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Sample records for sars-coronavirus mrna cap

  1. In vitro reconstitution of SARS-coronavirus mRNA cap methylation.

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    Mickaël Bouvet

    2010-04-01

    Full Text Available SARS-coronavirus (SARS-CoV genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16 constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet-dependent (guanine-N7-methyltransferase (N7-MTase activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 (7MeGpppA-RNAs. The latter are then selectively 2'O-methylated by the 2'O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 (7MeGpppA(2'OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC(50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design.

  2. In vitro reconstitution of SARS-coronavirus mRNA cap methylation.

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    Bouvet, Mickaël; Debarnot, Claire; Imbert, Isabelle; Selisko, Barbara; Snijder, Eric J; Canard, Bruno; Decroly, Etienne

    2010-04-22

    SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 (7Me)GpppA-RNAs. The latter are then selectively 2'O-methylated by the 2'O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 (7Me)GpppA(2'OMe)-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC(50) values in the micromolar range, providing a validated basis for anti-coronavirus drug design.

  3. Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus.

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    Sevajol, Marion; Subissi, Lorenzo; Decroly, Etienne; Canard, Bruno; Imbert, Isabelle

    2014-12-19

    The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3'-5' exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Crystallization and diffraction analysis of the SARS coronavirus nsp10–nsp16 complex

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    Debarnot, Claire; Imbert, Isabelle; Ferron, François; Gluais, Laure; Varlet, Isabelle; Papageorgiou, Nicolas; Bouvet, Mickaël; Lescar, Julien; Decroly, Etienne; Canard, Bruno

    2011-01-01

    To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1–16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2′-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress. PMID:21393853

  5. Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex.

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    Debarnot, Claire; Imbert, Isabelle; Ferron, François; Gluais, Laure; Varlet, Isabelle; Papageorgiou, Nicolas; Bouvet, Mickaël; Lescar, Julien; Decroly, Etienne; Canard, Bruno

    2011-03-01

    To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress.

  6. Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex

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    Decroly, Etienne; Debarnot, Claire; Ferron, François; Bouvet, Mickael; Coutard, Bruno; Imbert, Isabelle; Gluais, Laure; Papageorgiou, Nicolas; Sharff, Andrew; Bricogne, Gérard; Ortiz-Lombardia, Miguel; Lescar, Julien; Canard, Bruno

    2011-01-01

    Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses. PMID:21637813

  7. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

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    Etienne Decroly

    2011-05-01

    Full Text Available Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.

  8. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

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    Decroly, Etienne; Debarnot, Claire; Ferron, François; Bouvet, Mickael; Coutard, Bruno; Imbert, Isabelle; Gluais, Laure; Papageorgiou, Nicolas; Sharff, Andrew; Bricogne, Gérard; Ortiz-Lombardia, Miguel; Lescar, Julien; Canard, Bruno

    2011-05-01

    Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.

  9. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

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    ter Meulen, Jan; Bakker, Alexander B. H.; van den Brink, Edward N.; Weverling, Gerrit J.; Martina, Byron E. E.; Haagmans, Bart L.; Kuiken, Thijs; de Kruif, John; Preiser, Wolfgang; Spaan, Willy; Gelderblom, Hans R.; Goudsmit, Jaap; Osterhaus, Albert D. M. E.

    2004-01-01

    SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal

  10. VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.

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    Yu, Xiao; Chen, Shuliang; Hou, Panpan; Wang, Min; Chen, Yu; Guo, Deyin

    2015-04-03

    Eukaryotic cellular and most viral RNAs carry a 5'-terminal cap structure, a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2'-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.

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    Cheng Huang

    2011-12-01

    Full Text Available SARS coronavirus (SCoV nonstructural protein (nsp 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

  12. Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.

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    Yu Chen

    2011-10-01

    Full Text Available The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV encodes two S-adenosyl-L-methionine (SAM-dependent methyltransferases (MTase which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.

  13. Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.

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    Chen, Yu; Su, Ceyang; Ke, Min; Jin, Xu; Xu, Lirong; Zhang, Zhou; Wu, Andong; Sun, Ying; Yang, Zhouning; Tien, Po; Ahola, Tero; Liang, Yi; Liu, Xinqi; Guo, Deyin

    2011-10-01

    The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.

  14. A simple and rapid approach for screening of SARS-coronavirus genotypes: an evaluation study

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    Jin Yongjie

    2005-10-01

    Full Text Available Abstract Background The Severe Acute Respiratory Syndrome (SARS was a newly emerged infectious disease which caused a global epidemic in 2002–2003. Sequence analysis of SARS-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. This information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. However, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. We present here a simple and rapid assay for the screening of SARS-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination. Methods Thirty SARS patients were recruited. Allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (TaqMan. Genotyping of the SARS-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing. Results Genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. All of the 30 SARS-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. Seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort. Conclusion We have developed a simple and accurate method for the characterization and screening of SARS-coronavirus genotypes. It is a promising tool for the study of epidemiological relationships between documented cases during an outbreak.

  15. Understanding the T cell immune response in SARS coronavirus infection.

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    Janice Oh, Hsueh-Ling; Ken-En Gan, Samuel; Bertoletti, Antonio; Tan, Yee-Joo

    2012-09-01

    The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development.

  16. Understanding the T cell immune response in SARS coronavirus infection

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    Janice Oh, Hsueh-Ling; Ken-En Gan, Samuel; Bertoletti, Antonio; Tan, Yee-Joo

    2012-01-01

    The severe acute respiratory syndrome (SARS) epidemic started in late 2002 and swiftly spread across 5 continents with a mortality rate of around 10%. Although the epidemic was eventually controlled through the implementation of strict quarantine measures, there continues a need to investigate the SARS coronavirus (SARS-CoV) and develop interventions should it re-emerge. Numerous studies have shown that neutralizing antibodies against the virus can be found in patients infected with SARS-CoV within days upon the onset of illness and lasting up to several months. In contrast, there is little data on the kinetics of T cell responses during SARS-CoV infection and little is known about their role in the recovery process. However, recent studies in mice suggest the importance of T cells in viral clearance during SARS-CoV infection. Moreover, a growing number of studies have investigated the memory T cell responses in recovered SARS patients. This review covers the available literature on the emerging importance of T cell responses in SARS-CoV infection, particularly on the mapping of cytotoxic T lymphocyte (CTL) epitopes, longevity, polyfunctionality and human leukocyte antigen (HLA) association as well as their potential implications on treatment and vaccine development. PMID:26038429

  17. Conventional and unconventional mechanisms for capping viral mRNA.

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    Decroly, Etienne; Ferron, François; Lescar, Julien; Canard, Bruno

    2011-12-05

    In the eukaryotic cell, capping of mRNA 5' ends is an essential structural modification that allows efficient mRNA translation, directs pre-mRNA splicing and mRNA export from the nucleus, limits mRNA degradation by cellular 5'-3' exonucleases and allows recognition of foreign RNAs (including viral transcripts) as 'non-self'. However, viruses have evolved mechanisms to protect their RNA 5' ends with either a covalently attached peptide or a cap moiety (7-methyl-Gppp, in which p is a phosphate group) that is indistinguishable from cellular mRNA cap structures. Viral RNA caps can be stolen from cellular mRNAs or synthesized using either a host- or virus-encoded capping apparatus, and these capping assemblies exhibit a wide diversity in organization, structure and mechanism. Here, we review the strategies used by viruses of eukaryotic cells to produce functional mRNA 5'-caps and escape innate immunity.

  18. The search for a structural basis for therapeutic intervention against the SARS coronavirus

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    Bartlam, M.; Xue, X.; Rao, Z.

    2008-01-01

    The severe acute respiratory syndrome (SARS) coronavirus outbreak in 2003 had profound social and economic impacts worldwide. This review highlights the importance of structural biology and shows that structures for drug design can be rapidly determined in the event of an emerging infectious disease.

  19. Alisporivir inhibits MERS- and SARS-coronavirus replication in cell culture, but not SARS-coronavirus infection in a mouse model.

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    de Wilde, Adriaan H; Falzarano, Darryl; Zevenhoven-Dobbe, Jessika C; Beugeling, Corrine; Fett, Craig; Martellaro, Cynthia; Posthuma, Clara C; Feldmann, Heinz; Perlman, Stanley; Snijder, Eric J

    2017-01-15

    Currently, there is no registered treatment for infections with emerging zoonotic coronaviruses like SARS- and MERS-coronavirus. We here report that in cultured cells low-micromolar concentrations of alisporivir, a non-immunosuppressive cyclosporin A-analog, inhibit the replication of four different coronaviruses, including MERS- and SARS-coronavirus. Ribavirin was found to further potentiate the antiviral effect of alisporivir in these cell culture-based infection models, but this combination treatment was unable to improve the outcome of SARS-CoV infection in a mouse model. Nevertheless, our data provide a basis to further explore the potential of Cyp inhibitors as host-directed, broad-spectrum inhibitors of coronavirus replication. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

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    Lui Wing-bong

    2006-02-01

    Full Text Available Abstract Background We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR. In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. Methods An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen for quantitative performance, analytical sensitivity and precision. Results The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. Conclusion As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

  1. Development of chemical inhibitors of the SARS coronavirus: viral helicase as a potential target.

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    Keum, Young-Sam; Jeong, Yong-Joo

    2012-11-15

    Severe acute respiratory syndrome (SARS) was the first pandemic in the 21st century to claim more than 700 lives worldwide. However, effective anti-SARS vaccines or medications are currently unavailable despite being desperately needed to adequately prepare for a possible SARS outbreak. SARS is caused by a novel coronavirus, and one of its components, a viral helicase, is emerging as a promising target for the development of chemical SARS inhibitors. In the following review, we describe the characterization, family classification, and kinetic movement mechanisms of the SARS coronavirus (SCV) helicase-nsP13. We also discuss the recent progress in the identification of novel chemical inhibitors of nsP13 in the context of our recent discovery of the strong inhibition of the SARS helicase by natural flavonoids, myricetin and scutellarein. These compounds will serve as important resources for the future development of anti-SARS medications. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. The SARS-unique domain (SUD of SARS coronavirus contains two macrodomains that bind G-quadruplexes.

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    Jinzhi Tan

    2009-05-01

    Full Text Available Since the outbreak of severe acute respiratory syndrome (SARS in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV, the non-structural proteins (Nsps, have been determined. However, within the large Nsp3 (1922 amino-acid residues, the structure and function of the so-called SARS-unique domain (SUD have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core" of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively revealed that SUD(core forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins

  3. Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responses

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    Yang Moon-Sik

    2010-12-01

    Full Text Available Abstract Background Immunization with the spike protein (S of severe acute respiratory syndrome (SARS-coronavirus (CoV in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI is a cationic polymer which effectively delivers the plasmid DNA. Results In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n. route with SARS DNA vaccine (pci-S in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P + cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86 and class II major histocompatibility complex molecules (I-Ad were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice. Conclusion These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

  4. Memory T cell responses targeting the SARS coronavirus persist up to 11 years post-infection.

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    Ng, Oi-Wing; Chia, Adeline; Tan, Anthony T; Jadi, Ramesh S; Leong, Hoe Nam; Bertoletti, Antonio; Tan, Yee-Joo

    2016-04-12

    Severe acute respiratory syndrome (SARS) is a highly contagious infectious disease which first emerged in late 2002, caused by a then novel human coronavirus, SARS coronavirus (SARS-CoV). The virus is believed to have originated from bats and transmitted to human through intermediate animals such as civet cats. The re-emergence of SARS-CoV remains a valid concern due to the continual persistence of zoonotic SARS-CoVs and SARS-like CoVs (SL-CoVs) in bat reservoirs. In this study, the screening for the presence of SARS-specific T cells in a cohort of three SARS-recovered individuals at 9 and 11 years post-infection was carried out, and all memory T cell responses detected target the SARS-CoV structural proteins. Two CD8(+) T cell responses targeting the SARS-CoV membrane (M) and nucleocapsid (N) proteins were characterized by determining their HLA restriction and minimal T cell epitope regions. Furthermore, these responses were found to persist up to 11 years post-infection. An absence of cross-reactivity of these CD8(+) T cell responses against the newly-emerged Middle East respiratory syndrome coronavirus (MERS-CoV) was also demonstrated. The knowledge of the persistence of SARS-specific celullar immunity targeting the viral structural proteins in SARS-recovered individuals is important in the design and development of SARS vaccines, which are currently unavailable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. The role of epidermal growth factor receptor (EGFR) signaling in SARS coronavirus-induced pulmonary fibrosis.

    Science.gov (United States)

    Venkataraman, Thiagarajan; Frieman, Matthew B

    2017-07-01

    Many survivors of the 2003 outbreak of severe acute respiratory syndrome (SARS) developed residual pulmonary fibrosis with increased severity seen in older patients. Autopsies of patients that died from SARS also showed fibrosis to varying extents. Pulmonary fibrosis can be occasionally seen as a consequence to several respiratory viral infections but is much more common after a SARS coronavirus (SARS-CoV) infection. Given the threat of future outbreaks of severe coronavirus disease, including Middle East respiratory syndrome (MERS), it is important to understand the mechanisms responsible for pulmonary fibrosis, so as to support the development of therapeutic countermeasures and mitigate sequelae of infection. In this article, we summarize pulmonary fibrotic changes observed after a SARS-CoV infection, discuss the extent to which other respiratory viruses induce fibrosis, describe available animal models to study the development of SARS-CoV induced fibrosis and review evidence that pulmonary fibrosis is caused by a hyperactive host response to lung injury mediated by epidermal growth factor receptor (EGFR) signaling. We summarize work from our group and others indicating that inhibiting EGFR signaling may prevent an excessive fibrotic response to SARS-CoV and other respiratory viral infections and propose directions for future research. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected SARS coronavirus.

    Science.gov (United States)

    Yasui, Fumihiko; Kohara, Michinori; Kitabatake, Masahiro; Nishiwaki, Tetsu; Fujii, Hideki; Tateno, Chise; Yoneda, Misako; Morita, Kouichi; Matsushima, Kouji; Koyasu, Shigeo; Kai, Chieko

    2014-04-01

    While the 2002-2003 outbreak of severe acute respiratory syndrome (SARS) resulted in 774 deaths, patients who were affected with mild pulmonary symptoms successfully recovered. The objective of the present work was to identify, using SARS coronavirus (SARS-CoV) mouse infection models, immune factors responsible for clearing of the virus. The elimination of pulmonary SARS-CoV infection required the activation of B cells by CD4(+) T cells. Furthermore, passive immunization (post-infection) with homologous (murine) anti-SARS-CoV antiserum showed greater elimination efficacy against SARS-CoV than that with heterologous (rabbit) antiserum, despite the use of equivalent titers of neutralizing antibodies. This distinction was mediated by mouse phagocytic cells (monocyte-derived infiltrating macrophages and partially alveolar macrophages, but not neutrophils), as demonstrated both by adoptive transfer from donors and by immunological depletion of selected cell types. These results indicate that the cooperation of anti-SARS-CoV antibodies and phagocytic cells plays an important role in the elimination of SARS-CoV. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Peptide Mimicrying Between SARS Coronavirus Spike Protein and Human Proteins Reacts with SARS Patient Serum

    Directory of Open Access Journals (Sweden)

    K.-Y. Hwa

    2008-01-01

    Full Text Available Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. Severe acute respiratory syndrome (SARS is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV. The spike (S protein of SARS-CoV plays an important role in the virus entry into a cell. In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins. Two of the peptides D07 (residues 927–937 and D08 (residues 942–951 were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490–502 stimulated A549 to proliferate and secrete IL-8. The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.

  8. Cell Host Response to Infection with Novel Human Coronavirus EMC Predicts Potential Antivirals and Important Differences with SARS Coronavirus

    OpenAIRE

    Josset, Laurence; Menachery, Vineet D.; Gralinski, Lisa E.; Agnihothram, Sudhakar; Sova, Pavel; Carter, Victoria S.; Yount, Boyd L.; Graham, Rachel L.; Baric, Ralph S.; Katze, Michael G.

    2013-01-01

    ABSTRACT A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was abl...

  9. Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

    Science.gov (United States)

    Cameron, Mark J.; Kelvin, Alyson A.; Leon, Alberto J.; Cameron, Cheryl M.; Ran, Longsi; Xu, Luoling; Chu, Yong-Kyu; Danesh, Ali; Fang, Yuan; Li, Qianjun; Anderson, Austin; Couch, Ronald C.; Paquette, Stephane G.; Fomukong, Ndingsa G.; Kistner, Otfried; Lauchart, Manfred; Rowe, Thomas; Harrod, Kevin S.; Jonsson, Colleen B.; Kelvin, David J.

    2012-01-01

    In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)3-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines. PMID:23029269

  10. SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

    Directory of Open Access Journals (Sweden)

    Kèvin Knoops

    2008-09-01

    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.

  11. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.

    Directory of Open Access Journals (Sweden)

    I-Chueh Huang

    2011-01-01

    Full Text Available Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3 are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV hemagglutinin (HA protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2 of Marburg and Ebola filoviruses (MARV, EBOV. Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV and entry mediated by the SARS-CoV spike (S protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

  12. Long-term protection from SARS coronavirus infection conferred by a single immunization with an attenuated VSV-based vaccine.

    Science.gov (United States)

    Kapadia, Sagar U; Rose, John K; Lamirande, Elaine; Vogel, Leatrice; Subbarao, Kanta; Roberts, Anjeanette

    2005-09-30

    Although the recent SARS coronavirus (SARS-CoV) that appeared in 2002 has now been contained, the possibility of re-emergence of SARS-CoV remains. Due to the threat of re-emergence, the overall fatality rate of approximately 10%, and the rapid dispersion of the virus via international travel, viable vaccine candidates providing protection from SARS are clearly needed. We developed an attenuated VSV recombinant (VSV-S) expressing the SARS coronavirus (SARS-CoV) spike (S) protein. In cells infected with this recombinant, S protein was synthesized, glycosylated at approximately 17 Asn residues, and transported via the Golgi to the cell surface. Mice vaccinated with VSV-S developed SARS-neutralizing antibody and were able to control a challenge with SARS-CoV performed at 1 month or 4 months after a single vaccination. We also demonstrated, by passive antibody transfer, that the antibody response induced by the vaccine was sufficient for controlling SARS-CoV infection. A VSV-vectored SARS vaccine could have significant advantages over other SARS vaccine candidates described to date.

  13. Structure of a SARS coronavirus-derived peptide bound to the human major histocompatibility complex class I molecule HLA-B*1501

    DEFF Research Database (Denmark)

    Røder, Gustav; Kristensen, Ole; Kastrup, Jette S

    2008-01-01

    , the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 A). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making...

  14. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

    Science.gov (United States)

    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  15. Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003

    Directory of Open Access Journals (Sweden)

    Ooi Eng

    2004-09-01

    Full Text Available Abstract Background The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. Methods We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V, cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L, and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5 arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. Results Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day, or two mutations per human passage (adjusted R-square = 0.4014. Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are

  16. Development of a single nucleotide polymorphism DNA microarray for the detection and genotyping of the SARS coronavirus.

    Science.gov (United States)

    Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin

    2014-10-01

    Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.

  17. Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease.

    Science.gov (United States)

    Békés, Miklós; van der Heden van Noort, Gerbrand J; Ekkebus, Reggy; Ovaa, Huib; Huang, Tony T; Lima, Christopher D

    2016-05-19

    Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Identification of myricetin and scutellarein as novel chemical inhibitors of the SARS coronavirus helicase, nsP13.

    Science.gov (United States)

    Yu, Mi-Sun; Lee, June; Lee, Jin Moo; Kim, Younggyu; Chin, Young-Won; Jee, Jun-Goo; Keum, Young-Sam; Jeong, Yong-Joo

    2012-06-15

    Severe acute respiratory syndrome (SARS) is an infectious disease with a strong potential for transmission upon close personal contact and is caused by the SARS-coronavirus (CoV). However, there are no natural or synthetic compounds currently available that can inhibit SARS-CoV. We examined the inhibitory effects of 64 purified natural compounds against the activity of SARS helicase, nsP13, and the hepatitis C virus (HCV) helicase, NS3h, by conducting fluorescence resonance energy transfer (FRET)-based double-strand (ds) DNA unwinding assay or by using a colorimetry-based ATP hydrolysis assay. While none of the compounds, examined in our study inhibited the DNA unwinding activity or ATPase activity of human HCV helicase protein, we found that myricetin and scutellarein potently inhibit the SARS-CoV helicase protein in vitro by affecting the ATPase activity, but not the unwinding activity, nsP13. In addition, we observed that myricetin and scutellarein did not exhibit cytotoxicity against normal breast epithelial MCF10A cells. Our study demonstrates for the first time that selected naturally-occurring flavonoids, including myricetin and scultellarein might serve as SARS-CoV chemical inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. mRNA cap methylation influences pathogenesis of vesicular stomatitis virus in vivo.

    Science.gov (United States)

    Ma, Yuanmei; Wei, Yongwei; Zhang, Xiaodong; Zhang, Yu; Cai, Hui; Zhu, Yang; Shilo, Konstantin; Oglesbee, Michael; Krakowka, Steven; Whelan, Sean P J; Li, Jianrong

    2014-03-01

    One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2'-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452-456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2'-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2'-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many

  20. Triazole-containing monophosphate mRNA cap analogs as effective translation inhibitors.

    Science.gov (United States)

    Piecyk, Karolina; Lukaszewicz, Maciej; Darzynkiewicz, Edward; Jankowska-Anyszka, Marzena

    2014-10-01

    Synthetic analogs of the 5' end of mRNA (cap structure) are widely used in molecular studies on mechanisms of cellular processes such as translation, intracellular transport, splicing, and turnover. The best-characterized cap binding protein is translation initiation factor 4E (eIF4E). Recognition of the mRNA cap by eIF4E is a critical, rate-limiting step for efficient translation initiation and is considered a major target for anticancer therapy. Here, we report a facile methodology for the preparation of N2-triazole-containing monophosphate cap analogs and present their biological evaluation as inhibitors of protein synthesis. Five analogs possessing this unique hetero-cyclic ring spaced from the m7-guanine of the cap structure at a distance of one or three carbon atoms and/or additionally substituted by various groups containing the benzene ring were synthesized. All obtained compounds turned out to be effective translation inhibitors with IC50 similar to dinucleotide triphosphate m(7)GpppG. As these compounds possess a reduced number of phosphate groups and, thereby, a negative charge, which may support their cell penetration, this type of cap analog might be promising in terms of designing new potential therapeutic molecules. In addition, an exemplary dinucleotide from a corresponding mononucleotide containing benzyl substituted 1,2,3-triazole was prepared and examined. The superior inhibitory properties of this analog (10-fold vs. m(7)GpppG) suggest the usefulness of such compounds for the preparation of mRNA transcripts with high translational activity. © 2014 Piecyk et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  1. Reversible methylation of m6Am in the 5′ cap controls mRNA stability

    Science.gov (United States)

    Mauer, Jan; Luo, Xiaobing; Blanjoie, Alexandre; Jiao, Xinfu; Grozhik, Anya V.; Patil, Deepak P.; Linder, Bastian; Pickering, Brian F.; Vasseur, Jean-Jacques; Chen, Qiuying; Gross, Steven S.; Elemento, Olivier; Debart, Françoise; Kiledjian, Megerditch; Jaffrey, Samie R.

    2017-01-01

    Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5′ end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2′-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5′ cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability. PMID:28002401

  2. The SARS coronavirus papain like protease can inhibit IRF3 at a post activation step that requires deubiquitination activity.

    Science.gov (United States)

    Matthews, Krystal; Schäfer, Alexandra; Pham, Alissa; Frieman, Matthew

    2014-12-07

    The outcome of a viral infection is regulated by complex interactions of viral and host factors. SARS coronavirus (SARS-CoV) engages and regulates several innate immune response pathways during infection. We have previously shown that the SARS-CoV Papain-like Protease (PLpro) inhibits type I interferon (IFN) by inhibiting IRF3 phosphorylation thereby blocking downstream Interferon induction. This finding prompted us to identify other potential mechanisms of inhibition of PLpro on IFN induction. We have used plasmids expressing PLpro and IRF3 including an IRF3 mutant that is constitutively active, called IRF3(5D). In these experiments we utilize transfections, chromatin immunoprecipitation, Electro-mobility Shift Assays (EMSA) and protein localization to identify where IRF3 and IRF3(5D) are inhibited by PLpro. Here we show that PLpro also inhibits IRF3 activation at a step after phosphorylation and that this inhibition is dependent on the de-ubiquitination (DUB) activity of PLpro. We found that PLpro is able to block the type I IFN induction of a constitutively active IRF3, but does not inhibit IRF3 dimerization, nuclear localization or DNA binding. However, inhibition of PLpro's DUB activity by mutagenesis blocked the IRF3 inhibition activity of PLpro, suggesting a role for IRF3 ubiquitination in induction of a type I IFN innate immune response. These results demonstrate an additional mechanism that PLpro is able to inhibit IRF3 signaling. These data suggest novel innate immune antagonism activities of PLpro that may contribute to SARS-CoV pathogenesis.

  3. Inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with SARS coronavirus

    Directory of Open Access Journals (Sweden)

    Weber Friedemann

    2006-03-01

    Full Text Available Abstract Background SARS coronavirus (SARS-CoV is the etiologic agent of the severe acute respiratory syndrome. SARS-CoV mainly infects tissues of non-lymphatic origin, and the cytokine profile of those cells can determine the course of disease. Here, we investigated the cytokine response of two human non-lymphatic cell lines, Caco-2 and HEK 293, which are fully permissive for SARS-CoV. Results A comparison with established cytokine-inducing viruses revealed that SARS-CoV only weakly triggered a cytokine response. In particular, SARS-CoV did not activate significant transcription of the interferons IFN-α, IFN-β, IFN-λ1, IFN-λ2/3, as well as of the interferon-induced antiviral genes ISG56 and MxA, the chemokine RANTES and the interleukine IL-6. Interestingly, however, SARS-CoV strongly induced the chemokines IP-10 and IL-8 in the colon carcinoma cell line Caco-2, but not in the embryonic kidney cell line 293. Conclusion Our data indicate that SARS-CoV suppresses the antiviral cytokine system of non-immune cells to a large extent, thus buying time for dissemination in the host. However, synthesis of IP-10 and IL-8, which are established markers for acute-stage SARS, escapes the virus-induced silencing at least in some cell types. Therefore, the progressive infiltration of immune cells into the infected lungs observed in SARS patients could be due to the production of these chemokines by the infected tissue cells.

  4. Cell host response to infection with novel human coronavirus EMC predicts potential antivirals and important differences with SARS coronavirus.

    Science.gov (United States)

    Josset, Laurence; Menachery, Vineet D; Gralinski, Lisa E; Agnihothram, Sudhakar; Sova, Pavel; Carter, Victoria S; Yount, Boyd L; Graham, Rachel L; Baric, Ralph S; Katze, Michael G

    2013-04-30

    A novel human coronavirus (HCoV-EMC) was recently identified in the Middle East as the causative agent of a severe acute respiratory syndrome (SARS) resembling the illness caused by SARS coronavirus (SARS-CoV). Although derived from the CoV family, the two viruses are genetically distinct and do not use the same receptor. Here, we investigated whether HCoV-EMC and SARS-CoV induce similar or distinct host responses after infection of a human lung epithelial cell line. HCoV-EMC was able to replicate as efficiently as SARS-CoV in Calu-3 cells and similarly induced minimal transcriptomic changes before 12 h postinfection. Later in infection, HCoV-EMC induced a massive dysregulation of the host transcriptome, to a much greater extent than SARS-CoV. Both viruses induced a similar activation of pattern recognition receptors and the interleukin 17 (IL-17) pathway, but HCoV-EMC specifically down-regulated the expression of several genes within the antigen presentation pathway, including both type I and II major histocompatibility complex (MHC) genes. This could have an important impact on the ability of the host to mount an adaptive host response. A unique set of 207 genes was dysregulated early and permanently throughout infection with HCoV-EMC, and was used in a computational screen to predict potential antiviral compounds, including kinase inhibitors and glucocorticoids. Overall, HCoV-EMC and SARS-CoV elicit distinct host gene expression responses, which might impact in vivo pathogenesis and could orient therapeutic strategies against that emergent virus. Identification of a novel coronavirus causing fatal respiratory infection in humans raises concerns about a possible widespread outbreak of severe respiratory infection similar to the one caused by SARS-CoV. Using a human lung epithelial cell line and global transcriptomic profiling, we identified differences in the host response between HCoV-EMC and SARS-CoV. This enables rapid assessment of viral properties and the

  5. Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway.

    Science.gov (United States)

    Sui, Jianhua; Aird, Daniel R; Tamin, Azaibi; Murakami, Akikazu; Yan, Meiying; Yammanuru, Anuradha; Jing, Huaiqi; Kan, Biao; Liu, Xin; Zhu, Quan; Yuan, Qing-An; Adams, Gregory P; Bellini, William J; Xu, Jianguo; Anderson, Larry J; Marasco, Wayne A

    2008-11-01

    Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in

  6. eIF3d is an mRNA cap-binding protein required for specialized translation initiation

    Science.gov (United States)

    Lee, Amy S.Y.; Kranzusch, Philip J.; Doudna, Jennifer A.; Cate, Jamie H.D.

    2016-01-01

    Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation. PMID:27462815

  7. Adaptive evolution of the spike gene of SARS coronavirus: changes in positively selected sites in different epidemic groups

    Directory of Open Access Journals (Sweden)

    He Shao-Heng

    2006-10-01

    Full Text Available Abstract Background It is believed that animal-to-human transmission of severe acute respiratory syndrome (SARS coronavirus (CoV is the cause of the SARS outbreak worldwide. The spike (S protein is one of the best characterized proteins of SARS-CoV, which plays a key role in SARS-CoV overcoming species barrier and accomplishing interspecies transmission from animals to humans, suggesting that it may be the major target of selective pressure. However, the process of adaptive evolution of S protein and the exact positively selected sites associated with this process remain unknown. Results By investigating the adaptive evolution of S protein, we identified twelve amino acid sites (75, 239, 244, 311, 479, 609, 613, 743, 765, 778, 1148, and 1163 in the S protein under positive selective pressure. Based on phylogenetic tree and epidemiological investigation, SARS outbreak was divided into three epidemic groups: 02–04 interspecies, 03-early-mid, and 03-late epidemic groups in the present study. Positive selection was detected in the first two groups, which represent the course of SARS-CoV interspecies transmission and of viral adaptation to human host, respectively. In contrast, purifying selection was detected in 03-late group. These indicate that S protein experiences variable positive selective pressures before reaching stabilization. A total of 25 sites in 02–04 interspecies epidemic group and 16 sites in 03-early-mid epidemic group were identified under positive selection. The identified sites were different between these two groups except for site 239, which suggests that positively selected sites are changeable between groups. Moreover, it was showed that a larger proportion (24% of positively selected sites was located in receptor-binding domain (RBD than in heptad repeat (HR1-HR2 region in 02–04 interspecies epidemic group (p = 0.0208, and a greater percentage (25% of these sites occurred in HR1–HR2 region than in RBD in 03-early

  8. Interaction of human decapping scavenger with 5' mRNA cap analogues: structural requirements for catalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Darzynkiewicz, Zbigniew M [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Bojarska, Elzbieta [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kowalska, Joanna [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Lewdorowicz, Magdalena [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Jemielity, Jacek [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Kalek, Marcin [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Stepinski, Janusz [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland); Davis, Richard E [Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, 12801 East 17th Avenue, Aurora, CO 80045 (United States); Darzynkiewicz, Edward [Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury Street, 02-089 Warsaw (Poland)

    2007-07-18

    The cap structure is a specific feature of the 5' end of mRNA which plays an important role in the post-transcriptional control in gene expression. A major step of gene regulation occurs at the level of mRNA turnover. Degradation of most eukaryotic mRNAs entails the removal of the cap structure in the various pathways. A human scavenger decapping enzyme (hDcpS) catalyses the cleavage of the residual cap structure m{sup 7}GpppN and/or short oligonucleotides after the 3' to 5' exosom mediated digestion. In this paper we report a fluorescence study of association process of hDcpS with m{sup 7}GMP, m{sup 7}GDP and selected dinucleotide cap analogues resistant to enzymatic hydrolysis. The calculated values of association constants (K{sub as}) and corresponding Gibbs free energies ({delta}G{sup 0}) depend on the type of substituents and their positions in the cap molecule, indicating which structural modifications are crucial for the catalysis.

