Sample records for stalled replication sites

  1. RECQ5 Helicase Cooperates with MUS81 Endonuclease in Processing Stalled Replication Forks at Common Fragile Sites during Mitosis

    DEFF Research Database (Denmark)

    Di Marco, Stefano; Hasanova, Zdenka; Kanagaraj, Radhakrishnan


    The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent...

  2. PTEN Regulates DNA Replication Progression and Stalled Fork Recovery (United States)

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


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

  3. FBH1 Catalyzes Regression of Stalled Replication Forks

    DEFF Research Database (Denmark)

    Fugger, Kasper; Mistrik, Martin; Neelsen, Kai J


    DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression......, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose...

  4. Replisome stall events have shaped the distribution of replication origins in the genomes of yeasts (United States)

    Newman, Timothy J.; Mamun, Mohammed A.; Nieduszynski, Conrad A.; Blow, J. Julian


    During S phase, the entire genome must be precisely duplicated, with no sections of DNA left unreplicated. Here, we develop a simple mathematical model to describe the probability of replication failing due to the irreversible stalling of replication forks. We show that the probability of complete genome replication is maximized if replication origins are evenly spaced, the largest inter-origin distances are minimized, and the end-most origins are positioned close to chromosome ends. We show that origin positions in the yeast Saccharomyces cerevisiae genome conform to all three predictions thereby maximizing the probability of complete replication if replication forks stall. Origin positions in four other yeasts—Kluyveromyces lactis, Lachancea kluyveri, Lachancea waltii and Schizosaccharomyces pombe—also conform to these predictions. Equating failure rates at chromosome ends with those in chromosome interiors gives a mean per nucleotide fork stall rate of ∼5 × 10−8, which is consistent with experimental estimates. Using this value in our theoretical predictions gives replication failure rates that are consistent with data from replication origin knockout experiments. Our theory also predicts that significantly larger genomes, such as those of mammals, will experience a much greater probability of replication failure genome-wide, and therefore will likely require additional compensatory mechanisms. PMID:23963700

  5. Stalled replication forks generate a distinct mutational signature in yeast

    DEFF Research Database (Denmark)

    Larsen, Nicolai B.; Liberti, Sascha E.; Vogel, Ivan


    Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication-associ...

  6. Replication Stalling and Heteroduplex Formation within CAG/CTG Trinucleotide Repeats by Mismatch Repair

    KAUST Repository

    Viterbo, David


    Trinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

  7. 14-3-3 Proteins regulate exonuclease 1-dependent processing of stalled replication forks.

    Directory of Open Access Journals (Sweden)

    Kim Engels


    Full Text Available Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1 processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress.

  8. ETAA1 acts at stalled replication forks to maintain genome integrity (United States)

    Bass, Thomas E.; Luzwick, Jessica W.; Kavanaugh, Gina; Carroll, Clinton; Dungrawala, Huzefa; Glick, Gloria G.; Feldkamp, Michael D.; Putney, Reid; Chazin, Walter J.; Cortez, David


    The ATR checkpoint kinase coordinates cellular responses to DNA replication stress. Budding yeast contain three activators of Mec1 (the ATR orthologue); however, only TOPBP1 is known to activate ATR in vertebrates. We identified ETAA1 as a replication stress response protein in two proteomic screens. ETAA1-deficient cells accumulate double-strand breaks, sister chromatid exchanges, and other hallmarks of genome instability. They are also hyper-sensitive to replication stress and have increased frequencies of replication fork collapse. ETAA1 contains two RPA-interaction motifs that localize ETAA1 to stalled replication forks. It also interacts with several DNA damage response proteins including the BLM/TOP3α/RMI1/RMI2 and ATR/ATRIP complexes. It binds ATR/ATRIP directly using a motif with sequence similarity to the TOPBP1-ATR activation domain; and like TOPBP1, ETAA1 acts as a direct ATR activator. ETAA1 functions in parallel to the TOPBP1/RAD9/HUS1/RAD1 pathway to regulate ATR and maintain genome stability. Thus, vertebrate cells contain at least two ATR activating proteins. PMID:27723720

  9. Histone H2B mono-ubiquitylation maintains genomic integrity at stalled replication forks (United States)

    Northam, Matthew R.; Trujillo, Kelly M.


    Histone modifications play an important role in regulating access to DNA for transcription, DNA repair and DNA replication. A central player in these events is the mono-ubiquitylation of histone H2B (H2Bub1), which has been shown to regulate nucleosome dynamics. Previously, it was shown that H2Bub1 was important for nucleosome assembly onto nascent DNA at active replication forks. In the absence of H2Bub1, incomplete chromatin structures resulted in several replication defects. Here, we report new evidence, which shows that loss of H2Bub1 contributes to genomic instability in yeast. Specifically, we demonstrate that H2Bub1-deficient yeast accumulate mutations at a high frequency under conditions of replicative stress. This phenotype is due to an aberrant DNA Damage Tolerance (DDT) response upon fork stalling. We show that H2Bub1 normally functions to promote error-free translesion synthesis (TLS) mediated by DNA polymerase eta (Polη). Without H2Bub1, DNA polymerase zeta (Polζ) is responsible for a highly mutagenic alternative mechanism. While H2Bub1 does not appear to regulate other DDT pathways, error-free DDT mechanisms are employed by H2Bub1-deficient cells as another means for survival. However, in these instances, the anti-recombinase, Srs2, is essential to prevent the accumulation of toxic HR intermediates that arise in an unconstrained chromatin environment. PMID:27458205

  10. Stalled repair of lesions when present within a clustered DNA damage site

    International Nuclear Information System (INIS)

    Lomax, M.E.; Cunniffe, S.; O'Neill, P.


    Ionising radiation produces clustered DNA damages (two or more lesions within one or two helical turns of the DNA) which could challenge the repair mechanism(s) of the cell. Using purified base excision repair (BER) enzymes and synthetic oligonucleotides a number of recent studies have established the excision of a lesion within clustered damage sites is compromised. Evidence will be presented that the efficiency of repair of lesions within a clustered DNA damage site is reduced, relative to that of the isolated lesions, since the lifetime of both lesions is extended by up to four fold. Simple clustered damage sites, comprised of single-strand breaks, abasic sites and base damages, one or five bases 3' or 5' to each other, were synthesised in oligonucleotides and repair carried out in mammalian cell nuclear extracts. The rate of repair of the single-strand break/abasic site within these clustered damage sites is reduced, mainly due to inhibition of the DNA ligase. The mechanism of repair of the single-strand break/abasic site shows some asymmetry. Repair appears to be by the short-patch BER pathway when the lesions are 5' to each other. In contrast, when the lesions are 3' to each other repair appears to proceed along the long-patch BER pathway. The lesions within the cluster are processed sequentially, the single-strand break/abasic site being repaired before excision of 8-oxoG, limiting the formation of double-strand breaks to <2%. Stalled processing of clustered DNA damage extends the lifetime of the lesions to an extent that could have biological consequences, e.g. if the lesions are still present during transcription and/or at replication mutations could arise

  11. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

    DEFF Research Database (Denmark)

    Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete


    DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed...... knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...... replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling....

  12. Phenotypes of dnaXE145A Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks. (United States)

    Flåtten, Ingvild; Helgesen, Emily; Pedersen, Ida Benedikte; Waldminghaus, Torsten; Rothe, Christiane; Taipale, Riikka; Johnsen, Line; Skarstad, Kirsten


    The Escherichia coli dnaX E145A mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation parE10 The gene for the clamp loader subunits τ and γ, dnaX , but not the mutant dnaX E145A , was found to suppress parE10 (Ts) when overexpressed. Purified mutant protein was found to be functional in vitro , and few phenotypes were found in vivo apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at oriC never reach the terminus in dnaX E145A mutant cells. The SOS response was found to be induced, and a combination of the dnaX E145A mutation with recBC and recA mutations led to reduced viability. The mutant cells exhibited extensive chromosome fragmentation and degradation upon inactivation of recBC and recA , respectively. The results indicate that the dnaX E145A mutant cells suffer from broken replication forks and that these need to be repaired by homologous recombination. We suggest that the dnaX -encoded τ and γ subunits of the clamp loader, or the clamp loader complex itself, has a role in the restart of stalled replication forks without extensive homologous recombination. IMPORTANCE The E. coli clamp loader complex has a role in coordinating the activity of the replisome at the replication fork and loading β-clamps for lagging-strand synthesis. Replication forks frequently encounter obstacles, such as template lesions, secondary structures, and tightly bound protein complexes, which will lead to fork stalling. Some pathways of fork restart have been characterized, but much is still unknown about the actors and mechanisms involved. We have in this work characterized the dnaX E145A clamp loader mutant. We find that the naturally occurring obstacles encountered by a replication fork are not tackled in a proper way by the mutant clamp loader and suggest a role for the clamp loader in the restart of stalled

  13. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio


    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  14. An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication. (United States)

    Kamel, Wael; Akusjärvi, Göran


    Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3' end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host. © 2017 Kamel and Akusjärvi; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation

    International Nuclear Information System (INIS)

    Ikegami, Yosuke; Goto, Hidemasa; Kiyono, Tohru; Enomoto, Masato; Kasahara, Kousuke; Tomono, Yasuko; Tozawa, Keiichi; Morita, Akimichi; Kohri, Kenjiro; Inagaki, Masaki


    We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)-irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. In vitro analyses revealed Ser286 and Ser301 on Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2.

  16. Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage. (United States)

    Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle


    We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Replication Catastrophe

    DEFF Research Database (Denmark)

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


    Proliferating cells rely on the so-called DNA replication checkpoint to ensure orderly completion of genome duplication, and its malfunction may lead to catastrophic genome disruption, including unscheduled firing of replication origins, stalling and collapse of replication forks, massive DNA...... increased DNA replication stress....

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

    DEFF Research Database (Denmark)

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


    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tu...

  19. Inclusion bodies are a site of ebolavirus replication. (United States)

    Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz


    Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

  20. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W


    Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien......" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication......-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types....

  1. A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation. (United States)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W


    Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

  2. The Escherichia coli Tus-Ter replication fork barrier causes site-specific DNA replication perturbation in yeast. (United States)

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


    Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither Tof1-dependent nor counteracted by the Rrm3 helicase. Although the yeast replisome can overcome RF pausing at Tus-Ter modules, this event triggers site-specific homologous recombination that requires the RecQ helicase, Sgs1, for its timely resolution. We propose that Tus-Ter can be utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications.

  3. Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts. (United States)

    Thomas, D C; Veaute, X; Kunkel, T A; Fuchs, R P


    We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Replication product analyses reveal unique termination sites with each damaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct. We also observed a higher proportion of products representing replication of the undamaged strand compared to the damaged strand. This suggests that the undamaged strand is replicated more readily, either by uncoupling the first fork to encounter the lesion or by replication using the fork arriving from the other direction. Also included among replication products are covalently closed monomer-length molecules resistant to cleavage at the AAF-modified Nar I site. This resistance is characteristic of substrates containing the AAF adduct, suggesting that translesion bypass had occurred. Transformation of Escherichia coli cells with the replicated damaged DNA yielded lacZ alpha revertant frequencies significantly above values obtained with undamaged DNA or with damaged DNA not replicated in vitro. This increase was only seen with the substrate modified at the third guanine position. Analysis of mutant DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence. Generation of this position-dependent AAF-induced frameshift error in a human replication system is consistent with previous observations in E. coli suggesting that, after incorporation of dCMP opposite modified guanine in the third position, realignment of the template-primer occurs to form an intermediate with two

  4. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes. (United States)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W


    The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.

  5. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Cuesta-Geijo


    Full Text Available African swine fever virus (ASFV infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection.

  6. Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts.


    Thomas, D C; Veaute, X; Kunkel, T A; Fuchs, R P


    We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Replicati...

  7. Tus-Ter as a tool to study site-specific DNA replication perturbation in eukaryotes

    DEFF Research Database (Denmark)

    Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W


    have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding...... yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells....

  8. Disaster recovery using VMware vSphere Replication and vCenter Site Recovery Manager

    CERN Document Server

    GB, Abhilash


    This is a step-by-step guide that will help you understand disaster recovery using VMware vSphere Replication 5.5 and VMware vCenter Site Recovery Manager (SRM) 5.5. The topics and configuration procedures are accompanied with relevant screenshots, flowcharts, and logical diagrams that makes grasping the concepts easier. This book is a guide for anyone who is keen on using vSphere Replication or vCenter Site Recovery Manager as a disaster recovery solution. This is an excellent handbook for solution architects, administrators, on-field engineers, and support professionals. Although the book as

  9. Ultrastructure of the replication sites of positive-strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Harak, Christian; Lohmann, Volker, E-mail:


    Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.

  10. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M; Lovmand, J


    Two Akv murine leukemia virus-based retroviral vectors with primer binding sites matching tRNA(Gln-1) and tRNA(Lys-3) were constructed. The transduction efficiency of these mutated vectors was found to be comparable to that of a vector carrying the wild-type primer binding site matching t......RNA(Pro). Polymerase chain reaction amplification and sequence analysis of transduced proviruses confirmed the transfer of vectors with mutated primer binding sites and further showed that tRNA(Gln-2) may act efficiently in conjunction with the tRNA(Gln-1) primer binding site. We conclude that murine leukemia virus...... can replicate by using various tRNA molecules as primers and propose primer binding site-tRNA primer interactions to be of major importance for tRNA primer selection. However, efficient primer selection does not require perfect Watson-Crick base pairing at all 18 positions of the primer binding site....

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

    Directory of Open Access Journals (Sweden)

    Olivier Hyrien


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

  12. Theoretical analysis of transcription process with polymerase stalling (United States)

    Li, Jingwei; Zhang, Yunxin


    Experimental evidence shows that in gene transcription RNA polymerase has the possibility to be stalled at a certain position of the transcription template. This may be due to the template damage or protein barriers. Once stalled, polymerase may backtrack along the template to the previous nucleotide to wait for the repair of the damaged site, simply bypass the barrier or damaged site and consequently synthesize an incorrect messenger RNA, or degrade and detach from the template. Thus, the effective transcription rate (the rate to synthesize correct product mRNA) and the transcription effectiveness (the ratio of the effective transcription rate to the effective transcription initiation rate) are both influenced by polymerase stalling events. So far, no theoretical model has been given to discuss the gene transcription process including polymerase stalling. In this study, based on the totally asymmetric simple exclusion process, the transcription process including polymerase stalling is analyzed theoretically. The dependence of the effective transcription rate, effective transcription initiation rate, and transcription effectiveness on the transcription initiation rate, termination rate, as well as the backtracking rate, bypass rate, and detachment (degradation) rate when stalling, are discussed in detail. The results showed that backtracking restart after polymerase stalling is an ideal mechanism to increase both the effective transcription rate and the transcription effectiveness. Without backtracking, detachment of stalled polymerase can also help to increase the effective transcription rate and transcription effectiveness. Generally, the increase of the bypass rate of the stalled polymerase will lead to the decrease of the effective transcription rate and transcription effectiveness. However, when both detachment rate and backtracking rate of the stalled polymerase vanish, the effective transcription rate may also be increased by the bypass mechanism.

  13. PTEN regulates RPA1 and protects DNA replication forks (United States)

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


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

  14. Host DNA damage response factors localize to merkel cell polyomavirus DNA replication sites to support efficient viral DNA replication. (United States)

    Tsang, Sabrina H; Wang, Xin; Li, Jing; Buck, Christopher B; You, Jianxin


    Accumulating evidence indicates a role for Merkel cell polyomavirus (MCPyV) in the development of Merkel cell carcinoma (MCC), making MCPyV the first polyomavirus to be clearly associated with human cancer. With the high prevalence of MCPyV infection and the increasing amount of MCC diagnosis, there is a need to better understand the virus and its oncogenic potential. In this study, we examined the relationship between the host DNA damage response (DDR) and MCPyV replication. We found that components of the ATM- and ATR-mediated DDR pathways accumulate in MCPyV large T antigen (LT)-positive nuclear foci in cells infected with native MCPyV virions. To further study MCPyV replication, we employed our previously established system, in which recombinant MCPyV episomal DNA is autonomously replicated in cultured cells. Similar to native MCPyV infection, where both MCPyV origin and LT are present, the host DDR machinery colocalized with LT in distinct nuclear foci. Immunofluorescence in situ hybridization and bromodeoxyuridine (BrdU) incorporation analysis showed that these DDR proteins and MCPyV LT in fact colocalized at the actively replicating MCPyV replication complexes, which were absent when a replication-defective LT mutant or an MCPyV-origin mutant was introduced in place of wild-type LT or wild-type viral origin. Inhibition of DDR kinases using chemical inhibitors and ATR/ATM small interfering RNA (siRNA) knockdown reduced MCPyV DNA replication without significantly affecting LT expression or the host cell cycle. This study demonstrates that these host DDR factors are important for MCPyV DNA replication, providing new insight into the host machinery involved in the MCPyV life cycle. MCPyV is the first polyomavirus to be clearly associated with human cancer. However, the MCPyV life cycle and its oncogenic mechanism remain poorly understood. In this report, we show that, in cells infected with native MCPyV virions, components of the ATM- and ATR-mediated DDR

  15. Overcoming natural replication barriers: differential helicase requirements. (United States)

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


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

  16. Replicative bypass of abasic site in Escherichia coli and human cells: similarities and differences.

    Directory of Open Access Journals (Sweden)

    Savithri Weerasooriya

    Full Text Available Abasic [apurinic/apyrimidinic (AP] sites are the most common DNA damages, opposite which dAMP is frequently inserted ('A-rule' in Escherichia coli. Nucleotide insertion opposite the AP-site in eukaryotic cells depends on the assay system and the type of cells. Accordingly, a 'C-rule', 'A-rule', or the lack of specificity has been reported. DNA sequence context also modulates nucleotide insertion opposite AP-site. Herein, we have compared replication of tetrahydrofuran (Z, a stable analog of AP-site, in E. coli and human embryonic kidney 293T cells in two different sequences. The efficiency of translesion synthesis or viability of the AP-site construct in E. coli was less than 1%, but it was 7- to 8-fold higher in the GZGTC sequence than in the GTGZC sequence. The difference in viability increased even more in pol V-deficient strains. Targeted one-base deletions occurred in 63% frequency in the GZG and 68% frequency in GZC sequence, which dropped to 49% and 21%, respectively, upon induction of SOS. The full-length products with SOS primarily involved dAMP insertion opposite the AP-site, which occurred in 49% and 71% frequency, respectively, in the GZG and GZC sequence. dAMP insertion, largely carried out by pol V, was more efficient when the AP-site was a stronger replication block. In contrast to these results in E. coli, viability was 2 to 3 orders of magnitude higher in human cells, and the 'A-rule' was more rigidly followed. The AP-site in the GZG and GZC sequences gave 76% and 89%, respectively, Z → T substitutions. In human cells, targeted one-base deletion was undetectable, and dTMP>dCMP were the next preferred nucleotides inserted opposite Z. siRNA knockdown of Rev1 or pol ζ established that both these polymerases are vital for AP-site bypass, as demonstrated by 36-67% reduction in bypass efficiency. However, neither polymerase was indispensable, suggesting roles of additional DNA polymerases in AP-site bypass in human cells.

  17. Intrinsic bent DNA sites in the chromosomal replication origin of Xylella fastidiosa 9a5c

    Directory of Open Access Journals (Sweden)

    F. Gimenes


    Full Text Available The features of the nucleotide sequences in both replication and promoter regions have been investigated in many organisms. Intrinsically bent DNA sites associated with transcription have been described in several prokaryotic organisms. The aim of the present study was to investigate intrinsic bent DNA sites in the segment that holds the chromosomal replication origin, oriC, of Xylella fastidiosa 9a5c. Electrophoretic behavior analyses, as well as in silico analyses of both the 2-D projection and helical parameters, were performed. The chromosomal segment analyzed contains the initial sequence of the rpmH gene, an intergenic region, the dnaA gene, the oriC sequence, and the 5' partial sequence of the dnaN gene. The analysis revealed fragments with reduced electrophoretic mobility, which indicates the presence of curved DNA segments. The analysis of the helical parameter ENDS ratio revealed three bent DNA sites (b1, b2, and b3 located in the rpmH-dnaA intergenic region, the dnaA gene, and the oriC 5' end, respectively. The chromosomal segment of X. fastidiosa analyzed here is rich in phased AT tracts and in CAnT motifs. The 2-D projection indicated a segment whose structure was determined by the cumulative effect of all bent DNA sites. Further, the in silico analysis of the three different bacterial oriC sequences indicated similar negative roll and twist >34.00° values. The DnaA box sequences, and other motifs in them, may be associated with the intrinsic DNA curvature.

  18. Wider stall space affects behavior, lesion scores, and productivity of gestating sows. (United States)

    Salak-Johnson, J L; DeDecker, A E; Levitin, H A; McGarry, B M


    Limited space allowance within the standard gestation stall is an important welfare concern because it restricts the ability of the sow to make postural adjustments and hinders her ability to perform natural behaviors. Therefore, we evaluated the impacts of increasing stall space and/or providing sows the freedom to access a small pen area on sow well-being using multiple welfare metrics. A total of 96 primi- and multiparous crossbred sows were randomly assigned in groups of 4 sows/treatment across 8 replicates to 1 of 3 stall treatments (TRT): standard stall (CTL; dimensions: 61 by 216 cm), width-adjustable stall (flex stall [FLX]; dimensions: adjustable width of 56 to 79 cm by 216 cm), or an individual walk-in/lock-in stall with access to a small communal open-pen area at the rear of the stall (free-access stall [FAS]; dimensions: 69 by 226 cm). Lesion scores, behavior, and immune and productivity traits were measured at various gestational days throughout the study. Total lesion scores were greatest for sows in FAS and least for sows in FLX ( pregnancy progressed, lesion scores increased among sows in CTL ( postural behaviors and sham chew behavior were affected by TRT ( changes in postural behaviors, lesion severity scores, and other sow traits. Moreover, compromised welfare measures found among sows in various stall environments may be partly attributed to the specific constraints of each stall system such as restricted stall space in CTL, insufficient floor space in the open-pen area of the FAS system, and gate design of the FLX (e.g., direction of bars and feeder space). These results also indicate that parity and gestational day are additional factors that may exacerbate the effects of restricted stall space or insufficient pen space, further compromising sow well-being.

  19. Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements. (United States)

    Marschalek, Adriane; Finke, Stefan; Schwemmle, Martin; Mayer, Daniel; Heimrich, Bernd; Stitz, Lothar; Conzelmann, Karl-Klaus


    Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5'-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-beta) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-alpha receptor (IFNAR(-/-)). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR(-/-) mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.

  20. 14 CFR 25.103 - Stall speed. (United States)


    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stall speed. 25.103 Section 25.103... STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Performance § 25.103 Stall speed. (a) The reference stall speed, VSR, is a calibrated airspeed defined by the applicant. VSR may not be less than a 1-g stall...

  1. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmidt, J; Luz, A


    ) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary...... delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta......, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant....

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

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


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

  3. Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places. (United States)

    van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J


    DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

  4. RADX interacts with single-stranded DNA to promote replication fork stability

    DEFF Research Database (Denmark)

    Schubert, Lisa; Ho, Teresa; Hoffmann, Saskia


    has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA-binding factor in human...... cells. RADX binds ssDNA via an N-terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA...

  5. 14 CFR 25.203 - Stall characteristics. (United States)


    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Stall characteristics. 25.203 Section 25.203 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Flight Stalls § 25.203 Stall characteristics. (a) It must...

  6. A role for the weak DnaA binding sites in bacterial replication origins

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Løbner-Olesen, Anders


    between species. In the study by Taylor et al. (2011), new and unexpectedly weak DnaA-boxes were identified within the Caulobacter crescentus origin of replication (Cori). The position of weak and stronger DnaA-boxes follows a pattern seen in Escherichia coli oriC. This raises the possibility...... that bacterial origins might be more alike than previously thought....

  7. Ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells. (United States)

    Junjhon, Jiraphan; Pennington, Janice G; Edwards, Thomas J; Perera, Rushika; Lanman, Jason; Kuhn, Richard J


    During dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. Recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral RNA synthesis is lacking. Integrating techniques in electron tomography and molecular virology, we defined an early period in virus-infected mosquito cells during which the formation of a virus-modified membrane structure, the double-membrane vesicle, is proportional to the rate of viral RNA synthesis. Convoluted membranes were absent in dengue virus-infected C6/36 cells. Electron tomographic reconstructions elucidated a high-resolution view of the replication complexes inside vesicles and allowed us to identify distinct pathways of particle formation. Hence, our findings extend the structural details of dengue virus replication within mosquito cells and highlight their differences from mammalian cells. Dengue virus induces several distinct intracellular membrane structures within the endoplasmic reticulum of mammalian cells. These structures, including double-membrane vesicles and convoluted membranes, are linked, respectively, with viral replication and viral protein processing. However, dengue virus cycles between two disparate animal groups with differing physiologies: mammals and mosquitoes. Using techniques in electron microscopy, we examined the differences between intracellular structures induced by dengue virus in mosquito cells. Additionally, we utilized techniques in molecular virology to temporally link events in virus replication to the formation of these dengue virus-induced membrane structures.

  8. P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites.


    Pal, S K; Chattoraj, D K


    The unit-copy plasmid replicon mini-P1 consists of an origin, a gene for an initiator protein, RepA, and a control locus, incA. Both the origin and the incA locus contain repeat sequences that bind RepA. It has been proposed that the incA repeats control replication by sequestering the rate-limiting RepA initiator protein. Here we show that when the concentration of RepA was increased about fourfold beyond its normal physiological level from an inducible source in trans, the copy number of a ...

  9. Towards observing the encounter of the T7 DNA replication fork with a lesion site at the Single molecule level

    KAUST Repository

    Shirbini, Afnan


    Single-molecule DNA flow-stretching assays have been a powerful approach to study various aspects on the mechanism of DNA replication for more than a decade. This technique depends on flow-induced force on a bead attached to a surface-tethered DNA. The difference in the elastic property between double-strand DNA (long) and single-strand DNA (short) at low regime force allows the observation of the beads motion when the dsDNA is converted to ssDNA by the replisome machinery during DNA replication. Here, I aim to develop an assay to track in real-time the encounter of the bacteriophage T7 replisome with abasic lesion site inserted on the leading strand template. I optimized methods to construct the DNA substrate that contains the abasic site and established the T7 leading strand synthesis at the single molecule level. I also optimized various control experiments to remove any interference from the nonspecific interactions of the DNA with the surface. My work established the foundation to image the encounter of the T7 replisome with abasic site and to characterize how the interactions between the helicase and the polymerase could influence the polymerase proofreading ability and its direct bypass of this highly common DNA damage type.

  10. Clutch-Starting Stalled Research Students (United States)

    Ahern, Kathy; Manathunga, Catherine


    Many research students go through periods where their research seems to stall, their motivation drops, and they seem unable to make any progress. As supervisors, we attempt to remain alert to signs that our student's progress has stalled. Drawing on cognitive strategies, this article explores a problem-solving model supervisors can use to identify…

  11. Basis for an Active Stall Avoidance System

    Directory of Open Access Journals (Sweden)

    Richard Schulze


    Full Text Available A single-stage subsonic compressor was examined with respect to compressor instabilities. During the inception of rotating stall, the transients of the pressure rise and mass flow were measured as well as their hysteresis. The development of the stall cell and the characteristics of the unstable operating range were determined.

  12. Does resilience 'buffer' against depression in prostate cancer patients? A multi-site replication study. (United States)

    Sharpley, C F; Bitsika, V; Wootten, A C; Christie, D R H


    Although psychological resilience has been shown to 'buffer' against depression following major stressors, no studies have reported on this relationship within the prostate cancer (PCa) population, many of whom are at elevated risk of depression, health problems and suicide. To investigate the effects of resilience upon anxiety and depression in the PCa population, postal surveys of 425 PCa patients were collected from two sites: 189 PCa patients at site 1 and 236 at site 2. Background data plus responses to depression and resilience scales were collected. Results indicated that total resilience score was a significant buffer against depression across both sites. Resilience had different underlying component factor structures across sites, but only one (common) factor significantly (inversely) predicted depression. Within that factor, only some specific items significantly predicted depression scores, suggesting a focused relationship between resilience and depression. It may be concluded that measures of resilience may be used to screen depression at-risk PCa patients. These patients might benefit from resilience training to enhance their ability to cope effectively with the stress of their diagnosis and treatment. A focus upon specific aspects of overall resilience may be of further benefit in both these processes. © 2014 John Wiley & Sons Ltd.

  13. Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

    Directory of Open Access Journals (Sweden)

    Ting Wei

    Full Text Available The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa-long hydrophobic region (termed TM2. However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G. Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s oriented parallel to the membrane inner surface.

  14. DVC1 (C1orf124) is a DNA damage-targeting p97 adaptor that promotes ubiquitin-dependent responses to replication blocks

    DEFF Research Database (Denmark)

    Mosbech, Anna; Gibbs-Seymour, Ian; Kagias, Konstantinos


    Ubiquitin-mediated processes orchestrate critical DNA-damage signaling and repair pathways. We identify human DVC1 (C1orf124; Spartan) as a cell cycle-regulated anaphase-promoting complex (APC) substrate that accumulates at stalled replication forks. DVC1 recruitment to sites of replication stress...... synthesis (TLS) DNA polymerase η (Pol η) from monoubiquitylated PCNA. DVC1 knockdown enhances UV light-induced mutagenesis, and depletion of human DVC1 or the Caenorhabditis elegans ortholog DVC-1 causes hypersensitivity to replication stress-inducing agents. Our findings establish DVC1 as a DNA damage...

  15. Exploiting replicative stress to treat cancer

    DEFF Research Database (Denmark)

    Dobbelstein, Matthias; Sørensen, Claus Storgaard


    DNA replication in cancer cells is accompanied by stalling and collapse of the replication fork and signalling in response to DNA damage and/or premature mitosis; these processes are collectively known as 'replicative stress'. Progress is being made to increase our understanding of the mechanisms...... that govern replicative stress, thus providing ample opportunities to enhance replicative stress for therapeutic purposes. Rather than trying to halt cell cycle progression, cancer therapeutics could aim to increase replicative stress by further loosening the checkpoints that remain available to cancer cells...

  16. Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Schmidt, J; Luz, A


    -chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition...

  17. Anaphase onset before complete DNA replication with intact checkpoint responses

    DEFF Research Database (Denmark)

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


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

  18. Construction of hydrogenation stalls for explosions

    Energy Technology Data Exchange (ETDEWEB)

    Raichle, L.


    This report contained explanations for different questions that had been asked by the Association of Chemical Manufacturers. The first item discussed was the pressure occurring in hydrogenation stalls in hydrogen explosions. The pressures actually used were much smaller than the maximum design pressure due to burning gases being allowed to escape from the top and front of the stalls since these areas were open and it could not be assumed that the whole stall space was filled with a 32% hydrogen concentration at the beginning of an explosion. The second item discussed was specifications and rules for the building of hydrogenation stalls. These included the calculations for simple wind pressure according to the Building Code with the usual safety factors and the calculations for an inner pressure of 300 kg/m/sup 2/ with the usual safety factors. An explanation of a stall explosion in Poelitz and reinforced stall construction in Poelitz were two other items that were discussed. Appendix I of the report involved maximum pressures and temperature in hydrogen explosions. Diagram I was involved with this. Appendix II discussed the behavior of a hydrogen flame at high emerging velocities and Appendix III discussed stall construction at Poelitz.

  19. diffReps: detecting differential chromatin modification sites from ChIP-seq data with biological replicates.

    Directory of Open Access Journals (Sweden)

    Li Shen

    Full Text Available ChIP-seq is increasingly being used for genome-wide profiling of histone modification marks. It is of particular importance to compare ChIP-seq data of two different conditions, such as disease vs. control, and identify regions that show differences in ChIP enrichment. We have developed a powerful and easy to use program, called diffReps, to detect those differential sites from ChIP-seq data, with or without biological replicates. In addition, we have developed two useful tools for ChIP-seq analysis in the diffReps package: one for the annotation of the differential sites and the other for finding chromatin modification "hotspots". diffReps is developed in PERL programming language and runs on all platforms as a command line script. We tested diffReps on two different datasets. One is the comparison of H3K4me3 between two human cell lines from the ENCODE project. The other is the comparison of H3K9me3 in a discrete region of mouse brain between cocaine- and saline-treated conditions. The results indicated that diffReps is a highly sensitive program in detecting differential sites from ChIP-seq data.

  20. The Relevance of the Dynamic Stall Effect for Transient

    DEFF Research Database (Denmark)

    Jauch, Clemens; Sørensen, Poul; Bak-Jensen, Birgitte


    This article describes a methodology to quantify the influence of dynamic stall on transient fault operations of active-stall turbines. The model of the dynamic stall effect is introduced briefly. The behaviour of the dynamic stall model during a transient fault operation is described mathematica...

  1. Biomimetic Wind Turbine Design with Lift Enhancing Periodic Stall

    NARCIS (Netherlands)

    Stamhuis, Eize Jan


    A wind turbine includes a rotor; a blade; and a periodic stall system. The periodic stall system selectively moves at least part of the blade in an oscillating motion whereby an angle of incidence continuously varies to invoke periodic stall. The periodic stall system can move the entire blade or

  2. Human Cytomegalovirus UL44 Concentrates at the Periphery of Replication Compartments, the Site of Viral DNA Synthesis


    Strang, Blair L.; Boulant, Steeve; Chang, Lynne; Knipe, David M.; Kirchhausen, Tomas; Coen, Donald M.


    The formation of replication compartments, the subnuclear structures in which the viral DNA genome is replicated, is a hallmark of herpesvirus infections. The localization of proteins and viral DNA within human cytomegalovirus replication compartments is not well characterized. Immunofluorescence analysis demonstrated the accumulation of the viral DNA polymerase subunit UL44 at the periphery of replication compartments and the presence of different populations of UL44 in infected cells. In co...

  3. Prediction of induced vibrations in stall

    Energy Technology Data Exchange (ETDEWEB)

    Thirstrup Petersen, J.; Thomsen, K.; Aagaard Madsen, H. [Risoe National Lab., Wind Energy and Atmospheric Physics Dept., Roskilde (Denmark)


    The main results from recent research in stall induced vibrations are presented. The focus is on the edgewise blade vibrations, which during the last decade have turned out to be a potential threat against the stable operation of stall regulated wind turbines and a fact, which must be dealt with by the designer. The basic physical explanation for the phenomenon and examples of design precaution, which can be taken, are presented. (au)

  4. A Block to Efficient Replication of Simian Immunodeficiency Virus in C8166 Cells Can Be Overcome by Duplication of the NF-kappaB Binding Site. (United States)

    Bellas, R.E.; Li, Y.


    Sequence analysis of the acutely lethal pbj14 strain of simian immunodeficiency virus (SIVpbj14) clone revealed among other differences from its less pathogenic counterparts a duplication of its binding site for nuclear factor kappa B (NF-kappaB) in its long terminal repeats (LTR). We have investigated whether introducing a similar duplication into the pathogenic molecular clone SIV mac239 would alter its biological properties. We compared an SIV which possessed 2 NF-kappaB sites to the wild type, a single NF-kappaB site virus, with respect to its ability to replicate in vitro in established CD4+ T cell lines, primary peripheral blood mononuclear cells (PBMCs), and primary alveolar macrophages. The virus containing 2 NF-kappaB sites exhibited no apparent difference from wild type in established cell lines 174xCEM, MT-2 and MT-4, or in primary PBMC or tissue macrophage cultures. However, the 2 kappaB virus replicated well in the established cell line C8166, while the wild type, 1 kappaB virus replicated very poorly in this cell type, suggesting that duplication of the NF-kappaB site is capable of overcoming a block to efficient replication of SIVmac239 in C8166 cells. Interestingly, Em*, a macrophage tropic SIVmac that differs from SIVmac239 by 9 amino acids in the envelope region yet possesses only one NK-kappaB binding sites, also replicates well in C8166. The data suggest that the replication of wild type SIVmac239 is restricted in C8166 cells, but that this restriction can be overcome either by changes in the LTR or by changes in the envelope region. Copyright 1996 S. Karger AG, Basel

  5. Plasma-based Compressor Stall Control (United States)

    McGowan, Ryan; Corke, Thomas


    The use of dielectric barrier discharge (DBD) plasma actuator casing treatment to prevent or delay stall inception in an axial fan is examined. The actuators are powered by a pulsed-DC waveform which induces a larger peak velocity than a purely AC waveform such as a sine or sawtooth wave. With this system, a high-voltage DC source is supplied to both electrodes, remaining constant in time for the exposed electrode. Meanwhile, the covered electrode is periodically grounded for several microseconds and allowed to rise back to the source DC level. To test the actuators' ability to interact with and modify the formation of stall cells, a facility has been designed and constructed around nonconductive fan blades. The actuators are installed in the fan casing near the blade tips. The instrumentation allows for the measurement of rotating pressure disturbances (traveling stall cells) in this tip gap region as well as fan performance characteristics including pressure rise and flow rate. The casing plasma actuation is found to reduce the correlation of the rotating stall cells, thereby extending the stall margin of the fan. Various azimuthal arrangements of the plasma actuator casing treatment is explored, as well as input voltage levels to the actuator to determine optimum conditions. NASA SBIR Contract NNX14CC12C.

  6. The relevance of the dynamic stall effect for transient fault operations of active-stall wind turbines

    Energy Technology Data Exchange (ETDEWEB)

    Jauch, Clemens; Soerensen, Poul; Jensen, Birgitte Bak


    This article describes a methodology to quantify the influence of dynamic stall on transient fault operations of active-stall turbines. The model of the dynamic stall effect is introduced briefly. The behaviour of the dynamic stall model during a transient fault operation is described mathematically, and from this its effect quantified. Two quantities are chosen to describe the influence of the dynamic stall effect: one is active power and the other is time delay. Subsequently a transient fault scenario is simulated with and without the dynamic stall effect and the differences discussed. From this comparison, the conclusion is drawn that the dynamic stall effect has some influence on the post-fault behaviour of the wind turbine, and it is hence suggested that the dynamic stall effect is considered if an active-stall wind turbine is to be modelled realistically. (Author)

  7. Tissue Sites of Persistent Infection and Active Replication of Equine Infectious Anemia Virus during Acute Disease and Asymptomatic Infection in Experimentally Infected Equids (United States)

    Harrold, Sharon M.; Cook, Sheila J.; Cook, R. Frank; Rushlow, Keith E.; Issel, Charles J.; Montelaro, Ronald C.


    Equine infectious anemia virus (EIAV) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. The dynamics of EIAV viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of EIAV infection and replication in experimentally infected equids during acute disease episodes and during asymptomatic infections in long-term inapparent carriers. To characterize the in vivo site(s) of viral infection and replication, we developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral singly spliced mRNA. Animals were experimentally infected with one of two reference viruses: the animal-passaged field isolate designated EIAVWyo and the virulent cell-adapted strain designated EIAVPV. Tissues and blood cells were isolated during the initial acute disease or from asymptomatic animals and analyzed for viral DNA and RNA levels by the respective quantitative assays. The results of these experiments demonstrated that the appearance of clinical symptoms in experimentally infected equids coincided with rapid widespread seeding of viral infection and replication in a variety of tissues. During acute disease, the predominant cellular site of viral infection and replication was the spleen, which typically accounted for over 90% of the cellular viral burden. In asymptomatic animals, viral DNA and RNA persisted in virtually all tissues tested, but at extremely low levels, a finding indicative of tight but incomplete immune control of EIAV replication. During all disease states, peripheral blood mononuclear cells (PBMC) were found to harbor less than 1% of

  8. Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication (United States)

    Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.


    Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

  9. Load prediction of stall regulated wind turbines

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerck, A.; Dahlberg, J.Aa. [Aeronautical Research Inst. of Sweden, Bromma (Sweden); Carlen, I. [Chalmers Univ. of Technology, Goeteborg (Sweden). Div. of Marine Structural Engineering; Ganander, H. [Teknikgruppen AB, Sollentua (Sweden)


    Measurements of blade loads on a turbine situated in a small wind farm shows that the highest blade loads occur during operation close to the peak power i.e. when the turbine operates in the stall region. In this study the extensive experimental data base has been utilised to compare loads in selected campaigns with corresponding load predictions. The predictions are based on time domain simulations of the wind turbine structure, performed by the aeroelastic code VIDYN. In the calculations a model were adopted in order to include the effects of dynamic stall. This paper describes the work carried out so far within the project and key results. 5 refs, 10 figs

  10. Dynamic stall and 3D effects

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerck, A.; Thor, S.E. [Aeronautical Research Inst. of Sweden, Bromma (Sweden)


    The JOULE II project `Dynamic stall and 3D effects` started in January 1994 and was completed in September 1995. The objective of the project has been to increase the understanding of the three-dimensional and unsteady aerodynamics of stall controlled HAWT`s. The objectives have also been to develop `engineering models` suitable for inclusion into aero-elastic codes. The project included the participation of 13 parties within Europe. This paper describes an overview of the work carried out within the project and key results. 3 refs, 4 figs

  11. Building up and breaking down: mechanisms controlling recombination during replication. (United States)

    Branzei, Dana; Szakal, Barnabas


    The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.

  12. Education stalls and subsequent stalls in African fertility: A descriptive overview

    Directory of Open Access Journals (Sweden)

    Anne Goujon


    Full Text Available Background: Recent stalls in fertility decline have been observed in a few countries in sub-Saharan Africa, and so far no plausible common reason has been identified in the literature. This paper develops the hypothesis that these fertility stalls could be associated with stalls in the progress of education among the women of the relevant cohorts, possibly resulting partly from the Structural Adjustment Programs (SAPs of the 1980s. Methods: We descriptively link the change in the education composition of successive cohorts of young women in sub-Saharan Africa and the recent fertility stalls. We use reconstructed data on population by age, gender, and level of education from www.wittgenstein, and fertility rates from the United Nations. Results: In most sub-Saharan African countries, we observe that the same countries that had fertility stalls had a stall in the progress of education, particularly for young women who were of primary school age during the 1980s, when most of the countries were under structural adjustment. Conversely, stalls in fertility are less common in countries that did not have an education stall, possibly in relation to SAPs. Conclusions: The results point to the possibility of a link between the recent fertility stalls and discontinuities in the improvement of the education of the relevant cohorts, which in turn could be related to the SAPs in the 1980s. This descriptive finding now needs to be corroborated through more detailed cohort-specific fertility analysis. If the education-fertility link can be further established, it will have important implications for the projections of population growth in affected countries.

  13. Calculation of Rotor Performance and Loads Under Stalled Conditions

    National Research Council Canada - National Science Library

    Yeo, Hyeonsoo


    Rotor behavior in stalled conditions is investigated using wind tunnel test data of a 1/10-scale CH-47B/C type rotor, which provides a set of test conditions extending from unstalled to light stall...

