Science.gov

Sample records for revisionist replicon model

  1. Model-driven engineering of gene expression from RNA replicons.

    PubMed

    Beal, Jacob; Wagner, Tyler E; Kitada, Tasuku; Azizgolshani, Odisse; Parker, Jordan Moberg; Densmore, Douglas; Weiss, Ron

    2015-01-16

    RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. Here, we aim to enable the engineering of multigene expression from more than one species of replicon by creating a computational model based on our experimental observations of the expression dynamics in single- and multireplicon systems. To this end, we studied fluorescent protein expression in baby hamster kidney (BHK-21) cells using a replicon derived from Sindbis virus (SINV). We characterized expression dynamics for this platform based on the dose-response of a single species of replicon over 50 h and on a titration of two cotransfected replicons expressing different fluorescent proteins. From this data, we derive a quantitative model of multireplicon expression and validate it by designing a variety of three-replicon systems, with profiles that match desired expression levels. We achieved a mean error of 1.7-fold on a 1000-fold range, thus demonstrating how our model can be applied to precisely control expression levels of each Sindbis replicon species in a system. PMID:24877739

  2. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti

    PubMed Central

    diCenzo, George C.; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M.; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  3. Metabolic modelling reveals the specialization of secondary replicons for niche adaptation in Sinorhizobium meliloti.

    PubMed

    diCenzo, George C; Checcucci, Alice; Bazzicalupo, Marco; Mengoni, Alessio; Viti, Carlo; Dziewit, Lukasz; Finan, Turlough M; Galardini, Marco; Fondi, Marco

    2016-01-01

    The genome of about 10% of bacterial species is divided among two or more large chromosome-sized replicons. The contribution of each replicon to the microbial life cycle (for example, environmental adaptations and/or niche switching) remains unclear. Here we report a genome-scale metabolic model of the legume symbiont Sinorhizobium meliloti that is integrated with carbon utilization data for 1,500 genes with 192 carbon substrates. Growth of S. meliloti is modelled in three ecological niches (bulk soil, rhizosphere and nodule) with a focus on the role of each of its three replicons. We observe clear metabolic differences during growth in the tested ecological niches and an overall reprogramming following niche switching. In silico examination of the inferred fitness of gene deletion mutants suggests that secondary replicons evolved to fulfil a specialized function, particularly host-associated niche adaptation. Thus, genes on secondary replicons might potentially be manipulated to promote or suppress host interactions for biotechnological purposes. PMID:27447951

  4. Brecht's Galileo: A revisionist view

    NASA Astrophysics Data System (ADS)

    Schroeer, Dietrich

    1980-02-01

    Galileo is often claimed by scientists to be the first modern physicist, and because of his conflict with the Catholic Church is seen as a heroic figure fighting for the independence of pure science. Brecht, in his play Galileo, has presented a revisionist view of Galileo. This view developed over several versions of the play, and finally used him as a symbol for all scientists who reject social responsibility for their work. Is this revisionist view of Galileo any more distorted than the portrayal of him as the patron saint of modern science?

  5. Teaching the Revisionist Interpretation of the Cold War.

    ERIC Educational Resources Information Center

    O'Reilly, Kevin

    1986-01-01

    Provides a lesson plan, complete with readings and teaching procedures, for teaching students about the revisionist interpretations of Cold War history. Students are challenged to think critically about the differences in the traditional and revisionist interpretations. (JDH)

  6. The Cold War and Revisionist Historiography

    ERIC Educational Resources Information Center

    Hogeboom, Willard L.

    1970-01-01

    An important historiographic controversy exists between those who blame the Soviets for the origins of the Cold War (orthodox) and those who blame the U. S. (revisionist--New Left). While the latter criticize the orthodox historians' methods, they are often guilty of semilar biases and simplifications. (JB)

  7. A Revisionist View of Intervention.

    ERIC Educational Resources Information Center

    Veal, Mary Lou; And Others

    A study of professional development intervention programs in which schools choose their own issues and action plans is presented in this report. "Intervention" refers to university introduction of new professional development models into the school. A 3-year program involving five schools in collaboration with university staff members and…

  8. Chromosomal replicons of higher plants

    SciTech Connect

    Van't Hof, J.

    1987-03-16

    This brief discussion of replicons of higher plants offers a glimpse into the properties of chromosomal DNA replication. It gives evidence that the S phase of unrelated plant species is comprised of temporally ordered replicon families that increase in number with genome size. This orderly process, which assures a normal inheritance of genetic material to recipient daughter cells, is maintained at the level of replicon clusters by two mutually exclusive mechanisms, one involving the rate at which single replicons replicate their allotment of DNA, and another by means of the tempo-pause. The same two mechanisms are used by cells to alter the pattern of chromosomal DNA replication just prior to and during normal development. Both mechanisms are genetically determined and produce genetic effects when disturbed of disrupted by additional non-conforming DNAs. Further insight into how these two mechanisms operate requires more molecular information about the nature of replicons and the factors that govern when a replicon family replicates. Plant material is a rich and ideal source for this information just awaiting exploitation. 63 refs.

  9. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons

    PubMed Central

    Chagin, V. O.; Casas-Delucchi, C. S.; Reinhart, M.; Schermelleh, L.; Markaki, Y.; Maiser, A.; Bolius, J. J.; Bensimon, A.; Fillies, M.; Domaing, P.; Rozanov, Y. M.; Leonhardt, H.; Cardoso, M. C.

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  10. 4D Visualization of replication foci in mammalian cells corresponding to individual replicons.

    PubMed

    Chagin, V O; Casas-Delucchi, C S; Reinhart, M; Schermelleh, L; Markaki, Y; Maiser, A; Bolius, J J; Bensimon, A; Fillies, M; Domaing, P; Rozanov, Y M; Leonhardt, H; Cardoso, M C

    2016-01-01

    Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons. PMID:27052570

  11. DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection.

    PubMed

    Dalmia, Neha; Klimstra, William B; Mason, Carol; Ramsay, Alistair J

    2015-01-01

    There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters. PMID:26317509

  12. A personal reflection on the replicon theory: from R1 plasmid to replication timing regulation in human cells.

    PubMed

    Masai, Hisao

    2013-11-29

    Fifty years after the Replicon Theory was originally presented, detailed mechanistic insight into prokaryotic replicons has been obtained and rapid progress is being made to elucidate the more complex regulatory mechanisms of replicon regulation in eukaryotic cells. Here, I present my personal perspectives on how studies of model replicons have contributed to our understanding of the basic mechanisms of DNA replication as well as the evolution of replication regulation in human cells. I will also discuss how replication regulation contributes to the stable maintenance of the genome and how disruption of replication regulation leads to human diseases. PMID:23579064

  13. Hepatitis C Virus RNA Elimination and Development of Resistance in Replicon Cells Treated with BMS-790052

    PubMed Central

    Wang, Chunfu; Huang, Haichang; Valera, Lourdes; Sun, Jin-Hua; O'Boyle, Donald R.; Nower, Peter T.; Jia, Lingling; Qiu, Dike; Huang, Xin; Altaf, Aneela; Gao, Min

    2012-01-01

    BMS-790052, a first-in-class hepatitis C virus (HCV) replication complex inhibitor, targeting nonstructural protein 5A (NS5A), displays picomolar to nanomolar potency against genotypes 1 to 5. This exceptional potency translated into robust anti-HCV activity in clinical studies with HCV genotype 1-infected subjects. To date, all BMS-790052-associated resistance mutations have mapped to the N-terminal region of NS5A. To further characterize the antiviral activity of BMS-790052, HCV replicon elimination and colony formation assays were performed. Replicon was cleared from genotype 1a and 1b replicon cells in a time- and dose-dependent manner. Elimination of the genotype 1a replicon required longer treatment durations and higher concentrations of BMS-790052 than those for the genotype1b replicon. Single amino acid substitutions that conferred relatively low levels of resistance were observed at early time points and at low doses. Higher doses and longer treatment durations yielded mutations that conferred greater levels of resistance, including linked amino acid substitutions. Replicon cells that survived inhibitor treatment remained fully sensitivity to pegylated alpha interferon (pegIFN-α) and other HCV inhibitors. Moreover, genotype 1a replicon elimination was markedly enhanced when pegIFN-α and BMS-790052 were combined. Resistant variants observed in this study were very similar to those observed in a multiple ascending dose (MAD) monotherapy trial of BMS-790052, validating replicon elimination studies as a model to predict clinical resistance. Insights gained from the in vitro anti-HCV activity and resistance profiles of BMS-790052 will be used to help guide the clinical development of this novel HCV inhibitor. PMID:22214777

  14. Revisionist History in the Library: To Facilitate Access or Not to Facilitate Access?

    ERIC Educational Resources Information Center

    Katz, Jeffrey

    1991-01-01

    Discussion of the acquisition and dissemination of revisionist history materials by public libraries focuses on revisionism related to the Jewish Holocaust. Intellectual freedom is addressed, the role of the public library is considered, handling holocaust-denial literature is described, and an example from the Edmonton Public Library is…

  15. The Assessor Assessed: A "Revisionist" Looks at a Critique of the Sandia Report.

    ERIC Educational Resources Information Center

    Bracey, Gerald W.

    1995-01-01

    In critiquing the Sandia Report, Lawrence Stedman faulted it and other revisionist reports for emphasizing trend data rather than quality and not acknowledging educational requirements within a democratic society. The article addresses the issues, presenting evidence from additional studies to argue that none of Stedman's general contentions…

  16. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  17. Visualization of DNA replicons by image cytometry

    SciTech Connect

    Gratzner, H.G. )

    1993-01-01

    Replication of DNA in eukaryotic organisms proceeds bidirectionally along the double helix in replicon substructures. The process can be visualized by autoradiography of spreads of genomic DNA from lysed, whole cells which have been incubated with radioactive DNA precursors. The objective of the present study was to develop techniques to measure DNA strand initiation and elongation using immunofluorescence and image cytometry. Peripheral blood lymphocytes were cultured for 3 days with PHA and then pulsed for 5 or 15 minutes with iododeoxyuridine. Chromatin spreads were then produced on microscope slides by lysing with detergent and slides were immunofluorescently stained by an indirect anti-BrdU technique. Individual replicons of mammalian cells were visualized by immunofluorescence at high resolution and digital images were analyzed to determine the rates of elongation as well as initiation parameters. Elongation rates by the method were approximately 1 [mu]M/min. The methods are applicable to visualization and quantitation of the effects of radiation or other agents on DNA damage or repair.

  18. Construction and characterization of poliovirus subgenomic replicons.

    PubMed

    Kaplan, G; Racaniello, V R

    1988-05-01

    Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid

  19. Radiation effects on DNA synthesis in a defined chromosomal replicon

    SciTech Connect

    Larner, J.M.; Lee, H.; Hamlin, J.L. )

    1994-03-01

    It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G[sub 1] period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. The authors are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistance CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. They first show that the CHOC-400 cell line retains the classical acute-phase response but does not display the late G[sub 1] arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, they then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G[sub 1]/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation. 42 refs., 9 figs.

  20. Revisionist or simply wrong? A response to Armstrong's article on chronic illness

    PubMed Central

    Gilleard, Chris; Higgs, Paul

    2014-01-01

    This article is a response to David Armstrong's recent, revisionist account of the epidemiological transition which he claims replaced earlier discourses of ageing with new discourses of chronic disease. We argue (i) that he misrepresents a key element in Omran's account of the epidemiological transition, namely the decline in infant, child and maternal mortality; (ii) that he fails to acknowledge debates going back centuries in Western medicine over the distinctions between natural and accidental death and between endogenous and extrinsic causes of ageing and (iii) that he misrepresents the growth of medical interest in the everyday illnesses of old age over the course of the 20th century as a discourse of suppression rather than a process of inclusion. While we would acknowledge that the chronic illnesses of today are different from those of the past, this amounts to something more than the changing semantics of senility. PMID:25155775

  1. Lack of interference with immunogenicity of a chimeric alphavirus replicon particle-based influenza vaccine by preexisting antivector immunity.

    PubMed

    Uematsu, Yasushi; Vajdy, Michael; Lian, Ying; Perri, Silvia; Greer, Catherine E; Legg, Harold S; Galli, Grazia; Saletti, Giulietta; Otten, Gillis R; Rappuoli, Rino; Barnett, Susan W; Polo, John M

    2012-07-01

    Antivector immunity has been recognized as a potential caveat of using virus-based vaccines. In the present study, an alphavirus-based replicon particle vaccine platform, which has demonstrated robust immunogenicity in animal models, was tested for effects of antivector immunity on immunogenicity against hemagglutinin of influenza virus as a target antigen and efficacy for protection against lethal challenge with the virus. Chimeric alphavirus-based replicon particles, comprising Venezuelan equine encephalitis virus nonstructural and Sindbis virus structural components, induced efficient protective antibody responses, which were not adversely influenced after multiple immunizations with the same vector expressing various antigens. PMID:22623651

  2. Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates.

    PubMed

    Johnson, Timothy J; Wannemuehler, Yvonne M; Johnson, Sara J; Logue, Catherine M; White, David G; Doetkott, Curt; Nolan, Lisa K

    2007-03-01

    Despite the critical role of plasmids in horizontal gene transfer, few studies have characterized plasmid relatedness among different bacterial populations. Recently, a multiplex PCR replicon typing protocol was developed for classification of plasmids occurring in members of the Enterobacteriaceae. Here, a simplified version of this replicon typing procedure which requires only three multiplex panels to identify 18 plasmid replicons is described. This method was used to screen 1,015 Escherichia coli isolates of avian, human, and poultry meat origin for plasmid replicon types. Additionally, the isolates were assessed for their content of several colicin-associated genes. Overall, a high degree of plasmid variability was observed, with 221 different profiles occurring among the 1,015 isolates examined. IncFIB plasmids were the most common type identified, regardless of the source type of E. coli. IncFIB plasmids occurred significantly more often in avian pathogenic E. coli (APEC) and retail poultry E. coli (RPEC) than in uropathogenic E. coli (UPEC) and avian and human fecal commensal E. coli isolates (AFEC and HFEC, respectively). APEC and RPEC were also significantly more likely than UPEC, HFEC, and AFEC to possess the colicin-associated genes cvaC, cbi, and/or cma in conjunction with one or more plasmid replicons. The results suggest that E. coli isolates contaminating retail poultry are notably similar to APEC with regard to plasmid profiles, with both generally containing multiple plasmid replicon types in conjunction with colicin-related genes. In contrast, UPEC and human and avian commensal E. coli isolates generally lack the plasmid replicons and colicin-related genes seen in APEC and RPEC, suggesting limited dissemination of such plasmids among these bacterial populations. PMID:17277222

  3. Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

    PubMed Central

    Cheng, H P; Lessie, T G

    1994-01-01

    Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates. Images PMID:7517389

  4. Use of Recombinant Virus Replicon Particles for Vaccination against Mycobacterium ulcerans Disease

    PubMed Central

    Bolz, Miriam; Kerber, Sarah; Zimmer, Gert; Pluschke, Gerd

    2015-01-01

    Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans. PMID:26275222

  5. MULTIPLE REPLICONS CONSTITUTING THE GENOME OF PSEUDOMONAS CEPACIA 17616

    EPA Science Inventory

    Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction n enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 170-kb cryptic plas...

  6. Structural organization of replicon domains during DNA synthetic phase in the mammalian nucleus

    SciTech Connect

    Nakamura, H.; Morita, T.; Sato, C.

    1986-01-01

    In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with (/sup 3/H)thymidine at the limited resolution level of autoradiography. Bu immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.

  7. Morphine Enhances Hepatitis C Virus (HCV) Replicon Expression

    PubMed Central

    Li, Yuan; Zhang, Ting; Douglas, Steven D.; Lai, Jian-Ping; Xiao, Wei-Dong; Pleasure, David E.; Ho, Wen-Zhe

    2003-01-01

    Little information is available regarding whether substance abuse enhances hepatitis C virus (HCV) replication and promotes HCV disease progression. We investigated whether morphine alters HCV mRNA expression in HCV replicon-containing liver cells. Morphine significantly increased HCV mRNA expression, an effect which could be abolished by either of the opioid receptor antagonists, naltrexone or β-funaltrexamine. Investigation of the mechanism responsible for this enhancement of HCV replicon expression demonstrated that morphine activated NF-κB promoter and that caffeic acid phenethyl ester, a specific inhibitor of the activation of NF-κB, blocked morphine-activated HCV RNA expression. In addition, morphine compromised the anti-HCV effect of interferon alpha (IFN-α). Our in vitro data indicate that morphine may play an important role as a positive regulator of HCV replication in human hepatic cells and may compromise IFN-α therapy. PMID:12937158

  8. The replicon of pSW800 from Pantoea stewartii.

    PubMed

    Wu, C Y; Fu, J F; Liu, S T

    2001-10-01

    A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized. This replicon contains two genes--repA and repB, which encode a 36.5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively. These two genes overlap by 8 bases with repB situated upstream. The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB. The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem-loop structure, inhibiting the translation of repA. Furthermore, translation of repB is likely to disrupt the stem-loop structure, which is one of the criteria allowing the translation of repA to begin. A mutagenesis study revealed that a sequence (5'-GCACGGG-3') located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA. Additionally, this region and the stem-loop structure containing the RBS of repA may form an RNA pseudoknot. Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, IncIalpha and IncL/M groups also regulates pSW800 replication. PMID:11577155

  9. A PCR-based protocol for generating West Nile virus replicons.

    PubMed

    Maeda, Junko; Takagi, Hirotaka; Hashimoto, Shingo; Kurane, Ichiro; Maeda, Akihiko

    2008-03-01

    A new protocol for the generation of West Nile virus (WNV) replicons was developed. Fragmented cDNAs that covered the entire WNV RNA sequence, except the sequence corresponding to nucleotides 190-2379, were amplified separately by polymerase chain reactions (PCRs) using primer set franking with overlapping sequences of 40-50 bp at the 5'- and the 3'-ends of each fragment. All amplified fragments were mixed together and annealed to each other at the overlapping sequences. The annealed-DNA fragments were elongated by DNA polymerase and amplified by short-cycle PCRs to generate full-sized WNV replicon cDNAs. The WNV replicons were transcribed in vitro using the replicon cDNAs as templates. When the in vitro-transcribed replicon was introduced into mammalian cells, the viral envelope protein and viral positive- and negative-strand RNAs were detected in the replicon-transfected cells. It is noteworthy that the synthesis of the replicon cDNAs and the replicons took just 1 week, and that the use of a high-fidelity DNA polymerase afforded stability to the sequence of the synthetic replicon. PMID:18242719

  10. EVIDENCE FOR THE CHROMOSOMAL REPLICONS AS UNITS OF SISTER CHROMATID EXCHANGES

    EPA Science Inventory

    Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the FCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cycloph...

  11. Mucosal and systemic adjuvant activity of alphavirus replicon particles

    NASA Astrophysics Data System (ADS)

    Thompson, Joseph M.; Whitmore, Alan C.; Konopka, Jennifer L.; Collier, Martha L.; Richmond, Erin M. B.; Davis, Nancy L.; Staats, Herman F.; Johnston, Robert E.

    2006-03-01

    Vaccination represents the most effective control measure in the fight against infectious diseases. Local mucosal immune responses are critical for protection from, and resolution of, infection by numerous mucosal pathogens. Antigen processing across mucosal surfaces is the natural route by which mucosal immunity is generated, as peripheral antigen delivery typically fails to induce mucosal immune responses. However, we demonstrate in this article that mucosal immune responses are evident at multiple mucosal surfaces after parenteral delivery of Venezuelan equine encephalitis virus replicon particles (VRP). Moreover, coinoculation of null VRP (not expressing any transgene) with inactivated influenza virions, or ovalbumin, resulted in a significant increase in antigen-specific systemic IgG and fecal IgA antibodies, compared with antigen alone. Pretreatment of VRP with UV light largely abrogated this adjuvant effect. These results demonstrate that alphavirus replicon particles possess intrinsic systemic and mucosal adjuvant activity and suggest that VRP RNA replication is the trigger for this activity. We feel that these observations and the continued experimentation they stimulate will ultimately define the specific components of an alternative pathway for the induction of mucosal immunity, and if the activity is evident in humans, will enable new possibilities for safe and inexpensive subunit and inactivated vaccines. vaccine vector | Venezuelan equine encephalitis virus | viral immunology | RNA virus

  12. 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. PMID:26800776

  13. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    PubMed

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. PMID:25102364

  14. Antimicrobial Susceptibility and Plasmid Replicon Typing of Salmonella enterica serovar Kentucky isolates recovered from Broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella Kentucky has become the predominate serotype recovered from broiler slaughter in the United States and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serotype. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 ...

  15. The organization of repeated nucleotide sequences in the replicons of mammalian DNA.

    PubMed Central

    Mattern, M R; Painter, R B

    1977-01-01

    Chinese hamster ovary cells were irradiated with 100-5,000 rads of X-rays and inhibition of the initiation of replicons after irradiation was demonstrated by analyzing nascent DNA sedimented in alkaline sucrose gradients. The renaturation kinetics of DNA synthesized during 60 min of incubation after irradiation was compared with that of DNA synthesized during the 60 min after sham irradiation and with that of parental DNA. Nascent DNA from cells whose replicon initiation was inhibited renatured faster than nascent DNA from control cells in the COt range of repeated nucleotide sequences, suggesting that regions of the replicon not close to origins are enriched in repeated sequences and that regions close to origins are enriched in unique sequences. A class of repeated nucleotide sequences may be involved in the regulation of replicon initiation. PMID:880330

  16. Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

    PubMed Central

    Kaufmann, W K; Boyer, J C; Estabrooks, L L; Wilson, S J

    1991-01-01

    Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes. PMID:1646393

  17. Proteome Analysis of Liver Cells Expressing a Full- Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

    SciTech Connect

    Jacobs, Jon M.; Diamond, Deborah L.; Chan, Eric Y.; Gritsenko, Marina A.; Qian, Weijun; Stastna, Miroslava; Baas, Tracey; Camp, David G.; Carithers, Jr., Robert L.; Smith, Richard D.; Katze, Michael G.

    2005-06-01

    The development of a reproducible model system for the study of Hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full length HCV replicon. We detected > 4,400 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry (MS). The set of Huh-7.5 proteins confidently identified is, to our knowledge, the most comprehensive yet reported for a human cell line. Consistent with the literature, a comparison of Huh-7.5 cells (+) and (-) the HCV replicon identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where > 1,500 proteins were detected from 2 {micro}g protein lysate using the Huh-7.5 protein database and the accurate mass and time (AMT) tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.

  18. Antimicrobial Susceptibility and Plasmid Replicon Typing of Salmonella enterica Serovar Kentucky Isolates Recovered from Broilers.

    PubMed

    Ladely, Scott R; Meinersmann, Richard J; Ball, Takiyah A; Fedorka-Cray, Paula J

    2016-06-01

    Salmonella Kentucky has become the predominant serovar recovered from broilers slaughtered in the United States, and the prevalence of antimicrobial resistance (AMR) has increased dramatically in this serovar. Relationships between AMR, genotype, and plasmid replicon types were characterized for 600 Salmonella Kentucky isolates recovered from chicken carcasses from 2004 to 2013. Pulsed-field gel electrophoresis cluster analysis revealed 112 unique types sharing 79% similarity. Over half of the isolates studies were assigned to two large clusters (unique restriction patterns) consisting of 190 (A) and 151 (B) isolates. The remaining (n = 259) more diverse isolates (110 unique patterns) shall be designated cluster C for discussion. Clusters A had significantly more (p < 0.05) isolates resistant to streptomycin (68.4%) and tetracycline (91.6%) compared to cluster C (50.6% and 40.9% to streptomycin and tetracycline, respectively) or cluster B, which had the least (p < 0.05) resistance (11.9% and 13.2% to streptomycin and tetracycline, respectively). In addition, there was segregation of plasmid replicon types among clusters. Cluster A had significantly more (p < 0.05) replicon type FIB (90.5%) compared to cluster C (37.1%), which had significantly more compared to cluster B (10.6%). Cluster B had significantly more (p < 0.05) replicon type I1 (87.4%) compared to cluster C (68.7%), which had significantly more (p < 0.05) compared to cluster A (32.6%). Cluster C harbored significantly more (p < 0.05) HI2 replicon type (18.1%) compared to clonal clusters A (1.6%) or B (1.3%). The prevalence of plasmid replicon type A/C did not differ among clusters (A, 0.5%; B, 2.0%; C, 0.4%). Both streptomycin and tetracycline resistance were significantly linked (p < 0.05) to plasmid replicon type FIB. In addition, replicon type HI2 was also significantly linked (p < 0.05) to streptomycin resistance. We conclude that the dramatic increase in

  19. Functional analysis of the plasmid pM4 replicon from Lactobacillus plantarum M4: determination of the minimal replicon and functionality identification of the putative sso.