  9. 5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

    Science.gov (United States)

    Ogino, Minako; Ogino, Tomoaki

    2017-03-15

    The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential

  10. Methylation of vesicular stomatitis virus (VSV) mRNA 5'-cap structures in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, D.C.; Lesnaw, J.A.

    1987-05-01

    Monocistronic VSV mRNAs synthesized by subviral particles in vitro display the methylated 5'-cap structure m'G(5')ppp(5')Am. The authors have detected both monomethylated cap structures, m/sup 7/G(5')ppp(5')A and G(5')Am, in reactions containing suboptimal concentrations of AdoMet. To assess the putative precursor roles of these cap structures the authors devised dual label pulse-chase analyses employing S-(CH/sub 3/-/sup 3/H)-AdoMet and (..beta..-/sup 32/P)GTP. The labeled cap structures were analyzed by HPLC. The simultaneous chasing of both radiolabeled substrates allowed 1) the isolation of a specific set of caps labeled as (..beta..-/sup 32/P)-R/sup 7/G(5')ppp(5')AR (R=H or CH/sub 3/) and 2) the determination of the transcriptive fate of each intermediate cap structure within the set. The results demonstrated that both monomethylated cap structures serve as intermediates for the dimethylated cap and that the order of cap methylation is non-compulsory. These data, coupled with previous observations of hypomethylated cap structures in polyadenylated RNAs, have suggested that methylation occurs in a chain length dependent window.

  11. mRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research.

    Science.gov (United States)

    Li, Changqing; Guillén, Jaime; Rabah, Nadia; Blanjoie, Alexandre; Debart, Françoise; Vasseur, Jean-Jacques; Canard, Bruno; Decroly, Etienne; Coutard, Bruno

    2015-08-01

    Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m(7)GMP-nsP1 adduct. Subsequent transfer of m(7)GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m(7)G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m(7)GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research. Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap

  12. A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

    Science.gov (United States)

    Kusov, Yuri; Tan, Jinzhi; Alvarez, Enrique; Enjuanes, Luis; Hilgenfeld, Rolf

    2015-10-01

    The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

    Science.gov (United States)

    Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

    2012-08-01

    Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Acquisition of new protein domains by coronaviruses: analysis of overlapping genes coding for proteins N and 9b in SARS coronavirus.

    Science.gov (United States)

    Shukla, Aditi; Hilgenfeld, Rolf

    2015-02-01

    Acquisition of new proteins by viruses usually occurs through horizontal gene transfer or through gene duplication, but another, less common mechanism is the usage of completely or partially overlapping reading frames. A case of acquisition of a completely new protein through introduction of a start codon in an alternative reading frame is the protein encoded by open reading frame (orf) 9b of SARS coronavirus. This gene completely overlaps with the nucleocapsid (N) gene (orf9a). Our findings indicate that the orf9b gene features a discordant codon-usage pattern. We analyzed the evolution of orf9b in concert with orf9a using sequence data of betacoronavirus-lineage b and found that orf9b, which encodes the overprinting protein, evolved largely independent of the overprinted orf9a. We also examined the protein products of these genomic sequences for their structural flexibility and found that it is not necessary for a newly acquired, overlapping protein product to be intrinsically disordered, in contrast to earlier suggestions. Our findings contribute to characterizing sequence properties of newly acquired genes making use of overlapping reading frames.

  15. Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

    Science.gov (United States)

    Sun, Ying; Wang, Zidao; Tao, Jiali; Wang, Yi; Wu, Andong; Yang, Ziwen; Wang, Kaimei; Shi, Liqiao; Chen, Yu; Guo, Deyin

    2014-04-01

    The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Systematic analysis of the contribution of c-myc mRNA constituents upon cap and IRES mediated translation.

    Science.gov (United States)

    Meristoudis, Christos; Trangas, Theoni; Lambrianidou, Andromachi; Papadopoulos, Vasilios; Dimitriadis, Euthymios; Courtis, Nelly; Ioannidis, Panayotis

    2015-12-01

    Fine tuning of c-MYC expression is critical for its action and is achieved by several regulatory mechanisms. The contribution of c-myc mRNA regulatory sequences on its translational control has been investigated individually. However, putative interactions have not been addressed so far. The effect of these interactions upon the translatability of monocistronic and bicistronic chimaeric mRNAs, carrying combinations of the c-myc mRNA 5'-untranlated region (UTR), 3'-UTR, and coding region instability element (CRD) was investigated on this study. The presence of the 5'-UTR induced an increase in translatability of 50%. The presence of the CRD element, when in frame, reduced translatability by approximately 50%, regardless of the expression levels of the wild type CRD- binding protein (CRD-BP/IMP1). Conversely, overexpression of a mutated CRD-BP/IMP1 (Y396F) further impeded translation of the chimaeric mRNAs carrying its cognate sequences. The presence of the c-myc 3'-UTR increased translatability by approximately 300% affecting both cap and c-myc internal ribosome entry site (IRES) mediated translation. In addition, 3'-UTR rescued the cap mediated translation in the presence of the polyadenylation inhibitor cordycepin. Furthermore, the 3'-UTR rescued cap mediated translation under metabolic stress conditions and this was enhanced in the absence of a long poly (A) tail.

  17. Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: insights into mechanisms of general viral fusion and inhibitor design.

    Science.gov (United States)

    Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

    2014-05-01

    Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation=0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. © 2014 The Protein Society.

  18. SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex.

    Science.gov (United States)

    Chen, Xiaojuan; Yang, Xingxing; Zheng, Yang; Yang, Yudong; Xing, Yaling; Chen, Zhongbin

    2014-05-01

    SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

  19. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway.

    Science.gov (United States)

    Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2016-05-13

    SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

  20. SARS coronavirus papain-like protease up-regulates the collagen expression through non-Samd TGF-β1 signaling.

    Science.gov (United States)

    Wang, Ching-Ying; Lu, Chien-Yi; Li, Shih-Wen; Lai, Chien-Chen; Hua, Chun-Hung; Huang, Su-Hua; Lin, Ying-Ju; Hour, Mann-Jen; Lin, Cheng-Wen

    2017-05-02

    SARS coronavirus (CoV) papain-like protease (PLpro) reportedly induced the production of TGF-β1 through p38 MAPK/STAT3-meidated Egr-1-dependent activation (Sci. Rep. 6, 25754). This study investigated the correlation of PLpro-induced TGF-β1 with the expression of Type I collagen in human lung epithelial cells and mouse pulmonary tissues. Specific inhibitors for TGF-βRI, p38 MAPK, MEK, and STAT3 proved that SARS-CoV PLpro induced TGF-β1-dependent up-regulation of Type I collagen in vitro and in vivo. Subcellular localization analysis of SMAD3 and SMAD7 indicated that non-SMAD pathways in TGF-β1 signaling involved in the production of Type I collagen in transfected cells with pSARS-PLpro. Comprehensive analysis of ubiquitin-conjugated proteins using immunoprecipitation and nanoLC-MS/MS indicated that SARS-CoV PLpro caused the change in the ubiquitination profile of Rho GTPase family proteins, in which linked with the increase of Rho-like GTPase family proteins. Moreover, selective inhibitors TGF-βRI and STAT6 (AS1517499) ascertained that STAT6 activation was required for PLpro-induced TGF-β1-dependent up-regulation of Type I collagen in human lung epithelial cells. The results showed that SARS-CoV PLpro stimulated TGF-β1-dependent expression of Type I collagen via activating STAT6 pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Influence of hydrophobic and electrostatic residues on SARS-coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

    Science.gov (United States)

    Aydin, Halil; Al-Khooly, Dina; Lee, Jeffrey E

    2014-01-01

    Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS-coronavirus (SARS-CoV). SARS-CoV entry is facilitated by the spike protein (S), which consists of an N-terminal domain (S1) responsible for cellular attachment and a C-terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS-CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site-directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH-independent retroviral fusion proteins, SARS-CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS-CoV S2 analysis showed that specific hydrophobic positions at the C-terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C-terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C-terminal hydrophobic residues led us to identify a 42-residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections. PMID:24519901

  2. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation.

    Science.gov (United States)

    Lee, Amy S; Kranzusch, Philip J; Doudna, Jennifer A; Cate, Jamie H D

    2016-08-04

    Eukaryotic mRNAs contain a 5′ cap structure that is crucial for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression despite inactivation of eIF4E. Here we describe a cap-dependent pathway of translation initiation in human cells that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eIF3 complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modelling of cap recognition by eIF3d. eIF3d makes specific contacts with the cap, as exemplified by cap analogue competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs such as the cell proliferation regulator c-Jun (also known as JUN). The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a mechanism of cap-dependent translation that is independent of eIF4E, and illustrate how modular RNA elements work together to direct specialized forms of translation initiation.

  3. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus.

    Science.gov (United States)

    Hu, Ben; Zeng, Lei-Ping; Yang, Xing-Lou; Ge, Xing-Yi; Zhang, Wei; Li, Bei; Xie, Jia-Zheng; Shen, Xu-Rui; Zhang, Yun-Zhi; Wang, Ning; Luo, Dong-Sheng; Zheng, Xiao-Shuang; Wang, Mei-Niang; Daszak, Peter; Wang, Lin-Fa; Cui, Jie; Shi, Zheng-Li

    2017-11-01

    A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

  4. Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

    Science.gov (United States)

    Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

    2015-06-01

    Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Identification of a new region in the vesicular stomatitis virus L polymerase protein which is essential for mRNA cap methylation.

    Science.gov (United States)

    Grdzelishvili, Valery Z; Smallwood, Sherin; Tower, Dallas; Hall, Richard L; Hunt, D Margaret; Moyer, Sue A

    2006-07-05

    The vesicular stomatitis virus (VSV) L polymerase protein possesses two methyltransferase (MTase) activities, which catalyze the methylation of viral mRNA cap structures at the guanine-N7 and 2'-O-adenosine positions. To identify L sequences required for the MTase activities, we analyzed a host range (hr) and temperature-sensitive (ts) mutant of VSV, hr8, which was defective in mRNA cap methylation. Sequencing hr8 identified five amino acid substitutions, all residing in the L protein. Recombinant VSV were generated with each of the identified L mutations, and the presence of a single G1481R substitution in L, located between conserved domains V and VI, was sufficient to produce a dramatic reduction (about 90%) in overall mRNA methylation. Cap analysis showed residual guanine-N7 methylation and reduced 2'-O-adenosine methylation, identical to that of the original hr8 virus. When recombinant viruses were tested for virus growth under conditions that were permissive and nonpermissive for the hr8 mutant, the same single L mutation, G1481R, was solely responsible for both the hr and ts phenotypes. A spontaneous suppressor mutant of the rG1481R virus that restored both growth on nonpermissive cells and cap methylation was identified and mapped to a single change, L1450I, in L. Site-directed mutagenesis of the region between domains V and VI, amino acids 1419-1672 of L, followed by the rescue of recombinant viruses identified five additional virus mutants, K1468A, R1478A/D1479A, G1481A, G1481N, and G1672A, that were all hr and defective in mRNA cap methylation. Thus, in addition to the previously characterized domain VI [Grdzelishvili, V.Z., Smallwood, S., Tower, D., Hall, R.L., Hunt, D.M., Moyer, S.A., 2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol. 79, 7327-7337; Li, J., Fontaine-Rodriguez, E.C., Whelan, S.P., 2005. Amino acid residues within conserved domain VI of the

  6. A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

    Science.gov (United States)

    Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

    2016-01-01

    Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

  7. Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations.

    Science.gov (United States)

    Luzhkov, Victor; Decroly, Etienne; Canard, Bruno; Selisko, Barbara; Åqvist, Johan

    2013-02-01

    Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV . The docking simulations using GOLD, Glide, and Dock give consistent binding modes and binding affinities of adamantanes in the AdoMet-binding site of NS5MTaseDV and, in particular, yield similar positions for the previously found inhibitors. Combined, they perfectly correspond to the bioassay measurements of nucleoside-2'O-methyltransferase activity of NS5TaseDV , which confirmed inhibitory properties of the active urea adamantane but did not show inhibitory activity for amantadine and rimantadine. We also employed microscopic molecular dynamics (MD) simulations and a linear interaction energy (LIE) method to verify the docking results. The MD/LIE binding free energies of selected protein-inhibitor complexes agree overall with the binding affinities from docking and demonstrate that amantadine and rimantadine only weakly bind at the explored site. The MD simulations also demonstrated the flexible character of a protein loop that is located between the β2 and β3 strands and is part of the AdoMet-binding pocket of NS5MTaseDV . Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Ribose 2'-O methylation of the vesicular stomatitis virus mRNA cap precedes and facilitates subsequent guanine-N-7 methylation by the large polymerase protein.

    Science.gov (United States)

    Rahmeh, Amal A; Li, Jianrong; Kranzusch, Philip J; Whelan, Sean P J

    2009-11-01

    During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2'-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2'-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2'-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2'-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2'-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2'-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2'-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5' terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2'-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2'-O and G-N-7 methylation of the cap structure.

  9. The 5'-poly(A leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

    Directory of Open Access Journals (Sweden)

    Pragyesh Dhungel

    2017-08-01

    Full Text Available The poly(A leader at the 5'-untranslated region (5'-UTR is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs. These poly(A leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A leaders are 8-12 residues, the result suggests that the poly(A leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES. These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

  10. The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.

    Science.gov (United States)

    Dhungel, Pragyesh; Cao, Shuai; Yang, Zhilong

    2017-08-01

    The poly(A) leader at the 5'-untranslated region (5'-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5'-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5'-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5'-poly(A) leaders are 8-12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5'-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5'- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5'-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs.

  11. HIV-1 Gag Blocks Selenite-Induced Stress Granule Assembly by Altering the mRNA Cap-Binding Complex

    Directory of Open Access Journals (Sweden)

    Alessandro Cinti

    2016-03-01

    Full Text Available Stress granules (SGs are dynamic accumulations of stalled preinitiation complexes and translational machinery that assemble under stressful conditions. Sodium selenite (Se induces the assembly of noncanonical type II SGs that differ in morphology, composition, and mechanism of assembly from canonical SGs. Se inhibits translation initiation by altering the cap-binding activity of eukaryotic translation initiation factor 4E (eIF4E-binding protein 1 (4EBP1. In this work, we show that human immunodeficiency virus type 1 (HIV-1 Gag is able to block the assembly of type II noncanonical SGs to facilitate continued Gag protein synthesis. We demonstrate that expression of Gag reduces the amount of hypophosphorylated 4EBP1 associated with the 5′ cap potentially through an interaction with its target, eIF4E. These results suggest that the assembly of SGs is an important host antiviral defense that HIV-1 has evolved for inhibition through several distinct mechanisms.

  12. The 5'-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation

    Science.gov (United States)

    Dhungel, Pragyesh; Cao, Shuai

    2017-01-01

    The poly(A) leader at the 5’-untranslated region (5’-UTR) is an unusually striking feature of all poxvirus mRNAs transcribed after viral DNA replication (post-replicative mRNAs). These poly(A) leaders are non-templated and of heterogeneous lengths; and their function during poxvirus infection remains a long-standing question. Here, we discovered that a 5’-poly(A) leader conferred a selective translational advantage to mRNA in poxvirus-infected cells. A constitutive and uninterrupted 5’-poly(A) leader with 12 residues was optimal. Because the most frequent lengths of the 5’-poly(A) leaders are 8–12 residues, the result suggests that the poly(A) leader has been evolutionarily optimized to boost poxvirus protein production. A 5’-poly(A) leader also could increase protein production in the bacteriophage T7 promoter-based expression system of vaccinia virus, the prototypic member of poxviruses. Interestingly, although vaccinia virus post-replicative mRNAs do have 5’- methylated guanosine caps and can use cap-dependent translation, in vaccinia virus-infected cells, mRNA with a 5’-poly(A) leader could also be efficiently translated in cells with impaired cap-dependent translation. However, the translation was not mediated through an internal ribosome entry site (IRES). These results point to a fundamental mechanism poxvirus uses to efficiently translate its post-replicative mRNAs. PMID:28854224

  13. A Cap-to-Tail Guide to mRNA Translation Strategies in Virus-Infected Cells.

    Science.gov (United States)

    Jan, Eric; Mohr, Ian; Walsh, Derek

    2016-09-29

    Although viruses require cellular functions to replicate, their absolute dependence upon the host translation machinery to produce polypeptides indispensable for their reproduction is most conspicuous. Despite their incredible diversity, the mRNAs produced by all viruses must engage cellular ribosomes. This has proven to be anything but a passive process and has revealed a remarkable array of tactics for rapidly subverting control over and dominating cellular regulatory pathways that influence translation initiation, elongation, and termination. Besides enforcing viral mRNA translation, these processes profoundly impact host cell-intrinsic immune defenses at the ready to deny foreign mRNA access to ribosomes and block protein synthesis. Finally, genome size constraints have driven the evolution of resourceful strategies for maximizing viral coding capacity. Here, we review the amazing strategies that work to regulate translation in virus-infected cells, highlighting both virus-specific tactics and the tremendous insight they provide into fundamental translational control mechanisms in health and disease.

  14. The 5′ Untranslated Region of the Human T-Cell Lymphotropic Virus Type 1 mRNA Enables Cap-Independent Translation Initiation

    Science.gov (United States)

    Olivares, Eduardo; Landry, Dori M.; Cáceres, C. Joaquín; Pino, Karla; Rossi, Federico; Navarrete, Camilo; Huidobro-Toro, Juan Pablo; Thompson, Sunnie R.

    2014-01-01

    ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a complex human retrovirus that causes adult T cell leukemia and of HTLV-associated myelopathy/tropical spastic paraparesis. The mRNA of some complex retroviruses, including the human and simian immunodeficiency viruses (HIV and SIV), can initiate translation using a canonical cap-dependent mechanism or through an internal ribosome entry site (IRES). In this study, we present strong evidence showing that like HIV-1 and SIV, the 5′-untranslated region (5′UTR) of the HTLV-1 full-length mRNA harbors an IRES. Cap-independent translational activity was evaluated and demonstrated using dual luciferase bicistronic mRNAs in rabbit reticulocyte lysate, in mammalian cell culture, and in Xenopus laevis oocytes. Characterization of the HTLV-1 IRES shows that its activity is dependent on the ribosomal protein S25 (RPS25) and that its function is highly sensitive to the drug edeine. Together, these findings suggest that the 5′UTR of the HTLV-1 full-length mRNA enables internal recruitment of the eukaryotic translation initiation complex. However, the recognition of the initiation codon requires ribosome scanning. These results suggest that, after internal recruitment by the HTLV-1 IRES, a scanning step takes place for the 40S ribosomal subunit to be positioned at the translation initiation codon. IMPORTANCE The mechanism by which retroviral mRNAs recruit the 40S ribosomal subunit internally is not understood. This study provides new insights into the mechanism of translation initiation used by the human T-cell lymphotropic virus type 1 (HTLV-1). The results show that the HTLV-1 mRNA can initiate translation via a noncanonical mechanism mediated by an internal ribosome entry site (IRES). This study also provides evidence showing the involvement of cellular proteins in HTLV-1 IRES-mediated translation initiation. Together, the data presented in this report significantly contribute to the understanding of HTLV-1 gene

  15. The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor.

    Directory of Open Access Journals (Sweden)

    Rinki Minakshi

    2009-12-01

    Full Text Available The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR, which includes the inositol-requiring enzyme 1 (IRE-1, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1 increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha and inhibitory effects of a dominant-negative form of eIF2alpha on GRP78 promoter activity, (2 increased translation of activating transcription factor 4 (ATF4 mRNA, and (3 ATF4-dependent activation of the C/EBP homologous protein (CHOP gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1 degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.

  16. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination.

    Science.gov (United States)

    Lau, Susanna K P; Feng, Yun; Chen, Honglin; Luk, Hayes K H; Yang, Wei-Hong; Li, Kenneth S M; Zhang, Yu-Zhen; Huang, Yi; Song, Zhi-Zhong; Chow, Wang-Ngai; Fan, Rachel Y Y; Ahmed, Syed Shakeel; Yeung, Hazel C; Lam, Carol S F; Cai, Jian-Piao; Wong, Samson S Y; Chan, Jasper F W; Yuen, Kwok-Yung; Zhang, Hai-Lin; Woo, Patrick C Y

    2015-10-01

    Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8

  17. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination

    Science.gov (United States)

    Lau, Susanna K. P.; Feng, Yun; Chen, Honglin; Luk, Hayes K. H.; Yang, Wei-Hong; Li, Kenneth S. M.; Zhang, Yu-Zhen; Huang, Yi; Song, Zhi-Zhong; Chow, Wang-Ngai; Fan, Rachel Y. Y.; Ahmed, Syed Shakeel; Yeung, Hazel C.; Lam, Carol S. F.; Cai, Jian-Piao; Wong, Samson S. Y.; Chan, Jasper F. W.; Yuen, Kwok-Yung

    2015-01-01

    ABSTRACT Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS

  18. Ribose 2′-O Methylation of the Vesicular Stomatitis Virus mRNA Cap Precedes and Facilitates Subsequent Guanine-N-7 Methylation by the Large Polymerase Protein▿

    Science.gov (United States)

    Rahmeh, Amal A.; Li, Jianrong; Kranzusch, Philip J.; Whelan, Sean P. J.

    2009-01-01

    During conventional mRNA cap formation, two separate methyltransferases sequentially modify the cap structure, first at the guanine-N-7 (G-N-7) position and subsequently at the ribose 2′-O position. For vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses, the two methylase activities share a binding site for the methyl donor S-adenosyl-l-methionine and are inhibited by individual amino acid substitutions within the C-terminal domain of the large (L) polymerase protein. This led to the suggestion that a single methylase domain functions for both 2′-O and G-N-7 methylations. Here we report a trans-methylation assay that recapitulates both ribose 2′-O and G-N-7 modifications by using purified recombinant L and in vitro-synthesized RNA. Using this assay, we demonstrate that VSV L typically modifies the 2′-O position of the cap prior to the G-N-7 position and that G-N-7 methylation is diminished by pre-2′-O methylation of the substrate RNA. Amino acid substitutions in the C terminus of L that prevent all cap methylation in recombinant VSV (rVSV) partially retain the ability to G-N-7 methylate a pre-2′-O-methylated RNA, therefore uncoupling the effect of substitutions in the C terminus of the L protein on the two methylations. In addition, we show that the 2′-O and G-N-7 methylase activities act specifically on RNA substrates that contain the conserved elements of a VSV mRNA start at the 5′ terminus. This study provides new mechanistic insights into the mRNA cap methylase activities of VSV L, demonstrates that 2′-O methylation precedes and facilitates subsequent G-N-7 methylation, and reveals an RNA sequence and length requirement for the two methylase activities. We propose a model of regulation of the activity of the C terminus of L protein in 2′-O and G-N-7 methylation of the cap structure. PMID:19710136

  19. [Capping strategies in RNA viruses].

    Science.gov (United States)

    Bouvet, Mickaël; Ferron, François; Imbert, Isabelle; Gluais, Laure; Selisko, Barbara; Coutard, Bruno; Canard, Bruno; Decroly, Etienne

    2012-04-01

    Most viruses use the mRNA-cap dependent cellular translation machinery to translate their mRNAs into proteins. The addition of a cap structure at the 5' end of mRNA is therefore an essential step for the replication of many virus families. Additionally, the cap protects the viral RNA from degradation by cellular nucleases and prevents viral RNA recognition by innate immunity mechanisms. Viral RNAs acquire their cap structure either by using cellular capping enzymes, by stealing the cap of cellular mRNA in a process named "cap snatching", or using virus-encoded capping enzymes. Many viral enzymes involved in this process have recently been structurally and functionally characterized. These studies have revealed original cap synthesis mechanisms and pave the way towards the development of specific inhibitors bearing antiviral drug potential. © 2012 médecine/sciences – Inserm / SRMS.

  20. Cervical Cap

    Science.gov (United States)

    ... Sport for You Healthy School Lunch Planner Cervical Cap KidsHealth > For Teens > Cervical Cap Print A A ... and a female's egg. How Does a Cervical Cap Work? The cervical cap keeps sperm from entering ...

  1. Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay.

    Science.gov (United States)

    Aouadi, Wahiba; Eydoux, Cécilia; Coutard, Bruno; Martin, Baptiste; Debart, Françoise; Vasseur, Jean Jacques; Contreras, Jean Marie; Morice, Christophe; Quérat, Gilles; Jung, Marie-Louise; Canard, Bruno; Guillemot, Jean-Claude; Decroly, Etienne

    2017-08-01

    Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF ® ) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC 50 determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases. Copyright © 2017. Published by Elsevier B.V.

  2. Cradle cap

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007582.htm Cradle cap To use the sharing features on this page, please enable JavaScript. Cradle cap is seborrheic dermatitis that affects the scalp of ...

  3. mRNA display design of fibronectin-based intrabodies that detect and inhibit severe acute respiratory syndrome coronavirus nucleocapsid protein.

    Science.gov (United States)

    Liao, Hsiang-I; Olson, C Anders; Hwang, Seungmin; Deng, Hongyu; Wong, Elaine; Baric, Ralph S; Roberts, Richard W; Sun, Ren

    2009-06-26

    The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with Kd=1.7 nM. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication.

  4. mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein*

    Science.gov (United States)

    Liao, Hsiang-I.; Olson, C. Anders; Hwang, Seungmin; Deng, Hongyu; Wong, Elaine; Baric, Ralph S.; Roberts, Richard W.; Sun, Ren

    2009-01-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome (SARS) coronavirus plays important roles in both viral replication and modulation of host cell processes. New ligands that target the N protein may thus provide tools to track the protein inside cells, detect interaction hot spots on the protein surface, and discover sites that could be used to develop new anti-SARS therapies. Using mRNA display selection and directed evolution, we designed novel antibody-like protein affinity reagents that target SARS N protein with high affinity and selectivity. Our libraries were based on an 88-residue variant of the 10th fibronectin type III domain from human fibronectin (10Fn3). This selection resulted in eight independent 10Fn3 intrabodies, two that require the N-terminal domain for binding and six that recognize the C terminus, one with Kd = 1.7 nm. 10Fn3 intrabodies are well expressed in mammalian cells and are relocalized by N in SARS-infected cells. Seven of the selected intrabodies tested do not perturb cellular function when expressed singly in vivo and inhibit virus replication from 11- to 5900-fold when expressed in cells prior to infection. Targeting two sites on SARS-N simultaneously using two distinct 10Fn3s results in synergistic inhibition of virus replication. PMID:19364769

  5. Receptor recognition and cross-species infections of SARS coronavirus.

    Science.gov (United States)

    Li, Fang

    2013-10-01

    Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses". Copyright © 2013 Elsevier B.V. All rights reserved.

  6. The SARS coronavirus nucleocapsid protein--forms and functions.

    Science.gov (United States)

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses." Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Anti-SARS coronavirus agents: a patent review (2008 - present).

    Science.gov (United States)

    Kumar, Vathan; Jung, Young-Sik; Liang, Po-Huang

    2013-10-01

    A novel coronavirus (CoV), unlike previous typical human coronaviruses (HCoVs), was identified as causative agent for severe acute respiratory syndrome (SARS). SARS first surfaced as a pandemic in late 2002 and originated in southern China. SARS-CoV rapidly spread to > 30 countries by 2003, infecting nearly 8,000 people and causing around 800 fatalities. After 10 years of silence, a 2012 report alarmed researchers about the emergence of a new strain of CoV causing SARS-like disease. To combat SARS, scientists applied for patents on various therapeutic agents, including small-molecule inhibitors targeting the essential proteases, helicase and other proteins of the virus, natural products, approved drugs, molecules binding to the virus, neutralizing antibodies, vaccines, anti-sense RNA, siRNA and ribozyme against SARS-CoV. In this article, the patents published from 2008 to the present for the new therapeutics that could potentially be used in the prophylaxis and treatment of SARS are reviewed. The therapeutic interventions or prophylaxis discussed in this review seems to offer promising solutions to tackle SARS. Rather than being complacent about the results, we should envisage how to transform them into drug candidates that may be useful in combating SARS and related viral infections in the future.

  8. Receptor recognition and cross-species infections of SARS coronavirus

    Science.gov (United States)

    Li, Fang

    2013-01-01

    Receptor recognition is a major determinant of the host range, cross-species infections, and pathogenesis of the severe acute respiratory syndrome coronavirus (SARS-CoV). A defined receptor-binding domain (RBD) in the SARS-CoV spike protein specifically recognizes its host receptor, angiotensin-converting enzyme 2 (ACE2). This article reviews the latest knowledge about how RBDs from different SARS-CoV strains interact with ACE2 from several animal species. Detailed research on these RBD/ACE2 interactions has established important principles on host receptor adaptations, cross-species infections, and future evolution of SARS-CoV. These principles may apply to other emerging animal viruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV). This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.” PMID:23994189

  9. Cervical Cap

    Science.gov (United States)

    ... p020041. Accessed Nov. 11, 2014. Cervical cap About Advertisement Mayo Clinic does not endorse companies or products. ... a Job Site Map About This Site Twitter Facebook Google YouTube Pinterest Mayo Clinic is a not- ...

  10. Cervical Cap

    Science.gov (United States)

    ... pregnant during a year of typical use. This difference is due to the fact that the vagina and cervix are stretched by giving birth vaginally, which means the cervical cap may not fit as well. Inconsistent or incorrect ...

  11. Cradle Cap (For Parents)

    Science.gov (United States)

    ... Giving Teens a Voice in Health Care Decisions Cradle Cap (Infantile Seborrheic Dermatitis) KidsHealth > For Parents > Cradle Cap ( ... condition many babies develop called cradle cap. About Cradle Cap Cradle cap is the common term for seborrheic ...

  12. Cradle Cap

    Science.gov (United States)

    ... t contagious, and it's not caused by poor hygiene. Prevention Shampooing your baby's hair every few days can help prevent cradle cap. Stick with a mild baby shampoo unless your baby's doctor recommends something stronger. By Mayo Clinic Staff . Mayo Clinic Footer Legal Conditions and Terms Any ...

  13. Death cap

    DEFF Research Database (Denmark)

    Rudbæk, Torsten R; Kofoed, Pernille Bouteloup; Bove, Jeppe

    2014-01-01

    Death cap (Amanita phalloides) is commonly found and is one of the five most toxic fungi in Denmark. Toxicity is due to amatoxin, and poisoning is a serious medical condition, causing organ failure with potential fatal outcome. Acknowledgement and clarification of exposure, symptomatic and focused...

  14. Apical cap

    Energy Technology Data Exchange (ETDEWEB)

    McLoud, T.C.; Isler, R.J.; Novelline, R.A.; Putman, C.E.; Simeone, J.; Stark, P.

    1981-08-01

    Apical caps, either unilateral or bilateral, are a common feature of advancing age and are usually the result of subpleural scarring unassociated with other diseases. Pancoast (superior sulcus) tumors are a well recognized cause of unilateral asymmetric apical density. Other lesions arising in the lung, pleura, or extrapleural space may produce unilateral or bilateral apical caps. These include: (1) inflammatory: tuberculosis and extrapleural abscesses extending from the neck; (2) post radiation fibrosis after mantle therapy for Hodgkin disease or supraclavicular radiation in the treatment of breast carcinoma; (3) neoplasm: lymphoma extending from the neck or mediastinum, superior sulcus bronchogenic carcinoma, and metastases; (4) traumatic: extrapleural dissection of blood from a ruptured aorta, fractures of the ribs or spine, or hemorrhage due to subclavian line placement; (5) vascular: coarctation of the aorta with dilated collaterals over the apex, fistula between the subclavian artery and vein; and (6) miscellaneous: mediastinal lipomatosis with subcostal fat extending over the apices.