  14. Airfoil stall interpreted through linear stability analysis (United States)

    Busquet, Denis; Juniper, Matthew; Richez, Francois; Marquet, Olivier; Sipp, Denis


    Although airfoil stall has been widely investigated, the origin of this phenomenon, which manifests as a sudden drop of lift, is still not clearly understood. In the specific case of static stall, multiple steady solutions have been identified experimentally and numerically around the stall angle. We are interested here in investigating the stability of these steady solutions so as to first model and then control the dynamics. The study is performed on a 2D helicopter blade airfoil OA209 at low Mach number, M 0.2 and high Reynolds number, Re 1.8 ×106 . Steady RANS computation using a Spalart-Allmaras model is coupled with continuation methods (pseudo-arclength and Newton's method) to obtain steady states for several angles of incidence. The results show one upper branch (high lift), one lower branch (low lift) connected by a middle branch, characterizing an hysteresis phenomenon. A linear stability analysis performed around these equilibrium states highlights a mode responsible for stall, which starts with a low frequency oscillation. A bifurcation scenario is deduced from the behaviour of this mode. To shed light on the nonlinear behavior, a low order nonlinear model is created with the same linear stability behavior as that observed for that airfoil.

  15. 16 CFR 1505.50 - Stalled motor testing. (United States)


    ... 16 Commercial Practices 2 2010-01-01 2010-01-01 false Stalled motor testing. 1505.50 Section 1505... USE BY CHILDREN Policies and Interpretations § 1505.50 Stalled motor testing. (a) § 1505.6(e)(4)(ii) requires that a motor-operated toy be tested with the motor stalled if the construction of the toy is such...

  16. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases

    Directory of Open Access Journals (Sweden)

    Ryan Barnes


    Full Text Available Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome replication, but there are numerous situations in which one or more additional DNA polymerases are required to complete genome replication. Polymerases of the Y-family have been extensively studied in the bypass of DNA lesions; however, recent research has revealed that these polymerases play important roles in normal human physiology. Replication stress is widely cited as contributing to genome instability, and is caused by conditions leading to slowed or stalled DNA replication. Common Fragile Sites epitomize “difficult to replicate” genome regions that are particularly vulnerable to replication stress, and are associated with DNA breakage and structural variation. In this review, we summarize the roles of both the replicative and Y-family polymerases in human cells, and focus on how these activities are regulated during normal and perturbed genome replication.

  17. Investigation of the wind climate in connection with double-stall on wind turbines in Tarifa[Spain]; Undersoegelse af vindklima i forbindelse med dobbelt-stall paa vindmoeller i Tarifa

    Energy Technology Data Exchange (ETDEWEB)

    Christensen, T. [ELSAMPROJEKT A/S, Fredericia, (Denmark); Jensen, L.E. [LM Glasfiber A/S, Lunderskov (Denmark)


    This project has compiled data to allow the Danish manufacturers of wind turbines and blades to improve their knowledge of double stall. On the basis of the double stall difficulties different types of turbines using different makes of blades have encountered in the Tarifa area in Southern Spain, meteorological parameters and production data from two turbines have been measured for a local site. Part of the acquired data have been analysed to reach an understanding of why double stall occurs. The analysis strongly suggests that a change in power level due to double stall can be a result of several external factors: (1) Rain cleaning the blades. (2) A more or less random change in the wind speed components uv, or w, which in some cases can affect a - probably - fairly thick boundary layer. (3) A change in the high frequency turbulence where the vortex impact is too insignificant to affect an - almost - randomly - thick boundary layer. (au)

  18. The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for viral replication.

    Directory of Open Access Journals (Sweden)

    Laurence Colin

    Full Text Available Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1 genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105 both exhibit a phorbol 12-myristate 13-acetate (PMA-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

  19. The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication (United States)

    Colin, Laurence; Vandenhoudt, Nathalie; de Walque, Stéphane; Van Driessche, Benoît; Bergamaschi, Anna; Martinelli, Valérie; Cherrier, Thomas; Vanhulle, Caroline; Guiguen, Allan; David, Annie; Burny, Arsène; Herbein, Georges; Pancino, Gianfranco


    Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity. PMID:21526160

  20. Simulation model of an active stall wind turbine controller

    Energy Technology Data Exchange (ETDEWEB)

    Jauch, C.; Hansen, A.D.; Soerensen, P. [Risoe National Lab., Wind Energy Dept., Rosilde (Denmark); Blaabjerg, F. [Aalborg Univ., Inst. of Energy Technology (Denmark)


    This paper describes an active stall wind turbine controller. The objective is to develop a general model of an active stall controller in order to simulate the operation of grid connected active stall wind turbines. The active stall turbine concept and its control strategies are presented and evaluated on the basis of simulations. The presented controller is described for continuous operation under all wind speeds from start-up wind speed to shut doven wind speed. Due to its parametric implementation it is general i.e. it can represent different active stall wind turbine controllers and can be implemented in different simulation tools. (au)

  1. ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication (United States)

    Sowd, Gregory A.; Li, Nancy Yan; Fanning, Ellen


    Mutation of DNA damage checkpoint signaling kinases ataxia telangiectasia-mutated (ATM) or ATM- and Rad3-related (ATR) results in genomic instability disorders. However, it is not well understood how the instability observed in these syndromes relates to DNA replication/repair defects and failed checkpoint control of cell cycling. As a simple model to address this question, we have studied SV40 chromatin replication in infected cells in the presence of inhibitors of ATM and ATR activities. Two-dimensional gel electrophoresis and southern blotting of SV40 chromatin replication products reveal that ATM activity prevents accumulation of unidirectional replication products, implying that ATM promotes repair of replication-associated double strand breaks. ATR activity alleviates breakage of a functional fork as it converges with a stalled fork. The results suggest that during SV40 chromatin replication, endogenous replication stress activates ATM and ATR signaling, orchestrating the assembly of genome maintenance machinery on viral replication intermediates. PMID:23592994

  2. Numerical Investigations of Dynamic Stall Control

    Directory of Open Access Journals (Sweden)



    Full Text Available In this paper we investigated numerically the dynamic stall phenomenon and the possibilities to control it, with application to vertical axis wind turbines (for urban users. The Phenomenon appear at low tip speed ratio (TSR<4 and it has a great impact on structural integrity of the wind turbine and power performances. For this reason we performed a computational study of dynamic stall around NACA 0012 airfoil in pitching motion at relative low Reynolds number (105. Also, we performed the same analysis for four flow control methods: two passive (Gurney flap and slot and two active (blowing jet on the rounded trailing edge and synthetic jet periodically activated. The Results are compared to those of an existing experimental case test.

  3. Dynamic Stall Control Using Plasma Actuators (United States)

    Webb, Nathan; Singhal, Achal; Castaneda, David; Samimy, Mo


    Dynamic stall occurs in many applications, including sharp maneuvers of fixed wing aircraft, wind turbines, and rotorcraft and produces large unsteady aerodynamic loads that can lead to flutter and mechanical failure. This work uses flow control to reduce the unsteady loads by excitation of instabilities in the shear layer over the separated region using nanosecond pulse driven dielectric barrier discharge (NS-DBD) plasma actuators. These actuators have been shown to effectively delay or mitigate static stall. A wide range of flow parameters were explored in the current work: Reynolds number (Re = 167,000 to 500,000), reduced frequency (k = 0.025 to 0.075), and excitation Strouhal number (Ste = 0 to 10). Based on the results, three major conclusions were drawn: (a) Low Strouhal number excitation (Ste eliminated the dynamic stall vortex (DSV), thereby dramatically reducing the unsteady loading. The decrease in the strength of the DSV is achieved by the formation of shear layer coherent structures that bleed the leading-edge vorticity prior to the ejection of the DSV.

  4. DNA polymerases eta and kappa exchange with the polymerase delta holoenzyme to complete common fragile site synthesis. (United States)

    Barnes, Ryan P; Hile, Suzanne E; Lee, Marietta Y; Eckert, Kristin A


    Common fragile sites (CFSs) are inherently unstable genomic loci that are recurrently altered in human tumor cells. Despite their instability, CFS are ubiquitous throughout the human genome and associated with large tumor suppressor genes or oncogenes. CFSs are enriched with repetitive DNA sequences, one feature postulated to explain why these loci are inherently difficult to replicate, and sensitive to replication stress. We have shown that specialized DNA polymerases (Pols) η and κ replicate CFS-derived sequences more efficiently than the replicative Pol δ. However, we lacked an understanding of how these enzymes cooperate to ensure efficient CFS replication. Here, we designed a model of lagging strand replication with RFC loaded PCNA that allows for maximal activity of the four-subunit human Pol δ holoenzyme, Pol η, and Pol κ in polymerase mixing assays. We discovered that Pol η and κ are both able to exchange with Pol δ stalled at repetitive CFS sequences, enhancing Normalized Replication Efficiency. We used this model to test the impact of PCNA mono-ubiquitination on polymerase exchange, and found no change in polymerase cooperativity in CFS replication compared with unmodified PCNA. Finally, we modeled replication stress in vitro using aphidicolin and found that Pol δ holoenzyme synthesis was significantly inhibited in a dose-dependent manner, preventing any replication past the CFS. Importantly, Pol η and κ were still proficient in rescuing this stalled Pol δ synthesis, which may explain, in part, the CFS instability phenotype of aphidicolin-treated Pol η and Pol κ-deficient cells. In total, our data support a model wherein Pol δ stalling at CFSs allows for free exchange with a specialized polymerase that is not driven by PCNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes. (United States)

    Hilton, Benjamin A; Liu, Ji; Cartwright, Brian M; Liu, Yiyong; Breitman, Maya; Wang, Youjie; Jones, Rowdy; Tang, Hui; Rusinol, Antonio; Musich, Phillip R; Zou, Yue


    Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes. © FASEB.

  6. Recombination-dependent concatemeric viral DNA replication. (United States)

    Lo Piano, Ambra; Martínez-Jiménez, María I; Zecchi, Lisa; Ayora, Silvia


    The initiation of viral double stranded (ds) DNA replication involves proteins that recruit and load the replisome at the replication origin (ori). Any block in replication fork progression or a programmed barrier may act as a factor for ori-independent remodelling and assembly of a new replisome at the stalled fork. Then replication initiation becomes dependent on recombination proteins, a process called recombination-dependent replication (RDR). RDR, which is recognized as being important for replication restart and stability in all living organisms, plays an essential role in the replication cycle of many dsDNA viruses. The SPP1 virus, which infects Bacillus subtilis cells, serves as a paradigm to understand the links between replication and recombination in circular dsDNA viruses. SPP1-encoded initiator and replisome assembly proteins control the onset of viral replication and direct the recruitment of host-encoded replisomal components at viral oriL. SPP1 uses replication fork reactivation to switch from ori-dependent θ-type (circle-to-circle) replication to σ-type RDR. Replication fork arrest leads to a double strand break that is processed by viral-encoded factors to generate a D-loop into which a new replisome is assembled, leading to σ-type viral replication. SPP1 RDR proteins are compared with similar proteins encoded by other viruses and their possible in vivo roles are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Structure and Function of the PriC DNA Replication Restart Protein. (United States)

    Wessel, Sarah R; Cornilescu, Claudia C; Cornilescu, Gabriel; Metz, Alice; Leroux, Maxime; Hu, Kaifeng; Sandler, Steven J; Markley, John L; Keck, James L


    Collisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replication restart by recognizing and remodeling abandoned replication forks and reloading the replicative helicase. In vitro, the simplest of these pathways is mediated by the single-domain PriC protein, which, along with the DnaC helicase loader, can load the DnaB replicative helicase onto DNA bound by the single-stranded DNA (ssDNA)-binding protein (SSB). Previous biochemical studies have identified PriC residues that mediate interactions with ssDNA and SSB. However, the mechanisms by which PriC drives DNA replication restart have remained poorly defined due to the limited structural information available for PriC. Here, we report the NMR structure of full-length PriC from Cronobacter sakazakii PriC forms a compact bundle of α-helices that brings together residues involved in ssDNA and SSB binding at adjacent sites on the protein surface. Disruption of these interaction sites and of other conserved residues leads to decreased DnaB helicase loading onto SSB-bound DNA. We also demonstrate that PriC can directly interact with DnaB and the DnaB·DnaC complex. These data lead to a model in which PriC acts as a scaffold for recruiting DnaB·DnaC to SSB/ssDNA sites present at stalled replication forks. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    DEFF Research Database (Denmark)

    Stenfeldt, Anna Carolina; Belsham, Graham


    Foot-and-mouth disease (FMD) is a highly contagious viral infection of significant financial importance to the export and trade of agricultural products. The occurrence of persistently infected ‘‘carriers’’ of FMD-virus (FMDV) in ruminant species adds further complications to disease control....... There have been significant discrepancies in reports regarding the pathogenesis of FMDV infection in cattle with specific emphasis on the anatomical sites involved in early and persistent virus replication. In this study, collection of small biopsy samples from the dorsal soft palate (DSP) of live animals...... was used to investigate the level of FMDV RNA present at this site at sequential time points during the infection. Results were compared to measurements of virus excretion in samples of oropharyngeal fluid collected at corresponding time points. Possible sites of virus persistence were investigated through...

  9. The kissing-loop motif is a preferred site of 5' leader recombination during replication of SL3-3 murine leukemia viruses in mice

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Mikkelsen, J G; Schmidt, J


    , and the upstream part of the 5' untranslated region, enabled us to map recombination sites, guided by distinct scattered nucleotide differences. In 30 of 44 analyzed sequences, recombination was mapped to a 33-nucleotide similarity window coinciding with the kissing-loop stem-loop motif implicated in dimerization...... of the diploid genome. Interestingly, the recombination pattern preference found in replication-competent viruses from T-cell tumors is very similar to the pattern previously reported for retroviral vectors in cell culture experiments. The data therefore sustain the hypothesis that the kissing loop, presumably...

  10. Partial restoration of replication of simian immunodeficiency virus by point mutations in either the dimerization initiation site (DIS) or Gag region after deletion mutagenesis within the DIS. (United States)

    Guan, Y; Diallo, K; Detorio, M; Whitney, J B; Liang, C; Wainberg, M A


    We used the simian immunodeficiency virus (SIV) molecular clone SIVmac239 to generate a deletion construct, termed SD2, in which we eliminated 22 nucleotides at positions +398 to +418 within the putative dimerization initiation site (DIS) stem. This SD2 deletion severely impaired viral replication, due to adverse effects on the packaging of viral genomic RNA, the processing of Gag proteins, and viral protein patterns. However, long-term culture of SD2 in either C8166 or CEMx174 cells resulted in restoration of replication capacity, due to two different sets of three compensatory point mutations, located within both the DIS and Gag regions. In the case of C8166 cells, both a K197R and a E49K mutation were identified within the capsid (CA) protein and the p6 protein of Gag, respectively, while the other point mutation (A423G) was found within the putative DIS loop. In the case of CEMx174 cells, two compensatory mutations were present within the viral nucleocapsid (NC) protein, E18G and Q31K, in addition to the same A423G substitution as observed with C8166 cells. A set of all three mutations was required in each case for restoration of replication capacity, and either set of mutations could be substituted for the other in both the C8166 and CEMx174 cell lines.

  11. HuR and Ago2 Bind the Internal Ribosome Entry Site of Enterovirus 71 and Promote Virus Translation and Replication.

    Directory of Open Access Journals (Sweden)

    Jing-Yi Lin

    Full Text Available EV71 (enterovirus 71 RNA contains an internal ribosomal entry site (IRES that directs cap-independent initiation of translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs. We reported recently that mRNA decay factor AUF1 is a negative-acting ITAF that binds IRES stem-loop II. We also reported that the small RNA-processing enzyme Dicer produces at least four small RNAs (vsRNAs from the EV71 IRES. One of these, vsRNA1, derived from IRES stem-loop II, reduces IRES activity and virus replication. Since its mechanism of action is unknown, we hypothesized that it might control association of ITAFs with the IRES. Here, we identified the mRNA stability factor HuR and the RISC subunit Argonaute 2 (Ago2 as two ITAFs that bind stem-loop II. In contrast to AUF1, HuR and Ago2 promote EV71 IRES activity and virus replication. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with stem-loop II. This presents a possible mechanism by which vsRNA1 could control viral translation and replication.

  12. Impediment of Replication Forks by Long Non-coding RNA Provokes Chromosomal Rearrangements by Error-Prone Restart

    Directory of Open Access Journals (Sweden)

    Takaaki Watanabe


    Full Text Available Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs. Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

  13. Tonsillar crypt epithelium is an important extra-central nervous system site for viral replication in EV71 encephalomyelitis. (United States)

    He, Yaoxin; Ong, Kien Chai; Gao, Zifen; Zhao, Xishun; Anderson, Virginia M; McNutt, Michael A; Wong, Kum Thong; Lu, Min


    Enterovirus 71 (EV71; family Picornaviridae, species human Enterovirus A) usually causes hand, foot, and mouth disease, which may rarely be complicated by fatal encephalomyelitis. We investigated extra-central nervous system (extra-CNS) tissues capable of supporting EV71 infection and replication, and have correlated tissue infection with expression of putative viral entry receptors, scavenger receptor B2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL-1). Formalin-fixed, paraffin-embedded CNS and extra-CNS tissues from seven autopsy cases were examined by IHC and in situ hybridization to evaluate viral antigens and RNA. Viral receptors were identified with IHC. In all seven cases, the CNS showed stereotypical distribution of inflammation and neuronal localization of viral antigens and RNA, confirming the clinical diagnosis of EV71 encephalomyelitis. In six cases in which tonsillar tissues were available, viral antigens and/or RNA were localized to squamous epithelium lining the tonsillar crypts. Tissues from the gastrointestinal tract, pancreas, mesenteric nodes, spleen, and skin were all negative for viral antigens/RNA. Our novel findings strongly suggest that tonsillar crypt squamous epithelium supports active viral replication and represents an important source of viral shedding that facilitates person-to-person transmission by both the fecal-oral or oral-oral routes. It may also be a portal for viral entry. A correlation between viral infection and SCARB2 expression appears to be more significant than for PSGL-1 expression. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  14. The structure and function of replication protein A in DNA replication. (United States)

    Prakash, Aishwarya; Borgstahl, Gloria E O


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

  15. Inhibition of human immunodeficiency virus type 1 replication with artificial transcription factors targeting the highly conserved primer-binding site

    NARCIS (Netherlands)

    Eberhardy, Scott R.; Goncalves, Joao; Coelho, Sofia; Segal, David J.; Berkhout, Ben; Barbas, Carlos F.


    The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or

  16. 14 CFR 23.201 - Wings level stall. (United States)


    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Wings level stall. 23.201 Section 23.201 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS.... Starting from a speed at least 10 knots above the stall speed, the elevator control must be pulled back so...

  17. 14 CFR 33.65 - Surge and stall characteristics. (United States)


    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Surge and stall characteristics. 33.65 Section 33.65 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT... stall characteristics. When the engine is operated in accordance with operating instructions required by...

  18. Compressible dynamic stall vorticity flux control using a dynamic ...

    Indian Academy of Sciences (India)

    management of its unsteady vorticity using a variable droop leading edge (VDLE) airfoil. Through ... the pressure gradient term for the dynamic stall conditions encountered by a helicopter-rotor retreating blade. Thus ... This paper discusses control of compressible dynamic stall using the novel idea of variable droop leading ...

  19. DNA Virus Replication Compartments (United States)

    Schmid, Melanie; Speiseder, Thomas; Dobner, Thomas


    Viruses employ a variety of strategies to usurp and control cellular activities through the orchestrated recruitment of macromolecules to specific cytoplasmic or nuclear compartments. Formation of such specialized virus-induced cellular microenvironments, which have been termed viroplasms, virus factories, or virus replication centers, complexes, or compartments, depends on molecular interactions between viral and cellular factors that participate in viral genome expression and replication and are in some cases associated with sites of virion assembly. These virus-induced compartments function not only to recruit and concentrate factors required for essential steps of the viral replication cycle but also to control the cellular mechanisms of antiviral defense. In this review, we summarize characteristic features of viral replication compartments from different virus families and discuss similarities in the viral and cellular activities that are associated with their assembly and the functions they facilitate for viral replication. PMID:24257611

  20. Influence of anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture.


    Djian, P; Roncari, A K; Hollenberg, C H


    Using a propagating cell culture system of adipocyte precursors from 70-400-g rats, we explored the possibility that regional variations in properties of adipose tissue may reflect site-specific characteristics intrinsic to the cells, rather than extracellular influences. Initially, studies were made of the nature of the fibroblastlike cells from perirenal adipose tissue stroma. Using colony-forming techniques, it was shown that these cells were adipocyte precursors; each confluent colony tha...

  1. Stress induced by premature chromatin condensation triggers chromosome shattering and chromothripsis at DNA sites still replicating in micronuclei or multinucleate cells when primary nuclei enter mitosis. (United States)

    Terzoudi, Georgia I; Karakosta, Maria; Pantelias, Antonio; Hatzi, Vasiliki I; Karachristou, Ioanna; Pantelias, Gabriel


    Combination of next-generation DNA sequencing, single nucleotide polymorphism array analyses and bioinformatics has revealed the striking phenomenon of chromothripsis, described as complex genomic rearrangements acquired in a single catastrophic event affecting one or a few chromosomes. Via an unproven mechanism, it is postulated that mechanical stress causes chromosome shattering into small lengths of DNA, which are then randomly reassembled by DNA repair machinery. Chromothripsis is currently examined as an alternative mechanism of oncogenesis, in contrast to the present paradigm that considers a stepwise development of cancer. While evidence for the mechanism(s) underlying chromosome shattering during cancer development remains elusive, a number of hypotheses have been proposed to explain chromothripsis, including ionizing radiation, DNA replication stress, breakage-fusion-bridge cycles, micronuclei formation and premature chromosome compaction. In the present work, we provide experimental evidence on the mechanistic basis of chromothripsis and on how chromosomes can get locally shattered in a single catastrophic event. Considering the dynamic nature of chromatin nucleoprotein complex, capable of rapid unfolding, disassembling, assembling and refolding, we first show that chromatin condensation at repairing or replicating DNA sites induces the mechanical stress needed for chromosome shattering to ensue. Premature chromosome condensation is then used to visualize the dynamic nature of interphase chromatin and demonstrate that such mechanical stress and chromosome shattering can also occur in chromosomes within micronuclei or asynchronous multinucleate cells when primary nuclei enter mitosis. Following an aberrant mitosis, chromosomes could find themselves in the wrong place at the wrong time so that they may undergo massive DNA breakage and rearrangement in a single catastrophic event. Specifically, our results support the hypothesis that premature chromosome

  2. ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA

    DEFF Research Database (Denmark)

    Toledo Lazaro, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt


    ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled...... origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing...... commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors....

  3. Early events in the pathogenesis of foot-and-mouth disease in pigs; identification of oropharyngeal tonsils as sites of primary and sustained viral replication.

    Directory of Open Access Journals (Sweden)

    Carolina Stenfeldt

    Full Text Available A time-course study was performed to elucidate the early events of foot-and-mouth disease virus (FMDV infection in pigs subsequent to simulated natural, intra-oropharyngeal, inoculation. The earliest detectable event was primary infection in the lingual and paraepiglottic tonsils at 6 hours post inoculation (hpi characterized by regional localization of viral RNA, viral antigen, and infectious virus. At this time FMDV antigen was localized in cytokeratin-positive epithelial cells and CD172a-expressing leukocytes of the crypt epithelium of the paraepiglottic tonsils. De novo replication of FMDV was first detected in oropharyngeal swab samples at 12 hpi and viremia occurred at 18-24 hpi, approximately 24 hours prior to the appearance of vesicular lesions. From 12 through 78 hpi, microscopic detection of FMDV was consistently localized to cytokeratin-positive cells within morphologically characteristic segments of oropharyngeal tonsil crypt epithelium. During this period, leukocyte populations expressing CD172a, SLA-DQ class II and/or CD8 were found in close proximity to infected epithelial cells, but with little or no co-localization with viral proteins. Similarly, M-cells expressing cytokeratin-18 did not co-localize with FMDV proteins. Intra-epithelial micro-vesicles composed of acantholytic epithelial cells expressing large amounts of structural and non-structural FMDV proteins were present within crypts of the tonsil of the soft palate during peak clinical infection. These findings inculpate the paraepiglottic tonsils as the primary site of FMDV infection in pigs exposed via the gastrointestinal tract. Furthermore, the continuing replication of FMDV in the oropharyngeal tonsils during viremia and peak clinical infection with no concurrent amplification of virus occurring in the lower respiratory tract indicates that these sites are the major source of shedding of FMDV from pigs.

  4. Compartmental HBV evolution and replication in liver and extrahepatic sites after nucleos/tide analogue therapy in chronic hepatitis B carriers. (United States)

    Gao, Shan; Duan, Zhong-Ping; Chen, Yu; van der Meer, Frank; Lee, Samuel S; Osiowy, Carla; van Marle, Guido; Coffin, Carla S


    Hepatitis B virus (HBV) variants are associated with nucleos/tide analogue (NA) response and liver disease but it is unknown whether NA influences extrahepatic HBV persistence. To investigate HBV replication and genetic evolution in hepatic and extrahepatic sites of chronic hepatitis B (CHB) before and after NA therapy. A total of 13 paired plasma, peripheral blood mononuclear cells (PBMC), were collected from chronic HBV carriers at baseline and after a median 53 weeks NA therapy as well as liver biopsy (N=7 baseline, N=5 follow-up). HBV covalently closed circular DNA (cccDNA) and messenger (m) RNA in liver and PBMC were analyzed. HBV polymerase (P)/surface (S), basal core promoter (BCP)/pre-core (PC)/C gene clonal sequencing was done in plasma, peripheral blood mononuclear cells (PBMC), and liver. Compare to baseline, at ∼53 weeks follow-up, there was no significant change in HBV cccDNA levels in liver (0.2-0.08 copies/hepatocyte, p>0.05) or in PBMC 0.003-0.02 copies/PBMC, p>0.05), and HBV mRNA remained detectable in both sites. At baseline, BCP variants were higher in PBMC vs. liver and plasma. After therapy, drug resistant (DR) and immune escape (IE) variants increased in liver but IE and PC variants were more frequent in PBMC. HBV P/S diversity was significantly higher in PBMC compared to plasma. Continuous HBV replication occurs in liver and PBMC and shows compartmentalized evolution under selective pressure of potent NA therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Database Replication

    CERN Document Server

    Kemme, Bettina


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

  6. Dynamic Stall in Pitching Airfoils: Aerodynamic Damping and Compressibility Effects (United States)

    Corke, Thomas C.; Thomas, Flint O.


    Dynamic stall is an incredibly rich fluid dynamics problem that manifests itself on an airfoil during rapid, transient motion in which the angle of incidence surpasses the static stall limit. It is an important element of many manmade and natural flyers, including helicopters and supermaneuverable aircraft, and low-Reynolds number flapping-wing birds and insects. The fluid dynamic attributes that accompany dynamic stall include an eruption of vorticity that organizes into a well-defined dynamic stall vortex and massive excursions in aerodynamic loads that can couple with the airfoil structural dynamics. The dynamic stall process is highly sensitive to surface roughness that can influence turbulent transition and to local compressibility effects that occur at free-stream Mach numbers that are otherwise incompressible. Under some conditions, dynamic stall can result in negative aerodynamic damping that leads to limit-cycle growth of structural vibrations and rapid mechanical failure. The mechanisms leading to negative damping have been a principal interest of recent experiments and analysis. Computational fluid dynamic simulations and low-order models have not been good predictors so far. Large-eddy simulation could be a viable approach although it remains computationally intensive. The topic is technologically important owing to the desire to develop next-generation rotorcraft that employ adaptive rotor dynamic stall control.

  7. Database Replication

    Directory of Open Access Journals (Sweden)

    Marius Cristian MAZILU


    Full Text Available For someone who has worked in an environment in which the same database is used for data entry and reporting, or perhaps managed a single database server that was utilized by too many users, the advantages brought by data replication are clear. The main purpose of this paper is to emphasize those advantages as well as presenting the different types of Database Replication and the cases in which their use is recommended.

  8. The Polypyrimidine Tract-Binding Protein Affects Coronavirus RNA Accumulation Levels and Relocalizes Viral RNAs to Novel Cytoplasmic Domains Different from Replication-Transcription Sites (United States)

    Sola, Isabel; Galán, Carmen; Mateos-Gómez, Pedro A.; Palacio, Lorena; Zúñiga, Sonia; Cruz, Jazmina L.; Almazán, Fernando; Enjuanes, Luis


    The coronavirus (CoV) discontinuous transcription mechanism is driven by long-distance RNA-RNA interactions between transcription-regulating sequences (TRSs) located at the 5′ terminal leader (TRS-L) and also preceding each mRNA-coding sequence (TRS-B). The contribution of host cell proteins to CoV transcription needs additional information. Polypyrimidine tract-binding protein (PTB) was reproducibly identified in association with positive-sense RNAs of transmissible gastroenteritis coronavirus (TGEV) TRS-L and TRS-B by affinity chromatography and mass spectrometry. A temporal regulation of PTB cytoplasmic levels was observed during infection, with a significant increase from 7 to 16 h postinfection being inversely associated with a decrease in viral replication and transcription. Silencing the expression of PTB with small interfering RNA in two cell lines (Huh7 and HEK 293T) led to a significant increase of up to 4-fold in mRNA levels and virus titer, indicating a negative effect of PTB on CoV RNA accumulation. During CoV infection, PTB relocalized from the nucleus to novel cytoplasmic structures different from replication-transcription sites in which stress granule markers T-cell intracellular antigen-1 (TIA-1) and TIA-1-related protein (TIAR) colocalized. PTB was detected in these modified stress granules in TGEV-infected swine testis cells but not in stress granules induced by oxidative stress. Furthermore, viral genomic and subgenomic RNAs were detected in association with PTB and TIAR. These cytoplasmic ribonucleoprotein complexes might be involved in posttranscriptional regulation of virus gene expression. PMID:21411518

  9. Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites. (United States)

    Tamura, Tomokazu; Nagashima, Naofumi; Ruggli, Nicolas; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro


    Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

  10. Flap motion of helicopter rotors with novel, dynamic stall model

    Directory of Open Access Journals (Sweden)

    Han Wei


    Full Text Available In this paper, a nonlinear flapping equation for large inflow angles and flap angles is established by analyzing the aerodynamics of helicopter blade elements. In order to obtain a generalized flap equation, the Snel stall model was first applied to determine the lift coefficient of the helicopter rotor. A simulation experiment for specific airfoils was then conducted to verify the effectiveness of the Snel stall model as it applies to helicopters. Results show that the model requires no extraneous parameters compared to the traditional stall model and is highly accurate and practically applicable. Based on the model, the relationship between the flapping angle and the angle of attack was analyzed, as well as the advance ratio under the dynamic stall state.

  11. HAWT dynamic stall response asymmetries under yawed flow conditions

    Energy Technology Data Exchange (ETDEWEB)

    Schreck, S.; Robinson, M.; Hand, M.; Simms, D.


    Horizontal axis wind turbines can experience significant time varying aerodynamic loads, potentially causing adverse effects on structures, mechanical components, and power production. As designers attempt lighter and more flexible wind energy machines, greater accuracy and robustness will become even more critical in future aerodynamics models. Aerodynamics modeling advances, in turn, will rely on more thorough comprehension of the three-dimensional, unsteady, vortical flows that dominate wind turbine blade aerodynamics under high load conditions. To experimentally characterize these flows, turbine blade surface pressures were acquired at multiple span locations via the NREL Phase IV Unsteady Aerodynamics Experiment. Surface pressures and associated normal force histories were used to characterize dynamic stall vortex kinematics and normal force amplification. Dynamic stall vortices and normal force amplification were confirmed to occur in response to angle of attack excursions above the static stall threshold. Stall vortices occupied approximately one-half of the blade span and persisted for nearly one-fourth of the blade rotation cycle. Stall vortex convection varied along the blade, resulting in dramatic deformation of the vortex. Presence and deformation of the dynamic stall vortex produced corresponding amplification of normal forces. Analyses revealed consistent alterations to vortex kinematics in response to changes in reduced frequency, span location, and yaw error. Finally, vortex structures and kinematics not previously documented for wind turbine blades were isolated.

  12. Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1. (United States)

    Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun


    Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of

  13. Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells. (United States)

    Giraud, Matthieu; Yoshida, Hideyuki; Abramson, Jakub; Rahl, Peter B; Young, Richard A; Mathis, Diane; Benoist, Christophe


    Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

  14. Is Social Licence A Licence To Stall?

    Directory of Open Access Journals (Sweden)

    Mark Lowey


    Full Text Available The School of Public Policy at the University of Calgary organized a one-day symposium on Oct. 8, 2014 in Calgary, as part of the School’s TransCanada Corporation Energy Policy and Regulatory Frameworks Program. The symposium was titled “Is Social License a License to Stall?” Held at the Hotel Arts, the event attracted a full-capacity audience of about 110 people, including representatives from industry, government and environmental non-government organizations. The symposium included four moderated panel sessions and a keynote speaker at lunch. The School of Public Policy set the framework for discussion at the Calgary symposium with the following description: Canada’s regulators act in the public interest to review energy and infrastructure project applications. Regulators are guided by procedural fairness and follow a transparent application, review and hearing process with data filings and sworn testimony. But that’s changing. “Social license” is a relatively new term, which some interests are using to create a different standard for the approval of projects — especially energy projects. According to social license advocates, projects must meet often ill-defined requirements set up by non-governmental organizations, local residents or other interests — a new hurdle for project approval, but without the rigour and rule of law of a regulator. Is social license a meaningful addition to the regulatory process, or is it being used as a constantly moving goal-post designed to slow down regulatory processes, delay project implementation, frustrate energy infrastructure expansion and even enrich those advocates who promote it as a new model? This paper summarises the discussion and the themes that emerged throughout the day. Most notably, panellists concluded that “social licence” is a real and significant issue that presents both an opportunity and a problem, not only for regulators but for all parties involved in the

  15. Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane. (United States)

    Bailer, Susanne M.


    Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

  16. Dynamic Stall Characteristics of Drooped Leading Edge Airfoils (United States)

    Sankar, Lakshmi N.; Sahin, Mehmet; Gopal, Naveen


    Helicopters in high-speed forward flight usually experience large regions of dynamic stall over the retreating side of the rotor disk. The rapid variations in the lift and pitching moments associated with the stall process can result in vibratory loads, and can cause fatigue and failure of pitch links. In some instances, the large time lag between the aerodynamic forces and the blade motion can trigger stall flutter. A number of techniques for the alleviation of dynamic stall have been proposed and studied by researchers. Passive and active control techniques have both been explored. Passive techniques include the use of high solidity rotors that reduce the lift coefficients of individual blades, leading edge slots and leading edge slats. Active control techniques include steady and unsteady blowing, and dynamically deformable leading edge (DDLE) airfoils. Considerable amount of experimental and numerical data has been collected on the effectiveness of these concepts. One concept that has not received as much attention is the drooped-leading edge airfoil idea. It has been observed in wind tunnel studies and flight tests that drooped leading edge airfoils can have a milder dynamic stall, with a significantly milder load hysteresis. Drooped leading edge airfoils may not, however, be suitable at other conditions, e.g. in hover, or in transonic flow. Work needs to be done on the analysis and design of drooped leading edge airfoils for efficient operation in a variety of flight regimes (hover, dynamic stall, and transonic flow). One concept that is worthy of investigation is the dynamically drooping airfoil, where the leading edge shape is changed roughly once-per-rev to mitigate the dynamic stall.

  17. DNA replication origins in archaea

    Directory of Open Access Journals (Sweden)

    Zhenfang eWu


    Full Text Available DNA replication initiation, which starts at specific chromosomal site (known as replication origins, is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB that are located adjacent to a replication initiator gene. Both the ORB sequence and the adjacent initiator gene are considerably diverse among different replication origins, while in silico and genetic analyses have indicated the specificity between the initiator genes and their cognate origins. These replicator-initiator pairings are reminiscent of the oriC-dnaA system in bacteria, and a model for the negative regulation of origin activity by a downstream cluster of ORB elements has been recently proposed in haloarchaea. Moreover, comparative genomic analyses have revealed that the mosaics of replicator-initiator pairings in archaeal chromosomes originated from the integration of extrachromosomal elements. This review summarizes the research progress in understanding of archaeal replication origins with particular focus on the utilization, control and evolution of multiple replication origins in haloarchaea.

  18. Homologous regions of Fen1 and p21Cip1 compete for binding to the same site on PCNA: a potential mechanism to co-ordinate DNA replication and repair. (United States)

    Warbrick, E; Lane, D P; Glover, D M; Cox, L S


    Following genomic damage, the cessation of DNA replication is co-ordinated with onset of DNA repair; this co-ordination is essential to avoid mutation and genomic instability. To investigate these phenomena, we have analysed proteins that interact with PCNA, which is required for both DNA replication and repair. One such protein is p21Cip1, which inhibits DNA replication through its interaction with PCNA, while allowing repair to continue. We have identified an interaction between PCNA and the structure specific nuclease, Fen1, which is involved in DNA replication. Deletion analysis suggests that p21Cip1 and Fen1 bind to the same region of PCNA. Within Fen1 and its homologues a small region (10 amino acids) is sufficient for PCNA binding, which contains an 8 amino acid conserved PCNA-binding motif. This motif shares critical residues with the PCNA-binding region of p21Cip1. A PCNA binding peptide from p21Cip1 competes with Fen1 peptides for binding to PCNA, disrupts the Fen1-PCNA complex in replicating cell extracts, and concomitantly inhibits DNA synthesis. Competition between homologous regions of Fen1 and p21Cip1 for binding to the same site on PCNA may provide a mechanism to co-ordinate the functions of PCNA in DNA replication and repair.

  19. The Dynamics of SecM-Induced Translational Stalling

    Directory of Open Access Journals (Sweden)

    Albert Tsai


    Full Text Available SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation.

  20. The dynamics of SecM-induced translational stalling. (United States)

    Tsai, Albert; Kornberg, Guy; Johansson, Magnus; Chen, Jin; Puglisi, Joseph D


    SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  1. A Comparative Study of Three Methodologies for Modeling Dynamic Stall (United States)

    Sankar, L.; Rhee, M.; Tung, C.; ZibiBailly, J.; LeBalleur, J. C.; Blaise, D.; Rouzaud, O.


    During the past two decades, there has been an increased reliance on the use of computational fluid dynamics methods for modeling rotors in high speed forward flight. Computational methods are being developed for modeling the shock induced loads on the advancing side, first-principles based modeling of the trailing wake evolution, and for retreating blade stall. The retreating blade dynamic stall problem has received particular attention, because the large variations in lift and pitching moments encountered in dynamic stall can lead to blade vibrations and pitch link fatigue. Restricting to aerodynamics, the numerical prediction of dynamic stall is still a complex and challenging CFD problem, that, even in two dimensions at low speed, gathers the major difficulties of aerodynamics, such as the grid resolution requirements for the viscous phenomena at leading-edge bubbles or in mixing-layers, the bias of the numerical viscosity, and the major difficulties of the physical modeling, such as the turbulence models, the transition models, whose both determinant influences, already present in static maximal-lift or stall computations, are emphasized by the dynamic aspect of the phenomena.

  2. Time Accurate Unsteady Simulation of the Stall Inception Process in the Compression System of a US Army Helicopter Gas Turbine Engine

    National Research Council Canada - National Science Library

    Hathaway, Michael D; Herrick, Greg; Chen, Jenping; Webster, Robert


    .... Improved understanding of the stall inception process and how stall control technologies mitigate such will provide compressors with increased tolerance to stall, thereby expanding the operational...

  3. FANCJ couples replication past natural fork barriers with maintenance of chromatin structure. (United States)

    Schwab, Rebekka A; Nieminuszczy, Jadwiga; Shin-ya, Kazuo; Niedzwiedz, Wojciech


    Defective DNA repair causes Fanconi anemia (FA), a rare childhood cancer-predisposing syndrome. At least 15 genes are known to be mutated in FA; however, their role in DNA repair remains unclear. Here, we show that the FANCJ helicase promotes DNA replication in trans by counteracting fork stalling on replication barriers, such as G4 quadruplex structures. Accordingly, stabilization of G4 quadruplexes in ΔFANCJ cells restricts fork movements, uncouples leading- and lagging-strand synthesis and generates small single-stranded DNA gaps behind the fork. Unexpectedly, we also discovered that FANCJ suppresses heterochromatin spreading by coupling fork movement through replication barriers with maintenance of chromatin structure. We propose that FANCJ plays an essential role in counteracting chromatin compaction associated with unscheduled replication fork stalling and restart, and suppresses tumorigenesis, at least partially, in this replication-specific manner.

  4. Active Suppression of Rotating Stall Inception with Distributed Jet Actuation

    Directory of Open Access Journals (Sweden)

    Huu Duc Vo


    Full Text Available An analytical and experimental investigation of the effectiveness of full-span distributed jet actuation for active suppression of long length-scale rotating stall inception is carried out. Detailed modeling and experimental verification highlight the important effects of mass addition, discrete injectors, and feedback dynamics, which may be overlooked in preliminary theoretical studies of active control with jet injection. A model of the compression system incorporating nonideal injection and feedback dynamics is verified with forced response measurements to predict the right trends in the movement of the critical pole associated with the stall precursor. Active control experiments with proportional feedback control show that the predicted stall precursors are suppressed to give a 5.5% range extension in compressor flow coefficient. In addition, results suggest that the proposed model could be used to design a more sophisticated controller to further improve performance while reducing actuator bandwidth requirements.

  5. Stall Recovery Guidance Algorithms Based on Constrained Control Approaches (United States)

    Stepanyan, Vahram; Krishnakumar, Kalmanje; Kaneshige, John; Acosta, Diana


    Aircraft loss-of-control, in particular approach to stall or fully developed stall, is a major factor contributing to aircraft safety risks, which emphasizes the need to develop algorithms that are capable of assisting the pilots to identify the problem and providing guidance to recover the aircraft. In this paper we present several stall recovery guidance algorithms, which are implemented in the background without interfering with flight control system and altering the pilot's actions. They are using input and state constrained control methods to generate guidance signals, which are provided to the pilot in the form of visual cues. It is the pilot's decision to follow these signals. The algorithms are validated in the pilot-in-the loop medium fidelity simulation experiment.

  6. Managing Single-Stranded DNA during Replication Stress in Fission Yeast

    Directory of Open Access Journals (Sweden)

    Sarah A. Sabatinos


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


    Directory of Open Access Journals (Sweden)



    Full Text Available Thermoregulatory responses of horses subjected to summer-time road transport and stall confinement were investigated in this study. Six mature geldings were transported 168 km in a 4-horse trailer and were monitored while tethered in their stalls, on alternate days. Core body temperature (GT demonstrated negligible response during transport, but GT following transport was higher than GT for non-transport. GT tended to increase with increased temperature humidity index (THI. THI within the trailer was greatest for positions near the front, and was influenced by daily weather which varied over experiment days from heat stress conditions to moderate discomfort.