    PubMed

    Yin, Sheng; Hao, Yanling; Zhai, Zhengyuan; Zhang, Wei; Zhou, Hui; Wang, Guohong; Shi, Xianli; Luo, Yunbo

    2009-11-01

    In order to determine the minimal replicon and the single strand origin (sso) of the plasmid pM4, different fragments of pM4 were amplified by polymerase chain reaction (PCR) and cloned into pBEm, a replication probe vector for Lactobacillus. The deletion analysis results showed that the minimal replicon of pM4 could be determined within a 1280bp fragment consisting of double strand origin (dso) and rep gene encoding replication protein. Based on plasmid segregation stability assay and its ability to convert single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA) by Southern hybridization, an sso of replication was located at nucleotides -118-92 in the plasmid pM4, about 300bp upstream of dso. In addition, the host range assay indicated that plasmid pM4 could replicate in L. casei 05-21, L. rhamnosus AS 1.2466(T) and L. plantarum 05-19 of all the tested Lactobacillus strains. Analysis of the pM4 replicon will allow its use in constructing a food-grade vector for application in food industry. PMID:19651154

  20. Hepatitis C Virus Subgenomic Replicons Induce Endoplasmic Reticulum Stress Activating an Intracellular Signaling Pathway

    PubMed Central

    Tardif, Keith D.; Mori, Kazutoshi; Siddiqui, Aleem

    2002-01-01

    Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the α subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5′ noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis. PMID:12097557

  1. Cell-Free Replication of the Hepatitis C Virus Subgenomic Replicon

    PubMed Central

    Ali, Naushad; Tardif, Keith D.; Siddiqui, Aleem

    2002-01-01

    The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5′ noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3′ NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [α32P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents. PMID:12414942

  2. Do sister forks of bidirectionally growing replicons proceed at unequal rates

    SciTech Connect

    Dubey, D.D.; Raman, R.

    1987-02-01

    DNA fibre autoradiography in different tissues of the rodents Bandicota bengalensis and Nesokia indica reveals a high frequency of such bidirectionally replicating replicons whose sister hot tracks are of unequal size. These results suggest intrarepliconic difference in the rates of fork migration in the two directions.

  3. Replicon typing of plasmids encoding resistance to newer beta-lactams.

    PubMed

    Carattoli, Alessandra; Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M; Rossolini, Gian Maria

    2006-07-01

    Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  4. Replicon Typing of Plasmids Encoding Resistance to Newer β-Lactams

    PubMed Central

    Miriagou, Vivi; Bertini, Alessia; Loli, Alexandra; Colinon, Celine; Villa, Laura; Whichard, Jean M.; Rossolini, Gian Maria

    2006-01-01

    Polymerase chain reaction–based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems. PMID:16836838

  5. [IncP-7 plasmids' classification based on structural diversity of their basic replicons].

    PubMed

    Volkova, O V; Panov, A V; Kosheleva, I A; Boronin, A M

    2013-01-01

    The structural diversity of basic replicons and repB gene of the IncP-7 plasmids' collection was firstly assessed on the basis of PCR, restriction analysis and partial sequencing. It has been revealed that DNA fragment containing gene for UvrD-like helicase RepB is a part of all known P-7 replicons, but often serves as hot place for diverse IS-elements invasion. The first system of P-7 plasmids' classification has been worked out on the basis of determined repA-oriV-par WABC nucleotide divergency. Most degradation plasmids established to be belonging to large beta-subgroup, streptomycin resistance plasmid Rms148 (IncP-7 archetype)--to alpha-subgroup, carbazole degradation plasmid pCAR1 and NAH/SAL-plasmids from pY-line (Yamal oil deposits)--to gamma-subgroup and CAP-plasmid pBS270 with potentially reduced P-7 replicon--to delta-subgroup. It has been observed that the type of IncP-7 basic replicon molecular organization does not correlate with fixed phenotypic character in most cases, that is plasmids encoding different phenotypic markers could be members of the same P-7 subgroup. PMID:23808156

  6. Replicon typing of virulence plasmids of enterotoxigenic Escherichia coli isolates from cattle.

    PubMed Central

    Mainil, J G; Bex, F; Dreze, P; Kaeckenbeeck, A; Couturier, M

    1992-01-01

    Plasmid DNA hybridization with probes for virulence factors used for basic replicons of plasmids was used to identify the virulence plasmids of a collection of enterotoxigenic Escherichia coli isolates from cattle. The virulence probes were derived from the genes coding for the heat-stable enterotoxin STaP and for the F5 (K99) and F41 fimbrial adhesins. The replicon probes were derived from 16 different basic replicons of plasmids (probes repFIA, repFIB, repFIC, repFIIA, repI1, repHI1, repHI2, repL/M, repN, repP, repQ, repT, repU, repW, repX, and repY). The virulence genes coding for the STaP enterotoxin and for the F5 adhesin were located on a single plasmid band in each isolate. The sizes of most of these virulence plasmids were from 65 to 95 MDa. The F41 probe failed to hybridize with any plasmid band. The virulence plasmids had multireplicon types typical of plasmids of the IncF groups. The most common basic replicon association was the triple RepFIA-RepFIB-RepFIC family association. Images PMID:1639505

  7. Pea (Pisum sativum) cells arrested in G2 have nascent DNA with breaks between replicons and replication clusters

    SciTech Connect

    Van't Hof, J.

    1980-01-01

    DNA fiber autoradiography and alkaline sucrose sedimentation of DNA of cultured pea-root cells (Pisum sativum) arrested in G2 by carbohydrate starvation demonstrated that nascent DNA molecules of replicon (16 to 27 x 10/sup 6/D) and apparent cluster (approx. 330 x 10/sup 6/D) size were not joined. That the arrested cells were in G2 was confirmed by single-cell autoradiography and cytophotometry. In pea there are about 18 replicons per average cluster, 4.2 x 10/sup 3/ clusters, and 7.7 x 10/sup 4/ replicons per genome.

  8. 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

  9. Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315.

    PubMed

    Du, Wen-Li; Dubarry, Nelly; Passot, Fanny M; Kamgoué, Alain; Murray, Heath; Lane, David; Pasta, Franck

    2016-07-01

    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

  10. Characterization and replication mode determination of the minimal replicon of Tetragenococcus halophila ATCC33315 plasmid pUCL287.

    PubMed

    Benachour, A; Frère, J; Boutibonnes, P; Auffray, Y

    1995-01-01

    pUCL287 is a cryptic plasmid of Tetragenococcus halophila (formerly Pediococcus halophilus) ATCC33315 of relatively small size (8.7 kb). Its minimal replicon was located on a 1235 bp MamI-EcoRI fragment. This minimal replicon contains a non-translated region, followed by a gene encoding a putative 311 amino acid protein. Deletion experiments showed that the non-translated region corresponds to the replication origin. Determination of the replication mode was carried out in Enterococcus faecalis JH2-2 harboring pUCL287 minimal replicon. The replicating intermediates detected revealed that pUCL287 minimal replicon follows a bidirectional theta replicating mode. PMID:8824766

  11. Dengue Virus Reporter Replicon is a Valuable Tool for Antiviral Drug Discovery and Analysis of Virus Replication Mechanisms.

    PubMed

    Kato, Fumihiro; Hishiki, Takayuki

    2016-01-01

    Dengue, the most prevalent arthropod-borne viral disease, is caused by the dengue virus (DENV), a member of the Flaviviridae family, and is a considerable public health threat in over 100 countries, with 2.5 billion people living in high-risk areas. However, no specific antiviral drug or licensed vaccine currently targets DENV infection. The replicon system has all the factors needed for viral replication in cells. Since the development of replicon systems, transient and stable reporter replicons, as well as reporter viruses, have been used in the study of various virological aspects of DENV and in the identification of DENV inhibitors. In this review, we summarize the DENV reporter replicon system and its applications in high-throughput screening (HTS) for identification of anti-DENV inhibitors. We also describe the use of this system in elucidation of the mechanisms of virus replication and viral dynamics in vivo and in vitro. PMID:27164125

  12. Dengue Virus Reporter Replicon is a Valuable Tool for Antiviral Drug Discovery and Analysis of Virus Replication Mechanisms

    PubMed Central

    Kato, Fumihiro; Hishiki, Takayuki

    2016-01-01

    Dengue, the most prevalent arthropod-borne viral disease, is caused by the dengue virus (DENV), a member of the Flaviviridae family, and is a considerable public health threat in over 100 countries, with 2.5 billion people living in high-risk areas. However, no specific antiviral drug or licensed vaccine currently targets DENV infection. The replicon system has all the factors needed for viral replication in cells. Since the development of replicon systems, transient and stable reporter replicons, as well as reporter viruses, have been used in the study of various virological aspects of DENV and in the identification of DENV inhibitors. In this review, we summarize the DENV reporter replicon system and its applications in high-throughput screening (HTS) for identification of anti-DENV inhibitors. We also describe the use of this system in elucidation of the mechanisms of virus replication and viral dynamics in vivo and in vitro. PMID:27164125

  13. Natural selection of adaptive mutations in non-structural genes increases trans-encapsidation of hepatitis C virus replicons lacking envelope protein genes.

    PubMed

    Fournier, Carole; Helle, François; Descamps, Véronique; Morel, Virginie; François, Catherine; Dedeurwaerder, Sarah; Wychowski, Czeslaw; Duverlie, Gilles; Castelain, Sandrine

    2013-05-01

    A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a 100-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located at the NS5A-B cleavage site did not affect viral replication, but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines. PMID:23288424

  14. Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

    PubMed Central

    Wose Kinge, Constance N.; Espiritu, Christine; Prabdial-Sing, Nishi; Sithebe, Nomathamsaqa Patricia

    2014-01-01

    Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds. PMID:24982066

  15. Three Replicons of Rhizobium sp. Strain NGR234 Harbor Symbiotic Gene Sequences

    PubMed Central

    Flores, Margarita; Mavingui, Patrick; Girard, Lourdes; Perret, Xavier; Broughton, William J.; Martínez-Romero, Esperanza; Dávila, Guillermo; Palacios, Rafael

    1998-01-01

    Rhizobium sp. strain NGR234 contains three replicons: the symbiotic plasmid or pNGR234a, a megaplasmid (pNGR234b), and the chromosome. Symbiotic gene sequences not present in pNGR234a were analyzed by hybridization. DNA sequences homologous to the genes fixLJKNOPQGHIS were found on the chromosome, while sequences homologous to nodPQ and exoBDFLK were found on pNGR234b. PMID:9811668

  16. chr genes from adaptive replicons are responsible for chromate resistance by Burkholderia xenovorans LB400.

    PubMed

    Reyes-Gallegos, Rosa I; Ramírez-Díaz, Martha I; Cervantes, Carlos

    2016-03-01

    The chromate ion transporter (CHR) superfamily includes proteins that confer chromate resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain LB400 encodes six CHR homologues in its multireplicon genome and has been reported as highly chromate-resistant. The objective of this work was to analyze the involvement of chr redundant genes in chromate resistance by LB400. It was found that B. xenovorans plant rhizosphere strains lacking the megaplasmid are chromate-sensitive, suggesting that the chr gene present in this replicon is responsible for the chromate-resistance phenotype of the LB400 strain. Transformation of a chromate-sensitive B. xenovorans strain with each of the six cloned LB400 chr genes showed that genes from 'adaptive replicons' (chrA1b and chr1NCb from chromosome 2 and chrA2 from the megaplasmid) conferred higher chromate resistance levels than chr genes from 'central' chromosome 1 (chrA1a, chrA6, and chr1NCa). An LB400 insertion mutant affected in the chrA2 gene displayed a chromate-sensitive phenotype, which was fully reverted by transferring the chrA2 wild-type gene, and partially reverted by chrA1b or chr1NCb genes. These data indicate that chr genes from adaptive replicons, mainly chrA2 from the megaplasmid, are responsible for the B. xenovorans LB400 chromate-resistance phenotype. PMID:26873556

  17. Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

    PubMed Central

    2014-01-01

    Background Rhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids. Results Genome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones. Conclusion Variable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization

  18. Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

    PubMed Central

    2013-01-01

    Background Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs). Methods VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout. Results Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection. Conclusions A VRP based CHIKV neutralization assay using Gluc as readout

  19. Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

    PubMed Central

    Pang, Xiaowu; Zhang, Mingjie; Dayton, Andrew I

    2001-01-01

    Background Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons. Results We cloned into a Δpre-M/E dengue virus replicon genes for either green fluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA. Conclusions Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods. PMID:11747468

  20. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating...

  1. A Polyprotein-Expressing Salmonid Alphavirus Replicon Induces Modest Protection in Atlantic Salmon (Salmo Salar) Against Infectious Pancreatic Necrosis

    PubMed Central

    Abdullah, Azila; Olsen, Christel M.; Hodneland, Kjartan; Rimstad, Espen

    2015-01-01

    Vaccination is an important strategy for the control and prevention of infectious pancreatic necrosis (IPN) in farmed Atlantic salmon (Salmo salar) in the post-smolt stage in sea-water. In this study, a heterologous gene expression system, based on a replicon construct of salmonid alphavirus (SAV), was used for in vitro and in vivo expression of IPN virus proteins. The large open reading frame of segment A, encoding the polyprotein NH2-pVP2-VP4-VP3-COOH, as well as pVP2, were cloned and expressed by the SAV replicon in Chinook salmon embryo cells (CHSE-214) and epithelioma papulosum cyprini (EPC) cells. The replicon constructs pSAV/polyprotein (pSAV/PP) and pSAV/pVP2 were used to immunize Atlantic salmon (Salmo salar) by a single intramuscular injection and tested in a subsequent IPN virus (IPNV) challenge trial. A low to moderate protection against IPN was observed in fish immunized with the replicon vaccine that encoded the pSAV/PP, while the pSAV/pVP2 construct was not found to induce protection. PMID:25606973

  2. Development of porcine respiratory and reproductive syndrome virus replicon vector for foot-and-mouth disease vaccine

    PubMed Central

    Jeeva, Subbiah; Lee, Jung-Ah; Park, Seung-Yong; Song, Chang-Seon; Choi, In-Soo

    2014-01-01

    Purpose Foot-and-mouth disease (FMD) is an economically important global animal disease. To control FMD virus (FMDV) outbreaks, a lot of different novel approaches have been attempted. In this study, we proposed a novel porcine reproductive and respiratory syndrome virus (PRRSV) as a replicon vector to express FMDV structural protein. Materials and Methods PRRSV infectious clone (PRRSVK418DM) was used to develop an expression vector through the reverse genetic manipulation of PRRSV; FMDVP12A3C gene of serotype O was synthesized and used for an antigen. MARC-145 cells (African green monkey kidney epithelial cell line) were used for electroporation mediated transfection. The transfection or the expression of P12A3C and N protein of PRRSV was analyzed by either replicon containing PRRSV alone or by co-infection of helper PRRSV. Results We constructed PRRSVK418DM replicon vector containing FMDVP12A3C, and genome sequences were confirmed by subsequent sequence analysis. In vitro expression of P12A3C and PRRSV N protein was confirmed by immunofluorescence antibody assay using antibodies specific for PRRSV N protein (anti-PRRSV N MAb), FMDV-VP1 (anti-VP1 MAb). Conclusion The results indicate that PRRSV replicon vector can be a promising novel vector system to control FMDV and useful for vaccine development in the future. PMID:24427767

  3. Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

    PubMed Central

    Kotta-Loizou, Ioly; Karakasiliotis, Ioannis; Vassilaki, Niki; Sakellariou, Panagiotis; Bartenschlager, Ralf

    2015-01-01

    Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics. PMID:25694591

  4. Characterization of Multidrug-Resistant Escherichia coli by Plasmid Replicon Typing and Pulsed-Field Gel Electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Characterization of plasmids has particular clinical significance because genes encoding important traits such as antimicrobial resistance are frequently present in plasmids. Plasmid replicon typing is a multiplex PCR based method that can be used to classify 18 of the 26 known plasmid t...

  5. Effects of activated aflatoxin B/sub 1/ and caffeine on DNA replicon initiation in HeLa cells

    SciTech Connect

    Cramer, P.; Painter, R.B.

    1981-01-01

    Afatoxin B/sub 1/ (AFB/sub 1/) is activated by a rat microsomal extract (S-9) to form a product that inhibits DNA synthesis in HeLa cells. At 10/sup -7/ M, AFB/sub 1/ inhibited initiation of replicons, as shown in alkaline sucrose gradient profiles 30 min after incubation with the drug. Ninety minutes later, the profile of treated cells was similar to that of control, but 4 h later there was another effect on replicon initiation. At 10/sup -6/ M, the inhibition of initiation was greater than at 10/sup -7/ M and increased progressively. Four hours after removal of the drug, the gradient profile showed low amounts of radioactivity in all size classes of DNA. When cells were incubated in medium containing caffeine (2 mM) even as late as 60 min after incubation with AFB/sub 1/, the inhibition of replicon initiation was prevented. If caffeine was later removed from the medium, replicon initiation was then inhibited. At 10/sup -7/ M or 10/sup -6/ M, AFB/sub 1/ had little immediate effect on chain elongation, but at 10/sup -5/ M, the gradient profiles showed an accumulation of low molecular weight DNA molecules, with no radioactivity in the region of high molecular weight DNA, owing to a block to chain elongation; this was not affected by caffeine. These results suggest that AFB/sub 1/ induces damage that changes the fonformation of chromatin so that initiation of new replicons cannot occur; in the presence of caffeine this change does not occur and DNA replication is not inhibited.

  6. Mutations Conferring a Noncytotoxic Phenotype on Chikungunya Virus Replicons Compromise Enzymatic Properties of Nonstructural Protein 2

    PubMed Central

    Utt, Age; Das, Pratyush Kumar; Varjak, Margus; Lulla, Valeria; Lulla, Aleksei

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E116K (EK) substitution or a GEEGS sequence insertion after residue T648 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. IMPORTANCE CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the

  7. 5' and 3' Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves.

    PubMed

    Diamos, Andrew G; Rosenthal, Sun H; Mason, Hugh S

    2016-01-01

    We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5' untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5' UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5' UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody

  8. Antibiotic resistance due to an unusual ColE1-type replicon plasmid in Aeromonas salmonicida.

    PubMed

    Vincent, Antony T; Emond-Rheault, Jean-Guillaume; Barbeau, Xavier; Attéré, Sabrina A; Frenette, Michel; Lagüe, Patrick; Charette, Steve J

    2016-06-01

    Aeromonas salmonicida subsp. salmonicida is a fish pathogen known to have a rich plasmidome. In the present study, we discovered an isolate of this bacterium bearing an additional unidentified small plasmid. After having sequenced the DNA of that isolate by next-generation sequencing, it appeared that the new small plasmid is a ColE1-type replicon plasmid, named here pAsa7. This plasmid bears a functional chloramphenicol-acetyltransferase-encoding gene (cat-pAsa7) previously unknown in A. salmonicida and responsible for resistance to chloramphenicol. A comparison of pAsa7 with pAsa2, the only known ColE1-type replicon plasmid usually found in A. salmonicida subsp. salmonicida, revealed that even if both plasmids share a high structural similarity, it is still unclear if pAsa7 is a derivative of pAsa2 since they showed several mutations at the nucleotide level. Transcriptomic analysis revealed that the cat-pAsa4 gene, another chloramphenicol-acetyltransferase-encoding gene, found on the large plasmid pAsa4, was significantly more transcribed than cat-pAsa7. This was correlated with a higher chloramphenicol resistance for isolates bearing pAsa4 compared with the one having pAsa7. Finally, a phylogenetic analysis showed that both CAT-pAsa4 and CAT-pAsa7 proteins were in different clusters. The clustering was supported by the identity of residues involved in the catalytic site. In addition, to give a better understanding of the large drug-resistance panel of A. salmonicida, this study reinforces the hypothesis that A. salmonicida subsp. salmonicida is a considerable reservoir for mobile genetic elements such as plasmids. PMID:27028891

  9. A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge.

    PubMed

    Saxena, Sonal; Dahiya, Shyam S; Sonwane, Arvind A; Patel, Chhabi Lal; Saini, Mohini; Rai, A; Gupta, Praveen K

    2008-12-01

    A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies. PMID:18848857

  10. Selection and characterization of hepatitis C virus replicons dually resistant to the polymerase and protease inhibitors HCV-796 and boceprevir (SCH 503034).

    PubMed

    Flint, Mike; Mullen, Stanley; Deatly, Anne M; Chen, Wei; Miller, Lynn Z; Ralston, Robert; Broom, Colin; Emini, Emilio A; Howe, Anita Y M

    2009-02-01

    HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity. PMID:18936191

  11. Selection and Characterization of Hepatitis C Virus Replicons Dually Resistant to the Polymerase and Protease Inhibitors HCV-796 and Boceprevir (SCH 503034) ▿

    PubMed Central

    Flint, Mike; Mullen, Stanley; Deatly, Anne M.; Chen, Wei; Miller, Lynn Z.; Ralston, Robert; Broom, Colin; Emini, Emilio A.; Howe, Anita Y. M.

    2009-01-01

    HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity. PMID:18936191

  12. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

    PubMed Central

    McCullough, Kenneth C.; Milona, Panagiota; Thomann-Harwood, Lisa; Démoulins, Thomas; Englezou, Pavlos; Suter, Rolf; Ruggli, Nicolas

    2014-01-01

    Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future. PMID:26344889

  13. Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens

    PubMed Central

    Wilms, Ina; Overlöper, Aaron; Nowrousian, Minou; Sharma, Cynthia M.; Narberhaus, Franz

    2012-01-01

    Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium. PMID:22336765

  14. Novel diversity-oriented synthesis-derived respiratory syncytial virus inhibitors identified via a high throughput replicon-based screen.

    PubMed

    Duvall, Jeremy R; VerPlank, Lynn; Ludeke, Barbara; McLeod, Sarah M; Lee, Maurice D; Vishwanathan, Karthick; Mulrooney, Carol A; Le Quement, Sebastian; Yu, Qin; Palmer, Michelle A; Fleming, Paul; Fearns, Rachel; Foley, Michael A; Scherer, Christina A

    2016-07-01

    Respiratory syncytial virus (RSV) infections affect millions of children and adults every year. Despite the significant disease burden, there are currently no safe and effective vaccines or therapeutics. We employed a replicon-based high throughput screen combined with live-virus triaging assays to identify three novel diversity-oriented synthesis-derived scaffolds with activity against RSV. One of these small molecules is shown to target the RSV polymerase (L protein) to inhibit viral replication and transcription; the mechanisms of action of the other small molecules are currently unknown. The compounds described herein may provide attractive inhibitors for lead optimization campaigns. PMID:27059228

  15. Transformation of microorganisms with the plasmid vector with the replicon from pAC1 from Acetobacter pasteurianus.

    PubMed

    Grones, J; Turna, J

    1995-01-26

    A number of gram-negative and gram-positive bacteria species was screened for the expression of the gram-negative plasmid pACK5 and pACT72 with replicon of pAC1 plasmid from Acetobacter pasteurianus. As was described previously, both plasmids were expressed in Escherichia coli, Acetobacter pasteurianus, Acetobacter aceti, Shigella spp. and Citrobacter spp. Expressions of plasmids were successful in twelve species tested, Comamonas terrigena, Salmonella typhimurium, Serratia marcescens, Bacillus cereus, Bacillus megatericum, Bacillus subtilis, Lactobacillus helveticus, Micrococcus luteus, Sarcina lutea, Staphylococcus aureus, Staphylococcus epidermidis, Streptoccocus feacalis, and the stability of plasmid DNA was tested after cultivation in non-selective conditions. PMID:7832808

  16. pUCL287 plasmid from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315 represents a new theta-type replicon family of lactic acid bacteria.