  15. Cap-binding complex (CBC)

    Science.gov (United States)

    Gonatopoulos-Pournatzis, Thomas; Cowling, Victoria H.

    2013-01-01

    The 7mG (7-methylguanosine cap) formed on mRNA is fundamental to eukaryotic gene expression. Protein complexes recruited to 7mG mediate key processing events throughout the lifetime of the transcript. One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation. Gene expression can be regulated by signalling pathways which influence CBC function. The aim of the present review is to discuss the mechanisms by which CBC mediates and co-ordinates multiple gene expression events. PMID:24354960

  16. Molecular Basis of Transcription-Coupled Pre-mRNA Capping

    NARCIS (Netherlands)

    Martinez-Rucobo, Fuensanta W.; Kohler, Rebecca; van de Waterbeemd, Michiel|info:eu-repo/dai/nl/412537761; Heck, Albert J R|info:eu-repo/dai/nl/105189332; Hemann, Matthias; Herzog, Franz; Stark, Holger; Cramer, Patrick

    2015-01-01

    Capping is the first step in pre-mRNA processing, and the resulting 5'-RNA cap is required for mRNA splicing, export, translation, and stability. Capping is functionally coupled to transcription by RNA polymerase (Pol) II, but the coupling mechanism remains unclear. We show that efficient binding of

  17. Cradle Cap: Treatment

    Science.gov (United States)

    Cradle cap Treatment Cradle cap usually doesn't require medical treatment. It clears up on its own within a few months. In the meantime, ... care tips can help you control and manage cradle cap. Gently rub your baby's scalp with your fingers ...

  18. Cradle Cap: Treatment

    Science.gov (United States)

    Cradle cap Treatment Cradle cap usually doesn't require medical treatment. It clears up on its own within a few months. In the meantime, wash ... tips can help you control and manage cradle cap. Gently rub your baby's scalp with your fingers ...

  19. Biochemical principles and inhibitors to interfere with viral capping pathways.

    Science.gov (United States)

    Decroly, Etienne; Canard, Bruno

    2017-06-01

    Messenger RNAs are decorated by a cap structure, which is essential for their translation into proteins. Many viruses have developed strategies in order to cap their mRNAs. The cap is either synthetized by a subset of viral or cellular enzymes, or stolen from capped cellular mRNAs by viral endonucleases ('cap-snatching'). Reverse genetic studies provide evidence that inhibition of viral enzymes belonging to the capping pathway leads to inhibition of virus replication. The replication defect results from reduced protein synthesis as well as from detection of incompletely capped RNAs by cellular innate immunity sensors. Thus, it is now admitted that capping enzymes are validated antiviral targets, as their inhibition will support an antiviral response in addition to the attenuation of viral mRNA translation. In this review, we describe the different viral enzymes involved in mRNA capping together with relevant inhibitors, and their biochemical features useful in inhibitor discovery. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The cervical cap.

    Science.gov (United States)

    1988-10-07

    The US Food and Drug Administration has approved marketing of the Prentif cavity-rim cervical cap. This contraceptive device is being distributed in the US and Canada by Cervical Cap Ltd, Los Gatos, California. The Prentif cap is available in 4 sizes: 22, 25, 28, and 31 mm inside diameter, with a length of 1 1/4-1 1/2 inches. In a multicenter trial involving 522 diaphragm users and 581 cap users followed for 2 years, the cap was 82.6% effective and the diaphragm was 83.3% effective in preventing pregnancy. When pregnancies attributable to user failure were excluded, these rates were increased to 93.6% for the cap and 95.4% for the diaphragm. 4% of cap users compared with only 1.7% of diaphragm users in this study developed abnormal Pap smears after 3 months of use; in addition, a higher proportion of cap users became infected with Gardnerella vaginalis and Monilia. Theoretical hazards include toxic shock syndrome and endometriosis due to backflow of menstrual fluids. Cap users are advised to undergo a Pap test after 3 months of use and discontinue cap use if the results are abnormal. The cap should not be used during menstruation. Although the cap can be left in place for up to 48 hours, its position should be checked before and after each episode of intercourse. The cervical cap requires less spermicide than the diaphragm and is not as messy. In addition, it can be left in the vagina twice as long as the diaphragm, without additional spermicide. Since the cap is smaller than the diaphragm and does not cover the vaginal wall, some women find intercourse more pleasurable with this device.

  1. Molecular mechanisms of coronavirus RNA capping and methylation.

    Science.gov (United States)

    Chen, Yu; Guo, Deyin

    2016-02-01

    The 5'-cap structures of eukaryotic mRNAs are important for RNA stability, pre-mRNA splicing, mRNA export, and protein translation. Many viruses have evolved mechanisms for generating their own cap structures with methylation at the N7 position of the capped guanine and the ribose 2'-Oposition of the first nucleotide, which help viral RNAs escape recognition by the host innate immune system. The RNA genomes of coronavirus were identified to have 5'-caps in the early 1980s. However, for decades the RNA capping mechanisms of coronaviruses remained unknown. Since 2003, the outbreak of severe acute respiratory syndrome coronavirus has drawn increased attention and stimulated numerous studies on the molecular virology of coronaviruses. Here, we review the current understanding of the mechanisms adopted by coronaviruses to produce the 5'-cap structure and methylation modification of viral genomic RNAs.

  2. The viral RNA capping machinery as a target for antiviral drugs.

    Science.gov (United States)

    Ferron, François; Decroly, Etienne; Selisko, Barbara; Canard, Bruno

    2012-10-01

    Most viruses modify their genomic and mRNA 5'-ends with the addition of an RNA cap, allowing efficient mRNA translation, limiting degradation by cellular 5'-3' exonucleases, and avoiding its recognition as foreign RNA by the host cell. Viral RNA caps can be synthesized or acquired through the use of a capping machinery which exhibits a significant diversity in organization, structure and mechanism relative to that of their cellular host. Therefore, viral RNA capping has emerged as an interesting field for antiviral drug design. Here, we review the different pathways and mechanisms used to produce viral mRNA 5'-caps, and present current structures, mechanisms, and inhibitors known to act on viral RNA capping. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism.

    Directory of Open Access Journals (Sweden)

    Moheshwarnath Issur

    Full Text Available RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5' ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

  4. CENTRIFUGE END CAP

    Science.gov (United States)

    Beams, J.W.; Snoddy, L.B.

    1960-08-01

    An end cap for ultra-gas centrifuges is designed to impart or remove angular momentum to or from the gas and to bring the entering gas to the temperature of the gas inside the centrifuge. The end cap is provided with slots or fins for adjusting the temperature and the angular momentum of the entering gas to the temperature and momentum of the gas in the centrifuge and is constructed to introduce both the inner and the peripheral stream into the centrifuge.

  5. Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

    Science.gov (United States)

    2011-01-06

    with 1 U/ml neuraminidase from Clostridium perfringens (Sigma) for 24 hours or with 100 nM bafilomycin A1 (Baf A1, Sigma) for 6 hours before influenza...were incubated with infectious (A) MARV or (B) EBOV at indicated MOIs for 1 hour and then maintained in growth medium. After 72 hours, culture...HeLa cells were treated with 5000 U/ml IFN-b or maintained in growth medium for 48 hours before infection with the indicated pseudoviruses. Differences

  6. The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus.

    Science.gov (United States)

    McDermott, Jason E; Mitchell, Hugh D; Gralinski, Lisa E; Eisfeld, Amie J; Josset, Laurence; Bankhead, Armand; Neumann, Gabriele; Tilton, Susan C; Schäfer, Alexandra; Li, Chengjun; Fan, Shufang; McWeeney, Shannon; Baric, Ralph S; Katze, Michael G; Waters, Katrina M

    2016-09-23

    The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ anti-immune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine tumor necrosis factor alpha (TNFα) promote pathogenesis, presumably through excessive inflammation. The current study provides validation of network modeling approaches for identifying important players in virus infection pathogenesis, and a step forward in understanding the host response to an important infectious disease. The results presented here suggest the role of Kepi in the host response to SARS-CoV, as well as inflammatory activity driving pathogenesis through TNFα signaling in SARS-CoV infections. Though we have reported the utility of this approach in bacterial and cell culture studies previously, this is the first comprehensive study to confirm that network topology can be used to predict phenotypes in mice with experimental validation.

  7. Protection from SARS coronavirus conferred by live measles vaccine expressing the spike glycoprotein.

    Science.gov (United States)

    Escriou, Nicolas; Callendret, Benoît; Lorin, Valérie; Combredet, Chantal; Marianneau, Philippe; Février, Michèle; Tangy, Frédéric

    2014-03-01

    The recent identification of a novel human coronavirus responsible of a SARS-like illness in the Middle-East a decade after the SARS pandemic, demonstrates that reemergence of a SARS-like coronavirus from an animal reservoir remains a credible threat. Because SARS is contracted by aerosolized contamination of the respiratory tract, a vaccine inducing mucosal long-term protection would be an asset to control new epidemics. To this aim, we generated live attenuated recombinant measles vaccine (MV) candidates expressing either the membrane-anchored SARS-CoV spike (S) protein or its secreted soluble ectodomain (Ssol). In mice susceptible to measles virus, recombinant MV expressing the anchored full-length S induced the highest titers of neutralizing antibodies and fully protected immunized animals from intranasal infectious challenge with SARS-CoV. As compared to immunization with adjuvanted recombinant Ssol protein, recombinant MV induced stronger and Th1-biased responses, a hallmark of live attenuated viruses and a highly desirable feature for an antiviral vaccine. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants

    NARCIS (Netherlands)

    ter Meulen, Jan; van den Brink, Edward N.; Poon, Leo L. M.; Marissen, Wilfred E.; Leung, Cynthia S. W.; Cox, Freek; Cheung, Chung Y.; Bakker, Arjen Q.; Bogaards, Johannes A.; van Deventer, Els; Preiser, Wolfgang; Doerr, Hans Wilhelm; Chow, Vincent T.; de Kruif, John; Peiris, Joseph S. M.; Goudsmit, Jaap

    2006-01-01

    BACKGROUND: Experimental animal data show that protection against severe acute respiratory syndrome coronavirus (SARS-CoV) infection with human monoclonal antibodies (mAbs) is feasible. For an effective immune prophylaxis in humans, broad coverage of different strains of SARS-CoV and control of

  9. Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

    Science.gov (United States)

    Wu, Cheng Guo; Cheng, Shu Chun; Chen, Shiang Chuan; Li, Juo Yan; Fang, Yi Hsuan; Chen, Yau Hung; Chou, Chi Yuan

    2013-05-01

    The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

  10. Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant.

    Science.gov (United States)

    McPherson, Clifton; Chubet, Richard; Holtz, Kathy; Honda-Okubo, Yoshikazu; Barnard, Dale; Cox, Manon; Petrovsky, Nikolai

    2016-01-01

    Given periodic outbreaks of fatal human infections caused by coronaviruses, development of an optimal coronavirus vaccine platform capable of rapid production is an ongoing priority. This chapter describes the use of an insect cell expression system for rapid production of a recombinant vaccine against severe acute respiratory syndrome coronavirus (SARS). Detailed methods are presented for expression, purification, and release testing of SARS recombinant spike protein antigen, followed by adjuvant formulation and animal testing. The methods herein described for rapid development of a highly protective SARS vaccine are equally suited to rapid development of vaccines against other fatal human coronavirus infections, e.g., the MERS coronavirus.

  11. Ezrin Interacts with the SARS Coronavirus Spike Protein and Restrains Infection at the Entry Stage

    Science.gov (United States)

    Millet, Jean Kaoru; Kien, François; Cheung, Chung-Yan; Siu, Yu-Lam; Chan, Wing-Lim; Li, Huiying; Leung, Hiu-Lan; Jaume, Martial; Bruzzone, Roberto; Malik Peiris, Joseph S.; Altmeyer, Ralf Marius; Nal, Béatrice

    2012-01-01

    Background Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S). There are still many unknowns on the implication of cellular factors that regulate the entry process. Methodology/Principal Findings We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. Conclusions/Significance Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection. PMID:23185364

  12. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

    Science.gov (United States)

    Roh, Changhyun

    2012-01-01

    Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

  13. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    Science.gov (United States)

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses." Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Disulfiram can inhibit MERS and SARS coronavirus papain-like proteases via different modes.

    Science.gov (United States)

    Lin, Min-Han; Moses, David C; Hsieh, Chih-Hua; Cheng, Shu-Chun; Chen, Yau-Hung; Sun, Chiao-Yin; Chou, Chi-Yuan

    2017-12-28

    Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in southern China in late 2002 and caused a global outbreak with a fatality rate around 10% in 2003. Ten years later, a second highly pathogenic human CoV, MERS-CoV, emerged in the Middle East and has spread to other countries in Europe, North Africa, North America and Asia. As of November 2017, MERS-CoV had infected at least 2102 people with a fatality rate of about 35% globally, and hence there is an urgent need to identify antiviral drugs that are active against MERS-CoV. Here we show that a clinically available alcohol-aversive drug, disulfiram, can inhibit the papain-like proteases (PLpros) of MERS-CoV and SARS-CoV. Our findings suggest that disulfiram acts as an allosteric inhibitor of MERS-CoV PLpro but as a competitive (or mixed) inhibitor of SARS-CoV PLpro. The phenomenon of slow-binding inhibition and the irrecoverability of enzyme activity after removing unbound disulfiram indicate covalent inactivation of SARS-CoV PLpro by disulfiram, while synergistic inhibition of MERS-CoV PLpro by disulfiram and 6-thioguanine or mycophenolic acid implies the potential for combination treatments using these three clinically available drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.

    Science.gov (United States)

    Wang, Sheng-Fan; Tseng, Sung-Pin; Yen, Chia-Hung; Yang, Jyh-Yuan; Tsao, Ching-Han; Shen, Chun-Wei; Chen, Kuan-Hsuan; Liu, Fu-Tong; Liu, Wu-Tse; Chen, Yi-Ming Arthur; Huang, Jason C

    2014-08-22

    The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. The SARS Coronavirus 3a protein binds calcium in its cytoplasmic domain.

    Science.gov (United States)

    Minakshi, Rinki; Padhan, Kartika; Rehman, Safikur; Hassan, Md Imtaiyaz; Ahmad, Faizan

    2014-10-13

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a positive stranded RNA virus with ∼30kb genome. Among all open reading frames (orfs) of this virus, the orf3a is the largest, and encodes a protein of 274 amino acids, named as 3a protein. Sequence analysis suggests that the orf3a aligned to one calcium pump present in Plasmodium falciparum and the enzyme glutamine synthetase found in Leptospira interrogans. This sequence similarity was found to be limited only to amino acid residues 209-264 which form the cytoplasmic domain of the orf3a. Furthermore, this region was predicted to be involved in the calcium binding. Owing to this hypothesis, we were driven to establish its calcium binding property in vitro. Here, we expressed and purified the cytoplasmic domain of the 3a protein, called Cyto3a, as a recombinant His-tagged protein in the E. coli. The calcium binding nature was established by performing various staining methods such as ruthenium red and stains-all. (45)Ca overlay method was also done to further support the data. Since the 3a protein forms ion channels, we were interested to see any conformational changes occurring in the Cyot3a upon calcium binding, using fluorescence spectroscopy and circular dichroism. These studies clearly indicate a significant change in the conformation of the Cyto3a protein after binding with calcium. Our results strongly suggest that the cytoplasmic domain of the 3a protein of SARS-CoV binds calcium in vitro, causing a change in protein conformation. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage.

    Directory of Open Access Journals (Sweden)

    Jean Kaoru Millet

    Full Text Available BACKGROUND: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S. There are still many unknowns on the implication of cellular factors that regulate the entry process. METHODOLOGY/PRINCIPAL FINDINGS: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. CONCLUSIONS/SIGNIFICANCE: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.

  18. The SARS coronavirus spike glycoprotein is selectively recognized by lung surfactant protein D and activates macrophages

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Zhong, Fei; Chow, Vincent T K

    2007-01-01

    Da glycosylated protein. It was not secreted in the presence of tunicamycin and was detected as a 130 kDa protein in the cell lysate. The purified S-protein bound to Vero but not 293T cells and was itself recognized by lung surfactant protein D (SP-D), a collectin found in the lung alveoli. The binding required......The severe acute respiratory syndrome coronavirus (SARS-CoV) infects host cells with its surface glycosylated spike-protein (S-protein). Here we expressed the SARS-CoV S-protein to investigate its interactions with innate immune mechanisms in the lung. The purified S-protein was detected as a 210 k...... Ca(2+) and was inhibited by maltose. The serum collectin, mannan-binding lectin (MBL), exhibited no detectable binding to the purified S-protein. S-protein binds and activates macrophages but not dendritic cells (DCs). It suggests that SARS-CoV interacts with innate immune mechanisms in the lung...

  19. The 3a Protein of SARS-coronavirus Induces Apoptosis in Vero E6 Cells.

    Science.gov (United States)

    Y Waye, Mary; W Law, Patrick; Wong, Chi-Hang; C Au, Thomas; Chuck, Chi-Pang; Kong, Siu-Kai; S Chan, Paul; To, Ka-Fai; I Lo, Anthony; W Chan, Judy; Suen, Yick-Keung; Edwin Chan, H Y; Fung, Kwok-Pui; Y Sung, Joseph; Lo, Y M; W Tsui, Stephen

    2005-01-01

    An outbreak of severe acute respiratory syndrome (SARS) occurred in China and the first case emerged in mid November 2002. The etiologic agent of this disease was found to be a previously unknown coronavirus, SARS-CoV. The detailed pathology of SARS-CoV infection and the host response to the viral infection are still not known. The 3a gene encodes a non-structural viral protein which is predicted to be a transmembrane protein. In this study, we showed that the 3a protein was localized to the endoplasmic reticulum (ER) in 3a-transfected monkey kidney Vero E6 cells. In vitro experiments of chromatin condensation and DNA fragmentation suggest that the 3a protein may trigger apoptosis. Our data show that over-expression of a single SARS-CoV protein can induce apoptosis in vitro. Thus GFP-3a fusion protein could also be used as a biosensor for monitoring the cytopathic features of SARS infection, e.g. lymphopenia, in animal model systems, similar to nucleocapsid and 7a proteins.

  20. SARS coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon.

    Science.gov (United States)

    Totura, Allison L; Baric, Ralph S

    2012-06-01

    SARS-CoV is a pathogenic coronavirus that emerged from a zoonotic reservoir, leading to global dissemination of the virus. The association SARS-CoV with aberrant cytokine, chemokine, and Interferon Stimulated Gene (ISG) responses in patients provided evidence that SARS-CoV pathogenesis is at least partially controlled by innate immune signaling. Utilizing models for SARS-CoV infection, key components of innate immune signaling pathways have been identified as protective factors against SARS-CoV disease, including STAT1 and MyD88. Gene transcription signatures unique to SARS-CoV disease states have been identified, but host factors that regulate exacerbated disease phenotypes still remain largely undetermined. SARS-CoV encodes several proteins that modulate innate immune signaling through the antagonism of the induction of Interferon and by avoidance of ISG effector functions. Copyright © 2012. Published by Elsevier B.V.

  1. The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    McDermott, Jason E.; Mitchell, Hugh D.; Gralinski, Lisa E.; Eisfeld, Amie J.; Josset, Laurence; Bankhead, Armand; Neumann, Gabriele; Tilton, Susan C.; Schäfer, Alexandra; Li, Chengjun; Fan, Shufang; McWeeney, Shannon; Baric, Ralph S.; Katze, Michael G.; Waters, Katrina M.

    2016-09-23

    The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ antiimmune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), promote pathogenesis through a parallel feed-forward circuit that promotes inflammation. These results are consistent with previous studies showing the role of over-stimulation of the inflammatory response to SARS-CoV in pathogenesis. We conclude that the critical balance between immune response and inflammation can be manipulated to improve the outcome of the infection. Further, our study provides two potential therapeutic strategies for mitigating the effects of SARS-CoV infection, and may provide insight into treatment strategies for Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

  2. Inactivation of SARS coronavirus by means of povidone-iodine, physical conditions, and chemical reagents

    OpenAIRE

    Kariwa, Hiroaki; Fujii, Nobuhiro; TAKASHIMA, Ikuo

    2004-01-01

    products, a number of other chemical agents, and various physical conditions were evaluated for their ability to inactivate the severe acute respiratory syndrome coronavirus (SARS-CoV). Treatment of SARS-CoV with PVP-I products for 2 min reduced the virus infectivity from 1.17 x 10⁶ TCID₅₀/ml to below the detectable level. The efficacy of 70% ethanol was equivalent to that of PVP-I products. Fixation of SARS-CoV-infected Vero E6 cells with a fixative including formalin, glutaraldehyde, methan...

  3. Induction of neutralising antibodies and cellular immune responses against SARS coronavirus by recombinant measles viruses.

    Science.gov (United States)

    Liniger, Matthias; Zuniga, Armando; Tamin, Azaibi; Azzouz-Morin, Teldja N; Knuchel, Marlyse; Marty, Rene R; Wiegand, Marian; Weibel, Sara; Kelvin, David; Rota, Paul A; Naim, Hussein Y

    2008-04-16

    Live attenuated recombinant measles viruses (rMV) expressing a codon-optimised spike glycoprotein (S) or nucleocapsid protein (N) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) were generated (rMV-S and rMV-N). Both recombinant viruses stably expressed the corresponding SARS-CoV proteins, grew to similar end titres as the parental strain and induced high antibody titres against MV and the vectored SARS-CoV antigens (S and N) in transgenic mice susceptible to measles infection. The antibodies induced by rMV-S had a high neutralising effect on SARS-CoV as well as on MV. Moreover, significant N-specific cellular immune responses were measured by IFN-gamma ELISPOT assays. The pre-existence of anti-MV antibodies induced by the initial immunisation dose did not inhibit boost of anti-S and anti-N antibodies. Immunisations comprising a mixture of rMV-S and rMV-N induced immune responses similar in magnitude to that of vaccine components administered separately. These data support the suitability of MV as a bivalent candidate vaccine vector against MV and emerging viruses such as SARS-CoV.

  4. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.

    Directory of Open Access Journals (Sweden)

    Susanne Pfefferle

    2011-10-01

    Full Text Available Coronaviruses (CoVs are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS in 2002/2003 has demonstrated human vulnerability to (Coronavirus CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B as interaction partners of the CoV non-structural protein 1 (Nsp1. These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

  5. Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

    Directory of Open Access Journals (Sweden)

    Albrecht von Brunn

    2007-05-01

    Full Text Available The severe acute respiratory syndrome coronavirus (SARS-CoV genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

  6. Dynamics of SARS-coronavirus HR2 domain in the prefusion and transition states

    Science.gov (United States)

    McReynolds, Susanna; Jiang, Shaokai; Rong, Lijun; Caffrey, Michael

    2009-12-01

    The envelope glycoproteins S1 and S2 of severe acute respiratory syndrome coronavirus (SARS-CoV) mediate viral entry by conformational change from a prefusion state to a postfusion state that enables fusion of the viral and target membranes. In this work we present the characterization of the dynamic properties of the SARS-CoV S2-HR2 domain (residues 1141-1193 of S) in the prefusion and newly discovered transition states by NMR 15N relaxation studies. The dynamic properties of the different states, which are stabilized under different experimental conditions, extend the current model of viral membrane fusion and give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses such as HIV.

  7. Structure and inhibition of the SARS coronavirus envelope protein ion channel.

    Directory of Open Access Journals (Sweden)

    Konstantin Pervushin

    2009-07-01

    Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

  8. The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

    Science.gov (United States)

    McBride, Ruth; Fielding, Burtram C.

    2012-01-01

    A respiratory disease caused by a novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), was first reported in China in late 2002. The subsequent efficient human-to-human transmission of this virus eventually affected more than 30 countries worldwide, resulting in a mortality rate of ~10% of infected individuals. The spread of the virus was ultimately controlled by isolation of infected individuals and there has been no infections reported since April 2004. However, the natural reservoir of the virus was never identified and it is not known if this virus will re-emerge and, therefore, research on this virus continues. The SARS-CoV genome is about 30 kb in length and is predicted to contain 14 functional open reading frames (ORFs). The genome encodes for proteins that are homologous to known coronavirus proteins, such as the replicase proteins (ORFs 1a and 1b) and the four major structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). SARS-CoV also encodes for eight unique proteins, called accessory proteins, with no known homologues. This review will summarize the current knowledge on SARS-CoV accessory proteins and will include: (i) expression and processing; (ii) the effects on cellular processes; and (iii) functional studies. PMID:23202509

  9. Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

    Energy Technology Data Exchange (ETDEWEB)

    Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre, E-mail: marie-pierre.egloff@afmb.univ-mrs.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

    2006-04-01

    Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4{sub 1}32 or P4{sub 3}32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å.

  10. A Phrygian Cap

    Directory of Open Access Journals (Sweden)

    Marie-Janne S. van Kamp

    2013-08-01

    Full Text Available A Phrygian cap is a congenital anomaly of the gallbladder with an incidence of 4%. It can simulate a mass in the liver during hepatobiliary imaging and is sometimes mistaken for pathology. A Phrygian cap, however, has no pathological significance and normally causes no symptoms. A case will be presented where a Phrygian cap was found by coincidence during surgery. The patient was operated for colon cancer with liver metastasis in segment V. He underwent a simultaneous right hemicolectomy and wedge resection of the liver lesion. During perioperative inspection, a gallbladder with a folded fundus was seen. This deformity was, in retrospective, detected on the preoperative MRI scan. The patient underwent cholecystectomy to make the wedge resection easier to perform. Otherwise, cholecystectomy for a Phrygian cap is only indicated in case of symptoms. Radiographic imaging can be helpful in narrowing the differential diagnosis. To our knowledge, there is no recent literature about the Phrygian cap and its imaging aspects. Nowadays, multiphase MRI, or multiphase CT in case of MRI contraindication, are the first choices of hepatobiliary imaging.

  11. Microtubule's conformational cap

    DEFF Research Database (Denmark)

    Chretien, D.; Janosi, I.; Taveau, J.C.

    1999-01-01

    The molecular mechanisms that allow elongation of the unstable microtubule lattice remain unclear. It is usually thought that the GDP-liganded tubulin lattice is capped by a small layer of GTP- or GDP-P(i)-liganded molecules, the so called "GTP-cap". Here, we point-out that the elastic properties...... of the microtubule lattice cause a difference in stability between the elongating tubulin sheet and the completed microtubule wall. The implications of our observations for microtubule structure and dynamics are discussed....

  12. Synthesis of 5′ cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N7)-methyl transferase

    Science.gov (United States)

    Thillier, Yann; Decroly, Etienne; Morvan, François; Canard, Bruno; Vasseur, Jean-Jacques; Debart, Françoise

    2012-01-01

    The 5′ end of eukaryotic mRNA carries a N7-methylguanosine residue linked by a 5′-5′ triphosphate bond. This cap moiety (7mGpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 5′ exonucleases and avoiding its recognition as “nonself” by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 5′-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N7-methylguanosine, the synthesis of RNAs possessing a cap structure at the 5′ end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 2′-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 5′-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN2′-Om-RNAs were purified before the N7-methyl group was added by enzymatic means using the human (guanine-N7)-methyl transferase to yield 7mGpppN-RNAs (cap-0) or 7mGpppN2′-Om-RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies. PMID:22334760

  13. Synthesis of 5' cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase.

    Science.gov (United States)

    Thillier, Yann; Decroly, Etienne; Morvan, François; Canard, Bruno; Vasseur, Jean-Jacques; Debart, Françoise

    2012-04-01

    The 5' end of eukaryotic mRNA carries a N(7)-methylguanosine residue linked by a 5'-5' triphosphate bond. This cap moiety ((7m)GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 5' exonucleases and avoiding its recognition as "nonself" by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 5'-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N(7)-methylguanosine, the synthesis of RNAs possessing a cap structure at the 5' end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 2'-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 5'-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN(2'-Om)-RNAs were purified before the N(7)-methyl group was added by enzymatic means using the human (guanine-N(7))-methyl transferase to yield (7m)GpppN-RNAs (cap-0) or (7m)GpppN(2'-Om)-RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.

  14. Cradle Cap: Symptoms and Causes

    Science.gov (United States)

    Cradle cap Overview Cradle cap causes crusty or oily scaly patches on a baby's scalp. The condition isn't painful or itchy. But it ... yellow scales that aren't easy to remove. Cradle cap usually clears up on its own in a ...

  15. Designing Smart Charter School Caps

    Science.gov (United States)

    Dillon, Erin

    2010-01-01

    In 2007, Andrew J. Rotherham proposed a new approach to the contentious issue of charter school caps, the statutory limits on charter school growth in place in several states. Rotherham's proposal, termed "smart charter school caps," called for quality sensitive caps that allow the expansion of high-performing charter schools while also…

  16. Modeling Nanotube Caps: The Relationship Between Fullerenes and Caps.

    Science.gov (United States)

    Melle-Franco, Manuel; Brinkmann, Gunnar; Zerbetto, Francesco

    2015-12-24

    We present a novel method to calculate energies of nanotube caps with different levels of accuracy and a comprehensive study of its application to the IPR caps of the (10,0) carbon nanotube. The two most stable caps for (10,0) have 42 atoms, an energy of 8.7 eV, and correspond to sections of the third most abundant fullerene, C84. These caps are isoenergetic with a chemically unstable cap with 40 carbon atoms related to a C80 isomer that is also chemically unstable. Energies for the other caps are between 9.3 and 10 eV. A method to calculate cap energetics with fullerenes with an error less than 3% is also presented.

  17. Polar Cap Patch Dynamics

    Science.gov (United States)

    2013-04-25

    associated with the rotation of the extreme density may in itself lead to a stronger growth of ionospheric irregularities . These irregularities may...continue to grow all the way across the polar cap. The result is more efficient creation of ionospheric irregularities . Title 4: Motion of polar...the cusp ionosphere over Svalbard to investigate the production of decameter scale irregularities in the electron plasma associated with HF radar

  18. Saltstone Clean Cap Formulation

    Energy Technology Data Exchange (ETDEWEB)

    Langton, C

    2005-04-22

    The current operation strategy for using Saltstone Vault 4 to receive 0.2 Ci/gallon salt solution waste involves pouring a clean grout layer over the radioactive grout prior to initiating pour into another cell. This will minimize the radiating surface area and reduce the dose rate at the vault and surrounding area. The Clean Cap will be used to shield about four feet of Saltstone poured into a Z-Area vault cell prior to moving to another cell. The minimum thickness of the Clean Cap layer will be determined by the cesium concentration and resulting dose levels and it is expected to be about one foot thick based on current calculations for 0.1 Ci Saltstone that is produced in the Saltstone process by stabilization of 0.2 Ci salt solution. This report documents experiments performed to identify a formulation for the Clean Cap. Thermal transient calculations, adiabatic temperature rise measurements, pour height, time between pour calculations and shielding calculations were beyond the scope and time limitations of this study. However, data required for shielding calculations (composition and specific gravity) are provided for shielding calculations. The approach used to design a Clean Cap formulation was to produce a slurry from the reference premix (10/45/45 weight percent cement/slag/fly ash) and domestic water that resembled as closely as possible the properties of the Saltstone slurry. In addition, options were investigated that may offer advantages such as less bleed water and less heat generation. The options with less bleed water required addition of dispersants. The options with lower heat contained more fly ash and less slag. A mix containing 10/45/45 weight percent cement/slag/fly ash with a water to premix ratio of 0.60 is recommended for the Clean Cap. Although this mix may generate more than 3 volume percent standing water (bleed water), it has rheological, mixing and flow properties that are similar to previously processed Saltstone. The recommended

  19. Coupling mRNA processing with transcription in time and space.

    Science.gov (United States)

    Bentley, David L

    2014-03-01

    Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3' end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes.