  8. A dynamic stall model for airfoils with deformable trailing edges

    DEFF Research Database (Denmark)

    Andersen, Peter Bjørn; Gaunaa, Mac; Bak, Christian


    The present work contains an extension of the Beddoes-Leishman-type dynamic stall model. In this work, a deformable trailing-edge flap has been added to the dynamic stall model. The model predicts the unsteady aerodynamic forces and moments on an airfoil section undergoing arbitrary motion in hea...... for the attached flow region and Hansen et al. The model is compared qualitatively to wind tunnel measurements of a Riso/ B1-18 blade section equipped with deformable trailing-edge flap devices in the form of piezoelectric devices. Copyright © 2009 John Wiley & Sons, Ltd....

  9. Regulation of bacterial gene expression by ribosome stalling and rescuing. (United States)

    Jin, Yongxin; Jin, Shouguang; Wu, Weihui


    Ribosome is responsible for protein synthesis and is able to monitor the sequence and structure of the nascent peptide. Such ability plays an important role in determining overall gene expression profile of the bacteria through ribosome stalling and rescuing. In this review, we briefly summarize our current understanding of the regulation of gene expression through ribosome stalling and rescuing in bacteria, as well as mechanisms that modulate ribosome activity. Understanding the mechanisms of how bacteria modulate ribosome activity will provide not only fundamental insights into bacterial gene regulation, but also new candidate targets for the development of novel antimicrobial agents.

  10. A dynamic stall model for airfoils with deformable trailing edges

    DEFF Research Database (Denmark)

    Andersen, Peter Bjørn; Gaunaa, Mac; Bak, Dan Christian


    on an airfoil section undergoing arbitrary motion in heave, lead-lag, pitch, Trailing Edge (TE) flapping. In the linear region, the model reduces to the inviscid model of Gaunaa [4], which includes the aerodynamic effect of a thin airfoil with a deformable camberline in inviscid flow. Therefore, the proposed......The present work contains an extension of the Beddoes-Leishman (B-L) type dynamic stall model, as described by Hansen et al. [7]. In this work a Deformable Trailing Edge Geometry (DTEG) has been added to the dynamic stall model. The model predicts the unsteady aerodynamic forces and moments...

  11. Dynamic Stall Flow Control Through the Use of a Novel Plasma Based Actuator Technology, Phase I (United States)

    National Aeronautics and Space Administration — Lynntech proposes a novel flow control methodology for airfoils undergoing dynamic stall. Dynamic stall refers to an aerodynamic phenomenon that is experienced by...

  12. Unsteady Double Wake Model for the Simulation of Stalled Airfoils

    DEFF Research Database (Denmark)

    Ramos García, Néstor; Cayron, Antoine; Sørensen, Jens Nørkær


    separation and its dynamics. In this paper, the calculated integral forces have been successfully validated against wind tunnel measurements for the FFA-W3-211 airfoil. Furthermore, the computed highly unsteady flow field is analyzed in detail for a set of angles of attack ranging from light to deep stall...

  13. Dynamic Characteristics of Rotating Stall in Mixed Flow Pump

    Directory of Open Access Journals (Sweden)

    Xiaojun Li


    Full Text Available Rotating stall, a phenomenon that causes flow instabilities and pressure hysteresis by propagating at some fraction of the impeller rotational speed, can occur in centrifugal impellers, mixed impellers, radial diffusers, or axial diffusers. Despite considerable efforts devoted to the study of rotating stall in pumps, the mechanics of this phenomenon are not sufficiently understood. The propagation mechanism and onset of rotating stall are not only affected by inlet flow but also by outlet flow as well as the pressure gradient in the flow passage. As such, the complexity of these concepts is not covered by the classical explanation. To bridge this research gap, the current study investigated prerotation generated at the upstream of the impeller, leakage flow at the tip clearance between the casing and the impeller, and strong reserve flow at the inlet of the diffuser. Understanding these areas will clarify the origin of the positive slope of the head-flow performance curve for a mixed flow pump. Nonuniform pressure distribution and adverse pressure gradient were also introduced to evaluate the onset and development of rotating stall within the diffuser.

  14. The Mechanical Impact of Aerodynamic Stall on Tunnel Ventilation Fans

    Directory of Open Access Journals (Sweden)

    A. G. Sheard


    Full Text Available This paper describes work aimed at establishing the ability of a tunnel ventilation fan to operate without risk of mechanical failure in the event of aerodynamic stall. The research establishes the aerodynamic characteristics of a typical tunnel ventilation fan when operated in both stable and stalled aerodynamic conditions, with and without an anti-stall stabilisation ring, with and without a “nonstalling” blade angle and at full, half, and one quarter design speed. It also measures the fan’s peak stress, thus facilitating an analysis of the implications of the experimental results for mechanical design methodology. The paper concludes by presenting three different strategies for tunnel ventilation fan selection in applications where the selected fan will most likely stall. The first strategy selects a fan with a low-blade angle that is nonstalling. The second strategy selects a fan with a high-pressure developing capability. The third strategy selects a fan with a fitted stabilisation ring. Tunnel ventilation system designers each have their favoured fan selection strategy. However, all three strategies can produce system designs within which a tunnel ventilation fan performs reliably in-service. The paper considers the advantages and disadvantages of each selection strategy and considered the strengths and weaknesses of each.


    Directory of Open Access Journals (Sweden)

    Sabina Angrecka


    The obtained results allowed us to identify optimal orientation of barns and to suggest the simplest technical measures to protect sidewall stalls from solar heat gain deleterious to cows. The model analysis of stall shading demonstrated that extension of barn eaves to 1 m on the southern side reduced the insolation of stalls over even up to 90% of their area.

  16. A CFD Database for Airfoils and Wings at Post-Stall Angles of Attack (United States)

    Petrilli, Justin; Paul, Ryan; Gopalarathnam, Ashok; Frink, Neal T.


    This paper presents selected results from an ongoing effort to develop an aerodynamic database from Reynolds-Averaged Navier-Stokes (RANS) computational analysis of airfoils and wings at stall and post-stall angles of attack. The data obtained from this effort will be used for validation and refinement of a low-order post-stall prediction method developed at NCSU, and to fill existing gaps in high angle of attack data in the literature. Such data could have potential applications in post-stall flight dynamics, helicopter aerodynamics and wind turbine aerodynamics. An overview of the NASA TetrUSS CFD package used for the RANS computational approach is presented. Detailed results for three airfoils are presented to compare their stall and post-stall behavior. The results for finite wings at stall and post-stall conditions focus on the effects of taper-ratio and sweep angle, with particular attention to whether the sectional flows can be approximated using two-dimensional flow over a stalled airfoil. While this approximation seems reasonable for unswept wings even at post-stall conditions, significant spanwise flow on stalled swept wings preclude the use of two-dimensional data to model sectional flows on swept wings. Thus, further effort is needed in low-order aerodynamic modeling of swept wings at stalled conditions.

  17. Human Mitochondrial DNA Replication (United States)

    Holt, Ian J.; Reyes, Aurelio


    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808


    Mizukami, Ikuko; Gall, Joseph


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

  19. Prediction of RNA Polymerase II recruitment, elongation and stalling from histone modification data (United States)


    Background Initiation and elongation of RNA polymerase II (RNAPII) transcription is regulated by both DNA sequence and chromatin signals. Recent breakthroughs make it possible to measure the chromatin state and activity of core promoters genome-wide, but dedicated computational strategies are needed to progress from descriptive annotation of data to quantitative, predictive models. Results Here, we describe a computational framework which with high accuracy can predict the locations of core promoters, the amount of recruited RNAPII at the promoter, the amount of elongating RNAPII in the gene body, the mRNA production originating from the promoter and finally also the stalling characteristics of RNAPII by considering both quantitative and spatial features of histone modifications around the transcription start site (TSS). As the model framework can also pinpoint the signals that are the most influential for prediction, it can be used to infer underlying regulatory biology. For example, we show that the H3K4 di- and tri- methylation signals are strongly predictive for promoter location while the acetylation marks H3K9 and H3K27 are highly important in estimating the promoter usage. All of these four marks are found to be necessary for recruitment of RNAPII but not sufficient for the elongation. We also show that the spatial distributions of histone marks are almost as predictive as the signal strength and that a set of histone marks immediately downstream of the TSS is highly predictive of RNAPII stalling. Conclusions In this study we introduce a general framework to accurately predict the level of RNAPII recruitment, elongation, stalling and mRNA expression from chromatin signals. The versatility of the method also makes it ideally suited to investigate other genomic data. PMID:22047616

  20. Prediction of RNA Polymerase II recruitment, elongation and stalling from histone modification data

    Directory of Open Access Journals (Sweden)

    Chen Yun


    Full Text Available Abstract Background Initiation and elongation of RNA polymerase II (RNAPII transcription is regulated by both DNA sequence and chromatin signals. Recent breakthroughs make it possible to measure the chromatin state and activity of core promoters genome-wide, but dedicated computational strategies are needed to progress from descriptive annotation of data to quantitative, predictive models. Results Here, we describe a computational framework which with high accuracy can predict the locations of core promoters, the amount of recruited RNAPII at the promoter, the amount of elongating RNAPII in the gene body, the mRNA production originating from the promoter and finally also the stalling characteristics of RNAPII by considering both quantitative and spatial features of histone modifications around the transcription start site (TSS. As the model framework can also pinpoint the signals that are the most influential for prediction, it can be used to infer underlying regulatory biology. For example, we show that the H3K4 di- and tri- methylation signals are strongly predictive for promoter location while the acetylation marks H3K9 and H3K27 are highly important in estimating the promoter usage. All of these four marks are found to be necessary for recruitment of RNAPII but not sufficient for the elongation. We also show that the spatial distributions of histone marks are almost as predictive as the signal strength and that a set of histone marks immediately downstream of the TSS is highly predictive of RNAPII stalling. Conclusions In this study we introduce a general framework to accurately predict the level of RNAPII recruitment, elongation, stalling and mRNA expression from chromatin signals. The versatility of the method also makes it ideally suited to investigate other genomic data.

  1. LHCb experience with LFC replication

    CERN Document Server

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


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

  2. Simulation Model of an Active-stall Fixed-speed Wind Turbine Controller

    DEFF Research Database (Denmark)

    Jauch, Clemens; Hansen, Anca D.; Soerensen, Poul


    This paper describes an active-stall wind turbine controller. The objective is to develop a general model of an active stall controller in order to simulate the operation of grid connected active stall wind turbines. The active stall turbine concept and its control strategies are presented...... and evaluated by simulations. The presented controller is described for continuous operation under all wind speeds from start-up wind speed to shut down wind speed. Due to its parametric implementation it is general i.e. it can represent different active stall wind turbine controllers and can be implemented...

  3. Simulation model of an active-stall fixed-speed wind turbine controller

    Energy Technology Data Exchange (ETDEWEB)

    Jauch, C.; Hansen, A.D.; Sorensen, P.; Blaabjerg, F.


    This paper describes an active-stall wind turbine controller. The objective is to develop a general model of an active stall controller in order to simulate the operation of grid connected active stall wind turbines. The active stall turbine concept and its control strategies are presented and evaluated by simulations. The presented controller is described for continuous operation under all wind speeds from start-up wind speed to shut down wind speed. Due to its parametric implementation it is general i. e. it can represent different active stall wind turbine controllers and can be implemented in different simulation tools. (author)

  4. Synchronous replication of remote storage


    Mirzoev, Dr. Timur


    Storage replication is one of the essential requirements for network environments. While many forms of Network Attached Storage (NAS), Storage Area Networks (SAN) and other forms of network storage exist, there is a need for a reliable synchronous storage replication technique between distant sites (less than 1 mile). Such technology allows setting new standards for network failover and failback systems for virtual servers; specifically, addressing the growing need for effective disaster reco...

  5. Flow and Noise Characteristics of Centrifugal Fan under Different Stall Conditions

    Directory of Open Access Journals (Sweden)

    Lei Zhang


    Full Text Available An implicit, time-accurate 3D Reynolds-averaged Navier-Stokes (RANS solver is used to simulate the rotating stall phenomenon in a centrifugal fan. The goal of the present work is to shed light on the flow field and particularly the aerodynamic noise at different stall conditions. Aerodynamic characteristics, frequency domain characteristics, and the contours of sound power level under two different stall conditions are discussed in this paper. The results show that, with the decrease of valve opening, the amplitude of full pressure and flow fluctuations tends to be larger and the stall frequency remains the same. The flow field analysis indicates that the area occupied by stall cells expands with the decrease of flow rate. The noise calculation based on the simulation underlines the role of vortex noise after the occurrence of rotating stall, showing that the high noise area rotates along with the stall cell in the circumferential direction.

  6. DYNSTALL: Subroutine package with a dynamic stall model

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerck, Anders [Aeronautical Research Inst. of Sweden, Bromma (Sweden)


    A subroutine package, called DYNSTALL, for the calculation of 2D unsteady airfoil aerodynamics is described. The subroutines are written in FORTRAN. DYNSTALL is basically an implementation of the Beddoes-Leishman dynamic stall model. This model is a semi-empirical model for dynamic stall. It includes, however, also models for attached flow unsteady aerodynamics. It is complete in the sense that it treats attached flow as well as separated flow. Semi-empirical means that the model relies on empirically determined constants. Semi because the constants are constants in equations with some physical interpretation. It requires the input of 2D airfoil aerodynamic data via tables as function of angle of attack. The method is intended for use in an aeroelastic code with the aerodynamics solved by blade/element method. DYNSTALL was written to work for any 2D angles of attack relative to the airfoil, e.g. flow from the rear of an airfoil.

  7. Study and Control of a Radial Vaned Diffuser Stall

    Directory of Open Access Journals (Sweden)

    Aurélien Marsan


    Full Text Available The aim of the present study is to evaluate the efficiency of a boundary layer suction technique in case of a centrifugal compressor stage in order to extend its stable operating range. First, an analysis of the flow pattern within the radial vaned diffuser is presented. It highlights the stall of the diffuser vanes when reaching a low massflow. A boundary layer separation in the hub-suction side corner grows when decreasing the massflow from the nominal operating point to the surge and finally leads to a massive stall. An aspiration strategy is investigated in order to control the stall. The suction slot is put in the vicinity of the saddle that originates the main separating skin-friction line, identified thanks to the analysis of the skin-friction pattern. Several aspiration massflow rates are tested, and two different modelings of the aspiration are evaluated. Finally, an efficient control is reached with a removal of only 0,1% of the global massflow and leads—from a steady-state calculations point of view—to an increase by 40% of the compressor operating range extent.

  8. Vorticity Transport on a Flexible Wing in Stall Flutter (United States)

    Akkala, James; Buchholz, James; Farnsworth, John; McLaughlin, Thomas


    The circulation budget within dynamic stall vortices was investigated on a flexible NACA 0018 wing model of aspect ratio 6 undergoing stall flutter. The wing had an initial angle of attack of 6 degrees, Reynolds number of 1 . 5 ×105 and large-amplitude, primarily torsional, limit cycle oscillations were observed at a reduced frequency of k = πfc / U = 0 . 1 . Phase-locked stereo PIV measurements were obtained at multiple chordwise planes around the 62.5% and 75% spanwise locations to characterize the flow field within thin volumetric regions over the suction surface. Transient surface pressure measurements were used to estimate boundary vorticity flux. Recent analyses on plunging and rotating wings indicates that the magnitude of the pressure-gradient-driven boundary flux of secondary vorticity is a significant fraction of the magnitude of the convective flux from the separated leading-edge shear layer, suggesting that the secondary vorticity plays a significant role in regulating the strength of the primary vortex. This phenomenon is examined in the present case, and the physical mechanisms governing the growth and evolution of the dynamic stall vortices are explored. This work was supported by the Air Force Office of Scientific Research through the Flow Interactions and Control Program monitored by Dr. Douglas Smith, and through the 2014 AFOSR/ASEE Summer Faculty Fellowship Program (JA and JB).

  9. Simulasi Numerik Dynamic Stall Pada Airfoil Yang Berosilasi

    Directory of Open Access Journals (Sweden)

    Galih S.T.A. Bangga


    Full Text Available Kebutuhan analisa pada sudu helikopter, kompresor, kincir angin dan struktur streamline lainya yang beroperasi pada angle of attack yang tinggi dan melibatkan instationary effects yang disebut dynamic stall menjadi semakin penting. Fenomena ini ditandai dengan naiknya dynamic lift melewati static lift maksimum pada critical static stall angle, vortex yang terbentuk pada leading edge mengakibatkan naiknya suction contribution yang kemudian terkonveksi sepanjang permukaan hingga mencapai trailling edge diikuti terbentuknya trailling edge vortex yang menunjukkan terjadinya lift stall. Fenomena ini sangat berbahaya terhadap struktur airfoil itu sendiri. Secara umum, beban fatique yang ditimbulkan oleh adanya efek histerisis karena fluktuasi gaya lift akibat induksi vibrasi lebih besar dibandingkan kondisi statis. Simulasi numerik dilakukan secara 2D dengan menggunakan profil Boeing-Vertol V23010-1.58 pada α0 = 14.92°. Standard-kω dan SST-kω digunakan sebagai URANS turbulence modelling. Model osilasi dari airfoil disusun dalam suatu user defined function (UDF. Gerakan meshing beserta airfoil diakomodasi dengan menggunakan dynamic mesh approach. Simulasi numerik menunjukkan bahwa, model SST-kω menunjukkan performa yang lebih baik dibandingkan dengan Standard-kω. Fenomena travelling vortex yang terjadi mampu ditangkap dengan baik, meski pada angle of attack yang tinggi URANS turbulence model gagal memprediksikan fenomena yang terjadi karena dominasi efek 3D.

  10. Prediction of dynamic loads and induced vibrations in stall

    Energy Technology Data Exchange (ETDEWEB)

    Thirstrup Petersen, J.; Aagaard Madsen, H. [Risoe National Lab. (Denmark); Bjoerck, A. [Aeronautical Research Inst. of Sweden (Sweden); Enevoldsen, P. [Bonus Energy A/S (Denmark); Oeye, S. [The Technical Univ. of Denmark (Denmark); Ganander, H. [Teknikgruppen AB (Sweden); Winkelaar, D. [Netherlands Energy Research Foundation (Netherlands)


    Results from research in an EC Joule-III project and from national projects are presented. The objectives are improvement of design methods for stall regulated wind turbines with emphasis on stall induced vibrations and dynamic stall. The primary concern is limitation of the edgewise vibrations in the fundamental blade natural mode shape, which have caused trouble on modern wind turbines of approximate size 500 kW nominal power and 40 m rotor diameter. A theoretical study of quasi-steady aerodynamics confirms that the vibrations are driven basically by energy supplied from the aerodynamic forces during stalled operation. This energy exchange is equivalent to negative aerodynamic damping. The theoretical approach identifies the main parameters controlling the phenomenon. These parameters describe the steady and the dynamic airfoil characteristics, the overall aerodynamic layout of the blade, e.g. chord length and twist, the structural properties of the blade, e.g. structural damping and properties controlling the resulting vibration direction. Furthermore, full aeroelastic calculations and comparison with measurements show that the properties of the supporting structure, i.e. the main shaft, the nacelle and the tower, are important, as the global vibration of the rotor on its support may exchange energy with the blade vibration, when the blade natural frequency is close to one of the frequencies of the coupled rotor tilt-yaw mode shapes, usually denoted the global rotor whirl frequencies. It is confirmed that the influence of changing the primary design parameters can be determined by use of qualified aeroelastic calculations. Presented design guidelines therefore build on both the simple quasi-steady models, which can be used for the preliminary choice of the design variables mentioned above, and on full aeroelastic calculations. The aeroelastic calculations refine the design basis and should be used for choosing the final design variables and for final

  11. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    International Nuclear Information System (INIS)

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.


    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants

  12. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Roehrig, John T., E-mail: [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Butrapet, Siritorn; Liss, Nathan M. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Bennett, Susan L. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H. [Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)


    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  13. Climate trends account for stalled wheat yields in Australia since 1990. (United States)

    Hochman, Zvi; Gobbett, David L; Horan, Heidi


    Global food security requires that grain yields continue to increase to 2050, yet yields have stalled in many developed countries. This disturbing trend has so far been only partially explained. Here, we show that wheat yields in Australia have stalled since 1990 and investigate the extent to which climate trends account for this observation. Based on simulation of 50 sites with quality weather data, that are representative of the agro-ecological zones and of soil types in the grain zone, we show that water-limited yield potential declined by 27% over a 26 year period from 1990 to 2015. We attribute this decline to reduced rainfall and to rising temperatures while the positive effect of elevated atmospheric CO 2 concentrations prevented a further 4% loss relative to 1990 yields. Closer investigation of three sites revealed the nature of the simulated response of water-limited yield to water availability, water stress and maximum temperatures. At all three sites, maximum temperature hastened time from sowing to flowering and to maturity and reduced grain number per m 2 and average weight per grain. This 27% climate-driven decline in water-limited yield is not fully expressed in actual national yields. This is due to an unprecedented rate of technology-driven gains closing the gap between actual and water-limited potential yields by 25 kg ha -1  yr -1 enabling relative yields to increase from 39% in 1990 to 55% in 2015. It remains to be seen whether technology can continue to maintain current yields, let alone increase them to those required by 2050. © 2017 John Wiley & Sons Ltd.

  14. Performance augmentation with vortex generators: Design and testing for stall-regulated AWT-26 turbine

    Energy Technology Data Exchange (ETDEWEB)

    Griffin, D.A. [Advanced Wind Turbines Inc., Seattle, WA (United States)


    A study investigated the use of vortex generators (VGs) for performance augmentation of the stall-regulated AWT-26 wind turbine. Based on wind-tunnel results and analysis, a VG array was designed for and tested on the AWT-26 prototype, designated Pt. Performance and loads data were measured for P1, both with and without VGs installed. The turbine performance with VGs met most of the design requirements; power output was increased at moderate wind speeds with a minimal effect on peak power. However, VG drag penalties caused a loss in power output for low wind speeds, such that performance with VGs resulted in a net decrease in AEP for wind speed sites up to 8.5 m/s. 8 refs., 8 figs., 3 tabs.

  15. Precautions against axial fan stall in reactor building to Tianwan NPP

    International Nuclear Information System (INIS)

    Liu Chunlong; Pei Junmin


    The paper introduces the mechanism and harm of rotating stall of axial fans, analyzes the necessity for prevention against axial fan stall in reactor building of Tianwan NPP, introduces the precautions, and then makes an assessment on anti-stall effect of flow separators. It can provide reference for model-selection or reconstruction of similar fans in power stations, and for operation and maintenance of axial fans. (authors)

  16. Comparison of Different Stall Conditions in Axial Flow Compressor Using Analytic Wavelet Transform

    Directory of Open Access Journals (Sweden)

    Arshad Ali


    Full Text Available The rotating stall inception data analysis using Analytic Wavelet Transform (AWT in a low-speed axial compressor was presented in the authors’ previous studies [1], [2]. These studies focused on the detection of instability inception in an axial flow compressor when it enters into the instability regime due to the modal type of stall perturbation. In this paper, the effectiveness of AWT is further studied by applying it under different testing conditions. In order to examine the results of AWT on highly sampled data, at first, the stall data were acquired at a high sampling frequency and the results were compared with the conventional filtered signals. Secondly, the AWT analysis of stall data was carried out for the condition when compressor experienced a spike type rotating stall disturbance. The stall inception information obtained from the AWT analysis was then compared with the commonly used stall detection techniques. The results show that AWT is equally beneficial for the diagnostic of compressor instability regardless of the data sampling rate and represents an outstanding ability to detect stall disturbance irrespective of the type of stall precursor, i.e. the modal wave or spike.

  17. Enhancing BEM simulations of a stalled wind turbine using a 3D correction model (United States)

    Bangga, Galih; Hutomo, Go; Syawitri, Taurista; Kusumadewi, Tri; Oktavia, Winda; Sabila, Ahmad; Setiadi, Herlambang; Faisal, Muhamad; Hendranata, Yongki; Lastomo, Dwi; Putra, Louis; Kristiadi, Stefanus; Bumi, Ilmi


    Nowadays wind turbine rotors are usually employed with pitch control mechanisms to avoid deep stall conditions. Despite that, wind turbines often operate under pitch fault situation causing massive flow separation to occur. Pure Blade Element Momentum (BEM) approaches are not designed for this situation and inaccurate load predictions are already expected. In the present studies, BEM predictions are improved through the inclusion of a stall delay model for a wind turbine rotor operating under pitch fault situation of -2.3° towards stall. The accuracy of the stall delay model is assessed by comparing the results with available Computational Fluid Dynamics (CFD) simulations data.

  18. Numerical simulation of the RISOe1-airfoil dynamic stall

    Energy Technology Data Exchange (ETDEWEB)

    Bertagnolio, F.; Soerensen, N. [Risoe National Lab., Wind Energy and Atmospheric Physics Dept., Roskilde (Denmark)


    In this paper we are concerned with the numerical computation of the dynamic stall that occur in the viscous flowfield over an airfoil. These results are compared to experimental data that were obtained with the new designed RISOe1-airfoil, both for a motionless airfoil and for a pitching motion. Moreover, we present some numerical computations of the plunging and lead-lag motions. We also investigate the possibility of using the pitching motion to simulate the plunging and lead-lag situations. (au)

  19. El departamento musical Disney : las Silly Symphonies y Carl Stalling


    Duarte del Moral, Marina


    La historia de la animación tiene un nombre propio: Walt Disney. Gracias a su experimentación en diversos campos de esta materia, Disney consigue desarrollar la animación y su universo de una forma perseverante y continua, adaptándose a los diversos cambios producidos desde el nacimiento de ésta, reinventando una y otra vez el sector y añadiendo su toque mágico a la personalidad de cada personaje y cada obra. En este camino no está solo gracias al trabajo de genios como Carl Stalling, que apo...

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

    DEFF Research Database (Denmark)

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


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

  1. Replication and Comparison of the Newly Proposed ADOS-2, Module 4 Algorithm in ASD without ID: A Multi-Site Study (United States)

    Pugliese, Cara E.; Kenworthy, Lauren; Bal, Vanessa Hus; Wallace, Gregory L.; Yerys, Benjamin E.; Maddox, Brenna B.; White, Susan W.; Popal, Haroon; Armour, Anna Chelsea; Miller, Judith; Herrington, John D.; Schultz, Robert T.; Martin, Alex; Anthony, Laura Gutermuth


    Recent updates have been proposed to the Autism Diagnostic Observation Schedule-2 Module 4 diagnostic algorithm. This new algorithm, however, has not yet been validated in an independent sample without intellectual disability (ID). This multi-site study compared the original and revised algorithms in individuals with ASD without ID. The revised…

  2. The FFA dynamic stall model. The Beddoes-Leishman dynamic stall model modified for lead-lag oscillations

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerck, A. [FFA, The Aeronautical Research Institute of Sweden, Bromma (Sweden)


    For calculations of the dynamics of wind turbines the inclusion of a dynamic stall model is necessary in order to obtain reliable results at high winds. For blade vibrations in the lead-lag motion the velocity relative to the blade will vary in time. In the present paper modifications to the Beddoes-Leishman model is presented in order to improve the model for calculations of cases with a varying relative velocity. Comparisons with measurement are also shown and the influence on the calculated aerodynamic damping by the modifications are investigated. (au)

  3. Ingestive behavior of lambs confined in individual and group stalls. (United States)

    Filho, A Eustáquio; Carvalho, G G P; Pires, A J V; Silva, R R; Santos, P E F; Murta, R M; Pereira, F M


    The experiment was conducted to evaluate the ingestive behavior of lambs confined in individual and group stalls. We used thirty-four lambs in their growing phase, aged an average of three months, with mean initial live weight of 17.8±5.2 kg. They were allotted in a completely randomized design with 24 animals kept in individual stalls and 10 animals confined as a group. The experiment lasted for a total of 74 days, and the first 14 days were dedicated to the animals' adaption to the management, facilities and diets. The data collection period lasted 60 days, divided into three 20-d periods for the behavior evaluation. The animals were subjected to five days of visual observation during the experiment period, by the quantification of 24 h a day, with evaluations on the 15th day of each period and an interim evaluation consisting of two consecutive days on the 30th and 31st day of the experiment. The animals confined as a group consumed less (pbehavior.

  4. A.C. Plasma Anemometer for Axial Compressor Stall Warning (United States)

    Matlis, Eric; Cameron, Joshua; Morris, Scott; Corke, Thomas


    Compressor sections of turbo jet engines are subject to stall and surge as a result of flow instabilities that occur upstream of the compressor rotor. One of the instability modes that contributes to compressor surge is the so-called `spike' mode of stall inception. It has been shown that this mode of instability can be predicted before onset by performing real-time statistical auto-correlation measurements of the blade-passing pressure characteristic at the mid-chord location of the rotor. These measurements are performed with pressure sensors or hot-wires that are too fragile for a full-scale compressor. We have developed a sensor that can survive the vibration and temperatures of a full-scale rig while providing the bandwidth necessary to resolve the blade passage signature required by this coherence technique. This sensor, called the Plasma Anemometer, provides high-bandwith point measurements of velocity or pressure fluctuations with unparalleled mechanical robustness and resistance to vibration and thermal effects.

  5. Precise site-selective termination of DNA replication by caging the 3-position of thymidine and its application to polymerase chain reaction. (United States)

    Kuzuya, Akinori; Okada, Fuminori; Komiyama, Makoto


    A new caged thymidine, 3-N-(2-(2-nitrophenyl)propyloxymethyl)thymidine (T(NPPOM)) was synthesized and used as a site-selective terminator of DNA-polymerase reaction in light-assisted cohesive-ending PCR (LACE-PCR), which directly gives sticky-ended PCR products after brief UVA irradiation. Primer-extension experiments using a template involving T(NPPOM) have shown that this caged nucleotide efficiently and site-selectively blocks reactions of a variety of polymerases commonly used in PCR. Misincorporation of nucleobases, observed with the use of other previously reported caged thymidines, scarcely occurred. It has turned out that a slight structural difference of caging groups can significantly improve the termination yield of polymerase reactions. A LACE-PCR product coding GFP gene was prepared by using primers containing T(NPPOM) and was ligated with a vector fragment prepared using restriction enzymes. The resulting recombinant vector successfully transformed E. coli.

  6. Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase. (United States)

    Volkán-Kacsó, Sándor; Marcus, Rudolph A


    A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F 1 -ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.

  7. The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells. (United States)

    Gerhardt, Jeannine; Tomishima, Mark J; Zaninovic, Nikica; Colak, Dilek; Yan, Zi; Zhan, Qiansheng; Rosenwaks, Zev; Jaffrey, Samie R; Schildkraut, Carl L


    Fragile X syndrome (FXS) is caused by a CGG repeat expansion in the FMR1 gene that appears to occur during oogenesis and during early embryogenesis. One model proposes that repeat instability depends on the replication fork direction through the repeats such that (CNG)n hairpin-like structures form, causing DNA polymerase to stall and slip. Examining DNA replication fork progression on single DNA molecules at the endogenous FMR1 locus revealed that replication forks stall at CGG repeats in human cells. Furthermore, replication profiles of FXS human embryonic stem cells (hESCs) compared to nonaffected hESCs showed that fork direction through the repeats is altered at the FMR1 locus in FXS hESCs, such that predominantly the CCG strand serves as the lagging-strand template. This is due to the absence of replication initiation that would typically occur upstream of FMR1, suggesting that altered replication origin usage combined with fork stalling promotes repeat instability during early embryonic development. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. The Location of the Bacterial Origin of Replication is Critical for Initial Ciproflaxcin Antibiotic Resistance (United States)

    Bos, Julia; Nehring, Ralph; Cruz, Diane; Austin, Doug; Rosenberg, Susan; Austin, Robert

    By using E. coli cells in which the unique origin of replication has been moved to a ectopic chromosome location distant from the native one, we probe how perturbation of gene order near the origin of replication impacts genome stability and survival under genomic attack. We find that when challenged with sub-inhibitory doses of ciprofloxacin, an antibiotic that generates replication fork stalling, cells with the ectopic origin show significant fitness loss. We show that genes functionally relevant to the cipro-induced stress response are largely located near the native origin, even in distantly related species. We show that while cipro induces increased copy number of genes proximal to the origin of replication as a direct consequence of replication fork stalling, gene copy number variation was reduced near the ectopic origin. Altered gene dosage in cells with an ectopic origin resulted in impaired replication fork repair and chromosome instability. We propose that gene distribution in the origin region acts as a fundamental first line of defense when the integrity of the genome is threatened and that genes proximal to the origin of replication serve as a mechanism of genetic innovation and a driving force of genome evolution in the presence of genotoxic antibiotics. Lewis Sigler Institute for Integrative Genomics and the Physics Department at Princeton University.

  9. Evaluating Classroom Interaction with the iPad®: An Updated Stalling's Tool (United States)

    MacKinnon, Gregory; Schep, Lourens; Borden, Lisa Lunney; Murray-Orr, Anne; Orr, Jeff; MacKinnon, Paula


    A large study of classrooms in the Caribbean context necessitated the use of a validated classroom observation tool. In practice, the paper-version Stalling's instrument (Stallings & Kaskowitz 1974) presented specific challenges with respect to (a) facile data collection and (b) qualitative observations of classrooms. In response to these…

  10. Simulation of Entropy Generation under Stall Conditions in a Centrifugal Fan

    Directory of Open Access Journals (Sweden)

    Lei Zhang


    Full Text Available Rotating stalls are generally the first instability met in turbomachinery, before surges. This 3D phenomenon is characterized by one or more stalled flow cells which rotate at a fraction of the impeller speed. The goal of the present work is to shed some light on the entropy generation in a centrifugal fan under rotating stall conditions. A numerical simulation of entropy generation is carried out with the ANSYS Fluent software which solves the Navier-Stokes equations and user defined function (UDF. The entropy generation characteristics in the centrifugal fan for five typical conditions are presented and discussed, involving the design condition, conditions on occurrence and development of stall inception, the rotating stall conditions with two throttle coefficients. The results show that the entropy generation increases after the occurrence of stall inception. The high entropy generation areas move along the circumferential and axial directions, and finally merge into one stall cell. The entropy generation rate during circumferential propagation of the stall cell is also discussed, showing that the entropy generation history is similar to sine curves in impeller and volute, and the volute tongue has a great influence on entropy generation in the centrifugal fan.

  11. DNA replication and cancer

    DEFF Research Database (Denmark)

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


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

  12. Optimal parameters for the FFA-Beddoes dynamic stall model

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerck, A.; Mert, M. [FFA, The Aeronautical Research Institute of Sweden, Bromma (Sweden); Madsen, H.A. [Risoe National Lab., Roskilde (Denmark)


    Unsteady aerodynamic effects, like dynamic stall, must be considered in calculation of dynamic forces for wind turbines. Models incorporated in aero-elastic programs are of semi-empirical nature. Resulting aerodynamic forces therefore depend on values used for the semi-empiricial parameters. In this paper a study of finding appropriate parameters to use with the Beddoes-Leishman model is discussed. Minimisation of the `tracking error` between results from 2D wind tunnel tests and simulation with the model is used to find optimum values for the parameters. The resulting optimum parameters show a large variation from case to case. Using these different sets of optimum parameters in the calculation of blade vibrations, give rise to quite different predictions of aerodynamic damping which is discussed. (au)

  13. Proposed Chevron Tengiz venture stalls amid Soviet political squabble

    International Nuclear Information System (INIS)



    This paper reports on the status of foreign investment in Soviet oil and gas joint ventures which has reached a critical juncture. Just as the U.S. is considering granting most favored nation trade status to the U.S.S.R., the joint venture petroleum deal seen as the litmus test for such deals-Chevron Corp.'s proposed addition of supergiant Tengiz oil field to its Caspian Sea joint venture-has stalled amid controversy. Unconfirmed reports from Soviet officials and other foreign joint venture participants in the U.S.S.R. have Chevron pulling out of the long negotiated, multibillion dollar project after the Soviets rejected the company's terms. Chevron, however, insists the project is still alive

  14. The INO80 remodeller in transcription, replication and repair. (United States)

    Poli, Jérôme; Gasser, Susan M; Papamichos-Chronakis, Manolis


    The accessibility of eukaryotic genomes to the action of enzymes involved in transcription, replication and repair is maintained despite the organization of DNA into nucleosomes. This access is often regulated by the action of ATP-dependent nucleosome remodellers. The INO80 class of nucleosome remodellers has unique structural features and it is implicated in a diverse array of functions, including transcriptional regulation, DNA replication and DNA repair. Underlying these diverse functions is the catalytic activity of the main ATPase subunit, which in the context of a multisubunit complex can shift nucleosomes and carry out histone dimer exchange. In vitro studies showed that INO80 promotes replication fork progression on a chromatin template, while in vivo it was shown to facilitate replication fork restart after stalling and to help evict RNA polymerase II at transcribed genes following the collision of a replication fork with transcription. More recent work in yeast implicates INO80 in the general eviction and degradation of nucleosomes following high doses of oxidative DNA damage. Beyond these replication and repair functions, INO80 was shown to repress inappropriate transcription at promoters in the opposite direction to the coding sequence. Here we discuss the ways in which INO80's diverse functions help maintain genome integrity.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

  15. SMARCAL1 Resolves Replication Stress at ALT Telomeres. (United States)

    Cox, Kelli E; Maréchal, Alexandre; Flynn, Rachel Litman


    Cancer cells overcome replicative senescence by exploiting mechanisms of telomere elongation, a process often accomplished by reactivation of the enzyme telomerase. However, a subset of cancer cells lack telomerase activity and rely on the alternative lengthening of telomeres (ALT) pathway, a recombination-based mechanism of telomere elongation. Although the mechanisms regulating ALT are not fully defined, chronic replication stress at telomeres might prime these fragile regions for recombination. Here, we demonstrate that the replication stress response protein SMARCAL1 is a critical regulator of ALT activity. SMARCAL1 associates with ALT telomeres to resolve replication stress and ensure telomere stability. In the absence of SMARCAL1, persistently stalled replication forks at ALT telomeres deteriorate into DNA double-strand breaks promoting the formation of chromosome fusions. Our studies not only define a role for SMARCAL1 in ALT telomere maintenance, but also demonstrate that resolution of replication stress is a crucial step in the ALT mechanism. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Analysis of Low Speed Stall Aerodynamics of a Swept Wing with Laminar Flow Glove (United States)

    Bui, Trong T.


    Reynolds-Averaged Navier-Stokes (RANS) computational fluid dynamics (CFD) analysis was conducted to study the low-speed stall aerodynamics of a GIII aircraft's swept wing modified with a laminar-flow wing glove. The stall aerodynamics of the gloved wing were analyzed and compared with the unmodified wing for the flight speed of 120 knots and altitude of 2300 ft above mean sea level (MSL). The Star-CCM+ polyhedral unstructured CFD code was first validated for wing stall predictions using the wing-body geometry from the First American Institute of Aeronautics and Astronautics (AIAA) CFD High-Lift Prediction Workshop. It was found that the Star-CCM+ CFD code can produce results that are within the scattering of other CFD codes considered at the workshop. In particular, the Star-CCM+ CFD code was able to predict wing stall for the AIAA wing-body geometry to within 1 degree of angle of attack as compared to benchmark wind-tunnel test data. Current results show that the addition of the laminar-flow wing glove causes the gloved wing to stall much earlier than the unmodified wing. Furthermore, the gloved wing has a different stall characteristic than the clean wing, with no sharp lift drop-off at stall for the gloved wing.

  17. Analysis of Low-Speed Stall Aerodynamics of a Swept Wing with Laminar-Flow Glove (United States)

    Bui, Trong T.


    Reynolds-Averaged Navier-Stokes (RANS) computational fluid dynamics (CFD) analysis was conducted to study the low-speed stall aerodynamics of a GIII aircraft's swept wing modified with a laminar-flow wing glove. The stall aerodynamics of the gloved wing were analyzed and compared with the unmodified wing for the flight speed of 120 knots and altitude of 2300 ft above mean sea level (MSL). The Star-CCM+ polyhedral unstructured CFD code was first validated for wing stall predictions using the wing-body geometry from the First American Institute of Aeronautics and Astronautics (AIAA) CFD High-Lift Prediction Workshop. It was found that the Star-CCM+ CFD code can produce results that are within the scattering of other CFD codes considered at the workshop. In particular, the Star-CCM+ CFD code was able to predict wing stall for the AIAA wing-body geometry to within 1 degree of angle of attack as compared to benchmark wind-tunnel test data. Current results show that the addition of the laminar-flow wing glove causes the gloved wing to stall much earlier than the unmodified wing. Furthermore, the gloved wing has a different stall characteristic than the clean wing, with no sharp lift drop-off at stall for the gloved wing.

  18. Investigating Stall Flutter using a DS model-An application for HAWTs

    International Nuclear Information System (INIS)

    Nichols, James; Haans, Wouter; Witcher, David; Attorni, Andrea


    As wind turbine blades become larger there is a tendency for the blade torsional stiffness to reduce, producing the possibility of dynamic instability at moderate windspeeds. While linearised methods can assess the envelope of allowable blade properties for avoiding classical flutter with attached flow aerodynamics, wind turbine aerofoils can experience stalled flow. Therefore, it is necessary to explore the possible effects of stall-flutter on blade stability. This paper aims to address methods for judging the stability of blade designs during both attached flow and stalled flow behaviour. This paper covers the following areas: i) Attached flow model A Beddoes-Leishman indicial model is presented and the choice of coefficients is explained in the context of Theodorsen's theory for flat-plate aerofoils and experimental results by Beddoes and Leishman. Special attention is given to the differing dynamic behaviour of the pitching moment due to flapping motion, pitching motion and dynamically varying inflow. (ii) Classical flutter analysis The time domain attached flow model is verified against a linear flutter analysis by comparing time domain results for a 3D model of a representative multi-megawatt turbine blade, varying the position of the centre of mass along the chord. The results show agreement to within 6% for a range of flutter onset speeds. (iii) Dynamic stall model On entering the stalled region, damping of torsional motion of an aerofoil section can become negative. A dynamic stall model which encompasses the effects of trailing edge separation and leading edge vortex detachment is presented and validated against published experimental data. (iv) Stall flutter The resulting time domain model is used in simulations validating the prediction of reduced flutter onset for stalled aerofoils. Representative stalled conditions for a multi-megawatt wind turbine blade are investigated to assess the possible reduction in flutter speed. A maximum reduction of 17

  19. Replication fork stability confers chemoresistance in BRCA-deficient cells

    DEFF Research Database (Denmark)

    Chaudhuri, Arnab Ray; Callen, Elsa; Ding, Xia


    /4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11......Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3...... nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations...