    PubMed

    Benachour, A; Frère, J; Novel, G

    1995-05-01

    A cryptic plasmid, pUCL287, was isolated from Tetragenococcus halophila (Pediococcus halophilus) ATCC 33315. It had a theta-type mechanism of replication in its natural host. Its minimal replicon, Rep287, was isolated on a 1.6-kb EcoRI fragment. The Rep287 host range included the genera Pediococcus, Enterococcus, Lactobacillus and Leuconostoc but not genus Lactococcus. Plasmids hybridizing to pUCL287 are rare among lactic acid bacteria. As assessed by hybridization, Rep287 is dissimilar to pAM beta 1, pIP501 and pUCL22, representatives of the most common theta-type replicon groups in Gram-positive bacteria. Therefore, pUCL287 appears to represent a new theta-type replicon family from lactic acid bacteria. PMID:7750734

  17. Alphavirus replicon-based adjuvants enhance the immunogenicity and effectiveness of Fluzone ® in rhesus macaques.

    PubMed

    Carroll, Timothy D; Matzinger, Shannon R; Barro, Mario; Fritts, Linda; McChesney, Michael B; Miller, Christopher J; Johnston, Robert E

    2011-01-29

    Venezuelan equine encephalitis virus replicon particles (VRP) without a transgene (null VRP) have been used to adjuvant effective humoral [1], cellular [2], and mucosal [3] immune responses in mice. To assess the adjuvant activity of null VRP in the context of a licensed inactivated influenza virus vaccine, rhesus monkeys were immunized with Fluzone(®) alone or Fluzone(®) mixed with null VRP and then challenged with a human seasonal influenza isolate, A/Memphis/7/2001 (H1N1). Compared to Fluzone(®) alone, Fluzone(®)+null VRP immunized animals had stronger influenza-specific CD4(+) T cell responses (4.4 fold) with significantly higher levels of virus-specific IFN-γ (7.6 fold) and IL-2 (5.3 fold) producing CD4+ T cells. Fluzone(®)+null VRP immunized animals also had significantly higher plasma anti-influenza IgG (p<0.0001, 1.3 log) and IgA (p<0.05, 1.2 log) levels. In fact, the mean plasma anti-influenza IgG titers after one Fluzone(®)+null VRP immunization was 1.2 log greater (p<0.04) than after two immunizations with Fluzone(®) alone. After virus challenge, only Fluzone(®)+null VRP immunized monkeys had a significantly lower level of viral replication (p<0.001) relative to the unimmunized control animals. Although little anti-influenza antibody was detected in the respiratory secretions after immunization, strong anamnestic anti-influenza IgG and IgA responses were present in secretions of the Fluzone(®)+null VRP immunized monkeys immediately after challenge. There were significant inverse correlations between influenza RNA levels in tracheal lavages and plasma anti-influenza HI and IgG anti-influenza antibody titers prior to challenge. These results demonstrate that null VRP dramatically improve both the immunogenicity and protection elicited by a licensed inactivated influenza vaccine. PMID:21111777

  18. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    PubMed

    Sánchez-Puig, Juana M; Lorenzo, María M; Blasco, Rafael

    2013-01-01

    Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses. PMID:24130722

  19. Replicon typing of plasmids carrying blaCTX-M-1 in Enterobacteriaceae of animal, environmental and human origin

    PubMed Central

    Zurfluh, Katrin; Jakobi, Gianna; Stephan, Roger; Hächler, Herbert; Nüesch-Inderbinen, Magdalena

    2014-01-01

    Objectives: The aim of this work was to determine the plasmid replicon profiles of a collection of blaCTX-M-1-positive enterobacterial strains. The isolates originated from chicken in the production pyramid, healthy food-producing animals at slaughter (chicken, calves, and pigs), chicken retail meat, environmental isolates originating from water bodies, and isolates from humans. A selection of IncI and IncN plasmids were characterized by multilocus sequence typing in order to determine their epidemiological relatedness. Methods: Transconjugants of 74 blaCTX-M-1-positive isolates were analyzed by PCR-based replicon typing and by PCR-based plasmid multilocus sequence typing. Results: The incompatibility groups detected among the blaCTX-M-1-harboring plasmids included IncI1, IncN, IncHI1B, IncF, IncFIIS, IncFIB, and IncB/O, with plasmid lineage IncI1/ST3 predominating in isolates from chicken and from humans. Lineage IncN/ST1 was detected mainly in isolates from pigs. For the first time, blaCTX-M-1 genes encoded on IncHI1 plasmids were detected in isolates from cattle and from water bodies. Conclusions: This study identifies plasmid lineages that are contributing to the dissemination of blaCTX-M-1 genes in the food chain, the environment, and humans. PMID:25400623

  20. Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Protects Nonhuman Primates from Intramuscular and Aerosol Challenge with Ebolavirus

    PubMed Central

    Herbert, Andrew S.; Kuehne, Ana I.; Barth, James F.; Ortiz, Ramon A.; Nichols, Donald K.; Zak, Samantha E.; Stonier, Spencer W.; Muhammad, Majidat A.; Bakken, Russell R.; Prugar, Laura I.; Olinger, Gene G.; Groebner, Jennifer L.; Lee, John S.; Pratt, William D.; Custer, Max; Kamrud, Kurt I.; Smith, Jonathan F.; Hart, Mary Kate

    2013-01-01

    There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine. PMID:23408633

  1. The RepA_N replicons of Gram-positive bacteria: a family of broadly distributed but narrow host range plasmids

    PubMed Central

    Weaver, Keith E.; Kwong, Stephen M.; Firth, Neville; Francia, Maria Victoria

    2009-01-01

    The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multi-resistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families. PMID:19100285

  2. Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene.

    PubMed

    Ishikawa, Tomohiro; Abe, Makoto; Masuda, Michiaki

    2015-01-01

    Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, sudden emergence of the genotype V in China and Korea was reported, suggesting expansion of its geographical distribution. Although studies on the genotype V are becoming more important, they have been limited partly due to lack of its infectious molecular clone. In this study, a plasmid carrying cDNA corresponding to the entire genome of JEV Muar strain, which belongs to genotype V, in the downstream of T7 promoter was constructed. Electroporation of viral RNA transcribed by T7 RNA polymerase (T7RNAP) in vitro from the plasmid led to production of progeny viruses both in mammalian and mosquito cells. Also, transfection of the infectious clone plasmid into mammalian cells expressing T7RNAP transiently or stably was demonstrated to generate infectious progenies. When the viral structural protein genes were partially deleted from the full-length cDNA, the subgenomic RNA transcribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (Nakayama strain), the genotype V-derived replicon appeared to be more tolerant to introduction of a foreign gene. The infectious clone and the replicons constructed in this study may serve as useful tools for characterizing JEV genotype V. PMID:25451067

  3. A Kunjin Replicon Virus-like Particle Vaccine Provides Protection Against Ebola Virus Infection in Nonhuman Primates.

    PubMed

    Pyankov, Oleg V; Bodnev, Sergey A; Pyankova, Olga G; Solodkyi, Vladislav V; Pyankov, Stepan A; Setoh, Yin Xiang; Volchkova, Valentina A; Suhrbier, Andreas; Volchkov, Viktor V; Agafonov, Alexander A; Khromykh, Alexander A

    2015-10-01

    The current unprecedented outbreak of Ebola virus (EBOV) disease in West Africa has demonstrated the urgent need for a vaccine. Here, we describe the evaluation of an EBOV vaccine candidate based on Kunjin replicon virus-like particles (KUN VLPs) encoding EBOV glycoprotein with a D637L mutation (GP/D637L) in nonhuman primates. Four African green monkeys (Cercopithecus aethiops) were injected subcutaneously with a dose of 10(9) KUN VLPs per animal twice with an interval of 4 weeks, and animals were challenged 3 weeks later intramuscularly with 600 plaque-forming units of Zaire EBOV. Three animals were completely protected against EBOV challenge, while one vaccinated animal and the control animal died from infection. We suggest that KUN VLPs encoding GP/D637L represent a viable EBOV vaccine candidate. PMID:25732811

  4. Burkholderia xernovorans LB400 harbors a multi-replicon, 9.73-Mbp genome shaped for versatility

    SciTech Connect

    Chain, Patrick S. G.; Denef, Vincent; Konstantinidis, Konstantinos T; Vergez, Lisa; Agullo, Loreine; Reyes, Valeria Latorre; Hauser, Loren John; Cordova, Macarena; Gomez, Luis; Gonzalez, Myriam; Land, Miriam L; Lao, Victoria; Larimer, Frank W; LiPuma, John J; Mahenthiralingam, Eshwar; Malfatti, Stephanie; Marx, Christopher J; Parnell, J Jacob; Ramette, Alban; Richardson, P M; Seeger, Michael; Smith, Daryl; Spilker, Theodore; Sul, Woo Jun; Tsoi, Tamara V; Zhulin, Igor B; Tiedje, James M.

    2006-01-01

    Burkholderia xenovorans LB400 (LB400), a well studied, effective polychlorinated biphenyl-degrader, has one of the two largest known bacterial genomes and is the first nonpathogenic Burkholderia isolate sequenced. From an evolutionary perspective, we find significant differences in functional specialization between the three replicons of LB400, as well as a more relaxed selective pressure for genes located on the two smaller vs. the largest replicon. High genomic plasticity, diversity, and specialization within the Burkholderia genus are exemplified by the conservation of only 44% of the genes between LB400 and Burkholderia cepacia complex strain 383. Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp. The latter is largely explained by our findings that >20% of the LB400 sequence was recently acquired by means of lateral gene transfer. Although a range of genetic factors associated with in vivo survival and intercellular interactions are present, these genetic factors are likely related to niche breadth rather than determinants of pathogenicity. The presence of at least eleven 'central aromatic' and twenty 'peripheral aromatic' pathways in LB400, among the highest in any sequenced bacterial genome, supports this hypothesis. Finally, in addition to the experimentally observed redundancy in benzoate degradation and formaldehyde oxidation pathways, the fact that 17.6% of proteins have a better LB400 paralog than an ortholog in a different genome highlights the importance of gene duplication and repeated acquirement, which, coupled with their divergence, raises questions regarding the role of paralogs and potential functional redundancies in large-genome microbes.

  5. Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles

    PubMed Central

    Tonkin, Daniel R; Whitmore, Alan; Johnston, Robert E; Barro, Mario

    2012-01-01

    Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. PMID:22531556

  6. Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

    PubMed Central

    Halbherr, Stefan J.; Brostoff, Terza; Tippenhauer, Merve; Locher, Samira; Berger Rentsch, Marianne; Zimmer, Gert

    2013-01-01

    Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×108 infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry. PMID:23762463

  7. 5′ and 3′ Untranslated Regions Strongly Enhance Performance of Geminiviral Replicons in Nicotiana benthamiana Leaves

    PubMed Central

    Diamos, Andrew G.; Rosenthal, Sun H.; Mason, Hugh S.

    2016-01-01

    We previously reported a recombinant protein production system based on a geminivirus replicon that yields high levels of vaccine antigens and monoclonal antibodies in plants. The bean yellow dwarf virus (BeYDV) replicon generates massive amounts of DNA copies, which engage the plant transcription machinery. However, we noticed a disparity between transcript level and protein production, suggesting that mRNAs could be more efficiently utilized. In this study, we systematically evaluated genetic elements from human, viral, and plant sources for their potential to improve the BeYDV system. The tobacco extensin terminator enhanced transcript accumulation and protein production compared to other commonly used terminators, indicating that efficient transcript processing plays an important role in recombinant protein production. Evaluation of human-derived 5′ untranslated regions (UTRs) indicated that many provided high levels of protein production, supporting their cross-kingdom function. Among the viral 5′ UTRs tested, we found the greatest enhancement with the tobacco mosaic virus omega leader. An analysis of the 5′ UTRs from the Arabidopsis thaliana and Nicotinana benthamiana photosystem I K genes found that they were highly active when truncated to include only the near upstream region, providing a dramatic enhancement of transgene production that exceeded that of the tobacco mosaic virus omega leader. The tobacco Rb7 matrix attachment region inserted downstream from the gene of interest provided significant enhancement, which was correlated with a reduction in plant cell death. Evaluation of Agrobacterium strains found that EHA105 enhanced protein production and reduced cell death compared to LBA4301 and GV3101. We used these improvements to produce Norwalk virus capsid protein at >20% total soluble protein, corresponding to 1.8 mg/g leaf fresh weight, more than twice the highest level ever reported in a plant system. We also produced the monoclonal antibody

  8. Molecular Smallpox Vaccine Delivered by Alphavirus Replicons Elicits Protective Immunity in Mice and Non-human Primates

    PubMed Central

    Hooper, Jay W.; Ferro, Anthony M.; Golden, Joseph W.; Silvera, Peter; Dudek, Jeanne; Alterson, Kim; Custer, Max; Rivers, Bryan; Morris, John; Owens, Gary; Smith, Jonathan F.; Kamrud, Kurt I.

    2009-01-01

    Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 70s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRP) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 106 PFU of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine. PMID:19833247

  9. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

    PubMed

    Ricklin, Meret E; Vielle, Nathalie J; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4(+) T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value. PMID:27446083

  10. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies

    PubMed Central

    Ricklin, Meret E.; Vielle, Nathalie J.; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value. PMID:27446083

  11. Occurrence of 20S RNA and 23S RNA replicons in industrial yeast strains and their variation under nutritional stress conditions.

    PubMed

    López, Victoria; Gil, Rosario; Vicente Carbonell, José; Navarro, Alfonso

    2002-04-01

    We have characterized industrial yeast strains used in the brewing, baking, and winemaking industries for the presence or absence of cytoplasmic single-stranded 20S and 23S RNAs. Furthermore, the variation of intracellular concentrations of these replicons in brewing and laboratory strains under nutritional stress conditions was determined. Our results show a correlation between the relative abundance of these replicons and exposure of yeast to nutritionally stressful conditions, indicating that these RNAs could be employed as molecular probes to evaluate the exposure of 20S(+) and/or 23S(+) yeast strains to stress situations during industrial manipulation. During this study, several 20S(-)23S(+) Saccharomyces cerevisiae strains were isolated and identified. This is the first time that a yeast strain containing only 23S RNA has been reported, demonstrating that 20S RNA is not required for 23S RNA replication. PMID:11921103

  12. Combinations of various CpG motifs cloned into plasmid backbone modulate and enhance protective immunity of viral replicon DNA anthrax vaccines.

    PubMed

    Yu, Yun-Zhou; Ma, Yao; Xu, Wen-Hui; Wang, Shuang; Sun, Zhi-Wei

    2015-08-01

    DNA vaccines are generally weak stimulators of the immune system. Fortunately, their efficacy can be improved using a viral replicon vector or by the addition of immunostimulatory CpG motifs, although the design of these engineered DNA vectors requires optimization. Our results clearly suggest that multiple copies of three types of CpG motifs or combinations of various types of CpG motifs cloned into a viral replicon vector backbone with strong immunostimulatory activities on human PBMC are efficient adjuvants for these DNA vaccines to modulate and enhance protective immunity against anthrax, although modifications with these different CpG forms in vivo elicited inconsistent immune response profiles. Modification with more copies of CpG motifs elicited more potent adjuvant effects leading to the generation of enhanced immunity, which indicated a CpG motif dose-dependent enhancement of antigen-specific immune responses. Notably, the enhanced and/or synchronous adjuvant effects were observed in modification with combinations of two different types of CpG motifs, which provides not only a contribution to the knowledge base on the adjuvant activities of CpG motifs combinations but also implications for the rational design of optimal DNA vaccines with combinations of CpG motifs as "built-in" adjuvants. We describe an efficient strategy to design and optimize DNA vaccines by the addition of combined immunostimulatory CpG motifs in a viral replicon DNA plasmid to produce strong immune responses, which indicates that the CpG-modified viral replicon DNA plasmid may be desirable for use as vector of DNA vaccines. PMID:25265876

  13. RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

    PubMed Central

    Thakral, Deepshi; Joshi, Prashant; Durgapal, Hemlata; Panda, Subrat Kumar

    2014-01-01

    Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV. PMID:24505321

  14. Encoded library technology screening of hepatitis C virus NS4B yields a small-molecule compound series with in vitro replicon activity.

    PubMed

    Arico-Muendel, Christopher; Zhu, Zhengrong; Dickson, Hamilton; Parks, Derek; Keicher, Jesse; Deng, Jianghe; Aquilani, Leah; Coppo, Frank; Graybill, Todd; Lind, Kenneth; Peat, Andrew; Thomson, Michael

    2015-01-01

    To identify novel antivirals to the hepatitis C virus (HCV) NS4B protein, we utilized encoded library technology (ELT), which enables purified proteins not amenable to standard biochemical screening methods to be tested against large combinatorial libraries in a short period of time. We tested NS4B against several DNA-encoded combinatorial libraries (DEL) and identified a single DEL feature that was subsequently progressed to off-DNA synthesis. The most active of the initial synthesized compounds had 50% inhibitory concentrations (IC50s) of 50 to 130 nM in a NS4B radioligand binding assay and 300 to 500 nM in an HCV replicon assay. Chemical optimization yielded compounds with potencies as low as 20 nM in an HCV genotype 1b replicon assay, 500 nM against genotype 1a, and 5 μM against genotype 2a. Through testing against other genotypes and genotype 2a-1b chimeric replicons and from resistance passage using the genotype 1b replicon, we confirmed that these compounds were acting on the proposed first transmembrane region of NS4B. A single sequence change (F98L) was identified as responsible for resistance, and it was thought to largely explain the relative lack of potency of this series against genotype 2a. Unlike other published series that appear to interact with this region, we did not observe sensitivity to amino acid substitutions at positions 94 and 105. The discovery of this novel compound series highlights ELT as a valuable approach for identifying direct-acting antivirals to nonenzymatic targets. PMID:25824229

  15. Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

    DOE PAGESBeta

    Groom, Joseph; Chung, Daehwan; Olson, Daniel G.; Lynd, Lee R.; Guss, Adam M.; Westpheling, Janet

    2016-01-29

    Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproduciblymore » transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. Lastly, these results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.« less

  16. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    SciTech Connect

    Zheng Min; Jin Ningyi; Liu Qi; Huo Xiaowei; Li Yang; Hu Bo; Ma Haili; Zhu Zhanbo; Cong Yanzhao; Li Xiao; Jin Minglan; Zhu Guangze

    2009-08-15

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  17. An Alphavirus Replicon-Based Human Metapneumovirus Vaccine Is Immunogenic and Protective in Mice and Cotton Rats▿

    PubMed Central

    Mok, Hoyin; Tollefson, Sharon J.; Podsiad, Amy B.; Shepherd, Bryan E.; Polosukhin, Vasiliy V.; Johnston, Robert E.; Williams, John V.; Crowe, James E.

    2008-01-01

    Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that causes upper and lower respiratory tract infections in infants, the elderly, and immunocompromised individuals worldwide. Here, we developed Venezuelan equine encephalitis virus replicon particles (VRPs) encoding hMPV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and protective efficacy of these vaccine candidates in mice and cotton rats. VRPs encoding hMPV F protein, when administered intranasally, induced F-specific virus-neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. Challenge virus replication was reduced significantly in both the upper and lower respiratory tracts following intranasal hMPV challenge in these animals. However, vaccination with hMPV G protein VRPs did not induce neutralizing antibodies or protect animals from hMPV challenge. Close examination of the histopathology of the lungs of VRP-MPV F-vaccinated animals following hMPV challenge revealed no enhancement of inflammation or mucus production. Aberrant cytokine gene expression was not detected in these animals. Together, these results represent an important first step toward the use of VRPs encoding hMPV F proteins as a prophylactic vaccine for hMPV. PMID:18786987

  18. Profiling of antimicrobial resistance and plasmid replicon types in β-lactamase producing Escherichia coli isolated from Korean beef cattle

    PubMed Central

    Shin, Seung Won; Jung, Myunghwan; Shin, Min-Kyung

    2015-01-01

    In this study, 78 isolates of Escherichia coli isolated from Korean beef cattle farms were investigated for the production of extended-spectrum β-lactamase (ESBL) and/or AmpC β-lactamase. In the disc diffusion test with ampicillin, amoxicillin, cephalothin, ceftiofur, cefotaxime, ceftazidime, and cefoxitin, 38.5% of the isolates showed resistance to all of ampicillin, amoxicillin, and cephalothin. The double disc synergy method revealed that none of the isolates produced ESBL or AmpC β-lactamases. DNA sequencing showed that all isolates encoded genes for TEM-1-type β-lactamase. Moreover, 78.2% of the isolates transferred the TEM-1-type β-lactamase gene via conjugation. In plasmid replicon typing of all donors, IncFIB and IncFIA were identified in 71.4% and 41.0% of plasmids, respectively. In transconjugants, IncFIB and IncFIA were the most frequent types detected (61.5% and 41.0%, respectively). Overall, the present study indicates that selection pressures of antimicrobials on β-lactamases in beef cattle may be low relative to other livestock animals in Korea. Moreover, to reduce selection pressure and dissemination of β-lactamase, the long-term surveillance of antimicrobial use in domestic beef cattle should be established. PMID:26119172

  19. Development of a single-replicon miniBYV vector for co-expression of heterologous proteins.

    PubMed

    Prokhnevsky, Alex; Mamedov, Tarlan; Leffet, Brett; Rahimova, Rahila; Ghosh, Ananya; Mett, Vadim; Yusibov, Vidadi

    2015-02-01

    In planta production of recombinant proteins, including vaccine antigens and monoclonal antibodies, continues gaining acceptance. With the broadening range of target proteins, the need for vectors with higher performance is increasing. Here, we have developed a single-replicon vector based on beet yellows virus (BYV) that enables co-delivery of two target genes into the same host cell, resulting in transient expression of each target. This BYV vector maintained genetic stability during systemic spread throughout the host plant, Nicotiana benthamiana. Furthermore, we have engineered a miniBYV vector carrying the sequences encoding heavy and light chains of a monoclonal antibody (mAb) against protective antigen (PA) of Bacillius anthracis, and achieved the expression of the full-length functional anti-PA mAb at ~300 mg/kg of fresh leaf tissue. To demonstrate co-expression and functionality of two independent proteins, we cloned the sequences of the Pfs48/45 protein of Plasmodium falciparum and endoglycosidase F (PNGase F) from Flavobacterium meningosepticum into the miniBYV vector under the control of two subgenomic RNA promoters. Agroinfiltration of N. benthamiana with this miniBYV vector resulted in accumulation of biologically active Pfs48/45 that was devoid of N-linked glycosylation and had correct conformation and epitope display. Overall, our findings demonstrate that the new BYV-based vector is capable of co-expressing two functionally active recombinant proteins within the same host cell. PMID:25280556

  20. The host range of RK2 minimal replicon copy-up mutants is limited by species-specific differences in the maximum tolerable copy number.

    PubMed

    Haugan, K; Karunakaran, P; Tøndervik, A; Valla, S

    1995-01-01

    The minimal replicon of the broad-host-range plasmid RK2 consists of a gene, trfA (trans-acting replication), encoding a protein required for initiation of plasmid replication. The TrfA protein binds to iterons in the cis-acting origin of vegetative replication (oriV), but the exact mechanism by which TrfA-mediated replication initiation takes place is not known. We report here the isolation and characterization of five mini RK2 trfA mutant plasmids with an elevated plasmid copy number, four in Pseudomonas aeruginosa and one in Azotobacter vinelandii. The mutations are localized between or downstream of previously reported Escherichia coli copy-up mutations in trfA, and one of the mutations has been described earlier as an independent copy-up isolate in E. coli. The five mutant plasmids were all moderately copy up in both E. coli and their host of origin, in spite of the use of isolation procedures which were expected to select efficiently in favor of plasmid mutants specifying high copy numbers. In contrast, previously described high copy-up mutants isolated in E. coli could not be established in P. aeruginosa and A. vinelandii. These high copy-up mutants were shown to induce cell killing in E. coli under conditions where the plasmid copy number was increased as a physiological response to reduced growth rate. We propose that the reason for this killing effect is that the copy number under these conditions exceeds an upper tolerance level specific for E. coli. By assuming that the corresponding tolerance level is lower in P. aeruginosa and A. vinelandii than in E. coli, and that the mechanism of copy number regulation is similar, the model can explain the phenotypes of all tested copy up mutants in these two hosts. Analogous studies were also performed in Salmonella typhimurium and Acetobacter xylinum. The data obtained in these studies indicate that the above model is probably generally true for gram-negative bacteria, and the results also indicate that the

  1. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

    PubMed Central

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

    2016-01-01

    DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

  2. Incompatibility of Lactobacillus Vectors with Replicons Derived from Small Cryptic Lactobacillus Plasmids and Segregational Instability of the Introduced Vectors

    PubMed Central

    Posno, M.; Leer, R. J.; van Luijk, N.; van Giezen, M. J. F.; Heuvelmans, P. T. H. M.; Lokman, B. C.; Pouwels, P. H.