  20. Recognition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: implications for RNA-capping mechanisms in Flavivirus.

    Science.gov (United States)

    Bollati, Michela; Milani, Mario; Mastrangelo, Eloise; Ricagno, Stefano; Tedeschi, Gabriella; Nonnis, Simona; Decroly, Etienne; Selisko, Barbara; de Lamballerie, Xavier; Coutard, Bruno; Canard, Bruno; Bolognesi, Martino

    2009-01-09

    The mRNA-capping process starts with the conversion of a 5'-triphosphate end into a 5'-diphosphate by an RNA triphosphatase, followed by the addition of a guanosine monophosphate unit in a 5'-5' phosphodiester bond by a guanylyltransferase. Methyltransferases are involved in the third step of the process, transferring a methyl group from S-adenosyl-l-methionine to N7-guanine (cap 0) and to the ribose 2'OH group (cap 1) of the first RNA nucleotide; capping is essential for mRNA stability and proper replication. In the genus Flavivirus, N7-methyltransferase and 2'O-methyltransferase activities have been recently associated with the N-terminal domain of the viral NS5 protein. In order to further characterize the series of enzymatic reactions that support capping, we analyzed the crystal structures of Wesselsbron virus methyltransferase in complex with the S-adenosyl-l-methionine cofactor, S-adenosyl-l-homocysteine (the product of the methylation reaction), Sinefungin (a molecular analogue of the enzyme cofactor), and three different cap analogues (GpppG, (N7Me)GpppG, and (N7Me)GpppA). The structural results, together with those on other flaviviral methyltransferases, show that the capped RNA analogues all bind to an RNA high-affinity binding site. However, lack of specific interactions between the enzyme and the first nucleotide of the RNA chain suggests the requirement of a minimal number of nucleotides following the cap to strengthen protein/RNA interaction. Our data also show that, following incubation with guanosine triphosphate, Wesselsbron virus methyltransferase displays a guanosine monophosphate molecule covalently bound to residue Lys28, hinting at possible implications for the transfer of a guanine group to ppRNA. The structures of the Wesselsbron virus methyltransferase complexes obtained are discussed in the context of a model for N7-methyltransferase and 2'O-methyltransferase activities.

  1. Alternative splicing variations in mouse CAPS2: differential expression and functional properties of splicing variants

    Directory of Open Access Journals (Sweden)

    Furuichi Teiichi

    2007-04-01

    Full Text Available Abstract Background Ca2+-dependent activator protein 2 (CAPS2/CADPS2 is a secretory vesicle-associated protein involved in the release of neurotrophin. We recently reported that an aberrant, alternatively spliced CAPS2 mRNA that lacks exon 3 (CAPS2Δexon3 is detected in some patients with autism. Splicing variations in mouse CAPS2 and their expression and functions remain unclear. Results In this study, we defined 31 exons in the mouse CAPS2 gene and identified six alternative splicing variants, CAPS2a-f. CAPS2a is an isoform lacking exons 22 and 25, which encode part of the Munc13-1-homologous domain (MHD. CAPS2b lacks exon 25. CAPS2c lacks exons 11 and 22. CAPS2d, 2e, and 2f have C-terminal deletions from exon 14, exon 12, and exon 5, respectively. On the other hand, a mouse counterpart of CAPS2Δexon3 was not detected in the mouse tissues tested. CAPS2b was expressed exclusively in the brain, and the other isoforms were highly expressed in the brain, but also in some non-neural tissues. In the brain, all isoforms showed predominant expression patterns in the cerebellum. In the developing cerebellum, CAPS2b showed an up-regulated expression pattern, whereas the other isoforms exhibited transiently peaked expression patterns. CAPS2 proteins were mostly recovered in soluble fractions, but some were present in membrane fractions, except for CAPS2c and 2f, both of which lack the PH domain, suggesting that the PH domain is important for membrane association. In contrast to CAPS2a and 2b, CAPS2c showed slightly decreased BDNF-releasing activity, which is likely due to the C-terminal truncation of the PH domain in CAPS2c. Conclusion This study indicates that, in mouse, there are six splicing variants of CAPS2 (CAPS2a-f, and that these are subdivided into two groups: a long form containing the C-terminal MHD and a short form lacking the C-terminal MHD. These results demonstrate that the splicing variations correlate with their expression patterns and

  2. Genetics Home Reference: cap myopathy

    Science.gov (United States)

    ... expand/collapse boxes. Description Cap myopathy is a disorder that primarily affects skeletal muscles , which are muscles that the body uses for ... Ochala J. Thin filament proteins mutations associated with skeletal myopathies: ... in cap disease associated with beta-tropomyosin (TPM2) mutations. Neurology. 2008 ...

  3. ATLAS - End-Cap calorimeter

    CERN Multimedia

    CERN Audiovisual Unit

    2006-01-01

    The End-cap calorimeter was moved with the help of the rails and this calorimeter will measure the energy of particles close to the beam axis when protons collide. Cooling is important for maximum detector efficiency.

  4. Cap-binding complex (CBC)

    National Research Council Canada - National Science Library

    Gonatopoulos-Pournatzis, Thomas; Cowling, Victoria H

    2014-01-01

    .... One of the most important mediators of 7mG functions is CBC (cap-binding complex). CBC has a key role in several gene expression mechanisms, including transcription, splicing, transcript export and translation...

  5. Localized IRES-dependent translation of ER chaperone protein mRNA in sensory axons.

    Directory of Open Access Journals (Sweden)

    Almudena Pacheco

    Full Text Available Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.

  6. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    Energy Technology Data Exchange (ETDEWEB)

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  7. The North Zealand CAP Monitor

    DEFF Research Database (Denmark)

    Nielsen, Minna; Ravn, Pernille; Notander Clausen, Lise

    Contect We describe how we developed a monitoring system for community acquired pneumonia (CAP) at North Zealand Regional hospital. We serve 310.000 inhabitants and annually around 3200 patients with CAP are admitted. As part of a program of clinical pathways for common conditions, a pathway...... nurses and two senior doctors. Direct observations of the clinical processes revealed problems of coordination, complex disease trajectories that did not fit with the pneumonia pathway, unclear guidelines and variation in their interpretation. Intervention We designed a measurement system to monitor...

  8. Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates.

    Science.gov (United States)

    Wang, Qidi; Zhang, Lianfeng; Kuwahara, Kazuhiko; Li, Li; Liu, Zijie; Li, Taisheng; Zhu, Hua; Liu, Jiangning; Xu, Yanfeng; Xie, Jing; Morioka, Hiroshi; Sakaguchi, Nobuo; Qin, Chuan; Liu, Gang

    2016-05-13

    Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

  9. Allelic Variation in the Toll-Like Receptor Adaptor Protein Ticam2 Contributes to SARS-Coronavirus Pathogenesis in Mice.

    Science.gov (United States)

    Gralinski, Lisa E; Menachery, Vineet D; Morgan, Andrew P; Totura, Allison L; Beall, Anne; Kocher, Jacob; Plante, Jessica; Harrison-Shostak, D Corinne; Schäfer, Alexandra; Pardo-Manuel de Villena, Fernando; Ferris, Martin T; Baric, Ralph S

    2017-06-07

    Host genetic variation is known to contribute to differential pathogenesis following infection. Mouse models allow direct assessment of host genetic factors responsible for susceptibility to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). Based on an assessment of early stage lines from the Collaborative Cross mouse multi-parent population, we identified two lines showing highly divergent susceptibilities to SARS-CoV: the resistant CC003/Unc and the susceptible CC053/Unc. We generated 264 F2 mice between these strains, and infected them with SARS-CoV. Weight loss, pulmonary hemorrhage, and viral load were all highly correlated disease phenotypes. We identified a quantitative trait locus of major effect on chromosome 18 (27.1-58.6 Mb) which affected weight loss, viral titer and hemorrhage. Additionally, each of these three phenotypes had distinct quantitative trait loci [Chr 9 (weight loss), Chrs 7 and 12 (virus titer), and Chr 15 (hemorrhage)]. We identified Ticam2, an adaptor protein in the TLR signaling pathways, as a candidate driving differential disease at the Chr 18 locus. Ticam2-/- mice were highly susceptible to SARS-CoV infection, exhibiting increased weight loss and more pulmonary hemorrhage than control mice. These results indicate a critical role for Ticam2 in SARS-CoV disease, and highlight the importance of host genetic variation in disease responses. Copyright © 2017 Gralinski et al.

  10. Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease.

    Science.gov (United States)

    Konno, Hiroyuki; Wakabayashi, Masaki; Takanuma, Daiki; Saito, Yota; Akaji, Kenichi

    2016-03-15

    Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis

    NARCIS (Netherlands)

    Hamming, [No Value; Timens, W; Bulthuis, MLC; Lely, AT; Navis, GJ; van Goor, H

    Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Recently, a metallopeptidase named angiotensin-converting enzyme 2 (ACE2) has been

  12. Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

    Science.gov (United States)

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-12-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

    Science.gov (United States)

    Müller, Marcel A; Raj, V Stalin; Muth, Doreen; Meyer, Benjamin; Kallies, Stephan; Smits, Saskia L; Wollny, Robert; Bestebroer, Theo M; Specht, Sabine; Suliman, Tasnim; Zimmermann, Katrin; Binger, Tabea; Eckerle, Isabella; Tschapka, Marco; Zaki, Ali M; Osterhaus, Albert D M E; Fouchier, Ron A M; Haagmans, Bart L; Drosten, Christian

    2012-12-11

    A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is highly critical, because the SARS-CoV receptor influenced pathology, and its localization in the deep respiratory tract is thought to have restricted the transmissibility of SARS. Our data show that hCoV-EMC does not need the SARS-CoV receptor to infect human cells. Moreover, the virus is capable of infecting human, pig, and bat cells. This is remarkable, as human CoVs normally cannot replicate in bat cells as a consequence of host adaptation. Our results implicate that the new virus might use a receptor that is conserved between bats, pigs and humans suggesting a low barrier against cross-host transmission.

  14. Extraordinary GU-rich single-strand RNA identified from SARS coronavirus contributes an excessive innate immune response.

    Science.gov (United States)

    Li, Yan; Chen, Ming; Cao, Hongwei; Zhu, Yuanfeng; Zheng, Jiang; Zhou, Hong

    2013-02-01

    A dangerous cytokine storm occurs in the SARS involving in immune disorder, but many aspects of the pathogenetic mechanism remain obscure since its outbreak. To deeply reveal the interaction of host and SARS-CoV, based on the basic structural feature of pathogen-associated molecular pattern, we created a new bioinformatics method for searching potential pathogenic molecules and identified a set of SARS-CoV specific GU-rich ssRNA fragments with a high-density distribution in the genome. In vitro experiments, the result showed the representative SARS-CoV ssRNAs had powerful immunostimulatory activities to induce considerable level of pro-inflammatory cytokine TNF-a, IL-6 and IL-12 release via the TLR7 and TLR8, almost 2-fold higher than the strong stimulatory ssRNA40 that was found previously from other virus. Moreover, SARS-CoV ssRNA was able to cause acute lung injury in mice with a high mortality rate in vivo experiment. It suggests that SARS-CoV specific GU-rich ssRNA plays a very important role in the cytokine storm associated with a dysregulation of the innate immunity. This study not only presents new evidence about the immunopathologic damage caused by overactive inflammation during the SARS-CoV infection, but also provides a useful clue for a new therapeutic strategy. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  15. Proteolytic activation of the SARS-coronavirus spike protein: Cutting enzymes at the cutting edge of antiviral research

    Science.gov (United States)

    Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

    2013-01-01

    The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.” PMID:24121034

  16. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro.

    Directory of Open Access Journals (Sweden)

    Yiu-Wing Kam

    Full Text Available BACKGROUND: Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike of the severe acute respiratory syndrome coronavirus (SARS-CoV to cleavage by various airway proteases. METHODOLOGY/PRINCIPAL FINDINGS: PURIFIED TRISPIKE PROTEINS WERE READILY CLEAVED IN VITRO BY THREE DIFFERENT AIRWAY PROTEASES: trypsin, plasmin and TMPRSS11a. High Performance Liquid Chromatography (HPLC and amino acid sequencing analyses identified two arginine residues (R667 and R797 as potential protease cleavage site(s. The effect of protease-dependent enhancement of SARS-CoV infection was demonstrated with ACE2 expressing human bronchial epithelial cells 16HBE. Airway proteases regulate the infectivity of SARS-CoV in a fashion dependent on previous receptor binding. The role of arginine residues was further shown with mutant constructs (R667A, R797A or R797AR667A. Mutation of R667 or R797 did not affect the expression of S-protein but resulted in a differential efficacy of pseudotyping into SARS-CoVpp. The R667A SARS-CoVpp mutant exhibited a lack of virus entry enhancement following protease treatment. CONCLUSIONS/SIGNIFICANCE: These results suggest that SARS S-protein is susceptible to airway protease cleavage and, furthermore, that protease mediated enhancement of virus entry depends on specific conformation of SARS S-protein upon ACE2 binding. These data have direct implications for the cell entry mechanism of SARS-CoV along the respiratory system and, furthermore expand the possibility of identifying potential therapeutic agents against SARS-CoV.

  17. Toll-like receptors, chemokine receptors and death receptor ligands responses in SARS coronavirus infected human monocyte derived dendritic cells

    Directory of Open Access Journals (Sweden)

    Law Helen KW

    2009-06-01

    Full Text Available Abstract Background The SARS outbreak in 2003 provides a unique opportunity for the study of human responses to a novel virus. We have previously reported that dendritic cells (DCs might be involved in the immune escape mechanisms for SARS-CoV. In this study, we focussed on the gene expression of toll-like receptors (TLRs, chemokine receptors (CCRs and death receptor ligands in SARS-CoV infected DCs. We also compared adult and cord blood (CB DCs to find a possible explanation for the age-dependent severity of SARS. Results Our results demonstrates that SARS-CoV did not modulate TLR-1 to TLR-10 gene expression but significantly induced the expression of CCR-1, CCR-3, and CCR-5. There was also strong induction of TNF-related apoptosis-inducing ligand (TRAIL, but not Fas ligand gene expression in SARS-CoV infected DCs. Interestingly, the expressions of most genes studied were higher in CB DCs than adult DCs. Conclusion The upregulation of chemokines and CCRs may facilitate DC migration from the infection site to the lymph nodes, whereas the increase of TRAIL may induce lymphocyte apoptosis. These findings may explain the increased lung infiltrations and lymphoid depletion in SARS patients. Further explorations of the biological significance of these findings are warranted.

  18. Characterization of the PB2 Cap Binding Domain Accelerates Inhibitor Design

    Directory of Open Access Journals (Sweden)

    Amanda E. Constantinides

    2018-01-01

    Full Text Available X-ray crystallographic structural determinations of the PB2 cap binding domain (PB2cap have improved the conformational characterization of the RNA-dependent RNA polymerase machinery (PA, PB2, and PB1 of the influenza virus. Geometrically, the catalytic PB1 subunit resembles the palm of a human hand. PA lies near the thumb region, and PB2 lies near the finger region. PB2 binds the cap moiety in the pre-mRNA of the host cell, while the endonuclease of PA cleaves the pre-mRNA 10–13 nucleotides downstream. The truncated RNA piece performs as a primer for PB1 to synthesize the viral mRNA. Precisely targeting PB2cap with a small molecule inhibitor will halt viral proliferation via interference of the cap-snatching activity. Wild-type and mutant PB2cap from A/California/07/2009 H1N1 were expressed in Escherichia coli, purified by nickel affinity and size exclusion chromatography, crystallized, and subjected to X-ray diffraction experiments. The crystal of mutant PB2cap liganded with m7GTP was prepared by co-crystallization. Structures were solved by the molecular replacement method, refined, and deposited in the Protein Data Bank (PDB. Structural determination and comparative analyses of these structures revealed the functions of Glu361, Lys376, His357, Phe404, Phe323, Lys339, His432, Asn429, Gln406, and Met401 in PB2cap, and the dissociation of the influenza A PB2cap C-terminal subdomain (residues 446–479 upon ligand binding. Understanding the role of these residues will aid in the ultimate development of a small-molecule inhibitor that binds both Influenza A and B virus PB2cap.

  19. Capping Drugs: Development of Prodrugs

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 8; Issue 2. Capping Drugs: Development of Prodrugs. H Surya Prakash Rao. General Article Volume 8 Issue 2 February 2003 pp 19-27. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/008/02/0019-0027 ...

  20. From Blogs to Bottle Caps

    Science.gov (United States)

    Edinger, Ted

    2012-01-01

    There is a wonderful community of art educators connecting a once-isolated profession through blogging. Art educators around the world are sharing ideas and communicating with their peers through this amazing resource. In this article, the author describes the bottle cap mural at Tulip Grove Elementary School which was inspired by this exchange of…

  1. Spatially restricted translation of the xCR1 mRNA in Xenopus embryos.

    Science.gov (United States)

    Zhang, Yan; Forinash, Kara D; McGivern, Jered; Fritz, Brian; Dorey, Karel; Sheets, Michael D

    2009-07-01

    The xCR1 protein is a maternal determinant and cofactor for nodal signaling in vertebrate embryos. The xCR1 protein accumulates specifically in the animal cells of Xenopus embryos, but maternal xCR1 mRNA is distributed equally throughout all embryonic cells. Here, we show that vegetal cell-specific translational repression of xCR1 mRNA contributes to this spatially restricted accumulation of the xCR1 protein in Xenopus embryos. xCR1 mRNA was associated with polyribosomes in animal cells but not vegetal cells. A 351-nucleotide region of xCR1 mRNA's 3' untranslated region was sufficient to confer a spatially restricted pattern of translation to a luciferase reporter mRNA by repressing translation in vegetal cells. Repression depended upon the mRNA's 5' cap but not its 3' poly(A) tail. Furthermore, the region of xCR1 mRNA sufficient to confer vegetal cell-specific repression contained both Pumilio binding elements (PBEs) and binding sites for the CUG-BP1 protein. The PBEs and the CUG-BP1 sites were necessary but not sufficient for translation repression. Our studies of xCR1 mRNA document the first example of spatially regulated translation in controlling the asymmetric distribution of a maternal determinant in vertebrates.

  2. 21 CFR 884.5250 - Cervical cap.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cervical cap. 884.5250 Section 884.5250 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... cap. (a) Identification. A cervical cap is a flexible cuplike receptacle that fits over the cervix to...

  3. 21 CFR 888.3000 - Bone cap.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Bone cap. 888.3000 Section 888.3000 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES ORTHOPEDIC DEVICES Prosthetic Devices § 888.3000 Bone cap. (a) Identification. A bone cap is a mushroom...

  4. The cap-binding translation initiation factor, eIF4E, binds a pseudoknot in a viral cap-independent translation element

    Science.gov (United States)

    Wang, Zhaohui; Parisien, Marc; Scheets, Kay; Miller, W. Allen

    2011-01-01

    SUMMARY Eukaryotic initiation factor eIF4E performs a key early step in translation by specifically recognizing the m7GpppN cap structure at the 5' end of cellular mRNAs. Many viral mRNAs lack a 5' cap and thus bypass eIF4E. In contrast, we reported a novel cap-independent translation element (PTE) in Pea enation mosaic virus RNA2 that binds and requires eIF4E for translation initiation. To understand how this uncapped RNA is bound tightly by eIF4E we employ SHAPE probing, phylogenetic comparisons with new PTEs discovered in panico- and carmoviruses, footprinting of the eIF4E binding site, and 3D RNA modeling using NAST, MC-Fold, and MC-Sym to predict a compact, three-dimensional structure of the RNA. We propose that the cap-binding pocket of eIF4E clamps around a pseudoknot, placing a highly SHAPE-reactive guanosine in the pocket in place of the normal m7GpppN cap. This reveals a new mechanism of mRNA recognition by eIF4E. PMID:21645857

  5. The rebirth of the cervical cap.

    Science.gov (United States)

    Cappiello, J D; Grainger-Harrison, M

    1981-01-01

    In an effort to dispel myths surrounding the cervical cap, the historical and political factors affecting the cap's use in the U.S. are described. Clinical aspects of cap fitting are also included. The cervical cap has found only limited acceptance in the U.S. Skepticisms on the part of physicians may be the result of 2 factors: confusion of the cervical cap with intracervical devices used for artificial insemination and confusion with stem pessaries; and the lack of clinical research and statistical evaluation of efficacy rates. The latter factor prompted Tietze et al. to conduct the only U.S. statistical study of the cap in 1953. Of the 143 women studied, the pregnancy rate was 7.6/100 years of use. Of the 28 unplanned pregnancies, 6 were related to faulty technique or omission of a spermicide and 10 were instances of admittedly irregular use. When these failures are omitted, the theoretical effectiveness rate is about 98%. Some practitioners are concerned about an increased incidence of cervical erosion with cap use. Possibly currently conducted studies will show that cap and spermicide users have a lower incidence of cervical erosion than women using no contraceptive method. Study findings suggest that the cervical cap may afford protection without any spermicidal supplement, but the use of spermicides continues to be recommended to clients. Advantages of the cervical cap include the following: it can be left in place longer than a diaphragm without additional applications of spermicide in the vagina; and the insertion of the cap is unrelated to the time of intercourse. Despite research on toleration of the cap for 3 weeks at a time, it is recommended that the cap be worn for only a few days at a time. At this time there are no manufacturers of cervical caps for contraceptive use in the U.S. The cap is now being imported from England and it costs $6.00. A factor that has made the cap unpopular with many physicians is the lengthy time required for fitting. An

  6. Molecular characterization of a new member of the lariat capping twin-ribozyme introns

    DEFF Research Database (Denmark)

    Tang, Yunjia; Nielsen, Henrik; Masquida, Benoît

    2014-01-01

    BACKGROUND: Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA...... splicing and homing endonuclease mRNA maturation. We found Allovahlkampfia GIR2 to be a typical group IC1 splicing ribozyme responsible for addition of the exogenous guanosine cofactor (exoG), exon ligation and circularization of intron RNA. The Allovahlkampfia LC ribozyme, by contrast, represents...... an efficient self-cleaving ribozyme that generates a small 2',5' lariat cap at the 5' end of the homing endonuclease mRNA, and thus contributes to intron mobility. CONCLUSIONS: The discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify...

  7. Damage caps and defensive medicine, revisited.

    Science.gov (United States)

    Paik, Myungho; Black, Bernard; Hyman, David A

    2017-01-01

    Does tort reform reduce defensive medicine and thus healthcare spending? Several (though not all) prior studies, using a difference-in-differences (DiD) approach, find lower Medicare spending for hospital care after states adopt caps on non-economic or total damages ("damage caps"), during the "second" reform wave of the mid-1980s. We re-examine this issue in several ways. We study the nine states that adopted caps during the "third reform wave," from 2002 to 2005. We find that damage caps have no significant impact on Medicare Part A spending, but predict roughly 4% higher Medicare Part B spending. We then revisit the 1980s caps, and find no evidence of a post-adoption drop (or rise) in spending for these caps. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Ocular injuries from flying bottle caps.

    Science.gov (United States)

    Fonseka, C

    1993-12-01

    Three cases of serious eye injury are described from flying metal caps of carbonated drink bottles. The injuries occurred while attempting to open the bottle in an unconventional and dangerous way. Though injuries from flying bottle caps have been described before, they have occurred when the bottle exploded. This is the first report of eye injuries caused by bottle caps while opening and are similar to the injuries caused by champagne corks.

  9. Translation of maternal histone mRNAs in sea urchin embryos: a test of control by 5' cap methylation.

    Science.gov (United States)

    Showman, R M; Leaf, D S; Anstrom, J A; Raff, R A

    1987-05-01

    Recent results have demonstrated the occurrence of mRNA cap methylation in the sea urchin embryo following fertilization. It has been suggested that this methylation event is responsible for the translational activation of maternal histone mRNAs in these embryos. We have used aphidicolin, an effective inhibitor of both DNA synthesis and cap methylation in cleavage stage sea urchin embryos, to examine the relationship between cap methylation and translation. At 5 micrograms/ml, a dose which rapidly abolishes DNA replication and blocks cleavage, we note no effect on recruitment or translation of maternal alpha-subtype histone mRNAs. This suggests that a postfertilization cap methylation event is not critical to the process of regulation of the translation of stored alpha-subtype histone mRNAs.

  10. m6A Facilitates eIF4F-Independent mRNA Translation.

    Science.gov (United States)

    Coots, Ryan A; Liu, Xiao-Min; Mao, Yuanhui; Dong, Leiming; Zhou, Jun; Wan, Ji; Zhang, Xingqian; Qian, Shu-Bing

    2017-10-23

    In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Assured information flow capping architecture

    Science.gov (United States)

    Black, M. D.; Carvin, N. A.

    1985-05-01

    The Tactical Air Control System (TACS) is that set of Tactical Air Force assets used to assess the air and ground situation, and to plan, allocate, commit, and control assigned resources. Previous studies noted that the TACS elements should be more highly distributed to improve survivability in the battlefield of the future. This document reports on the results of the Assured Information Flow Capping Architecture study, which developed governing concepts for communications architectures that can support the information flow requirements of a future, distributed TACS. Architecture comprising existing and planned communications equipment were postulated and compared with a set of goals to identify deficiencies. Architectures using new equipment that resolve many of the deficiencies were then postulated, and areas needing further investigation were identified.

  12. Unbonded capping for concrete masonry units

    Energy Technology Data Exchange (ETDEWEB)

    Crouch, L.K.; Knight, M.L.; Henderson, R.C.; Sneed, W.A. Jr.

    1999-07-01

    Due to the manufacturing process, the bearing surfaces of concrete masonry units are often somewhat rough and uneven. Therefore, concrete masonry units must be capped when tested in compression according to ASTM C 140-96, Standard Test Methods of Sampling and Testing Concrete Masonry Units. Capping of concrete masonry units is time consuming and expensive. Several studies of compression tests on concrete cylinders indicate that use of elastic pads in rigid retaining caps give similar compressive strength results to approved capping methods.An unbonded capping system for concrete masonry units similar to that described in ASTM C 1231-93, Standard Practice for Use of Unbonded Caps in Determination of Compressive Strength of Hardened Concrete Cylinders, was developed. The average compressive strength results obtained when using the unbonded capping system ranged from 92--94% of the average compressive strength results obtained when using ASTM C 140-96 approved methods. Further, use of the unbonded capping system was found to increase productivity and substantially reduce testing cost.

  13. Nature mangement, landscape and the CAP

    NARCIS (Netherlands)

    Brouwer, F.M.; Godeschalk, F.E.

    2004-01-01

    The integration of nature management, landscape and environmental concerns into the Common Agricultural Policy (CAP) has gained momentum with the CAP reforms adopted in June 2003. The report explores instruments and approaches that contribute to the inte-gration of nature conservation and landscape

  14. CAP binding proteins associated with the nucleus.

    Science.gov (United States)

    Patzelt, E; Blaas, D; Kuechler, E

    1983-01-01

    Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in initiation factors as well as in preparations of crude nuclei. It was released from nuclei by washing with buffer of moderate salt concentration. Three high molecular weight cap binding proteins (approximately 120 000, approximately 89 000, approximately 80 000 dalton) were found in the nuclear fraction and were only partly released upon nuclease digestion and high salt extraction. Images PMID:6889136

  15. Dynamic m6A mRNA methylation directs translational control of heat shock response

    Science.gov (United States)

    Zhou, Jun; Wan, Ji; Gao, Xiangwei; Zhang, Xingqian; Qian, Shu-Bing

    2015-01-01

    The most abundant mRNA post-transcriptional modification is N6-methyladenosine (m6A) that has broad roles in RNA biology1-5. In mammalian cells, the asymmetric distribution of m6A along mRNAs leaves relatively less methylation in the 5′ untranslated region (5′UTR) compared to other regions6,7. However, whether and how 5′UTR methylation is regulated is poorly understood. Despite the crucial role of the 5′UTR in translation initiation, very little is known whether m6A modification influences mRNA translation. Here we show that in response to heat shock stress, m6A is preferentially deposited to the 5′UTR of newly transcribed mRNAs. We found that the dynamic 5′UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well characterized m6A “reader”. Upon heat shock stress, the nuclear YTHDF2 preserves 5′UTR methylation of stress-induced transcripts by limiting the m6A “eraser” FTO from demethylation. Remarkably, the increased 5′UTR methylation in the form of m6A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single site m6A modification in the 5′UTR enables translation initiation independent of the 5′ end m7G cap. The elucidation of the dynamic feature of 5′UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m6A, but also uncovers a previously unappreciated translational control mechanism in heat shock response. PMID:26458103

  16. Analyses of hydraulic performance of velocity caps

    DEFF Research Database (Denmark)

    Christensen, Erik Damgaard; Degn Eskesen, Mark Chr.; Buhrkall, Jeppe

    2014-01-01

    The hydraulic performance of a velocity cap has been investigated. Velocity caps are often used in connection with offshore intakes. CFD (computational fluid dynamics) examined the flow through the cap openings and further down into the intake pipes. This was combined with dimension analyses...... in order to analyse the effect of different layouts on the flow characteristics. In particular, flow configurations going all the way through the structure were revealed. A couple of suggestions to minimize the risk for flow through have been tested....

  17. C-CAP Niihau 2005 Land Cover

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set consists of land cover derived from high resolution imagery according to the Coastal Change Analysis Program (C-CAP) protocol. This data set utilized 1...

  18. Circumpolar Active-Layer Permafrost System (CAPS)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Circumpolar Active-Layer Permafrost System (CAPS) contains over 100 data sets pertaining to permafrost and frozen ground topics. It also contains detailed...

  19. DESIGN CONSIDERATION INVOLVING ACTIVE SEDIMENT CAPS (PRESENTATION)

    Science.gov (United States)

    When contaminated sediments pose unacceptable risks to human health and the environment, management activities such as removal, treatment, or isolation of contaminated sediments may be required. Various capping designs are being considered for isolating contaminated sediment are...

  20. Mapping of p140Cap phosphorylation sites

    DEFF Research Database (Denmark)

    Repetto, Daniele; Aramu, Simona; Boeri Erba, Elisabetta

    2013-01-01

    Protein phosphorylation tightly regulates specific binding of effector proteins that control many diverse biological functions of cells (e. g. signaling, migration and proliferation). p140Cap is an adaptor protein, specifically expressed in brain, testis and epithelial cells, that undergoes...... phosphorylation and tunes its interactions with other regulatory molecules via post-translation modification. In this work, using mass spectrometry, we found that p140Cap is in vivo phosphorylated on tyrosine (Y) within the peptide GEGLpYADPYGLLHEGR (from now on referred to as EGLYA) as well as on three serine...... residues. Consistently, EGLYA has the highest score of in silico prediction of p140Cap phosphorylation. To further investigate the p140Cap function, we performed site specific mutagenesis on tyrosines inserted in EGLYA and EPLYA, a second sequence with the same highest score of phosphorylation. The mutant...

  1. C-CAP Land Cover, Maui, Hawaii

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set consists of land derived from high resolution imagery and was analyzed according to the Coastal Change Analysis Program (C-CAP) protocol to determine...

  2. DESIGN CONSIDERATION INVOLVING ACTIVE SEDIMENT CAPS

    Science.gov (United States)

    When contaminated sediments pose unacceptable risks to human health and the environment, management activities such as removal, treatment, or isolation of contaminated sediments may be required. Various capping designs are being considered for isolating contaminated sediment are...