  20. Analysis of Low-Speed Stall Aerodynamics of a Business Jets Wing Using STAR-CCM+ (United States)

    Bui, Trong


    Reynolds-Averaged Navier-Stokes (RANS) computational fluid dynamics (CFD) analysis was conducted: to study the low-speed stall aerodynamics of a GIII aircrafts swept wing modified with (1) a laminar-flow wing glove, or (2) a seamless flap. The stall aerodynamics of these two different wing configurations were analyzed and compared with the unmodified baseline wing for low-speed flight. The Star-CCM+ polyhedral unstructured CFD code was first validated for wing stall predictions using the wing-body geometry from the First AIAA CFD High-Lift Prediction Workshop.

  1. Manual of Cupule Replication Technology

    Directory of Open Access Journals (Sweden)

    Giriraj Kumar


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

  2. Stalling HIV through social marketing: prospects in Pakistan. (United States)

    Husain, Sara; Shaikh, Babar T


    Over the last two decades HIV/AIDS has evolved from a series of interesting case-reports to a growing epidemic that threatens the entire world. It is feared to cause devastation among large pockets of populations and may roll back more than thirty years of public health achievements. This killer disease has been more amenable to behavioral change than by provision of curative services and attempts are being made to educate the public about this threat. Various techniques of promotion have been tried through out the world including television dramas/soaps, mass media and school curricula. Social marketing is an evolving strategy used to influence human behavior and choices. By using the principles of marketing and promoting behavior as a product, social marketers attempt to understand the dynamics of human behaviour and devise messages and products to change, modify, accept or reject unsafe behaviors or practices. Thus, social marketers provide an effective force to combat the spread of HIV and may serve to be invaluable allies in health promotion efforts. In a complex and diversified cultural milieu of Pakistan, social marketing can have a significant impact on health determinants and the conditions that will facilitate the adoption of health-oriented behaviors and practices. This paper gives an account of the elements needed for the success of a health promotion strategy adopted in a developing country and makes a case for social marketing to be adopted as the lead strategy for stalling HIV/AIDS in Pakistan.

  3. Initial design of a stall-controlled wind turbine rotor

    Energy Technology Data Exchange (ETDEWEB)

    Nygaard, T.A. [Inst. for Energiteknikk, Kjeller (Norway)


    A model intended for initial design of stall-controlled wind turbine rotors is described. The user specifies relative radial position of an arbitrary number of airfoil sections, referring to a data file containing lift-and drag curves. The data file is on the same format as used in the commercial blade-element code BLADES-/2/, where lift- and drag coefficients are interpolated from tables as function of Reynolds number, relative thickness and angle of attack. The user can set constraints on a selection of the following: Maximum power; Maximum thrust in operation; Maximum root bending moment in operation; Extreme root bending moment, parked rotor; Tip speed; Upper and lower bounds on optimisation variables. The optimisation variables can be selected from: Blade radius; Rotational speed; Chord and twist at an arbitrary number of radial positions. The user can chose linear chord distribution and a hyperbola-like twist distribution to ensure smooth planform and twist, or cubic spline interpolation for one or both. The aerodynamic model is based on classical strip theory with Prandtl tip loss correction, supplemented by empirical data for high induction factors. (EG)

  4. Cow preference and usage of free stalls compared with an open pack area. (United States)

    Fregonesi, J A; von Keyserlingk, M A G; Weary, D M


    Free-stall housing systems are designed to provide a comfortable and hygienic lying area, but some aspects of stall design may restrict usage by cows. The aim of this study was to compare free-stall housing with a comparable lying area (open pack) without stall partitions. We predicted that cows would spend more time lying down and standing in the bedded area when provided access to an open pack than when in free stalls. We also predicted that cows would spend less time standing outside of the lying area and less time perching with the front 2 hooves in the lying area when using the open pack. Groups (n = 8) of 12 cows each were provided access to either the open pack or stalls. After a 7-d adaptation period, each group was tested sequentially in the 2 treatments for 3 d each. This no-choice phase was followed by an 8-d choice phase during which cows had simultaneous access to both treatments. During the no-choice phase, cows spent more time lying down (13.03 +/- 0.24 vs. 12.48 +/- 0.24 h/d) and standing with all 4 hooves in the bedded area (0.96 +/- 0.12 vs. 0.41 +/- 0.12 h/d) of the open pack than in the stalls. During the choice phase, cows spent more time lying down (7.20 +/- 0.29 vs. 5.86 +/- 0.29 h/d) and standing with all 4 hooves in the bedded area (0.58 +/- 0.07 vs. 0.12 +/- 0.07 h/d) of the open pack than in the stalls. In both the no-choice (1.66 +/- 0.24 vs. 0.55 +/- 0.24 h/d) and choice (0.55 +/- 0.07 vs. 0.29 +/- 0.07 h/d) phases, cows spent more time standing with just 2 hooves in the stalls than in the open pack. In conclusion, cows spent more time lying and standing with all 4 hooves in the bedded open pack than in the stalls. Additionally, cows spent more time standing in the alley and standing with just the front 2 hooves on the bedding in the stalls than in the bedded open pack; increased standing time on wet concrete is a known risk factor for lameness.

  5. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney


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

  6. Replication Clamps and Clamp Loaders (United States)

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


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

  7. Dynamic stall characterization using modal analysis of phase-averaged pressure distributions (United States)

    Harms, Tanner; Nikoueeyan, Pourya; Naughton, Jonathan


    Dynamic stall characterization by means of surface pressure measurements can simplify the time and cost associated with experimental investigation of unsteady airfoil aerodynamics. A unique test capability has been developed at University of Wyoming over the past few years that allows for time and cost efficient measurement of dynamic stall. A variety of rotorcraft and wind turbine airfoils have been tested under a variety of pitch oscillation conditions resulting in a range of dynamic stall behavior. Formation, development and separation of different flow structures are responsible for the complex aerodynamic loading behavior experienced during dynamic stall. These structures have unique signatures on the pressure distribution over the airfoil. This work investigates the statistical behavior of phase-averaged pressure distribution for different types of dynamic stall by means of modal analysis. The use of different modes to identify specific flow structures is being investigated. The use of these modes for different types of dynamic stall can provide a new approach for understanding and categorizing these flows. This work uses airfoil data acquired under Army contract W911W60160C-0021, DOE Grant DE-SC0001261, and a gift from BP Alternative Energy North America, Inc.

  8. High-Speed Experiments on Combustion-Powered Actuation for Dynamic Stall Suppression (United States)

    Matalanis, Claude; Bowles, Patrick; Lorber, Peter; Crittenden, Thomas; Glezer, Ari; Schaeffler, Norman; Min, Byung-Young; Jee, Solkeun; Kuczek, Andrzej; Wake, Brian


    This work documents high-speed wind tunnel experiments conducted on a pitching airfoil equipped with an array of combustion-powered actuators (COMPACT). The main objective of these experiments was to demonstrate the stall-suppression capability of COMPACT on a high-lift rotorcraft airfoil, the VR-12, at relevant Mach numbers. Through dynamic pressure measurements at the airfoil surface it was shown that COMPACT can positively affect the stall behavior of the VR-12 at Mach numbers up to 0.4. Static airfoil results demonstrated 25% and 50% increases in post-stall lift at Mach numbers of 0.4 and 0.3, respectively. Deep dynamic stall results showed cycle-averaged lift coefficient increases up to 11% at Mach 0.4. Furthermore, it was shown that these benefits could be achieved with relatively few pulses during down-stroke and with no need to pre-anticipate the stall event. The flow mechanisms responsible for stall suppression were investigated using particle image velocimetry.

  9. Experimental Investigation of Stall Inception Mechanisms of Low Speed Contra Rotating Axial Flow Fan Stage

    Directory of Open Access Journals (Sweden)

    Tegegn Dejene Toge


    Full Text Available The present paper is an attempt in understanding the stall inception mechanism in a low speed, contra rotating axial flow fan stage, using wavelet transforms. The rotors used in this study have relatively large tip gap (about 3% of the blade span and aspect ratio of 3. The study was carried out near stall and at stall mass flow conditions for different speed ratios of rotor-2 to rotor-1. Unsteady pressure data from the casing wall mounted sensors are used to understand the stall inception mechanism. The wavelet transform clearly indicates that stall inception occurs mainly through long length scale disturbances for both rotors. It also reveals that short length disturbances occur simultaneously or intermittently in the case of rotor-1. The analysis shows the presence of a strong modal disturbance with 25–80% of the rotor frequency in the case of rotor-1 at the stall mass flow for all the speed combinations studied. The most interesting thing observed in the present study is that the frequency amplitude of the disturbance level is very small for both rotors.

  10. Adenovirus DNA Replication (United States)

    Hoeben, Rob C.; Uil, Taco G.


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

  11. Intracellular Detection of Viral Transcription and Replication Using RNA FISH (United States)


    Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...resolution. However, viral RNA tends to cluster in specific subcellular sites (e.g. viral replication factories). Thus while true single-molecule...assays [4]. Detection of viral RNA allows for in depth interrogation of the subcellular sites of viral replication and such experiments will help further

  12. A Method to Predict Compressor Stall in the TF34-100 Turbofan Engine Utilizing Real-Time Performance Data (United States)


    pass turbo –fan engine sensor data to seek its deterioration modelling and prognostics capability. In futurity this will allow for achievement of...preventive maintenance for the TF34-100 jet engine to prevent engine compressor stalls for the A-10 aircraft. Due to their destructive nature, compressor...stalls are a significant concern in axial flow compressor jet engines. A compressor stall is caused by air approaching the compressor blades at an

  13. Mode of transgene expression after fusion to early or late viral genes of a conditionally replicating adenovirus via an optimized internal ribosome entry site in vitro and in vivo

    International Nuclear Information System (INIS)

    Rivera, Angel A.; Wang Minghui; Suzuki, Kaori; Uil, Taco G.; Krasnykh, Victor; Curiel, David T.; Nettelbeck, Dirk M.


    The expression of therapeutic genes by oncolytic viruses is a promising strategy to improve viral oncolysis, to augment gene transfer compared with a nonreplicating adenoviral vector, or to combine virotherapy and gene therapy. Both the mode of transgene expression and the locale of transgene insertion into the virus genome critically determine the efficacy of this approach. We report here on the properties of oncolytic adenoviruses which contain the luciferase cDNA fused via an optimized internal ribosome entry site (IRES) to the immediate early adenoviral gene E1A (AdΔE1AIL), the early gene E2B (AdΔE2BIL), or the late fiber gene (AdΔfiberIL). These viruses showed distinct kinetics of transgene expression and luciferase activity. Early after infection, luciferase activities were lower for these viruses, especially for AdΔE2BIL, compared with nonreplicating AdTL, which contained the luciferase gene expressed from the strong CMV promoter. However, 6 days after infection, luciferase activities were approximately four (AdΔE1AIL) to six (AdΔfiberIL) orders of magnitude higher than for AdTL, reflecting virus replication and efficient transgene expression. Similar results were obtained in vivo after intratumoral injection of AdΔE2BIL, AdΔfiberIL, and AdTL. AdΔfiberIL and the parental virus, Ad5-Δ24, resulted in similar cytotoxicity, but AdΔE2BIL and AdΔE1AIL were slightly attenuated. Disruption of the expression of neighboring viral genes by insertion of the transgene was minimal for AdΔE2BIL and AdΔfiberIL, but substantial for AdΔE1AIL. Our observations suggest that insertion of IRES-transgene cassettes into viral transcription units is an attractive strategy for the development of armed oncolytic adenoviruses with defined kinetics and strength of transgene expression

  14. An archival analysis of stall warning system effectiveness during airborne icing encounters (United States)

    Maris, John Michael

    An archival study was conducted to determine the influence of stall warning system performance on aircrew decision-making outcomes during airborne icing encounters. A Conservative Icing Response Bias (CIRB) model was developed to explain the historical variability in aircrew performance in the face of airframe icing. The model combined Bayes' Theorem with Signal Detection Theory (SDT) concepts to yield testable predictions that were evaluated using a Binary Logistic Regression (BLR) multivariate technique applied to two archives: the NASA Aviation Safety Reporting System (ASRS) incident database, and the National Transportation Safety Board (NTSB) accident databases, both covering the period January 1, 1988 to October 2, 2015. The CIRB model predicted that aircrew would experience more incorrect response outcomes in the face of missed stall warnings than with stall warning False Alarms. These predicted outcomes were observed at high significance levels in the final sample of 132 NASA/NTSB cases. The CIRB model had high sensitivity and specificity, and explained 71.5% (Nagelkerke R2) of the variance of aircrew decision-making outcomes during the icing encounters. The reliability and validity metrics derived from this study suggest indicate that the findings are generalizable to the population of U.S. registered turbine-powered aircraft. These findings suggest that icing-related stall events could be reduced if the incidence of stall warning Misses could be minimized. Observed stall warning Misses stemmed from three principal causes: aerodynamic icing effects, which reduced the stall angle-of-attack (AoA) to below the stall warning calibration threshold; tail stalls, which are not monitored by contemporary protection systems; and icing-induced system issues (such as frozen pitot tubes), which compromised stall warning system effectiveness and airframe envelope protections. Each of these sources of missed stall warnings could be addressed by Aerodynamic Performance

  15. Stall Margin Improvement in a Centrifugal Compressor through Inducer Casing Treatment

    Directory of Open Access Journals (Sweden)

    V. V. N. K. Satish Koyyalamudi


    Full Text Available The increasing trend of high stage pressure ratio with increased aerodynamic loading has led to reduction in stable operating range of centrifugal compressors with stall and surge initiating at relatively higher mass flow rates. The casing treatment technique of stall control is found to be effective in axial compressors, but very limited research work is published on the application of this technique in centrifugal compressors. Present research was aimed to investigate the effect of casing treatment on the performance and stall margin of a high speed, 4 : 1 pressure ratio centrifugal compressor through numerical simulations using ANSYS CFX software. Three casing treatment configurations were developed and incorporated in the shroud over the inducer of the impeller. The predicted performance of baseline compressor (without casing treatment was in good agreement with published experimental data. The compressor with different inducer casing treatment geometries showed varying levels of stall margin improvement, up to a maximum of 18%. While the peak efficiency of the compressor with casing treatment dropped by 0.8%–1% compared to the baseline compressor, the choke mass flow rate was improved by 9.5%, thus enhancing the total stable operating range. The inlet configuration of the casing treatment was found to play an important role in stall margin improvement.

  16. An airloads theory for morphing airfoils in dynamic stall with experimental correlation (United States)

    Ahaus, Loren A.

    Helicopter rotor blades frequently encounter dynamic stall during normal flight conditions, limiting the applicability of classical thin-airfoil theory at large angles of attack. Also, it is evident that because of the largely different conditions on the advancing and retreating sides of the rotor, future rotorcraft may incorporate dynamically morphing airfoils (trailing-edge aps, dynamic camber, dynamic droop, etc.). Reduced-order aerodynamic models are needed for preliminary design and ight simulation. A unified model for predicting the airloads on a morphing airfoil in dynamic stall is presented, consisting of three components. First, a linear airloads theory allows for arbitrary airfoil deformations consistent with a morphing airfoil. Second, to capture the effects of the wake, the airloads theory is coupled to an induced ow model. Third, the overshoot and time delay associated with dynamic stall are modeled by a second-order dynamic filter, along the lines of the ONERA dynamic stall model. This paper presents a unified airloads model that allows arbitrary airfoil morphing with dynamic stall. Correlations with experimental data validate the theory.

  17. A time-varying subjective quality model for mobile streaming videos with stalling events (United States)

    Ghadiyaram, Deepti; Pan, Janice; Bovik, Alan C.


    Over-the-top mobile video streaming is invariably influenced by volatile network conditions which cause playback interruptions (stalling events), thereby impairing users' quality of experience (QoE). Developing models that can accurately predict users' QoE could enable the more efficient design of quality-control protocols for video streaming networks that reduce network operational costs while still delivering high-quality video content to the customers. Existing objective models that predict QoE are based on global video features, such as the number of stall events and their lengths, and are trained and validated on a small pool of ad hoc video datasets, most of which are not publicly available. The model we propose in this work goes beyond previous models as it also accounts for the fundamental effect that a viewer's recent level of satisfaction or dissatisfaction has on their overall viewing experience. In other words, the proposed model accounts for and adapts to the recency, or hysteresis effect caused by a stall event in addition to accounting for the lengths, frequency of occurrence, and the positions of stall events - factors that interact in a complex way to affect a user's QoE. On the recently introduced LIVE-Avvasi Mobile Video Database, which consists of 180 distorted videos of varied content that are afflicted solely with over 25 unique realistic stalling events, we trained and validated our model to accurately predict the QoE, attaining standout QoE prediction performance.

  18. Animal Mitochondrial DNA Replication (United States)

    Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.


    Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

  19. Psychology, replication & beyond. (United States)

    Laws, Keith R


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

  20. Registered Replication Report

    DEFF Research Database (Denmark)

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


    In an anonymous 4-person economic game, participants contributed more money to a common project (i.e., cooperated) when required to decide quickly than when forced to delay their decision (Rand, Greene & Nowak, 2012), a pattern consistent with the social heuristics hypothesis proposed by Rand...... and colleagues. The results of studies using time pressure have been mixed, with some replication attempts observing similar patterns (e.g., Rand et al., 2014) and others observing null effects (e.g., Tinghög et al., 2013; Verkoeijen & Bouwmeester, 2014). This Registered Replication Report (RRR) assessed...... the size and variability of the effect of time pressure on cooperative decisions by combining 21 separate, preregistered replications of the critical conditions from Study 7 of the original article (Rand et al., 2012). The primary planned analysis used data from all participants who were randomly assigned...

  1. The role of tip clearance in high-speed fan stall

    Energy Technology Data Exchange (ETDEWEB)

    Adamczyk, J.J. (NASA Lewis Research Center, Cleveland, OH (United States)); Celestina, M.L. (Sverdrup Tech., Inc., Cleveland, OH (United States)); Greitzer, E.M. (Massachusetts Institute of Technology, Cambridge, MA (United States))


    A numerical experiment has been carried out to define the near-stall casing endwall flow field of a high-speed fan rotor. The experiment used a simulation code incorporating a simple clearance model, whose calibration is presented. The results of the simulation show that the interaction of the tip leakage vortex and the in-passage shock plays a major role in determining the fan flow range. More specifically, the computations imply that it is the area increase of this vortex as it passes through the in-passage shock that is the source of the blockage associated with stall. In addition, for fans of this type, it is the clearance over the forward portion of the fan blade that controls the flow processes leading to stall.

  2. The role of tip clearance in high-speed fan stall (United States)

    Adamczyk, J. J.; Celestina, M. L.; Greitzer, E. M.


    A numerical experiment has been carried out to define the near-stall casing endwall flowfield of a high-speed fan rotor. The experiment used a simulation code incorporating a simple clearance model, whose calibration is presented. The results of the simulation show that the interaction of the tip leakage vortex and the in-pasage shock plays a major role in determining the fan flow range. More specifically, the computations imply that it is the area increase of this vortex as it passes through the in-passage shock, which is the source of the blockage associated with stall. In addition, for fans of this type, it is the clearance over the forward portion of the fan blade which controls the flow processes leading to stall.

  3. The computation of the post-stall behavior of a circulation controlled airfoil (United States)

    Linton, Samuel W.


    The physics of the circulation controlled airfoil is complex and poorly understood, particularly with regards to jet stall, which is the eventual breakdown of lift augmentation by the jet at some sufficiently high blowing rate. The present paper describes the numerical simulation of stalled and unstalled flows over a two-dimensional circulation controlled airfoil using a fully implicit Navier-Stokes code, and the comparison with experimental results. Mach numbers of 0.3 and 0.5 and jet total to freestream pressure ratios of 1.4 and 1.8 are investigated. The Baldwin-Lomax and k-epsilon turbulence models are used, each modified to include the effect of strong streamline curvature. The numerical solutions of the post-stall circulation controlled airfoil show a highly regular unsteady periodic flowfield. This is the result of an alternation between adverse pressure gradient and shock induced separation of the boundary layer on the airfoil trailing edge.

  4. Replication, refinement & reachability

    DEFF Research Database (Denmark)

    Debois, Søren; Hildebrandt, Thomas T.; Slaats, Tijs


    We explore the complexity of reachability and run-time refinement under safety and liveness constraints in event-based process models. Our study is framed in the DCR? process language, which supports modular specification through a compositional operational semantics. DCR? encompasses the “Dynamic...... Condition Response (DCR) graphs” declarative process model for analysis, execution and safe run-time refinement of process-aware information systems; including replication of sub-processes. We prove that event-reachability and refinement are np-hard for DCR? processes without replication...

  5. Effect of summer grazing on welfare of dairy cows reared in mountain tie-stall barns

    Directory of Open Access Journals (Sweden)

    Simonetta Dovier


    Full Text Available Traditional mountain farms have an important economic, social and environmental role. The Alps management system for dairy cows consists of animals kept indoors from autumn to spring, mostly in tie-stalls, and moved to mountain pasture in summer. The aim of our study was to assess the effect of mountain summer grazing on the welfare of dairy cows housed in tie-stall barns. Twenty-four farms were considered. In twelve of them, animals were reared in tie-stalls and moved to mountain pasture for three months in summer; they were visited three times: (i four weeks before grazing during the indoor period in the stall; (ii about three weeks after the start of grazing; and (iii in the stall, in autumn, at least three weeks after returning from grazing. The other twelve farms kept the animals in tie-stalls all year; they were visited once in autumn. Data were collected following a protocol that considers animal-based measures and structure information on the basis of Quality Welfare Consortium® indications. Data allowed the calculation of both the Animal Needs Index score (ANI 35L and an overall assessment of the cows’ welfare obtained from three general aspects: housing, animal’s physical condition, and animal’s behaviour. Summer grazing had a significant positive effect on injuries, lameness and animal’s rising duration but a negative effect on faeces consistency. Moreover, a reduction of tongue playing was observed. The ANI 35L and the overall assessment did not show significant differences linked to summer grazing, which tended to have a positive but temporary effect on animal behaviour.

  6. Prediction of RNA Polymerase II recruitment, elongation and stalling from histone modification data

    DEFF Research Database (Denmark)

    Chen, Yun; Jørgensen, Mette; Kolde, Raivo


    strategies are needed to progress from descriptive annotation of data to quantitative, predictive models. RESULTS: Here, we describe a computational framework which with high accuracy can predict the locations of core promoters, the amount of recruited RNAPII at the promoter, the amount of elongating RNAPII...... of RNAPII stalling. CONCLUSIONS: In this study we introduce a general framework to accurately predict the level of RNAPII recruitment, elongation, stalling and mRNA expression from chromatin signals. The versatility of the method also makes it ideally suited to investigate other genomic data....

  7. Analysis of compressible light dynamic stall flow at transitional Reynolds numbers

    DEFF Research Database (Denmark)

    Dyken, R.D. Van; Ekaterinaris, John A.; Chandrasekhara, M.S.


    Numerical and experimental results of steady and light dynamic stall flow over an oscillating NACA 0012 airfoil at a freestream Mach number of 0.3 and Reynolds number of 0.54 x 10(6) are compared, The experimental observation that dynamic stall is induced from the bursting of a laminar separation...... point is specified suitably and a simple transition length model is incorporated to determine the extent of the laminar separation bubble. The thin-layer approximations of compressible, Reynolds-averaged, Navier-Stokes equations are used for the numerical solution, with an implicit, upwind-biased, third...

  8. The influence of elevated feed stalls on feeding behaviour of lactating dairy cows

    Directory of Open Access Journals (Sweden)

    Barbara Benz


    Full Text Available The performance level of high yielding cows can only be guaranteed by high quality forage and high feed intake. An about 15–20 cm elevated and 160 cm long feed stall with rubber flooring doesn’t only offer undisturbed meals but also a yielding and dry standing surface. In a pilot stable with 130 dairy cows (German Simmental the feeding alley was subsequently equipped with elevated feed stalls. The results show that animals frequented the feeding barn less often while the duration of single meals prolonged. The specific behavioural changes differed depending on milk yield and number of lactation.

  9. Diagnosis of voltage collapse due to induction motor stalling using static analysis

    International Nuclear Information System (INIS)

    Karbalaei, F.; Kalantar, M.; Kazemi, A.


    Induction motor stalling is one of the important reasons for voltage collapse. This paper presents that, for induction motor stalling diagnosis, it is not necessary to use a third or first order dynamic model of induction motors. Instead, a method is presented based on algebraic calculations for which the steady state model of the induction motor considering different kinds of mechanical loads (constant and variable torque) is added to the power flow equations. Simulation results for a simple system confirm the correctness of the proposed method as compared to dynamic simulation results

  10. Power control of a wind farm with active stall wind turbines and AC grid connection

    DEFF Research Database (Denmark)

    Hansen, Anca Daniela; Sørensen, Poul; Iov, Florin

    This paper describes the design of a centralised wind farm controller for a wind farm made-up exclusively of active stall wind turbines with AC grid connection. The overall aim of such controller is to enable the wind farms to provide the best grid support. The designed wind farm control involves...... both the control on wind turbine level as well as the central control on the wind farm level. The ability of active stall wind farms with AC grid connection to regulate the power production to the reference power ordered by the operators is assessed and discussed by means of simulations....

  11. Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors

    Directory of Open Access Journals (Sweden)

    Moss Bernard


    Full Text Available Abstract Background Replication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins. DNA synthesis appears to start near the ends of the genome, but specific origin sequences have not been defined. Surprisingly, transfected circular DNA lacking specific viral sequences is also replicated in poxvirus-infected cells. Origin-independent plasmid replication depends on the viral DNA polymerase, but neither the number of additional viral proteins nor the site of replication has been determined. Results Using a novel real-time polymerase chain reaction assay, we detected a >400-fold increase in newly replicated plasmid in cells infected with vaccinia virus. Studies with conditional lethal mutants of vaccinia virus indicated that each of the five proteins known to be required for viral genome replication was also required for plasmid replication. The intracellular site of replication was determined using a plasmid containing 256 repeats of the Escherichia coli lac operator and staining with an E. coli lac repressor-maltose binding fusion protein followed by an antibody to the maltose binding protein. The lac operator plasmid was localized in cytoplasmic viral factories delineated by DNA staining and binding of antibody to the viral uracil DNA glycosylase, an essential replication protein. In addition, replication of the lac operator plasmid was visualized continuously in living cells infected with a recombinant vaccinia virus that expresses the lac repressor fused to enhanced green fluorescent protein. Discrete cytoplasmic fluorescence was detected in cytoplasmic juxtanuclear sites at 6 h after infection and the area and intensity of fluorescence increased over the next several hours. Conclusion Replication of a circular plasmid lacking specific poxvirus DNA sequences mimics viral genome replication by occurring in cytoplasmic viral factories

  12. Evolution of Replication Machines (United States)

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


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

  13. Replication studies in longevity

    DEFF Research Database (Denmark)

    Varcasia, O; Garasto, S; Rizza, T


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

  14. Validation of the Beddoes-Leishman Dynamic Stall Model for Horizontal Axis Wind Turbines using MEXICO data

    NARCIS (Netherlands)

    Pereira, R.; Schepers, G.; Pavel, M.D.


    The aim of this study is to assess the load predicting capability of a classical Beddoes-Leishman dynamic stall model in a horizontal axis wind turbine (HAWT) environment, in the presence of yaw-misalignment. The dynamic stall model was tailored to the HAWT environment, and validated against

  15. ATR-Chk1-APC/C-dependent stabilization of Cdc7-ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

    DEFF Research Database (Denmark)

    Yamada, M.; Watanabe, K.; Mistrik, M.


    replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosomeCdh1 (APC/C) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C through...... degradation of Cdh1 upon replication block, thereby stabilizing APC/C substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4...

  16. Loss of Caenorhabditis elegans BRCA1 Promotes Genome Stability During Replication in smc-5 Mutants (United States)

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


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

  17. A stochastic model for the simulation of wind turbine blades in static stall

    DEFF Research Database (Denmark)

    Bertagnolio, Franck; Rasmussen, Flemming; Sørensen, Niels N.


    The aim of this work is to improve aeroelastic simulation codes by accounting for the unsteady aerodynamic forces that a blade experiences in static stall. A model based on a spectral representation of the aerodynamic lift force is defined. The drag and pitching moment are derived using...

  18. Physics of Prestall Propagating Disturbances in Axial Compressors and Their Potential as a Stall Warning Indicator

    Directory of Open Access Journals (Sweden)

    Mario Eck


    Full Text Available Axial compressors in aero engines are prone to suffering a breakdown of orderly flow when operating at the peak of the pressure rise characteristic. The damaging potential of separated flows is why a safe distance has to be left between every possible operating point and an operating point at which stall occurs. During earlier investigations of stall inception mechanisms, a new type of prestall instability has been found. In this study, it could be demonstrated that the prestall instability characterised by discrete flow disturbances can be clearly assigned to the subject of “Rotating Instabilities”. Propagating disturbances are responsible for the rise in blade passing irregularity. If the mass flow is reduced successively, the level of irregularity increases until the prestall condition devolves into rotating stall. The primary objective of the current work is to highlight the basic physics behind these prestall disturbances by complementary experimental and numerical investigations. Before reaching the peak of the pressure rise characteristic flow, disturbances appear as small vortex tubes with one end attached to the casing and the other attached to the suction surface of the rotor blade. These vortex structures arise when the entire tip region is affected by blockage and at the same time the critical rotor incidence is not exceeded in this flow regime. Furthermore, a new stall indicator was developed by applying statistical methods to the unsteady pressure signal measured over the rotor blade tips, thus granting a better control of the safety margin.

  19. Numerical Study on the Acoustic Characteristics of an Axial Fan under Rotating Stall Condition

    Directory of Open Access Journals (Sweden)

    Lei Zhang


    Full Text Available Axial fan is an important piece of equipment in the thermal power plant that provides enough air for combustion of coal. This paper focuses on the aerodynamic noise characteristics of an axial fan in the development from stall inception to stall cells. The aerodynamic noise characteristic of monitoring region in time and frequency domains was simulated employing the large-eddy simulation (LES, with the addition of throttle setting and the Ffowcs Williams-Hawkings (FW-H noise model. The numerical results show that, under the design condition, the acoustic pressure presents regular periodicity along with the time. The noise energy is concentrated with high energy of the fundamental frequency and high order harmonics. During the stall inception stage, the acoustic pressure amplitude starts fluctuating and discrete frequencies are increased significantly in the low frequency; among them, there are three obvious discrete frequencies: 27.66 Hz, 46.10 Hz and 64.55 Hz. On the rotating stall condition, the fluctuation of the acoustic pressure level and amplitude are more serious than that mentioned above. During the whole evolution process, the acoustic pressure peak is difficult to keep stable all the time, and a sudden increase of the peak value at the 34.5th revolution corresponds to the relative velocity’s first sudden increase at the time when the valve coefficient is 0.780.

  20. Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation. (United States)

    Artieri, Carlo G; Fraser, Hunter B


    The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling. © 2014 Artieri and Fraser; Published by Cold Spring Harbor Laboratory Press.


    The report gives results of emissions measurements carried out at a representative facility (Eljer Plumbingware in Wilson, NC) that manufactures polyester-resin-reinforced shower stalls and bathtubs by spraying styrene-based resins onto molds in vented, open, spray booths. Styren...

  2. 14 CFR 23.203 - Turning flight and accelerated turning stalls. (United States)


    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Turning flight and accelerated turning stalls. 23.203 Section 23.203 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF... a 30 degree bank. Reduce speed by steadily and progressively tightening the turn with the elevator...

  3. Power reduction and the radial limit of stall delay in revolving wings of different aspect ratio

    NARCIS (Netherlands)

    Kruyt, J.W.; Heijst, Van G.F.; Altshuler, D.L.; Lentink, David


    Airplanes and helicopters use high aspect ratio wings to reduce the power required to fly, but must operate at low angle of attack to prevent flow separation and stall. Animals capable of slow sustained flight, such as hummingbirds, have low aspect ratio wings and flap their wings at high angle

  4. Vortical structures responsible for delayed stall in an idealized humpback whale flipper model (United States)

    Kim, Heesu; Kim, Jooha; Choi, Haecheon


    In this study, we investigate how the tubercles on the leading edge of an idealized humpback whale flipper model delay the stall. Oil-surface visualization is performed to see the surface flow pattern on the suction surface, and PIV is conducted in several streamwise and crossflow planes at different attack angles (α). Without tubercles, leading edge separation first occurs near the tip region and progresses inboard with increasing α. With tubercles, however, two types of vortical motions are observed at the mid-span. The first is streamwise vortex arrays which are dominant at α 9° , and these structures appear near the trailing edge. These two types of vortical motions delay flow separation at the peak regions of the mid-span, eliminating the spanwise stall progression and resulting in delayed stall. At α = 16° at which the tubercle model stalls, a large-scale streamwise vortex is originated from flow separation near the root region. This structure delays flow separation at the mid-span, leading to higher lift coefficient. Supported by NRF-2014M3C1B1033848.

  5. Identification of phlebovirus and arenavirus RNA sequences that stall and repress the exoribonuclease XRN1. (United States)

    Charley, Phillida A; Wilusz, Carol J; Wilusz, Jeffrey


    Regulated mRNA decay plays a vital role in determining both the level and quality of cellular gene expression. Viral RNAs must successfully evade this host RNA decay machinery to establish a productive infection. One way for RNA viruses to accomplish this is to target the cellular exoribonuclease XRN1, because this enzyme is accessible in the cytoplasm and plays a major role in mRNA decay. Members of the Flaviviridae use RNA structures in their 5'- or 3'-untranslated regions to stall and repress XRN1, effectively stabilizing viral RNAs while also causing significant dysregulation of host cell mRNA stability. Here, we use a series of biochemical assays to demonstrate that the 3'-terminal portion of the nucleocapsid (N) mRNA of Rift Valley fever virus, a phlebovirus of the Bunyaviridae family, also can effectively stall and repress XRN1. The region responsible for impeding XRN1 includes a G-rich portion that likely forms a G-quadruplex structure. The 3'-terminal portions of ambisense-derived transcripts of multiple arenaviruses also stalled XRN1. Therefore, we conclude that RNAs from two additional families of mammalian RNA viruses stall and repress XRN1. This observation. emphasizes the importance and commonality of this viral strategy to interfere with the 5'-to-3'-exoribonuclease component of the cytoplasmic RNA decay machinery. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Theory/test correlation of helicopter rotor blade element airloads in the blade stall regime (United States)

    Bobo, C. J.


    The effects of stall on a rotor blade element in a three-dimensional rotating environment was investigated. The model rotor test provided blade element airloads and local boundary layer flow characteristics at the three-quarter blade radius position for a wide range of rotor operating conditions. A description of the test program and the test results are presented.

  7. Field rotor measurements. Data sets prepared for analysis of stall hysteresis

    Energy Technology Data Exchange (ETDEWEB)

    Aagaard Madsen, H.; Thirstrup Petersen, J. [Risoe National Lab. (Denmark); Bruining, A. [Delft Univ. of Technology (Netherlands); Brand, A. [ECN (Netherlands); Graham, M. [Imperical College (United Kingdom)


    As part of the JOULE-3 project `STALLVIB` an analysis and synthesis of the data from the field rotor experiments at ECN, Delft University, Imperial College, NREL and Risoe has been carried out. This has been done in order to see to what extent the data could be used for further development and validation of engineering dynamic stall models. A detailed investigation of the influence of the post-processing of the different data sets has been performed. Further, important statistical functions such as PSD spectra, coherence and transfer functions have been derived for the data sets which can be used as basis for evaluation of the quality of the data seen relative to actual application of the data. The importance of using an appropriate low-pass filtering to remove high frequency noise has been demonstrated when the relation between instantaneous values of e.g. {alpha} and C{sub N} is considered. In general, the complicated measurement on a rotor of {alpha} and w and the interpretation of these parameters combined with the strongly three-dimensional, turbulent flow field around the rotating blade has the consequence that it seems difficult to derive systematic information from the different data sets about stall hysteresis. In particular, the measurement of {alpha}, which determination of the stagnation point gives reasonable data below stall but fails in stall. On the other hand, measurements of {alpha} with a five hole pitot tube can be used also in the stall region. Another main problem is the non-dimensionalization of the coefficients C{sub N} and C{sub r}. If the dynamic pressure used for the non-dimensionalization is not fully correlated with the aerodynamic pressure over the considered airfoil section due to e.g. influence of the gravity on the pressure pipes, the hysteresis loops will be distorted. However, using the data with caution and applying a suitable post-processing as described by the different participants, it will probably be possible to obtain some

  8. Modeling dynamic stall on wind turbine blades under rotationally augmented flow fields

    Energy Technology Data Exchange (ETDEWEB)

    Guntur, S. [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Schreck, S. [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Sorensen, N. N. [Technical Univ. of Denmark, Lyngby (Denmark); Bergami, L. [Technical Univ. of Denmark, Lyngby (Denmark)


    It is well known that airfoils under unsteady flow conditions with a periodically varying angle of attack exhibit aerodynamic characteristics different from those under steady flow conditions, a phenomenon commonly known as dynamic stall. It is also well known that the steady aerodynamic characteristics of airfoils in the inboard region of a rotating blade differ from those under steady two-dimensional (2D) flow conditions, a phenomenon commonly known as rotational augmentation. This paper presents an investigation of these two phenomena together in the inboard parts of wind turbine blades. This analysis is carried out using data from three sources: (1) the National Renewable Energy Laboratory’s Unsteady Aerodynamics Experiment Phase VI experimental data, including constant as well as continuously pitching blade conditions during axial operation, (2) data from unsteady Delayed Detached Eddy Simulations (DDES) carried out using the Technical University of Denmark’s in-house flow solver Ellipsys3D, and (3) data from a simplified model based on the blade element momentum method with a dynamic stall subroutine that uses rotationally augmented steady-state polars obtained from steady Phase VI experimental sequences, instead of the traditional 2D nonrotating data. The aim of this work is twofold. First, the blade loads estimated by the DDES simulations are compared to three select cases of the N sequence experimental data, which serves as a validation of the DDES method. Results show reasonable agreement between the two data in two out of three cases studied. Second, the dynamic time series of the lift and the moment polars obtained from the experiments are compared to those from the dynamic stall subroutine that uses the rotationally augmented steady polars. This allowed the differences between the stall phenomenon on the inboard parts of harmonically pitching blades on a rotating wind turbine and the classic dynamic stall representation in 2D flow to be

  9. Parametric analyses on dynamic stall control of rotor airfoil via synthetic jet

    Directory of Open Access Journals (Sweden)

    Qijun ZHAO


    Full Text Available The effects of synthetic jet control on unsteady dynamic stall over rotor airfoil are investigated numerically. A moving-embedded grid method and an Unsteady Reynolds Averaged Navier-Stokes (URANS solver coupled with k-ω Shear Stress Transport (SST turbulence model are established for predicting the complex flowfields of oscillatory airfoil under jet control. Additionally, a velocity boundary condition modeled by sinusoidal function has been developed to fulfill the perturbation effect of periodic jet. The validity of present CFD method is evaluated by comparisons of the calculated results of baseline dynamic stall case for rotor airfoil and jet control case for VR-7B airfoil with experimental data. Then, parametric analyses are conducted emphatically for an OA212 rotor airfoil to investigate the effects of jet control parameters (jet location, dimensionless frequency, momentum coefficient, jet angle, jet type and dual-jet on dynamic stall characteristics of rotor airfoil. It is demonstrated by the calculated results that efficiency of jet control could be improved with specific momentum coefficient and jet angle when the jet is located near separation point of rotor airfoil. Furthermore, the dual-jet could improve control efficiency more obviously on dynamic stall of rotor airfoil with respect to the unique jet, and the influence laws of dual-jet’s angles and momentum coefficients on control effects are similar to those of the unique jet. Finally, unsteady aerodynamic characteristics of rotor via synthetic jet which is located on the upper surface of rotor blade in forward flight are calculated, and as a result, the aerodynamic characteristics of rotor are improved compared with the baseline. The results indicate that synthetic jet has the capability in improving aerodynamic characteristics of rotor. Keywords: Airfoil, Dynamic stall characteristics, Flow control, Moving-embedded grid methodology, Navier-Stokes equations, Parametric

  10. Power reduction and the radial limit of stall delay in revolving wings of different aspect ratio. (United States)

    Kruyt, Jan W; van Heijst, GertJan F; Altshuler, Douglas L; Lentink, David


    Airplanes and helicopters use high aspect ratio wings to reduce the power required to fly, but must operate at low angle of attack to prevent flow separation and stall. Animals capable of slow sustained flight, such as hummingbirds, have low aspect ratio wings and flap their wings at high angle of attack without stalling. Instead, they generate an attached vortex along the leading edge of the wing that elevates lift. Previous studies have demonstrated that this vortex and high lift can be reproduced by revolving the animal wing at the same angle of attack. How do flapping and revolving animal wings delay stall and reduce power? It has been hypothesized that stall delay derives from having a short radial distance between the shoulder joint and wing tip, measured in chord lengths. This non-dimensional measure of wing length represents the relative magnitude of inertial forces versus rotational accelerations operating in the boundary layer of revolving and flapping wings. Here we show for a suite of aspect ratios, which represent both animal and aircraft wings, that the attachment of the leading edge vortex on a revolving wing is determined by wing aspect ratio, defined with respect to the centre of revolution. At high angle of attack, the vortex remains attached when the local radius is shorter than four chord lengths and separates outboard on higher aspect ratio wings. This radial stall limit explains why revolving high aspect ratio wings (of helicopters) require less power compared with low aspect ratio wings (of hummingbirds) at low angle of attack and vice versa at high angle of attack. © 2015 The Author(s) Published by the Royal Society. All rights reserved.

  11. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon. (United States)

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S


    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. © 2015 von der Malsburg et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (

  12. Flooding of S. Dakota mine stalls plans for laboratory

    CERN Document Server

    Chang, K


    The owner of a former gold mine in South Dakota turned off the pumps allowing water to begin accumulating in the tunnels below ground. The site had previously been proposed as the location for a new underground particle physics and astronomy laboratory (1 page).

  13. Asynchronous Replication, Mono-Allelic Expression, and Long Range Cis-Effects of ASAR6


    Donley, Nathan; Stoffregen, Eric P.; Smith, Leslie; Montagna, Christina; Thayer, Mathew J.


    Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encod...

  14. Genome Replication in Thermococcus kodakarensis Independent of Cdc6 and an Origin of Replication

    Directory of Open Access Journals (Sweden)

    Alexandra M. Gehring


    Full Text Available The initiation of DNA replication is typically tightly regulated by proteins that form initiation complexes at specific sequences known as replication origins. In Archaea and Eukaryotes, Cdc6, a near-universally conserved protein binds and facilitates the origin-dependent assembly of the replicative apparatus. TK1901 encodes Cdc6 in Thermococcus kodakarensis but, as we report here, TK1901 and the presumed origin of replication can be deleted from the genome of this hyperthermophilic Archaeon without any detectable effects on growth, genetic competence or the ability to support autonomous plasmid replication. All regions of the genome were equally represented in the sequences generated by whole genome sequencing of DNA isolated from T. kodakarensis strains with or without TK1901, inconsistent with DNA initiation occurring at one or few origins, and instead suggestive of replication initiating at many sites distributed throughout the genome. We were unable to generate strains lacking the recombination factors, RadA or RadB, consistent with T. kodakarensis cells, that are oligoploid (7–19 genomes per cell, employing a recombination-based mechanism of DNA replication. Deletion of the previously presumed origin region reduced the long-term viability of cultures supporting the possibility that retaining an origin-based mechanism of DNA initiation provides a survival mechanism for stationary phase cells with only one genome.