    1991-01-01

    Three new Lactobacillus vectors based on cryptic Lactobacillus plasmids were constructed. The shuttle vector pLP3537 consists of a 2.3-kb plasmid from Lactobacillus pentosus MD353, an erythromycin resistance gene from Staphylococcus aureus plasmid pE194, and pUC19 as a replicon for Escherichia coli. The vectors pLPE317 and pLPE323, which do not contain E. coli sequences, were generated by introducing the erythromycin resistance gene of pE194 into a 1.7- and a 2.3-kb plasmid from L. pentosus MD353, respectively. These vectors and the shuttle vector pLP825 (M. Posno, R. J. Leer, J. M. M. van Rijn, B. C. Lokman, and P. H. Pouwels, p. 397-401, in A. T. Ganesan and J. A. Hoch, ed., Genetics and biotechnology of bacilli, vol. 2, 1988) could be introduced by electroporation into Lactobacillus casei, L. pentosus, L. plantarum, L. acidophilus, L. fermentum, and L. brevis strains with similar efficiencies. Transformation efficiencies were strain dependent and varied from 102 to 107 transformants per μg of DNA. Plasmid DNA analysis of L. pentosus MD353 transformants revealed that the introduction of pLP3537 or pLPE323 was invariably accompanied by loss of the endogenous 2.3-kb plasmid. Remarkably, pLPE317 could only be introduced into an L. pentosus MD353 strain that had been previously cured of its endogenous 1.7-kb plasmid. The curing phenomena are most likely to be explained by the incompatibility of the vectors and resident plasmids. Lactobacillus vectors are generally rapidly lost when cells are cultivated in the absence of selective pressure. However, pLPE323 is stable in three of four Lactobacillus strains tested so far. Images PMID:16348515

  3. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  4. The Economics of Education: A Revisionist's View.

    ERIC Educational Resources Information Center

    Mace, John

    1984-01-01

    A review of the state of the art of educational economics considers recent seemingly conflicting or disparate forms of economic analysis such as human capital theory, the study of education and economic growth, distribution of income, internal system efficiency, and education's effects on crime, savings, and consumption. (MSE)

  5. Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.

    PubMed

    Chiang, Chung-Jen; Chen, Po Ting; Chao, Yun-Peng

    2008-12-01

    Genetic manipulation of cells for desired traits is the most appreciable strategy implemented in the field of bioengineering. However, this approach closely relies on the use of plasmids and is commonly afflicted by the potential problem of plasmid instability and safety caution. Meanwhile, it may also lead to the spread of antibiotic-resistant markers with replicons of plasmids to the environment. However, this issue has long been neglected. In this study, we have addressed these subjects by developing replicon-free and markerless methods for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. For the former application, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. Their utility was illustrated by site-specific insertion of target genes, the endogenous dxs gene and three heterologous genes consisting of gps, crtI, and crtB, fused to T7 promoter into E. coli genome. For the latter application, the template vectors for promoter replacement were constructed to carry a DNA cassette containing the T7 promoter linked to a selective marker flanked with the FRT site. Subsequently, it was illustrated by replacement of the native promoter of chromosomal pckA by the T7 promoter. Finally, with the aid of FLP recombinase supplied from a helper plasmid, the regions containing replicon and/or selective markers in inserted DNAs were eliminated from integrants for both approaches. As a consequence, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, resulting in a high and stable production of lycopene in the cell. This result indicates the promise of developed methods for genome engineering in E. coli. PMID:18553504

  6. Unique plasmids generated via pUC replicon mutagenesis in an error-prone thermophile derived from Geobacillus kaustophilus HTA426.

    PubMed

    Kobayashi, Jyumpei; Tanabiki, Misaki; Doi, Shohei; Kondo, Akihiko; Ohshiro, Takashi; Suzuki, Hirokazu

    2015-11-01

    The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75(αβ)-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75(αβ)-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75(αβ)-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75(αβ)-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75(αβ)-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli. PMID:26319877

  7. Unique Plasmids Generated via pUC Replicon Mutagenesis in an Error-Prone Thermophile Derived from Geobacillus kaustophilus HTA426

    PubMed Central

    Kobayashi, Jyumpei; Tanabiki, Misaki; Doi, Shohei; Kondo, Akihiko; Ohshiro, Takashi

    2015-01-01

    The plasmid pGKE75-catA138T, which comprises pUC18 and the catA138T gene encoding thermostable chloramphenicol acetyltransferase with an A138T amino acid replacement (CATA138T), serves as an Escherichia coli-Geobacillus kaustophilus shuttle plasmid that confers moderate chloramphenicol resistance on G. kaustophilus HTA426. The present study examined the thermoadaptation-directed mutagenesis of pGKE75-catA138T in an error-prone thermophile, generating the mutant plasmid pGKE75αβ-catA138T responsible for substantial chloramphenicol resistance at 65°C. pGKE75αβ-catA138T contained no mutation in the catA138T gene but had two mutations in the pUC replicon, even though the replicon has no apparent role in G. kaustophilus. Biochemical characterization suggested that the efficient chloramphenicol resistance conferred by pGKE75αβ-catA138T is attributable to increases in intracellular CATA138T and acetyl-coenzyme A following a decrease in incomplete forms of pGKE75αβ-catA138T. The decrease in incomplete plasmids may be due to optimization of plasmid replication by RNA species transcribed from the mutant pUC replicon, which were actually produced in G. kaustophilus. It is noteworthy that G. kaustophilus was transformed with pGKE75αβ-catA138T using chloramphenicol selection at 60°C. In addition, a pUC18 derivative with the two mutations propagated in E. coli at a high copy number independently of the culture temperature and high plasmid stability. Since these properties have not been observed in known plasmids, the outcomes extend the genetic toolboxes for G. kaustophilus and E. coli. PMID:26319877

  8. Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo

    PubMed Central

    McCullough, Kenneth C; Bassi, Isabelle; Milona, Panagiota; Suter, Rolf; Thomann-Harwood, Lisa; Englezou, Pavlos; Démoulins, Thomas; Ruggli, Nicolas

    2014-01-01

    Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements—slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein. PMID:25004099

  9. The Combination of Grazoprevir, a Hepatitis C Virus (HCV) NS3/4A Protease Inhibitor, and Elbasvir, an HCV NS5A Inhibitor, Demonstrates a High Genetic Barrier to Resistance in HCV Genotype 1a Replicons.

    PubMed

    Lahser, Frederick C; Bystol, Karin; Curry, Stephanie; McMonagle, Patricia; Xia, Ellen; Ingravallo, Paul; Chase, Robert; Liu, Rong; Black, Todd; Hazuda, Daria; Howe, Anita Y M; Asante-Appiah, Ernest

    2016-05-01

    The selection of resistance-associated variants (RAVs) against single agents administered to patients chronically infected with hepatitis C virus (HCV) necessitates that direct-acting antiviral agents (DAAs) targeting multiple viral proteins be developed to overcome failure resulting from emergence of resistance. The combination of grazoprevir (formerly MK-5172), an NS3/4A protease inhibitor, and elbasvir (formerly MK-8742), an NS5A inhibitor, was therefore studied in genotype 1a (GT1a) replicon cells. Both compounds were independently highly potent in GT1a wild-type replicon cells, with 90% effective concentration (EC90) values of 0.9 nM and 0.006 nM for grazoprevir and elbasvir, respectively. No cross-resistance was observed when clinically relevant NS5A and NS3 RAVs were profiled against grazoprevir and elbasvir, respectively. Kinetic analyses of HCV RNA reduction over 14 days showed that grazoprevir and elbasvir inhibited prototypic NS5A Y93H and NS3 R155K RAVs, respectively, with kinetics comparable to those for the wild-type GT1a replicon. In combination, grazoprevir and elbasvir interacted additively in GT1a replicon cells. Colony formation assays with a 10-fold multiple of the EC90 values of the grazoprevir-elbasvir inhibitor combination suppressed emergence of resistant colonies, compared to a 100-fold multiple for the independent agents. The selected resistant colonies with the combination harbored RAVs that required two or more nucleotide changes in the codons. Mutations in the cognate gene caused greater potency losses for elbasvir than for grazoprevir. Replicons bearing RAVs identified from resistant colonies showed reduced fitness for several cell lines and may contribute to the activity of the combination. These studies demonstrate that the combination of grazoprevir and elbasvir exerts a potent effect on HCV RNA replication and presents a high genetic barrier to resistance. The combination of grazoprevir and elbasvir is currently approved for

  10. Phosphorylation of eIF2α is responsible for the failure of the picornavirus internal ribosome entry site to direct translation from Sindbis virus replicons.

    PubMed

    Sanz, Miguel Angel; Redondo, Natalia; García-Moreno, Manuel; Carrasco, Luis

    2013-04-01

    Translation directed by the poliovirus (PV) or encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) is very inefficient when expressed from Sindbis virus (SV) replicons. This inhibition can be rescued by co-expression of PV 2A protease (2A(pro)). Inhibition correlates with the extensive phosphorylation of eukaryotic initiation factor (eIF) 2α induced by SV replication. Confirmation that PV or EMCV IRES-driven translation can function when eIF2α is not phosphorylated was obtained in dsRNA-activated protein kinase knockout mouse embryonic fibroblasts (PKR(-/-) MEFs), where SV replication cannot induce eIF2α phosphorylation, and in variant S51A MEFs that express an unphosphorylatable eIF2α. In these cells, PV or EMCV IRES-dependent translation operated more efficiently than in wild-type MEFs. However, this translation was potently blocked when eIF2α was phosphorylated by the addition of thapsigargin to PKR(-/-) MEFs. In addition, when wild-type eIF2α was expressed in S51A MEFs or PKR was expressed in PKR(-/-) MEFs, PV IRES-dependent translation decreased. In both cases, the decrease in PV IRES-dependent translation correlated with the phosphorylation of eIF2α. Notably, PV 2A(pro) expression rescued PV IRES-driven translation in thapsigargin-treated PKR(-/-) MEFs. Taken together, these results demonstrated that PV IRES-driven translation can take place from SV replicons if eIF2α remains unphosphorylated. Remarkably, PV IRES-dependent translation was fully functional in this system when PV 2A(pro) was present, even if eIF2α was phosphorylated. PMID:23255624

  11. Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

    PubMed

    Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

    2016-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection. PMID:26895704

  12. Diagnostic Potential and Antigenic Properties of Recombinant Tick-Borne Encephalitis Virus Subviral Particles Expressed in Mammalian Cells from Semliki Forest Virus Replicons

    PubMed Central

    Kuivanen, Suvi; Matveev, Andrey; Swaminathan, Sathyamangalam; Jääskeläinen-Hakala, Anu; Vapalahti, Olli

    2014-01-01

    The precursor membrane envelope (prME) proteins of all three tick-borne encephalitis virus (TBEV) subtypes were produced based on expression from Semliki Forest virus (SFV) replicons transcribed from recombinant plasmids. Vero E6 cells transfected by these plasmids showed specific reactivities in immunofluorescence and immunoblot assays by monoclonal antibodies against European and Far-Eastern subtype strains of TBEV, indicating proper folding of the expressed glycoproteins. The prME glycoproteins were secreted into the cell culture supernatant, forming TBEV subviral particles of 20 to 30 nm in diameter. IgM μ-capture and IgG monoclonal antibody (MAb)-capture enzyme immunoassays (EIAs) were developed based on prME Karelia-94 (Siberian subtype) particles. Altogether, 140 human serum samples were tested using these assays, and the results were compared to those obtained with a commercial IgM EIA, an in-house μ-capture IgM assay based on baculovirus-expressed antigen, a commercial IgG EIA, and a hemagglutination inhibition test. Compared to reference enzyme-linked immunosorbent assays (ELISAs), the sensitivities of the generated μ-capture IgM SFV-prME and IgG MAb-capture SFV-prME EIAs were 97.4 to 100% and 98.7%, respectively, and the specificities of the two assays were 100%. IgM and IgG immunofluorescence assays (IFAs) were created based on Vero E6 cells transfected with the recombinant plasmid carrying the TBEV Karelia-94 prME glycoproteins. The IgM IFA was 100% concordant with the μ-capture IgM bac-prME ELISA. The IgG IFA sensitivity and specificity were 98.7% and 100%, respectively, compared to those of the commercial ELISA. In conclusion, the tests developed based on SFV replicon-driven expression of TBEV glycoproteins provide safe and robust alternatives for conducting TBEV serology. PMID:24371235

  13. Relicts and models of the RNA world

    NASA Astrophysics Data System (ADS)

    Lehto, Kirsi; Karetnikov, Alexey

    2005-01-01

    It is widely believed that the current DNA-RNA-protein-based life forms have evolved from preceding RNA-protein-based life forms, and these again, from mere RNA replicons. By rationale, it can be assumed that the early RNA replicons were fully heterotrophic in terms of obtaining all their building blocks from their environment. In the absence of protein catalysts, their essential life functions had to be mediated by simple functional structures and mechanisms, such as RNA secondary structures, RNA-RNA interactions and RNA-mediated catalysis, and possibly by catalytic minerals or clays. The central role of RNA catalysts in early life forms is supported by the fact that several catalytic RNAs still perform central biological functions in current life forms, and at least some of these may be derived as molecular relicts from the early RNA-based life. The RNA-catalysed metabolic reactions and molecular fossils are more conserved in the eukaryotic life forms than in the prokaryotes, suggesting that the linear eukaryote genomes may more closely resemble the structure and function of the early RNA replicons, than what do the circular prokaryote genomes. Present-day RNA viruses and viroids utilize ultimately simple life strategies, which may be similar to those used by the early RNA replicons. Thus, molecular and functional properties of viruses and viroids may be considered as examples or models of the structures and replication mechanisms, which might have been used for the replication of the early biopolymers.

  14. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

    PubMed

    Witteveldt, Jeroen; Martin-Gans, Marion; Simmonds, Peter

    2016-05-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  15. Depeptidization efforts on P[subscript 3]-P[prime subscript 2] [alpha]-ketoamide inhibitors of HCV NS3-4A serine protease: Effect on HCV replicon activity

    SciTech Connect

    Bogen, Stephane L.; Ruan, Sumei; Liu, Rong; Agrawal, Sony; Pichardo, John; Prongay, Andrew; Baroudy, Bahige; Saksena, Anil K.; Girijavallabhan, Viyyoor; Njoroge, F. George

    2008-06-30

    Depeptidization efforts of the P{sub 3}-P{sub 2} region of P{sub 3} capped {alpha}-ketoamide inhibitor of HCV NS3 serine protease 1 are reported. We clearly established that N-methylation of the P{sub 2} nitrogen and modification of the P{prime}{sub 2} carboxylic acid terminus were essential for activity in the replicon assay.

  16. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing

    PubMed Central

    Witteveldt, Jeroen; Martin-Gans, Marion

    2016-01-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  17. Sequence analysis and minimal replicon determination of a new haloarchaeal plasmid pHF2 isolated from Haloferax sp. strain Q22.

    PubMed

    Chen, Shaoxing; Wang, Chuangming; Xiang, Hua

    2016-01-01

    A new cryptic plasmid, pHF2 (2520 bp), was isolated from Haloferax sp. strain Q22 (CGMCC 1.15317), a haloarchaeal strain living in a subterranean halite deposit. Sequence analysis revealed that it is the smallest plasmid in the genus Haloferax so far, and three syntropic open reading frames (ORF1, ORF2, and ORF3) were identified on the same strand. ORF1 encodes a putative replication initiation protein (Rep). Three typical motifs (I, II, and III) were presented in the Rep proteins of rolling-circle replicating (RCR) plasmids. The amino acid sequence of the Rep protein is very similar to that of another haloarchaeal plasmid pNB101 in Natronobacterium sp. AS-7091 (coverage 97%, identity 56%). The minimal replicon (~1000 bp) of pHF2 was determined through the construction of a series of truncated plasmids. Interestingly, we also found that the incomplete rep gene still can drive plasmid replication. This plasmid has provided another valuable extra-chromosomal genetic resource, and deepened our knowledge in DNA replication. PMID:26601892

  18. PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

    PubMed

    Ricker, N; Shen, S Y; Goordial, J; Jin, S; Fulthorpe, R R

    2016-07-25

    We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies. PMID:27063562

  19. Overexpression and self-assembly of virus-like particles in Nicotiana benthamiana by a single-vector DNA replicon system.

    PubMed

    Moon, Ki-Beom; Lee, Jisu; Kang, Sebyung; Kim, Moonil; Mason, Hugh S; Jeon, Jae-Heung; Kim, Hyun-Soon

    2014-10-01

    Based on recent developments, virus-like particles (VLPs) are considered to be perfect candidates as nanoplatforms for applications in materials science and medicine. To succeed, mass production of VLPs and self-assembly into a correct form in plant systems are key factors. Here, we report expression of synthesized coat proteins of the three viruses, Brome mosaic virus, Cucumber mosaic virus, and Maize rayado fino virus, in Nicotiana benthamiana and production of self-assembled VLPs by transient expression system using agroinfiltration. Each coat protein was synthesized and cloned into a pBYR2fp single replicon vector. Target protein expression in cells containing p19 was fourfold higher than that of cells lacking p19. After agroinfiltration, protein expression was analyzed by SDS-PAGE and quantitative image analyzer. Quantitative analysis showed that BMVCP, CMVCP, and MRFVCP concentrations were 0.5, 1.0, and 0.8 mg · g(-1) leaf fresh weight, respectively. VLPs were purified by sucrose cushion ultracentrifugation and then analyzed by transmission electron microscopy. Our results suggested that BMVCP and CMVCP proteins expressed in N. benthamiana leaves were able to correctly self-assemble into particles. Moreover, we evaluated internal cavity accessibility of VLPs to load foreign molecules. Finally, plant growth conditions after agroinfiltration are critical for increasing heterologous protein expression levels in a transient expression system. PMID:24965559

  20. Structure and Immunogenicity of Alternative Forms of the Simian Immunodeficiency Virus Gag Protein Expressed Using Venezuelan Equine Encephalitis Virus Replicon Particles

    PubMed Central

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T.; Swanstrom, Ronald; Davis, Nancy L.

    2007-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-), or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infected primary mouse embryo fibroblasts, mouse L929 cells and primate Vero cells showed comparable expression levels for each protein, as well as extracellular virus-like particles (VRP-Gagmyr+), and distinctive cytoplasmic aggregates (VRP-Gagmyr-) with each cell type. VPR were used to immunize BALB/c mice, and immune responses were compared using an interferon (IFN)-γ ELISPOT assay and a serum antibody ELISA. Although all three VRP generated similar levels of IFN-γ-producing cells at 1 week post-boost, at 10 weeks post-boost the MA/CA-VRP-induced response was maintained at a significantly higher level relative to that induced by Gagmyr+-VRP. Antibody responses to MA/CA-VRP and Gagmyr+-VRP were not significantly different. PMID:17275057

  1. Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection.

    PubMed

    Flores, R; Minoia, S; Carbonell, A; Gisel, A; Delgado, S; López-Carrasco, A; Navarro, B; Di Serio, F

    2015-11-01

    The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously-and to incite diseases in certain plants-without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features-they are circular molecules that fold upon themselves adopting compact secondary conformations-and then to how they manipulate their hosts to be propagated. Replication of viroids-in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands-dealt three surprises: (i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, (ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and (iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world. PMID:25738582

  2. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

    PubMed

    Osada, Takuya; Berglund, Peter; Morse, Michael A; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Yang, Xiao Yi; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R; Clay, Timothy M; Smith, Jonathan; Kim Lyerly, H

    2012-11-01

    We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted. PMID:22488274

  3. Venezuelan Equine Encephalitis Virus Replicon Particles Encoding Respiratory Syncytial Virus Surface Glycoproteins Induce Protective Mucosal Responses in Mice and Cotton Rats▿

    PubMed Central

    Mok, Hoyin; Lee, Sujin; Utley, Thomas J.; Shepherd, Bryan E.; Polosukhin, Vasiliy V.; Collier, Martha L.; Davis, Nancy L.; Johnston, Robert E.; Crowe, James E.

    2007-01-01

    Respiratory syncytial virus (RSV) is an important viral pathogen that causes severe lower respiratory tract infection in infants, the elderly, and immunocompromised individuals. There are no licensed RSV vaccines to date. To prevent RSV infection, immune responses in both the upper and lower respiratory tracts are required. Previously, immunization with Venezuelan equine encephalitis virus replicon particles (VRPs) demonstrated effectiveness in inducing mucosal protection against various pathogens. In this study, we developed VRPs encoding RSV fusion (F) or attachment (G) glycoproteins and evaluated the immunogenicity and efficacy of these vaccine candidates in mice and cotton rats. VRPs, when administered intranasally, induced surface glycoprotein-specific virus neutralizing antibodies in serum and immunoglobulin A (IgA) antibodies in secretions at the respiratory mucosa. In addition, fusion protein-encoding VRPs induced gamma interferon (IFN-γ)-secreting T cells in the lungs and spleen, as measured by reaction with an H-2Kd-restricted CD8+ T-cell epitope. In animals vaccinated with F protein VRPs, challenge virus replication was reduced below the level of detection in both the upper and lower respiratory tracts following intranasal RSV challenge, while in those vaccinated with G protein VRPs, challenge virus was detected in the upper but not the lower respiratory tract. Close examination of histopathology of the lungs of vaccinated animals following RSV challenge revealed no enhanced inflammation. Immunization with VRPs induced balanced Th1/Th2 immune responses, as measured by the cytokine profile in the lungs and antibody isotype of the humoral immune response. These results represent an important first step toward the use of VRPs encoding RSV proteins as a prophylactic vaccine for RSV. PMID:17928349

  4. Regulation of the CYP1A1 gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin but not by beta-naphthoflavone or 3-methylcholanthrene is altered in hepatitis C virus replicon-expressing cells.