  3. The C. elegans mRNA decapping enzyme shapes morphology of cilia.

    Science.gov (United States)

    Adachi, Takeshi; Nagahama, Keigo; Izumi, Susumu

    2017-11-04

    Cilia and flagella are evolutionarily conserved organelles that protrude from cell surfaces. Most cilia and flagella are single rod-shaped but some cilia show a variety of shapes. For example, human airway epithelial cells are multiciliated, flagella of crayfish spermatozoon are star-like shaped, and fruit fly spermatozoon extends long flagella. In Caenorhabditis elegans, cilia display morphological diversity of shapes (single, dual rod-type and wing-like and highly-branched shapes). Here we show that DCAP-1 and DCAP-2, which are the homologues of mammalian DCP1 and DCP2 mRNA decapping enzymes, respectively, are involved in formation of dual rod-type and wing-like shaped cilia in C. elegans. mRNA decapping enzyme catalyzes hydrolysis of 5' cap structure of mRNA, which leads to degradation of mRNA. Rescue experiments showed that DCAP-2 acts not in glial cells surrounding cilia but in neurons. This is the first evidence to demonstrate that mRNA decapping is involved in ciliary shape formation. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. The structure of the SOLE element of oskar mRNA.

    Science.gov (United States)

    Simon, Bernd; Masiewicz, Pawel; Ephrussi, Anne; Carlomagno, Teresa

    2015-08-01

    mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance. © 2015 Simon et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  5. Righting an ATLAS end-cap

    CERN Document Server

    Maximilien Brice

    2003-01-01

    Each component of the giant ATLAS detector must be moved and transported with great caution so that delicate components are not damaged. Therefore, tests are performed, like the one shown. The end-cap weighs 160 tonnes, making the delicate moving process a non-trivial feat. A special crane and supports have been designed so that the end-cap can be raised using a pivot.

  6. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells.

    Science.gov (United States)

    Nakagawa, K; Lokugamage, K G; Makino, S

    2016-01-01

    Coronaviruses have large positive-strand RNA genomes that are 5' capped and 3' polyadenylated. The 5'-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15-16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3'-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5' leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. © 2016 Elsevier Inc. All rights reserved.

  7. Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment.

    Science.gov (United States)

    Tanaka, T; Nishihara, M; Seki, M; Sakamoto, A; Tanaka, K; Irifune, K; Morikawa, H

    1995-05-01

    Gold particles coated with beta-glucuronidase (GUS) mRNA with a 5' cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.

  8. Capped poly(A) leaders of variable lengths at the 5' ends of vaccinia virus late mRNAs

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, B.Y.; Moss, B.

    1989-01-01

    Evidence for capped poly(A) leaders of variable lengths located immediately upstream of the translation initiation codon was obtained by direct analyses of a major late mRNA species. A decapping-recapping method was used to specifically substitute a radioactively labeled phosphate for an unlabeled one within the cap structure. RNase H-susceptible sites were made by hybridizing synthetic oligodeoxyribonucleotides to the mRNA encoding a late major structural protein of 11 kilodaltons. Sequences of the type m/sup 7/G(5')pppAmp (Ap)/sub n/UpG /hor elipsis/, where n varies from a few to more than 40 nucleotides, were deduced by analysis of the length and sequence of RNase, H, RNase T/sub 1/, and RNase U2 digestion products.

  9. 75 FR 49527 - Caps Visual Communications, LLC; Black Dot Group; Formerly Known as Caps Group Acquisition, LLC...

    Science.gov (United States)

    2010-08-13

    ... Employment and Training Administration Caps Visual Communications, LLC; Black Dot Group; Formerly Known as... Adjustment Assistance on June 24, 2010, applicable to workers of Caps Visual Communications, LLC, Black Dot..., Caps Visual Communications, LLC, Black Dot Group, formerly known as Caps Group Acquisition, LLC...

  10. Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA

    DEFF Research Database (Denmark)

    Birgisdottir, Asa B; Nielsen, Henrik; Beckert, Bertrand

    2011-01-01

    Abstract RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin......-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal...

  11. Seismic explosion sources on an ice cap

    DEFF Research Database (Denmark)

    Shulgin, Alexey; Thybo, Hans

    2015-01-01

    Controlled source seismic investigation of crustal structure below ice covers is an emerging technique. We have recently conducted an explosive refraction/wide-angle reflection seismic experiment on the ice cap in east-central Greenland. The data-quality is high for all shot points and a full...... crustal model can be modelled. A crucial challenge for applying the technique is to control the sources. Here, we present data that describe the efficiency of explosive sources in the ice cover. Analysis of the data shows, that the ice cap traps a significant amount of energy, which is observed...... as a strong ice wave. The ice cap leads to low transmission of energy into the crust such that charges need be larger than in conventional onshore experiments to obtain reliable seismic signals. The strong reflection coefficient at the base of the ice generates strong multiples which may mask for secondary...

  12. Martian north polar cap summer water cycle

    Science.gov (United States)

    Brown, Adrian J.; Calvin, Wendy M.; Becerra, Patricio; Byrne, Shane

    2016-10-01

    A key outstanding question in Martian science is "are the polar caps gaining or losing mass and what are the implications for past, current and future climate?" To address this question, we use observations from the Compact Reconnaissance Imaging Spectrometer for Mars (CRISM) of the north polar cap during late summer for multiple Martian years, to monitor the summertime water cycle in order to place quantitative limits on the amount of water ice deposited and sublimed in late summer. We establish here for the first time the summer cycle of water ice absorption band signatures on the north polar cap. We show that in a key region in the interior of the north polar cap, the absorption band depths grow until Ls = 120, when they begin to shrink, until they are obscured at the end of summer by the north polar hood. This behavior is transferable over the entire north polar cap, where in late summer regions 'flip' from being net sublimating into net condensation mode. This transition or 'mode flip' happens earlier for regions closer to the pole, and later for regions close to the periphery of the cap. The observations and calculations presented herein estimate that on average a water ice layer ∼70 microns thick is deposited during the Ls = 135-164 period. This is far larger than the results of deposition on the south pole during summer, where an average layer 0.6-6 microns deep has been estimated by Brown et al. (2014) Earth Planet. Sci. Lett., 406, 102-109.

  13. Assembling the CMS yoke end-caps

    CERN Multimedia

    Laurent Guiraud

    2001-01-01

    A crane is used to piece together one of the end-caps that will provide the path for magnetic flux return on the CMS experiment. A total of six end-cap discs will be assembled before being positioned on the barrel yoke to complete the huge 12 500 tonne cylinder yoke. The magnetic field produced will be greater than any other solenoid created to date at 4 T, 100 000 times greater than the Earth's natural magnetic field, and will store enough energy to melt 18 tonnes of gold.

  14. Does uncertainty justify intensity emission caps?

    OpenAIRE

    Quirion, Philippe

    2005-01-01

    International audience; Environmental policies often set ‘‘relative'' or ‘‘intensity'' emission caps, i.e. emission limits proportional to the polluting firm's output. One of the arguments put forth in favour of relative caps is based on the uncertainty on business-as-usual output: if the firm's production level is higher than expected, so will be business-as-usual emissions, hence reaching a given level of emissions will be more costly than expected.As a consequence, it is argued, a higher e...

  15. A hybrid approach to direct pulp capping by using emdogain with a capping material.

    Science.gov (United States)

    Al-Hezaimi, Khalid; Al-Tayar, Badr Abdullah; Bajuaifer, Yaseer Salim; Salameh, Ziad; Al-Fouzan, Khalid; Tay, Franklin R

    2011-05-01

    This study evaluated the formation of reparative hard tissues in baboon pulps after Emdogain (EMD) application in conjunction with 3 pulp-capping materials. Thirty-two premolars in four 3-year-old baboons were randomly assigned to 1 of 3 pulp-capping materials. A split-mouth design and intra-animal side randomization were applied to 3 experimental groups (calcium hydroxide, ProRoot White mineral trioxide aggregate, white Portland cement) and the control group (no pulp-capping material). In the hybrid EMD approach, a small drop of EMD was placed over the exposure site after arrest of hemorrhage. The designated pulp-capping material was placed over the EMD, followed by placement of resin-modified glass ionomer cement over the set/unset pulp-capping material. The animals were killed after 4 months. Histomorphometric analysis and micro-computed tomography were performed on the retrieved specimens. All groups capped with EMD and 1 of the 3 capping materials exhibited similar reparative tissue thickness (P > .05). Dentin tunnel defects were absent in the mineral trioxide aggregate and Portland cement groups after the use of EMD. However, only a tubular was identified from all specimens. Mineral trioxide aggregate produces a better quality reparative hard tissue response with the adjunctive use of Emdogain, when compared with the use of calcium hydroxide. Copyright © 2011 American Association of Endodontists. All rights reserved.

  16. Scrap the cap or jeopardise patient safety.

    Science.gov (United States)

    2017-05-17

    After the general election, for the sake of patient safety, the government must scrap the cap on nurses' pay and help to fill the 40,000 vacant jobs. These are not my words, but those of RCN general secretary Janet Davies, and summarise the college's key message to the political parties in the run-up to polling day on 8 June.

  17. The Effectiveness of Caps on Political Lobbying

    NARCIS (Netherlands)

    Matejka, M.; Onderstal, A.M.; De Waegenaere, A.M.B.

    2002-01-01

    In this paper, we analyze a lobby game, modelled as an all-pay auction in which interest groups submit bids in order to obtain a political prize.The bids are restricted to be below a cap imposed by the government.For both an incomplete and a complete information setting we show the following

  18. INTERNATIONAL WHEAT PRICE TRANSMISSION AND CAP REFORM

    OpenAIRE

    Thompson,Stanley R.; Bohl, Martin T.

    1999-01-01

    We illustrate how CAP policy reforms influence the transmission of world prices to domestic markets. Monthly wheat price data in Germany are used to obtain price transmission elasticities. Correctly accounting for structural breaks and the time series properties of the data yield transmission elasticities that differ dramatically among policy regimes.

  19. CAP Reform and the Doha Development Agenda

    NARCIS (Netherlands)

    van Dijck, P.; Faber, G.|info:eu-repo/dai/nl/069308187

    2004-01-01

    The CAP reforms that the EU accepted in June 2003 will partially decouple direct income payments to farmers from production and make these payments conditional on cross-compliance. The reforms are driven by enlargement of EU membership, budgetary constraints, mounting pressures from diverse animal

  20. Arrays of magnetic nanoparticles capped with alkylamines

    Indian Academy of Sciences (India)

    Magnetic metal and metal oxide nanoparticles capped with alkylamines have been synthesized and characterized by transmission electron microscopy, X-ray diffraction, energy dispersive X-ray analysis and magnetization measurements. Core-shell Pd–Ni particles with composition, Pd561Ni3000, (diameter ∼ 3.3 nm) are ...

  1. CAPS Capsule. Volume 5, Number 2.

    Science.gov (United States)

    Walz, Garry, Ed.; And Others

    Published 3 times yearly by the ERIC Clearinghouse on Counseling and Personnel Services, CAPS Capsule acquaints the reader with recent projects, meetings, publications and materials which are of interest those in the helping professions. This issue features: (1) New Career Development Projects; (2) Supreme Court Decision Ramifications; (3)…

  2. ATLAS: End-cap Toroid assembly

    CERN Multimedia

    2006-01-01

    In building 191 and building 180- assembly of this massive piece.To reach the top of the end-cap the cranes has to be used and during the assembly you can see welding and hear many tools running background.

  3. Combining different mRNA capture methods to analyze the transcriptome: analysis of the Xenopus laevis transcriptome.

    Directory of Open Access Journals (Sweden)

    Michael D Blower

    Full Text Available mRNA sequencing (mRNA-seq is a commonly used technique to survey gene expression from organisms with fully sequenced genomes. Successful mRNA-seq requires purification of mRNA away from the much more abundant ribosomal RNA, which is typically accomplished by oligo-dT selection. However, mRNAs with short poly-A tails are captured poorly by oligo-dT based methods. We demonstrate that combining mRNA capture via oligo-dT with mRNA capture by the 5' 7-methyl guanosine cap provides a more complete view of the transcriptome and can be used to assay changes in mRNA poly-A tail length on a genome-wide scale. We also show that using mRNA-seq reads from both capture methods as input for de novo assemblers provides a more complete reconstruction of the transcriptome than either method used alone. We apply these methods of mRNA capture and de novo assembly to the transcriptome of Xenopus laevis, a well-studied frog that currently lacks a finished sequenced genome, to discover transcript sequences for thousands of mRNAs that are currently absent from public databases. The methods we describe here will be broadly applicable to many organisms and will provide insight into the transcriptomes of organisms with sequenced and unsequenced genomes.

  4. Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

    Science.gov (United States)

    Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

    2017-08-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus.IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

  5. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Development of an unbonded capping system for clay masonry prisms

    Energy Technology Data Exchange (ETDEWEB)

    Crouch, L.K.; Henderson, R.C.; Sneed, W.A. Jr.

    1999-07-01

    To ascertain if an unbonded capping system was feasible for clay masonry prisms, the compressive strengths of thirty clay masonry prisms capped with an unbonded capping system modeled after ASTM C 1231 were compared with those of thirty masonry prisms capped with ASTM C 67 approved high-strength gypsum cement at the ages of 7 and 28 days. All prisms were constructed by a professional mason using Grade SW, Type FBS cored face brick from the same lot and ASTM C 270 Type S PC-lime mortar. There was no significant difference in mean compressive strength for the two capping methods at either age. In addition, capping with the unbonded capping system was faster and easier. Further, 28-day results obtained using the unbonded capping system had a lower coefficient of variation and higher mean compressive strength than those obtained with high-strength gypsum.

  7. Facially amphiphilic thiol capped gold and silver nanoparticles

    Indian Academy of Sciences (India)

    Abstract. A series of bile acid-derived facially amphiphilic thiols have been used to cap sliver and gold nanoparticles. The self-assembling properties of these steroid-capped nanoparticles have been investigated and reported in this article.

  8. NAMMA CLOUD MICROPHYSICS (CAPS-PIP) V1

    Data.gov (United States)

    National Aeronautics and Space Administration — The NAMMA Cloud Microphysics (CAPS-PIP) dataset consists of particle size distributions from the Clouds, Aerosol and Preciptaition Spectrometer (CAPS) and the...

  9. Exploring plasmonic coupling in hole-cap arrays

    OpenAIRE

    Schmidt, Thomas M.; Maj Frederiksen; Vladimir Bochenkov; Duncan S. Sutherland

    2015-01-01

    Summary The plasmonic coupling between gold caps and holes in thin films was investigated experimentally and through finite-difference time-domain (FDTD) calculations. Sparse colloidal lithography combined with a novel thermal treatment was used to control the vertical spacing between caps and hole arrays and compared to separated arrays of holes or caps. Optical spectroscopy and FDTD simulations reveal strong coupling between the gold caps and both Bloch Wave-surface plasmon polariton (BW-SP...

  10. The Nuclear Cap-Binding Complex Mediates Meiotic Silencing by Unpaired DNA

    Directory of Open Access Journals (Sweden)

    Logan M. Decker

    2017-04-01

    Full Text Available In the filamentous fungus Neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish DNAs. Accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. One of these defense mechanisms is called meiotic silencing by unpaired DNA (MSUD, which identifies and silences unpaired genes during meiosis. Utilizing common RNA interference (RNAi proteins, such as Dicer and Argonaute, MSUD targets mRNAs homologous to the unpaired sequence to achieve silencing. In this study, we have identified an additional silencing component, namely the cap-binding complex (CBC. Made up of cap-binding proteins CBP20 and CBP80, CBC associates with the 5′ cap of mRNA transcripts in eukaryotes. The loss of CBC leads to a deficiency in MSUD activity, suggesting its role in mediating silencing. As confirmed in this study, CBC is predominantly nuclear, although it is known to travel in and out of the nucleus to facilitate RNA transport. As seen in animals but not in plants, CBP20’s robust nuclear import depends on CBP80 in Neurospora. CBC interacts with a component (Argonaute of the perinuclear meiotic silencing complex (MSC, directly linking the two cellular factors.

  11. Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases.

    Directory of Open Access Journals (Sweden)

    Juan Reguera

    2016-06-01

    Full Text Available Segmented negative strand RNA viruses of the arena-, bunya- and orthomyxovirus families uniquely carry out viral mRNA transcription by the cap-snatching mechanism. This involves cleavage of host mRNAs close to their capped 5' end by an endonuclease (EN domain located in the N-terminal region of the viral polymerase. We present the structure of the cap-snatching EN of Hantaan virus, a bunyavirus belonging to hantavirus genus. Hantaan EN has an active site configuration, including a metal co-ordinating histidine, and nuclease activity similar to the previously reported La Crosse virus and Influenza virus ENs (orthobunyavirus and orthomyxovirus respectively, but is more active in cleaving a double stranded RNA substrate. In contrast, Lassa arenavirus EN has only acidic metal co-ordinating residues. We present three high resolution structures of Lassa virus EN with different bound ion configurations and show in comparative biophysical and biochemical experiments with Hantaan, La Crosse and influenza ENs that the isolated Lassa EN is essentially inactive. The results are discussed in the light of EN activation mechanisms revealed by recent structures of full-length influenza virus polymerase.

  12. CMS end-cap yoke at the detector's assembly site.

    CERN Multimedia

    Patrice Loïez

    2002-01-01

    The magnetic flux generated by the superconducting coil in the CMS detector is returned via an iron yoke comprising three end-cap discs at each end (end-cap yoke) and five concentric cylinders (barrel yoke). This picture shows the first of three end-cap discs (red) seen through the outer cylinder of the vacuum tank which will house the superconducting coil.

  13. 20 CFR 606.22 - Application for cap.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Application for cap. 606.22 Section 606.22... Reduction § 606.22 Application for cap. (a) Application. (1) The Governor of the State shall make... a State requests a cap on tax credit reduction. The Governor is required to notify the Department on...

  14. Greening CAP payments: a missed opportunity?

    Energy Technology Data Exchange (ETDEWEB)

    Matthews, Alan

    2013-01-15

    At an important point in the current reform of the Common Agricultural Policy (CAP), a new IIEA policy brief by Professor Alan Matthews, one of the EU’s foremost experts on the topic, considers proposals to green direct farm payments. Professor Matthews argues that proposed greening of direct payments – the key innovation in the current round of CAP Reform – look likely to fail. While greening may survive as a concept, the likely outcome of the negotiations between Agriculture Ministers and the European Parliament will deliver little practical environmental benefit. The paper examines the rationale underpinning greening, arguing that it exists to justify the continuation of a large agricultural budget, explores reasons for the apparent failure of the proposals, and reflects on the implications for future efforts to better integrate environmental objectives into EU agriculture policy. This is the first in a series of Environment Nexus policy briefs by leading experts in the fields of agriculture, energy, climate change and water.

  15. CAp 2017 challenge: Twitter Named Entity Recognition

    OpenAIRE

    Lopez, Cédric; Partalas, Ioannis; Balikas, Georgios; Derbas, Nadia; Martin, Amélie; Reutenauer, Coralie; Segond, Frédérique; Amini, Massih-Reza

    2017-01-01

    The paper describes the CAp 2017 challenge. The challenge concerns the problem of Named Entity Recognition (NER) for tweets written in French. We first present the data preparation steps we followed for constructing the dataset released in the framework of the challenge. We begin by demonstrating why NER for tweets is a challenging problem especially when the number of entities increases. We detail the annotation process and the necessary decisions we made. We provide statistics on the inter-...

  16. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody.

    Science.gov (United States)

    Ng, Oi-Wing; Keng, Choong-Tat; Leung, Cynthia Sau-Wai; Peiris, J S Malik; Poon, Leo Lit Man; Tan, Yee-Joo

    2014-01-01

    The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is the etiological agent for the infectious disease, SARS, which first emerged 10 years ago. SARS-CoV is a zoonotic virus that has crossed the species barriers to infect humans. Bats, which harbour a diverse pool of SARS-like CoVs (SL-CoVs), are believed to be the natural reservoir. The SARS-CoV surface Spike (S) protein is a major antigenic determinant in eliciting neutralizing antibody production during SARS-CoV infection. In our previous work, we showed that a panel of murine monoclonal antibodies (mAbs) that target the S2 subunit of the S protein are capable of neutralizing SARS-CoV infection in vitro (Lip KM et al, J Virol. 2006 Jan; 80(2): 941-50). In this study, we report our findings on the characterization of one of these mAbs, known as 1A9, which binds to the S protein at a novel epitope within the S2 subunit at amino acids 1111-1130. MAb 1A9 is a broadly neutralizing mAb that prevents viral entry mediated by the S proteins of human and civet SARS-CoVs as well as bat SL-CoVs. By generating mutant SARS-CoV that escapes the neutralization by mAb 1A9, the residue D1128 in S was found to be crucial for its interaction with mAb 1A9. S protein containing the substitution of D1128 with alanine (D1128A) exhibited a significant decrease in binding capability to mAb 1A9 compared to wild-type S protein. By using a pseudotyped viral entry assay, it was shown that the D1128A substitution in the escape virus allows it to overcome the viral entry blockage by mAb 1A9. In addition, the D1128A mutation was found to exert no effects on the S protein cell surface expression and incorporation into virion particles, suggesting that the escape virus retains the same viral entry property as the wild-type virus.

  17. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1.

    Science.gov (United States)

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Hein, Marco Y; Müller, Marcel A; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-08-30

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PL(pro)), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95-144 of RCHY1 and 389-652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PL(pro)s from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD-PL(pro) fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PL(pro) alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes.

  18. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    Science.gov (United States)

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu, Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Pöhlmann, Stefan

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S-activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation. PMID:21435673

  19. SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6.

    Science.gov (United States)

    Li, Shih-Wen; Wang, Ching-Ying; Jou, Yu-Jen; Huang, Su-Hua; Hsiao, Li-Hsin; Wan, Lei; Lin, Ying-Ju; Kung, Szu-Hao; Lin, Cheng-Wen

    2016-05-05

    Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

  20. SARS-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the MAVS/TRAF3/TRAF6 signalosome.

    Science.gov (United States)

    Shi, Chong-Shan; Qi, Hai-Yan; Boularan, Cedric; Huang, Ning-Na; Abu-Asab, Mones; Shelhamer, James H; Kehrl, John H

    2014-09-15

    Coronaviruses (CoV) have recently emerged as potentially serious pathogens that can cause significant human morbidity and death. The severe acute respiratory syndrome (SARS)-CoV was identified as the etiologic agent of the 2002-2003 international SARS outbreak. Yet, how SARS evades innate immune responses to cause human disease remains poorly understood. In this study, we show that a protein encoded by SARS-CoV designated as open reading frame-9b (ORF-9b) localizes to mitochondria and causes mitochondrial elongation by triggering ubiquitination and proteasomal degradation of dynamin-like protein 1, a host protein involved in mitochondrial fission. Also, acting on mitochondria, ORF-9b targets the mitochondrial-associated adaptor molecule MAVS signalosome by usurping PCBP2 and the HECT domain E3 ligase AIP4 to trigger the degradation of MAVS, TRAF3, and TRAF 6. This severely limits host cell IFN responses. Reducing either PCBP2 or AIP4 expression substantially reversed the ORF-9b-mediated reduction of MAVS and the suppression of antiviral transcriptional responses. Finally, transient ORF-9b expression led to a strong induction of autophagy in cells. The induction of autophagy depended upon ATG5, a critical autophagy regulator, but the inhibition of MAVS signaling did not. These results indicate that SARS-CoV ORF-9b manipulates host cell mitochondria and mitochondrial function to help evade host innate immunity. This study has uncovered an important clue to the pathogenesis of SARS-CoV infection and illustrates the havoc that a small ORF can cause in cells.

  1. p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

    Science.gov (United States)

    Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Müller, Marcel A.; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

    2016-01-01

    Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes. PMID:27519799

  2. High-dose hydrocortisone reduces expression of the pro-inflammatory chemokines CXCL8 and CXCL10 in SARS coronavirus-infected intestinal cells.

    Science.gov (United States)

    Cinatl, Jindrich; Michaelis, Martin; Morgenstern, Birgit; Doerr, Hans Wilhelm

    2005-02-01

    Clinical observations and our high-density oligonucleotide microarray results demonstrated increased expression of proinflammatory chemokines after SARS-CoV infection. Here, we investigated the influence of SARS-CoV infection on CXCL8 (interleukin 8) and CXCL10 (interferon-gamma-inducible protein 10) in human intestinal epithelial (Caco2) cells. RT-PCR and ELISA showed time-dependent up-regulation of both chemokines after SARS-CoV infection. Electric mobility shift assay revealed increased DNA binding activity of the cellular transcription factors activator protein 1 (AP-1) and nuclear factor (B (NF-kappaB) in SARS-CoV infected cells. High hydrocortisone concentrations (> or =50 microg/ml) completely prevented increased DNA binding activity of AP-1 and NF-kappaB and inhibited up-regulation of CXCL8 and CXCL10, but did not reduce chemokine expression to basal levels. Ribavirin that does not inhibit SARS-CoV replication in Vero cells inhibited SARS-CoV replication in Caco2 cells at therapeutical concentrations. Hydrocortisone neither influenced SARS-CoV titres alone nor in combination with ribavirin. Our results show that corticosteroids may be of limited benefit in the suppression of chemokine production by SARS-CoV-infected cells.

  3. The Severe Acute Respiratory Syndrome (SARS-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein

    Directory of Open Access Journals (Sweden)

    Tan Yee-Joo

    2005-02-01

    Full Text Available Abstract Background A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV, resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro. Presentation of the hypothesis It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression. Testing the hypothesis The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology. Implication of the hypothesis If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe acute respiratory syndrome-coronavirus.

  4. Cap analog and Potato virus A HC-Pro silencing suppressor improve GFP transient expression using an infectious virus vector in Nicotiana benthamiana.

    Science.gov (United States)

    Tahmasebi, Amin-Alah; Afsharifar, Alireza

    2017-06-01

    Transient expression of proteins in plants has become a choice to facilitate recombinant protein production with its fast and easy application. On the other hand, host defensive mechanisms have been reported to reduce the efficiency of transient expression in plants. Hence, this study was designed to evaluate the effect of cap analog and Potato virus A helper component proteinase (PVA HC-Pro) on green fluorescent protein (GFP) expression efficiency. N. benthamiana leaves were inoculated with capped or un-capped RNA transcripts of a Turnip crinkle virus (TCV) construct containing a green fluorescent protein reporter gene (TCV-sGFP) in place of its coat protein (CP) ORF. PVA HC-Pro as a viral suppressor of RNA silencing was infiltrated in trans by Agrobacterium tumefaciens, increased the GFP foci diameter to six and even more cells in both capped and un capped treatments. The expression level of GFP in inoculated plants with TCV-sGFP transcript pre-infiltrated with PVA HC-Pro was 12.97-fold higher than the GFP accumulation level in pre-infiltrated leaves with empty plasmid (EP) control. Also, the yield of GFP in inoculated N. benthamiana plants with capped TCV-sGFP transcript pre-infiltrated with EP and PVA HC-Pro was 1.54 and 1.2-fold respectively, greater than the level of GFP expressed without cap analog application at 5 days post inoculation (dpi). In addition, the movement of TCV-sGFP was increased in some cells of inoculated leaves with capped transcripts. Results of this study indicated that PVA HC-Pro and mRNA capping can increase GFP expression and its cell to cell movement in N. benthamiana.

  5. SAPCE CAP linkage methodology and preliminary results

    Energy Technology Data Exchange (ETDEWEB)

    Choo, Yeon Joon; Hong, Soon Joon; Lee, Byung Chul [FNC Tech., Seoul (Korea, Republic of); Ha, Sang Jun; Choi, Hoon [KHNP Centeral research institute, Daejeon (Korea, Republic of); Lee, Tae Beom [KEPCO Nuclear Fuel, Daejeon (Korea, Republic of)

    2012-10-15

    As a part of the development project of the domestic design code for licensing purposes of pressurized water reactors, the SPACE (Safety and Performance Calculation Code) and CAP (Containment Analysis Package) code are under development in Korea. The SPACE code has applicability to analysis of the system behavior of nuclear power plant under the various accident conditions. On the order hand, CAP code is focused on the calculation of pressure and temperature of containment compartments. Two codes, of course, were designed to deal with the general thermal hydraulic problems also. In other to evaluate conservatively the performance of ECCS equipment; minimum containment back pressure is conservative, the containment pressure is important boundary condition. In this respect, linking two codes; RELAP5/MOD3 for system code and CONTEMPT4/MOD5 for containment code was tried to calculate the minimum containment back pressure in the framework of KREM (KEPRI Realistic Evaluation Methodology). These results are used in FSAR of Kori3/4. For the above same purpose, linking the SPACE and CAP code has tried based on KREM in this research. The target plant is the Shinkori 3/4 nuclear plant; APR1400 type. This plant has quite different design concept from other operating plants such as OPR1000 and WH 3 loop plant. Major feature of APR1400 is that RWST (Refueling Water Storage Tank) is located in plant.

  6. Pulp-Capping with Mineral Trioxide Aggregate

    Directory of Open Access Journals (Sweden)

    Peycheva Kalina

    2015-11-01

    Full Text Available There are two considerations for direct pulp capping - accidental mechanical pulp exposure and exposure caused by caries. Mineral trioxide aggregate (MTA was used as pulp-capping material to preserve the vitality of the pulpal tissues. Follow-up examinations revealed that treatment was successful in preserving pulpal vitality and continued development of the tooth. On the basis of available information, it appears that MTA is the material of choice for some clinical applications. Material and methods: Cases 18 - 8 teeth with grey MTA, 10 teeth with white MTA; diagnose: Pulpitis chronica ulcerosa, Electro pulpal test (EOD - 30-35 μA, pre-clinical X-ray - without changes in the structures, follow ups for 4 years. Successful treatments: without clinical symptoms and changes in the X-rays: 5 teeth with grey MTA, 8 teeth with white MTA for period of 4 years. Unsuccessful treatments: Clinical symptoms and sometimes changes in the X-ray: 3 with grey MTA, 2 with white MTA. MTA is an appropriate material for pulp-capping and follow-up examinations revealed that the treatment was successful in preserving pulpal vitality.

  7. MycoCAP - Mycobacterium Comparative Analysis Platform.

    Science.gov (United States)

    Choo, Siew Woh; Ang, Mia Yang; Dutta, Avirup; Tan, Shi Yang; Siow, Cheuk Chuen; Heydari, Hamed; Mutha, Naresh V R; Wee, Wei Yee; Wong, Guat Jah

    2015-12-15

    Mycobacterium spp. are renowned for being the causative agent of diseases like leprosy, Buruli ulcer and tuberculosis in human beings. With more and more mycobacterial genomes being sequenced, any knowledge generated from comparative genomic analysis would provide better insights into the biology, evolution, phylogeny and pathogenicity of this genus, thus helping in better management of diseases caused by Mycobacterium spp.With this motivation, we constructed MycoCAP, a new comparative analysis platform dedicated to the important genus Mycobacterium. This platform currently provides information of 2108 genome sequences of at least 55 Mycobacterium spp. A number of intuitive web-based tools have been integrated in MycoCAP particularly for comparative analysis including the PGC tool for comparison between two genomes, PathoProT for comparing the virulence genes among the Mycobacterium strains and the SuperClassification tool for the phylogenic classification of the Mycobacterium strains and a specialized classification system for strains of Mycobacterium abscessus. We hope the broad range of functions and easy-to-use tools provided in MycoCAP makes it an invaluable analysis platform to speed up the research discovery on mycobacteria for researchers. Database URL: http://mycobacterium.um.edu.my.