  15. Internal Flow of a High Specific-Speed Diagonal-Flow Fan (Rotor Outlet Flow Fields with Rotating Stall

    Directory of Open Access Journals (Sweden)

    Norimasa Shiomi


    Full Text Available We carried out investigations for the purpose of clarifying the rotor outlet flow fields with rotating stall cell in a diagonal-flow fan. The test fan was a high–specific-speed (ns=1620 type of diagonal-flow fan that had 6 rotor blades and 11 stator blades. It has been shown that the number of the stall cell is 1, and its propagating speed is approximately 80% of its rotor speed, although little has been known about the behavior of the stall cell because a flow field with a rotating stall cell is essentially unsteady. In order to capture the behavior of the stall cell at the rotor outlet flow fields, hot-wire surveys were performed using a single-slant hotwire probe. The data obtained by these surveys were processed by means of a double phase-locked averaging technique, which enabled us to capture the flow field with the rotating stall cell in the reference coordinate system fixed to the rotor. As a result, time-dependent ensemble averages of the three-dimensional velocity components at the rotor outlet flow fields were obtained. The behavior of the stall cell was shown for each velocity component, and the flow patterns on the meridional planes were illustrated.

  16. USP7 is a SUMO deubiquitinase essential for DNA replication

    DEFF Research Database (Denmark)

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


    to the accumulation of Ub on SUMOylated proteins, which are displaced away from replisomes. Our findings provide a model explaining the differential accumulation of SUMO and Ub at replication forks and identify an essential role of USP7 in DNA replication that should be considered in the development of USP7...... is maintained at sites of DNA replication in mammalian cells remains unexplored. Here we identify USP7 as a replisome-enriched SUMO deubiquitinase that is essential for DNA replication. By acting on SUMO and SUMOylated proteins, USP7 counteracts their ubiquitination. Inhibition or genetic deletion of USP7 leads...

  17. RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress

    DEFF Research Database (Denmark)

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


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

  18. Budding Yeast Rif1 Controls Genome Integrity by Inhibiting rDNA Replication.

    Directory of Open Access Journals (Sweden)

    Maksym Shyian


    Full Text Available The Rif1 protein is a negative regulator of DNA replication initiation in eukaryotes. Here we show that budding yeast Rif1 inhibits DNA replication initiation at the rDNA locus. Absence of Rif1, or disruption of its interaction with PP1/Glc7 phosphatase, leads to more intensive rDNA replication. The effect of Rif1-Glc7 on rDNA replication is similar to that of the Sir2 deacetylase, and the two would appear to act in the same pathway, since the rif1Δ sir2Δ double mutant shows no further increase in rDNA replication. Loss of Rif1-Glc7 activity is also accompanied by an increase in rDNA repeat instability that again is not additive with the effect of sir2Δ. We find, in addition, that the viability of rif1Δ cells is severely compromised in combination with disruption of the MRX or Ctf4-Mms22 complexes, both of which are implicated in stabilization of stalled replication forks. Significantly, we show that removal of the rDNA replication fork barrier (RFB protein Fob1, alleviation of replisome pausing by deletion of the Tof1/Csm3 complex, or a large deletion of the rDNA repeat array all rescue this synthetic growth defect of rif1Δ cells lacking in addition either MRX or Ctf4-Mms22 activity. These data suggest that the repression of origin activation by Rif1-Glc7 is important to avoid the deleterious accumulation of stalled replication forks at the rDNA RFB, which become lethal when fork stability is compromised. Finally, we show that Rif1-Glc7, unlike Sir2, has an important effect on origin firing outside of the rDNA locus that serves to prevent activation of the DNA replication checkpoint. Our results thus provide insights into a mechanism of replication control within a large repetitive chromosomal domain and its importance for the maintenance of genome stability. These findings may have important implications for metazoans, where large blocks of repetitive sequences are much more common.

  19. Replication Research and Special Education (United States)

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


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

  20. Effect of timing of relocation of replacement gilts from group pens to individual stalls before breeding on fertility and well-being. (United States)

    Knox, R V; Shen, J; Greiner, L L; Connor, J F


    Variation in gilt fertility is associated with increased replacement and reduced longevity. Stress before breeding is hypothesized to be involved in reduced fertility. This study tested the timing of gilt relocation from pens to individual stalls before breeding on fertility and well-being. The experiment was performed in replicates on a commercial research farm. After detection of first estrus, gilts ( = 563) were assigned to treatment for relocation into stalls 3 wk (REL3wk), 2 wk (REL2wk), or 1 wk (REL1wk) before breeding at second estrus. Subsets of gilts from each treatment ( = 60) were selected for assessment of follicles at second estrus. Data included interestrus interval, number of services, conception, farrowing, total born, and wean to service interval. Piglet birth weight was obtained on subsets of litters ( = 42/treatment). Measures of well-being included BW, backfat, BCS, lesions, and lameness from wk 1 after first estrus until wk 16. Gilt BW at wk 5 (158.4 kg) was not affected ( > 0.10) by treatment. Measures of BCS, lameness, and lesions at breeding and throughout gestation did not differ ( > 0.10). Treatment did not affect ( > 0.10) gilts expressing a normal interestrus interval of 18 to 24 d (83.4%) but did influence ( Gilts in REL3wk had a shorter ( Gilts with shorter intervals ( = 24) had fewer total born while gilts expressing longer cycles ( = 65) had reduced farrowing rates. The number of services (1.9) and number of follicles (19.7) at breeding were not affected ( > 0.10) by relocation. There was no effect of treatment on farrowing rate (85.2%), born alive (12.6), or any litter birth weight measures ( > 0.10). The percentage of sows bred within 7 d after weaning (94.4%) was also not affected by treatment ( > 0.10). These results suggest that the timing of relocation before breeding had no effect on well-being or on the majority of gilts with normal estrous cycles and their subsequent fertility. However, a smaller proportion of the gilts

  1. Modeling inhomogeneous DNA replication kinetics.

    Directory of Open Access Journals (Sweden)

    Michel G Gauthier

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

  2. Why Do Promising Therapies Stall in Development and How Can We Move Them Forward? (United States)

    Wegner, Craig D; Goodwin, Andrew; Cook, Jon C; Allamneni, Krishna; Sohn, Jane; McVean, Maralee

    There are many reasons that molecules fail to progress to market and various principles of risk-benefit decisions that can help drive the molecule through development. This symposium included discussions on global strategies involved in pushing promising molecules to market, what to do when a molecule stalls in its progress to market, and options for rescuing the molecule and pushing it forward again. Innovative partnerships that bring stalled drugs back into clinical development were also addressed. A regulatory perspective on common reasons for a molecule to fail in its forward progress was presented. In addition, situations arise when a third-party advisory committee can provide input to help overcome issues identified by a regulatory agency. Using examples from the private and public domain, presentations centered on how to repurpose a molecule and when more science is needed.

  3. A Beddoes-Leishman type dynamic stall model in state-space and indicial formulations

    DEFF Research Database (Denmark)

    Hansen, M.H.; Gaunaa, Mac; Aagaard Madsen, Helge


    This report contains a description of a Beddoes-Leishman type dynamic stall model in both a state-space and an indicial function formulation. The model predicts the unsteady aerodynamic forces and moment on an airfoil section undergoing arbitrary motionin heave, lead-lag, and pitch. The model...... features, such as overshoot of the lift, in the stall region. The linearized model is shown to give identicalresults to the full model for small amplitude oscillations. Furthermore, it is shown that the response of finite thichkness airfoils can be reproduced to a high accuracy by the use of specific...... is carried out by comparing the response of the model with inviscid solutions and observing the general behavior of the model using known airfoil data as input. Theproposed dynamic model gives results identical to inviscid solutions within the attached-flow region; and it exhibits the expected dynamic...

  4. Grid support of a wind farm with active stall wind turbines and AC grid connection

    DEFF Research Database (Denmark)

    Hansen, Anca Daniela; Sørensen, Poul Ejnar; Iov, F.


    grid connection. The designed control system has the task of enabling such a wind farm to provide the best grid support. It is based on two control levels: a supervisory control level, which controls the power production of the whole farm by sending out reference signals to each individual wind turbine......, and a local control level, which ensures that the reference power signals at the wind turbine level are reached. The ability of active stall wind farms with AC grid connection to control the power production to the reference power ordered by the operators is assessed and discussed by means of simulations.......One of the main concerns in the grid integration of large wind farms is their ability to behave as active controllable components in the power system. This article presents the design of a new integrated power control system for a wind farm made up exclusively of active stall wind turbines with AC...

  5. Simulation model of a transient fault controller for an active-stall wind turbine

    Energy Technology Data Exchange (ETDEWEB)

    Jauch, C.; Soerensen, P.; Bak Jensen, B.


    This paper describes the simulation model of a controller that enables an active-stall wind turbine to ride through transient faults. The simulated wind turbine is connected to a simple model of a power system. Certain fault scenarios are specified and the turbine shall be able to sustain operation in case of such faults. The design of the controller is described and its performance assessed by simulations. The control strategies are explained and the behaviour of the turbine discussed. (author)

  6. SUMO and KSHV Replication

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Pei-Ching [Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan (China); Kung, Hsing-Jien, E-mail: [Institute for Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan (China); Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616 (United States); UC Davis Cancer Center, University of California, Davis, CA 95616 (United States); Division of Molecular and Genomic Medicine, National Health Research Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan (China)


    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets represents a potential therapeutic strategy when treating KSHV, as well as other oncogenic herpesviruses. Here, we summarize the different ways KSHV exploits and manipulates the cellular SUMO system and explore the multi-faceted functions of SUMO during KSHV’s life cycle and pathogenesis.

  7. DksA guards elongating RNA polymerase against ribosome-stalling-induced arrest. (United States)

    Zhang, Yan; Mooney, Rachel A; Grass, Jeffrey A; Sivaramakrishnan, Priya; Herman, Christophe; Landick, Robert; Wang, Jue D


    In bacteria, translation-transcription coupling inhibits RNA polymerase (RNAP) stalling. We present evidence suggesting that, upon amino acid starvation, inactive ribosomes promote rather than inhibit RNAP stalling. We developed an algorithm to evaluate genome-wide polymerase progression independently of local noise and used it to reveal that the transcription factor DksA inhibits promoter-proximal pausing and increases RNAP elongation when uncoupled from translation by depletion of charged tRNAs. DksA has minimal effect on RNAP elongation in vitro and on untranslated RNAs in vivo. In these cases, transcripts can form RNA structures that prevent backtracking. Thus, the effect of DksA on transcript elongation may occur primarily upon ribosome slowing/stalling or at promoter-proximal locations that limit the potential for RNA structure. We propose that inactive ribosomes prevent formation of backtrack-blocking mRNA structures and that, in this circumstance, DksA acts as a transcription elongation factor in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. An automatic system for the detection of dairy cows lying behaviour in free-stall barns

    Directory of Open Access Journals (Sweden)

    Simona M.C. Porto


    Full Text Available In this paper, a method for the automatic detection of dairy cow lying behaviour in free-stall barns is proposed. A computer visionbased system (CVBS composed of a video-recording system and a cow lying behaviour detector based on the Viola Jones algorithm was developed. The CVBS performance was tested in a head-to-head free stall barn. Two classifiers were implemented in the software component of the CVBS to obtain the cow lying behaviour detector. The CVBS was validated by comparing its detection results with those generated from visual recognition. This comparison allowed the following accuracy indices to be calculated: the branching factor (BF, the miss factor (MF, the sensitivity, and the quality percentage (QP. The MF value of approximately 0.09 showed that the CVBS missed one cow every 11 well detected cows. Conversely, the BF value of approximately 0.08 indicated that one false positive was detected every 13 well detected cows. The high value of approximately 0.92 obtained for the sensitivity index and that obtained for QP of about 0.85 revealed the ability of the proposed system to detect cows lying in the stalls.

  9. Piloted Simulator Evaluation Results of Flight Physics Based Stall Recovery Guidance (United States)

    Lombaerts, Thomas; Schuet, Stefan; Stepanyan, Vahram; Kaneshige, John; Hardy, Gordon; Shish, Kimberlee; Robinson, Peter


    In recent studies, it has been observed that loss of control in flight is the most frequent primary cause of accidents. A significant share of accidents in this category can be remedied by upset prevention if possible, and by upset recovery if necessary, in this order of priorities. One of the most important upsets to be recovered from is stall. Recent accidents have shown that a correct stall recovery maneuver remains a big challenge in civil aviation, partly due to a lack of pilot training. A possible strategy to support the flight crew in this demanding context is calculating a recovery guidance signal, and showing this signal in an intuitive way on one of the cockpit displays, for example by means of the flight director. Different methods for calculating the recovery signal, one based on fast model predictive control and another using an energy based approach, have been evaluated in four relevant operational scenarios by experienced commercial as well as test pilots in the Vertical Motion Simulator at NASA Ames Research Center. Evaluation results show that this approach could be able to assist the pilots in executing a correct stall recovery maneuver.

  10. The Relationships between Selection and Processing Food with Escherichia coli Contaminant on Food Stall Serving

    Directory of Open Access Journals (Sweden)

    Tris Eryando


    Full Text Available Escherichia coli in food stalls surrounding the X Campuss in Depok, year 2012. The research conducted to examine food safety, which were served in surrounding the campus X in Depok. Escherichia coli (E. coli existence was used to indicate the quality of hygiene and sanitation of the food that was served. Using the cross sectional method, the research examined the persons who served the food to be sold in the food stalls in the campus. There were 173 food servers chosen as the respondents from 10 different food stalls around the university. The existence of E. coli examined in the microbiology laboratory in the Faculty of Public Health. Using the most probable number (MPN method found that 59.54% of the food served in the campus were contaminated E. coli. Factors affecting the existence of E. coli were the raw materials (vegetables treated and the length of cooking of the materials (rice/beens. The improper treatment such as washing with no running water or even unwashed vegetables had 5 times risk of the E. coli contamination. Cooking less than 15 minutes was also more risky than cooking more than 15 minutes. As a result, this is very important to find a method to improve knowledge and to increase practical skills in food safety. Furthermore, in this research area may give contribution to avoid E. coli contamination which will prevent unnecessary illness among students in the campus.

  11. Near Stall Flow Analysis in the Transonic Fan of the RTA Propulsion System (United States)

    Hah, Chunill


    Turbine-based propulsion systems for access to space have been investigated at NASA Glenn Research center. A ground demonstrator engine for validation testing has been developed as a part of the program. The demonstrator, the Revolutionary Turbine Accelerator (RTA-1), is a variable cycle turbofan ramjet designed to transition from an augmented turbofan to a ramjet that produces the thrust required to accelerate the vehicle to Mach 4. The RTA-1 is designed to accommodate a large variation in bypass ratio from sea level static to Mach 4 flight condition. A key component of this engine is a new fan stage that accommodates these large variations in bypass ratio and flow ranges. In the present study, unsteady flow behavior in the fan of the RTA-1 is studied in detail with large eddy simulation (LES) and the numerical results are compared with measured data. During the experimental study of the fan stage, humming sound was detected at 100 % speed near stall operation. The main purpose of the study is to investigate details of the unsteady flow behavior at near stall operation and to identify a possible cause of the hum. The large eddy simulation of the current flow field reproduces main features of the measured flow very well. The LES simulation indicates that non-synchronous flow instability develops as the fan operates toward the stall limit. The FFT analysis of the calculated wall pressure shows that the rotating flow instability has the characteristic frequency that is about 50% of the blade passing frequency.

  12. Short revolving wings enable hovering animals to avoid stall and reduce drag (United States)

    Lentink, David; Kruyt, Jan W.; Heijst, Gertjan F.; Altshuler, Douglas L.


    Long and slender wings reduce the drag of airplanes, helicopters, and gliding animals, which operate at low angle of attack (incidence). Remarkably, there is no evidence for such influence of wing aspect ratio on the energetics of hovering animals that operate their wings at much higher incidence. High incidence causes aircraft wings to stall, hovering animals avoid stall by generating an attached vortex along the leading edge of their wings that elevates lift. Hypotheses that explain this capability include the necessity for a short radial distance between the shoulder joint and wing tip, measured in chord lengths, instead of the long tip-to-tip distance that elevates aircraft performance. This stems from how hovering animals revolve their wings around a joint, a condition for which the precise effect of aspect ratio on stall performance is unknown. Here we show that the attachment of the leading edge vortex is determined by wing aspect ratio with respect to the center of rotation-for a suite of aspect ratios that represent both animal and aircraft wings. The vortex remains attached when the local radius is shorter than 4 chord lengths, and separates outboard on more slender wings. Like most other hovering animals, hummingbirds have wing aspect ratios between 3 and 4, much stubbier than helicopters. Our results show this makes their wings robust against flow separation, which reduces drag below values obtained with more slender wings. This revises our understanding of how aspect ratio improves performance at low Reynolds numbers.

  13. Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis. (United States)

    Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf


    Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

  14. Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity. (United States)

    Olivares, Isabel; Gutiérrez-Rivas, Mónica; López-Galíndez, Cecilio; Menéndez-Arias, Luis


    The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.

  15. Exploiting translational stalling peptides in an effort to extend azithromycin interaction within the prokaryotic ribosome nascent peptide exit tunnel. (United States)

    Washington, Arren Z; Tapadar, Subhasish; George, Alex; Oyelere, Adegboyega K


    The ribosome is the primary protein synthesis machine in the cell and is a target for treatment of a variety of diseases including bacterial infection and cancer. The ribosomal peptide exit tunnel, the route of egress for the nascent peptide, is an inviting site for drug design. Toward a rational engagement of the nascent peptide components for the design of small molecule inhibitors of ribosome function, we designed and disclosed herein a set of N-10 indole functionalized azithromycin analogs. The indole moiety of these compounds is designed to mimic the translation stalling interaction of SecM W155 side-chain with the prokaryotic (Escherichia coli) ribosome A751 residue. Many of these N-10 functionalized compounds have enhanced translation inhibition activities against E. coli ribosome relative to azithromycin while a subset inhibited the growth of representative susceptible bacteria strains to about the same extent as azithromycin. Moreover, the inclusion of bovine serum in the bacterial growth media enhanced the anti-bacterial potency of the N-10 functionalized azithromycin analogs by as high as 10-fold. Published by Elsevier Ltd.

  16. Dynamics and Control of Three-Dimensional Perching Maneuver under Dynamic Stall Influence (United States)

    Feroskhan, Mir Alikhan Bin Mohammad

    Perching is a type of aggressive maneuver performed by the class 'Aves' species to attain precision point landing with a generally short landing distance. Perching capability is desirable on unmanned aerial vehicles (UAVs) due to its efficient deceleration process that potentially expands the functionality and flight envelope of the aircraft. This dissertation extends the previous works on perching, which is mostly limited to two-dimensional (2D) cases, to its state-of-the-art threedimensional (3D) variety. This dissertation presents the aerodynamic modeling and optimization framework adopted to generate unprecedented variants of the 3D perching maneuver that include the sideslip perching trajectory, which ameliorates the existing 2D perching concept by eliminating the undesirable undershoot and reliance on gravity. The sideslip perching technique methodically utilizes the lateral and longitudinal drag mechanisms through consecutive phases of yawing and pitching-up motion. Since perching maneuver involves high rates of change in the angles of attack and large turn rates, introduction of three internal variables thus becomes necessary for addressing the influence of dynamic stall delay on the UAV's transient post-stall behavior. These variables are then integrated into a static nonlinear aerodynamic model, developed using empirical and analytical methods, and into an optimization framework that generates a trajectory of sideslip perching maneuver, acquiring over 70% velocity reduction. An impact study of the dynamic stall influence on the optimal perching trajectories suggests that consideration of dynamic stall delay is essential due to the significant discrepancies in the corresponding control inputs required. A comparative study between 2D and 3D perching is also conducted to examine the different drag mechanisms employed by 2D and 3D perching respectively. 3D perching is presented as a more efficient deceleration technique with respect to spatial costs and



    Hendro Nindito; Evaristus Didik Madyatmadja; Albert Verasius Dian Sano


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

  18. Chromatin replication and epigenome maintenance

    DEFF Research Database (Denmark)

    Alabert, Constance; Groth, Anja


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

  19. A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex

    Energy Technology Data Exchange (ETDEWEB)

    Aller, Pierre; Duclos, Stéphanie; Wallace, Susan S.; Doublié, Sylvie (Vermont)


    Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5'-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5'-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. and Doublie S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.]. Several studies showed that in the sequence context 5'-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5'-A-Tg-G, 5'-T-Tg-G, and 5'-C-Tg-G. A combination of several factors - including the associated exonuclease activity, the nature of the 3' and 5' bases surrounding Tg, and the cis-trans interconversion of Tg - influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.

  20. The effect of alphacypermethrin-treated mesh protection against African horse sickness virus vectors on jet stall microclimate, clinical variables and faecal glucocorticoid metabolites of horses. (United States)

    Page, Patrick; Ganswindt, Andre; Schoeman, Johan; Venter, Gert; Guthrie, Alan


    African horse sickness (AHS) is of importance to health and international trade in horses worldwide. During export from and transit through AHS endemic countries or zones, physical and chemical measures to protect horses from the vectors of AHS virus (AHSV) are recommended by the World Organization for Animal Health. Protection of containerized air transport systems for horses (jet stalls) with alphacypermethrin insecticide-treated high density polyethylene mesh is effective in reducing the Culicoides midge vector attack rate. In order to determine the effect of this mesh on jet stall ventilation and horse welfare under temperate climatic conditions, jet stall microclimate, clinical variables and faecal glucocorticoid metabolite (FGM) levels of 12 horses were monitored during overnight housing in either a treated or untreated stall in two blocks of a 2 × 3 randomized crossover design. Temperature difference between the treated stall and outside was significantly higher than the difference between the untreated stall and outside at 1/15 time points only (P = 0.045, r = 0.70). Relative humidity (RH) difference between the treated stall and outside did not differ from the untreated stall and outside. Temperature and RH in the treated stall were highly and significantly correlated with outside temperature (r = 0.96, P < 0.001) and RH (r = 0.95, P < 0.001), respectively. No significant differences were detected between rectal temperatures, pulse and respiratory rates of horses in the treated stall compared to the untreated stall. Mean FGM concentrations for horses housed in the treated stall peaked earlier (24 h) and at a higher concentration than horses housed in the untreated stall (48 h), but were not significantly different from baseline. No significant difference was detected in FGM concentrations when the treated and untreated stall groups were compared at individual time points up to 72 h after exiting the jet stall. Alphacypermethrin

  1. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides (United States)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.


    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  2. Replication of bacteriophage lambda DNA

    International Nuclear Information System (INIS)

    Tsurimoto, T.; Matsubara, K.


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

  3. DNA breaks early in replication in B cell cancers (United States)

    Research by scientists at the NCI has identified a new class of DNA sites in cells that break early in the replication process. They found that these break sites correlate with damage often seen in B cell cancers, such as diffuse large B cell lymphoma.

  4. Human CST Facilitates Genome-wide RAD51 Recruitment to GC-Rich Repetitive Sequences in Response to Replication Stress. (United States)

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


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

  5. Animal hygiene assessment of microclimate in semi open free-stall barns for dairy cows

    Directory of Open Access Journals (Sweden)

    D. Dimov


    Full Text Available Abstract. The study was conducted in three semi open free-stall barns (B1, B2, and B3 for dairy cows with capacities for 120, 120 and 500 cows, respectively, from three different dairy farms (F-1, F-2 and F-3, situated in Central Southern Bulgaria. The investigated farms had the same production system – loose housing in semi open free-stall dairy barn. For each of the farms the main microclimatic parameters – air temperature, relative humidity and speed of airflow were recorded twice a month at 10.00 h 12.00 h, 14.00 h, 16.00 h and 18.00 h of the day inside the barns in three main technological zones - above the stalls, above manure and feed alleys and outside the buildings. It was found that: a Microclimatic parameters (air temperature, air relative humidity and speed of airflow in technological zones (above the stalls, the manure and feed alleys of three semi open free-stall dairy barns meet the animal hygienic requirements for all seasons according to Regulation No. 44 (2006. Exceptions are some values of relative humidity in B1 and B2 in the spring, and in B1 in winter and summer, which are lower than the minimum humidity (50% according to the standard. b The investigated barns are characterized with poor insulation and do not provide enough isolation from the external ambient temperatures. With the exception of winter, the temperature of the air inside the buildings was lower than that outside, with minor differences for all seasons. The fans in the barns have no effect on the inside air temperature, especially in summer. There was a risk of higher temperatures mainly during the summer period. c There is no significant difference between the average temperatures, air humidity and speed of airflow in all technological zones of the investigated barns. d The largest and statistically significant is the difference between the relative air humidity outside and inside the building in Farm 3, followed by buildings in Farm 1 and 2, where the

  6. Identification of the adenovirus E4orf4 protein binding site on the B55α and Cdc55 regulatory subunits of PP2A: Implications for PP2A function, tumor cell killing and viral replication.

    Directory of Open Access Journals (Sweden)

    Melissa Z Mui

    Full Text Available Adenovirus E4orf4 protein induces the death of human cancer cells and Saccharomyces cerevisiae. Binding of E4orf4 to the B/B55/Cdc55 regulatory subunit of protein phosphatase 2A (PP2A is required, and such binding inhibits PP2A(B55 activity leading to dose-dependent cell death. We found that E4orf4 binds across the putative substrate binding groove predicted from the crystal structure of B55α such that the substrate p107 can no longer interact with PP2A(B55α. We propose that E4orf4 inhibits PP2A(B55 activity by preventing access of substrates and that at high E4orf4 levels this inhibition results in cell death through the failure to dephosphorylate substrates required for cell cycle progression. However, E4orf4 is expressed at much lower and less toxic levels during a normal adenovirus infection. We suggest that in this context E4orf4 largely serves to recruit novel substrates such as ASF/SF2/SRSF1 to PP2A(B55 to enhance adenovirus replication. Thus E4orf4 toxicity probably represents an artifact of overexpression and does not reflect the evolutionary function of this viral product.

  7. Stall, Spiculate, or Run Away: The Fate of Fibers Growing towards Fluctuating Membranes (United States)

    Daniels, D. R.; Marenduzzo, D.; Turner, M. S.


    We study the dynamics of a growing semiflexible fiber approaching a membrane at an angle. At late times we find three regimes: fiber stalling, when growth stops, runaway, in which the fiber bends away from the membrane, and another regime in which spicules form. We discuss which regions of the resulting “phase diagram” are explored by (i) single and bundled actin fibers in living cells, (ii) sickle hemoglobin fibers, and (iii) microtubules inside vesicles. We complement our analysis with 3D stochastic simulations.

  8. Prediction of H.A.W.T. blade stall and performance

    Energy Technology Data Exchange (ETDEWEB)

    Giannakidis, G.; Graham, J.M.R. [Imperial College, Dept. of Aeronautics, London (United Kingdom)


    A model is being developed for the prediction of Horizontal Axis Wind Turbine blade stall and performance coupled with a simple aeroelastic analysis model. For the aerodynamic calculation a two dimensional unsteady Navier-Stokes solver on a sectional basis on the blade is coupled with a three dimensional vortex lattice wake. Pressure coefficient distributions are calculated from the two dimensional viscous flow in each blade section. The aerodynamic computations are coupled with a vibrating beam model in order to incorporate flapwise deformations of the blade. (au) 17 refs.

  9. Modeling dynamic stall on wind turbine blades under rotationally augmented flow fields

    DEFF Research Database (Denmark)

    Guntur, Srinivas; Sørensen, Niels N.; Schreck, Scott


    Experiment Phase VI experimental data, including constant as well as continuously pitching blade conditions during axial operation; (2) data from unsteady delayed detached eddy simulations (DDES) carried out using the Technical University of Denmark’s in-house flow solver Ellipsys3D; and (3) data from...... agreement between the model and the experimental data in many cases, which suggests that the current two-dimensional dynamic stall model as used in blade element momentum-based aeroelastic codes may provide a reasonably accurate representation of three-dimensional rotor aerodynamics when used in combination...

  10. CTCF driven TERRA transcription facilitates completion of telomere DNA replication. (United States)

    Beishline, Kate; Vladimirova, Olga; Tutton, Stephen; Wang, Zhuo; Deng, Zhong; Lieberman, Paul M


    Telomere repeat DNA forms a nucleo-protein structure that can obstruct chromosomal DNA replication, especially under conditions of replication stress. Transcription of telomere repeats can initiate at subtelomeric CTCF-binding sites to generate telomere repeat-encoding RNA (TERRA), but the role of transcription, CTCF, and TERRA in telomere replication is not known. Here, we have used CRISPR/Cas9 gene editing to mutate CTCF-binding sites at the putative start site of TERRA transcripts for a class of subtelomeres. Under replication stress, telomeres lacking CTCF-driven TERRA exhibit sister-telomere loss and upon entry into mitosis, exhibit the formation of ultra-fine anaphase bridges and micronuclei. Importantly, these phenotypes could be rescued by the forced transcription of TERRA independent of CTCF binding. Our findings indicate that subtelomeric CTCF facilitates telomeric DNA replication by promoting TERRA transcription. Our findings also demonstrate that CTCF-driven TERRA transcription acts in cis to facilitate telomere repeat replication and chromosome stability.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  12. Replication-uncoupled histone deposition during adenovirus DNA replication. (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke


    In infected cells, the chromatin structure of the adenovirus genome DNA plays critical roles in its genome functions. Previously, we reported that in early phases of infection, incoming viral DNA is associated with both viral core protein VII and cellular histones. Here we show that in late phases of infection, newly synthesized viral DNA is also associated with histones. We also found that the knockdown of CAF-1, a histone chaperone that functions in the replication-coupled deposition of histones, does not affect the level of histone H3 bound on viral chromatin, although CAF-1 is accumulated at viral DNA replication foci together with PCNA. Chromatin immunoprecipitation assays using epitope-tagged histone H3 demonstrated that histone variant H3.3, which is deposited onto the cellular genome in a replication-independent manner, is selectively associated with both incoming and newly synthesized viral DNAs. Microscopic analyses indicated that histones but not USF1, a transcription factor that regulates viral late gene expression, are excluded from viral DNA replication foci and that this is achieved by the oligomerization of the DNA binding protein (DBP). Taken together, these results suggest that histone deposition onto newly synthesized viral DNA is most likely uncoupled with viral DNA replication, and a possible role of DBP oligomerization in this replication-uncoupled histone deposition is discussed.

  13. Chromatin replication and histone dynamics

    DEFF Research Database (Denmark)

    Alabert, Constance; Jasencakova, Zuzana; Groth, Anja


    organization into chromatin. We reveal how specialized replication-coupled mechanisms rapidly assemble newly synthesized DNA into nucleosomes, while the complete restoration of chromatin organization including histone marks is a continuous process taking place throughout the cell cycle. Because failure...

  14. Modelling of multiple short-length-scale stall cells in an axial compressor using evolved GMDH neural networks

    International Nuclear Information System (INIS)

    Amanifard, N.; Nariman-Zadeh, N.; Farahani, M.H.; Khalkhali, A.


    Over the past 15 years there have been several research efforts to capture the stall inception nature in axial flow compressors. However previous analytical models could not explain the formation of short-length-scale stall cells. This paper provides a new model based on evolved GMDH neural network for transient evolution of multiple short-length-scale stall cells in an axial compressor. Genetic Algorithms (GAs) are also employed for optimal design of connectivity configuration of such GMDH-type neural networks. In this way, low-pass filter (LPF) pressure trace near the rotor leading edge is modelled with respect to the variation of pressure coefficient, flow rate coefficient, and number of rotor rotations which are defined as inputs


    Directory of Open Access Journals (Sweden)



    Full Text Available Although individual and social behavior of cattle has been studied extensively under pasture and loose housing conditions, similar behavioral data for partial or total confinement housing are limited. Essentially, the 24- h time budget represents the net response of a cow to her environment (Grant, 2003. Daily time budget was first introduced by Grant and Albright (in 2000 for cows housed in free-stall environment. Choices in stabling and management affect the behavior, health, longevity and performance of cows. Behavior while resting, eating, ruminating, urinating or defecating provides additional information about comfort. In the current study were used ten Romanian Black and White multiparous cows, housed in a tie stall barn 24 hours per day. Experiments were carried out during the cold season, in February 2008. Cows monitored were in their first hundred days of lactation. In our study cows spent resting on average 379.9 minutes (6.33 hours, value that represents 26.38 % from the days interval. Time devoted to feeding was on average 341.9 minutes (5.69 hours, in 17.5 periods. Rumination had place on average in 17.3 periods and a total time of 517.5 minutes (8.62 hours. Cows adopted lying position on average 581.2 minutes (40.36% from 24-h and standing position on average in 858.7 minutes (59.63% from 24-h.

  16. Numerical study on a single bladed vertical axis wind turbine under dynamic stall

    Energy Technology Data Exchange (ETDEWEB)

    Bangga, Galih [Institute of Aerodynamics and Gas Dynamics, University of Stuttgart, Stuttgart (Germany); Hutomo, Go; Sasongko, Herman [Dept. of Mechanical Engineering, Institut Teknologi Sepuluh Nopember, Surabaya (Indonesia); Wiranegara, Raditya [School of Mechanical Aerospace and Civil Engineering, University of Manchester, Manchester (United Kingdom)


    The aim of this study is to investigate the flow development of a single bladed vertical axis wind turbine using Computational fluid dynamics (CFD) methods. The blade is constructed using the NACA 0012 profile and is operating under stalled conditions at tip speed ratio of 2. Two dimensional simulations are performed using a commercial CFD package, ANSYS Fluent 15.0, employing the Menter-SST turbulence model. For the preliminary study, simulations of the NACA 0012 airfoil under static conditions are carried out and compared with available measurement data and calculations using the boundary layer code XFOIL. The CFD results under the dynamic case are presented and the resulting aerodynamic forces are evaluated. The turbine is observed to generate negative power at certain azimuth angles which can be divided into three main zones. The blade vortex interaction is observed to strongly influence the flow behavior near the blade and contributes to the power production loss. However, the impact is considered small since it covers only 6.4 % of the azimuth angle range where the power is negative compared to the dynamic stall impact which covers almost 22 % of the azimuth angle range.

  17. Wind-up of a spanwise vortex in deepening transition and stall

    Energy Technology Data Exchange (ETDEWEB)

    Smith, F.T.; Bowles, R.I. [University Coll., London (United Kingdom). Dept. of Mathematics; Walker, J.D.A. [Mechanical Engineering Department, Packard Laboratory No. 19, Lehigh University, Bethlehem, PA 18015 (United States)


    A fundamental flow problem of unsteady wind-up of a spanwise vortex is studied in this theoretical work on deepening dynamic stall and transition in a boundary layer, internal layer or related unsteady motion. It examines the nonlinear evolution of the spanwise vortex produced when the local wall pressure develops a maximum or minimum, subsequent to the finite-time break-up of an interacting layer and the impact of normal pressure gradients. The evolution is controlled by an inner-outer interaction between the effects of the normal pressure gradient and the momentum jumps across and outside the vortex, which is situated near the strong inflexion point induced in the mean flow. Although the work concentrates on a particular internal-flow context, many of the flow properties found are generic and in particular apply for a more general case including external flows. Analysis and associated computations point to two main distinct trends in the vortex response, depending to a large extent on a parameter gauging the relative strengths of the above effects. The response is either an explosive one, provoking enhanced wind-up, growth and pressure in the vortex, or it is implosive, causing the vortex to shrink and virtually empty itself through unwinding, leaving little local pressure variation. A further discussion includes the after-effects of this vortex response and some of the connections with experiments and direct computations on deepening stall and transition. (orig.)

  18. Experimental Methods Applied in a Study of Stall Flutter in an Axial Flow Fan

    Directory of Open Access Journals (Sweden)

    John D. Gill


    Full Text Available Flutter testing is an integral part of aircraft gas turbine engine development. In typical flutter testing blade mounted sensors in the form of strain gages and casing mounted sensors in the form of light probes (NSMS are used. Casing mounted sensors have the advantage of being non-intrusive and can detect the vibratory response of each rotating blade. Other types of casing mounted sensors can also be used to detect flutter of rotating blades. In this investigation casing mounted high frequency response pressure transducers are used to characterize the part-speed stall flutter response of a single stage unshrouded axial-flow fan. These dynamic pressure transducers are evenly spaced around the circumference at a constant axial location upstream of the fan blade leading edge plane. The pre-recorded experimental data at 70% corrected speed is analyzed for the case where the fan is back-pressured into the stall flutter zone. The experimental data is analyzed using two probe and multi-probe techniques. The analysis techniques for each method are presented. Results from these two analysis methods indicate that flutter occurred at a frequency of 411 Hz with a dominant nodal diameter of 2. The multi-probe analysis technique is a valuable method that can be used to investigate the initiation of flutter in turbomachines.

  19. Active flow control of the laminar separation bubble on a plunging airfoil near stall (United States)

    Pande, Arth; Agate, Mark; Little, Jesse; Fasel, Hermann


    The effects of small amplitude (A/c = 0.048) high frequency (πfc/U∞ = 0.70) plunging motion on the X-56A airfoil are examined experimentally at Re = 200,000 for 12° angle of attack (CL,MAX = 12.25°) . The purpose of this research is to study the aerodynamic influence of structural motion when the wing is vibrating close to its eigenfrequency near static stall. Specific focus is placed on the laminar separation bubble (LSB) near the leading edge and its control via plasma actuation. In the baseline case, the leading edge bubble bursts during the oscillation cycle causing moment stall. A collaborative computational effort has shown that small amplitude forcing at a frequency that is most amplified by the primary instability of the LSB (FLSB+= 1, Fc+= 52) generates coherent spanwise vortices that entrain freestream momentum, thus reducing separation all while maintaining a laminar flow state. Results (PIV and surface pressure) indicate that a similar control mechanism is effective in the experiments. This is significant given the existence of freestream turbulence in the wind tunnel which has been shown to limit the efficacy of this active flow control technique in a model problem using Direct Numerical Simulation. The implications of these results are discussed.

  20. Compliance with NAGCAT work practices recommendations for youth cleaning service alleys in stall barns. (United States)

    Canan, B D; Asti, L; Heaney, C; Ashida, S; Renick, K; Xiang, H; Stallones, L; Jepsen, S D; Crawford, J M; Wilkins, J R


    Unintentional injury is the leading cause of death in the U.S. among persons 1 to 44 years of age. Over one million children and adolescents in the U.S. live, work, and/or play on farms, where injury risk is relatively high compared to other settings. In an attempt to reduce the number of childhood agricultural injuries occurring on farms, the North American Guidelines for Children's Agricultural Tasks (NAGCAT) was developed to assist parents or other caregivers in assigning developmentally appropriate chores to youth exposed to agricultural hazards. The results presented here are from a longitudinal study in which we obtained (self-reported) daily chore, injury, and safety behavior data from children and adolescents. We focused on one NAGCAT chore, cleaning a service alley in a stall barn, in order to estimate the extent of compliance with specific work practice recommendations contained in the NAGCAT. Our results indicated that among the four NAGCAT-recommended safety practices for cleaning service alleys in stall barns (wearing nonskid shoes, leather gloves, a respirator, and eye protection), wearing non-skid shoes was the only safety practice reported with any degree of regularity. Overall, boys were more likely to wear non-skid shoes compared to girls. In addition, older youth were generally more likely to report higher work practice compliance compared to younger youth.

  1. Effects of reducing dietary nitrogen on ammonia emissions from manure on the floor of a naturally ventilated free stall dairy barn at low (0-20 degrees C) temperatures. (United States)

    Li, Lifeng; Cyriac, Joby; Knowlton, Katharine F; Marr, Linsey C; Gay, Susan W; Hanigan, Mark D; Ogejo, Jactone Arogo


    This study was conducted to determine the potential for reducing ammonia (NH3) emissions from manure deposited on the floor of a naturally ventilated free stall barn by mid-lactation dairy cows fed reduced or normal N diets. Two crude protein (CP) diets (178 g kg(-1) [high] and 159 g kg(-1) [low] dry matter ), were used. The diets were fed to 48 Holstein cows in a replicated crossover design with two pens per diet. The NH3 emitted from the manure deposited on the floor was measured using a dynamic flux chamber. The NH3 emissions were 2.7 (+/-2.0) and 2.9 (+/-1.8) g N cow(-1) d(-1) for high and low CP diets, respectively. Ammonia emission rates were significantly affected by manure pH, TKN, and ambient air temperature (Pammoniacal N (TAN) in manure and had no significant effect on the ammonia emission rates from the barn floor.

  2. Autonomous replication of plasmids bearing monkey DNA origin-enriched sequences

    Energy Technology Data Exchange (ETDEWEB)

    Frappier, L.; Zannis-Hadjopoulos, M.


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

  3. Ethnic Dimensions of Guatemala's Stalled Transition: A Parity-Specific Analysis of Ladino and Indigenous Fertility Regimes. (United States)

    Grace, Kathryn; Sweeney, Stuart


    In some contemporary populations, fertility levels appear to plateau, with women maintaining a consistently high level of fertility for a relatively extended period. Because this plateau does not reflect the historical patterns observed in Europe, the focus of most studies on fertility patterns, mechanisms underlying the plateau and the reinstatement of a decline have not been fully explored and are not fully understood. Through the construction of fertility histories of 25,000 women using multiple years of health survey data, we analyze some of the components of stalled fertility as they pertain to Guatemala, the only Central American country to have experienced a stalled fertility decline.

  4. Non‐Canonical Replication Initiation: You’re Fired!

    Directory of Open Access Journals (Sweden)

    Bazilė Ravoitytė


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

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

    DEFF Research Database (Denmark)

    Østergaard, Vibe Hallundbæk; Lisby, Michael


    Collision between the molecular machineries responsible for transcription and replication is an important source of genome instability. Certain transcribed regions known as chromosomal fragile sites are particularly prone to recombine and mutate in a manner that correlates with specific transcrip......Collision between the molecular machineries responsible for transcription and replication is an important source of genome instability. Certain transcribed regions known as chromosomal fragile sites are particularly prone to recombine and mutate in a manner that correlates with specific...... transcription and replication patterns. At the same time, these chromosomal fragile sites engage in aberrant DNA structures in mitosis. Here, we discuss the mechanistic details of transcription–replication conflicts including putative scenarios for R-loop-induced replication inhibition to understand how...... transcription–replication conflicts transition from S phase into various aberrant DNA structures in mitosis....