    PubMed

    Anderson, Garret R; Hasan, Aliya; Yin, Hao; Qadri, Ishtiaq; Quattrochi, Linda C

    2006-09-01

    Exposure to hepatitis C virus (HCV) can lead to the development of cirrhosis and hepatocellular carcinoma. To examine the effects of long-term HCV infection on hepatic cytochrome P450 1A1 (CYP1A1) expression and function, we used a human hepatoma cell line expressing the HCV subgenomic replicon (Huh.8) to evaluate CYP1A1 induction by the aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we demonstrate that the induction of CYP1A1 expression in Huh.8 cells by TCDD but not by beta-naphthoflavone or 3-methylcholanthrene was significantly diminished. TCDD exposure of Huh.8 cells resulted in greater than 55% suppression of CYP1A1 transcription compared with the parent cell line Huh7, whereas protein levels and enzyme activities were further diminished. Suppression of CYP1A1 mRNA expression in TCDD-treated Huh.8 cells was partially reversed after pretreatment with the antioxidants N-acetylcysteine and nordihydroguaiaretic acid, suggesting a role for oxidative stress. Induced CYP1A1 message, protein, and enzyme activity were partially restored in an Huh7 cell line expressing the HCV replicon containing a deletion in the nonstructural protein NS5A. Furthermore, adenoviral expression of NS5A in Huh7 partially suppressed TCDD-induced CYP1A1 protein and enzyme activity, implicating this protein in the mechanism of suppression. These findings demonstrate that TCDD-mediated AhR signaling is impaired in hepatocytes in which HCV is present and that NS5A alone or in the presence of other nonstructural proteins of the subgenomic replicon is in part responsible. PMID:16788090

  5. A zebrafish model for subgenomic hepatitis C virus replication.

    PubMed

    Ding, Cun-Bao; Zhao, Ye; Zhang, Jing-Pu; Peng, Zong-Gen; Song, Dan-Qing; Jiang, Jian-Dong

    2015-03-01

    Persistent infection with hepatitis C virus (HCV) is a major risk factor in the development of hepatocellular carcinoma. The elucidation of the pathogenesis of HCV-associated liver disease is hampered by the absence of an appropriate small animal model. Zebrafish exhibits high genetic homology to mammals, and is easily manipulated experimentally. In this study, we describe the use of a zebrafish model for the analysis of HCV replication mechanisms. As the 5' untranslated region (UTR), the core protein, the non-structural protein 5B (NS5B) and the 3'UTR are essential for HCV replication, we constructed a HCV sub-replicon gene construct including the 4 gene sequences and the enhanced green fluorescent protein (EGFP) reporter gene; these genes were transcribed through the mouse hepatocyte nuclear factor 4 (mHNF4) promoter. By microinjection of the subgenomic replicon vector into zebrafish larvae, the virus was easily detected by observing EGFP fluorescence in the liver. The positive core and NS5B signals showed positive expression of the HCV gene construct in zebrafish by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. Importantly, the negative strand sequence of the HCV subgenomic RNA was detected by RT-PCR and hybridization in situ, demonstrating that the HCV sub-replicon has positive replication activity. Furthermore, the hybridization signal mainly appeared in the liver region of larvae, as detected by the sense probe of the core protein or NS5B, which confirmed that the sub-replicon amplification occurred in the zebrafish liver. The amplification of the sub-replicon caused alterations in the expression of certain genes, which is similar to HCV infection in human liver cells. To verify the use of this zebrafish model in drug evaluation, two drugs against HCV used in clinical practice, ribavirin and oxymatrine, were tested and these drugs showed significant inhibition of replication of the HCV sub-replicon in the larvae. In

  6. Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1

    PubMed Central

    Ku, Hye-Jin; Park, Myeong Soo; Lee, Ju-Hoon

    2015-01-01

    A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori), a potential ribosomal binding site (RBS), and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, four different PCR products (MR1, ori+RBS+repA; MR2, RBS+repA; MR2’, repA; MR3, fragment of repA) were obtained and cloned into pUC19 (pKUCm1, pKUCm2, pKUCm2’, and pKUCm3, respectively) containing the chloramphenicol acetyltransferase (CAT) gene. These constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5 × 105 CFU/μg, 1.3 × 101 CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria. PMID:25691882

  7. Characterization of a minimal pKW2124 replicon from Weissella cibaria KLC140 and its application for the construction of the Weissella expression vector pKUCm1.

    PubMed

    Ku, Hye-Jin; Park, Myeong Soo; Lee, Ju-Hoon

    2015-01-01

    A 2.1-kb plasmid was previously isolated from Weissella cibaria KLC140 in kimchi and cloned into pUC19 along with the slpA and gfp genes, resulting in an 8.6-kb pKWCSLGFP construct for use as a novel surface display vector. To reduce the size of the vector, the minimal replicon of pKW2124 was determined. The pKW2124 plasmid contains a putative origin of replication (ori), a potential ribosomal binding site (RBS), and the repA gene encoding a plasmid replication protein. To conduct the minimal replicon experiment, four different PCR products (MR1, ori+RBS+repA; MR2, RBS+repA; MR2', repA; MR3, fragment of repA) were obtained and cloned into pUC19 (pKUCm1, pKUCm2, pKUCm2', and pKUCm3, respectively) containing the chloramphenicol acetyltransferase (CAT) gene. These constructed vectors were electroporated into W. confusa ATCC 10881 with different transformation efficiencies of 1.5 × 10(5) CFU/μg, 1.3 × 10(1) CFU/μg, and no transformation, respectively, suggesting that the putative ori, RBS, and repA gene are essential for optimum plasmid replication. Subsequent segregational plasmid stability testing of pKUCm1 and pKUCm2 showed that the vector pKUCm1 is highly stable up to 100 generations but pKUCm2 was completely lost after 60 generations, suggesting that the putative ori may be important for plasmid stability in the host strain. In addition, a host range test of pKUCm1 revealed that it has a broad host range spectrum including Weissella, Lactococcus, Leuconostoc, and even Lactobacillus. To verify the application of pKUCm1, the β-galactosidase gene and its promoter region from W. cibaria KSD1 were cloned in the vector, resulting in pKUGal. Expression of the β-galactosidase gene was confirmed using blue-white screening after IPTG induction. The small and stable pKUGal vector will be useful for gene transfer, expression, and manipulation in the Weissella genome and in other lactic acid bacteria. PMID:25691882

  8. Structure-based optimization and derivatization of 2-substituted quinolone-based non-nucleoside HCV NS5B inhibitors with submicromolar cellular replicon potency.

    PubMed

    Cheng, Yu; Shen, Jian; Peng, Run-Ze; Wang, Gui-Feng; Zuo, Jian-Ping; Long, Ya-Qiu

    2016-06-15

    HCV NS5B polymerase is an attractive and validated target for anti-HCV therapy. Starting from our previously identified 2-aryl quinolones as novel non-nucleoside NS5B polymerase inhibitors, structure-based optimization furnished 2-alkyl-N-benzyl quinolones with improved antiviral potency by employing privileged fragment hybridization strategy. The N-(4-chlorobenzyl)-2-(methoxymethyl)quinolone derivative 5f proved to be the best compound of this series, exhibiting a selective sub-micromolar antiviral effect (EC50=0.4μM, SI=10.8) in Huh7.5.1 cells carrying a HCV genotype 2a. Considering the undesirable pharmacokinetic property of the highly substituted quinolones, a novel chemotype of 1,6-naphthyridine-4,5-diones were evolved via scaffold hopping, affording brand new structure HCV inhibitors with compound 6h (EC50 (gt2a)=2.5μM, SI=7.2) as a promising hit. Molecular modeling studies suggest that both of 2-alkyl quinolones and 1,6-naphthyridine-4,5-diones function as HCV NS5B thumb pocket II inhibitors. PMID:27133482

  9. Divergent Evolution of the repFII Replicon of IncF Plasmids Carrying Cytotoxic Necrotizing Factor cnf2, Cytolethal Distending Toxin cdtIII, and f17Ae Fimbrial Variant Genes in Type 2 Necrotoxigenic Escherichia coli Isolates from Calves.

    PubMed

    Bihannic, Morgan; Haenni, Marisa; Oswald, Eric; Madec, Jean-Yves

    2016-01-01

    Among the pathovars of Escherichia coli in cattle, necrotoxigenic E. coli (NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of the cnf2, f17Ae, and cdtIII genes in a collection of fecal E. coli isolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread of cnf2, f17Ae, and cdtIII on similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carrying cnf2 alone. These results suggest two divergent lineages of cnf2-carrying IncF plasmids depending on the presence of the f17Ae and cdtIII genes. This partition was observed in E. coli strains of unrelated backgrounds, suggesting two different evolutionary paths of cnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants in E. coli, in particular, plasmids of the IncF family. PMID:26546422

  10. Divergent Evolution of the repFII Replicon of IncF Plasmids Carrying Cytotoxic Necrotizing Factor cnf2, Cytolethal Distending Toxin cdtIII, and f17Ae Fimbrial Variant Genes in Type 2 Necrotoxigenic Escherichia coli Isolates from Calves

    PubMed Central

    Bihannic, Morgan; Haenni, Marisa; Oswald, Eric

    2015-01-01

    Among the pathovars of Escherichia coli in cattle, necrotoxigenic E. coli (NTEC) is defined by the production of cytotoxic necrotizing factors (CNFs). In particular, type 2 NTEC (NTEC2) strains are frequent in diarrheic and septicemic calves and usually coproduce CNF type 2 (CNF2), cytolethal distending toxin type III (CDTIII), and fimbrial adhesins of the F17 family, whose genetic determinants have frequently been reported on the same Vir-like plasmid. In this study, we investigated the genetic environment of the cnf2, f17Ae, and cdtIII genes in a collection of fecal E. coli isolates recovered from 484 French and 58 Iranian calves. In particular, we highlighted the spread of cnf2, f17Ae, and cdtIII on similar 150-kb IncF plasmids harboring the newly assigned repFII replicon allele F74 in NTEC2 isolates. Interestingly, this 150-kb IncF plasmid differed from the 140-kb IncF plasmid harboring the newly assigned repFII replicon allele F75 and carrying cnf2 alone. These results suggest two divergent lineages of cnf2-carrying IncF plasmids depending on the presence of the f17Ae and cdtIII genes. This partition was observed in E. coli strains of unrelated backgrounds, suggesting two different evolutionary paths of cnf2-carrying IncF plasmids rather than divergent evolutions of NTEC2 clones. The driving forces for such divergent evolutions are not known, and further studies are required to clarify the selection of plasmid subtypes spreading virulence determinants in E. coli, in particular, plasmids of the IncF family. PMID:26546422

  11. A mathematical model of biological evolution.

    PubMed

    Ishii, K; Matsuda, H; Ogita, N

    1982-01-01

    In order to understand generally how the biological evolution rate depends on relevant parameters such as mutation rate, intensity of selection pressure and its persistence time, the following mathematical model is proposed: dNn(t)/dt = (mn(t) - mu)Nn(t) + muNn-1(t) (n = 0,1,2,3,...), where Nn(t) and mn(t) are respectively the number and Malthusian parameter of replicons with step number n in a population at time t and mean is the mutation rate, assumed to be a positive constant. The step number of each replicon is defined as either equal to or larger by one than that of its parent, the latter case occurring when and only when mutation has taken place. The average evolution rate defined by v infinity identical to lim t leads to infinity sigma infinity n = o nNn(t)/t sigma infinity n = o Nn(t) is rigorously obtained for the case (i) mn(t) = mn is independent of t (constant fitness model), where mn is essentially periodic with respect to n, and for the case (ii) mn(t) = s(-1) n+[t/tau] (periodic fitness model), together with the long time average -m infinity of the average Malthusian parameter -m identical to sigma infinity n = o mn(t)Nn(t)/sigma infinity n = o Nn(t). The biological meaning of the results is discussed, comparing them with the features of actual molecular evolution and with some results of computer simulation of the model for finite populations. PMID:7119589

  12. Living in the Past: Some Revisionist Thoughts on the Historiography of Art and Design Education

    ERIC Educational Resources Information Center

    Romans, Mervyn

    2004-01-01

    There is a dominant history of art and design education in Britain. This has been established by five books published in the 1960s and 1970s. They are Quentin Bell's The Schools of Design (1963), Gordon Sutton's Artisan or Artist(1967), Richard Carline's Draw They Must(1968), Stuart Macdonald's The History and Philosophy of Art Education (1970),…

  13. The Treatment of the Working Class in American Educational Historiography: Continuing Theoretical Constructs for Revisionist History.

    ERIC Educational Resources Information Center

    Morgart, Robert A.; Mihalik, Gregory

    A rationale is presented for a new direction in educational historiography which reevaluates the treatment of the working class as active creators of history. Presently the impact of the working class on the public school history curriculum is not fully assessed or explained in light of other social and economic circumstances in the twentieth…

  14. Grease, anthraxgate, and kennel cough: a revisionist history of early veterinary vaccines.

    PubMed

    Tizard, I

    1999-01-01

    In conclusion, it is remarkable just how farsighted many of the early vaccine investigators were. Jenner was apparently very comfortable with contagion and even recognized that infectious agents could gradually change and adapt to a new species. Pasteur, long before his fowl cholera experiment, dreamed that attenuation could yield safe vaccines and it took him no time at all therefore to recognize the significance of that serendipitous experiment. The fact that two other investigators were also developing anthrax vaccines simultaneously is yet another example of how the times favor certain discoveries. Finally Ferry, while constrained by the fact that he had no idea that distemper was caused by a virus, recognized well the concept of secondary infection and rationalized, not unreasonably, that his vaccine might assist in controlling this. It is also clear that we must look skeptically at the accepted historical record. Thus, it is clear that Jenner used horse-derived material as a source of vaccine material and that vaccinia may in fact be the long-lost agent of horsepox. Certainly this would not be news to many nineteenth-century investigators and veterinarians. Individuals planning to use live vaccinia in recombinant vaccines may wish to keep this in mind. Who discovered anthrax vaccine? Burdon-Sanderson clearly recognized that he could attenuate the organism. Greenfield showed that this could protect against disease although he was far from developing an effective vaccine. Poor Henri Toussaint was probably the first to develop an effective product but did not publicize his results widely. It was left to Louis Pasteur to take the risks inherent in a high-profile public experiment and win. I believe that he richly deserves the prize. Finally, who deserves the credit for distemper vaccine? First, Carré deserves much more credit than hitherto for discovering that distemper was caused by a virus. Second, Ferry, although misled by his identification of B. bronchiseptica deserves credit for realizing that his vaccine could play a role in controlling secondary infections. The true discoverer of an effective distemper vaccine was the Italian, Puntoni, but once again the publicity went to others, Laidlaw and Dunkin. Thus a pattern emerges that prior discovery matters little in the face of aggressive publicity. If nobody knows you did the experiment you might as well have never done it in the first place. Publish or perish is by no means a new phenomenon. PMID:9890006

  15. The medium and the message: a revisionist view of image quality

    NASA Astrophysics Data System (ADS)

    Ferwerda, James A.

    2010-02-01

    In his book "Understanding Media" social theorist Marshall McLuhan declared: "The medium is the message." The thesis of this paper is that with respect to image quality, imaging system developers have taken McLuhan's dictum too much to heart. Efforts focus on improving the technical specifications of the media (e.g. dynamic range, color gamut, resolution, temporal response) with little regard for the visual messages the media will be used to communicate. We present a series of psychophysical studies that investigate the visual system's ability to "see through" the limitations of imaging media to perceive the messages (object and scene properties) the images represent. The purpose of these studies is to understand the relationships between the signal characteristics of an image and the fidelity of the visual information the image conveys. The results of these studies provide a new perspective on image quality that shows that images that may be very different in "quality", can be visually equivalent as realistic representations of objects and scenes.

  16. O'Connor v. Donaldson: retelling a classic and finding some revisionist history.

    PubMed

    Behnke, S H

    1999-01-01

    This article discusses the 1975 Supreme Court opinion O'Connor v. Donaldson. The article first examines the due process clause of the Fourteenth Amendment, the basis for the O'Connor ruling. It then looks carefully at the Court's conclusions, with particular attention to the Court's reasoning and analysis. The article goes on to look at how the Supreme Court has interpreted O'Connor on subsequent occasions and suggests that the Court has often misconstrued its own decision. The article concludes by offering thoughts and commentary on the O'Connor opinion and its progeny. PMID:10212031

  17. The Great School Legend: A Revisionist Interpretation of American Public Education.

    ERIC Educational Resources Information Center

    Greer, Colin

    American schools have been credited with building American democracy. This is a myth which has been largely responsible for the resistance of today's schools to needed change. Exposing this myth are records of several major urban school systems which show the high rate of school failure among the urban poor since before 1900. These statistics have…

  18. Romantic Rhetoric, Revisionist Reality: The Effectiveness of Regulation in Maritime Education and Training

    ERIC Educational Resources Information Center

    Sampson, Helen

    2004-01-01

    This article offers an insight into the regulation and conduct of training and education in a globalised industry, and across an international labour market. Focusing on the cargo shipping sector of the maritime industry, it considers the provision of training and education for modern merchant officers within the context of an international…

  19. Spin-glass phase transition and behavior of nonlinear susceptibility in the Sherrington-Kirkpatrick model with random fields

    NASA Astrophysics Data System (ADS)

    Morais, C. V.; Zimmer, F. M.; Lazo, M. J.; Magalhães, S. G.; Nobre, F. D.

    2016-06-01

    The behavior of the nonlinear susceptibility χ3 and its relation to the spin-glass transition temperature Tf in the presence of random fields are investigated. To accomplish this task, the Sherrington-Kirkpatrick model is studied through the replica formalism, within a one-step replica-symmetry-breaking procedure. In addition, the dependence of the Almeida-Thouless eigenvalue λAT (replicon) on the random fields is analyzed. Particularly, in the absence of random fields, the temperature Tf can be traced by a divergence in the spin-glass susceptibility χSG, which presents a term inversely proportional to the replicon λAT. As a result of a relation between χSG and χ3, the latter also presents a divergence at Tf, which comes as a direct consequence of λAT=0 at Tf. However, our results show that, in the presence of random fields, χ3 presents a rounded maximum at a temperature T* which does not coincide with the spin-glass transition temperature Tf (i.e., T*>Tf for a given applied random field). Thus, the maximum value of χ3 at T* reflects the effects of the random fields in the paramagnetic phase instead of the nontrivial ergodicity breaking associated with the spin-glass phase transition. It is also shown that χ3 still maintains a dependence on the replicon λAT, although in a more complicated way as compared with the case without random fields. These results are discussed in view of recent observations in the LiHoxY1 -xF4 compound.

  20. Management of multipartite genomes: the Vibrio cholerae model.

    PubMed

    Val, Marie-Eve; Soler-Bistué, Alfonso; Bland, Michael J; Mazel, Didier

    2014-12-01

    A minority of bacterial species has been found to carry a genome divided among several chromosomes. Among these, all Vibrio species harbor a genome split into two chromosomes of uneven size, with distinctive replication origins whose replication firing involves common and specific factors. Most of our current knowledge on replication and segregation in multi-chromosome bacteria has come from the study of Vibrio cholerae, which is now the model organism for this field. It has been firmly established that replication of the two V. cholerae chromosomes is temporally regulated and coupled to the cell cycle, but the mediators of these processes are as yet mostly unknown. The two chromosomes are also organized along different patterns within the cell and occupy different subcellular domains. The selective advantages provided by this partitioning into two replicons are still unclear and are a key motivation for these studies. PMID:25460805

  1. CORONAVIRUS REVERSE GENETIC SYSTEMS: INFECTIOUS CLONES AND REPLICONS

    PubMed Central

    Almazán, Fernando; Sola, Isabel; Zuñiga, Sonia; Marquez-Jurado, Silvia; Morales, Lucia; Becares, Martina; Enjuanes, Luis

    2016-01-01

    Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last thirteen years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV). PMID:24930446

  2. The Rationalization of Unethical Research: Revisionist Accounts of the Tuskegee Syphilis Study and the New Zealand "Unfortunate Experiment".

    PubMed

    Paul, Charlotte; Brookes, Barbara

    2015-10-01

    Two studies, widely condemned in the 1970s and 1980s-the Tuskegee study of men with untreated syphilis and the New Zealand study of women with untreated carcinoma in situ of the cervix-received new defenses in the 21st century. We noted remarkable similarities in both the studies and their defenses. Here we evaluate the scientific, political, and moral claims of the defenders. The scientific claims are largely based on incomplete or misinterpreted evidence and exaggeration of the uncertainties of science. The defenders' political arguments mistakenly claim that identity politics clouded the original critiques; in fact such politics opened the eyes of the public to exploitation. The moral defenses demonstrate an overreliance on codes of conduct and have implications for research ethics today. PMID:26270295

  3. Oral Histories of Education and the Relevance of Theory: Claiming New Spaces in a Post-Revisionist Era

    ERIC Educational Resources Information Center

    Eick, Caroline

    2011-01-01

    The essential nature of oral historical inquiry "naturally" and "relentlessly" brings an oral historian to theory. In this essay, the author argues for the relevance of theory in oral historical research that explores generational transformations in the relational experiences of youth attending desegregated schools in the latter part of the…

  4. Education in Capitalist Society: Toward a Reconsideration of the State in Educational Policy.

    ERIC Educational Resources Information Center

    Shapiro, H. Svi

    1982-01-01

    Critical and revisionist historians of American education have generated a model of education in a capitalist society that has a functionalist orientation. However, this model does not properly acknowledge the importance of society's contradictions, disjunctions, and incoherence in social and educational change. (FG)

  5. Modeling

    SciTech Connect

    Loth, E.; Tryggvason, G.; Tsuji, Y.; Elghobashi, S. E.; Crowe, Clayton T.; Berlemont, A.; Reeks, M.; Simonin, O.; Frank, Th; Onishi, Yasuo; Van Wachem, B.

    2005-09-01

    Slurry flows occur in many circumstances, including chemical manufacturing processes, pipeline transfer of coal, sand, and minerals; mud flows; and disposal of dredged materials. In this section we discuss slurry flow applications related to radioactive waste management. The Hanford tank waste solids and interstitial liquids will be mixed to form a slurry so it can be pumped out for retrieval and treatment. The waste is very complex chemically and physically. The ARIEL code is used to model the chemical interactions and fluid dynamics of the waste.

  6. Recent contributions of in vitro models to our understanding of hepatitis C virus life cycle.

    PubMed

    Régeard, Morgane; Lepère, Charlotte; Trotard, Maud; Gripon, Philippe; Le Seyec, Jacques

    2007-09-01

    Hepatitis C virus is a human pathogen responsible for liver diseases including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Its high prevalence, the absence of a prophylactic vaccine and the poor efficiency of current therapies are huge medical problems. Since the discovery of the hepatitis C virus, our knowledge of its biology has been largely punctuated by the development of original models of research. At the end of the 1980s, the chimpanzee model led to cloning of the viral genome and the definition of infectious molecular clones. In 1999, a breakthrough was achieved with the development of a robust in vitro replication model named 'replicon'. This system allowed intensive research into replication mechanisms and drug discovery. Later, in 2003, pseudotyped retroviruses harbouring surface proteins of hepatitis C virus were produced to specifically investigate the viral entry process. It was only in 2005 that infectious viruses were produced in vitro, enabling intensive investigations into the entire life cycle of the hepatitis C virus. This review describes the different in vitro models developed to study hepatitis C virus, their contribution to current knowledge of the virus biology and their future research applications. PMID:17824957

  7. A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours

    PubMed Central

    Bogaert, Lies; Woodham, Andrew W.; Da Silva, Diane M.; Martens, Ann; Meyer, Evelyne

    2015-01-01

    Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials. PMID:26044793

  8. Experimental models for hepatitis C virus (HCV): new opportunities for combating hepatitis C.

    PubMed

    Trujillo-Murillo, Karina del Carmen; Garza-Rodríguez, María del Lourdes; Martínez-Rodríguez, Herminia Guadalupe; Barrera-Saldaña, Hugo Alberto; Bosques-Padilla, Francisco; Ramos-Jiménez, Javier; Rivas-Estilla, Ana María

    2004-01-01

    Infection with hepatitis C virus (HCV) is a global public health issue. More than 200 million people in the world are infected with HCV. Hepatitis C is considered one of the main causes of chronic hepatitis, cirrhosis, hepatocellular carcinoma and liver transplantation. The identification of the viral genome quickly allowed delineation of genomic organization, and the structure and biochemical characterization of the proteins of HCV. However, it has been difficult to study its life cycle, as well as the development of antiviral agents due to the lack of a system of permissible culture. Numerous attempts have been reported to establish an in vitro system for the study of HCV. Recently, a system of efficient culture was established that allows replication of subgenomic molecules of HCV in a cell line of human hepatoma. In this revision, after a brief description of the molecular biology, means of transmission and clinical characteristics of hepatitis C, some of the experimental models are described that have been developed to date, focusing mainly on the subgenomic replicon system and their use in the development of new antiviral treatments. PMID:15257247

  9. Coenzyme world model of the origin of life.

    PubMed

    Sharov, Alexei A

    2016-06-01

    The origin of life means the emergence of heritable and evolvable self-reproduction. However the mechanisms of primordial heredity were different from those in contemporary cells. Here I argue that primordial life had no nucleic acids; instead heritable signs were represented by isolated catalytically active self-reproducing molecules, similar to extant coenzymes, which presumably colonized surfaces of oil droplets in water. The model further assumes that coenzyme-like molecules (CLMs) changed surface properties of oil droplets (e.g., by oxidizing terminal carbons), and in this way created and sustained favorable conditions for their own self-reproduction. Such niche-dependent self-reproduction is a necessary condition for cooperation between different kinds of CLMs because they have to coexist in the same oil droplets and either succeed or perish together. Additional kinds of hereditary molecules were acquired via coalescence of oil droplets carrying different kinds of CLMs or via modification of already existing CLMs. Eventually, polymerization of CLMs became controlled by other polymers used as templates; and this kind of template-based synthesis eventually resulted in the emergence of RNA-like replicons. Apparently, oil droplets transformed into the outer membrane of cells via engulfing water, stabilization of the surface, and osmoregulation. In result, the metabolism was internalized allowing cells to accumulate free-floating resources (e.g., animoacids, ATP), which was a necessary condition for the development of protein synthesis. Thus, life originated from simple but already functional molecules, and its gradual evolution towards higher complexity was driven by cooperation and natural selection. PMID:26968100

  10. The wide distribution of endornaviruses, large double-stranded RNA replicons with plasmid-like properties.

    PubMed

    Fukuhara, T; Koga, R; Aoki, N; Yuki, C; Yamamoto, N; Oyama, N; Udagawa, T; Horiuchi, H; Miyazaki, S; Higashi, Y; Takeshita, M; Ikeda, K; Arakawa, M; Matsumoto, N; Moriyama, H

    2006-05-01

    The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms. PMID:16341944

  11. Application of a plasmid classification system to determine prevalence of replicon families among multidrug resistant enterococci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The presence and transfer of plasmids from commensal bacteria to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. However, prevalence of plasmids from commensal bacteria in food animals such as the enterococci remains largely unknown. In this study, the prevale...