  8. Blowout recovery operations : the capping operation

    Energy Technology Data Exchange (ETDEWEB)

    Miller, M.; Badick, M. [Safety BOSS, Calgary, AB (Canada)

    2001-07-01

    Capping is generally the final work done at a wellhead. A properly planned capping operation is one of the easiest aspects of a recovery process that should be completed in one or two days. The objective is to establish a safe and reliable casing attachment and seal that is designed to suit the activities that follow. The movement of equipment on and off the wellhead is among the higher risk objectives of a recovery operation, so it is crucial that the job be done right the first time. This paper focused on blowout recovery operations and covered the operations from the point where a sound pipe or a reliable casing flange has been established to the point of installing a conventional wellhead, a diverter system blowout preventer (BOP) stack suitable for shutting in the well, continuing to flow the well, or proceeding with a killing operation. The advantages and disadvantages of many capping options were discussed along with the procedures for installing a wellhead or BOP. The choices for wellheads, diverter systems and BOP stack configurations depend on whether the well can be shut-in, killed or if it must be flowed while an offset well is drilled. The choices presented in this paper included, slip rams, casing bowls, and snubbing.

  9. An mRNA is capped by a 2', 5' lariat catalyzed by a group I-like ribozyme

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Westhof, Eric; Johansen, Steinar

    2005-01-01

    Twin-ribozyme introns are formed by two ribozymes belonging to the group I family and occur in some ribosomal RNA transcripts. The group I-like ribozyme, GIR1, liberates the 5' end of a homing endonuclease messenger RNA in the slime mold Didymium iridis. We demonstrate that this cleavage occurs...

  10. Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase.

    Science.gov (United States)

    Luzhkov, Victor B; Selisko, Barbara; Nordqvist, Anneli; Peyrane, Frédéric; Decroly, Etienne; Alvarez, Karine; Karlen, Anders; Canard, Bruno; Qvist, Johan

    2007-12-15

    We report high-throughput structure-based virtual screening of putative Flavivirus 2'-O-methyltransferase inhibitors together with results from subsequent bioassay tests of selected compounds. Potential inhibitors for the S-adenosylmethionine binding site were explored using 2D similarity searching, pharmacophore filtering and docking. The inhibitory activities of 15 top-ranking compounds from the docking calculations were tested on a recombinant methyltransferase with the RNA substrate (7Me)GpppAC(5). Local and global docking simulations were combined to estimate the ligand selectivity for the target site. The results of the combined computational and experimental screening identified a novel inhibitor, with a previously unknown scaffold, that has an IC(50) value of 60 microM.

  11. Partitions of AG(4,3) into Maximal Caps

    OpenAIRE

    Follett, Michael; Kalail, Kyle; McMahon, Elizabeth; Pelland, Catherine; Won, Robert

    2013-01-01

    In a geometry, a maximal cap is a collection of points of largest size containing no lines. In AG(4,3), maximal caps contain 20 points. The 81 points of AG(4,3) can be partitioned into 4 mutually disjoint maximal caps together with a single point P, where every pair of points that makes a line with P lies entirely inside one of those caps. The caps in a partition can be paired up so that both pairs are either in exactly one partition or they are both in two different partitions. This differen...

  12. What Lies Below a Martian Ice Cap

    Science.gov (United States)

    2008-01-01

    [figure removed for brevity, see original site] Click on image for larger annotated version This image (top) taken by the Shallow Radar instrument on NASA's Mars Reconnaissance Orbiter reveals the layers of ice, sand and dust that make up the north polar ice cap on Mars. It is the most detailed look to date at the insides of this ice cap. The colored map below the radar picture shows the topography of the corresponding Martian terrain (red and white represent higher ground, and green and yellow lower). The radar image reveals four never-before-seen thick layers of ice and dust separated by layers of nearly pure ice. According to scientists, these thick ice-free layers represent approximately one-million-year-long cycles of climate change on Mars caused by variations in the planet's tilted axis and its eccentric orbit around the sun. Adding up the entire stack of ice gives an estimated age for the north polar ice cap of about 4 million years a finding that agrees with previous theoretical estimates. The ice cap is about 2 kilometers (1.2 miles) thick. The radar picture also shows that the boundary between the ice layers and the surface of Mars underneath is relatively flat (bottom white line on the right). This implies that the surface of Mars is not sagging, or bending, under the weight of the ice cap and this, in turn, suggests that the planet's lithosphere, a combination of the crust and the strong parts of the upper mantle, is thicker than previously thought. A thicker lithosphere on Mars means that temperatures increase more gradually with depth toward the interior. Temperatures warm enough for water to be liquid are therefore deeper than previously thought. Likewise, if liquid water does exist in aquifers below the surface of Mars, and if there are any organisms living in that water, they would have to be located deeper in the planet. The topography data are from Mars Orbiter Laser Altimeter, which was flown on NASA's Mars Global Surveyor mission. NPLD stands

  13. To be or not to be: the regulation of mRNA fate as a survival strategy during mammalian hibernation.

    Science.gov (United States)

    Tessier, Shannon N; Storey, Kenneth B

    2014-11-01

    Mammalian hibernators undergo profound behavioral, physiological, and biochemical changes in order to cope with hypothermia, ischemia-reperfusion, and finite fuel reserves over days or weeks of continuous torpor. Against a backdrop of global reductions in energy-expensive processes such as transcription and translation, a subset of genes/proteins are strategically upregulated in order to meet challenges associated with hibernation. Consequently, hibernation involves substantial transcriptional and posttranscriptional regulatory mechanisms and provides a phenomenon with which to understand how a set of common genes/proteins can be differentially regulated in order to enhance stress tolerance beyond that which is possible for nonhibernators. The present review focuses on the involvement of messenger RNA (mRNA) interacting factors that play a role in the regulation of gene/protein expression programs that define the hibernating phenotype. These include proteins involved in mRNA processing (i.e., capping, splicing, and polyadenylation) and the possible role of alternative splicing as a means of enhancing protein diversity. Since the total pool of mRNA remains constant throughout torpor, mechanisms which enhance mRNA stability are discussed in the context of RNA binding proteins and mRNA decay pathways. Furthermore, mechanisms which control the global reduction of cap-dependent translation and the involvement of internal ribosome entry sites in mRNAs encoding stress response proteins are also discussed. Finally, the concept of regulating each of these factors in discrete subcellular compartments for enhanced efficiency is addressed. The analysis draws on recent research from several well-studied mammalian hibernators including ground squirrels, bats, and bears.

  14. Pore Water PAH Transport in Amended Sediment Caps

    Science.gov (United States)

    Gidley, P. T.; Kwon, S.; Ghosh, U.

    2009-05-01

    Capping is a common remediation strategy for contaminated sediments that creates a physical barrier between contaminated sediments and the water column. Diffusive flux of contaminants through a sediment cap is small. However, under certain hydrodynamic conditions such as groundwater potential and tidal pumping, groundwater advection can accelerate contaminant transport. Hydrophobic organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) could be transported through the cap under advective conditions. To better understand PAH migration under these conditions, physical models of sediment caps were evaluated in the laboratory through direct measurement of pore water using solid phase micro-extraction with gas chromatography and mass spectrometry. Contaminated sediment and capping material was obtained from an existing Superfund site that was capped at Eagle Harbor, Washington. A PAH dissolution model linked to an advection-dispersion equation with retardation using published organic carbon-water partitioning coefficients (Koc) was compared to measured PAHs in the sediment and cap porewater of the physical model.

  15. A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Einvik, Christer; Lentz, Thomas E

    2009-01-01

    DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5'-end of the 3'-cleavage product. The 3'-cleavage product...... reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5'-UTR as a direct consequence of the DiGIR1 branching reaction...

  16. GPS scintillations associated with cusp dynamics and polar cap patches

    Science.gov (United States)

    Jin, Yaqi; Moen, Jøran I.; Oksavik, Kjellmar; Spicher, Andres; Clausen, Lasse B. N.; Miloch, Wojciech J.

    2017-10-01

    This paper investigates the relative scintillation level associated with cusp dynamics (including precipitation, flow shears, etc.) with and without the formation of polar cap patches around the cusp inflow region by the EISCAT Svalbard radar (ESR) and two GPS scintillation receivers. A series of polar cap patches were observed by the ESR between 8:40 and 10:20 UT on December 3, 2011. The polar cap patches combined with the auroral dynamics were associated with a significantly higher GPS phase scintillation level (up to 0.6 rad) than those observed for the other two alternatives, i.e., cusp dynamics without polar cap patches, and polar cap patches without cusp aurora. The cusp auroral dynamics without plasma patches were indeed related to GPS phase scintillations at a moderate level (up to 0.3 rad). The polar cap patches away from the active cusp were associated with sporadic and moderate GPS phase scintillations (up to 0.2 rad). The main conclusion is that the worst global navigation satellite system space weather events on the dayside occur when polar cap patches enter the polar cap and are subject to particle precipitation and flow shears, which is analogous to the nightside when polar cap patches exit the polar cap and enter the auroral oval.

  17. An anilinoquinazoline derivative inhibits tumor growth through interaction with hCAP-G2, a subunit of condensin II.

    Directory of Open Access Journals (Sweden)

    Hirokazu Shiheido

    Full Text Available We screened 46 novel anilinoquinazoline derivatives for activity to inhibit proliferation of a panel of human cancer cell lines. Among them, Q15 showed potent in vitro growth-inhibitory activity towards cancer cell lines derived from colorectal cancer, lung cancer and multiple myeloma. It also showed antitumor activity towards multiple myeloma KMS34 tumor xenografts in lcr/scid mice in vivo. Unlike the known anilinoquinazoline derivative gefitinib, Q15 did not inhibit cytokine-mediated intracellular tyrosine phosphorylation. Using our mRNA display technology, we identified hCAP-G2, a subunit of condensin II complex, which is regarded as a key player in mitotic chromosome condensation, as a Q15 binding partner. Immunofluorescence study indicated that Q15 compromises normal segregation of chromosomes, and therefore might induce apoptosis. Thus, our results indicate that hCAP-G2 is a novel therapeutic target for development of drugs active against currently intractable neoplasms.

  18. Effective polar cap area and multi-station basis for Polar Cap (PC) indices

    Science.gov (United States)

    Stauning, Peter

    2017-04-01

    The Polar Cap (PC) indices are useful indices for Space Weather forecasts and analyses. The PC indices have been used to monitor the interplanetary geoeffective electric field and solar wind pressure pulses, to analyze cross polar cap voltages, polar cap diameter, and general polar cap dynamics. Furthermore, the PC indices have been used to monitor auroral electrojet intensities, ionospheric Joule heating, and global auroral power, and to predict ring current intensities. For specific Space Weather warning forecasts the PC indices can be used to predict substorm development and the associated risk of power line disturbances in the subauroral regions. The PC indices, PCN (North) and PCS (South), are derived from geomagnetic observations at Thule in Greenland and Vostok in Antarctica, respectively. In order to provide reliable forecast services based on PC indices, it would be advantageous to have available back-up suppliers of index values. The presentation provides an analysis of the effective area for useful PC index derivation and suggests observatories that could provide back-up data for PC index calculations should the primary sources fail due to instrument or communication problems.

  19. Control of mRNA turnover as a mechanism of glucose repression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Scheffler, I E; de la Cruz, B J; Prieto, S

    1998-11-01

    The phenomenon of glucose repression in yeast is concerned with the repression of a large number of genes when glucose is an abundant carbon source and almost all of the energy requirements of the cell can be satisfied from glycolysis. Prominent among the repressed genes are those encoding mitochondrial proteins required for respiration and oxidative phosphorylation. Past studies have characterized a pathway by which a signal generated from extracellular glucose is transmitted to the nucleus. The ultimate outcome is the repression of transcription of numerous genes, but also the induction of a limited number of others. The emphasis has been almost exclusively on transcriptional control mechanisms. A discovery made originally with the transcript of the SDH2 gene prompted an investigation of post-transcriptional mechanisms, and more specifically a study of the turnover rate of this mRNA in the absence and presence of glucose. SDH2 mRNA has a very short half-life in medium with glucose (YPD) and a significantly longer half-life in medium with glycerol (YPG). Experimental evidence and recent progress in understanding of (1) mRNA turnover in yeast and (2) initiation of translation on the 5' untranslated region of mRNAs, lead to a working hypothesis with the following major features: the carbon source, via a signaling pathway involving kinase/phosphatase activities, controls the rate of initiation, and thus influences a competition between eukaryotic initiation factors (prominently eIF4E, eIF4G, eIF3) binding to the capped mRNA and a decapping activity (DCP1) which is one of the rate limiting activities in the turnover of such mRNAs.

  20. MFTF-. cap alpha. + T progress report

    Energy Technology Data Exchange (ETDEWEB)

    Nelson, W.D. (ed.)

    1985-04-01

    Early in FY 1983, several upgrades of the Mirror Fusion Test Facility (MFTF-B) at Lawrence Livermore National Laboratory (LLNL) were proposed to the fusion community. The one most favorably received was designated MFTF-..cap alpha..+T. The engineering design of this device, guided by LLNL, has been a principal activity of the Fusion Engineering Design Center during FY 1983. This interim progress report represents a snapshot of the device design, which was begun in FY 1983 and will continue for several years. The report is organized as a complete design description. Because it is an interim report, some parts are incomplete; they will be supplied as the design study proceeds. As described in this report, MFTF-..cap alpha..+T uses existing facilities, many MFTF-B components, and a number of innovations to improve on the physics parameters of MFTF-B. It burns deuterium-tritium and has a central-cell Q of 2, a wall loading GAMMA/sub n/ of 2 MW/m/sup 2/ (with a central-cell insert module), and an availability of 10%. The machine is fully shielded, allows hands-on maintenance of components outside the vacuum vessel 24 h after shutdown, and has provisions for repair of all operating components.

  1. Incorporating Wind Generation in Cap and Trade Programs

    Energy Technology Data Exchange (ETDEWEB)

    Bluestein, J.; Salerno, E.; Bird, L.; Vimmerstedt, L.

    2006-07-01

    Cap and trade programs are increasingly being used to reduce emissions from electricity generation in the United States. Cap and trade programs primarily target emitting generators, but programs have also included renewable generators, such as wind generators. States cite several reasons why they have considered the policy option of including renewable generators in cap and trade programs: to provide an incentive for lower-emitting generation, to achieve emissions reductions in non-capped pollutants, and to gain local economic benefits associated with renewable energy projects. The U.S. Environmental Protection Agency also notes these rationales for considering this policy alternative, and the National Association of Regulatory Commissioners (NARUC) passed a resolution supporting the inclusion of renewable energy in cap and trade programs. This report explores why states consider this policy option, what participation could mean for wind generators, and how wind generation can most effectively be included in state, federal, and regional cap and trade programs.

  2. Acoustic Monitoring of the Arctic Ice Cap

    Science.gov (United States)

    Porter, D. L.; Goemmer, S. A.; Chayes, D. N.

    2012-12-01

    Introduction The monitoring of the Arctic Ice Cap is important economically, tactically, and strategically. In the scenario of ice cap retreat, new paths of commerce open, e.g. waterways from Northern Europe to the Far East. Where ship-going commerce is conducted, the U.S. Navy and U.S. Coast Guard have always stood guard and been prepared to assist from acts of nature and of man. It is imperative that in addition to measuring the ice from satellites, e.g. Icesat, that we have an ability to measure the ice extent, its thickness, and roughness. These parameters play an important part in the modeling of the ice and the processes that control its growth or shrinking and its thickness. The proposed system consists of three subsystems. The first subsystem is an acoustic source, the second is an array of geophones and the third is a system to supply energy and transmit the results back to the analysis laboratory. The subsystems are described below. We conclude with a plan on how to tackle this project and the payoff to the ice cap modeler and hence the users, i.e. commerce and defense. System Two historically tested methods to generate a large amplitude multi-frequency sound source include explosives and air guns. A new method developed and tested by the University of Texas, ARL is a combustive Sound Source [Wilson, et al., 1995]. The combustive sound source is a submerged combustion chamber that is filled with the byproducts of the electrolysis of sea water, i.e. Hydrogen and Oxygen, an explosive mixture which is ignited via a spark. Thus, no additional compressors, gases, or explosives need to be transported to the Arctic to generate an acoustic pulse capable of the sediment and the ice. The second subsystem would be geophones capable of listening in the O(10 Hz) range and transmitting that data back to the laboratory. Thus two single arrays of geophones arranged orthogonal to each other with a range of 1000's of kilometers and a combustive sound source where the two

  3. Increased 5. cap alpha. -reductase activity in idiopathic hirsutism

    Energy Technology Data Exchange (ETDEWEB)

    Serafini, P.; Lobo, R.A.

    1985-01-01

    In vitro, genital skin 5..cap alpha..-reductase activity (5..cap alpha..-RA) was measured in ten hirsute women with normal androgen levels (idiopathic hirsutism (IH)) and in ten hirsute women with elevated androgen levels (polycystic ovary syndrome (PCO)) in order to determine the influence of secreted androgens on 5..cap alpha..-RA. In vitro 5..cap alpha..-RA was assessed by incubations of skin with /sup 14/C-testosterone (T) for 2 hours, after which steroids were separated and the radioactivity of dihydrotestosterone (DHT) and 5..cap alpha..-androstane 3..cap alpha..-17..beta..-estradiol (3..cap alpha..-diol) in specific eluates were determined. All androgens were normal in IH with the exception of higher levels of 3..cap alpha..-diol glucuronide which were similar to the levels of PCO. The conversion ratio (CR) of T to DHT in IH and PCO were similar, yet significantly greater than the CR of control subjects. The CR of T to 3..cap alpha..-diol in IH and PCO were similar, yet higher than in control subjects. Serum androgens showed no correlation with 5..cap alpha..-RA, while the CR of T to DHT showed a significant positive correlation with the Ferriman and Gallwey score. The increased 5..cap alpha..-RA in IH appears to be independent of serum androgen levels and is, therefore, an inherent abnormality. The term idiopathic is a misnomer, because hirsutism in these patients may be explained on the basis of increased skin 5..cap alpha..-RA.

  4. Preform spar cap for a wind turbine rotor blade

    Science.gov (United States)

    Livingston, Jamie T [Simpsonville, SC; Driver, Howard D [Greer, SC; van Breugel, Sjef [Enschede, NL; Jenkins, Thomas B [Cantonment, FL; Bakhuis, Jan Willem [Nijverdal, NL; Billen, Andrew J [Daarlerveen, NL; Riahi, Amir [Pensacola, FL

    2011-07-12

    A spar cap for a wind turbine rotor blade. The spar cap may include multiple preform components. The multiple preform components may be planar sheets having a swept shape with a first end and a second end. The multiple preform components may be joined by mating the first end of a first preform component to the second end of a next preform component, forming the spar cap.

  5. Capped bit patterned media for high density magnetic recording

    Science.gov (United States)

    Li, Shaojing; Livshitz, Boris; Bertram, H. Neal; Inomata, Akihiro; Fullerton, Eric E.; Lomakin, Vitaliy

    2009-04-01

    A capped composite patterned medium design is described which comprises an array of hard elements exchange coupled to a continuous cap layer. The role of the cap layer is to lower the write field of the individual hard element and introduce ferromagnetic exchange interactions between hard elements to compensate the magnetostatic interactions. Modeling results show significant reduction in the reversal field distributions caused by the magnetization states in the array which is important to prevent bit errors and increase achievable recording densities.

  6. Lowering the YE+1 end-cap for CMS

    CERN Multimedia

    Maximilien Brice

    2007-01-01

    On 9 January 2007, the massive YE+1 end-cap was lowered into the CMS cavern. This is a very precise process as the crane must lower the end-cap through minimal clearance without tilt or sway. Once in the cavern, the end-cap is then positioned over the end of the barrel to detect particles produced in collisions that travel close to the axis of the beams.

  7. The potential of targeted antibody prophylaxis in SARS outbreak control: a mathematic analysis

    NARCIS (Netherlands)

    Bogaards, Johannes Antonie; Putter, Hein; Jan Weverling, Gerrit; ter Meulen, Jan; Goudsmit, Jaap

    2007-01-01

    BACKGROUND: Severe acute respiratory syndrome (SARS) coronavirus-like viruses continue to circulate in animal reservoirs. If new mutants of SARS coronavirus do initiate another epidemic, administration of prophylactic antibodies to risk groups may supplement the stringent isolation procedures that

  8. Update: Mechanisms underlying N6-methyladenosine modification of eukaryotic mRNA

    Science.gov (United States)

    Wang, Yang; Zhao, Jing Crystal

    2016-01-01

    Summary Eukaryotic messenger RNA (mRNA) undergoes chemical modification both at the 5′cap [1, 2] and internally [3–14]. Among internal modifications, m6A, by far the most abundant, is present in all eukaryotes examined, including mammals [3–6], flies [15], plants [16, 17] and yeast [18, 19]. m6A modification plays an essential role in diverse biological processes. Over the past few years, our knowledge relevant to establishment and function of this modification has grown rapidly. This review focuses on technologies that have facilitated m6A detection in mRNAs, identification of m6A methylation enzymes and binding proteins, and potential functions of the modification at the molecular level. Regarding m6A function at cellular or organismal levels or in disease, please refer to other recent reviews [20–23]. PMID:27793360

  9. The CAP Theorem Versus Databases with Relaxed ACID properties

    DEFF Research Database (Denmark)

    Frank, Lars; Ulslev Pedersen, Rasmus; Frank, Christian Havnø

    2014-01-01

    The CAP theorem combines the three desirable properties C (data consistency), A (data availability), and P (partition-tolerance: tolerance of inconsistencies between data stored in a distributed database where partitions are allowed). The CAP theorem asserts that any distributed system that uses ...... data from different locations can have at most two of the three desirable CAP properties [5]. The NoSQL movement has applied the CAP theorem as an argument against traditional ACID (atomicity, consistency, isolation, and durability) databases, which prioritize consistency and partition...

  10. NAMMA CLOUD MICROPHYSICS (CAPS-PIP) V1

    Data.gov (United States)

    National Aeronautics and Space Administration — The Cloud Microphysics dataset consists of particle size distributions from the Clouds, Aerosol and Preciptaition Spectrometer (CAPS) and the Precipitaiton Imaging...

  11. CAP1 expression is developmentally regulated in Xenopus.

    Science.gov (United States)

    KhosrowShahian, F; Hubberstey, A V; Crawford, M J

    2002-05-01

    We have cloned and characterized a Xenopus member of the cyclase associated protein (CAP) gene family. xCAP1 is expressed as a maternal transcript, but is up-regulated prior to gastrulation and subsequently localizes to head mesenchyme, lens, otic vesicle, and trunk mesoderm including the pronephros. At different stages, the gene also appears to differentiate surface from deep (sensorial) ectoderm. As in Drosophila, Xenopus CAP1 is expressed in the developing eye, specifically in the differentiating lens. However, in distinction to Drosophila, Xenopus CAP1 does not express in periodically arrayed neural bands.

  12. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  13. Stress granules: sites of mRNA triage that regulate mRNA stability and translatability.

    Science.gov (United States)

    Kedersha, N; Anderson, P

    2002-11-01

    Mammalian stress granules (SGs) are cytoplasmic domains into which mRNAs are sorted dynamically in response to phosphorylation of eukaryotic initiation factor (eIF) 2alpha, a key regulatory step in translational initiation. The activation of one or more of the eIF2alpha kinases leads to SG assembly by decreasing the levels of eIF2-GTP-tRNA(Met), the ternary complex that is normally required for loading the initiator methionine onto the 48 S preinitiation complex to begin translation. This stress-induced scarcity of eIF2-GTP-tRNA(Met) allows the RNA-binding proteins TIA-1 (T-cell internal antigen-1) and TIAR (TIA-1-related protein) to bind the 48 S complex in lieu of the ternary complex, thereby promoting polysome disassembly and the concurrent routing of the mRNA into a SG. The actual formation of SGs occurs upon auto-aggregation of the prion-like C-termini of TIA-1 proteins; this aggregation is reversed in vivo by overexpression of the heat-shock protein (HSP) chaperone HSP70. Remarkably, HSP70 mRNA is excluded from SGs and is preferentially translated during stress, indicating that the RNA composition of the SG is selective. Moreover, the effects of HSP70 on TIA aggregation suggest a feedback loop whereby HSP70 synthesis is auto-regulated. Proteins that promote mRNA stability [e.g. HuR (Hu protein R)] and destabilize mRNA [i.e. tristetraprolin (TTP)] are also recruited to SGs, suggesting that SGs effect a process of mRNA triage, by promoting polysome disassembly and routing mRNAs to cytoplasmic domains enriched for HuR and TTP. This model reveals connections between the eIF2alpha kinase system, mRNA stability and cellular chaperone levels.

  14. mRNA quality control pathways in Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    2013-07-10

    Jul 10, 2013 ... mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional .... Figure 1. mRNA life-cycle in eukaryotic cell: Schematic view of the nuclear and cytoplasmic phases of mRNA life cycle, namely ..... structure is characteristic and critical feature of an mRNA.

  15. Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5.

    Science.gov (United States)

    Egloff, Marie-Pierre; Decroly, Etienne; Malet, Hélène; Selisko, Barbara; Benarroch, Delphine; Ferron, François; Canard, Bruno

    2007-09-21

    The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.

  16. Electroporated Antigen-Encoding mRNA Is Not a Danger Signal to Human Mature Monocyte-Derived Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Stefanie Hoyer

    2015-01-01

    Full Text Available For therapeutic cancer vaccination, the adoptive transfer of mRNA-electroporated dendritic cells (DCs is frequently performed, usually with monocyte-derived, cytokine-matured DCs (moDCs. However, DCs are rich in danger-sensing receptors which could recognize the exogenously delivered mRNA and induce DC activation, hence influencing the DCs’ immunogenicity. Therefore, we examined whether electroporation of mRNA with a proper cap and a poly-A tail of at least 64 adenosines had any influence on cocktail-matured moDCs. We used 16 different RNAs, encoding tumor antigens (MelanA, NRAS, BRAF, GNAQ, GNA11, and WT1, and variants thereof. None of those RNAs induced changes in the expression of CD25, CD40, CD83, CD86, and CD70 or the secretion of the cytokines IL-8, IL-6, and TNFα of more than 1.5-fold compared to the control condition, while an mRNA encoding an NF-κB-activation protein as positive control induced massive secretion of the cytokines. To determine whether mRNA electroporation had any effect on the whole transcriptome of the DCs, we performed microarray analyses of DCs of 6 different donors. None of 60,000 probes was significantly different between mock-electroporated DCs and MelanA-transfected DCs. Hence, we conclude that no transcriptional programs were induced within cocktail-matured DCs by electroporation of single tumor-antigen-encoding mRNAs.

  17. Carpentier, la otra novela (cap. IV

    Directory of Open Access Journals (Sweden)

    Urbano Martínez Carmenate

    2014-07-01

    Full Text Available El presente ensayo está extraído de la obra inédita de Urbano Martínez Carmenate, Carpentier, la otra novela (cap. IV. Ofrece una mirada sobre los primeros años de la trayectoria profesional de Alejo Carpentier. Se muestra cómo, durante la década de 1920, se inicia como periodista y cronista teatral. A la vez, comienzan sus conexiones con el Grupo Minorista, que desempeña un papel destacado en la etapa republicana en Cuba. También se contempla su viaje a México en 1926, que constituye su primer contacto directo con la naturaleza y la cultura americanas.

  18. Carpentier, la otra novela (cap. IV

    Directory of Open Access Journals (Sweden)

    Urbano Martínez Carmenate

    2014-06-01

    Full Text Available El presente ensayo está extraído de la obra inédita de Urbano Martínez Carmenate, Carpentier, la otra novela (cap. IV. Ofrece una mirada sobre los primeros años de la trayectoria profesional de Alejo Carpentier. Se muestra cómo, durante la década de 1920, se inicia como periodista y cronista teatral. A la vez, comienzan sus conexiones con el Grupo Minorista, que desempeña un papel destacado en la etapa republicana en Cuba. También se contempla su viaje a México en 1926, que constituye su primer contacto directo con la naturaleza y la cultura americanas.

  19. Cytocompatibility and Antibacterial Properties of Capping Materials

    Directory of Open Access Journals (Sweden)

    Claudio Poggio

    2014-01-01

    Full Text Available The aim of this study was to evaluate and compare the antimicrobial activity and cytocompatibility of six different pulp-capping materials: Dycal (Dentsply, Calcicur (Voco, Calcimol LC (Voco, TheraCal LC (Bisco, MTA Angelus (Angelus, and Biodentine (Septodont. To evaluate antimicrobial activity, materials were challenged in vitro with Streptococcus mutans, Streptococcus salivarius, and Streptococcus sanguis in the agar disc diffusion test. Cytocompatibility of the assayed materials towards rat MDPC-23 cells was evaluated at different times by both MTT and apoptosis assays. Results significantly differed among the different materials tested. Both bacterial growth inhibition halos and cytocompatibility performances were significantly different among materials with different composition. MTA-based products showed lower cytotoxicity and valuable antibacterial activity, different from calcium hydroxide-based materials, which exhibited not only higher antibacterial activity but also higher cytotoxicity.

  20. Cytocompatibility and Antibacterial Properties of Capping Materials

    Science.gov (United States)

    Arciola, Carla Renata; Monaco, Annachiara; Lombardini, Marco

    2014-01-01

    The aim of this study was to evaluate and compare the antimicrobial activity and cytocompatibility of six different pulp-capping materials: Dycal (Dentsply), Calcicur (Voco), Calcimol LC (Voco), TheraCal LC (Bisco), MTA Angelus (Angelus), and Biodentine (Septodont). To evaluate antimicrobial activity, materials were challenged in vitro with Streptococcus mutans, Streptococcus salivarius, and Streptococcus sanguis in the agar disc diffusion test. Cytocompatibility of the assayed materials towards rat MDPC-23 cells was evaluated at different times by both MTT and apoptosis assays. Results significantly differed among the different materials tested. Both bacterial growth inhibition halos and cytocompatibility performances were significantly different among materials with different composition. MTA-based products showed lower cytotoxicity and valuable antibacterial activity, different from calcium hydroxide-based materials, which exhibited not only higher antibacterial activity but also higher cytotoxicity. PMID:24959601

  1. Microtubule dynamics: Caps, catastrophes, and coupled hydrolysis

    DEFF Research Database (Denmark)

    Flyvbjerg, H.; Holy, T.E.; Leibler, S.

    1996-01-01

    and probability distributions relating to available experimental data are derived. Caps are found to be short and the total rate of hydrolysis at a microtubule end is found to be dynamically coupled to growth. The so-called catastrophe rate is a simple function of the microtubule growth rare and fits experimental...... data. A constant nonzero catastrophe rare, identical for both microtubule ends, is predicted at large growth rates. The delay time for dilution-induced catastrophes is stochastic with a simple distribution that fits the experimental one and, like the experimental one, does not depend on the rate...... description of several apparently contradictory experimental data. Experimental results for the catastrophe rate at different concentrations of magnesium ions and of microtubule associated proteins are discussed in terms of the model. Feasible experiments are suggested that can provide decisive tests...

  2. Glaciers and ice caps outside Greenland

    Science.gov (United States)

    Sharp, Marin; Wolken, G.; Burgess, D.; Cogley, J.G.; Copland, L.; Thomson, L.; Arendt, A.; Wouters, B.; Kohler, J.; Andreassen, L.M.; O'Neel, Shad; Pelto, M.

    2015-01-01

    Mountain glaciers and ice caps cover an area of over 400 000 km2 in the Arctic, and are a major influence on global sea level (Gardner et al. 2011, 2013; Jacob et al. 2012). They gain mass by snow accumulation and lose mass by meltwater runoff. Where they terminate in water (ocean or lake), they also lose mass by iceberg calving. The climatic mass balance (Bclim, the difference between annual snow accumulation and annual meltwater runoff) is a widely used index of how glaciers respond to climate variability and change. The total mass balance (ΔM) is defined as the difference between annual snow accumulation and annual mass losses (by iceberg calving plus runoff).

  3. Pulsar gamma rays from polar cap regions

    Science.gov (United States)

    Chiang, James; Romani, Roger W.