  6. Histone Modification Associated with Initiation of DNA Replication | Center for Cancer Research (United States)

    Before cells are able to divide, they must first duplicate their chromosomes accurately. DNA replication and packaging of DNA into chromosomes by histone proteins need to be coordinated by the cell to ensure proper transmission of genetic and epigenetic information to the next generation. Mammalian DNA replication begins at specific chromosomal sites, called replication origins, which are located throughout the genome. The replication origins are tightly regulated to start replication only once per cell division so that genomic stability is maintained and cancer development is prevented.

  7. Parametrised Constants and Replication for Spatial Mobility

    DEFF Research Database (Denmark)

    Hüttel, Hans; Haagensen, Bjørn


    and the calculus of mobile ambients. Here, processes are located at sites and can migrate between them. In this paper we say that an encoding is local if it does not introduce extra migration. We first study this property for the distributed π-calculus where locations can be dynamically created. If the set...... of reachable sites is static an encoding exists, but we also show that parametrised constants can not be encoded in the full calculus. The locality requirement supplements widely accepted encoding criteria. It appears to be a natural property in spatial calculi where links and locations can fail. The versions...... of the distributed π-calculus with parametrised constants and replication are incomparable. On the other hand, we shall see that there exists a simple encoding of recursion in mobile ambients....

  8. SV40 utilizes ATM kinase activity to prevent non-homologous end joining of broken viral DNA replication products. (United States)

    Sowd, Gregory A; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L; Fanning, Ellen


    Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PK(cs) kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PK(cs) and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5' to 3' end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication.

  9. A dimeric Rep protein initiates replication of a linear archaeal virus genome: implications for the Rep mechanism and viral replication

    DEFF Research Database (Denmark)

    Oke, Muse; Kerou, Melina; Liu, Huanting


    that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between...... positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral......The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report...

  10. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis


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

  11. Defects of mitochondrial DNA replication. (United States)

    Copeland, William C


    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease. © The Author(s) 2014.

  12. Biomarkers of replicative senescence revisited

    DEFF Research Database (Denmark)

    Nehlin, Jan


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

  13. Personality and Academic Motivation: Replication, Extension, and Replication (United States)

    Jones, Martin H.; McMichael, Stephanie N.


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

  14. International Expansion through Flexible Replication

    DEFF Research Database (Denmark)

    Jonsson, Anna; Foss, Nicolai Juul


    to local environments and under the impact of new learning. To illuminate these issues, we draw on a longitudinal in-depth study of Swedish home furnishing giant IKEA, involving more than 70 interviews. We find that IKEA has developed organizational mechanisms that support an ongoing learning process aimed...... at frequent modification of the format for replication. Another finding is that IKEA treats replication as hierarchical: lower-level features (marketing efforts, pricing, etc.) are allowed to vary across IKEA stores in response to market-based learning, while higher-level features (fundamental values, vision...

  15. Spins, Stalls, and Shutdowns: Pitfalls of Qualitative Policing and Security Research

    Directory of Open Access Journals (Sweden)

    Randy K. Lippert


    Full Text Available This article explores key elements of qualitative research on policing and security agencies, including barriers encountered and strategies to prevent them. While it is oft-assumed that policing/security agencies are difficult to access due to their clandestine or bureaucratic nature, this article demonstrates this is not necessarily the case, as access was gained for three distinct qualitative research projects. Yet, access and subsequent research were not without pitfalls, which we term security spins, security stalls, and security shutdowns. We illustrate how each was encountered and argue these pitfalls are akin to researchers falling into risk categories, not unlike those used by policing/security agents in their work. Before concluding we discuss methodological strategies for scholars to avoid these pitfalls and to advance research that critically interrogates the immense policing/security realm. URN:

  16. URANS simulations of separated flow with stall cells over an NREL S826 airfoil (United States)

    Sarlak, H.; Nishino, T.; Sørensen, J. N.


    A series of wind tunnel measurements and oil flow visualization was recently carried out at the Technical University of Denmark in order to investigate flow characteristics over a 14% thick NREL S826 airfoil at low Reynolds numbers. This paper aims at presenting numerical simulations of the same airfoil using unsteady Reynolds-averaged Navier-Stokes (URANS) approach. Results of the simulations are demonstrated in terms of mean flow velocity, lift and drag, as well as pressure distribution, and validated against available experimental data. The simulations are carried out with a wide computational domain (with a span-to-chord ratio of 5) and it is illustrated that the URANS approach is capable of predicting 3D spanwise structures, known as stall cells.

  17. Fixed-speed active-stall wind turbines in offshore applications

    DEFF Research Database (Denmark)

    Akhmatov, Vladislav; Nielsen, Arne Hejde


    A large offshore wind farm in the East Danish power system was commissioned in 2003 at Rodsand. The power capacity of the wind farm is 165 MW divided between 72 wind turbines. For this large offshore application, robust and well-known wind technology has been chosen in the form of fixed-speed, ac......, active-stall wind turbines equipped with induction generators. In this paper, maintaining and improving the short-term voltage stability is discussed and systematized in terms of this wind technology. Copyright (C) 2004 John Wiley Sons, Ltd.......A large offshore wind farm in the East Danish power system was commissioned in 2003 at Rodsand. The power capacity of the wind farm is 165 MW divided between 72 wind turbines. For this large offshore application, robust and well-known wind technology has been chosen in the form of fixed-speed...

  18. Listeria phospholipases subvert host autophagic defenses by stalling pre-autophagosomal structures (United States)

    Tattoli, Ivan; Sorbara, Matthew T; Yang, Chloe; Tooze, Sharon A; Philpott, Dana J; Girardin, Stephen E


    Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)-dependent cytosolic escape of Listeria triggered a transient amino-acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro-autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co-infection experiments, wild-type Listeria protected PlcA/B-deficient bacteria from autophagy-mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense. PMID:24162724

  19. Study of Near-Stall Flow Behavior in a Modern Transonic Fan with Composite Sweep (United States)

    Hah, Chunill; Shin, Hyoun-Woo


    Detailed flow behavior in a modern transonic fan with a composite sweep is investigated in this paper. Both unsteady Reynolds-averaged Navier-Stokes (URANS) and Large Eddy Simulation (LES) methods are applied to investigate the flow field over a wide operating range. The calculated flow fields are compared with the data from an array of high-frequency response pressure transducers embedded in the fan casing. The current study shows that a relatively fine computational grid is required to resolve the flow field adequately and to calculate the pressure rise across the fan correctly. The calculated flow field shows detailed flow structure near the fan rotor tip region. Due to the introduction of composite sweep toward the rotor tip, the flow structure at the rotor tip is much more stable compared to that of the conventional blade design. The passage shock stays very close to the leading edge at the rotor tip even at the throttle limit. On the other hand, the passage shock becomes stronger and detaches earlier from the blade passage at the radius where the blade sweep is in the opposite direction. The interaction between the tip clearance vortex and the passage shock becomes intense as the fan operates toward the stall limit, and tip clearance vortex breakdown occurs at near-stall operation. URANS calculates the time-averaged flow field fairly well. Details of measured RMS static pressure are not calculated with sufficient accuracy with URANS. On the other hand, LES calculates details of the measured unsteady flow features in the current transonic fan with composite sweep fairly well and reveals the flow mechanism behind the measured unsteady flow field.

  20. Benchmarking welfare indicators in 73 free-stall dairy farms in north-western Spain (United States)

    Trillo, Yolanda; Quintela, Luis Angel; Barrio, Mónica; Becerra, Juan José; Peña, Ana Isabel; Vigo, Marcos; Garcia Herradon, Pedro


    The aim of this study was to describe the status of body condition score (BCS), hock injuries prevalence, locomotion and body hygiene score as animal welfare measures in 73 free-stall dairy cattle farms in Lugo (Spain). A benchmarking process was established across farms: (1) the animal-based indicators were ordered from low to high values; (2) The farms were classified into three categories based on the number of indicators within less than the 25th percentile, 25th to 75th percentile and above the 75th percentile. The median prevalence of unsuitable BCS, hock injuries and clinical lameness was (median (range)) 51.7 per cent (13.3 to 89.5 per cent), 40.0 per cent (7.0per cent to 100 per cent) and 9.0 per cent (0per cent to 60.0 per cent) respectively. The dirtiness of the cow’s coat had a high prevalence (73.0 per cent (37.5per cent to 100 per cent)). Most farms did not display consistently good or poor animal-based indicators and each farm had its own set of strong and weak points. Moreover, facilities design and management practices were described to understand source of the observations made of the cows. The incidence of overstocking was 31.5 per cent for stalls and 26.0 per cent for headlocks. The front lunge space was reduced (farms and they could benefit from others by changing management practices related to facilities and herds. PMID:29018530

  1. Reducing Respiratory Health Risks to Horses and Workers: A Comparison of Two Stall Bedding Materials

    Directory of Open Access Journals (Sweden)

    Markku Saastamoinen


    Full Text Available Stable air quality and the choice of bedding material are an important health issue both in horses and people working or visiting horse stables. Risks of impaired respiratory health are those that can especially be avoided by improving air quality in the stable. The choice of bedding material is particularly important in cold climate conditions; where horses are kept most of the day and year indoors throughout their life. This study examined the effect of two bedding materials; wood shavings and peat; on stable air quality and health of horses. Ammonia and dust levels were also measured to assess conditions in the stable. Ammonia was not detected or was at very low levels (<0.25 ppm in the boxes in which peat was used as bedding; but its concentration was clearly higher (1.5–7.0 ppm in stalls with wood shavings as bedding. Personal measurements of workers revealed quite high ammonia exposure (5.9 ppm8h in the boxes in which wood shavings were used; but no exposure was Animals 2015, 5 966 observed in stalls bedded with peat. The respiratory symptoms in horses increased regardless of the bedding material at the beginning of the study. The health status of the horses in the peat bedding group returned to the initial level in the end of the trial but horses bedded with wood shavings continued to be symptomatic. The hooves of the horses with peat bedding had a better moisture content than those of the horses bedded with wood shavings. The results suggest that peat is a better bedding material for horses than wood shavings regarding the health of both horses and stable workers.

  2. Low-Order Modeling of Dynamic Stall on Airfoils in Incompressible Flow (United States)

    Narsipur, Shreyas

    Unsteady aerodynamics has been a topic of research since the late 1930's and has increased in popularity among researchers studying dynamic stall in helicopters, insect/bird flight, micro air vehicles, wind-turbine aerodynamics, and ow-energy harvesting devices. Several experimental and computational studies have helped researchers gain a good understanding of the unsteady ow phenomena, but have proved to be expensive and time-intensive for rapid design and analysis purposes. Since the early 1970's, the push to develop low-order models to solve unsteady ow problems has resulted in several semi-empirical models capable of effectively analyzing unsteady aerodynamics in a fraction of the time required by high-order methods. However, due to the various complexities associated with time-dependent flows, several empirical constants and curve fits derived from existing experimental and computational results are required by the semi-empirical models to be an effective analysis tool. The aim of the current work is to develop a low-order model capable of simulating incompressible dynamic-stall type ow problems with a focus on accurately modeling the unsteady ow physics with the aim of reducing empirical dependencies. The lumped-vortex-element (LVE) algorithm is used as the baseline unsteady inviscid model to which augmentations are applied to model unsteady viscous effects. The current research is divided into two phases. The first phase focused on augmentations aimed at modeling pure unsteady trailing-edge boundary-layer separation and stall without leading-edge vortex (LEV) formation. The second phase is targeted at including LEV shedding capabilities to the LVE algorithm and combining with the trailing-edge separation model from phase one to realize a holistic, optimized, and robust low-order dynamic stall model. In phase one, initial augmentations to theory were focused on modeling the effects of steady trailing-edge separation by implementing a non-linear decambering

  3. Effect of stall design on dairy calf transition to voluntary feeding on an automatic milk feeder after introduction to group housing. (United States)

    Wilson, Tanya R; LeBlanc, Stephen J; DeVries, Trevor J; Haley, Derek B


    Automatic milk feeders (AMF) for young dairy calves are widely used in the dairy industry. These feeders are thought to have benefits for calf health and welfare and may reduce labor required for feeding; however, little is known about how calves adapt to feeding with AMF. The objective of this study was to observe the effects of feeding stall design on calves learning to use the AMF. The hypothesis was that solid side stalls, compared with steel bar stalls, would result in a longer latency to approach and feed from the AMF without assistance. A total of 147 Holstein calves (80 male and 67 female) were enrolled at 4 d of age, introduced to a group pen, and, at the same time, trained on an AMF. For training, calves were allowed to suck on the trainer's fingers and guided to the teat. Calves were allocated to 1 of 2 stall designs at the pen level, depending on which treatment cohort they were born into, either with steel bar stall walls (n = 46 male, 34 female calves) or with solid side stall walls (n = 34 male, 33 female calves). For 72 h after introductory training on the AMF, data from the feeders were collected and calf behavior was monitored by video. Outcomes measured included latency to first voluntary visit to the feeder and to first feeding, time spent in the feeder, amount of milk consumed over 72 h, number of retraining sessions required (retrained if linear regression models or a Poisson model for the outcome of retraining. For certain outcomes the effects of stall design interacted with difficulty of training (willingness to enter feeder and drink); for the 38% of calves that were scored as moderately difficult to train on a scale of easy, moderate, or difficult, treatment (stall design) differences were detected. These calves took 2× longer to lick or bite toward the nipple, 2× longer to first voluntarily feeding, and consumed less milk over 72 h following training when trained on the steel bar stall design. These results suggest simple features of a

  4. Hyperthermia stimulates HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Ferdinand Roesch

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

  5. Chameleon Chasing II: A Replication. (United States)

    Newsom, Doug A.; And Others


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

  6. Dissection of a replication origin of Xenopus DNA

    Energy Technology Data Exchange (ETDEWEB)

    Chambers, J.C.; Watanabe, S.; Taylor, J.H.


    A previously cloned 503-base pair (bp) EcoRI segment of genomic DNA from Xenopus laevis selected for enhancement of replication of its vector plasmid was moved to the EcoRI site of pBR322. This plasmid designated pJCC31 and five other clones, which were made by cleaving the 503-bp segment in relation to a dispersed repeated sequence and subcloning, were compared with pBR322 for replication by microinjection into Xenopus eggs. The replication measured by incorporation of a /sup 32/P-labeled nucleotide as well as semiconservative segregation and dilution of N/sup 6/-methyladenine at the EcoRI sites showed pJCC31 to be about 15 times as efficient as pBR322. The next most efficient subclone, pJCC31-2, contains an insert with a complete 320-bp dispersed repeated sequence bracketed by an 8-bp direct repeat. This observation, along with the authors' previous report that repeated sequences of the Alu family in the human genome enhanced replication of the vector plasmid nearly as much as that of the presumptive Xenopus origin, leads to the hypothesis that members of a subset of the short dispersed repeated sequences in vertebrates function as origins for chromosomal replication. Preliminary studies also show that the presumptive Xenopus origin contains a RNA polymerase promoter that increases the transcription of the plasmid when it is microinjected into Xenopus oocytes.

  7. The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units.

    Directory of Open Access Journals (Sweden)

    Christopher N Merrikh


    Full Text Available In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes or co-directional (leading strand genes orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though Escherichia coli accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential Bacillus subtilis accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA's ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA's C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life

  8. Dissection of the beta-globin replication-initiation region reveals specific requirements for replicator elements during gene amplification.

    Directory of Open Access Journals (Sweden)

    Naoya Okada

    Full Text Available Gene amplification plays a pivotal role in malignant transformation of human cells. A plasmid with both a mammalian replication-initiation region (IR/origin/replicator and a nuclear matrix-attachment region (MAR is spontaneously amplified in transfected cells by a mechanism that involves amplification at the extrachromosomal site, followed by amplification at the chromosomal arm, ultimately generating a long homogeneously staining region (HSR. Several observations suggest that replication initiation from IR sequences might mediate amplification. To test this idea, we previously dissected c-myc and DHFR IRs to identify the minimum sequence required to support amplification. In this study, we applied an improved analysis that discriminates between two amplification steps to the ß-globin RepP IR, which contains separate elements already known to be essential for initiation on the chromosome arm. The IR sequence was required at least for the extrachromosomal amplification step. In addition to the vector-encoded MAR, amplification also required an AT-rich region and a MAR-like element, consistent with the results regarding replicator activity on the chromosome. However, amplification did not require the AG-rich tract necessary for replicator activity, but instead required a novel sequence containing another AG-rich tract. The differential sequence requirement might be a consequence of extrachromosomal replication.

  9. Replication strategy of human hepatitis B virus

    International Nuclear Information System (INIS)

    Will, H.; Reiser, W.; Weimer, T.; Pfaff, E.; Buescher, M.; Sprengel, R.; Cattaneo, R.; Schaller, H.


    To study the replication strategy of the human hepatitis B virus, the 5' end of the RNA pregenome and the initiation sites of DNA plus and minus strands have been mapped. The RNA pregenome was found to be terminally redundant by 120 nucleotides; it is initiated within the pre-C region and may also function as mRNA for synthesis of the major core protein and the hepatitis B virus reverse transcriptase. The hepatitis B virus DNA minus strand is initiated within the direct repeat sequence DR1, it contains a terminal redundancy of up to eight nucleotides, and its synthesis does not require any template switch. The DNA plus strand is primed by a short oligoribonucleotide probably derived from the 5' end of the RNA pregenome, and its synthesis is initiated close to the direct repeat sequence DR2. For its elongation to pass the discontinuity in the DNA minus strand an intramolecular template switch occurs using the terminal redundancy of this template. Thus, the route of reverse transcription and DNA replication of hepatitis B viruses is fundamentally different from that of retroviruses

  10. Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork. (United States)

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


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

  11. Power and precision of replicated helicopter surveys in mixed bushveld

    Directory of Open Access Journals (Sweden)

    B.K. Reilly


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

  12. Replicator dynamics in value chains

    DEFF Research Database (Denmark)

    Cantner, Uwe; Savin, Ivan; Vannuccini, Simone


    The pure model of replicator dynamics though providing important insights in the evolution of markets has not found much of empirical support. This paper extends the model to the case of firms vertically integrated in value chains. We show that i) by taking value chains into account, the replicator...... dynamics may revert its effect. In these regressive developments of market selection, firms with low fitness expand because of being integrated with highly fit partners, and the other way around; ii) allowing partner's switching within a value chain illustrates that periods of instability in the early...... stage of industry life-cycle may be the result of an 'optimization' of partners within a value chain providing a novel and simple explanation to the evidence discussed by Mazzucato (1998); iii) there are distinct differences in the contribution to market selection between the layers of a value chain...

  13. Security in a Replicated Metadata Catalogue

    CERN Document Server

    Koblitz, B


    The gLite-AMGA metadata has been developed by NA4 to provide simple relational metadata access for the EGEE user community. As advanced features, which will be the focus of this presentation, AMGA provides very fine-grained security also in connection with the built-in support for replication and federation of metadata. AMGA is extensively used by the biomedical community to store medical images metadata, digital libraries, in HEP for logging and bookkeeping data and in the climate community. The biomedical community intends to deploy a distributed metadata system for medical images consisting of various sites, which range from hospitals to computing centres. Only safe sharing of the highly sensitive metadata as provided in AMGA makes such a scenario possible. Other scenarios are digital libraries, which federate copyright protected (meta-) data into a common catalogue. The biomedical and digital libraries have been deployed using a centralized structure already for some time. They now intend to decentralize ...

  14. Data Service: Distributed Data Capture and Replication (United States)

    Warner, P. B.; Pietrowicz, S. R.


    Data Service is a critical component of the NOAO Data Management and Science Support (DMaSS) Solutions Platform, which is based on a service-oriented architecture, and is to replace the current NOAO Data Transport System. Its responsibilities include capturing data from NOAO and partner telescopes and instruments and replicating the data across multiple (currently six) storage sites. Java 5 was chosen as the implementation language, and Java EE as the underlying enterprise framework. Application metadata persistence is performed using EJB and Hibernate on the JBoss Application Server, with PostgreSQL as the persistence back-end. Although potentially any underlying mass storage system may be used as the Data Service file persistence technology, DTS deployments and Data Service test deployments currently use the Storage Resource Broker from SDSC. This paper presents an overview and high-level design of the Data Service, including aspects of deployment, e.g., for the LSST Data Challenge at the NCSA computing facilities.

  15. A Signature of Genomic Instability Resulting from Deficient Replication Licensing.

    Directory of Open Access Journals (Sweden)

    Steven C Pruitt


    Full Text Available Insufficient licensing of DNA replication origins has been shown to result in genome instability, stem cell deficiency, and cancers. However, it is unclear whether the DNA damage resulting from deficient replication licensing occurs generally or if specific sites are preferentially affected. To map locations of ongoing DNA damage in vivo, the DNAs present in red blood cell micronuclei were sequenced. Many micronuclei are the product of DNA breaks that leave acentromeric remnants that failed to segregate during mitosis and should reflect the locations of breaks. To validate the approach we show that micronuclear sequences identify known common fragile sites under conditions that induce breaks at these locations (hydroxyurea. In MCM2 deficient mice a different set of preferred breakage sites is identified that includes the tumor suppressor gene Tcf3, which is known to contribute to T-lymphocytic leukemias that arise in these mice, and the 45S rRNA gene repeats.

  16. Asynchronous replication, mono-allelic expression, and long range Cis-effects of ASAR6. (United States)

    Donley, Nathan; Stoffregen, Eric P; Smith, Leslie; Montagna, Christina; Thayer, Mathew J


    Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.

  17. Asynchronous replication, mono-allelic expression, and long range Cis-effects of ASAR6.

    Directory of Open Access Journals (Sweden)

    Nathan Donley


    Full Text Available Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.

  18. Viral rewiring of cellular lipid metabolism to create membranous replication compartments

    NARCIS (Netherlands)

    Strating, Jeroen Rpm; van Kuppeveld, Frank Jm


    Positive-strand RNA (+RNA) viruses (e.g. poliovirus, hepatitis C virus, dengue virus, SARS-coronavirus) remodel cellular membranes to form so-called viral replication compartments (VRCs), which are the sites where viral RNA genome replication takes place. To induce VRC formation, these viruses

  19. Has Uganda experienced any stalled fertility transitions? Reflecting on the last four decades (1973-2011). (United States)

    Kabagenyi, Allen; Reid, Alice; Rutaremwa, Gideon; Atuyambe, Lynn M; Ntozi, James P M


    Persistent high fertility is associated with mother and child mortality. While most regions in the world have experienced declines in fertility rates, there are conflicting views as to whether Uganda has entered a period of fertility transition. There are limited data available that explicitly detail the fertility trends and patterns in Uganda over the last four decades, from 1973 to 2011. Total fertility rate (TFR) is number of live births that a woman would have throughout her reproductive years if she were subject to the prevailing age specific fertility patterns. The current TFR for Uganda stands at 6.2 children born per woman, which is one of the highest in the region. This study therefore sought to examine whether there has been a fertility stall in Uganda using all existing Demographic Health Survey data, to provide estimates for the current fertility levels and trends in Uganda, and finally to examine the demographic and socioeconomic factors responsible for fertility levels in Uganda. This is a secondary analysis of data from five consecutive Ugandan Demographic Health Surveys (UDHS); 1988/1989, 1995, 2000/2001, 2006 and 2011. Using pooled data to estimate for fertility levels, patterns and trends, we applied a recently developed fertility estimation approach. A Poisson regression model was also used to analyze fertility differentials over the study period. Over the studied period, fertility trends and levels fluctuated from highs of 8.8 to lows of 5.7, with no specific lag over the study period. These findings suggest Uganda is at the pre-transitional stage, with indications of imminent fertility rate reductions in forthcoming years. Marital status remained a strong predictor for number of children born, even after controlling for other variables. This study suggests there is no evidence of a fertility stall in Uganda, but demonstrates an onset of fertility transition in the country. If this trend continues, Uganda will experience a low fertility rate in

  20. Design of a 21 m blade with Risø-A1 airfoils for active stall controlled wind turbines

    DEFF Research Database (Denmark)

    Fuglsang, Peter; Sangill, O.; Hansen, P.


    This is the final report, from the project, "Design of a Rotor/Airfoil Family for Active Stall-regulated Wind Turbines by Use of Multi-point Optimization". It describes the full scale testing of a 21 m wind turbine blade specially designed for active stallregulation. Design objectives were...... increased ratio of produced energy to turbine loads and more stable power control characteristics. Both were taken directly into account during the design of the blade using numerical optimization. The blade used theRisø-A1 airfoil family, which was specially designed for operation on wind turbine blades...... be concluded that the new LM 21.0 ASR blade could replace the LM 21.0P leading to improved cost efficiency and that the Risø-A1 airfoils were well suited for active stall control. With the newestablished knowledge of the actual airfoil characteristics, a possible future blade design could be made also...

  1. Experimental investigation of flow-induced vibration of a pitch-plunge NACA 0015 airfoil under deep dynamic stall

    Czech Academy of Sciences Publication Activity Database

    Šidlof, Petr; Vlček, Václav; Štěpán, M.


    Roč. 67, November (2016), s. 48-59 ISSN 0889-9746 R&D Projects: GA ČR GA13-10527S Institutional support: RVO:61388998 Keywords : NACA 0015 airfoil * aeroelasticity * stall flutter * dynamic stall * limit cycle oscillation * schlieren Subject RIV: BI - Acoustics Impact factor: 2.021, year: 2016

  2. Advance Ratio Effects on the Dynamic-stall Vortex of a Rotating Blade in Steady Forward Flight (United States)


    of the wind tunnel and is illustrated in Fig. 1. In order to simplify the operations, a two-bladed rotor design was preferred. The setup had wind turbines, compressors, helicopter rotors , and even insect wing aerodynamics. Dynamic stall occurs on rotating blades of a helicopter in forward...between the flow structure on helicopter rotor blades, wind turbine blades, and insect wings. Due to these wide engineering implications there has

  3. Replication of micro and nano surface geometries

    DEFF Research Database (Denmark)

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


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

  4. Evaluating replicability of laboratory experiments in economics. (United States)

    Camerer, Colin F; Dreber, Anna; Forsell, Eskil; Ho, Teck-Hua; Huber, Jürgen; Johannesson, Magnus; Kirchler, Michael; Almenberg, Johan; Altmejd, Adam; Chan, Taizan; Heikensten, Emma; Holzmeister, Felix; Imai, Taisuke; Isaksson, Siri; Nave, Gideon; Pfeiffer, Thomas; Razen, Michael; Wu, Hang


    The replicability of some scientific findings has recently been called into question. To contribute data about replicability in economics, we replicated 18 studies published in the American Economic Review and the Quarterly Journal of Economics between 2011 and 2014. All of these replications followed predefined analysis plans that were made publicly available beforehand, and they all have a statistical power of at least 90% to detect the original effect size at the 5% significance level. We found a significant effect in the same direction as in the original study for 11 replications (61%); on average, the replicated effect size is 66% of the original. The replicability rate varies between 67% and 78% for four additional replicability indicators, including a prediction market measure of peer beliefs. Copyright © 2016, American Association for the Advancement of Science.

  5. Parametric analyses for synthetic jet control on separation and stall over rotor airfoil

    Directory of Open Access Journals (Sweden)

    Zhao Guoqing


    Full Text Available Numerical simulations are performed to investigate the effects of synthetic jet control on separation and stall over rotor airfoils. The preconditioned and unsteady Reynolds-averaged Navier–Stokes equations coupled with a k − ω shear stream transport turbulence model are employed to accomplish the flowfield simulation of rotor airfoils under jet control. Additionally, a velocity boundary condition modeled by a sinusoidal function is developed to fulfill the perturbation effect of periodic jets. The validity of the present CFD procedure is evaluated by the simulated results of an isolated synthetic jet and the jet control case for airfoil NACA0015. Then, parametric analyses are conducted specifically for an OA213 rotor airfoil to investigate the effects of jet parameters (forcing frequency, jet location and momentum coefficient, jet direction, and distribution of jet arrays on the control effect of the aerodynamic characteristics of a rotor airfoil. Preliminary results indicate that the efficiency of jet control can be improved with specific frequencies (the best lift-drag ratio at F+ = 2.0 and jet angles (40° or 75° when the jets are located near the separation point of the rotor airfoil. Furthermore, as a result of a suitable combination of jet arrays, the lift coefficient of the airfoil can be improved by nearly 100%, and the corresponding drag coefficient decreased by 26.5% in comparison with the single point control case.

  6. Analysis of the grid connection sequence of stall- and pitch-controlled wind turbines

    Energy Technology Data Exchange (ETDEWEB)

    Quinonez-Varela, G.; Cruden, A.; Anaya-Lara, O.; Tumilty, R.; McDonald, J.R. [Univ. of Strathclyde, Inst. for Energy and Environment (United Kingdom)


    The realistic modelling of wind turbines and wind farms is crucial in any form of power system analysis, and consequently, knowledge about their electrical characteristics and performance is also vital. One of the operating conditions producing major transient interaction between a wind turbine generator and the local grid is the grid connection sequence itself, which is particularly significant in fixed-speed turbines. This paper presents experimental measurements of the grid connection sequence of both types of fixed speed wind turbines, i.e. stall- and pitch-controlled via a soft-start device performed at two existing wind farms. Some of the results evidenced significant discrepancies between the actual soft-start operating intervals and those stated/suggested by open literature. The discussion of the paper focuses on highlighting the importance of accurate modelling of the grid connection sequence in order to avoid erroneous estimations of the interaction between the turbine and the grid during this operating state, or inappropriate design of the grid connection. (au)

  7. Why commercialization of gene therapy stalled; examining the life cycles of gene therapy technologies. (United States)

    Ledley, F D; McNamee, L M; Uzdil, V; Morgan, I W


    This report examines the commercialization of gene therapy in the context of innovation theories that posit a relationship between the maturation of a technology through its life cycle and prospects for successful product development. We show that the field of gene therapy has matured steadily since the 1980s, with the congruent accumulation of >35 000 papers, >16 000 US patents, >1800 clinical trials and >$4.3 billion in capital investment in gene therapy companies. Gene therapy technologies comprise a series of dissimilar approaches for gene delivery, each of which has introduced a distinct product architecture. Using bibliometric methods, we quantify the maturation of each technology through a characteristic life cycle S-curve, from a Nascent stage, through a Growing stage of exponential advance, toward an Established stage and projected limit. Capital investment in gene therapy is shown to have occurred predominantly in Nascent stage technologies and to be negatively correlated with maturity. Gene therapy technologies are now achieving the level of maturity that innovation research and biotechnology experience suggest may be requisite for efficient product development. Asynchrony between the maturation of gene therapy technologies and capital investment in development-focused business models may have stalled the commercialization of gene therapy.

  8. Flow-around modes for a rhomboid wing with a stall vortex in the shock layer (United States)

    Zubin, M. A.; Maximov, F. A.; Ostapenko, N. A.


    The results of theoretical and experimental investigation of an asymmetrical hypersonic flow around a V-shaped wing with the opening angle larger than π on the modes with attached shockwaves on forward edges, when the stall flow is implemented on the leeward wing cantilever behind the kink point of the cross contour. In this case, a vortex of nonviscous nature is formed in which the velocities on the sphere exceeding the speed of sound and resulting in the occurrence of pressure shocks with an intensity sufficient for the separation of the turbulent boundary layer take place in the reverse flow according to the calculations within the framework of the ideal gas. It is experimentally established that a separation boundary layer can exist in the reverse flow, and its structure is subject to the laws inherent to the reverse flow in the separation region of the turbulent boundary layer arising in the supersonic conic flow under the action of a shockwave incident to the boundary layer.

  9. Exploring farmers’ seasonal and full year adoption of stall feeding of livestock in Tigrai region, Ethiopia

    Directory of Open Access Journals (Sweden)

    Hadush Muuz


    Full Text Available Adoption of stall feeding (SF of livestock was assessed in northern Ethiopia based on a household survey conducted in 2015. The study covered 21 communities in Tigrai to account for differences in agroecology. The purpose of this study was to understand the driving factors of full or seasonal SF adoption and its intensity. A Heckman selection model was used to estimate adoption and extent of adoption based on a model of technology adoption within an agricultural household framework, and Poisson Model for explaining the number of SF adopting seasons. The descriptive results indicate that 36% of the farmers were actually practicing SF in a full year whereas 55.6% were seasonal adopters in the study area. Empirical results of this study showed that our result is in favor of the Boserupian hypothesis indicating that small grazing land and large exclosure are associated with a higher probability of use of SF and with a higher number of SF adopting seasons. In a similar vein, small average village farm size stimulated SF adoption and adopting seasons, Availability of labor and a number of breed cows significantly increased the probability of using SF by 0.01% and 66% respectively. While animal shock had a marginal effect of 14%, factors such as access to information and early exposure increased SF adoption by about 18% and 6%. Similarly, the positive marginal effect of real milk price is 15%. However, SF appears to be less attractive to those farmers with more herd size and less crop residue.

  10. Quorum-based Data Replication in Grid Environment

    Directory of Open Access Journals (Sweden)

    Rohaya Latip


    Full Text Available Replication is a useful technique for distributed database systems and can be implemented in a grid computation environment to provide a high availability, fault tolerant, and enhance the performance of the system. This paper discusses a new protocol named Diagonal Data Replication in 2D Mesh structure (DR2M protocol where the performance addressed are data availability which is compared with the previous replication protocols, Read-One Write-All (ROWA, Voting (VT, Tree Quorum (TQ, Grid Configuration (GC, and Neighbor Replication on Grid (NRG. DR2M protocol is organized in a logical two dimensional mesh structure and by using quorums and voting techniques to improve the performance and availability of the replication protocol where it reduce the number of copies of data replication for read or write operations. The data file is copied at the selected node of the diagonal site in a quorum. The selection of a replica depends on the diagonal location of the structured two dimensional mesh quorum where the middle node is selected because it is the best location to get a copy of the data if every node has the equal number of request and data accessing in the network. The algorithm in this paper also calculates the best number of nodes in each quorum and how many quorums are needed for N number of nodes in a network. DR2M protocol also ensures that the data for read and write operations is consistency, by proofing the quorum must not have a nonempty intersection quorum. To evaluate DR2M protocol, we developed a simulation model in Java. Our results prove that DR2M protocol improves the performance of the data availability compare to the previous data replication protocol, ROWA, VT, TQ, GC and NRG.

  11. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation. (United States)

    Devlin, Rebecca; Marques, Catarina A; Paape, Daniel; Prorocic, Marko; Zurita-Leal, Andrea C; Campbell, Samantha J; Lapsley, Craig; Dickens, Nicholas; McCulloch, Richard


    Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating - a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility.

  12. Control of bacterial chromosome replication by non-coding regions outside the origin

    DEFF Research Database (Denmark)

    Frimodt-Møller, Jakob; Charbon, Godefroid; Løbner-Olesen, Anders


    Chromosome replication in Eubacteria is initiated by initiator protein(s) binding to specific sites within the replication origin, oriC. Recently, initiator protein binding to chromosomal regions outside the origin has attracted renewed attention; as such binding sites contribute to control...... the frequency of initiations. These outside-oriC binding sites function in several different ways: by steric hindrances of replication fork assembly, by titration of initiator proteins away from the origin, by performing a chaperone-like activity for inactivation- or activation of initiator proteins...... or by mediating crosstalk between chromosomes. Here, we discuss initiator binding to outside-oriC sites in a broad range of different taxonomic groups, to highlight the significance of such regions for regulation of bacterial chromosome replication. For Escherichia coli, it was recently shown that the genomic...

  13. Transonic Wind Tunnel Modernization for Experimental Investigation of Dynamic Stall in a Wide Range of Mach Numbers by Plasma Actuators with Combined Energy/Momentum Action (United States)


    wind tunnel for the study of plasma based methods for the control of dynamic stall for helicopter rotor blades. The tunnel has a 3D positioning...SECURITY CLASSIFICATION OF: This equipment grant supported the design and construction of a subsonic variable speed wind tunnel for the study of...plasma based methods for the control of dynamic stall for helicopter rotor blades. The tunnel has a 3D positioning system and servomotor mounted below

  14. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    International Nuclear Information System (INIS)

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


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

  15. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Directory of Open Access Journals (Sweden)

    Jason Diaz


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

  16. Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Energy Technology Data Exchange (ETDEWEB)

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


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

  17. Variance Swap Replication: Discrete or Continuous?

    Directory of Open Access Journals (Sweden)

    Fabien Le Floc’h


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

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

    Directory of Open Access Journals (Sweden)

    Majid Eshaghi

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

  19. Broken silence restored-remodeling primes for deacetylation at replication forks

    DEFF Research Database (Denmark)

    Jasencakova, Zuzana; Groth, Anja


    Faithful propagation of chromatin structures requires assimilation of new histones to the modification profile of individual loci. In this issue of Molecular Cell, Rowbotham and colleagues identify a remodeler, SMARCAD1, acting at replication sites to facilitate histone deacetylation and restorat......Faithful propagation of chromatin structures requires assimilation of new histones to the modification profile of individual loci. In this issue of Molecular Cell, Rowbotham and colleagues identify a remodeler, SMARCAD1, acting at replication sites to facilitate histone deacetylation...

  20. Chromatin-remodelling factors and the maintenance of transcriptional states through DNA replication. (United States)

    Aligianni, Sofia; Varga-Weisz, Patrick


    At the replication fork, nucleosomes, transcription factors and RNA polymerases are stripped off the DNA, the DNA double strands are unzipped and DNA methylation marks may be erased. Therefore DNA replication is both a 'curse' and 'bliss' for the epigenome, as it disrupts its stability by causing chromatin perturbations, yet it offers an opportunity to initiate changes in chromatin architecture and gene expression patterns, especially during development. Thus the DNA replication site is a critical point for regulation. It has become apparent that there is a close functional relationship between those factors that regulate transcriptional competence and the DNA replication programme. In this review we discuss novel insights into how chromatin-remodelling factors at replication sites are involved in both the maintenance and regulation of transcriptional states.

  1. Bromodomain Protein Brd4 Plays a Key Role in Merkel Cell Polyomavirus DNA Replication (United States)

    Wang, Xin; Li, Jing; Schowalter, Rachel M.; Jiao, Jing; Buck, Christopher B.; You, Jianxin


    Merkel cell polyomavirus (MCV or MCPyV) is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4) interacts with MCV large T antigen (LT) and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC) to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+) leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus. PMID:23144621

  2. Bromodomain protein Brd4 plays a key role in Merkel cell polyomavirus DNA replication.

    Directory of Open Access Journals (Sweden)

    Xin Wang

    Full Text Available Merkel cell polyomavirus (MCV or MCPyV is the first human polyomavirus to be definitively linked to cancer. The mechanisms of MCV-induced oncogenesis and much of MCV biology are largely unexplored. In this study, we demonstrate that bromodomain protein 4 (Brd4 interacts with MCV large T antigen (LT and plays a critical role in viral DNA replication. Brd4 knockdown inhibits MCV replication, which can be rescued by recombinant Brd4. Brd4 colocalizes with the MCV LT/replication origin complex in the nucleus and recruits replication factor C (RFC to the viral replication sites. A dominant negative inhibitor of the Brd4-MCV LT interaction can dissociate Brd4 and RFC from the viral replication complex and abrogate MCV replication. Furthermore, obstructing the physiologic interaction between Brd4 and host chromatin with the chemical compound JQ1(+ leads to enhanced MCV DNA replication, demonstrating that the role of Brd4 in MCV replication is distinct from its role in chromatin-associated transcriptional regulation. Our findings demonstrate mechanistic details of the MCV replication machinery; providing novel insight to elucidate the life cycle of this newly discovered oncogenic DNA virus.

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

    Energy Technology Data Exchange (ETDEWEB)

    Slor, H.; Cleaver, J.E.


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

  4. NIPBL rearrangements in Cornelia de Lange syndrome: evidence for replicative mechanism and genotype–phenotype correlation (United States)

    Pehlivan, Davut; Hullings, Melanie; Carvalho, Claudia M.B.; Gonzaga-Jauregui, Claudia G.; Loy, Elizabeth; Jackson, Laird G.; Krantz, Ian D.; Deardorff, Matthew A.; Lupski, James R.


    Purpose Cornelia de Lange syndrome (CdLS) is a multisystem congenital anomaly disorder characterized by mental retardation, limb abnormalities, distinctive facial features, and hirsutism. Mutations in three genes involved in sister chromatid cohesion, NIPBL, SMC1A, and SMC3, account for ~55% of CdLS cases. The molecular etiology of a significant fraction of CdLS cases remains unknown. We hypothesized that large genomic rearrangements of cohesin complex subunit genes may play a role in the molecular etiology of this disorder. Methods Custom high-resolution oligonucleotide array comparative genomic hybridization analyses interrogating candidate cohesin genes and breakpoint junction sequencing of identified genomic variants were performed. Results Of the 162 patients with CdLS, for whom mutations in known CdLS genes were previously negative by sequencing, deletions containing NIPBL exons were observed in 7 subjects (~5%). Breakpoint sequences in five patients implicated microhomology-mediated replicative mechanisms—such as serial replication slippage and fork stalling and template switching/microhomology-mediated break-induced replication—as a potential predominant contributor to these copy number variations. Most deletions are predicted to result in haploinsuflciency due to heterozygous loss-of-function mutations; such mutations may result in a more severe CdLS phenotype. Conclusion Our findings suggest a potential clinical utility to testing for copy number variations involving NIPBL when clinically diagnosed CdLS cases are mutation-negative by DNA-sequencing studies. PMID:22241092

  5. Evaluation of free-stall mattress bedding treatments to reduce mastitis bacterial growth

    Energy Technology Data Exchange (ETDEWEB)

    Kristula, M.A.; Dou, Z.; Toth, J.D.; Smith, B.I.; Harvey, N.; Sabo, M. [University of Penn, Kennett Square, PA (United States)


    Bacterial counts were compared in free-stall mattresses and teat ends exposed to 5 treatments in a factorial study design on 1 dairy farm. Mattresses in five 30-cow groups were subjected to 1 of 5 bedding treatments every other day: 0.5 kg of hydrated limestone, 120 mL of commercial acidic conditioner, 1 kg of coal fly ash, 1 kg of kiln-dried wood shavings, and control (no bedding). Counts of coliforms, Klebsiella spp., Escherichia coli, and Streptococcus spp. were lowest on mattresses bedded with lime. Mattresses bedded with the commercial acidic conditioner had the next lowest counts for coliforms, Klebsiella spp., and Streptococcus spp. Wood shavings and the no-bedding control had the highest counts for coliform and Klebsiella spp. Compared with wood shavings or control, fly ash reduced the counts of coliforms, whereas for the other 3 bacterial groups, the reduction was not always significant. Streptococcus spp. counts were greatest in the control group and did not differ among the shavings and fly ash groups. Teat swab results indicated that hydrated lime was the only bedding treatment that significantly decreased the counts of both coliforms and Klebsiella spp. There were no differences in Streptococcus spp. numbers on the teats between any of the bedding treatments. Bacterial populations grew steadily on mattresses and were generally higher at 36 to 48 h than at 12 to 24 h, whereas bacterial populations on teats grew rapidly by 12 h and then remained constant. Hydrated lime was the only treatment that significantly reduced bacterial counts on both mattresses and teat ends, but it caused some skin irritation.