  12. Antimicrobial susceptibility and plasmid replicon typing of Salmonella enterica serovar Kentucky isolates recovered from broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the United States, Salmonella enterica serotype Kentucky has become the predominate serotype recovered from broiler slaughter samples and the prevalence of resistance to streptomycin and tetracycline has increased dramatically in this serotype. To characterize the relationships between antimicro...

  13. Rapid generation of a mouse model for Middle East respiratory syndrome

    PubMed Central

    Zhao, Jincun; Li, Kun; Wohlford-Lenane, Christine; Agnihothram, Sudhakar S.; Fett, Craig; Zhao, Jingxian; Gale, Michael J.; Baric, Ralph S.; Enjuanes, Luis; Gallagher, Tom; McCray, Paul B.; Perlman, Stanley

    2014-01-01

    In this era of continued emergence of zoonotic virus infections, the rapid development of rodent models represents a critical barrier to public health preparedness, including the testing of antivirus therapy and vaccines. The Middle East respiratory syndrome coronavirus (MERS-CoV) was recently identified as the causative agent of a severe pneumonia. Given the ability of coronavirus to rapidly adapt to new hosts, a major public health concern is that MERS-CoV will further adapt to replication in humans, triggering a pandemic. No small-animal model for this infection is currently available, but studies suggest that virus entry factors can confer virus susceptibility. Here, we show that mice were sensitized to MERS-CoV infection by prior transduction with adenoviral vectors expressing the human host-cell receptor dipeptidyl peptidase 4. Mice developed a pneumonia characterized by extensive inflammatory-cell infiltration with virus clearance occurring 6–8 d after infection. Clinical disease and histopathological changes were more severe in the absence of type-I IFN signaling whereas the T-cell response was required for virus clearance. Using these mice, we demonstrated the efficacy of a therapeutic intervention (poly I:C) and a potential vaccine [Venezuelan equine encephalitis replicon particles expressing MERS-CoV spike protein]. We also found little protective cross-reactivity between MERS-CoV and the severe acute respiratory syndrome-CoV. Our results demonstrate that this system will be useful for MERS-CoV studies and for the rapid development of relevant animal models for emerging respiratory viral infections. PMID:24599590

  14. Complete Genome Sequence of the Model Rhizosphere Strain Azospirillum brasilense Az39, Successfully Applied in Agriculture.

    PubMed

    Rivera, Diego; Revale, Santiago; Molina, Romina; Gualpa, José; Puente, Mariana; Maroniche, Guillermo; Paris, Gastón; Baker, David; Clavijo, Bernardo; McLay, Kirsten; Spaepen, Stijn; Perticari, Alejandro; Vazquez, Martín; Wisniewski-Dyé, Florence; Watkins, Chris; Martínez-Abarca, Francisco; Vanderleyden, Jos; Cassán, Fabricio

    2014-01-01

    We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids. PMID:25059863

  15. Mosaic gene network modelling identified new regulatory mechanisms in HCV infection.

    PubMed

    Popik, Olga V; Petrovskiy, Evgeny D; Mishchenko, Elena L; Lavrik, Inna N; Ivanisenko, Vladimir A

    2016-06-15

    , the mosaic model integrates the model of HCV subgenomic replicon replication with the model of TNF-induced apoptosis and NF-κB induction. Analysis of the mosaic model revealed that the regulation of TNF-induced signaling by the HCV network is crucially dependent on the RIP1, TRADD, TRAF2, FADD, IKK, IκBα, c-FLIP, and BAR genes. Overall, the developed mosaic gene network modelling approach demonstrated good predictive power and allowed the prediction of new regulatory nodes in HCV action on apoptosis and the NF-κB pathway. Those theoretical predictions could be a basis for further experimental verification. PMID:26481968

  16. A Nonhuman Primate Scrub Typhus Model: Protective Immune Responses Induced by pKarp47 DNA Vaccination in Cynomolgus Macaques

    PubMed Central

    Chattopadhyay, Suchismita; Jiang, Ju; Nawtaisong, Pruksa; Lee, John S.; Tan, Esterlina; Dela Cruz, Eduardo; Burgos, Jasmin; Abalos, Rodolfo; Blacksell, Stuart D.; Lombardini, Eric; Turner, Gareth D.; Day, Nicholas P. J.; Richards, Allen L.

    2015-01-01

    We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi–specific, IFN-γ–producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine

  17. A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques.

    PubMed

    Paris, Daniel H; Chattopadhyay, Suchismita; Jiang, Ju; Nawtaisong, Pruksa; Lee, John S; Tan, Esterlina; Dela Cruz, Eduardo; Burgos, Jasmin; Abalos, Rodolfo; Blacksell, Stuart D; Lombardini, Eric; Turner, Gareth D; Day, Nicholas P J; Richards, Allen L

    2015-02-15

    We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi-specific, IFN-γ-producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune

  18. A tetravalent alphavirus-vector based dengue vaccine provides effective immunity in an early life mouse model.

    PubMed

    Khalil, Syed Muaz; Tonkin, Daniel R; Mattocks, Melissa D; Snead, Andrew T; Johnston, Robert E; White, Laura J

    2014-07-01

    Dengue viruses (DENV1-4) cause 390 million clinical infections every year, several hundred thousand of which progress to severe hemorrhagic and shock syndromes. Preexisting immunity resulting from a previous DENV infection is the major risk factor for severe dengue during secondary heterologous infections. During primary infections in infants, maternal antibodies pose an analogous risk. At the same time, maternal antibodies are likely to prevent induction of endogenous anti-DENV antibodies in response to current live, attenuated virus (LAV) vaccine candidates. Any effective early life dengue vaccine has to overcome maternal antibody interference (leading to ineffective vaccination) and poor induction of antibody responses (increasing the risk of severe dengue disease upon primary infection). In a previous study, we demonstrated that a non-propagating Venezuelan equine encephalitis virus replicon expression vector (VRP), expressing the ectodomain of DENV E protein (E85), overcomes maternal interference in a BALB/c mouse model. We report here that a single immunization with a tetravalent VRP vaccine induced NAb and T-cell responses to each serotype at a level equivalent to the monovalent vaccine components, suggesting that this vaccine modality can overcome serotype interference. Furthermore, neonatal immunization was durable and could be boosted later in life to further increase NAb and T-cell responses. Although the neonatal immune response was lower in magnitude than responses in adult BALB/c mice, we demonstrate that VRP vaccines generated protective immunity from a lethal challenge after a single neonatal immunization. In summary, VRP vaccines expressing DENV antigens were immunogenic and protective in neonates, and hence are promising candidates for safe and effective vaccination in early life. PMID:24882043

  19. Development of a nanoparticle-based oral vaccine for Atlantic salmon against ISAV using an alphavirus replicon as adjuvant.

    PubMed

    Rivas-Aravena, Andrea; Fuentes, Yazmin; Cartagena, Julio; Brito, Tania; Poggio, Verónica; La Torre, José; Mendoza, Hegaly; Gonzalez-Nilo, Fernando; Sandino, Ana María; Spencer, Eugenio

    2015-07-01

    Adjuvants used in vaccine aquaculture are frequently harmful for the fish, causing melanosis, granulomas and kidney damage. Along with that, vaccines are mostly administered by injection, causing pain and stress to the fish. We used the DNA coding for the replicase of alphavirus as adjuvant (Ad) of a vaccine against ISAV. The Ad and an inactivated ISAV (V) were loaded in chitosan nanoparticles (NPs) to be administered orally to Atlantic salmon. NP-Ad was able to deliver the DNA ex vivo and in vivo. Oral administration of the NPs stimulated the expression of immune molecules, but did not stimulate the humoral response. Although the vaccination with NP-V results in a modest protection of fish against ISAV, NP-V administered together with NP-Ad caused a protection of 77%. Therefore, the DNA coding for the replicase of alphavirus could be administered orally and can potentiate the immuneprotection of a virine against infection. PMID:25862072

  20. Fifty Years after the Replicon Hypothesis: Cell-Specific Master Regulators as New Players in Chromosome Replication Control

    PubMed Central

    Wolański, Marcin; Jakimowicz, Dagmara

    2014-01-01

    Numerous free-living bacteria undergo complex differentiation in response to unfavorable environmental conditions or as part of their natural cell cycle. Developmental programs require the de novo expression of several sets of genes responsible for morphological, physiological, and metabolic changes, such as spore/endospore formation, the generation of flagella, and the synthesis of antibiotics. Notably, the frequency of chromosomal replication initiation events must also be adjusted with respect to the developmental stage in order to ensure that each nascent cell receives a single copy of the chromosomal DNA. In this review, we focus on the master transcriptional factors, Spo0A, CtrA, and AdpA, which coordinate developmental program and which were recently demonstrated to control chromosome replication. We summarize the current state of knowledge on the role of these developmental regulators in synchronizing the replication with cell differentiation in Bacillus subtilis, Caulobacter crescentus, and Streptomyces coelicolor, respectively. PMID:24914187

  1. EXPRESSION AND SELF-ASSEMBLY OF NORWALK VIRUS CAPSID PROTEIN FROM VENEZUELAN EQUINE ENCEPHALITIS VIRUS REPLICONS. (R826139)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  2. Venezuelan Equine Encephalitis Virus replicon particles can induce rapid protection against Foot-and-Mouth Disease Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously shown that swine pretreated with a replication-defective human adenovirus vector (Ad5) containing the porcine type I interferon gene (poIFN-alpha/Beta) are sterilely protected when challenged one day later with Foot-and-Mouth Disease Virus (FMDV), but the dose required is relativ...

  3. Characterization of multidrug-resistant Escherichia coli by antimicrobial resistance profiles, plasmid replicon typing, and pulsed-field gel electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aim: Plasmid characterization has particular clinical importance because genes encoding significant traits including antimicrobial resistance are frequently carried on plasmids. The objective of this study was to examine the distribution of multidrug resistance (MDR) in Escherichia coli in relation ...

  4. From Chaos to Order and Back? A Revisionist Reflection on the California Master Plan for Higher Education@50 and Thoughts about Its Future. Research & Occasional Paper Series: CSHE.7.10

    ERIC Educational Resources Information Center

    Douglass, John Aubrey

    2010-01-01

    In 1960, California developed a "master plan" for its already famed public higher education system. It was and continues to be arguably the single most influential effort to plan the future of a system of higher education in the annals of American higher education. Despite popular belief, however, the California Master Plan for Higher Education is…

  5. Leadership Models.

    ERIC Educational Resources Information Center

    Freeman, Thomas J.

    This paper discusses six different models of organizational structure and leadership, including the scalar chain or pyramid model, the continuum model, the grid model, the linking pin model, the contingency model, and the circle or democratic model. Each model is examined in a separate section that describes the model and its development, lists…

  6. A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant

    PubMed Central

    Agnihothram, Sudhakar; Yount, Boyd L.; Donaldson, Eric F.; Huynh, Jeremy; Menachery, Vineet D.; Gralinski, Lisa E.; Graham, Rachel L.; Becker, Michelle M.; Tomar, Sakshi; Scobey, Trevor D.; Osswald, Heather L.; Whitmore, Alan; Gopal, Robin; Ghosh, Arun K.; Mesecar, Andrew; Zambon, Maria; Heise, Mark; Denison, Mark R.; Baric, Ralph S.

    2014-01-01

    ABSTRACT Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. PMID:24667706

  7. Bench-to-bedside review: Fundamental principles of acid-base physiology

    PubMed Central

    Corey, Howard E

    2005-01-01

    Complex acid–base disorders arise frequently in critically ill patients, especially in those with multiorgan failure. In order to diagnose and treat these disorders better, some intensivists have abandoned traditional theories in favor of revisionist models of acid–base balance. With claimed superiority over the traditional approach, the new methods have rekindled debate over the fundmental principles of acid–base physiology. In order to shed light on this controversy, we review the derivation and application of new models of acid–base balance. PMID:15774076

  8. Models and role models.

    PubMed

    ten Cate, Jacob M

    2015-01-01

    Developing experimental models to understand dental caries has been the theme in our research group. Our first, the pH-cycling model, was developed to investigate the chemical reactions in enamel or dentine, which lead to dental caries. It aimed to leverage our understanding of the fluoride mode of action and was also utilized for the formulation of oral care products. In addition, we made use of intra-oral (in situ) models to study other features of the oral environment that drive the de/remineralization balance in individual patients. This model addressed basic questions, such as how enamel and dentine are affected by challenges in the oral cavity, as well as practical issues related to fluoride toothpaste efficacy. The observation that perhaps fluoride is not sufficiently potent to reduce dental caries in the present-day society triggered us to expand our knowledge in the bacterial aetiology of dental caries. For this we developed the Amsterdam Active Attachment biofilm model. Different from studies on planktonic ('single') bacteria, this biofilm model captures bacteria in a habitat similar to dental plaque. With data from the combination of these models, it should be possible to study separate processes which together may lead to dental caries. Also products and novel agents could be evaluated that interfere with either of the processes. Having these separate models in place, a suggestion is made to design computer models to encompass the available information. Models but also role models are of the utmost importance in bringing and guiding research and researchers. PMID:25871413

  9. MODEL DEVELOPMENT - DOSE MODELS

    EPA Science Inventory

    Model Development

    Humans are exposed to mixtures of chemicals from multiple pathways and routes. These exposures may result from a single event or may accumulate over time if multiple exposure events occur. The traditional approach of assessing risk from a single chemica...

  10. Promoting Models

    NASA Astrophysics Data System (ADS)

    Li, Qin; Zhao, Yongxin; Wu, Xiaofeng; Liu, Si

    There can be multitudinous models specifying aspects of the same system. Each model has a bias towards one aspect. These models often override in specific aspects though they have different expressions. A specification written in one model can be refined by introducing additional information from other models. The paper proposes a concept of promoting models which is a methodology to obtain refinements with support from cooperating models. It refines a primary model by integrating the information from a secondary model. The promotion principle is not merely an academic point, but also a reliable and robust engineering technique which can be used to develop software and hardware systems. It can also check the consistency between two specifications from different models. A case of modeling a simple online shopping system with the cooperation of the guarded design model and CSP model illustrates the practicability of the promotion principle.

  11. Models, Part IV: Inquiry Models.

    ERIC Educational Resources Information Center

    Callison, Daniel

    2002-01-01

    Discusses models for information skills that include inquiry-oriented activities. Highlights include WebQuest, which uses Internet resources supplemented with videoconferencing; Minnesota's Inquiry Process based on the Big Six model for information problem-solving; Indiana's Student Inquiry Model; constructivist learning models for inquiry; and…

  12. Supermatrix models

    SciTech Connect

    Yost, S.A.

    1991-05-01

    Radom matrix models based on an integral over supermatrices are proposed as a natural extension of bosonic matrix models. The subtle nature of superspace integration allows these models to have very different properties from the analogous bosonic models. Two choices of integration slice are investigated. One leads to a perturbative structure which is reminiscent of, and perhaps identical to, the usual Hermitian matrix models. Another leads to an eigenvalue reduction which can be described by a two component plasma in one dimension. A stationary point of the model is described.

  13. Supermatrix models

    SciTech Connect

    Yost, S.A. . Dept. of Physics and Astronomy)

    1992-09-30

    In this paper, random matrix models based on an integral over supermatrices are proposed as a natural extension of bosonic matrix models. The subtle nature of superspace integration allows these models to have very different properties from the analogous bosonic models. Two choices of integration slice are investigated. One leads to a perturbative structure which is reminiscent of, and perhaps identical to, the usual Hermitian matrix models. Another leads to an eigenvalue reduction which can be described by a two-component plasma in one dimension. A stationary point of the model is described.

  14. ENTRAINMENT MODELS

    EPA Science Inventory

    This presentation presented information on entrainment models. Entrainment models use entrainment hypotheses to express the continuity equation. The advantage is that plume boundaries are known. A major disadvantage is that the problems that can be solved are rather simple. The ...

  15. MODELS - 3

    EPA Science Inventory

    Models-3 is a third generation air quality modeling system that contains a variety of tools to perform research and analysis of critical environmental questions and problems. These tools provide regulatory analysts and scientists with quicker results, greater scientific accuracy ...

  16. Construction of a model secretion system for oral streptococci.

    PubMed Central

    Shiroza, T; Kuramitsu, H K

    1993-01-01

    A DNA fragment corresponding to the secretory domain from the Streptococcus mutans GS-5 gtfB gene, which encodes the putative 38-amino-acid signal peptide of the glucosyltransferase I (GTF-I) enzyme product, has been constructed. This fragment was fused with the alpha-amylase structural gene from alkalophilic Bacillus sp. strain 707. This hybrid gene as well as the intact amylase gene were introduced into an Escherichia coli-streptococcus shuttle vector consisting of three components: the E. coli replicon p15Aori from pACYC177, an erythromycin resistance gene from pAM beta-1, and the streptococcal replicon from pVA838. Transformation of the oral noncariogenic bacterium Streptococcus gordonii with the chimeric plasmid harboring the hybrid amylase gene resulted in strong extracellular amylase production. By contrast, transformants containing the intact amylase gene exhibited only trace amounts of amylase activity in culture fluids. Since the two signal peptide structures of the GTF-I enzyme and the Bacillus amylase are distinct from each other, these differences might result from the inability of S. gordonii to correctly process the Bacillus signal peptide. Furthermore, culture fluids from transformants of S. mutans as well as Streptococcus milleri harboring the hybrid amylase gene showed only weak amylase activity. Deletion of the gtfB, gtfC, or ftf gene from S. mutans GS-5 did not increase amylase secretion following transformation with the hybrid amylase gene. These results suggest that in contrast to S. gordonii, the inability of S. mutans and S. milleri to secrete hybrid amylase molecules could result from incorrect interaction of the secretory components of these organisms with amylase precursor molecules. Images PMID:7689539

  17. Turbulence modeling

    NASA Technical Reports Server (NTRS)

    Rubesin, Morris W.

    1987-01-01

    Recent developments at several levels of statistical turbulence modeling applicable to aerodynamics are briefly surveyed. Emphasis is on examples of model improvements for transonic, two-dimensional flows. Experience with the development of these improved models is cited to suggest methods of accelerating the modeling process necessary to keep abreast of the rapid movement of computational fluid dynamics into the computation of complex three-dimensional flows.

  18. Doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against HER 2/neu in a murine mammary carcinoma model

    PubMed Central

    Eralp, Yesim; Wang, Xiaoyan; Wang, Jian-Ping; Maughan, Maureen F; Polo, John M; Lachman, Lawrence B

    2004-01-01

    Introduction The purpose of the present study was to determine whether cytotoxic chemotherapeutic agents administered prior to immunotherapy with gene vaccines could augment the efficacy of the vaccines. Methods Mice were injected in the mammary fat pad with an aggressive breast tumor cell line that expresses HER2/neu. The mice were treated 3 days later with a noncurative dose of either doxorubicin or paclitaxel, and the following day with a gene vaccine to HER2/neu. Two more doses of vaccine were given 14 days apart. Two types of gene vaccines were tested: a plasmid vaccine encoding a self-replicating RNA (replicon) of Sindbis virus (SINCP), in which the viral structural proteins were replaced by the gene for neu; and a viral replicon particle derived from an attenuated strain of Venezuelan equine encephalitis virus, containing a replicon RNA in which the Venezuelan equine encephalitis virus structural proteins were replaced by the gene for neu. Results Neither vaccination alone nor chemotherapy alone significantly reduced the growth of the mammary carcinoma. In contrast, chemotherapy followed by vaccination reduced tumor growth by a small, but significant amount. Antigen-specific CD8+ T lymphocytes were induced by the combined treatment, indicating that the control of tumor growth was most probably due to an immunological mechanism. The results demonstrated that doxorubicin and paclitaxel, commonly used chemotherapeutic agents for the treatment of breast cancer, when used at immunomodulating doses augmented the antitumor efficacy of gene vaccines directed against HER2/neu. Conclusions The combination of chemotherapeutic agents plus vaccine immunotherapy may induce a tumor-specific immune response that could be beneficial for the adjuvant treatment of patients with minimal residual disease. The regimen warrants further evaluation in a clinical setting. PMID:15217493

  19. Phoenix model

    EPA Science Inventory

    Phoenix (formerly referred to as the Second Generation Model or SGM) is a global general equilibrium model designed to analyze energy-economy-climate related questions and policy implications in the medium- to long-term. This model disaggregates the global economy into 26 industr...

  20. Waveguide model

    NASA Technical Reports Server (NTRS)

    1981-01-01

    A model is presented which quantifies the electromagnetic modes (field configurations) in the immediate vicinity of the rectenna element. Specifically, the waveguide model characterizes the electromagnetic modes generated by planar waves normal to the array. The model applies only to incidence normal to the array.

  1. Radiation Models

    ERIC Educational Resources Information Center

    James, W. G. G.

    1970-01-01

    Discusses the historical development of both the wave and the corpuscular photon model of light. Suggests that students should be informed that the two models are complementary and that each model successfully describes a wide range of radiation phenomena. Cites 19 references which might be of interest to physics teachers and students. (LC)

  2. Genome Sequence of the Saprophyte Leptospira Biflexa Provides Insights into the Evolution of Leptospira and the Pathogenesis of Leptospirosis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leptospira biflexa is a free-living spirochete found broadly distributed in aquatic environments, and is the primary model for studying gene function in Leptospira spp. The L. biflexa genome has 3,590 protein-coding genes (excluding transposases) distributed across three circular replicons: two of w...

  3. Whole-genome Sequencing of the Saprophyte L. biflexa and Comparative Genomics of Pathogoenic and Saprophytic Leptospira Species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We present the complete genome sequences of Leptospira biflexa, a saprophyte that is used as a model for gene functional analysis in Leptospira spp. The L. biflexa genome has 3,730 protein-coding genes distributed across three circular replicons: two of which are chromosomes (3,600- and 278-kb in si...

  4. Hydrological models are mediating models

    NASA Astrophysics Data System (ADS)

    Babel, L. V.; Karssenberg, D.

    2013-08-01

    Despite the increasing role of models in hydrological research and decision-making processes, only few accounts of the nature and function of models exist in hydrology. Earlier considerations have traditionally been conducted while making a clear distinction between physically-based and conceptual models. A new philosophical account, primarily based on the fields of physics and economics, transcends classes of models and scientific disciplines by considering models as "mediators" between theory and observations. The core of this approach lies in identifying models as (1) being only partially dependent on theory and observations, (2) integrating non-deductive elements in their construction, and (3) carrying the role of instruments of scientific enquiry about both theory and the world. The applicability of this approach to hydrology is evaluated in the present article. Three widely used hydrological models, each showing a different degree of apparent physicality, are confronted to the main characteristics of the "mediating models" concept. We argue that irrespective of their kind, hydrological models depend on both theory and observations, rather than merely on one of these two domains. Their construction is additionally involving a large number of miscellaneous, external ingredients, such as past experiences, model objectives, knowledge and preferences of the modeller, as well as hardware and software resources. We show that hydrological models convey the role of instruments in scientific practice by mediating between theory and the world. It results from these considerations that the traditional distinction between physically-based and conceptual models is necessarily too simplistic and refers at best to the stage at which theory and observations are steering model construction. The large variety of ingredients involved in model construction would deserve closer attention, for being rarely explicitly presented in peer-reviewed literature. We believe that devoting

  5. Model Reduction in Groundwater Modeling

    NASA Astrophysics Data System (ADS)

    Yeh, W. W. G.