    1992-01-01

    The production is studied of pulsar gamma rays by energetic electrons flowing in the open field region above pulsar polar caps. The propagation was followed of curvature radiation from primary electrons, as well as hard synchrotron radiation generated by secondary pairs, through the pulsar magnetosphere for vacuum dipole open field geometries. Using data from radio and optical observations, models were constructed for the specific geometries and viewing angles appropriate to particular pulsars. These detailed models produce normalized spectra above 10 MeV, pulse profiles, beaming fractions and phase resolved spectra appropriate for direct comparison with COS-B and GRO data. Models are given for the Crab, Vela, and other potentially detectable pulsars; general agreement with existing data is good, although perturbations to the simplified models are needed for close matches. The calculations were extended to the millisecond pulsar range, which allows the production of predictions for the flux and spectra of populations of recycled pulsars and search strategies are pointed out.

  4. Diagnostic criteria for cryopyrin-associated periodic syndrome (CAPS).

    Science.gov (United States)

    Kuemmerle-Deschner, Jasmin B; Ozen, Seza; Tyrrell, Pascal N; Kone-Paut, Isabelle; Goldbach-Mansky, Raphaela; Lachmann, Helen; Blank, Norbert; Hoffman, Hal M; Weissbarth-Riedel, Elisabeth; Hugle, Boris; Kallinich, Tilmann; Gattorno, Marco; Gul, Ahmet; Ter Haar, Nienke; Oswald, Marlen; Dedeoglu, Fatma; Cantarini, Luca; Benseler, Susanne M

    2017-06-01

    Cryopyrin-associated periodic syndrome (CAPS) is a rare, heterogeneous disease entity associated with NLRP3 gene mutations and increased interleukin-1 (IL-1) secretion. Early diagnosis and rapid initiation of IL-1 inhibition prevent organ damage. The aim of the study was to develop and validate diagnostic criteria for CAPS. An innovative process was followed including interdisciplinary team building, item generation: review of CAPS registries, systematic literature review, expert surveys, consensus conferences for item refinement, item reduction and weighting using 1000Minds decision software. Resulting CAPS criteria were tested in large cohorts of CAPS cases and controls using correspondence analysis. Diagnostic models were explored using sensitivity analyses. The international team included 16 experts. Systematic literature and registry review identified 33 CAPS-typical items; the consensus conferences reduced these to 14. 1000Minds exercises ranked variables based on importance for the diagnosis. Correspondence analysis determined variables consistently associated with the diagnosis of CAPS using 284 cases and 837 controls. Seven variables were significantly associated with CAPS (pCAPS-typical symptoms: urticaria-like rash, cold-triggered episodes, sensorineural hearing loss, musculoskeletal symptoms, chronic aseptic meningitis and skeletal abnormalities. Sensitivity was 81%, specificity 94%. It performed well for all CAPS subtypes and regardless of NLRP3 mutation. The novel approach integrated traditional methods of evidence synthesis with expert consensus, web-based decision tools and innovative statistical methods and may serve as model for other rare diseases. These criteria will enable a rapid diagnosis for children and adults with CAPS. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  5. Messenger RNA (mRNA) nanoparticle tumour vaccination

    Science.gov (United States)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  6. Preparing an ATLAS toroid magnet end-cap for lowering

    CERN Multimedia

    Claudia Marcelloni

    2007-01-01

    One of the two 13-m high toroid magnet end-caps for the ATLAS experiment being transported from the construction hall to the experimental area. The end-cap will be lowered into the ATLAS cavern and attached to an end of the detector.

  7. Relative symplectic caps, 4-genus and fibered knots

    Indian Academy of Sciences (India)

    proof presented here does not invoke sophisticated machinery such as the Seiberg–Witten theory. We shall apply Theorem 1.2 in .... Construction of relative symplectic caps. In this section, we construct ... case we prefer to denote it by K. By inductively applying the following construction of the cap for each component of L, ...

  8. ATLAS end-caps 
on the move

    CERN Multimedia

    2007-01-01

    Two delicate and spectacular transport operations have been performed for ATLAS in recent weeks: the first end-cap tracker was installed in its final position, and one of the huge end-caps of the toroid magnet was moved to the top of the experiment’s shaft.

  9. Polar cap index as a proxy for hemispheric Joule heating

    DEFF Research Database (Denmark)

    Chun, F.K.; Knipp, D.J.; McHarg, M.G.

    1999-01-01

    The polar cap (PC) index measures the level of geomagnetic activity in the polar cap based on magnetic perturbations from overhead ionospheric currents and distant field-aligned currents on the poleward edge of the nightside auroral oval. Because PC essentially measures the main sources of energy...

  10. 7 CFR 1714.7 - Interest rate cap.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Interest rate cap. 1714.7 Section 1714.7 Agriculture... PRE-LOAN POLICIES AND PROCEDURES FOR INSURED ELECTRIC LOANS General § 1714.7 Interest rate cap. Except as provided in paragraph (c) of this section, the municipal interest rate may not exceed 7 percent on...

  11. Wooden beverage cases cause little damage to bottle caps

    Science.gov (United States)

    R. Bruce Anderson; William C. Miller

    1973-01-01

    Wooden beverage cases cause little damage to aluminum resealable caps during distribution. A study at bottling plants and distribution warehouses showed that an average of 1 bottle out of 4,000 has cap damage. Most of the damage was attributed to handling at the warehouse and in transit. Some recommendations are given for improvement of wooden beverage cases to prevent...

  12. Effect of capping agents on optical and antibacterial properties of ...

    Indian Academy of Sciences (India)

    Home; Journals; Bulletin of Materials Science; Volume 38; Issue 5. Effect of ... Capping agents; quantum confinement; poly-dispersed; luminescent. Abstract. Cadmium selenide quantum dots (CdSe QDs) were synthesized in aqueous phase by the freezing temperature injection technique using different capping agents (viz.

  13. 20 CFR 606.21 - Criteria for cap.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Criteria for cap. 606.21 Section 606.21 Employees' Benefits EMPLOYMENT AND TRAINING ADMINISTRATION, DEPARTMENT OF LABOR TAX CREDITS UNDER THE... Reduction § 606.21 Criteria for cap. (a) Reduction in unemployment tax effort. (1) For purposes of paragraph...

  14. Survival of perennial carbon dioxide ice caps on Mars

    Science.gov (United States)

    Byrne, S.; Hayne, P. O.; Becerra, P.

    2017-09-01

    Perennial CO2 ice caps on Mars roughen unstably generating expanding pits that are observed by spacecraft. We tie observations of the surface and atmosphere together with landscape evolution models to explain this phenomenon and predict a polar cap that waxes and wanes over timescales of 100 yrs.

  15. Progress in LAr EndCap Calorimetry: News from the Hadronic EndCap Group.

    CERN Multimedia

    Oram, C.J.

    With module production and testing completed for the Hadronic EndCap calorimeter, the attention of the HEC group is heavily directed towards wheel assembly in building 180. Three of the four HEC wheels are now assembled and rotated, and work is progressing on assembling the final wheel. This year has been a busy year for the installation of components in the EndCap C cryostat: the signal feedthrough installation was completed April 22nd, the pre-sampler shortly thereafter and the Electro-Magnetic EndCap August 13th. This allowed the HEC group to start transferring the HEC wheels from the T6A storage cradle into the cryostat. The operation started in mid-September and has progressed, on or ahead of schedule, since then with the major milestones being: Insertion of 67 ton front HEC wheel October 3rd Insertion of 90 ton rear HEC wheel October 22nd. The wheel alignment has proved to be excellent, with the position of the centre of the front(rear) wheel with respect to the nominal position being displaced b...

  16. [Radiation protection provided by tungsten bismuth caps during interventional cardiology].

    Science.gov (United States)

    Ramos-Avasola, Sergio; Díaz, Natalia; Roldán, Reynaldo; Gamarra, Jorge; Catalán, Mónica

    2016-07-01

    The effectiveness against radiation of tungsten bismuth caps, used in interventional cardiology is not well known. To determine the degree of radiation protection conferred by these caps in real work conditions. We compared the gross electric charges received at brain lobe levels by three occupationally exposed professionals who participated in 22 consecutive procedures, inside and outside of the tungsten bismuth cap. The median electric charges outside and inside the cap were 3.71 (range 1.46-5.62) and 2.2 (range 1.29-3.93) nC, which correspond to a 40% radiation attenuation. However, the protection was heterogeneous. Tungsten bismuth caps provide an adequate attenuation, but its degree is heterogeneous.

  17. Exploring plasmonic coupling in hole-cap arrays

    Directory of Open Access Journals (Sweden)

    Thomas M. Schmidt

    2015-01-01

    Full Text Available The plasmonic coupling between gold caps and holes in thin films was investigated experimentally and through finite-difference time-domain (FDTD calculations. Sparse colloidal lithography combined with a novel thermal treatment was used to control the vertical spacing between caps and hole arrays and compared to separated arrays of holes or caps. Optical spectroscopy and FDTD simulations reveal strong coupling between the gold caps and both Bloch Wave-surface plasmon polariton (BW-SPP modes and localized surface plasmon resonance (LSPR-type resonances in hole arrays when they are in close proximity. The interesting and complex coupling between caps and hole arrays reveals the details of the field distribution for these simple to fabricate structures.

  18. Exploring plasmonic coupling in hole-cap arrays.

    Science.gov (United States)

    Schmidt, Thomas M; Frederiksen, Maj; Bochenkov, Vladimir; Sutherland, Duncan S

    2015-01-01

    The plasmonic coupling between gold caps and holes in thin films was investigated experimentally and through finite-difference time-domain (FDTD) calculations. Sparse colloidal lithography combined with a novel thermal treatment was used to control the vertical spacing between caps and hole arrays and compared to separated arrays of holes or caps. Optical spectroscopy and FDTD simulations reveal strong coupling between the gold caps and both Bloch Wave-surface plasmon polariton (BW-SPP) modes and localized surface plasmon resonance (LSPR)-type resonances in hole arrays when they are in close proximity. The interesting and complex coupling between caps and hole arrays reveals the details of the field distribution for these simple to fabricate structures.

  19. 30 CFR 250.1157 - How do I receive approval to produce gas-cap gas from an oil reservoir with an associated gas cap?

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 2 2010-07-01 2010-07-01 false How do I receive approval to produce gas-cap gas from an oil reservoir with an associated gas cap? 250.1157 Section 250.1157 Mineral Resources... do I receive approval to produce gas-cap gas from an oil reservoir with an associated gas cap? (a...

  20. The influence of connecting pile cap-column in the mechanisms of break in the two pile caps

    Directory of Open Access Journals (Sweden)

    A. C. MESQUITA

    Full Text Available Abstract The paper analyzes the two pile caps with partially embedded socket and subject a center load. Three models were experimentally tested, varying the type of conformation of the column and walls of the socket, with a smooth, the other rough, and a monolithic two pile cap, used for reference. The roughening of the column-socket interface was examined with the aim of verifying the difference of the distribution of compressive and tensile stresses in the strut an tie model used for design. The experimental test to show that the two pile caps with conformation rough of the column and walls of the socket, support more load in comparison with two pile caps with smooth of the column and walls of the socket. Both however underperformed the monolithic two pile cap, with values of 66% and 36% respectively.

  1. Cap0037, a Novel Global Regulator of Clostridium acetobutylicum Metabolism

    Directory of Open Access Journals (Sweden)

    Ngoc-Phuong-Thao Nguyen

    2016-10-01

    Full Text Available An operon comprising two genes, CA_P0037 and CA_P0036, that encode proteins of unknown function that were previously shown to be highly expressed in acidogenic cells and repressed in solventogenic and alcohologenic cells is located on the pSOL1 megaplasmid of Clostridium acetobutylicum upstream of adhE2. A CA_P0037::int (189/190s mutant in which an intron was inserted at position 189/190 in the sense strand of CA_P0037 was successfully generated by the Targetron technique. The resultant mutant showed significantly different metabolic flux patterns in acidogenic (producing mainly lactate, butyrate, and butanol and alcohologenic (producing mainly butyrate, acetate, and lactate chemostat cultures but not in solventogenic or batch cultures. Transcriptomic investigation of the CA_P0037::int (189/190s mutant showed that inactivation of CA_P0037 significantly affected the expression of more than 258 genes under acidogenic conditions. Surprisingly, genes belonging to the Fur regulon, involved in iron transport (CA_C1029-CA_C1032, or coding for the main flavodoxin (CA_C0587 were the most significantly expressed genes under all conditions, whereas fur (coding for the ferric uptake regulator gene expression remained unchanged. Furthermore, most of the genes of the Rex regulon, such as the adhE2 and ldhA genes, and of the PerR regulon, such as rbr3A-rbr3B and dfx, were overexpressed in the mutant. In addition, the whole CA_P0037-CA_P0036 operon was highly expressed under all conditions in the CA_P0037::int (189/190s mutant, suggesting a self-regulated expression mechanism. Cap0037 was shown to bind to the CA_P0037-CA_P0036 operon, sol operon, and adc promoters, and the binding sites were determined by DNA footprinting. Finally, a putative Cap0037 regulon was generated using a bioinformatic approach.

  2. Options for In Situ Capping of Palos Verdes Shelf Contaminated Sediments

    National Research Council Canada - National Science Library

    Palermo, Michael; Schroeder, Paul; Rivera, Yilda; Ruiz, Carlos; Clarke, Doug; Gailani, Joe; Clausner, James; Hynes, Mary; Fredette, Thomas; Tardy, Barbara

    1999-01-01

    .... This study included prioritizing areas of the PV shelf to be capped, determining an appropriate cap design or designs, developing an equipment selection and operations plan for placement of the cap...

  3. Casimir effect in hemisphere capped tubes

    CERN Document Server

    de Mello, E R Bezerra

    2014-01-01

    In this paper we investigate the vacuum densities for a massive scalar field with general curvature coupling in background of a (2+1)-dimensional spacetime corresponding to a cylindrical tube with a hemispherical cap. A complete set of mode functions is constructed and the positive-frequency Wightman function is evaluated for both the cylindrical and hemispherical subspaces. On the base of this, the vacuum expectation values of the field squared and energy-momentum tensor are investigated. The mean field squared and the normal stress are finite on the boundary separating two subspaces, whereas the energy density and the parallel stress diverge as the inverse power of the distance from the boundary. For a conformally coupled field, the vacuum energy density is negative on the cylindrical part of the space. On the hemisphere, it is negative near the top and positive close to the boundary. In the case of minimal coupling the energy density on the cup is negative. On the tube it is positive near the boundary and ...

  4. Temperature Distribution within a Cold Cap during Nuclear Waste Vitrification.

    Science.gov (United States)

    Dixon, Derek R; Schweiger, Michael J; Riley, Brian J; Pokorny, Richard; Hrma, Pavel

    2015-07-21

    The kinetics of the feed-to-glass conversion affects the waste vitrification rate in an electric glass melter. The primary area of interest in this conversion process is the cold cap, a layer of reacting feed on top of the molten glass. The work presented here provides an experimental determination of the temperature distribution within the cold cap. Because direct measurement of the temperature field within the cold cap is impracticable, an indirect method was developed in which the textural features in a laboratory-made cold cap with a simulated high-level waste feed were mapped as a function of position using optical microscopy, scanning electron microscopy, energy dispersive spectroscopy, and X-ray diffraction. The temperature distribution within the cold cap was established by correlating microstructures of cold-cap regions with heat-treated feed samples of nearly identical structures at known temperatures. This temperature profile was compared with a mathematically simulated profile generated by a cold-cap model that has been developed to assess the rate of glass production in a melter.

  5. Protection of Drosophila chromosome ends through minimal telomere capping.

    Science.gov (United States)

    Dubruille, Raphaëlle; Loppin, Benjamin

    2015-05-15

    In Drosophila, telomere-capping proteins have the remarkable capacity to recognize chromosome ends in a sequence-independent manner. This epigenetic protection is essential to prevent catastrophic ligations of chromosome extremities. Interestingly, capping proteins occupy a large telomere chromatin domain of several kilobases; however, the functional relevance of this to end protection is unknown. Here, we investigate the role of the large capping domain by manipulating HOAP (encoded by caravaggio) capping-protein expression in the male germ cells, where telomere protection can be challenged without compromising viability. We show that the exhaustion of HOAP results in a dramatic reduction of other capping proteins at telomeres, including K81 [encoded by ms(3)K81], which is essential for male fertility. Strikingly however, we demonstrate that, although capping complexes are barely detected in HOAP-depleted male germ cells, telomere protection and male fertility are not dramatically affected. Our study thus demonstrates that efficient protection of Drosophila telomeres can be achieved with surprisingly low amounts of capping complexes. We propose that these complexes prevent fusions by acting at the very extremity of chromosomes, reminiscent of the protection conferred by extremely short telomeric arrays in yeast or mammalian systems. © 2015. Published by The Company of Biologists Ltd.

  6. Vitamin D3 analog maxacalcitol (OCT) induces hCAP-18/LL-37 production in human oral epithelial cells.

    Science.gov (United States)

    Tada, Hiroyuki; Shimizu, Takamitsu; Nagaoka, Isao; Takada, Haruhiko

    2016-01-01

    Maxacalcitol (22-oxacalcitriol: OCT) is a synthetic vitamin D3 analog with a limited calcemic effect. In this study, we investigated whether OCT increases the production of LL-37/CAP-18, a human cathelicidin antimicrobial peptide, in human gingival/oral epithelial cells. A human gingival epithelial cell line (Ca9-22) and human oral epithelial cell lines (HSC-2, HSC-3, and HSC-4) exhibited the enhanced expression of LL-37 mRNA upon stimulation with OCT as well as active metabolites of vitamins D3 and D2. Among the human epithelial cell lines, Ca9-22 exhibited the strongest response to these vitamin D-related compounds. OCT induced the higher production of CAP-18 (ng/mL order) until 6 days time-dependently in Ca9-22 cells in culture. The periodontal pathogen Porphyromonas gingivalis was killed by treatment with the LL-37 peptide. These findings suggest that OCT induces the production of hCAP-18/LL-37 in a manner similar to that induced by the active metabolite of vitamin D3.

  7. Numerical modeling of experimental human fibrous cap delamination.

    Science.gov (United States)

    Leng, Xiaochang; Davis, Lindsey A; Deng, Xiaomin; Sutton, Michael A; Lessner, Susan M

    2016-06-01

    Fibrous cap delamination is a critical process during the rupture of atherosclerotic plaque, which often leads to severe life-threatening clinical consequences such as myocardial infarction or stroke. In this study a finite element modeling and simulation approach is presented that enables the study of fibrous cap delamination experiments for the purpose of understanding the fibrous cap delamination process. A cohesive zone model (CZM) approach is applied to simulate delamination of the fibrous cap from the underlying plaque tissue. A viscoelastic anisotropic (VA) model for the bulk arterial material behavior is extended from existing studies so that the hysteresis phenomenon observed in the fibrous cap delamination experiments can be captured. A finite element model is developed for the fibrous cap delamination experiments, in which arterial layers (including the fibrous cap and the underlying plaque tissue) are represented by solid elements based on the VA model and the fibrous cap-underlying plaque tissue interface is characterized by interfacial CZM elements. In the CZM, the delamination process is governed by an exponential traction-separation law which utilizes critical energy release rates obtained directly from the fibrous cap delamination experiments. A set of VA model parameter values and CZM parameter values is determined based on values suggested in the literature and through matching simulation predictions of the load vs. load-point displacement curve with one set of experimental measurements. Using this set of parameter values, simulation predictions for other sets of experimental measurements are obtained and good agreement between simulation predictions and experimental measurements is observed. Results of this study demonstrate the applicability of the viscoelastic anisotropic model and the CZM approach for the simulation of diseased arterial tissue failure processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. A helicase-independent activity of eIF4A in promoting mRNA recruitment to the human ribosome.

    Science.gov (United States)

    Sokabe, Masaaki; Fraser, Christopher S

    2017-06-13

    In the scanning model of translation initiation, the decoding site and latch of the 40S subunit must open to allow the recruitment and migration of messenger RNA (mRNA); however, the precise molecular details for how initiation factors regulate mRNA accommodation into the decoding site have not yet been elucidated. Eukaryotic initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA entry channel and A-site of the 40S subunit. Previous studies have shown that a reduced affinity of eIF3j for the 43S preinitiation complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j affinity likely reflects a state of full accommodation of mRNA into the decoding site. Here, we have used a fluorescence-based anisotropy assay to quantitatively determine how initiation components coordinate their activities to reduce the affinity of eIF3j during the recruitment of mRNA to the 43S PIC. Unexpectedly, we show that a full reduction in eIF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of eIF4A. This result suggests that in addition to its helicase activity, eIF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during mRNA recruitment. Therefore, our results define eIF4A as a universal initiation factor in cap-dependent translation initiation that functions beyond its role in RNA unwinding.

  9. Biomolecularly capped uniformly sized nanocrystalline materials: glutathione-capped ZnS nanocrystals

    Science.gov (United States)

    Torres-Martínez, Claudia L.; Nguyen, Liem; Kho, Richard; Bae, Weon; Bozhilov, Krassimir; Klimov, Victor; Mehra, Rajesh K.

    1999-09-01

    Micro-organisms such as bacteria and yeasts form CdS to detoxify toxic cadmium ions. Frequently, CdS particles formed in yeasts and bacteria were found to be associated with specific biomolecules. It was later determined that these biomolecules were present at the surface of CdS. This coating caused a restriction in the growth of CdS particles and resulted in the formation of nanometre-sized semiconductors (NCs) that exhibited typical quantum confinement properties. Glutathione and related phytochelatin peptides were shown to be the biomolecules that capped CdS nanocrystallites synthesized by yeasts Candida glabrata and Schizosaccharomyces pombe. Although early studies showed the existence of specific biochemical pathways for the synthesis of biomolecularly capped CdS NCs, these NCs could be formed in vitro under appropriate conditions. We have recently shown that cysteine and cysteine-containing peptides such as glutathione and phytochelatins can be used in vitro to dictate the formation of discrete sizes of CdS and ZnS nanocrystals. We have evolved protocols for the synthesis of ZnS or CdS nanocrystals within a narrow size distribution range. These procedures involve three steps: (1) formation of metallo-complexes of cysteine or cysteine-containing peptides, (2) introduction of stoichiometric amounts of inorganic sulfide into the metallo-complexes to initiate the formation of nanocrystallites and finally (3) size-selective precipitation of NCs with ethanol in the presence of Na+. The resulting NCs were characterized by optical spectroscopy, high-resolution transmission electron microscopy (HRTEM), x-ray diffraction and electron diffraction. HRTEM showed that the diameter of the ZnS-glutathione nanocrystals was 3.45+/-0.5 nm. X-ray diffraction and electron diffraction analyses indicated ZnS-glutathione to be hexagonal. Photocatalytic studies suggest that glutathione-capped ZnS nanocrystals prepared by our procedure are highly efficient in degrading a test model

  10. 5' cap-independent translation of dengue virus genomic RNA

    OpenAIRE

    Pérez Dominguez, Mariela; Facultad de Odontología. Universidad de Carabobo. Valencia, Venezuela. Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Rojas, Roselbis; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Requena, Dayana; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Ferreras, Ana C.; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Triana, Juana L.; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.; Triana-Alonso, Francisco; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Sede Aragua, Maracay, Venezuela.

    2015-01-01

    Objetives. To analyze the involvement of methyl guanosine triphosphate cap (5’cap) and the start site of the genomic RNA ofDengue virus serotype 2 (DENV-2) American genotype in translation, using a cell-free system prepared from human placenta.Materials and methods. The recombinant plasmid pTZ18R-D2 was prepared containing DNA encoding the 5’UTR and thefirst 201 nucleotides of the viral capsid. This plasmid was used to transcribe the corresponding RNA (RNA-D2) without the 5’cap. The RNA-D2 wa...

  11. Antioxidant Properties of Cap and Stipe from Coprinus comatus

    OpenAIRE

    Haijuan Nan; Xiaomin Suo; Fei Lu; Bo Li; Bin Li

    2010-01-01

    Coprinus comatus, also called chicken drumstick mushroom, is currently commercially available in China. Hot water and ethanolic extracts were prepared from cap and stipe of C. comatus fruit bodies and their antioxidant properties were studied. Ethanolic extract from stipe showed high antioxidant activity (80.6%) at 1 mg/mL. Reducing power of hot water extracts from cap was 1.653 at 10 mg/mL. Extracts from cap showed better scavenging ability on DPPH (57.9% at 1 mg/mL) than stipe ones. Ethanol...

  12. Shelf life of pie caps with biodegradable films as spacers

    OpenAIRE

    Daniela Verónica Escobar Gianni; Angelina Sala; Carlos Silvera; Rodrigo Harispe; Rosa Marquez Romero

    2013-01-01

    Commonly pie caps at market use polyethylene films as spacers between them. This paper studies the conventional spacers replacement with edible and biodegradable films made with whey protein isolate (WPI) and potassium sorbate as a preservative. Besides facilitating the separation of pie caps, with this application is intended to increase their shelf life. The films made by the compression molding method were used as spacers in pie caps without preservative in their formula (A) and with prese...

  13. Thin-film structure of semiconducting end-capped oligothiophenes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimoto, N [Graduate School of Engineering, Iwate University, Ueda Morioka 020-8551 (Japan); Li, W Y [JST Innovation Satellite Iwate, Iiokashinden Morioka 020-0852 (Japan); Omote, K [Rigaku Corporation, 3-9-12 Matsubara-cho Akishima, Tokyo 196-8666 (Japan); Ackermann, J [Laboratoire des Materiaux Moleculaires et des Biomateriaux, GCOM2 UMR CNRS 6114 Faculte des Sciences de Luminy, Case 901, F-13288, Marseille cedex 09 (France); Videlot-Ackermann, C [Laboratoire des Materiaux Moleculaires et des Biomateriaux, GCOM2 UMR CNRS 6114 Faculte des Sciences de Luminy, Case 901, F-13288, Marseille cedex 09 (France); Brisset, H [Laboratoire des Materiaux Moleculaires et des Biomateriaux, GCOM2 UMR CNRS 6114 Faculte des Sciences de Luminy, Case 901, F-13288, Marseille cedex 09 (France); Fages, F [Laboratoire des Materiaux Moleculaires et des Biomateriaux, GCOM2 UMR CNRS 6114 Faculte des Sciences de Luminy, Case 901, F-13288, Marseille cedex 09 (France)

    2007-10-15

    Distyryl-oligothiophenes (DS-nT) is one of the promising semiconducting materials that use for organic thin-film transistors (OTFTs). The in-plane structures of vapor deposited ultrathin films of DS-4T, and its derivatives with different end-cap groups, on SiO{sub 2}substrate were characterized by grazing incidence x-ray diffractometry (GIXD). The morphology and film structure change with the nature of end-cap groups. The increase in volume of end-cap group causes the decrease in crystallinity and increase in frequency in nucleation. These characteristics could affect to the transport properties in OTFTs.

  14. Cap-dependent Endonuclease Inhibitor S-033188 for the Treatment of Influenza: Results from a Phase 3, Randomized, Double-Blind, Placebo- and Active-Controlled Study in Otherwise Healthy Adolescents and Adults with Seasonal Influenza

    OpenAIRE

    Portsmouth, Simon; Kawaguchi, Keiko; Arai, Masatsugu; Tsuchiya, Kenji; Uehara, Takeki

    2017-01-01

    Abstract Background Cap-dependent endonuclease (CEN) resides in the PA subunit of influenza virus polymerase and mediates the “cap-snatching” process during viral mRNA biosynthesis. S-033188 is a potent, selective, small molecule inhibitor of CEN. Here we report clinical and virologic outcomes from a global Phase 3 study CAPSTONE-1. Method This was a multicenter, randomized, double-blind, placebo- and active-controlled study. Key eligibility criteria included 12–64 years of age, fever (axilla...

  15. mRNA vaccines - a new era in vaccinology.

    Science.gov (United States)

    Pardi, Norbert; Hogan, Michael J; Porter, Frederick W; Weissman, Drew

    2018-01-12

    mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.

  16. eIF4E Phosphorylation Influences Bdnf mRNA Translation in Mouse Dorsal Root Ganglion Neurons

    Directory of Open Access Journals (Sweden)

    Jamie K. Moy

    2018-02-01

    Full Text Available Plasticity in dorsal root ganglion (DRG neurons that promotes pain requires activity-dependent mRNA translation. Protein synthesis inhibitors block the ability of many pain-promoting molecules to enhance excitability in DRG neurons and attenuate behavioral signs of pain plasticity. In line with this, we have recently shown that phosphorylation of the 5′ cap-binding protein, eIF4E, plays a pivotal role in plasticity of DRG nociceptors in models of hyperalgesic priming. However, mRNA targets of eIF4E phosphorylation have not been elucidated in the DRG. Brain-derived neurotrophic factor (BDNF signaling from nociceptors in the DRG to spinal dorsal horn neurons is an important mediator of hyperalgesic priming. Regulatory mechanisms that promote pain plasticity via controlling BDNF expression that is involved in promoting pain plasticity have not been identified. We show that phosphorylation of eIF4E is paramount for Bdnf mRNA translation in the DRG. Bdnf mRNA translation is reduced in mice lacking eIF4E phosphorylation (eIF4ES209A and pro-nociceptive factors fail to increase BDNF protein levels in the DRGs of these mice despite robust upregulation of Bdnf-201 mRNA levels. Importantly, bypassing the DRG by giving intrathecal injection of BDNF in eIF4ES209A mice creates a strong hyperalgesic priming response that is normally absent or reduced in these mice. We conclude that eIF4E phosphorylation-mediated translational control of BDNF expression is a key mechanism for nociceptor plasticity leading to hyperalgesic priming.

  17. Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells.

    Science.gov (United States)

    Etchison, D; Hansen, J; Ehrenfeld, E; Edery, I; Sonenberg, N; Milburn, S; Hershey, J W

    1984-01-01

    Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex. Images PMID:6088805

  18. 47 CFR 61.41 - Price cap requirements generally.

    Science.gov (United States)

    2010-10-01

    ... acquires, is acquired by, merges with, or otherwise becomes affiliated with a telephone company that is not...), when a telephone company subject to price cap regulation acquires, is acquired by, merges with, or...

  19. The General Design and Technology Innovations of CAP1400

    Directory of Open Access Journals (Sweden)

    Mingguang Zheng

    2016-03-01

    Full Text Available The pressurized water reactor CAP1400 is one of the sixteen National Science and Technology Major Projects. Developed from China's nuclear R&D system and manufacturing capability, as well as AP1000 technology introduction and assimilation, CAP1400 is an advanced large passive nuclear power plant with independent intellectual property rights. By discussing the top design principle, main performance objectives, general parameters, safety design, and important improvements in safety, economy, and other advanced features, this paper reveals the technology innovation and competitiveness of CAP1400 as an internationally promising Gen-III PWR model. Moreover, the R&D of CAP1400 has greatly promoted China's domestic nuclear power industry from the Gen-II to the Gen-III level.

  20. Philosophical enquiry as a pedagogical tool to implement the CAPS ...

    African Journals Online (AJOL)

    Windows User

    2016-02-22

    ) if the principles that underlie the curriculum are to ... Keywords: CAPS curriculum; citizenship education; cognitive education; community of enquiry pedagogy; critical thinking ..... I think the one thing that really breaks the ice is.

  1. Aspiration of a perforated pen cap: complete tracheal obstruction ...

    African Journals Online (AJOL)

    org. Received 19 September 2011 accepted 23 November 2011. Introduction ... Inflation of the distal balloon allowed controlled extraction of the cap past the vocal cords (Figs 1 and 2). The patient received two postoperative doses of intra-.

  2. ATLAS - End-Cap calorimeter lowered in to the cavern

    CERN Multimedia

    2006-01-01

    The End-cap calorimeter was lowered into the ATLAS cavern at POINT1. This calorimeter will measure the energy of particles close to the beam axis when protons collide. Cooling is important for maximum detector efficiency.

  3. C-CAP Land Cover, Kauai, Hawaii 2005

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set consists of land derived from high resolution imagery and was analyzed according to the Coastal Change Analysis Program (C-CAP) protocol to determine...

