highly variable among replicates of the same instar and between instars in free-feeding bioassays. Mortality in second instar larvae...

  5. Protozoa and human macrophages infection by Legionella pneumophila environmental strains belonging to different serogroups.

    PubMed

    Messi, Patrizia; Patrizia, Messi; Bargellini, Annalisa; Annalisa, Bargellini; Anacarso, Immacolata; Immacolata, Anacarso; Marchesi, Isabella; Isabella, Marchesi; de Niederhäusern, Simona; Bondi, Moreno; Moreno, Bondi

    2013-02-01

    Three Legionella pneumophila strains isolated from municipal hot tap water during a multicentric Italian survey and belonging to serogroups 1, 6, 9 and the reference strain Philadelphia-1 were studied to determine the intracellular replication capability and the cytopathogenicity in human monocyte cell line U937 and in an Acanthamoeba polyphaga strain. Our results show that both serogroups 1 and Philadelphia-1 were able to multiply into macrophages inducing cytopathogenicity, while serogroup 6 and ever more serogroup 9 were less efficient in leading to death of the infected macrophages. Both serogroups 1 and 6 displayed a quite good capability of intracellular replication in A. polyphaga, although serogroup 1 was less cytopathogenic than serogroup 6. Serogroup 9, like Philadelphia-1 strain, showed a reduced efficiency of infection and replication and a low cytopathogenicity towards the protozoan. Our study suggests that bacterial pathogenesis is linked to the difference in the virulence expression of L. pneumophila serogroups in both hosts, as demonstrated by the fact that only L. pneumophila serogroup 1 shows the contextual expression of the two virulence traits. Serogroup 6 proves to be a good candidate as pathogen since it shows a good capacity for intracellular replication in protozoan.

  6. A new system to measure and compare hepatitis C virus replication capacity using full-length, replication competent viruses.

    PubMed

    Lassmann, Britta; Arumugaswami, Vaithilingaraja; Chew, Kara W; Lewis, Martha J

    2013-12-01

    Measuring the in vitro replication capacity of viruses is an important tool for assessing the effects of selective pressure of immune responses and drug therapy. Measuring hepatitis C virus (HCV) replication capacity utilizing primarily sub-genomic reporter constructs is limited. To overcome some of these limitations a quantitative reverse transcriptase PCR (RT-qPCR) was designed to measure simultaneously the growth rate of 2 whole genome HCV variants under identical culture conditions. The assay demonstrates 100% specificity of detection of each variant and a linear detection range from 200 to 2×10(8) copies. The system was validated using a panel of HCV mutants, including the NS3 protease inhibitor drug resistance mutants R155K and T54A. The creation of a unique sequence tag results in highly sensitive and specific discrimination of parental JFH-FNX and modified clones using distinct probes in a RT-qPCR allowing for comparison of the effect of drug resistance or immune escape mutations on HCV replication. This system has advantages over existing methods both by permitting direct comparison of the replication capacity of fully replication-competent HCV mutants under identical culture conditions and by measuring effects on replication capacity due to mutations affecting all stages of the viral life cycle including entry and egress. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin.

    PubMed

    Pellegrino, Stefania; Michelena, Jone; Teloni, Federico; Imhof, Ralph; Altmeyer, Matthias

    2017-05-30

    The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1's target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Radio frequency strain monitor

    NASA Technical Reports Server (NTRS)

    Heyman, Joseph S. (Inventor); Rogowski, Robert S. (Inventor); Holben, Jr., Milford S. (Inventor)

    1989-01-01

    A radio frequency strain monitor includes a voltage controlled oscillator for generating an oscillating signal that is input into a propagation path. The propagation path is preferably bonded to the surface of a structure to be monitored and produces a propagated signal. A phase difference between the oscillating and propagated signals is detected and maintained at a substantially constant value which is preferably a multiple of 90.degree. by changing the frequency of the oscillating signal. Any change in frequency of the oscillating signal provides an indication of strain in the structure to which the propagation path is bonded.

  9. Generation of influenza A viruses as live but replication-incompetent virus vaccines.

    PubMed

    Si, Longlong; Xu, Huan; Zhou, Xueying; Zhang, Ziwei; Tian, Zhenyu; Wang, Yan; Wu, Yiming; Zhang, Bo; Niu, Zhenlan; Zhang, Chuanling; Fu, Ge; Xiao, Sulong; Xia, Qing; Zhang, Lihe; Zhou, Demin

    2016-12-02

    The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell-mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus. Copyright © 2016, American Association for the Advancement of Science.

  10. Transformation and Tumorigenicity Testing of Simian Cell Lines and Evaluation of Poliovirus Replication

    PubMed Central

    Dotti, Silvia; Lombardo