    2014-12-01

    Model reduction has been shown to be a very effective method for reducing the computational burden of large-scale simulations. Model reduction techniques preserve much of the physical knowledge of the system and primarily seek to remove components from the model that do not provide significant information of interest. Proper Orthogonal Decomposition (POD) is a model reduction technique by which a system of ordinary equations is projected onto a much smaller subspace in such a way that the span of the subspace is equal to the span of the original full model space. Basically, the POD technique selects a small number of orthonormal basis functions (principal components) that span the spatial variability of the solutions. In this way the state variable (head) is approximated by a linear combination of these basis functions and, using a Galerkin projection, the dimension of the problem is significantly reduced. It has been shown that for a highly discritized model, the reduced model can be two to three orders of magnitude smaller than the original model and runs 1,000 faster. More importantly, the reduced model captures the dominating characteristics of the full model and produces sufficiently accurate solutions. One of the major tasks in the development of the reduced model is the selection of snapshots which are used to determine the dominant eigenvectors. This paper discusses ways to optimize the snapshot selection. Additionally, the paper also discusses applications of the reduced model to parameter estimation, Monte Carlo simulation and experimental design in groundwater modeling.

  6. Model Experiments and Model Descriptions

    NASA Technical Reports Server (NTRS)

    Jackman, Charles H.; Ko, Malcolm K. W.; Weisenstein, Debra; Scott, Courtney J.; Shia, Run-Lie; Rodriguez, Jose; Sze, N. D.; Vohralik, Peter; Randeniya, Lakshman; Plumb, Ian

    1999-01-01

    The Second Workshop on Stratospheric Models and Measurements Workshop (M&M II) is the continuation of the effort previously started in the first Workshop (M&M I, Prather and Remsberg [1993]) held in 1992. As originally stated, the aim of M&M is to provide a foundation for establishing the credibility of stratospheric models used in environmental assessments of the ozone response to chlorofluorocarbons, aircraft emissions, and other climate-chemistry interactions. To accomplish this, a set of measurements of the present day atmosphere was selected. The intent was that successful simulations of the set of measurements should become the prerequisite for the acceptance of these models as having a reliable prediction for future ozone behavior. This section is divided into two: model experiment and model descriptions. In the model experiment, participant were given the charge to design a number of experiments that would use observations to test whether models are using the correct mechanisms to simulate the distributions of ozone and other trace gases in the atmosphere. The purpose is closely tied to the needs to reduce the uncertainties in the model predicted responses of stratospheric ozone to perturbations. The specifications for the experiments were sent out to the modeling community in June 1997. Twenty eight modeling groups responded to the requests for input. The first part of this section discusses the different modeling group, along with the experiments performed. Part two of this section, gives brief descriptions of each model as provided by the individual modeling groups.

  7. Modeling Pharmacokinetics.

    PubMed

    Bois, Frederic Y; Brochot, Céline

    2016-01-01

    Pharmacokinetics is the study of the fate of xenobiotics in a living organism. Physiologically based pharmacokinetic (PBPK) models provide realistic descriptions of xenobiotics' absorption, distribution, metabolism, and excretion processes. They model the body as a set of homogeneous compartments representing organs, and their parameters refer to anatomical, physiological, biochemical, and physicochemical entities. They offer a quantitative mechanistic framework to understand and simulate the time-course of the concentration of a substance in various organs and body fluids. These models are well suited for performing extrapolations inherent to toxicology and pharmacology (e.g., between species or doses) and for integrating data obtained from various sources (e.g., in vitro or in vivo experiments, structure-activity models). In this chapter, we describe the practical development and basic use of a PBPK model from model building to model simulations, through implementation with an easily accessible free software. PMID:27311461

  8. Bioactive Cembrane Derivatives from the Indian Ocean Soft Coral, Sinularia kavarattiensis

    PubMed Central

    Lillsunde, Katja-Emilia; Festa, Carmen; Adel, Harshada; De Marino, Simona; Lombardi, Valter; Tilvi, Supriya; Nawrot, Dorota A.; Zampella, Angela; D’Souza, Lisette; D’Auria, Maria Valeria; Tammela, Päivi

    2014-01-01

    Marine organisms and their metabolites represent a unique source of potential pharmaceutical substances. In this study, we examined marine-derived substances for their bioactive properties in a cell-based Chikungunya virus (CHIKV) replicon model and for in vitro anti-inflammatory activity. In the screening of a marine sample library, crude extracts from the Indian soft coral, Sinularia kavarattiensis, showed promising activity against the CHIKV replicon. Bioassay-guided chemical fractionation of S. kavarattiensis resulted in the isolation of six known norcembranoids (1–6) and one new compound, named kavaranolide (7). The structures were elucidated on the basis of NMR and MS spectroscopic data. Compounds 1–3 and 5–7 were evaluated for their replicon-inhibiting potential in the CHIKV model by using a luminescence-based detection technique and live cell imaging. Compounds 1 and 2 showed moderate inhibition of the CHIKV replicon, but imaging studies also revealed cytotoxic properties. Moreover, the effects of the isolated compounds on primary microglial cells, an experimental model for neuroinflammation, were evaluated. Compound 2 was shown to modulate the immune response in microglial cells and to possess potential anti-inflammatory properties by dose-dependently reducing the release of pro- and anti-inflammatory cytokines. PMID:25056629

  9. Ventilation Model

    SciTech Connect

    H. Yang

    1999-11-04

    The purpose of this analysis and model report (AMR) for the Ventilation Model is to analyze the effects of pre-closure continuous ventilation in the Engineered Barrier System (EBS) emplacement drifts and provide heat removal data to support EBS design. It will also provide input data (initial conditions, and time varying boundary conditions) for the EBS post-closure performance assessment and the EBS Water Distribution and Removal Process Model. The objective of the analysis is to develop, describe, and apply calculation methods and models that can be used to predict thermal conditions within emplacement drifts under forced ventilation during the pre-closure period. The scope of this analysis includes: (1) Provide a general description of effects and heat transfer process of emplacement drift ventilation. (2) Develop a modeling approach to simulate the impacts of pre-closure ventilation on the thermal conditions in emplacement drifts. (3) Identify and document inputs to be used for modeling emplacement ventilation. (4) Perform calculations of temperatures and heat removal in the emplacement drift. (5) Address general considerations of the effect of water/moisture removal by ventilation on the repository thermal conditions. The numerical modeling in this document will be limited to heat-only modeling and calculations. Only a preliminary assessment of the heat/moisture ventilation effects and modeling method will be performed in this revision. Modeling of moisture effects on heat removal and emplacement drift temperature may be performed in the future.

  10. Climate Models

    NASA Technical Reports Server (NTRS)

    Druyan, Leonard M.

    2012-01-01

    Climate models is a very broad topic, so a single volume can only offer a small sampling of relevant research activities. This volume of 14 chapters includes descriptions of a variety of modeling studies for a variety of geographic regions by an international roster of authors. The climate research community generally uses the rubric climate models to refer to organized sets of computer instructions that produce simulations of climate evolution. The code is based on physical relationships that describe the shared variability of meteorological parameters such as temperature, humidity, precipitation rate, circulation, radiation fluxes, etc. Three-dimensional climate models are integrated over time in order to compute the temporal and spatial variations of these parameters. Model domains can be global or regional and the horizontal and vertical resolutions of the computational grid vary from model to model. Considering the entire climate system requires accounting for interactions between solar insolation, atmospheric, oceanic and continental processes, the latter including land hydrology and vegetation. Model simulations may concentrate on one or more of these components, but the most sophisticated models will estimate the mutual interactions of all of these environments. Advances in computer technology have prompted investments in more complex model configurations that consider more phenomena interactions than were possible with yesterday s computers. However, not every attempt to add to the computational layers is rewarded by better model performance. Extensive research is required to test and document any advantages gained by greater sophistication in model formulation. One purpose for publishing climate model research results is to present purported advances for evaluation by the scientific community.

  11. Phenomenological models

    SciTech Connect

    Braby, L.A.

    1990-09-01

    The biological effects of ionizing radiation exposure are the result of a complex sequence of physical, chemical, biochemical, and physiological interactions. One way to begin a search for an understanding of health effects of radiation is through the development of phenomenological models of the response. Many models have been presented and tested in the slowly evolving process of characterizing cellular response. A range of models covering different endpoints and phenomena has developed in parallel. Many of these models employ similar assumptions about some underlying processes while differing about the nature of others. An attempt is made to organize many of the models into groups with similar features and to compare the consequences of those features with the actual experimental observations. It is assumed that by showing that some assumptions are inconsistent with experimental observations, the job of devising and testing mechanistic models can be simplified. 43 refs., 13 figs.

  12. Building models

    SciTech Connect

    Burr, M.T.

    1995-04-01

    As developers make progress on independent power projects around the world, models for success are beginning to emerge. Different models are evolving to create ownership structures that accomoate a complex system of regulatory requirements. Other frameworks make use of previously untapped fuel resources, or establish new sources of financing; however, not all models may be applied to a given project. This article explores how developers are finding new alternatives for overcoming development challenges that are common to projects in many countries.

  13. Ventilation Model

    SciTech Connect

    V. Chipman

    2002-10-05

    The purpose of the Ventilation Model is to simulate the heat transfer processes in and around waste emplacement drifts during periods of forced ventilation. The model evaluates the effects of emplacement drift ventilation on the thermal conditions in the emplacement drifts and surrounding rock mass, and calculates the heat removal by ventilation as a measure of the viability of ventilation to delay the onset of peak repository temperature and reduce its magnitude. The heat removal by ventilation is temporally and spatially dependent, and is expressed as the fraction of heat carried away by the ventilation air compared to the fraction of heat produced by radionuclide decay. One minus the heat removal is called the wall heat fraction, or the remaining amount of heat that is transferred via conduction to the surrounding rock mass. Downstream models, such as the ''Multiscale Thermohydrologic Model'' (BSC 2001), use the wall heat fractions as outputted from the Ventilation Model to initialize their post-closure analyses. The Ventilation Model report was initially developed to analyze the effects of preclosure continuous ventilation in the Engineered Barrier System (EBS) emplacement drifts, and to provide heat removal data to support EBS design. Revision 00 of the Ventilation Model included documentation of the modeling results from the ANSYS-based heat transfer model. The purposes of Revision 01 of the Ventilation Model are: (1) To validate the conceptual model for preclosure ventilation of emplacement drifts and verify its numerical application in accordance with new procedural requirements as outlined in AP-SIII-10Q, Models (Section 7.0). (2) To satisfy technical issues posed in KTI agreement RDTME 3.14 (Reamer and Williams 2001a). Specifically to demonstrate, with respect to the ANSYS ventilation model, the adequacy of the discretization (Section 6.2.3.1), and the downstream applicability of the model results (i.e. wall heat fractions) to initialize post

  14. Model Selection for Geostatistical Models

    SciTech Connect

    Hoeting, Jennifer A.; Davis, Richard A.; Merton, Andrew A.; Thompson, Sandra E.

    2006-02-01

    We consider the problem of model selection for geospatial data. Spatial correlation is typically ignored in the selection of explanatory variables and this can influence model selection results. For example, the inclusion or exclusion of particular explanatory variables may not be apparent when spatial correlation is ignored. To address this problem, we consider the Akaike Information Criterion (AIC) as applied to a geostatistical model. We offer a heuristic derivation of the AIC in this context and provide simulation results that show that using AIC for a geostatistical model is superior to the often used approach of ignoring spatial correlation in the selection of explanatory variables. These ideas are further demonstrated via a model for lizard abundance. We also employ the principle of minimum description length (MDL) to variable selection for the geostatistical model. The effect of sampling design on the selection of explanatory covariates is also explored.

  15. Budget Model.

    ERIC Educational Resources Information Center

    Washington State Board for Community Coll. Education, Olympia.

    Computerized formula-driven budget models are used by the Washington community college system to define resource needs for legislative budget requests and to distribute legislative appropriations among 22 community college districts. This manual outlines the sources of information needed to operate the model and illustrates the principles on which…

  16. Dispersion Modeling.

    ERIC Educational Resources Information Center

    Budiansky, Stephen

    1980-01-01

    This article discusses the need for more accurate and complete input data and field verification of the various models of air pollutant dispension. Consideration should be given to changing the form of air quality standards based on enhanced dispersion modeling techniques. (Author/RE)

  17. Modeling Sunspots

    ERIC Educational Resources Information Center

    Oh, Phil Seok; Oh, Sung Jin

    2013-01-01

    Modeling in science has been studied by education researchers for decades and is now being applied broadly in school. It is among the scientific practices featured in the "Next Generation Science Standards" ("NGSS") (Achieve Inc. 2013). This article describes modeling activities in an extracurricular science club in a high…

  18. Turbulence modeling

    NASA Technical Reports Server (NTRS)

    Bardina, Jorge E.

    1995-01-01

    The objective of this work is to develop, verify, and incorporate the baseline two-equation turbulence models which account for the effects of compressibility into the three-dimensional Reynolds averaged Navier-Stokes (RANS) code and to provide documented descriptions of the models and their numerical procedures so that they can be implemented into 3-D CFD codes for engineering applications.

  19. Zitterbewegung modeling

    SciTech Connect

    Hestenes, D. )

    1993-03-01

    Guidelines for constructing point particle models of the electron with [ital zitterbewegung] and other features of the Dirac theory are discussed. Such models may at least be useful approximations to the Dirac theory, but the more exciting possibility is that this approach may lead to a more fundamental reality. 6 refs.

  20. Phonological Models.

    ERIC Educational Resources Information Center

    Ballard, W.L.

    1968-01-01

    The article discusses models of synchronic and diachronic phonology and suggests changes in them. The basic generative model of phonology is outlined with the author's reinterpretations. The systematic phonemic level is questioned in terms of its unreality with respect to linguistic performance and its lack of validity with respect to historical…

  1. OSPREY Model

    SciTech Connect

    Veronica J. Rutledge

    2013-01-01

    The absence of industrial scale nuclear fuel reprocessing in the U.S. has precluded the necessary driver for developing the advanced simulation capability now prevalent in so many other countries. Thus, it is essential to model complex series of unit operations to simulate, understand, and predict inherent transient behavior and feedback loops. A capability of accurately simulating the dynamic behavior of advanced fuel cycle separation processes will provide substantial cost savings and many technical benefits. The specific fuel cycle separation process discussed in this report is the off-gas treatment system. The off-gas separation consists of a series of scrubbers and adsorption beds to capture constituents of interest. Dynamic models are being developed to simulate each unit operation involved so each unit operation can be used as a stand-alone model and in series with multiple others. Currently, an adsorption model has been developed within Multi-physics Object Oriented Simulation Environment (MOOSE) developed at the Idaho National Laboratory (INL). Off-gas Separation and REcoverY (OSPREY) models the adsorption of off-gas constituents for dispersed plug flow in a packed bed under non-isothermal and non-isobaric conditions. Inputs to the model include gas, sorbent, and column properties, equilibrium and kinetic data, and inlet conditions. The simulation outputs component concentrations along the column length as a function of time from which breakthrough data is obtained. The breakthrough data can be used to determine bed capacity, which in turn can be used to size columns. It also outputs temperature along the column length as a function of time and pressure drop along the column length. Experimental data and parameters were input into the adsorption model to develop models specific for krypton adsorption. The same can be done for iodine, xenon, and tritium. The model will be validated with experimental breakthrough curves. Customers will be given access to

  2. SYSTEMIC, MUCOSAL AND HETEROTYPIC IMMUNE INDUCTION IN MICE INOCULATED WITH VENEZUELAN EQUINE ENCEPHALITIS REPLICONS EXPRESSING NORWALK VIRUS-LIKE PARTICLES. (R826139)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  3. Phenomenological models.

    PubMed

    Braby, L A

    1991-01-01

    The biological effects of ionizing radiation exposure are the result of a complex sequence of physical, chemical, biochemical, and physiological interactions which are modified by characteristics of the radiation, the timing of its administration, the chemical and physical environment, and the nature of the biological system. However, it is generally agreed that the health effects in animals originate from changes in individual cells, or possibly small groups of cells, and that these cellular changes are initiated by ionizations and excitations produced by the passage of charged particles through the cells. One way to begin a search for an understanding of health effects of radiation is through the development of phenomenological models of the response. Many models have been presented and tested in the slowly evolving process of characterizing cellular response. Different phenomena (LET dependence, dose rate effect, oxygen effect etc.) and different end points (cell survival, aberration formation, transformation, etc.) have been observed, and no single model has been developed to cover all of them. Instead, a range of models covering different end points and phenomena have developed in parallel. Many of these models employ similar assumptions about some underlying processes while differing about the nature of others. An attempt is made to organize many of the models into groups with similar features and to compare the consequences of those features with the actual experimental observations. It is assumed that by showing that some assumptions are inconsistent with experimental observations, the job of devising and testing mechanistic models can be simplified. PMID:1811477

  4. Stereometric Modelling

    NASA Astrophysics Data System (ADS)

    Grimaldi, P.

    2012-07-01

    These mandatory guidelines are provided for preparation of papers accepted for publication in the series of Volumes of The The stereometric modelling means modelling achieved with : - the use of a pair of virtual cameras, with parallel axes and positioned at a mutual distance average of 1/10 of the distance camera-object (in practice the realization and use of a stereometric camera in the modeling program); - the shot visualization in two distinct windows - the stereoscopic viewing of the shot while modelling. Since the definition of "3D vision" is inaccurately referred to as the simple perspective of an object, it is required to add the word stereo so that "3D stereo vision " shall stand for "three-dimensional view" and ,therefore, measure the width, height and depth of the surveyed image. Thanks to the development of a stereo metric model , either real or virtual, through the "materialization", either real or virtual, of the optical-stereo metric model made visible with a stereoscope. It is feasible a continuous on line updating of the cultural heritage with the help of photogrammetry and stereometric modelling. The catalogue of the Architectonic Photogrammetry Laboratory of Politecnico di Bari is available on line at: http://rappresentazione.stereofot.it:591/StereoFot/FMPro?-db=StereoFot.fp5&-lay=Scheda&-format=cerca.htm&-view

  5. Model hydrographs

    USGS Publications Warehouse

    Mitchell, W.D.

    1972-01-01

    Model hydrographs are composed of pairs of dimensionless ratios, arrayed in tabular form, which, when modified by the appropriate values of rainfall exceed and by the time and areal characteristics of the drainage basin, satisfactorily represent the flood hydrograph for the basin. Model bydrographs are developed from a dimensionless translation hydrograph, having a time base of T hours and appropriately modified for storm duration by routing through reservoir storage, S=kOx. Models fall into two distinct classes: (1) those for which the value of x is unity and which have all the characteristics of true unit hydrographs and (2) those for which the value of x is other than unity and to which the unit-hydrograph principles of proportionality and superposition do not apply. Twenty-six families of linear models and eight families of nonlinear models in tabular form from the principal subject of this report. Supplemental discussions describe the development of the models and illustrate their application. Other sections of the report, supplemental to the tables, describe methods of determining the hydrograph characteristics, T, k, and x, both from observed hydrograph and from the physical characteristics of the drainage basin. Five illustrative examples of use show that the models, when properly converted to incorporate actual rainfall excess and the time and areal characteristics of the drainage basins, do indeed satisfactorily represent the observed flood hydrographs for the basins.

  6. Energy Models

    EPA Science Inventory

    Energy models characterize the energy system, its evolution, and its interactions with the broader economy. The energy system consists of primary resources, including both fossil fuels and renewables; power plants, refineries, and other technologies to process and convert these r...

  7. Programming models

    SciTech Connect

    Daniel, David J; Mc Pherson, Allen; Thorp, John R; Barrett, Richard; Clay, Robert; De Supinski, Bronis; Dube, Evi; Heroux, Mike; Janssen, Curtis; Langer, Steve; Laros, Jim

    2011-01-14

    A programming model is a set of software technologies that support the expression of algorithms and provide applications with an abstract representation of the capabilities of the underlying hardware architecture. The primary goals are productivity, portability and performance.

  8. The French Revolution and "Revisionism."

    ERIC Educational Resources Information Center

    Langlois, Claude

    1990-01-01

    Outlines revisionist interpretations of the French Revolution that challenged the dominant historiographical tradition during the 1950s and 1960s. Distinguishes four central characteristics of revisionist works. Identifies a key split in current French Revolution historiography between reflection on nineteenth-century…

  9. Mechanistic models

    SciTech Connect

    Curtis, S.B.

    1990-09-01

    Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) ``interaction`` of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

  10. Mechanistic models

    SciTech Connect

    Curtis, S.B.

    1990-09-01

    Several models and theories are reviewed that incorporate the idea of radiation-induced lesions (repairable and/or irreparable) that can be related to molecular lesions in the DNA molecule. Usually the DNA double-strand or chromatin break is suggested as the critical lesion. In the models, the shoulder on the low-LET survival curve is hypothesized as being due to one (or more) of the following three mechanisms: (1) interaction'' of lesions produced by statistically independent particle tracks; (2) nonlinear (i.e., linear-quadratic) increase in the yield of initial lesions, and (3) saturation of repair processes at high dose. Comparisons are made between the various approaches. Several significant advances in model development are discussed; in particular, a description of the matrix formulation of the Markov versions of the RMR and LPL models is given. The more advanced theories have incorporated statistical fluctuations in various aspects of the energy-loss and lesion-formation process. An important direction is the inclusion of physical and chemical processes into the formulations by incorporating relevant track structure theory (Monte Carlo track simulations) and chemical reactions of radiation-induced radicals. At the biological end, identification of repair genes and how they operate as well as a better understanding of how DNA misjoinings lead to lethal chromosome aberrations are needed for appropriate inclusion into the theories. More effort is necessary to model the complex end point of radiation-induced carcinogenesis.

  11. Modeling reality

    NASA Technical Reports Server (NTRS)

    Denning, Peter J.

    1990-01-01

    Although powerful computers have allowed complex physical and manmade hardware systems to be modeled successfully, we have encountered persistent problems with the reliability of computer models for systems involving human learning, human action, and human organizations. This is not a misfortune; unlike physical and manmade systems, human systems do not operate under a fixed set of laws. The rules governing the actions allowable in the system can be changed without warning at any moment, and can evolve over time. That the governing laws are inherently unpredictable raises serious questions about the reliability of models when applied to human situations. In these domains, computers are better used, not for prediction and planning, but for aiding humans. Examples are systems that help humans speculate about possible futures, offer advice about possible actions in a domain, systems that gather information from the networks, and systems that track and support work flows in organizations.

  12. Supernova models

    SciTech Connect

    Woosley, S.E.; Weaver, T.A.

    1980-01-01

    Recent progress in understanding the observed properties of Type I supernovae as a consequence of the thermonuclear detonation of white dwarf stars and the ensuing decay of the /sup 56/Ni produced therein is reviewed. Within the context of this model for Type I explosions and the 1978 model for Type II explosions, the expected nucleosynthesis and gamma-line spectra from both kinds of supernovae are presented. Finally, a qualitatively new approach to the problem of massive star death and Type II supernovae based upon a combination of rotation and thermonuclear burning is discussed.

  13. Molecular Modeling

    NASA Astrophysics Data System (ADS)

    Holmes, Jon L.

    1999-06-01

    Molecular modeling has trickled down from the realm of pharmaceutical and research laboratories into the realm of undergraduate chemistry instruction. It has opened avenues for the visualization of chemical concepts that previously were difficult or impossible to convey. I am sure that many of you have developed exercises using the various molecular modeling tools. It is the desire of this Journal to become an avenue for you to share these exercises among your colleagues. It is to this end that Ron Starkey has agreed to edit such a column and to publish not only the description of such exercises, but also the software documents they use. The WWW is the obvious medium to distribute this combination and so accepted submissions will appear online as a feature of JCE Internet. Typical molecular modeling exercise: finding conformation energies. Molecular Modeling Exercises and Experiments is the latest feature column of JCE Internet, joining Conceptual Questions and Challenge Problems, Hal's Picks, and Mathcad in the Chemistry Curriculum. JCE Internet continues to seek submissions in these areas of interest and submissions of general interest. If you have developed materials and would like to submit them, please see our Guide to Submissions for more information. The Chemical Education Resource Shelf, Equipment Buyers Guide, and WWW Site Review would also like to hear about chemistry textbooks and software, equipment, and WWW sites, respectively. Please consult JCE Internet Features to learn more about these resources at JCE Online. Email Announcements Would you like to be informed by email when the latest issue of the Journal is available online? when a new JCE Software title is shipping? when a new JCE Internet article has been published or is available for Open Review? when your subscription is about to expire? A new feature of JCE Online makes this possible. Visit our Guestbook to learn how. When

  14. Entrepreneurship Models.

    ERIC Educational Resources Information Center

    Finger Lakes Regional Education Center for Economic Development, Mount Morris, NY.