  6. Evaluation of rice mutant lines for resistance to brown planthopper, nilaparvata lugens stall

    International Nuclear Information System (INIS)



    The most important and common insect in rice cultivation in South East Asia is brown planthopper, nilaparvata lugens stall. Seven rice mutant lines produced by the National Atomic Energy Agency, Indonesia, were tested at IRRI, the Philippines for resistance to brown planthopper. Those mutant lines were Atomita 1, 627/10-3/PsJ, Atomita 2 and 627/4-E/PsJ originated from Pelita 1/1 which was irradiated with 0.2 kGy of gamma rays and A227/2/PsJ, A227/3/PsJ and A227/5/PsJ, originated from early maturing mutant A23/PsJ/72K from irradiated Pelita 1/1 which was irradiated with 0.1 kGy of gamma rays. Evaluation of resistance was carried out by seedling bulk screening, honeydew excretion, survival and population build up tests by using brown planthopper biotype 1, 2 and 3. Results of these tests showed that the seven tested mutant lines were resistant to biotype 1 but susceptible to biotype 2. Reaction to biotype 3 showed that six mutant lines tested were moderately resistant and only one mutant of 627/4-E/PsJ was susceptible. Reactions of the mutant lines to biotype 1, 2 and 3 were different from the resistant varieties, Mudgo or ASD-7. This indicated that mutant lines might have gene(s) for resistance which differed from those of resistant varieties. The results showed that resistance to brown planthopper is possible to be introduced in Indonesian rice varieties by means of mutations. (author)

  7. Hepatitis C virus translation preferentially depends on active RNA replication.

    Directory of Open Access Journals (Sweden)

    Helene Minyi Liu

    Full Text Available Hepatitis C virus (HCV RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.

  8. Adressing Replication and Model Uncertainty

    DEFF Research Database (Denmark)

    Ebersberger, Bernd; Galia, Fabrice; Laursen, Keld

    innovation survey data for France, Germany and the UK, we conduct a ‘large-scale’ replication using the Bayesian averaging approach of classical estimators. Our method tests a wide range of determinants of innovation suggested in the prior literature, and establishes a robust set of findings on the variables...... which shape the introduction of new to the firm and new to the world innovations. We provide some implications for innovation research, and explore the potential application of our approach to other domains of research in strategic management.......Many fields of strategic management are subject to an important degree of model uncertainty. This is because the true model, and therefore the selection of appropriate explanatory variables, is essentially unknown. Drawing on the literature on the determinants of innovation, and by analyzing...

  9. Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Vos, J.M.; Avalosse, B.; Su, Z.Z.; Rommelaere, J.


    Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated by cell UV-irradiation.

  10. Initiation of chromosomal replication in predatory bacterium Bdellovibrio bacteriovorus

    Directory of Open Access Journals (Sweden)

    Lukasz Makowski


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

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

    Directory of Open Access Journals (Sweden)

    Paolo Norio


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

  12. An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents.

    Directory of Open Access Journals (Sweden)

    Ryan W Benson

    Full Text Available DinB (DNA Pol IV is a translesion (TLS DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ, a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS. Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.

  13. Opposite replication polarities of transcribed and nontranscribed histone H5 genes

    International Nuclear Information System (INIS)

    Trempe, J.P.; Lindstrom, Y.I.; Leffak, M.


    The authors used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocyctes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, the authors conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction

  14. The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication

    International Nuclear Information System (INIS)

    Mathew, Shomita S.; Bridge, Eileen


    Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci

  15. Mcm2 phosphorylation and the response to replicative stress

    Directory of Open Access Journals (Sweden)

    Stead Brent E


    Full Text Available Abstract Background The replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm proteins 2 through 7 (Mcm2-7 and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK. In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress. Results We show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU and to the base analogue 5-fluorouracil (5-FU but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate. Conclusions Together these observations point to a role for DDK-mediated phosphorylation

  16. Replicating chromatin: a tale of histones

    DEFF Research Database (Denmark)

    Groth, Anja


    framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures...... reassembly on nascent DNA strands. The aim of this review is to discuss how histones - new and old - are handled at the replication fork, highlighting new mechanistic insights and revisiting old paradigms....

  17. Effect of paddock vs. stall housing on 24 hour gastric pH within the proximal and ventral equine stomach. (United States)

    Husted, L; Sanchez, L C; Olsen, S N; Baptiste, K E; Merritt, A M


    Stall housing has been suggested as a risk factor for ulcer development in the equine stomach; however, the exact pathogenesis for this has not been established. To investigate the effect of 3 environmental situations (grass paddock, stall alone or stall with adjacent companion) on pH in the proximal and the ventral stomach. Six horses with permanently implanted gastric cannulae were used in a randomised, cross-over, block design. Each horse rotated through each of three 24 h environmental situations. Horses remained on their normal diet (grass hay ad libitum and grain b.i.d.) throughout the study. Intragastric pH was measured continuously for 72 h just inside the lower oesophageal sphincter (proximal stomach) and via a pH probe in the gastric cannula (ventral stomach). Neither proximal nor ventral 24 h gastric pH changed significantly between the 3 environmental situations. Mean hourly proximal gastric pH decreased significantly in the interval from 01.00-09.00 h compared to the interval from 13.00-20.00 h, regardless of environmental situation. Median hourly proximal pH only differed in the interval from 06.00-07.00 h compared to the interval 14.00-19.00 h. Neither mean nor median hourly ventral gastric pH varied significantly with the time of day. The change in housing status used in the current study did not affect acid exposure within either region of the equine stomach. The pH in the ventral stomach was uniformly stable throughout the study, while the proximal pH demonstrated a 24 h circadian pattern.

  18. Timeless links replication termination to mitotic kinase activation.

    Directory of Open Access Journals (Sweden)

    Jayaraju Dheekollu


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

  19. Enzymatic recognition of DNA replication origins

    International Nuclear Information System (INIS)

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


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

  20. The Effects of Gilts Housed Either in Group with the Electronic Sow Feeding System or Conventional Stall. (United States)

    Jang, J C; Jung, S W; Jin, S S; Ohh, S J; Kim, J E; Kim, Y Y


    This experiment was conducted to assess the welfare and productivity of gestating gilts in groups with the electronic sow feeding (ESF) system compared to conventional stalls. A total of 83 gilts (Yorkshire×Landrace) were housed into individual stalls to be artificially inseminated. Gilts confirmed pregnant were introduced to their treatment, conventional stalls (ST) or groups with the ESF system. All gilts were taken to the farrowing crates one week prior to their expected farrowing date. In the gestation period, there were no significant differences between gilts allocated to ST and ESF on growth performance. However, backfat thickness gain (p = 0.08) and body condition score (BCS) at 110 days of gestation (p = 0.10) tended to be higher in ESF gilts than ST. Likewise, gilts housed in group showed significantly higher estimated body muscle contents at 110 days of gestation (p = 0.02) and body muscle change during gestation (p = 0.01). There was a trend for a shorter parturition time in ESF gilts (p = 0.07). In the lactation period, group housed gilts showed a tendency to increased BCS changes (p = 0.06). Reproductive performance did not differ with the exception of piglet mortality (ST = 0.2 no. of piglets vs ESF = 0.4 no. of piglets; p = 0.01). In blood profiles, ST gilts showed a higher cortisol level at 110 days of gestation (p = 0.01). Weaning to estrus interval was shorter in gilts housed in ESF than ST (p = 0.01). In locomotory behaviors, ESF gilts recorded a tendency to elevate locomotion score at 36, 70, and 110 days of gestation (p = 0.07, p = 0.06, and p = 0.06, respectively). Similarly, ESF gilts showed significantly higher incidence of scratches at 36, 70, and 110 days of gestation (p = 0.01). Moreover, farrowing rates were higher in stall treatment (97.6%) compare to group housing treatment (95.2%). In conclusion, while group housed gilts with ESF system positively affected welfare status in combination with less physiologically stressful

  1. Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture (United States)

    Lohmann, Volker; Hoffmann, Sandra; Herian, Ulrike; Penin, Francois; Bartenschlager, Ralf


    Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself. PMID:12584326

  2. Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome.

    Directory of Open Access Journals (Sweden)

    Jared M Peace

    Full Text Available Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1's role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4Δ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1Δ cells, and checkpoint-defective mec1Δ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres.

  3. Visualization and measurement of ATP levels in living cells replicating hepatitis C virus genome RNA.

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

    Full Text Available Adenosine 5'-triphosphate (ATP is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV, a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome.

  4. Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. (United States)

    Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J


    Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

  5. Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

    Directory of Open Access Journals (Sweden)

    Stephanie Munk


    Full Text Available The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity.

  6. Host factors involved in chikungunya virus replication

    NARCIS (Netherlands)

    Scholte, Florine Elisabeth Maria


    In this thesis the interplay of CHIKV with cellular (host) factors involved in its replication is addressed. An in-depth understanding of the interactions between the viral proteins and those of their host is required for the elucidation of molecular mechanisms underlying viral replication. A

  7. Using Replication Projects in Teaching Research Methods (United States)

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


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

  8. Replication and Robustness in Developmental Research (United States)

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


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

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


    Klein, Richard A.; Ratliff, Kate A; Vianello, Michelangelo; Adams Jr., Reginald B; Bahník, Stĕpán; Bernstein, Michael J; Bocian, Konrad; Brandt, Mark J; Brooks, Beach; Brumbaugh, Claudia Chloe; Cemalcilar, Zeynep; Chandler, Jesse; Cheong, Winnee; Davis, William E; Devos, Thierry


    This dataset is from the Many Labs Replication Project in which 13 effects were replicated across 36 samples and over 6,000 participants. Data from the replications are included, along with demographic variables about the participants and contextual information about the environment in which the replication was conducted. Data were collected in-lab and online through a standardized procedure administered via an online link. The dataset is stored on the Open Science Framework website. These da...

  10. Chromosome length influences replication-induced topological stress. (United States)

    Kegel, Andreas; Betts-Lindroos, Hanna; Kanno, Takaharu; Jeppsson, Kristian; Ström, Lena; Katou, Yuki; Itoh, Takehiko; Shirahige, Katsuhiko; Sjögren, Camilla


    During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.

  11. Suppression of Poxvirus Replication by Resveratrol

    Directory of Open Access Journals (Sweden)

    Shuai Cao


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

  12. Rescue from replication stress during mitosis. (United States)

    Fragkos, Michalis; Naim, Valeria


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

  13. Characteristics of the poliovirus replication complex. (United States)

    Bienz, K; Egger, D; Pfister, T


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

  14. Recommendations for Replication Research in Special Education: A Framework of Systematic, Conceptual Replications (United States)

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


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

  15. Replication and efficiency in experiments for marketable emissions permits

    Energy Technology Data Exchange (ETDEWEB)

    Cason, T.N. [Univ. of Southern California, Los Angeles, CA (United States). Dept. of Economics; Elliott, S.R. [Oak Ridge National Lab., TN (United States); Kundra, I. [Dept. of Energy, Washington, DC (United States). Energy Information Administration; Van Boening, M.V. [Univ. of Mississippi, Oxford, MS (United States). Dept. of Economics and Finance


    The Energy Information Administration (EIA) funded the universities of Colorado and Arizona to define an experimental institution that captures the salient features of the sulfur dioxide allowance market created by the Clean Air Act Amendments of 1990 (CAAA); to develop and document a transportable software that implements the experimental institutions; and to replicate experiments. Subsequently, EIA, in conjunction with the Oak Ridge National Laboratory (ORNL) funded the universities of Mississippi and Southern California to test the replicability of these experiments using statistically sound experimental design and the standardized software developed by the University of Arizona. The present experiment is designed to identify any differences in the results of the two laboratory sites. It is designed to determine whether market outcomes are reproducible across different laboratories and experimenters and to determine if any behavior patterns exist across a large set of independent experimental sessions.

  16. Roles for Dam methylation in bacterial chromosome replication

    DEFF Research Database (Denmark)

    Charbon, Godefroid; Koch, Birgit; Skovgaard, Ole

    . Second, new DnaA binding sites outside oriC are generated by replication which serve to titrate free DNA protein. Third, after initiation, DnaA-ATP is converted to inactive DnaA-ADP by a process called RIDA (regulatory inactivation of DnaA), which is dependent on the beta-clamp of DNA polymerase III...... of DnaA-ADP to DnaA-ATP by the DARS1 sequence was inhibited. Second, excess SeqA reduced the overall level of DnaA by about 30% through dnaA gene transcription. However, this reduced initiator level did not negatively affect initiation of replication. This suggests that the amount of DnaA available...

  17. A model for roll stall and the inherent stability modes of low aspect ratio wings at low Reynolds numbers (United States)

    Shields, Matt

    The development of Micro Aerial Vehicles has been hindered by the poor understanding of the aerodynamic loading and stability and control properties of the low Reynolds number regime in which the inherent low aspect ratio (LAR) wings operate. This thesis experimentally evaluates the static and damping aerodynamic stability derivatives to provide a complete aerodynamic model for canonical flat plate wings of aspect ratios near unity at Reynolds numbers under 1 x 105. This permits the complete functionality of the aerodynamic forces and moments to be expressed and the equations of motion to solved, thereby identifying the inherent stability properties of the wing. This provides a basis for characterizing the stability of full vehicles. The influence of the tip vortices during sideslip perturbations is found to induce a loading condition referred to as roll stall, a significant roll moment created by the spanwise induced velocity asymmetry related to the displacement of the vortex cores relative to the wing. Roll stall is manifested by a linearly increasing roll moment with low to moderate angles of attack and a subsequent stall event similar to a lift polar; this behavior is not experienced by conventional (high aspect ratio) wings. The resulting large magnitude of the roll stability derivative, Cl,beta and lack of roll damping, Cl ,rho, create significant modal responses of the lateral state variables; a linear model used to evaluate these modes is shown to accurately reflect the solution obtained by numerically integrating the nonlinear equations. An unstable Dutch roll mode dominates the behavior of the wing for small perturbations from equilibrium, and in the presence of angle of attack oscillations a previously unconsidered coupled mode, referred to as roll resonance, is seen develop and drive the bank angle? away from equilibrium. Roll resonance requires a linear time variant (LTV) model to capture the behavior of the bank angle, which is attributed to the

  18. Differential Requirement of Human Cytomegalovirus UL112-113 Protein Isoforms for Viral Replication. (United States)

    Schommartz, Tim; Tang, Jiajia; Brost, Rebekka; Brune, Wolfram


    The UL112-113 gene is one of the few alternatively spliced genes of human cytomegalovirus (HCMV). It codes for four phosphoproteins, p34, p43, p50, and p84, all of which are expressed with early kinetics and accumulate at sites of viral DNA replication within the host cell nucleus. Although these proteins are known to play important, possibly essential, roles in the viral replication cycle, little is known about the contribution of individual UL112-113 protein products. Here we used splice site mutagenesis, intron deletion and substitution, and nonsense mutagenesis to prevent the individual expression of each UL112-113 protein isoform and to investigate the importance of each isoform for viral replication. We show that HCMV mutants lacking p34 or p50 expression replicated to high titers in human fibroblasts and endothelial cells, indicating that these proteins are nonessential for viral replication, while mutant viruses carrying a stop mutation within the p84 coding sequence were severely growth impaired. Viral replication could not be detected upon the inactivation of p43 expression, indicating that this UL112-113 protein is essential for viral replication. We also analyzed the ability of UL112-113 proteins to recruit other viral proteins to intranuclear prereplication compartments. While UL112-113 expression was sufficient to recruit the UL44-encoded viral DNA polymerase processivity factor, it was not sufficient for the recruitment of the viral UL84 and UL117 proteins. Remarkably, both the p43 and p84 isoforms were required for the efficient recruitment of pUL44, which is consistent with their critical role in the viral life cycle. IMPORTANCE Human cytomegalovirus requires gene products from 11 genetic loci for the lytic replication of its genome. One of these loci, UL112-113, encodes four proteins with common N termini by alternative splicing. In this study, we inactivated the expression of each of the four UL112-113 proteins individually and determined their

  19. Nucleotide sequence analysis of regions of adenovirus 5 DNA containing the origins of DNA replication

    International Nuclear Information System (INIS)

    Steenbergh, P.H.


    The purpose of the investigations described is the determination of nucleotide sequences at the molecular ends of the linear adenovirus type 5 DNA. Knowledge of the primary structure at the termini of this DNA molecule is of particular interest in the study of the mechanism of replication of adenovirus DNA. The initiation- and termination sites of adenovirus DNA replication are located at the ends of the DNA molecule. (Auth.)

  20. Exploratory study of the effects of wing-leading-edge modifications on the stall/spin behavior of a light general aviation airplane (United States)


    Configurations with full-span and segmented leading-edge flaps and full-span and segmented leading-edge droop were tested. Studies were conducted with wind-tunnel models, with an outdoor radio-controlled model, and with a full-scale airplane. Results show that wing-leading-edge modifications can produce large effects on stall/spin characteristics, particularly on spin resistance. One outboard wing-leading-edge modification tested significantly improved lateral stability at stall, spin resistance, and developed spin characteristics.

  1. How Xenopus laevis embryos replicate reliably: Investigating the random-completion problem (United States)

    Yang, Scott Cheng-Hsin; Bechhoefer, John


    DNA synthesis in Xenopus frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the time to complete and may lead to cell death if replication takes longer than the cell cycle time (≈25min) . Surprisingly, although the typical replication time is about 20min , in vivo experiments show that replication fails to complete only about 1 in 300 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two solutions to this “random-completion problem.” The first solution uses randomly located origins but increases their rate of initiation as S phase proceeds, while the second uses regularly spaced origins. In this paper, we investigate the random-completion problem using a type of model first developed to describe the kinetics of first-order phase transitions. Using methods from the field of extreme-value statistics, we derive the distribution of replication-completion times for a finite genome. We then argue that the biologists’ first solution to the problem is not only consistent with experiment but also nearly optimizes the use of replicative proteins. We also show that spatial regularity in origin placement does not alter significantly the distribution of replication times and, thus, is not needed for the control of replication timing.

  2. Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential. (United States)

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


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

  3. Targeting DNA Replication Stress for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Jun Zhang


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

  4. Factors influencing microinjection molding replication quality (United States)

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


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

  5. Ultrastructural Characterization of Zika Virus Replication Factories

    Directory of Open Access Journals (Sweden)

    Mirko Cortese


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

  6. Hypotension and Environmental Noise: A Replication Study

    Directory of Open Access Journals (Sweden)

    Peter Lercher


    Full Text Available Up to now, traffic noise effect studies focused on hypertension as health outcome. Hypotension has not been considered as a potential health outcome although in experiments some people also responded to noise with decreases of blood pressure. Currently, the characteristics of these persons are not known and whether this down regulation of blood pressure is an experimental artifact, selection, or can also be observed in population studies is unanswered. In a cross-sectional replication study, we randomly sampled participants (age 20–75, N = 807 from circular areas (radius = 500 m around 31 noise measurement sites from four noise exposure strata (35–44, 45–54, 55–64, >64 Leq, dBA. Repeated blood pressure measurements were available for a smaller sample (N = 570. Standardized information on socio-demographics, housing, life style and health was obtained by door to door visits including anthropometric measurements. Noise and air pollution exposure was assigned by GIS based on both calculation and measurements. Reported hypotension or hypotension medication past year was the main outcome studied. Exposure-effect relationships were modeled with multiple non-linear logistic regression techniques using separate noise estimations for total, highway and rail exposure. Reported hypotension was significantly associated with rail and total noise exposure and strongly modified by weather sensitivity. Reported hypotension medication showed associations of similar size with rail and total noise exposure without effect modification by weather sensitivity. The size of the associations in the smaller sample with BMI as additional covariate was similar. Other important cofactors (sex, age, BMI, health and moderators (weather sensitivity, adjacent main roads and associated annoyance need to be considered as indispensible part of the observed relationship. This study confirms a potential new noise effect pathway and discusses potential patho

  7. Replicated Data Management for Mobile Computing

    CERN Document Server

    Douglas, Terry


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

  8. Replication and Analysis of Ebbinghaus' Forgetting Curve. (United States)

    Murre, Jaap M J; Dros, Joeri


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

  9. Replication and Analysis of Ebbinghaus’ Forgetting Curve (United States)

    Murre, Jaap M. J.; Dros, Joeri


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

  10. Replication and Analysis of Ebbinghaus' Forgetting Curve.

    Directory of Open Access Journals (Sweden)

    Jaap M J Murre

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

  11. Replication-Competent Controlled Herpes Simplex Virus. (United States)

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


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

  12. LHCb Data Replication During SC3

    CERN Multimedia

    Smith, A


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

  13. Surface Microstructure Replication in Injection Moulding

    DEFF Research Database (Denmark)

    Hansen, Hans Nørgaard; Arlø, Uffe Rolf


    topography is transcribed onto the plastic part through complex mechanisms. This replication however, is not perfect, and the replication quality depends on the plastic material properties, the topography itself, and the process conditions. This paper describes and discusses an investigation of injection......In recent years polymer components with surface microstructures have been in rising demand for applications such as lab-on-a-chip and optical components. Injection moulding has proven to be a feasible and efficient way to manufacture such components. In injection moulding the mould surface...... moulding of surface microstructures. Emphasis is put on the ability to replicate surface microstructures under normal injection moulding conditions, notably with low cost materials at low mould temperatures. The replication of surface microstructures in injection moulding has been explored...

  14. Lipid Tales of Viral Replication and Transmission. (United States)

    Altan-Bonnet, Nihal


    Positive-strand RNA viruses are the largest group of RNA viruses on Earth and cellular membranes are critical for all aspects of their life cycle, from entry and replication to exit. In particular, membranes serve as platforms for replication and as carriers to transmit these viruses to other cells, the latter either as an envelope surrounding a single virus or as the vesicle containing a population of viruses. Notably, many animal and human viruses appear to induce and exploit phosphatidylinositol 4-phosphate/cholesterol-enriched membranes for replication, whereas many plant and insect-vectored animal viruses utilize phosphatidylethanolamine/cholesterol-enriched membranes for the same purpose; and phosphatidylserine-enriched membrane carriers are widely used by both single and populations of viruses for transmission. Here I discuss the implications for viral pathogenesis and therapeutic development of this remarkable convergence on specific membrane lipid blueprints for replication and transmission. Published by Elsevier Ltd.

  15. Surface Micro Topography Replication in Injection Moulding

    DEFF Research Database (Denmark)

    Arlø, Uffe Rolf; Hansen, Hans Nørgaard; Kjær, Erik Michael


    carried out with rough EDM (electrical discharge machining) mould surfaces, a PS grade, and by applying established three-dimensional topography parameters. Significant quantitative relationships between process parameters and topography parameters were established. It further appeared that replication...

  16. Molecular Mechanisms of DNA Replication Checkpoint Activation

    Directory of Open Access Journals (Sweden)

    Bénédicte Recolin


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

  17. Potential biomarkers of DNA replication stress in cancer

    DEFF Research Database (Denmark)

    Ren, Liqun; Chen, Long; Wu, Wei


    oncogene-induced RS and CIN, with a particular emphasis on regions of the human genome that show enhanced sensitivity to the destabilizing effects of RS, such as common fragile sites. Because RS exists in a wide range of cancer types, we propose that the proteins involved counteracting this stress......Oncogene activation is an established driver of tumorigenesis. An apparently inevitable consequence of oncogene activation is the generation of DNA replication stress (RS), a feature common to most cancer cells. RS, in turn, is a causal factor in the development of chromosome instability (CIN...

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

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, D.C.; Kunkel, T.A. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States))


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

  20. Replication and Analysis of Ebbinghaus? Forgetting Curve


    Murre, Jaap M. J.; Dros, Joeri


    We present a successful replication of Ebbinghaus’ classic forgetting curve from 1880 based on the method of savings. One subject spent 70 hours learning lists and relearning them after 20 min, 1 hour, 9 hours, 1 day, 2 days, or 31 days. The results are similar to Ebbinghaus' original data. We analyze the effects of serial position on forgetting and investigate what mathematical equations present a good fit to the Ebbinghaus forgetting curve and its replications. We conclude that the Ebbingha...

  1. Evolution of Database Replication Technologies for WLCG


    Baranowski, Zbigniew; Pardavila, Lorena Lobato; Blaszczyk, Marcin; Dimitrov, Gancho; Canali, Luca


    In this article we summarize several years of experience on database replication technologies used at WLCG and we provide a short review of the available Oracle technologies and their key characteristics. One of the notable changes and improvement in this area in recent past has been the introduction of Oracle GoldenGate as a replacement of Oracle Streams. We report in this article on the preparation and later upgrades for remote replication done in collaboration with ATLAS and Tier 1 databas...

  2. The Legal Road To Replicating Silicon Valley


    John Armour; Douglas Cumming


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

  3. Lymphatic endothelial cells are a replicative niche for Mycobacterium tuberculosis (United States)

    Lerner, Thomas R.; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Russell, Matthew R.G.; Borel, Sophie; Diedrich, Collin R.; Rohde, Manfred; Wainwright, Helen; Collinson, Lucy M.; Wilkinson, Robert J.; Griffiths, Gareth; Gutierrez, Maximiliano G.


    In extrapulmonary tuberculosis, the most common site of infection is within the lymphatic system, and there is growing recognition that lymphatic endothelial cells (LECs) are involved in immune function. Here, we identified LECs, which line the lymphatic vessels, as a niche for Mycobacterium tuberculosis in the lymph nodes of patients with tuberculosis. In cultured primary human LECs (hLECs), we determined that M. tuberculosis replicates both in the cytosol and within autophagosomes, but the bacteria failed to replicate when the virulence locus RD1 was deleted. Activation by IFN-γ induced a cell-autonomous response in hLECs via autophagy and NO production that restricted M. tuberculosis growth. Thus, depending on the activation status of LECs, autophagy can both promote and restrict replication. Together, these findings reveal a previously unrecognized role for hLECs and autophagy in tuberculosis pathogenesis and suggest that hLECs are a potential niche for M. tuberculosis that allows establishment of persistent infection in lymph nodes. PMID:26901813

  4. Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

    Energy Technology Data Exchange (ETDEWEB)

    Khalil, Mohamed I., E-mail: [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States); Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo (Egypt); Che, Xibing; Sung, Phillip; Sommer, Marvin H. [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States); Hay, John [Department of Microbiology and Immunology, School of Medicine and Biomedical Science, University at Buffalo, Buffalo, NY (United States); Arvin, Ann M. [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States)


    VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.

  5. Commercial Building Partnerships Replication and Diffusion

    Energy Technology Data Exchange (ETDEWEB)

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


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

  6. Effects of the inlet boundary layer thickness on the flow in an axial compressor (I) : hub corner stall and tip leakage flow

    International Nuclear Information System (INIS)

    Choi, Min Suk; Baek, Je Hyun; Park, Jun Young


    A three-dimensional computation was conducted to understand effects of the inlet boundary layer thickness on the internal flow in a low-speed axial compressor operating at the design condition(φ=85%) and near stall condition(φ=65%). At the design condition, the flows in the axial compressor show, independent of the inlet boundary layer thickness, similar characteristics such as the pressure distribution, size of the hub corner-stall, tip leakage flow trajectory, limiting streamlines on the blade suction surface, etc. However, as the load is increased, the hub corner-stall grows to make a large separation region at the junction of the hub and suction surface for the inlet condition with thick boundary layers at the hub and casing. Moreover, the tip leakage flow is more vortical than that observed in case of the thin inlet boundary layer and has the critical point where the trajectory of the tip leakage flow is abruptly turned into the downstream. For the inlet condition with thin boundary layers, the hub corner-stall is diminished so it is indistinguishable from the wake. The tip leakage flow leans to the leading edge more than at the design condition but has no critical point. In addition to these, the severe reverse flow, induced by both boundary layer on the blade surface and the tip leakage flow, can be found to act as the blockage of flows near the casing, resulting in heavy loss

  7. Stall/surge dynamics of a multi-stage air compressor in response to a load transient of a hybrid solid oxide fuel cell-gas turbine system (United States)

    Azizi, Mohammad Ali; Brouwer, Jacob


    A better understanding of turbulent unsteady flows in gas turbine systems is necessary to design and control compressors for hybrid fuel cell-gas turbine systems. Compressor stall/surge analysis for a 4 MW hybrid solid oxide fuel cell-gas turbine system for locomotive applications is performed based upon a 1.7 MW multi-stage air compressor. Control strategies are applied to prevent operation of the hybrid SOFC-GT beyond the stall/surge lines of the compressor. Computational fluid dynamics tools are used to simulate the flow distribution and instabilities near the stall/surge line. The results show that a 1.7 MW system compressor like that of a Kawasaki gas turbine is an appropriate choice among the industrial compressors to be used in a 4 MW locomotive SOFC-GT with topping cycle design. The multi-stage radial design of the compressor enhances the ability of the compressor to maintain air flow rate during transient step-load changes. These transient step-load changes are exhibited in many potential applications for SOFC/GT systems. The compressor provides sustained air flow rate during the mild stall/surge event that occurs due to the transient step-load change that is applied, indicating that this type of compressor is well-suited for this hybrid application.

  8. Analysis of the Unsteady Flow Field in a Centrifugal Compressor from Peak Efficiency to Near Stall with Full-Annulus Simulations

    Directory of Open Access Journals (Sweden)

    Yannick Bousquet


    Full Text Available This study concerns a 2.5 pressure ratio centrifugal compressor stage consisting of a splittered unshrouded impeller and a vaned diffuser. The aim of this paper is to investigate the modifications of the flow structure when the operating point moves from peak efficiency to near stall. The investigations are based on the results of unsteady three-dimensional simulations, in a calculation domain comprising all the blade. A detailed analysis is given in the impeller inducer and in the vaned diffuser entry region through time-averaged and unsteady flow field. In the impeller inducer, this study demonstrates that the mass flow reduction from peak efficiency to near stall leads to intensification of the secondary flow effects. The low momentum fluid accumulated near the shroud interacts with the main flow through a shear layer zone. At near stall condition, the interface between the two flow structures becomes unstable leading to vortices development. In the diffuser entry region, by reducing the mass flow, the high incidence angle from the impeller exit induces a separation on the diffuser vane suction side. At near stall operating point, vorticity from the separation is shed into vortex cores which are periodically formed and convected downstream along the suction side.

  9. Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication. (United States)

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


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

  10. Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication.

    Directory of Open Access Journals (Sweden)

    Katrin Schenk


    Full Text Available DnaA is a conserved key regulator of replication initiation in bacteria, and is homologous to ORC proteins in archaea and in eukaryotic cells. The ATPase binds to several high affinity binding sites at the origin region and upon an unknown molecular trigger, spreads to several adjacent sites, inducing the formation of a helical super structure leading to initiation of replication. Using FRAP analysis of a functional YFP-DnaA allele in Bacillus subtilis, we show that DnaA is bound to oriC with a half-time of 2.5 seconds. DnaA shows similarly high turnover at the replication machinery, where DnaA is bound to DNA polymerase via YabA. The absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Likewise, a deletion of soj (encoding a ParA protein leads to an increase in residence time and to overinitiation, while a mutation in DnaA that leads to lowered initiation frequency, due to a reduced ATPase activity, shows a decreased residence time on binding sites. Finally, our single molecule tracking experiments show that DnaA rapidly moves between chromosomal binding sites, and does not arrest for more than few hundreds of milliseconds. In Escherichia coli, DnaA also shows low residence times in the range of 200 ms and oscillates between spatially opposite chromosome regions in a time frame of one to two seconds, independently of ongoing transcription. Thus, DnaA shows extremely rapid binding turnover on the chromosome including oriC regions in two bacterial species, which is influenced by Soj and YabA proteins in B. subtilis, and is crucial for balanced initiation control, likely preventing fatal premature multimerization and strand opening of DnaA at oriC.

  11. Intermolecular RNA Recombination Occurs at Different Frequencies in Alternate Forms of Brome Mosaic Virus RNA Replication Compartments

    Directory of Open Access Journals (Sweden)

    Hernan Garcia-Ruiz


    Full Text Available Positive-strand RNA viruses replicate their genomes in membrane-bound replication compartments. Brome mosaic virus (BMV replicates in vesicular invaginations of the endoplasmic reticulum membrane. BMV has served as a productive model system to study processes like virus-host interactions, RNA replication and recombination. Here we present multiple lines of evidence showing that the structure of the viral RNA replication compartments plays a fundamental role and that recruitment of parental RNAs to a common replication compartment is a limiting step in intermolecular RNA recombination. We show that a previously defined requirement for an RNA recruitment element on both parental RNAs is not to function as a preferred crossover site, but in order for individual RNAs to be recruited into the replication compartments. Moreover, modulating the form of the replication compartments from spherular vesicles (spherules to more expansive membrane layers increased intermolecular RNA recombination frequency by 200- to 1000-fold. We propose that intermolecular RNA recombination requires parental RNAs to be recruited into replication compartments as monomers, and that recruitment of multiple RNAs into a contiguous space is much more common for layers than for spherules. These results could explain differences in recombination frequencies between viruses that replicate in association with smaller spherules versus larger double-membrane vesicles and convoluted membranes.

  12. New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria

    DEFF Research Database (Denmark)

    Ioannidis, Panagiotis; Dunning Hotopp, Julie C; Sapountzis, Panagiotis


    as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria. RESULTS: Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus...... bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes...

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


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


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

  14. Dynamics of Escherichia coli Chromosome Segregation during Multifork Replication

    DEFF Research Database (Denmark)

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


    Slowly growing Escherichia coli cells have a simple cell cycle, with replication and progressive segregation of the chromosome completed before cell division. In rapidly growing cells, initiation of replication occurs before the previous replication rounds are complete. At cell division...

  15. Insights into the Determination of the Templating Nucleotide at the Initiation of φ29 DNA Replication. (United States)

    del Prado, Alicia; Lázaro, José M; Longás, Elisa; Villar, Laurentino; de Vega, Miguel; Salas, Margarita


    Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the replication origins, placed at both 5' ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser-232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3' end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser-232 is located indicate that the aromatic residue Phe-230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerization active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe-230 in limiting the internalization of the template strand in the polymerization active site is discussed. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. miR-370 suppresses HBV gene expression and replication by targeting nuclear factor IA. (United States)

    Fan, Hongxia; Lv, Ping; Lv, Jing; Zhao, Xiaopei; Liu, Min; Zhang, Guangling; Tang, Hua


    Hepatitis B virus (HBV) infection is a major health problem worldwide. The roles of microRNAs in the regulation of HBV expression are being increasingly recognized. In this study, we found that overexpression of miR-370 suppressed HBV gene expression and replication in Huh7 cells, whereas antisense knockdown of endogenous miR-370 enhanced HBV gene expression and replication in Huh7 cells and HepG2.2.15 cells. Further, we identified the transcription factor nuclear factor IA (NFIA) as a new host target of miR-370. Overexpression and knockdown studies showed that NFIA stimulated HBV gene expression and replication. Importantly, overexpression of NFIA counteracted the effect of miR-370 on HBV gene expression and replication. Further mechanistic studies showed that miR-370 suppressed HBV replication and gene expression by repressing HBV Enhancer I activity, and one of the NFIA binding site in the Enhancer I element was responsible for the repressive effect of miR-370 on HBV Enhancer I activity. Altogether, our results demonstrated that miR-370 suppressed HBV gene expression and replication through repressing NFIA expression, which stimulates HBV replication via direct regulation on HBV Enhancer I activities. Our findings may provide a new antiviral strategy for HBV infection. J. Med. Virol. 89:834-844, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Recent advances in the genome-wide study of DNA replication origins in yeast (United States)

    Peng, Chong; Luo, Hao; Zhang, Xi; Gao, Feng


    DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve yeast replication origins prediction. PMID:25745419

  18. Recent advances in the genome-wide study of DNA replication origins in yeast

    Directory of Open Access Journals (Sweden)

    Chong ePeng


    Full Text Available DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs. Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genome. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some nonconventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve the replication origins prediction.

  19. Replication ofVibrio choleraeclassical CTX phage. (United States)

    Kim, Eun Jin; Yu, Hyun Jin; Lee, Je Hee; Kim, Jae-Ouk; Han, Seung Hyun; Yun, Cheol-Heui; Chun, Jongsik; Nair, G Balakrish; Kim, Dong Wook


    The toxigenic classical and El Tor biotype Vibrio cholerae serogroup O1 strains are generated by lysogenization of host-type-specific cholera toxin phages (CTX phages). Experimental evidence of the replication and transmission of an El Tor biotype-specific CTX phage, CTX-1, has explained the evolution of V. cholerae El Tor biotype strains. The generation of classical biotype strains has not been demonstrated in the laboratory, and the classical biotype-specific CTX phage, CTX-cla, is considered to be defective with regard to replication. However, the identification of atypical El Tor strains that contain CTX-cla-like phage, CTX-2, indicates that CTX-cla and CTX-2 replicate and can be transmitted to V. cholerae strains. The replication of CTX-cla and CTX-2 phages and the transduction of El Tor biotype strains by various CTX phages under laboratory conditions are demonstrated in this report. We have established a plasmid-based CTX phage replication system that supports the replication of CTX-1, CTX-cla, CTX-2, and CTX-O139. The replication of CTX-2 from the tandem repeat of lysogenic CTX-2 in Wave 2 El Tor strains is also presented. El Tor biotype strains can be transduced by CTX phages in vitro by introducing a point mutation in toxT , the transcriptional activator of the tcp (toxin coregulated pilus) gene cluster and the cholera toxin gene. This mutation also increases the expression of cholera toxin in El Tor strains in a sample single-phase culture. Our results thus constitute experimental evidence of the genetic mechanism of the evolution of V. cholerae .

  20. Ribosome stalling regulates IRES-mediated translation in eukaryotes, a parallel to prokaryotic attenuation

    NARCIS (Netherlands)

    Fernandez, James; Yaman, Ibrahim; Huang, Charles; Liu, Haiyan; Lopez, Alex B.; Komar, Anton A.; Caprara, Mark G.; Merrick, William C.; Snider, Martin D.; Kaufman, Randal J.; Lamers, Wouter H.; Hatzoglou, Maria


    It was previously shown that the mRNA for the cat-1 Arg/Lys transporter is translated from an internal ribosome entry site (IRES) that is regulated by cellular stress. Amino acid starvation stimulated cat-1 translation via a mechanism that requires translation of an ORF in the mRNA leader and

  1. ATLAS Replica Management in Rucio: Replication Rules and Subscriptions

    CERN Document Server

    Barisits, M; The ATLAS collaboration; Garonne, V; Lassnig, M; Stewart, G; Beermann, T; Vigne, R; Goossens, L; Nairz, A; Molfetas, A


    The ATLAS Distributed Data Management system stores more than 150PB of physics data across 120 sites globally. To cope with the anticipated ATLAS workload of the coming decade, Rucio, the next-generation data management system has been developed. Replica management, as one of the keys aspects of the system, has to satisfy critical performance requirements in order to keep pace with the experiment’s high rate of continual data generation. The challenge lies in meeting these performance objectives while still giving the system users and applications a powerful toolkit to control their data workflows. In this work we present the concept, design and implementation of the replica management in Rucio. We will specifically introduce the workflows behind replication rules, their formal language definition, weighting and site selection. Furthermore we will present the subscription component, which offers functionality for users to proclaim interest in data that has not been created yet. This contribution describes t...

  2. ATLAS Replica Management in Rucio: Replication Rules and Subscriptions

    CERN Document Server

    Barisits, M; The ATLAS collaboration; Garonne, V; Lassnig, M; Stewart, G; Beermann, T; Vigne, R; Goossens, L; Nairz, A; Molfetas, A


    The ATLAS Distributed Data Management system stores more than 150PB of physics data across 120 sites globally. To cope with the anticipated ATLAS workload of the coming decade, Rucio, the next-generation data management system has been developed. Replica management, as one of the keys aspects of the system, has to satisfy critical performance requirements in order to keep pace with the experiment’s high rate of continual data generation. The challenge lies in meeting these performance objectives while still giving the system users and applications a powerful toolkit to control their data workflows. In this work we present the concept, design and implementation of the replica management in Rucio. We will specifically introduce the workflows behind replication rules, their formal language definition, weighting and site selection. Furthermore we will present the subscription component, which offers functionality for users to proclaim interest in data that has not been created yet. This contribution describes t...

  3. Optical tweezers reveal how proteins alter replication (United States)

    Chaurasiya, Kathy

    Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic

  4. Suppression of inducer stall based on inlet recirculation in a centrifugal impeller. 1st Report. Improvement in stall limit by ring groove arrangement; Enshin haneguruma iriguchi junkanryu ni yoru inducer shissoku no yokusei. 1. kanjoko ni yoru shissoku genkai no kaizen

    Energy Technology Data Exchange (ETDEWEB)

    Ueki, H.; Ishida, M.; Sakaguchi, D.; Sun, Z. [Nagasaki University, Nagasaki (Japan). Faculty of Engineering


    A ring groove arrangement is proposed to suppress unstable flow or surge in a centrifugal blower. The ring groove arrangement connects the upstream position of impeller inlet and the inducer throat tip through a bypass. The recirculation flow 'vas formed by the pressure difference between the two positions, and the recirculation flow rate was changed by increasing the ring groove widths. The inlet recirculation results in a decrease in the flow rate of unstable flow inception, and an up to 800 improvement in stall limit was obtained by the ring groove arrangement at a small expense of the delivery pressure drop. The improvement of stall limit in the present experiment seems to be mainly due to decrease in flow incidence based on the inlet recirculation flow. Tre flow incidence decreases more as the recirculation flow rate increases, thus resulting in a larger improvement in stall limit. (author)

  5. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication. (United States)

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


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

  6. Methadone enhances human influenza A virus replication. (United States)

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


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

  7. Extremal dynamics in random replicator ecosystems

    Energy Technology Data Exchange (ETDEWEB)

    Kärenlampi, Petri P., E-mail:


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

  8. Replicating viruses for gynecologic cancer therapy. (United States)

    Park, J W; Kim, M


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

  9. COPI is required for enterovirus 71 replication.

    Directory of Open Access Journals (Sweden)

    Jianmin Wang

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

  10. Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli*


    Holberger, Laura E.; Hayes, Christopher S.


    Translational pausing in Escherichia coli can lead to mRNA cleavage within the ribosomal A-site. A-site mRNA cleavage is thought to facilitate transfer-messenger RNA (tmRNA)·SmpB- mediated recycling of stalled ribosome complexes. Here, we demonstrate that the aminoglycosides paromomycin and streptomycin inhibit A-site cleavage of stop codons during inefficient translation termination. Aminoglycosides also induced stop codon read-through, suggesting that these antibiotics alleviate ribosome pa...