    This guide describes seven model programs that were developed by the Finger Lakes Regional Center for Economic Development (New York) to meet the training needs of female and minority entrepreneurs to help their businesses survive and grow and to assist disabled and dislocated workers and youth in beginning small businesses. The first three models…

  15. Atmospheric Modeling

    EPA Science Inventory

    Although air quality models have been applied historically to address issues specific to ambient air quality standards (i.e., one criteria pollutant at a time) or welfare (e.g.. acid deposition or visibility impairment). they are inherently multipollutant based. Therefore. in pri...

  16. Ensemble Models

    EPA Science Inventory

    Ensemble forecasting has been used for operational numerical weather prediction in the United States and Europe since the early 1990s. An ensemble of weather or climate forecasts is used to characterize the two main sources of uncertainty in computer models of physical systems: ...

  17. Modeling Convection

    ERIC Educational Resources Information Center

    Ebert, James R.; Elliott, Nancy A.; Hurteau, Laura; Schulz, Amanda

    2004-01-01

    Students must understand the fundamental process of convection before they can grasp a wide variety of Earth processes, many of which may seem abstract because of the scales on which they operate. Presentation of a very visual, concrete model prior to instruction on these topics may facilitate students' understanding of processes that are largely…

  18. Painting models

    NASA Astrophysics Data System (ADS)

    Baart, F.; Donchyts, G.; van Dam, A.; Plieger, M.

    2015-12-01

    The emergence of interactive art has blurred the line between electronic, computer graphics and art. Here we apply this art form to numerical models. Here we show how the transformation of a numerical model into an interactive painting can both provide insights and solve real world problems. The cases that are used as an example include forensic reconstructions, dredging optimization, barrier design. The system can be fed using any source of time varying vector fields, such as hydrodynamic models. The cases used here, the Indian Ocean (HYCOM), the Wadden Sea (Delft3D Curvilinear), San Francisco Bay (3Di subgrid and Delft3D Flexible Mesh), show that the method used is suitable for different time and spatial scales. High resolution numerical models become interactive paintings by exchanging their velocity fields with a high resolution (>=1M cells) image based flow visualization that runs in a html5 compatible web browser. The image based flow visualization combines three images into a new image: the current image, a drawing, and a uv + mask field. The advection scheme that computes the resultant image is executed in the graphics card using WebGL, allowing for 1M grid cells at 60Hz performance on mediocre graphic cards. The software is provided as open source software. By using different sources for a drawing one can gain insight into several aspects of the velocity fields. These aspects include not only the commonly represented magnitude and direction, but also divergence, topology and turbulence .

  19. Modeling Muscles

    ERIC Educational Resources Information Center

    Goodwyn, Lauren; Salm, Sarah

    2007-01-01

    Teaching the anatomy of the muscle system to high school students can be challenging. Students often learn about muscle anatomy by memorizing information from textbooks or by observing plastic, inflexible models. Although these mediums help students learn about muscle placement, the mediums do not facilitate understanding regarding integration of…

  20. Modeling Lessons

    ERIC Educational Resources Information Center

    Casey, Katherine

    2011-01-01

    As teachers learn new pedagogical strategies, they crave explicit demonstrations that show them how the new strategies will work with their students in their classrooms. Successful instructional coaches, therefore, understand the importance of modeling lessons to help teachers develop a vision of effective instruction. The author, an experienced…

  1. Criticality Model

    SciTech Connect

    A. Alsaed

    2004-09-14

    The ''Disposal Criticality Analysis Methodology Topical Report'' (YMP 2003) presents the methodology for evaluating potential criticality situations in the monitored geologic repository. As stated in the referenced Topical Report, the detailed methodology for performing the disposal criticality analyses will be documented in model reports. Many of the models developed in support of the Topical Report differ from the definition of models as given in the Office of Civilian Radioactive Waste Management procedure AP-SIII.10Q, ''Models'', in that they are procedural, rather than mathematical. These model reports document the detailed methodology necessary to implement the approach presented in the Disposal Criticality Analysis Methodology Topical Report and provide calculations utilizing the methodology. Thus, the governing procedure for this type of report is AP-3.12Q, ''Design Calculations and Analyses''. The ''Criticality Model'' is of this latter type, providing a process evaluating the criticality potential of in-package and external configurations. The purpose of this analysis is to layout the process for calculating the criticality potential for various in-package and external configurations and to calculate lower-bound tolerance limit (LBTL) values and determine range of applicability (ROA) parameters. The LBTL calculations and the ROA determinations are performed using selected benchmark experiments that are applicable to various waste forms and various in-package and external configurations. The waste forms considered in this calculation are pressurized water reactor (PWR), boiling water reactor (BWR), Fast Flux Test Facility (FFTF), Training Research Isotope General Atomic (TRIGA), Enrico Fermi, Shippingport pressurized water reactor, Shippingport light water breeder reactor (LWBR), N-Reactor, Melt and Dilute, and Fort Saint Vrain Reactor spent nuclear fuel (SNF). The scope of this analysis is to document the criticality computational method. The criticality

  2. Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

    PubMed Central

    Villarreal, L P

    1991-01-01

    The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and

  3. Modeling Molecules

    NASA Technical Reports Server (NTRS)

    2000-01-01

    The molecule modeling method known as Multibody Order (N) Dynamics, or MBO(N)D, was developed by Moldyn, Inc. at Goddard Space Flight Center through funding provided by the SBIR program. The software can model the dynamics of molecules through technology which stimulates low-frequency molecular motions and properties, such as movements among a molecule's constituent parts. With MBO(N)D, a molecule is substructured into a set of interconnected rigid and flexible bodies. These bodies replace the computation burden of mapping individual atoms. Moldyn's technology cuts computation time while increasing accuracy. The MBO(N)D technology is available as Insight II 97.0 from Molecular Simulations, Inc. Currently the technology is used to account for forces on spacecraft parts and to perform molecular analyses for pharmaceutical purposes. It permits the solution of molecular dynamics problems on a moderate workstation, as opposed to on a supercomputer.

  4. Dendrite Model

    NASA Technical Reports Server (NTRS)

    1999-01-01

    Dr. Donald Gilles, the Discipline Scientist for Materials Science in NASA's Microgravity Materials Science and Applications Department, demonstrates to Carl Dohrman a model of dendrites, the branch-like structures found in many metals and alloys. Dohrman was recently selected by the American Society for Metals International as their 1999 ASM International Foundation National Merit Scholar. The University of Illinois at Urbana-Champaign freshman recently toured NASA's materials science facilities at the Marshall Space Flight Center.

  5. Model checking

    NASA Technical Reports Server (NTRS)

    Dill, David L.

    1995-01-01

    Automatic formal verification methods for finite-state systems, also known as model-checking, successfully reduce labor costs since they are mostly automatic. Model checkers explicitly or implicitly enumerate the reachable state space of a system, whose behavior is described implicitly, perhaps by a program or a collection of finite automata. Simple properties, such as mutual exclusion or absence of deadlock, can be checked by inspecting individual states. More complex properties, such as lack of starvation, require search for cycles in the state graph with particular properties. Specifications to be checked may consist of built-in properties, such as deadlock or 'unspecified receptions' of messages, another program or implicit description, to be compared with a simulation, bisimulation, or language inclusion relation, or an assertion in one of several temporal logics. Finite-state verification tools are beginning to have a significant impact in commercial designs. There are many success stories of verification tools finding bugs in protocols or hardware controllers. In some cases, these tools have been incorporated into design methodology. Research in finite-state verification has been advancing rapidly, and is showing no signs of slowing down. Recent results include probabilistic algorithms for verification, exploitation of symmetry and independent events, and the use symbolic representations for Boolean functions and systems of linear inequalities. One of the most exciting areas for further research is the combination of model-checking with theorem-proving methods.

  6. Modeling biomembranes.

    SciTech Connect

    Plimpton, Steven James; Heffernan, Julieanne; Sasaki, Darryl Yoshio; Frischknecht, Amalie Lucile; Stevens, Mark Jackson; Frink, Laura J. Douglas

    2005-11-01

    Understanding the properties and behavior of biomembranes is fundamental to many biological processes and technologies. Microdomains in biomembranes or ''lipid rafts'' are now known to be an integral part of cell signaling, vesicle formation, fusion processes, protein trafficking, and viral and toxin infection processes. Understanding how microdomains form, how they depend on membrane constituents, and how they act not only has biological implications, but also will impact Sandia's effort in development of membranes that structurally adapt to their environment in a controlled manner. To provide such understanding, we created physically-based models of biomembranes. Molecular dynamics (MD) simulations and classical density functional theory (DFT) calculations using these models were applied to phenomena such as microdomain formation, membrane fusion, pattern formation, and protein insertion. Because lipid dynamics and self-organization in membranes occur on length and time scales beyond atomistic MD, we used coarse-grained models of double tail lipid molecules that spontaneously self-assemble into bilayers. DFT provided equilibrium information on membrane structure. Experimental work was performed to further help elucidate the fundamental membrane organization principles.

  7. 10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. MOVABLE BED SEDIMENTATION MODELS. DOGTOOTH BEND MODEL (MODEL SCALE: 1' = 400' HORIZONTAL, 1' = 100' VERTICAL), AND GREENVILLE BRIDGE MODEL (MODEL SCALE: 1' = 360' HORIZONTAL, 1' = 100' VERTICAL). - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  8. Students' Models of Curve Fitting: A Models and Modeling Perspective

    ERIC Educational Resources Information Center

    Gupta, Shweta

    2010-01-01

    The Models and Modeling Perspectives (MMP) has evolved out of research that began 26 years ago. MMP researchers use Model Eliciting Activities (MEAs) to elicit students' mental models. In this study MMP was used as the conceptual framework to investigate the nature of students' models of curve fitting in a problem-solving environment consisting of…

  9. Biomimetic modelling.

    PubMed Central

    Vincent, Julian F V

    2003-01-01

    Biomimetics is seen as a path from biology to engineering. The only path from engineering to biology in current use is the application of engineering concepts and models to biological systems. However, there is another pathway: the verification of biological mechanisms by manufacture, leading to an iterative process between biology and engineering in which the new understanding that the engineering implementation of a biological system can bring is fed back into biology, allowing a more complete and certain understanding and the possibility of further revelations for application in engineering. This is a pathway as yet unformalized, and one that offers the possibility that engineers can also be scientists. PMID:14561351

  10. Modeling fatigue.

    PubMed Central

    Sumner, Walton; Xu, Jin Zhong

    2002-01-01

    The American Board of Family Practice is developing a patient simulation program to evaluate diagnostic and management skills. The simulator must give temporally and physiologically reasonable answers to symptom questions such as "Have you been tired?" A three-step process generates symptom histories. In the first step, the simulator determines points in time where it should calculate instantaneous symptom status. In the second step, a Bayesian network implementing a roughly physiologic model of the symptom generates a value on a severity scale at each sampling time. Positive, zero, and negative values represent increased, normal, and decreased status, as applicable. The simulator plots these values over time. In the third step, another Bayesian network inspects this plot and reports how the symptom changed over time. This mechanism handles major trends, multiple and concurrent symptom causes, and gradually effective treatments. Other temporal insights, such as observations about short-term symptom relief, require complimentary mechanisms. PMID:12463924

  11. Existing and Future Drugs for the Treatment of the Dark Side of Addiction.

    PubMed

    Koob, George F; Mason, Barbara J

    2016-01-01

    The identification of a heuristic framework for the stages of the addiction cycle that are linked to neurocircuitry changes in pathophysiology includes the binge/intoxication stage, the withdrawal/negative affect stage, and the preoccupation/anticipation (craving) stage, which represent neuroadaptations in three neurocircuits (basal ganglia, extended amygdala, and frontal cortex, respectively). The identification of excellent and validated animal models, the development of human laboratory models, and an enormous surge in our understanding of neurocircuitry and neuropharmacological mechanisms have provided a revisionist view of addiction that emphasizes the loss of brain reward function and gain of stress function that drive negative reinforcement (the dark side of addiction) as a key to compulsive drug seeking. Reversing the dark side of addiction not only explains much of the existing successful pharmacotherapies for addiction but also points to vast new opportunities for future medications to alleviate this major source of human suffering. PMID:26514207

  12. Modeling uncertainty: quicksand for water temperature modeling

    USGS Publications Warehouse

    Bartholow, John M.

    2003-01-01

    Uncertainty has been a hot topic relative to science generally, and modeling specifically. Modeling uncertainty comes in various forms: measured data, limited model domain, model parameter estimation, model structure, sensitivity to inputs, modelers themselves, and users of the results. This paper will address important components of uncertainty in modeling water temperatures, and discuss several areas that need attention as the modeling community grapples with how to incorporate uncertainty into modeling without getting stuck in the quicksand that prevents constructive contributions to policy making. The material, and in particular the reference, are meant to supplement the presentation given at this conference.

  13. Pre-Modeling Ensures Accurate Solid Models

    ERIC Educational Resources Information Center

    Gow, George

    2010-01-01

    Successful solid modeling requires a well-organized design tree. The design tree is a list of all the object's features and the sequential order in which they are modeled. The solid-modeling process is faster and less prone to modeling errors when the design tree is a simple and geometrically logical definition of the modeled object. Few high…

  14. Skylab Model

    NASA Technical Reports Server (NTRS)

    1967-01-01

    This photograph is of a model of the Skylab with the Command/Service Module being docked. In an early effort to extend the use of Apollo for further applications, NASA established the Apollo Applications Program (AAP) in August of 1965. The AAP was to include long duration Earth orbital missions during which astronauts would carry out scientific, technological, and engineering experiments in space by utilizing modified Saturn launch vehicles and the Apollo spacecraft. Established in 1970, the Skylab Program was the forerurner of the AAP. The goals of the Skylab were to enrich our scientific knowledge of the Earth, the Sun, the stars, and cosmic space; to study the effects of weightlessness on living organisms, including man; to study the effects of the processing and manufacturing of materials utilizing the absence of gravity; and to conduct Earth resource observations. The Skylab also conducted 19 selected experiments submitted by high school students. Skylab's 3 different 3-man crews spent up to 84 days in Earth orbit. The Marshall Space Flight Center (MSFC) had responsibility for developing and integrating most of the major components of the Skylab: the Orbital Workshop (OWS), Airlock Module (AM), Multiple Docking Adapter (MDA), Apollo Telescope Mount (ATM), Payload Shroud (PS), and most of the experiments. MSFC was also responsible for providing the Saturn IB launch vehicles for three Apollo spacecraft and crews and a Saturn V launch vehicle for the Skylab.

  15. CISNET lung models: Comparison of model assumptions and model structures

    PubMed Central

    McMahon, Pamela M.; Hazelton, William; Kimmel, Marek; Clarke, Lauren

    2012-01-01

    Sophisticated modeling techniques can be powerful tools to help us understand the effects of cancer control interventions on population trends in cancer incidence and mortality. Readers of journal articles are however rarely supplied with modeling details. Six modeling groups collaborated as part of the National Cancer Institute’s Cancer Intervention and Surveillance Modeling Network (CISNET) to investigate the contribution of US tobacco control efforts towards reducing lung cancer deaths over the period 1975 to 2000. The models included in this monograph were developed independently and use distinct, complementary approaches towards modeling the natural history of lung cancer. The models used the same data for inputs and agreed on the design of the analysis and the outcome measures. This article highlights aspects of the models that are most relevant to similarities of or differences between the results. Structured comparisons can increase the transparency of these complex models. PMID:22882887

  16. Biologically-supported structural model for a viral satellite RNA

    PubMed Central

    Ashton, Peter; Wu, Baodong; D'Angelo, Jessica; Grigull, Jörg; White, K. Andrew

    2015-01-01

    Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons, and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified throughout the length of the satRNA. Notably, a 3′-terminal segment was found to adopt two mutually-exclusive RNA secondary structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small parasitic RNA and provide a valuable structural framework for detailed mechanistic studies. PMID:26384416

  17. Building Mental Models by Dissecting Physical Models

    ERIC Educational Resources Information Center

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to…

  18. I&C Modeling in SPAR Models

    SciTech Connect

    John A. Schroeder

    2012-06-01

    The Standardized Plant Analysis Risk (SPAR) models for the U.S. commercial nuclear power plants currently have very limited instrumentation and control (I&C) modeling [1]. Most of the I&C components in the operating plant SPAR models are related to the reactor protection system. This was identified as a finding during the industry peer review of SPAR models. While the Emergency Safeguard Features (ESF) actuation and control system was incorporated into the Peach Bottom Unit 2 SPAR model in a recent effort [2], various approaches to expend resources for detailed I&C modeling in other SPAR models are investigated.

  19. Comparative Protein Structure Modeling Using Modeller

    PubMed Central

    Eswar, Narayanan; Marti-Renom, Marc A.; Madhusudhan, M.S.; Eramian, David; Shen, Min-yi; Pieper, Ursula

    2014-01-01

    Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:18428767

  20. Forward model nonlinearity versus inverse model nonlinearity

    USGS Publications Warehouse

    Mehl, S.

    2007-01-01

    The issue of concern is the impact of forward model nonlinearity on the nonlinearity of the inverse model. The question posed is, "Does increased nonlinearity in the head solution (forward model) always result in increased nonlinearity in the inverse solution (estimation of hydraulic conductivity)?" It is shown that the two nonlinearities are separate, and it is not universally true that increased forward model nonlinearity increases inverse model nonlinearity. ?? 2007 National Ground Water Association.

  1. Modeling cholera outbreaks

    PubMed Central

    Longini, Ira M.; Morris, J. Glenn

    2014-01-01

    Mathematical modeling can be a valuable tool for studying infectious disease outbreak dynamics and simulating the effects of possible interventions. Here, we describe approaches to modeling cholera outbreaks and how models have been applied to explore intervention strategies, particularly in Haiti. Mathematical models can play an important role in formulating and evaluating complex cholera outbreak response options. Major challenges to cholera modeling are insufficient data for calibrating models and the need to tailor models for different outbreak scenarios. PMID:23412687

  2. Uncertainty Modeling Via Frequency Domain Model Validation

    NASA Technical Reports Server (NTRS)

    Waszak, Martin R.; Andrisani, Dominick, II

    1999-01-01

    Abstract The majority of literature on robust control assumes that a design model is available and that the uncertainty model bounds the actual variations about the nominal model. However, methods for generating accurate design models have not received as much attention in the literature. The influence of the level of accuracy of the uncertainty model on closed loop performance has received even less attention. The research reported herein is an initial step in applying and extending the concept of model validation to the problem of obtaining practical uncertainty models for robust control analysis and design applications. An extension of model validation called 'sequential validation' is presented and applied to a simple spring-mass-damper system to establish the feasibility of the approach and demonstrate the benefits of the new developments.

  3. Model selection for logistic regression models

    NASA Astrophysics Data System (ADS)

    Duller, Christine

    2012-09-01

    Model selection for logistic regression models decides which of some given potential regressors have an effect and hence should be included in the final model. The second interesting question is whether a certain factor is heterogeneous among some subsets, i.e. whether the model should include a random intercept or not. In this paper these questions will be answered with classical as well as with Bayesian methods. The application show some results of recent research projects in medicine and business administration.

  4. China model: Energy modeling the modern dynasty

    SciTech Connect

    Shaw, Jason

    1996-05-01

    In this paper a node-based microeconomic analysis is used to model the Chinese energy system. This model is run across multiple periods employing Lagrangian Relaxation techniques to achieve general equilibrium. Later, carbon dioxide emissions are added and the model is run to answer the question, {open_quotes}How can greenhouse gas emissions be reduced{close_quotes}?

  5. Modeling transient rootzone salinity (SWS Model)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The combined, water quality criteria for irrigation, water and ion processes in soils, and plant and soil response is sufficiently complex that adequate analysis requires computer models. Models for management are also needed but these models must consider that the input requirements must be reasona...

  6. "Bohr's Atomic Model."

    ERIC Educational Resources Information Center

    Willden, Jeff

    2001-01-01

    "Bohr's Atomic Model" is a small interactive multimedia program that introduces the viewer to a simplified model of the atom. This interactive simulation lets students build an atom using an atomic construction set. The underlying design methodology for "Bohr's Atomic Model" is model-centered instruction, which means the central model of the…

  7. Multilevel Model Prediction

    ERIC Educational Resources Information Center

    Frees, Edward W.; Kim, Jee-Seon

    2006-01-01

    Multilevel models are proven tools in social research for modeling complex, hierarchical systems. In multilevel modeling, statistical inference is based largely on quantification of random variables. This paper distinguishes among three types of random variables in multilevel modeling--model disturbances, random coefficients, and future response…

  8. Building mental models by dissecting physical models.

    PubMed

    Srivastava, Anveshna

    2016-01-01

    When students build physical models from prefabricated components to learn about model systems, there is an implicit trade-off between the physical degrees of freedom in building the model and the intensity of instructor supervision needed. Models that are too flexible, permitting multiple possible constructions require greater supervision to ensure focused learning; models that are too constrained require less supervision, but can be constructed mechanically, with little to no conceptual engagement. We propose "model-dissection" as an alternative to "model-building," whereby instructors could make efficient use of supervisory resources, while simultaneously promoting focused learning. We report empirical results from a study conducted with biology undergraduate students, where we demonstrate that asking them to "dissect" out specific conceptual structures from an already built 3D physical model leads to a significant improvement in performance than asking them to build the 3D model from simpler components. Using questionnaires to measure understanding both before and after model-based interventions for two cohorts of students, we find that both the "builders" and the "dissectors" improve in the post-test, but it is the latter group who show statistically significant improvement. These results, in addition to the intrinsic time-efficiency of "model dissection," suggest that it could be a valuable pedagogical tool. PMID:26712513

  9. Geologic Framework Model Analysis Model Report

    SciTech Connect

    R. Clayton

    2000-12-19

    The purpose of this report is to document the Geologic Framework Model (GFM), Version 3.1 (GFM3.1) with regard to data input, modeling methods, assumptions, uncertainties, limitations, and validation of the model results, qualification status of the model, and the differences between Version 3.1 and previous versions. The GFM represents a three-dimensional interpretation of the stratigraphy and structural features of the location of the potential Yucca Mountain radioactive waste repository. The GFM encompasses an area of 65 square miles (170 square kilometers) and a volume of 185 cubic miles (771 cubic kilometers). The boundaries of the GFM were chosen to encompass the most widely distributed set of exploratory boreholes (the Water Table or WT series) and to provide a geologic framework over the area of interest for hydrologic flow and radionuclide transport modeling through the unsaturated zone (UZ). The depth of the model is constrained by the inferred depth of the Tertiary-Paleozoic unconformity. The GFM was constructed from geologic map and borehole data. Additional information from measured stratigraphy sections, gravity profiles, and seismic profiles was also considered. This interim change notice (ICN) was prepared in accordance with the Technical Work Plan for the Integrated Site Model Process Model Report Revision 01 (CRWMS M&O 2000). The constraints, caveats, and limitations associated with this model are discussed in the appropriate text sections that follow. The GFM is one component of the Integrated Site Model (ISM) (Figure l), which has been developed to provide a consistent volumetric portrayal of the rock layers, rock properties, and mineralogy of the Yucca Mountain site. The ISM consists of three components: (1) Geologic Framework Model (GFM); (2) Rock Properties Model (RPM); and (3) Mineralogic Model (MM). The ISM merges the detailed project stratigraphy into model stratigraphic units that are most useful for the primary downstream models and the

  10. Educating with Aircraft Models

    ERIC Educational Resources Information Center

    Steele, Hobie

    1976-01-01

    Described is utilization of aircraft models, model aircraft clubs, and model aircraft magazines to promote student interest in aerospace education. The addresses for clubs and magazines are included. (SL)