  11. The "enemies within": regions of the genome that are inherently difficult to replicate [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Rahul Bhowmick


    Full Text Available An unusual feature of many eukaryotic genomes is the presence of regions that appear intrinsically difficult to copy during the process of DNA replication. Curiously, the location of these difficult-to-replicate regions is often conserved between species, implying a valuable role in some aspect of genome organization or maintenance. The most prominent class of these regions in mammalian cells is defined as chromosome fragile sites, which acquired their name because of a propensity to form visible gaps/breaks on otherwise-condensed chromosomes in mitosis. This fragility is particularly apparent following perturbation of DNA replication—a phenomenon often referred to as “replication stress”. Here, we review recent data on the molecular basis for chromosome fragility and the role of fragile sites in the etiology of cancer. In particular, we highlight how studies on fragile sites have provided unexpected insights into how the DNA repair machinery assists in the completion of DNA replication.

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

    Directory of Open Access Journals (Sweden)

    Amnon Koren


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

  13. Dynamics of viral replication in blood and lymphoid tissues during SIVmac251 infection of macaques

    Directory of Open Access Journals (Sweden)

    Mannioui Abdelkrim


    Full Text Available Abstract Background Extensive studies of primary infection are crucial to our understanding of the course of HIV disease. In SIV-infected macaques, a model closely mimicking HIV pathogenesis, we used a combination of three markers -- viral RNA, 2LTR circles and viral DNA -- to evaluate viral replication and dissemination simultaneously in blood, secondary lymphoid tissues, and the gut during primary and chronic infections. Subsequent viral compartmentalization in the main target cells of the virus in peripheral blood during the chronic phase of infection was evaluated by cell sorting and viral quantification with the three markers studied. Results The evolutions of viral RNA, 2LTR circles and DNA levels were correlated in a given tissue during primary and early chronic infection. The decrease in plasma viral load principally reflects a large decrease in viral replication in gut-associated lymphoid tissue (GALT, with viral RNA and DNA levels remaining stable in the spleen and peripheral lymph nodes. Later, during chronic infection, a progressive depletion of central memory CD4+ T cells from the peripheral blood was observed, accompanied by high levels of viral replication in the cells of this subtype. The virus was also found to replicate at this point in the infection in naive CD4+ T cells. Viral RNA was frequently detected in monocytes, but no SIV replication appeared to occur in these cells, as no viral DNA or 2LTR circles were detected. Conclusion We demonstrated the persistence of viral replication and dissemination, mostly in secondary lymphoid tissues, during primary and early chronic infection. During chronic infection, the central memory CD4+ T cells were the major site of viral replication in peripheral blood, but viral replication also occurred in naive CD4+ T cells. The role of monocytes seemed to be limited to carrying the virus as a cargo because there was an observed lack of replication in these cells. These data may have important

  14. DNA replication stress and cancer chemotherapy. (United States)

    Kitao, Hiroyuki; Iimori, Makoto; Kataoka, Yuki; Wakasa, Takeshi; Tokunaga, Eriko; Saeki, Hiroshi; Oki, Eiji; Maehara, Yoshihiko


    DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  15. Towards scalable Byzantine fault-tolerant replication (United States)

    Zbierski, Maciej


    Byzantine fault-tolerant (BFT) replication is a powerful technique, enabling distributed systems to remain available and correct even in the presence of arbitrary faults. Unfortunately, existing BFT replication protocols are mostly load-unscalable, i.e. they fail to respond with adequate performance increase whenever new computational resources are introduced into the system. This article proposes a universal architecture facilitating the creation of load-scalable distributed services based on BFT replication. The suggested approach exploits parallel request processing to fully utilize the available resources, and uses a load balancer module to dynamically adapt to the properties of the observed client workload. The article additionally provides a discussion on selected deployment scenarios, and explains how the proposed architecture could be used to increase the dependability of contemporary large-scale distributed systems.

  16. The replication of expansive production knowledge

    DEFF Research Database (Denmark)

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


    Purpose – With the aim to support offshore production line replication, this paper specifically aims to explore the use of templates and principles to transfer expansive productive knowledge embedded in a production line and understand the contingencies that influence the mix of these approaches...... exploration, the small sample size is an obvious limitation for generalisation. Practical implications – A roadmap for knowledge transfer within the replication of a production line is suggested, which, together with four managerial suggestions, provides strong support and clear directions to managers....... Originality/value – Research in replication to date has mostly focused on templates and has mainly taken an organizational perspective. This paper shows its potential contribution on bridging the relevant theoretical gaps by (1) addressing the effects of principles; and (2) exploring how to use templates...

  17. The replication of expansive production knowledge

    DEFF Research Database (Denmark)

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


    Purpose – With the aim to support offshore production line replication, this paper specifically aims to explore the use of templates and principles to transfer expansive productive knowledge embedded in a production line and understand the contingencies that influence the mix of these approaches....... Design/methodology/approach – Two case studies are introduced. Empirical data were collected over a period of two years based on interviews and participating observations. Findings – The findings show that (1) knowledge transfer within the replication of a production line is a stepwise expansive process......; and (2) rather than being viewed as alternative approaches, templates and principles should be seen as complementary once the transfer motive moves beyond pure replication. Research limitations – The concepts introduced in this paper were derived from two Danish cases. While acceptable for theory...

  18. Evolution of Database Replication Technologies for WLCG

    CERN Document Server

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


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

  19. Chromatin structure and replication origins: determinants of chromosome replication and nuclear organization. (United States)

    Smith, Owen K; Aladjem, Mirit I


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

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

    NARCIS (Netherlands)

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


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

  1. Signal replication in a DNA nanostructure (United States)

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


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

  2. Chromatin challenges during DNA replication and repair

    DEFF Research Database (Denmark)

    Groth, Anja; Rocha, Walter; Verreault, Alain


    Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...... landscape may be stably maintained even in the face of dramatic changes in chromatin structure....

  3. Temporal organization of cellular self-replication (United States)

    Alexandrov, Victor; Pugatch, Rami

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

  4. Involvement of Autophagy in Coronavirus Replication

    Directory of Open Access Journals (Sweden)

    Paul Britton


    Full Text Available Coronaviruses are single stranded, positive sense RNA viruses, which induce the rearrangement of cellular membranes upon infection of a host cell. This provides the virus with a platform for the assembly of viral replication complexes, improving efficiency of RNA synthesis. The membranes observed in coronavirus infected cells include double membrane vesicles. By nature of their double membrane, these vesicles resemble cellular autophagosomes, generated during the cellular autophagy pathway. In addition, coronavirus infection has been demonstrated to induce autophagy. Here we review current knowledge of coronavirus induced membrane rearrangements and the involvement of autophagy or autophagy protein microtubule associated protein 1B light chain 3 (LC3 in coronavirus replication.

  5. Replication protocol analysis: a method for the study of real-world design thinking

    DEFF Research Database (Denmark)

    Galle, Per; Kovacs, L. B.


    ’ is refined into a method called ‘replication protocol analysis’ (RPA), and discussed from a methodological perspective of design research. It is argued that for the study of real-world design thinking this method offers distinct advantages over traditional ‘design protocol analysis’, which seeks to capture......Given the brief of an architectural competition on site planning, and the design awarded the first prize, the first author (trained as an architect but not a participant in the competition) produced a line of reasoning that might have led from brief to design. In the paper, such ‘design replication...... the designer’s authentic line of reasoning. To illustrate how RPA can be used, the site planning case is briefly presented, and part of the replicated line of reasoning analysed. One result of the analysis is a glimpse of a ‘logic of design’; another is an insight which sheds new light on Darke’s classical...

  6. Replication of HIV-1 in vivo and in vitro. (United States)

    Orenstein, Jan Marc


    formation of LE/MVB, the MVB was first identified as a site of HIV replication by macrophages many years ago, but the full implication of this observation was not appreciated at the time. As in many other areas of HIV research, what has been totally lacking is an in vivo confirmation of the in vitro phenomenon. Herein, the ultrastructure of HIV interaction with cells in vitro and in vivo is explored. It is shown that while HIV is regularly found in LE/MVB in vitro, it is infrequently the case in vivo. Therefore, the results challenge the "Trojan horse" concept.

  7. Gene- and protein-delivered zinc finger-staphylococcal nuclease hybrid for inhibition of DNA replication of human papillomavirus. (United States)

    Mino, Takashi; Mori, Tomoaki; Aoyama, Yasuhiro; Sera, Takashi


    Previously, we reported that artificial zinc-finger proteins (AZPs) inhibited virus DNA replication in planta and in mammalian cells by blocking binding of a viral replication protein to its replication origin. However, the replication mechanisms of viruses of interest need to be disentangled for the application. To develop more widely applicable methods for antiviral therapy, we explored the feasibility of inhibition of HPV-18 replication as a model system by cleaving its viral genome. To this end, we fused the staphylococcal nuclease cleaving DNA as a monomer to an AZP that binds to the viral genome. The resulting hybrid nuclease (designated AZP-SNase) cleaved its target DNA plasmid efficiently and sequence-specifically in vitro. Then, we confirmed that transfection with a plasmid expressing AZP-SNase inhibited HPV-18 DNA replication in transient replication assays using mammalian cells. Linker-mediated PCR analysis revealed that the AZP-SNase cleaved an HPV-18 ori plasmid around its binding site. Finally, we demonstrated that the protein-delivered AZP-SNase inhibited HPV-18 DNA replication as well and did not show any significant cytotoxicity. Thus, both gene- and protein-delivered hybrid nucleases efficiently inhibited HPV-18 DNA replication, leading to development of a more universal antiviral therapy for human DNA viruses.

  8. HIV-1 clade promoters strongly influence spatial and temporal dynamics of viral replication in vivo

    NARCIS (Netherlands)

    Centlivre, Mireille; Sommer, Peter; Michel, Marie; Ho Tsong Fang, Raphaël; Gofflo, Sandrine; Valladeau, Jenny; Schmitt, Nathalie; Thierry, Françoise; Hurtrel, Bruno; Wain-Hobson, Simon; Sala, Monica


    Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of

  9. BRPF3-HBO1 regulates replication origin activation and histone H3K14 acetylation

    DEFF Research Database (Denmark)

    Feng, Yunpeng; Vlassis, Arsenios; Roques, Céline


    that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45...

  10. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L


    We describe replication competent retroviruses capable of expressing heterologous genes during multiple rounds of infection. An internal ribosome entry site (IRES) from encephalomyocarditis virus was inserted in the U3 region of Akv- and SL3-3-murine leukemia viruses (MLV) to direct translation...

  11. Suppression of Coronavirus Replication by Cyclophilin Inhibitors

    Directory of Open Access Journals (Sweden)

    Takashi Sasaki


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

  12. Replication and analysis of Ebbinghaus' forgetting curve

    NARCIS (Netherlands)

    Murre, J.M.J.; Dros, J.


    We present a successful replication of Ebbinghaus’ classic forgetting curve from 1880 based on the method of savings. One subject spent 70 hours learning lists and relearning them after 20 min, 1 hour, 9 hours, 1 day, 2 days, or 31 days. The results are similar to Ebbinghaus' original data. We

  13. Optical replication techniques for image slicers

    Czech Academy of Sciences Publication Activity Database

    Schmoll, J.; Robertson, D.J.; Dubbeldam, C.M.; Bortoletto, F.; Pína, L.; Hudec, René; Prieto, E.; Norrie, C.; Ramsay- Howat, S.


    Roč. 50, 4-5 (2006), s. 263-266 ISSN 1387-6473 Institutional research plan: CEZ:AV0Z10030501 Keywords : smart focal planes * image slicers * replication Subject RIV: BN - Astronomy, Celestial Mechanics, Astrophysics Impact factor: 1.914, year: 2006

  14. Surface microstructure replication in injection molding

    DEFF Research Database (Denmark)

    Theilade, Uffe Arlø; Hansen, Hans Nørgaard


    molding of surface microstructures. The fundamental problem of surface microstructure replication has been studied. The research is based on specific microstructures as found in lab-on-a-chip products and on rough surfaces generated from EDM (electro discharge machining) mold cavities. Emphasis is put...

  15. Conditionally replicating HIV and SIV variants

    NARCIS (Netherlands)

    Das, Atze T.; Berkhout, Ben


    Conditionally replicating human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) variants that can be switched on and off at will are attractive tools for HIV and SIV research. We constructed HIV and SIV variants in which the natural transcription control mechanism was replaced

  16. Inhibition of DNA replication by ultraviolet light

    Energy Technology Data Exchange (ETDEWEB)

    Edenberg, H.J.


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

  17. Origins of DNA Replication and Amplification in the Breast Cancer Genome (United States)


    somatic mutations or other genomic changes. We wish to ask if a genomic change (genetic or epigenetic) might produce novel binding site(s) for the...bacteriophages, animal viruses , and unicellular eukaryotes studied thus far have high AT content, presumably to facilitate unwinding of the DNA. Task (3...have the GC skew, which is more consistent with the base composition of replication origins from bacteria, viruses and yeast.  Performed

  18. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. (United States)

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


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

  19. Bacteriophage SPP1 DNA replication strategies promote viral and disable host replication in vitro. (United States)

    Seco, Elena M; Zinder, John C; Manhart, Carol M; Lo Piano, Ambra; McHenry, Charles S; Ayora, Silvia


    Complex viruses that encode their own initiation proteins and subvert the host's elongation apparatus have provided valuable insights into DNA replication. Using purified bacteriophage SPP1 and Bacillus subtilis proteins, we have reconstituted a rolling circle replication system that recapitulates genetically defined protein requirements. Eleven proteins are required: phage-encoded helicase (G40P), helicase loader (G39P), origin binding protein (G38P) and G36P single-stranded DNA-binding protein (SSB); and host-encoded PolC and DnaE polymerases, processivity factor (β(2)), clamp loader (τ-δ-δ') and primase (DnaG). This study revealed a new role for the SPP1 origin binding protein. In the presence of SSB, it is required for initiation on replication forks that lack origin sequences, mimicking the activity of the PriA replication restart protein in bacteria. The SPP1 replisome is supported by both host and viral SSBs, but phage SSB is unable to support B. subtilis replication, likely owing to its inability to stimulate the PolC holoenzyme in the B. subtilis context. Moreover, phage SSB inhibits host replication, defining a new mechanism by which bacterial replication could be regulated by a viral factor.

  20. Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

    Directory of Open Access Journals (Sweden)

    Duensing Stefan


    Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

  1. Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

    Energy Technology Data Exchange (ETDEWEB)

    Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)


    Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

  2. Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA. (United States)

    Gros, Julien; Kumar, Charanya; Lynch, Gerard; Yadav, Tejas; Whitehouse, Iestyn; Remus, Dirk


    Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. Similarly, in yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a redistribution of Mcm2-7 complexes along the chromosomes, resulting in a corresponding shift in DNA replication initiation sites. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Cui, Hongguang; Wang, Aiming


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

  4. Differential Tus-Ter binding and lock formation: implications for DNA replication termination in Escherichia coli. (United States)

    Moreau, Morgane J J; Schaeffer, Patrick M


    In E. coli, DNA replication termination occurs at Ter sites and is mediated by Tus. Two clusters of five Ter sites are located on each side of the terminus region and constrain replication forks in a polar manner. The polarity is due to the formation of the Tus-Ter-lock intermediate. Recently, it has been shown that DnaB helicase which unwinds DNA at the replication fork is preferentially stopped at the non-permissive face of a Tus-Ter complex without formation of the Tus-Ter-lock and that fork pausing efficiency is sequence dependent, raising two essential questions: Does the affinity of Tus for the different Ter sites correlate with fork pausing efficiency? Is formation of the Tus-Ter-lock the key factor in fork pausing? The combined use of surface plasmon resonance and GFP-Basta showed that Tus binds strongly to TerA-E and G, moderately to TerH-J and weakly to TerF. Out of these ten Ter sites only two, TerF and H, were not able to form significant Tus-Ter-locks. Finally, Tus's resistance to dissociation from Ter sites and the strength of the Tus-Ter-locks correlate with the differences in fork pausing efficiency observed for the different Ter sites by Duggin and Bell (2009).

  5. Replisome speed determines the efficiency of the Tus−Ter replication termination barrier

    KAUST Repository

    Elshenawy, Mohamed


    In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites. Escherichia coli sets a \\'replication fork trap\\' that allows the first arriving fork to enter but not to leave the terminus region. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction. However, the efficiency of fork blockage by Tus-Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus-Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes. ©2015 Macmillan Publishers Limited. All rights reserved.

  6. Nuclear safety aspects of exported replicate nuclear power plants and associated problems

    International Nuclear Information System (INIS)

    Kern, H.G.


    The standardization of the export nuclear power plant is being pursued with the concept of replication. This concept entails using another exported Nuclear Power Plant (NPP) as the base design and adapting it to a new site. The general ground rule applied to this concept is upgrading the design where necessary and duplicating the design where it is superior. Such continuous improvement will result in a standard export NPP that incorporates design features which will make it essentially acceptable for any suitable site. The advantages of replication are, therefore, boundless. However, the replication mode requires superior design control by the engineer to assure that only improvements alter the base design. With this concept, the replicating engineer is essentially assigned the responsiblity of safeguarding the standard export plant design. He is delegated the task of filtering the design such that only the conservative aspects prevail. Tight control of design changes via properly administered procedures is necessary to assure that no unforeseen compromises are made in designs which have already achieved optimization. Techniques to accomplish successful replication include, among others, the use of PCNs, system cognizant engineers, design verfication review, and the participation of all engineering disciplines in the development of the project schedule. (author)

  7. Operating systems: Internals and design principles, 5th ed. William Stallings : Prentice Hall, 2005. ISBN: 0-13-147954-7


    Molinari, Lía Hebe


    As a professor of the Operating Systems subject, I can say that for many years the book “Operating Systems Concepts” by Avi Silberschatz et al. was considered as the unquestionable number one in the subject’s bibliography. The book we are talking about now, the new edition of “Operating Systems” by William Stallings, is thought to be the possible replacement of the first one.

  8. The elastic transfer model of angular rate modulation in F1-ATPase stalling and controlled rotation experiments (United States)

    Volkán-Kacsó, S.


    The recent experimental, theoretical and computational advances in the field of F1-ATPase single-molecule microscopy are briefly surveyed. The role of theory is revealed in the statistical analysis, interpretation and prediction of single-molecule experimental trajectories, and in linking them with atomistic simulations. In particular, a theoretical model of elastically coupled molecular group transfer is reviewed and a detailed method for its application in stalling and controlled rotation experiments is provided. It is shown how the model can predict, using previous experiments, the rates of ligand binding/release processes (steps) and their exponential dependence on rotor angle in these experiments. The concept of Brønsted slopes is reviewed in the context of the single-molecule experiments, and the rate versus rotor angle relations are explained using the elastic model. These experimental data are treated in terms of the effect of thermodynamic driving forces on the rates assuming that the rotor shaft is elastically coupled to stator ring subunits in which the steps occur. In the application of the group transfer model on an extended angular range processes leading up to the transfer are discussed. Implications for large-scale atomistic simulation are suggested for the treatment of torque-generating steps.

  9. A study of the drooped leading edge airfoil. [on wind tunnel models to reduce spin entry after stall (United States)

    Anderson, J. D., Jr.; Barlow, J. B.


    Wind tunnel tests were conducted to examine various aspects of the drooped-leading edge airfoil which reduces the tendency for an airplane to enter a spin after stall occurs. Three baseline models were used for tests of two dimensional models: NACA 0015, 0014.6, and 0014.2. The 14.6% and 14.2% models were derived from NACA 0015 sections by increasing the chord and matching the profiles aft section. Force, balance data (lift, drag, pitching moment) were obtained for each model at a free-steam Reynold's number of 2.66 x 10 to the 6th power/m. In addition, oil flow visualization tests were performed at various angles of attack. An existing NACA 64 sub 1 A211 airfoil was used in a second series of tests. The leading edge flap was segmented in three parts which allowed various baseline/drooped leading edge configurations to be tested. Force balance and flow visualization tests were completer at chord Renolds numbers of 0.44 x 10 to the 6th power, 1.4 x 10 to the 6th power, and 2.11 x 10 to the 6th power. Test results are included.

  10. Using Nature-Based Rehabilitation to Restart a Stalled Process of Rehabilitation in Individuals with Stress-Related Mental Illness

    Directory of Open Access Journals (Sweden)

    Eva Sahlin


    Full Text Available After a period of decrease, sick leave in Sweden due to psychiatric diagnoses is on the increase. The lack of established rehabilitation programmes for patients with stress-related mental disorders (SRMD has opened up for the use of garden/nature in a multimodal rehabilitation context (Nature-Based Rehabilitation, NBR. Region Västra Götaland (VGR started an NBR to offer additional rehabilitation for its employees on long-term sick leave due to SRMD, where initial care had not been sufficient. The aim was to explore whether the mental health and well-being of NBR participants had improved at the end of the NBR and at three follow-ups, and to explore the development of sick leave and health care utilization according to the NBR model (n = 57 and an occupational health service (OHS model (n = 45. Self-assessment instruments for measuring burnout, depression, anxiety and wellbeing, and data from regional and national registers were used. Results showed decreased scores on burnout, depression and anxiety, and increased well-being scores and significantly reduced health care utilization in the NBR group. A large movement from ordinary sickness benefit to rehabilitation benefit was observed, which was not observed in the OHS group. The two groups were in different rehabilitation phases, which limited comparisons. The results point to beneficial effects of using NBR for this patient group and for enhancing a stalled rehabilitation process.

  11. Regulatory cross-talk links Vibrio cholerae chromosome II replication and segregation.

    Directory of Open Access Journals (Sweden)

    Yoshiharu Yamaichi


    Full Text Available There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products that surround the origin of replication (oriCII of Vibrio cholerae chromosome II (chrII are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for chrII partitioning, is located immediately downstream of rctA. Here, we explored how rctA exerts negative control over chrII replication. Our observations suggest that RctB has at least two DNA binding domains--one for binding to oriCII and initiating replication and the other for binding to rctA and thereby inhibiting RctB's ability to initiate replication. Notably, the inhibitory effect of rctA could be alleviated by binding of ParB2 to a centromere-like parS site within rctA. Furthermore, by binding to rctA, ParB2 and RctB inversely regulate expression of the parAB2 genes. Together, our findings suggest that fluctuations in binding of the partitioning protein ParB2 and the chrII initiator RctB to rctA underlie a regulatory network controlling both oriCII firing and the production of the essential chrII partitioning proteins. Thus, by binding both RctB and ParB2, rctA serves as a nexus for regulatory cross-talk coordinating chrII replication and segregation.

  12. Impaired replication stress response in cells from immunodeficiency patients carrying Cernunnos/XLF mutations.

    Directory of Open Access Journals (Sweden)

    Michal Schwartz

    Full Text Available Non-Homologous End Joining (NHEJ is one of the two major pathways of DNA Double Strand Breaks (DSBs repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(DJ recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(DJ recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis.

  13. FANCM, BRCA1, and BLM cooperatively resolve the replication stress at the ALT telomeres. (United States)

    Pan, Xiaolei; Drosopoulos, William C; Sethi, Louisa; Madireddy, Advaitha; Schildkraut, Carl L; Zhang, Dong


    In the mammalian genome, certain genomic loci/regions pose greater challenges to the DNA replication machinery (i.e., the replisome) than others. Such known genomic loci/regions include centromeres, common fragile sites, subtelomeres, and telomeres. However, the detailed mechanism of how mammalian cells cope with the replication stress at these loci/regions is largely unknown. Here we show that depletion of FANCM, or of one of its obligatory binding partners, FAAP24, MHF1, and MHF2, induces replication stress primarily at the telomeres of cells that use the alternative lengthening of telomeres (ALT) pathway as their telomere maintenance mechanism. Using the telomere-specific single-molecule analysis of replicated DNA technique, we found that depletion of FANCM dramatically reduces the replication efficiency at ALT telomeres. We further show that FANCM, BRCA1, and BLM are actively recruited to the ALT telomeres that are experiencing replication stress and that the recruitment of BRCA1 and BLM to these damaged telomeres is interdependent and is regulated by both ATR and Chk1. Mechanistically, we demonstrated that, in FANCM-depleted ALT cells, BRCA1 and BLM help to resolve the telomeric replication stress by stimulating DNA end resection and homologous recombination (HR). Consistent with their roles in resolving the replication stress induced by FANCM deficiency, simultaneous depletion of BLM and FANCM, or of BRCA1 and FANCM, leads to increased micronuclei formation and synthetic lethality in ALT cells. We propose that these synthetic lethal interactions can be explored for targeting the ALT cancers.

  14. Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication (United States)

    Friebe, Peter; Lohmann, Volker; Krieger, Nicole; Bartenschlager, Ralf


    Sequences in the 5′ and 3′ termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5′ end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5′ NTR required for RNA replication. We show that deletions introduced into the 5′ terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5′ NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5′ NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5′ NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus. PMID:11711595

  15. Investigating dynamic stall, 3-D and rotational effects on wind turbine blades by means of an unsteady quasi-3D Navier-Stokes solver

    Energy Technology Data Exchange (ETDEWEB)

    Chaviaropoulos, P.K. [CRES-Center for Renewable Energy Sources, Pikermi Attiki (Greece)


    The blade element codes provide surprisingly accurate predictions of the aerodynamic loads provided that they are `fed` with proper lift and drag - incidence curves for the profiles mounted on the rotor blades. The evident question is how one can obtain such data. It is common experience that the use of the mostly available steady two-dimensional profile data may lead to serious discrepancies between measured and simulated loads. Although several correction techniques have been proposed as a remedy during the last years, from simplified dynamic stall models suitably tuned for wind turbines to 3-D correction schemes for profile data, the problem is by no means over-passed. Especially for the three-dimensional effects it seems that part of the difficulty is due to our limited understanding of the physical mechanism which is responsible for the extra loading of the inner part of the blades. Recognizing the importance of the above aspects two relevant Joule projects have been launched, the concluded `Dynamic Stall and 3-D Effects` JOU2-CT93-0345 and the ongoing `VISCWIND` JOR3-CT95-0007 project. Part of the activities in the first and all the activities in the second project are devoted to the identification and quantification of the dynamic stall and three-dimensional effects experienced by the wind turbine blades using Navier-Stokes computations. The contribution of CRES in these two projects is briefly presented in this paper. (EG)

  16. Mechanisms Governing DDK Regulation of the Initiation of DNA Replication

    Directory of Open Access Journals (Sweden)



    Full Text Available The budding yeast Dbf4-dependent kinase (DDK complex—comprised of cell division cycle (Cdc7 kinase and its regulatory subunit dumbbell former 4 (Dbf4—is required to trigger the initiation of DNA replication through the phosphorylation of multiple minichromosome maintenance complex subunits 2-7 (Mcm2-7. DDK is also a target of the radiation sensitive 53 (Rad53 checkpoint kinase in response to replication stress. Numerous investigations have determined mechanistic details, including the regions of Mcm2, Mcm4, and Mcm6 phosphorylated by DDK, and a number of DDK docking sites. Similarly, the way in which the Rad53 forkhead-associated 1 (FHA1 domain binds to DDK—involving both canonical and non-canonical interactions—has been elucidated. Recent work has revealed mutual promotion of DDK and synthetic lethal with dpb11-1 3 (Sld3 roles. While DDK phosphorylation of Mcm2-7 subunits facilitates their interaction with Sld3 at origins, Sld3 in turn stimulates DDK phosphorylation of Mcm2. Details of a mutually antagonistic relationship between DDK and Rap1-interacting factor 1 (Rif1 have also recently come to light. While Rif1 is able to reverse DDK-mediated Mcm2-7 complex phosphorylation by targeting the protein phosphatase glycogen 7 (Glc7 to origins, there is evidence to suggest that DDK can counteract this activity by binding to and phosphorylating Rif1.

  17. DNA transformations of Candida tropicalis with replicating and integrative vectors. (United States)

    Sanglard, D; Fiechter, A


    The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

  18. Investigation of Break-Induced Replication in Yeast. (United States)

    Elango, Rajula; Kockler, Zachary; Liu, Liping; Malkova, Anna


    Break-induced replication (BIR) is an important mechanism aimed to repair one-ended double-strand DNA breaks. BIR is initiated by invasion of a broken DNA end into a homologous template followed by DNA synthesis that can proceed for hundreds of kilobases to the end of the chromosome. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. The unusual mode of DNA synthesis during BIR leads to an increased level of genetic instabilities including increased mutagenesis and chromosomal rearrangements. Here, we describe our experimental system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break and where the repair involves two copies of chromosome III. This system allows investigation of BIR using genetic and molecular biology approaches, and can be used for characterization of the BIR mechanism, roles of individual proteins in BIR, and for the analysis of genetic instabilities associated with BIR. © 2018 Elsevier Inc. All rights reserved.

  19. An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies. (United States)

    Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald


    Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and

  20. Caracterização do microambiente em secção transversal de um galpão do tipo "free-stall" orientado na direção norte-sul Environment characterization in transversal direction in a free-stall housing oriented to north-south direction

    Directory of Open Access Journals (Sweden)

    Alessandro T. Campos


    Full Text Available O trabalho teve o objetivo de caracterizar o microambiente climático das baias de um galpão de confinamento para gado leiteiro, modelo "free-stall", no sentido transversal da instalação. O "free-stall" é orientado na direção norte-sul, localizado em Marechal Cândido Rondon, região Oeste do Paraná, com capacidade para abrigar 40 vacas em lactação (40 baias. Visando à determinação do Índice de Temperatura do Globo e Umidade (ITGU e da Carga Térmica de Radiação (CTR, foram instalados quatro globos negros, no centro de baias dispostas no sent ido transversal, dispondo dois globos no lado oeste e dois globos no lado leste (separados pelo corredor de alimentação. Lateralmente ao galpão, no lado leste, havia vegetação a 4 m da instalação, que promovia sombreamento nas primeiras horas do dia. Pode-se concluir que, nas primeiras horas do dia, não há diferença significativa entre os valores de ITGU e CTR encontrados, indicando que o sombreamento, devido à vegetação, foi capaz de amenizar os efeitos da radiação solar. Às 17 h 30 min, verificou-se que há ocorrência de maiores valores, tanto de ITGU quanto de CTR, nas baias localizadas no lado oeste do "free-stall".This work aimed to characterize the microclimatic conditions of the stalls of a free-stall model confinement building for dairy cattle in the transversal direction of the installation. Free-stall building was oriented to north-south direction, located in Marechal Cândido Rondon, west of Paraná State -Brazil, with a capacity of 40 dairy cows (40 stalls. In order to determine the Black-Globe-Humidity Index (BGHI and Radiant Heat Load (RHL, four black globes were installed in the center of stalls disposed in the transversal direction, disposing two globes in the west side and two globes in the east side (separated by the feed alley. At the building sidelong, east side, there was a four meter distant vegetation, that promoted shading in the first hours of the day

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

    Directory of Open Access Journals (Sweden)

    Michelle Hawkins


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

  2. DNA Replication in Engineered Escherichia coli Genomes with Extra Replication Origins. (United States)

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


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

  3. Purification of highly active alphavirus replication complexes demonstrates altered fractionation of multiple cellular membranes. (United States)

    Pietilä, Maija K; van Hemert, Martijn J; Ahola, Tero


    Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER) and late endosome markers enriched in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the co-fractionation of the RC-carrying plasma membrane and ER suggests that SFV may recruit ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs. IMPORTANCE Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories but little is known about the arrangement of viral replicase proteins and the

  4. DNA replication and repair in Tilapia cells

    International Nuclear Information System (INIS)

    Yew, F.H.; Chang, L.M.


    The effect of ultraviolet radiation on a cell line established from the warm water fish Tilapia has been assessed by measuring the rate of DNA synthesis, excision repair, post-replication repair and cell survival. The cells tolerate ultraviolet radiation better than mammalian cells with respect to DNA synthesis, post-replication repair and cell survival. They are also efficient in excision repair, which in other fish cell lines has been found to be at a low level or absent. Their response to the inhibitors hydroxyurea and 1-β-D-arabinofuranosylcytosine is less sensitive than that of other cell lines, yet the cells seem to have very small pools of DNA precursor. (author)

  5. Perdas econômicas ocasionadas pelas enfermidades podais em vacas leiteiras confinadas em sistema free stall Economic losses caused by sequels of lameness in free-stall-housed dairy cows

    Directory of Open Access Journals (Sweden)

    R.C. Souza


    Full Text Available Avaliaram-se 55 casos clínicos de manqueira em um rebanho de 100 vacas em lactação confinadas em sistema de free stall, durante um ano. As afecções mais observadas foram abscessos de sola e talão, úlcera de sola e dermatite digital que representaram 87,3% (48/55 das ocorrências. O tratamento empregado mostrou-se satisfatório com recuperação de todos os animais tratados com 24,5 dias, em média, sem ocorrência de descarte. O custo com tratamento e redução na produção de leite foi de US$95.80/vaca, o que representou US$52.69 por vaca alojada/ano. Quando se computaram as perdas reprodutivas e com mastite, obtiveram-se US$227.94 adicionais em vaca com problema de manqueira. O custo adicional anual total no rebanho decorrente de seqüelas de manqueira foi de US$12,536.70, que representou US$125.36 por vaca alojada/ano. O período de serviço e o número de serviços por concepção em vacas com problemas de manqueira e normais foram 266 e 200,5 dias e 4,3 e 3,3 serviços, respectivamente. As incidências de mastite e metrite na mesma ordem de citação anterior foram 60% e 25% e 29% e 12,5%.Fifty-five clinical cases of lameness were evaluated in 100 lactating cows housed in a free-stall system during one year. The most observed affections were sole and heel abscesses, sole ulcers and digital dermatitis that accounted for 87.3% (48/55 of the occurrences. The treated cows showed satisfactory recovery (24.5 average days without culling. The total individual cost including treatment and reduction of milk production was US$95.80/cow or US$52.69 per housed cow/year. When reproductive losses and mastitis were considered an additional cost of US$227.94 per lameness cow was estimated. The total additional annual cost from sequels of lameness was US$12.536.70 or US$152.36 per housed cow/year. The number of days open and the number of services per conception in lameness and normal cows were, respectively, 266 and 200.5 days and 4.3 and 3

  6. Business Model and Replication Study of BIG HIT

    DEFF Research Database (Denmark)

    Zhao, Guangling; Ravn Nielsen, Eva

    . The BIG HIT project is creating a replicable hydrogen territory in Orkney (An island archipelago six miles offshore from North of Mainland Scotland.) by implementing a fully integrated model of hydrogen production, storage, distribution of the hydrogen across Orkney and utilised for mobility, heat...... and power. The BIG HIT project will use otherwise curtailed electricity from one wind turbine on Shapinsay and one wind turbine and a tidal test sites on Eday, and use 1.5 MW of Polymer Electrolyte Membrane (PEM) electrolyser to convert it into ~50 t pa of hydrogen. This will be used to provide heat...... and infrastructure investment and operation cost of electricity and water consumption. The functional unit is 1 kg hydrogen produced and consumed. The data collected from the project patterns and suppliers. The current analysis is based on the estimation of hydrogen production and consumption on both Shapinsay...

  7. Replicable effects of primes on human behavior. (United States)

    Payne, B Keith; Brown-Iannuzzi, Jazmin L; Loersch, Chris


    [Correction Notice: An Erratum for this article was reported online in Journal of Experimental Psychology: General on Oct 31 2016 (see record 2016-52334-001). ] The effect of primes (i.e., incidental cues) on human behavior has become controversial. Early studies reported counterintuitive findings, suggesting that primes can shape a wide range of human behaviors. Recently, several studies failed to replicate some earlier priming results, raising doubts about the reliability of those effects. We present a within-subjects procedure for priming behavior, in which participants decide whether to bet or pass on each trial of a gambling game. We report 6 replications (N = 988) showing that primes consistently affected gambling decisions when the decision was uncertain. Decisions were influenced by primes presented visibly, with a warning to ignore the primes (Experiments 1 through 3) and with subliminally presented masked primes (Experiment 4). Using a process dissociation procedure, we found evidence that primes influenced responses through both automatic and controlled processes (Experiments 5 and 6). Results provide evidence that primes can reliably affect behavior, under at least some conditions, without intention. The findings suggest that the psychological question of whether behavior priming effects are real should be separated from methodological issues affecting how easily particular experimental designs will replicate. PsycINFO Database Record (c) 2016 APA, all rights reserved

  8. Ultrastructural Characterization of Zika Virus Replication Factories. (United States)

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


    A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Molecular Biology of Rotavirus Entry and Replication (United States)

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


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

  10. The molecular biology of Bluetongue virus replication. (United States)

    Patel, Avnish; Roy, Polly


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

  11. Connecting the dots of the bacterial cell cycle: Coordinating chromosome replication and segregation with cell division. (United States)

    Hajduk, Isabella V; Rodrigues, Christopher D A; Harry, Elizabeth J


    Proper division site selection is crucial for the survival of all organisms. What still eludes us is how bacteria position their division site with high precision, and in tight coordination with chromosome replication and segregation. Until recently, the general belief, at least in the model organisms Bacillus subtilis and Escherichia coli, was that spatial regulation of division comes about by the combined negative regulatory mechanisms of the Min system and nucleoid occlusion. However, as we review here, these two systems cannot be solely responsible for division site selection and we highlight additional regulatory mechanisms that are at play. In this review, we put forward evidence of how chromosome replication and segregation may have direct links with cell division in these bacteria and the benefit of recent advances in chromosome conformation capture techniques in providing important information about how these three processes mechanistically work together to achieve accurate generation of progenitor cells. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  12. Pressure tube replication techniques using the advanced NDE system

    International Nuclear Information System (INIS)

    Isherwood, A.; Jarron, D.; Travers, J.; Hanley, K.


    rotary position of the flaw, as determined by ultrasonics, is entered and the tool is positioned in the channel and locked in place. A twenty minute auto-sequence is then initiated which injects material into the flaw and allows it to cure. Once complete, the delivery machine comes off channel and the tool is pushed out of the machine onto a loading trough. The operators remove the replica and recharge the tool for the next set of indications. Once the removed replica has been examined by trained technicians, it is moved to an on-site laser profilometry device to convert the positive of the flaw to a 3D computer image. This image is then curve fitted to determine the smallest radius of the flaw. The combination of in-vault recharging and a two plate tool has reduced critical path times for this phase of the outages. The ANDE Replication System has achieved rates of 10 replicas in just 42 hours. The ANDE Replication System has been successfully used at four different CANDU stations on over 30 flaws. It has been used with both the UDM and the ADM (Advanced Delivery Machine). This paper will present details of the new tool, control system, and field execution of the ANDE Replication System at Ontario Power Generation reactors. (author)

  13. DNA replication is the target for the antibacterial effects of nonsteroidal anti-inflammatory drugs. (United States)

    Yin, Zhou; Wang, Yao; Whittell, Louise R; Jergic, Slobodan; Liu, Michael; Harry, Elizabeth; Dixon, Nicholas E; Kelso, Michael J; Beck, Jennifer L; Oakley, Aaron J


    Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding clamp at a common binding site required for partner binding. Inhibition of interaction of the clamp loader and/or the replicative polymerase α subunit with the sliding clamp is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding clamp. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Systematic determination of replication activity type highlights interconnections between replication, chromatin structure and nuclear localization. (United States)

    Farkash-Amar, Shlomit; David, Yaara; Polten, Andreas; Hezroni, Hadas; Eldar, Yonina C; Meshorer, Eran; Yakhini, Zohar; Simon, Itamar


    DNA replication is a highly regulated process, with each genomic locus replicating at a distinct time of replication (ToR). Advances in ToR measurement technology enabled several genome-wide profiling studies that revealed tight associations between ToR and general genomic features and a remarkable ToR conservation in mammals. Genome wide studies further showed that at the hundreds kb-to-megabase scale the genome can be divided into constant ToR regions (CTRs) in which the replication process propagates at a faster pace due to the activation of multiple origins and temporal transition regions (TTRs) in which the replication process propagates at a slower pace. We developed a computational tool that assigns a ToR to every measured locus and determines its replication activity type (CTR versus TTR). Our algorithm, ARTO (Analysis of Replication Timing and Organization), uses signal processing methods to fit a constant piece-wise linear curve to the measured raw data. We tested our algorithm and provide performance and usability results. A Matlab implementation of ARTO is available at Applying our algorithm to ToR data measured in multiple mouse and human samples allowed precise genome-wide ToR determination and replication activity type characterization. Analysis of the results highlighted the plasticity of the replication program. For example, we observed significant ToR differences in 10-25% of the genome when comparing different tissue types. Our analyses also provide evidence for activity type differences in up to 30% of the probes. Integration of the ToR data with multiple aspects of chromosome organization characteristics suggests that ToR plays a role in shaping the regional chromatin structure. Namely, repressive chromatin marks, are associated with late ToR both in TTRs and CTRs. Finally, characterization of the differences between TTRs and CTRs, with matching ToR, revealed that TTRs are associated with

  15. Systematic determination of replication activity type highlights interconnections between replication, chromatin structure and nuclear localization.

    Directory of Open Access Journals (Sweden)

    Shlomit Farkash-Amar

    Full Text Available DNA replication is a highly regulated process, with each genomic locus replicating at a distinct time of replication (ToR. Advances in ToR measurement technology enabled several genome-wide profiling studies that revealed tight associations between ToR and general genomic features and a remarkable ToR conservation in mammals. Genome wide studies further showed that at the hundreds kb-to-megabase scale the genome can be divided into constant ToR regions (CTRs in which the replication process propagates at a faster pace due to the activation of multiple origins and temporal transition regions (TTRs in which the replication process propagates at a slower pace. We developed a computational tool that assigns a ToR to every measured locus and determines its replication activity type (CTR versus TTR. Our algorithm, ARTO (Analysis of Replication Timing and Organization, uses signal processing methods to fit a constant piece-wise linear curve to the measured raw data. We tested our algorithm and provide performance and usability results. A Matlab implementation of ARTO is available at Applying our algorithm to ToR data measured in multiple mouse and human samples allowed precise genome-wide ToR determination and replication activity type characterization. Analysis of the results highlighted the plasticity of the replication program. For example, we observed significant ToR differences in 10-25% of the genome when comparing different tissue types. Our analyses also provide evidence for activity type differences in up to 30% of the probes. Integration of the ToR data with multiple aspects of chromosome organization characteristics suggests that ToR plays a role in shaping the regional chromatin structure. Namely, repressive chromatin marks, are associated with late ToR both in TTRs and CTRs. Finally, characterization of the differences between TTRs and CTRs, with matching ToR, revealed that TTRs are

  16. Replication stress, a source of epigenetic aberrations in cancer?

    DEFF Research Database (Denmark)

    Jasencakova, Zusana; Groth, Anja


    . Chromatin organization is transiently disrupted during DNA replication and maintenance of epigenetic information thus relies on faithful restoration of chromatin on the new daughter strands. Acute replication stress challenges proper chromatin restoration by deregulating histone H3 lysine 9 mono...

  17. The progression of replication forks at natural replication barriers in live bacteria. (United States)

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


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

  18. Addressing the "Replication Crisis": Using Original Studies to Design Replication Studies with Appropriate Statistical Power. (United States)

    Anderson, Samantha F; Maxwell, Scott E


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

  19. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

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    Kèvin Knoops


    Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

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

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    Nunya Chotiwan


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