  4. Analyses of Current And Wave Forces on Velocity Caps

    DEFF Research Database (Denmark)

    Christensen, Erik Damgaard; Buhrkall, Jeppe; Eskesen, Mark C. D.

    2015-01-01

    leads the water into another pipe or tunnel system. A pressure gradient generated by the water level difference between the sea and basin drives the flow through the tunnel system. The tunnel system is often in the order of a couple kilometers long. Based on CFD analyses (computational fluid dynamics......Velocity caps are often used in connection with for instance offshore intake sea water for the use of for cooling water for power plants or as a source for desalinization plants. The intakes can also be used for river intakes. The velocity cap is placed on top of a vertical pipe. The vertical pipe......) this paper investigates the current and wave forces on the velocity cap and the vertical cylinder. The Morison’s force model was used in the analyses of the extracted force time series in from the CFD model. Further the distribution of the inlet velocities around the velocity cap was also analyzed in detail...

  5. Wind blade spar cap and method of making

    Science.gov (United States)

    Mohamed, Mansour H [Raleigh, NC

    2008-05-27

    A wind blade spar cap for strengthening a wind blade including an integral, unitary three-dimensional woven material having a first end and a second end, corresponding to a root end of the blade and a tip end of the blade, wherein the material tapers in width from the first to the second end while maintaining a constant thickness and decreasing weight therebetween, the cap being capable of being affixed to the blade for providing increased strength with controlled variation in weight from the root end to the tip end based upon the tapered width of the material thereof. The present inventions also include the method of making the wind blade spar cap and a wind blade including the wind blade spar cap.

  6. C-CAP Land Cover, Big Island, Hawaii

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set consists of land derived from high resolution imagery and was analyzed according to the Coastal Change Analysis Program (C-CAP) protocol to determine...

  7. Download - RGP caps | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...About This Database Database Description Download License Update History of This Database Site Policy | Contact Us Download - RGP caps | LSDB Archive ...

  8. Interleukine-1-remming in cryopyrinegeassocieerd periodiek syndroom (CAPS) en schnitzlersyndroom

    NARCIS (Netherlands)

    Koning, H.D. de; Schalkwijk, J.; Meer, J.W. van der; Simon, A.

    2012-01-01

    Cryopyrin-associated periodic syndrome (CAPS) is a hereditary autoinflammatory disorder. Patients suffer from chronic systemic inflammation involving the skin (urticaria), joints arthritis) and in some cases also peritoneum (peritonitis) and meninges (meningitis). Recently, a causative mutation was

  9. Dynamic passive pressure on abutments and pile caps.

    Science.gov (United States)

    2010-08-01

    This study investigated the lateral load response of a full-scale pile cap with nine different backfill conditions, more specifically being: 1) no backfill present (baseline response), 2) densely compacted clean sand, 3) loosely compacted clean sand,...

  10. Unintended consequences of caps on Medicare drug benefits.

    Science.gov (United States)

    Hsu, John; Price, Mary; Huang, Jie; Brand, Richard; Fung, Vicki; Hui, Rita; Fireman, Bruce; Newhouse, Joseph P; Selby, Joseph V

    2006-06-01

    Little information exists about the consequences of limits on prescription-drug benefits for Medicare beneficiaries. We compared the clinical and economic outcomes in 2003 among 157,275 Medicare+Choice beneficiaries whose annual drug benefits were capped at 1,000 dollars and 41,904 beneficiaries whose drug benefits were unlimited because of employer supplements. After adjusting for individual characteristics, we found that subjects whose benefits were capped had pharmacy costs for drugs applicable to the cap that were lower by 31 percent than subjects whose benefits were not capped (95 percent confidence interval, 29 to 33 percent) but had total medical costs that were only 1 percent lower (95 percent confidence interval, -4 to 6 percent). Subjects whose benefits were capped had higher relative rates of visits to the emergency department (relative rate, 1.09 [95 percent confidence interval, 1.04 to 1.14]), nonelective hospitalizations (relative rate, 1.13 [1.05 to 1.21]), and death (relative rate, 1.22 [1.07 to 1.38]; difference, 0.68 per 100 person-years [0.30 to 1.07]). Among subjects who used drugs for hypertension, hyperlipidemia, or diabetes in 2002, those whose benefits were capped were more likely to be nonadherent to long-term drug therapy in 2003; the respective odds ratios were 1.30 (95 percent confidence interval, 1.23 to 1.38), 1.27 (1.19 to 1.34), and 1.33 (1.18 to 1.48) for subjects using drugs for hypertension, hyperlipidemia, and diabetes. In each subgroup, the physiological outcomes were worse for subjects whose drug benefits were capped than for those whose benefits were not capped; the odds ratios were 1.05 (95 percent confidence interval, 1.00 to 1.09), 1.13 (1.03 to 1.25), and 1.23 (1.03 to 1.46), respectively, for subjects with a systolic blood pressure of 140 mm Hg or more, a serum low-density-lipoprotein cholesterol level of 130 mg per deciliter or more, and a glycated hemoglobin level of 8 percent or more. A cap on drug benefits was

  11. Density functional study of condensation in capped capillaries.

    Science.gov (United States)

    Yatsyshin, P; Savva, N; Kalliadasis, S

    2015-07-15

    We study liquid adsorption in narrow rectangular capped capillaries formed by capping two parallel planar walls (a slit pore) with a third wall orthogonal to the two planar walls. The most important transition in confined fluids is arguably condensation, where the pore becomes filled with the liquid phase which is metastable in the bulk. Depending on the temperature T, the condensation in capped capillaries can be first-order (at T≤Tcw) or continuous (at T>Tcw), where Tcw is the capillary wetting temperature. At T>Tcw, the capping wall can adsorb mesoscopic amounts of metastable under-condensed liquid. The onset of condensation is then manifested by the continuous unbinding of the interface between the liquid adsorbed on the capping wall and the gas filling the rest of the capillary volume. In wide capped capillaries there may be a remnant of wedge filling transition, which is manifested by the adsorption of liquid drops in the corners. Our classical statistical mechanical treatment predicts a possibility of three-phase coexistence between gas, corner drops and liquid slabs adsorbed on the capping wall. In sufficiently wide capillaries we find that thick prewetting films of finite length may be nucleated at the capping wall below the boundary of the prewetting transition. Prewetting then proceeds in a continuous manner manifested by the unbinding interface between the thick and thin films adsorbed on the side walls. Our analysis is based on a detailed numerical investigation of the density functional theory for the fluid equilibria for a number of illustrative case studies.

  12. The General Design and Technology Innovations of CAP1400

    OpenAIRE

    Zheng, Mingguang; Yan, Jinquan; Jun, Shentu; Tian, Lin; Wang, Xujia; Qiu, Zhongming

    2016-01-01

    The pressurized water reactor CAP1400 is one of the sixteen National Science and Technology Major Projects. Developed from China's nuclear R&D system and manufacturing capability, as well as AP1000 technology introduction and assimilation, CAP1400 is an advanced large passive nuclear power plant with independent intellectual property rights. By discussing the top design principle, main performance objectives, general parameters, safety design, and important improvements in safety, economy, an...

  13. Drying Characteristics Of Cap And Stem Of Mushroom | Addo ...

    African Journals Online (AJOL)

    Thin-layer drying of cap and stem of mushroom was studied at temperatures of 40, 50 and 60°C. Drying took place in the falling rate period, and the drying behaviour was adequately described by the Page's equation. The activation energy values of cap and stem were determined to be 26.96 and 26.85 kJ/mol, respectively.

  14. Identification of an RNase J ortholog in Sulfolobus solfataricus: implications for 5'-to-3' directional decay and 5'-end protection of mRNA in Crenarchaeota.

    Science.gov (United States)

    Hasenöhrl, David; Konrat, Robert; Bläsi, Udo

    2011-01-01

    In both Bacteria and Eukaryotes, degradation is known to start at the 5' and at the 3' extremities of mRNAs. Until the recent discovery of 5'-to-3' exoribonucleases in hyperthermophilic Euryarchaeota, the exosome was assumed to be the key enzyme in mRNA degradation in Archaea. By means of zymogram assays and bioinformatics, we have identified a 5'-to-3' exoribonuclease activity in the crenarchaeum Sulfolobus solfataricus (Sso), which is affected by the phosphorylation state of the 5'-end of the mRNA. The protein comprises typical signature motifs of the β-CASP family of metallo-β-lactamases and was termed Sso-RNAse J. Thus, our study provides the first evidence for a 5'-to-3' directional mRNA decay pathway in the crenarchaeal clade of Archaea. In Bacteria the 5'-end of mRNAs is often protected by a tri-phosphorylated 5'-terminus and/or by stem-loop structures, while in Eukaryotes the cap-binding complex is responsible for this task. Here, we show that binding of translation initiation factor a/eIF2(γ) to the 5'-end of mRNA counteracts the 5'-to-3' exoribonucleolytic activity of Sso-RNase J in vitro. Hence, 5'-to-3' directional decay and 5'-end protection appear to be conserved features of mRNA turnover in all kingdoms of life.

  15. A decision tool for selecting trench cap designs

    Energy Technology Data Exchange (ETDEWEB)

    Paige, G.B.; Stone, J.J.; Lane, L.J. [USDA-ARS, Tucson, AZ (United States)] [and others

    1995-12-31

    A computer based prototype decision support system (PDSS) is being developed to assist the risk manager in selecting an appropriate trench cap design for waste disposal sites. The selection of the {open_quote}best{close_quote} design among feasible alternatives requires consideration of multiple and often conflicting objectives. The methodology used in the selection process consists of: selecting and parameterizing decision variables using data, simulation models, or expert opinion; selecting feasible trench cap design alternatives; ordering the decision variables and ranking the design alternatives. The decision model is based on multi-objective decision theory and uses a unique approach to order the decision variables and rank the design alternatives. Trench cap designs are evaluated based on federal regulations, hydrologic performance, cover stability and cost. Four trench cap designs, which were monitored for a four year period at Hill Air Force Base in Utah, are used to demonstrate the application of the PDSS and evaluate the results of the decision model. The results of the PDSS, using both data and simulations, illustrate the relative advantages of each of the cap designs and which cap is the {open_quotes}best{close_quotes} alternative for a given set of criteria and a particular importance order of those decision criteria.

  16. CAPS--pathogenesis, presentation and treatment of an autoinflammatory disease.

    Science.gov (United States)

    Kuemmerle-Deschner, Jasmin B

    2015-07-01

    The cryopyrin-associated periodic syndrome (CAPS) is a severity spectrum of rare diseases. CAPS comprises the three conditions previously described as familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disorder (NOMID), also known as chronic infantile neurologic, cutaneous, and articular (CINCA) syndrome. The clinical phenotype of CAPS is characterized by systemic inflammation. General symptoms are fatigue and fever. Local manifestations affect multiple tissues such as skin, joints, muscles, eyes, and the central nervous system. Distinct clinical features are characteristic for each subphenotype. In FCAS, these are cold-induced urticaria and fever, in MWS systemic amyloidosis and hearing loss and in NOMID/CINCA central nervous system inflammation and bone deformities. CAPS is caused by single heterozygous germline or somatic gain of function mutations in the NLRP3 gene encoding the protein cryopyrin. Cryopyrin nucleates an NLRP3 inflammasome, which regulates the activation and cleavage of caspase-1 that cleaves the pro-inflammatory cytokines, IL-1β and IL-18. IL-1β plays the key role in the induction of inflammation in CAPS. This has been confirmed by the application of IL-1 blocking agents, which lead not only to a rapid and sustained reversal of daily symptoms but also to some extent of long-term disease sequelae. To prevent CAPS-induced organ damage, early diagnosis and swift initiation of effective treatment are mandatory.

  17. Integrin beta 4 MRNA expression levels in bronchial asthma patients ...

    African Journals Online (AJOL)

    Serum total IgE was measured by ELISA and mRNA expression of ITGβ4 was assessed by reverse transcriptase PCR (RT-PCR) using real time PCR.. ITGβ4 mRNA expression was significantly down regulated with increased serum total IgE in patients with asthma compared to controls. Moreover, ITGβ4 expression was ...

  18. Functional Integration of mRNA Translational Control Programs

    Directory of Open Access Journals (Sweden)

    Melanie C. MacNicol

    2015-07-01

    Full Text Available Regulated mRNA translation plays a key role in control of cell cycle progression in a variety of physiological and pathological processes, including in the self-renewal and survival of stem cells and cancer stem cells. While targeting mRNA translation presents an attractive strategy for control of aberrant cell cycle progression, mRNA translation is an underdeveloped therapeutic target. Regulated mRNAs are typically controlled through interaction with multiple RNA binding proteins (RBPs but the mechanisms by which the functions of distinct RBPs bound to a common target mRNA are coordinated are poorly understood. The challenge now is to gain insight into these mechanisms of coordination and to identify the molecular mediators that integrate multiple, often conflicting, inputs. A first step includes the identification of altered mRNA ribonucleoprotein complex components that assemble on mRNAs bound by multiple, distinct RBPs compared to those recruited by individual RBPs. This review builds upon our knowledge of combinatorial control of mRNA translation during the maturation of oocytes from Xenopus laevis, to address molecular strategies that may mediate RBP diplomacy and conflict resolution for coordinated control of mRNA translational output. Continued study of regulated ribonucleoprotein complex dynamics promises valuable new insights into mRNA translational control and may suggest novel therapeutic strategies for the treatment of disease.

  19. Phytochrome B mRNA expression enhances biomass yield and ...

    African Journals Online (AJOL)

    The present study shows successful transformation and mRNA expression in Phytochrome B transformed CIM 482 cotton plants. Transgenic cotton plants expressing Phytochrome B mRNA have showed more than two times increase in relative leaf growth rate (RLGR) and photosynthetic rate, more than one time increase in ...

  20. mRNA pseudoknot structures can act as ribosomal roadblocks

    DEFF Research Database (Denmark)

    Hansen, Jesper Tholstrup; Oddershede, Lene Broeng; Sørensen, Michael Askvad

    2012-01-01

    Several viruses utilize programmed ribosomal frameshifting mediated by mRNA pseudoknots in combination with a slippery sequence to produce a well defined stochiometric ratio of the upstream encoded to the downstream-encoded protein. A correlation between the mechanical strength of mRNA pseudoknots...

  1. High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2'O positions.

    Science.gov (United States)

    Peyrane, F; Selisko, B; Decroly, E; Vasseur, J J; Benarroch, D; Canard, B; Alvarez, K

    2007-01-01

    Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.

  2. Epithelial Sodium Channel-Mediated Sodium Transport Is Not Dependent on the Membrane-Bound Serine Protease CAP2/Tmprss4.

    Directory of Open Access Journals (Sweden)

    Anna Keppner

    Full Text Available The membrane-bound serine protease CAP2/Tmprss4 has been previously identified in vitro as a positive regulator of the epithelial sodium channel (ENaC. To study its in vivo implication in ENaC-mediated sodium absorption, we generated a knockout mouse model for CAP2/Tmprss4. Mice deficient in CAP2/Tmprss4 were viable, fertile, and did not show any obvious histological abnormalities. Unexpectedly, when challenged with sodium-deficient diet, these mice did not develop any impairment in renal sodium handling as evidenced by normal plasma and urinary sodium and potassium electrolytes, as well as normal aldosterone levels. Despite minor alterations in ENaC mRNA expression, we found no evidence for altered proteolytic cleavage of ENaC subunits. In consequence, ENaC activity, as monitored by the amiloride-sensitive rectal potential difference (ΔPD, was not altered even under dietary sodium restriction. In summary, ENaC-mediated sodium balance is not affected by lack of CAP2/Tmprss4 expression and thus, does not seem to directly control ENaC expression and activity in vivo.

  3. 22 CFR 121.11 - Military demolition blocks and blasting caps.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Military demolition blocks and blasting caps... blasting caps. Military demolition blocks and blasting caps referred to in Category IV(a) do not include the following articles: (a) Electric squibs. (b) No. 6 and No. 8 blasting caps, including electric...

  4. An add-on cap for ATR-IR spectroscopy studies

    DEFF Research Database (Denmark)

    2014-01-01

    The invention relates to a cap (300B) for an attenuated total reflectance infrared (ATR-IR) spectrometer, the ATR-IR spectrometer comprising an ATR-IR plate (200). The cap (300B) comprises an ATR- IR plate facing cap surface. When the ATR-IR plate facing cap surface is placed on the sample surface...

  5. Role of mRNA Methylation in Prostate Cancer

    Science.gov (United States)

    2015-02-01

    regulation of mRNA. Recent technological advances have made it possible to detect mRNA methylation . The m6A was found near regulatory regions and...TERMS mRNA methylation , FTO, MeRIP-seq, RNA-seq, m6A 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...10 9. Appendices……………………………………………………………10 1. INTRODUCTION: Methylation at the N6 position of adenosine ( m6A ) is a post-transcriptional modification of

  6. Parametric study of propeller boss cap fins for container ships

    Directory of Open Access Journals (Sweden)

    Sang-Seop Lim

    2014-06-01

    Full Text Available The global price of oil, which is both finite and limited in quantity, has been rising steadily because of the increasing requirements for energy in both developing and developed countries. Furthermore, regulations have been strengthened across all industries to address global warming. Many studies of hull resistance, propulsion and operation of ships have been performed to reduce fuel consumption and emissions. This study examined the design parameters of the propeller boss cap fin (PBCF and hub cap for 6,000TEU container ships to improve the propulsion efficiency. The design parameters of PBCF have been selected based on the geometrical shape. Computational fluid dynamics (CFD analysis with a propeller open water (POW test was performed to check the validity of CFD analysis. The design of experiment (DOE case was selected as a full factorial design, and the experiment was analyzed by POW and CFD analysis. Analysis of variance (ANOVA was performed to determine the correlation among design parameters. Four design alternatives of PBCF were selected from the DOE. The shape of a propeller hub cap was selected as a divergent shape, and the divergent angle was determined by the DOE. Four design alternatives of PBCF were attached to the divergent hub cap, and the POW was estimated by CFD. As a result, the divergent hub cap with PBCF has a negative effect on the POW, which is induced by an increase in torque coefficient. A POW test and cavitation test were performed with a divergent hub cap with PBCF to verify the CFD result. The POW test result showed that the open water efficiency was increased approximately 2% with a divergent hub cap compared to a normal cap. The POW test result was similar to the CFD result, and the divergent hub cap with the PBCF models showed lower open water efficiency. This was attributed to an increase in the torque coefficient just like the CFD results. A cavitation test was performed using the 2 models selected. The test

  7. CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis.

    Science.gov (United States)

    Miyake, Kotaro; Ohta, Toshio; Nakayama, Hisako; Doe, Nobutaka; Terao, Yuri; Oiki, Eiji; Nagatomo, Izumi; Yamashita, Yui; Abe, Takaya; Nishikura, Kazuko; Kumanogoh, Atsushi; Hashimoto, Kouichi; Kawahara, Yukio

    2016-11-15

    Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  8. Assessing the value of price caps and floors

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2009-07-01

    This publication assesses the long-term economic and climatic effects of introducing price caps and price floors in hypothetical global climate change mitigation policy. Based on emission trends, abatement costs and equilibrium climate sensitivity from IPCC and IEA reports, this quantitative analysis confirms that price caps could significantly reduce economic uncertainty. This uncertainty stems primarily from unpredictable economic growth and energy prices, and ultimately unabated emission trends. In addition, the development of abatement technologies is uncertain. Furthermore,this analysis shows that rigid targets may entail greater economic risks with little or no comparative advantage for the climate. More ambitious emission objectives, combined with price caps and price floors, could still entail significantly lower expected costs while driving similar, or even slightly better, climatic outcomes in probabilistic terms.

  9. Caps Seal Boltholes On Vacuum-System Flanges

    Science.gov (United States)

    Roman, Robert F.

    1993-01-01

    Sealing caps devised for boltholes on vacuum-system flanges. Used in place of leak-prone gaskets, and provide solid metal-to-metal interfaces. Each sealing cap contains square-cut circular groove in which O-ring placed. Mounted on studs protruding into access ports, providing positive seal around each bolthole. Each cap mates directly with surface of flange, in solid metal-to-metal fit, with O-ring completely captured in groove. Assembly immune to misalignment, leakage caused by vibration, and creeping distortion caused by weight of port. O-ring material chosen for resistance to high temperature; with appropriate choice of material, temperature raised to as much as 315 degrees C.

  10. Buy badges and help finance Cap Loisirs activities!

    CERN Multimedia

    2008-01-01

    Please give a warm welcome to Cap Loisirs’ volunteers who will be manning a stand at the main entrance on Friday 8 February! In honour of St. Valentine’s Day, they will be selling a selection of 5 badges designed by artist Anna Sommer, depicting declarations of love by our friends from the animal kingdom. Price : Frs. 3.- per badge The aim of Fondation Cap Loisirs is to provide leisure activities for mentally handicapped children, adolescents and adults. The Foundation offers a wide range of activities which allow the mentally handicapped to enjoy quality leisure in areas as varied as culture, sport, tourism, travel and artistic expression. Fondation Cap Loisirs Rue de Monthoux 66 – 1201 Geneva – Tel: 022 731 86 00 – CCP Genève 12-5587-5 – caploisirs@caploisirs.ch – http://www.Caploisirs.ch

  11. Validation of CAP with SETH-PANDA Test-9

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hoon; Ha, Sang Jun; Jun, Hwang Yong [Korea Hydro and Nuclear Power Co., Daejeon (Korea, Republic of); Bae, Sung Won [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2012-05-15

    As part of the project of the development of domestic design codes, the CAP(Containment Analysis Package) code has been under development and an evaluation version was released for verification and validation at the end of 2011. To validate the gas mixing and stratification simulation ability of the CAP code, the SETH-PANDA test 9 was chosen. The results of test 9 show the 3-dimensional gas distribution in the test facility and were used to verify the simulation capabilities of the CFD codes. Though the CAP code doesn't have a 3-D calculation module yet, gas mixing simulation has been attempted by dividing into many lumped parameter volumes

  12. NDE Process Development Specification for SRB Composite Nose Cap

    Science.gov (United States)

    Suits, M.

    1999-01-01

    The Shuttle Upgrade program is a continuing improvement process to enable the Space Shuttle to be an effective space transportation vehicle for the next few decades. The Solid Rocket Booster (SRB), as a component of that system, is currently undergoing such an improvement. Advanced materials, such as composites, have given us a chance to improve performance and to reduce weight. The SRB Composite Nose Cap (CNC) program aims to replace the current aluminum nose cap, which is coated with a Thermal Protection System and poses a possible debris hazard, with a lighter, stronger, CNC. For the next 2 years, this program will evaluate the design, material selection, properties, and verification of the CNC. This particular process specification cites the methods and techniques for verifying the integrity of such a nose cap with nondestructive evaluation.

  13. Surface Plasmon Coupling and Control Using Spherical Cap Structures

    Energy Technology Data Exchange (ETDEWEB)

    Gong, Yu; Joly, Alan G.; Zhang, Xin; El-Khoury, Patrick Z.; Hess, Wayne P.

    2017-06-05

    Propagating surface plasmons (PSPs) launched from a protruded silver spherical cap structure are investigated using photoemission electron microscopy (PEEM) and finite difference time domain (FDTD) calculations. Our combined experimental and theoretical findings reveal that PSP coupling efficiency is comparable to conventional etched-in plasmonic coupling structures. Additionally, plasmon propagation direction can be varied by a linear rotation of the driving laser polarization. A simple geometric model is proposed in which the plasmon direction selectivity is proportional to the projection of the linear laser polarization on the surface normal. An application for the spherical cap coupler as a gate device is proposed. Overall, our results indicate that protruded cap structures hold great promise as elements in emerging surface plasmon applications.

  14. Economic evaluation of closure cap barrier materials study

    Energy Technology Data Exchange (ETDEWEB)

    Serrato, M.G.; Bhutani, J.S.; Mead, S.M.

    1993-09-01

    Volume II of the Economic Evaluation of the Closure Cap Barrier Materials, Revision I contains detailed cost estimates for closure cap barrier materials. The cost estimates incorporate the life cycle costs for a generic hazardous waste seepage basin closure cap under the RCRA Post Closure Period of thirty years. The economic evaluation assessed six barrier material categories. Each of these categories consists of several composite cover system configurations, which were used to develop individual cost estimates. The information contained in this report is not intended to be used as a cost estimating manual. This information provides the decision makers with the ability to screen barrier materials, cover system configurations, and identify cost-effective materials for further consideration.

  15. CAPS and Munc13: CATCHRs that SNARE vesicles

    Directory of Open Access Journals (Sweden)

    Declan J James

    2013-12-01

    Full Text Available Abstract. CAPS (Calcium-dependent Activator Protein for Secretion, aka CADPS and Munc13 (Mammalian Unc-13 proteins function to prime vesicles for Ca2+-triggered exocytosis in neurons and neuroendocrine cells. CAPS and Munc13 proteins contain conserved C-terminal domains that promote the assembly of SNARE complexes for vesicle priming. Similarities of the C-terminal domains of CAPS/Munc13 proteins with CATCHR (Complex Associated with Tethering Containing Helical Rods domains in multi-subunit tethering complexes have been reported. Multi-subunit tethering complexes coordinate multiple interactions for SNARE complex assembly at constitutive membrane fusion steps. We review aspects of these diverse tethering and priming factors to identify common operating principles.

  16. Mutual relationships between transcription and pre-mRNA processing in the synthesis of mRNA.

    Science.gov (United States)

    Lenasi, Tina; Barboric, Matjaz

    2013-01-01

    The generation of messenger RNA (mRNA) in eukaryotes is achieved by transcription from the DNA template and pre-mRNA processing reactions of capping, splicing, and polyadenylation. Although RNA polymerase II (RNAPII) catalyzes the synthesis of pre-mRNA, it also serves as a principal coordinator of the processing reactions in the course of transcription. In this review, we focus on the interplay between transcription and cotranscriptional pre-mRNA maturation events, mediated by the recruitment of RNA processing factors to differentially phosphorylated C-terminal domain of Rbp1, the largest subunit of RNAPII. Furthermore, we highlight the bidirectional nature of the interplay by discussing the impact of RNAPII kinetics on pre-mRNA processing as well as how the processing events reach back to different phases of gene transcription. Copyright © 2012 John Wiley & Sons, Ltd.

  17. Empiric penicillin monotherapy of CAP is not associated with increased mortality; experiences from the retrospective CAP-North cohort

    DEFF Research Database (Denmark)

    Baunbæk-Knudsen, Getrud; Vestergaard Jensen, Andreas; Andersen, Stine

    2016-01-01

    Background: Community-acquired pneumonia (CAP) is a severe infection, with high morbidity and mortality. The antibiotic strategies for CAP differ across Europe. Objective: To assess the usage of Penicillin monotherapy in a real-life cohort and to evaluate predictors of treatment duration and the ......Background: Community-acquired pneumonia (CAP) is a severe infection, with high morbidity and mortality. The antibiotic strategies for CAP differ across Europe. Objective: To assess the usage of Penicillin monotherapy in a real-life cohort and to evaluate predictors of treatment duration......, and evaluated predictors of treatment duration by linear regression. Mortality of patients receiving empiric penicillin-G/V was compared to others by logistic regression analysis. The CAPNETZ database technology was used for data-capture. Results: We included 1320 patients. The incidence of hospitalized CAP...... was 3.1 per 1000 inhabitants. The median age was 71 years (IQR; 58.81). In-hospital mortality was 8%. Patients treated with penicillin-G/V as empiric monotherapy (45%) did not have a higher mortality than those treated with broader spectrum antibiotics (OR 1.30, CI 95% 0.84-2-02). The median duration...

  18. CapsID: a web-based tool for developing parsimonious sets of CAPS molecular markers for genotyping

    Directory of Open Access Journals (Sweden)

    Provart Nicholas J

    2006-05-01

    Full Text Available Abstract Background Genotyping may be carried out by a number of different methods including direct sequencing and polymorphism analysis. For a number of reasons, PCR-based polymorphism analysis may be desirable, owing to the fact that only small amounts of genetic material are required, and that the costs are low. One popular and cheap method for detecting polymorphisms is by using cleaved amplified polymorphic sequence, or CAPS, molecular markers. These are also known as PCR-RFLP markers. Results We have developed a program, called CapsID, that identifies snip-SNPs (single nucleotide polymorphisms that alter restriction endonuclease cut sites within a set or sets of reference sequences, designs PCR primers around these, and then suggests the most parsimonious combination of markers for genotyping any individual who is not a member of the reference set. The output page includes biologist-friendly features, such as images of virtual gels to assist in genotyping efforts. CapsID is freely available at http://bbc.botany.utoronto.ca/capsid. Conclusion CapsID is a tool that can rapidly provide minimal sets of CAPS markers for molecular identification purposes for any biologist working in genetics, community genetics, plant and animal breeding, forensics and other fields.

  19. Analyses of Current And Wave Forces on Velocity Caps

    OpenAIRE

    Christensen, Erik Damgaard; Buhrkall, Jeppe; Eskesen, Mark C. D.; Jensen, Bjarne

    2015-01-01

    Velocity caps are often used in connection with for instance offshore intake sea water for the use of for cooling water for power plants or as a source for desalinization plants. The intakes can also be used for river intakes. The velocity cap is placed on top of a vertical pipe. The vertical pipe leads the water into another pipe or tunnel system. A pressure gradient generated by the water level difference between the sea and basin drives the flow through the tunnel system. The tunnel system...

  20. The Drosophila HOAP protein is required for telomere capping.

    Science.gov (United States)

    Cenci, Giovanni; Siriaco, Giorgia; Raffa, Grazia D; Kellum, Rebecca; Gatti, Maurizio

    2003-01-01

    HOAP (HP1/ORC-associated protein) has recently been isolated from Drosophila melanogaster embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). Here, we show that caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. Our analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2.

  1. P3 cap modified Phe*-Ala series BACE inhibitors.

    Science.gov (United States)

    Chen, Shu-Hui; Lamar, Jason; Guo, Deqi; Kohn, Todd; Yang, Hsiu-Chiung; McGee, James; Timm, David; Erickson, Jon; Yip, Yvonne; May, Patrick; McCarthy, James

    2004-01-05

    With the aim of reducing molecular weight and adjusting log D value of BACE inhibitors to more favorable range for BBB penetration and better bioavailability, we synthesized and evaluated several series of P3 cap modified BACE inhibitors obtained via replacement of the P3NHBoc moiety as seen in 3 with other polar functional groups such as amino, hydroxyl and fluorine. Several promising inhibitors emerging from this P3 cap SAR study (e.g., 15 and 19) demonstrated good enzyme inhibitory potencies (BACE-1 IC(50) <50 nM) and whole cell activities (IC(50) approximately 1 microM).

  2. High-energy accelerators above pulsar polar caps

    Science.gov (United States)

    Xu, R. X.; Qiao, G. J.

    Similar to the terrestrial collision accelerators of electron-antielectron, another kind of accelerator is above a positively or negatively charged pulsar polar cap. In the case of pulsars with magnetic axis parallel (anti-parallel) to rotational axis, relativistic antielectron (electron) with Lorentz factor about 10^6 hit the electrons in the polar caps. These scenarios are investigated both for pulsars being bare strange stars and for pulsars being neutron stars. Such a study may be valuable to differentiate neutron stars and bare strange stars observationally.

  3. Collapse mechanisms and strength prediction of reinforced concrete pile caps

    DEFF Research Database (Denmark)

    Jensen, Uffe G.; Hoang, Linh Cao

    2012-01-01

    This paper describes an upper bound plasticity approach for strength prediction of reinforced concrete pile caps. A number of collapse mechanisms are identified and analysed. The procedure leads to an estimate of the load-carrying capacity and an identification of the critical collapse mechanism....... Calculations have been compared with nearly 200 test results found in the literature. Satisfactory agreement has been found. The analyses are conducted on concentrically loaded caps supported by four piles. The paper briefly outlines how the approach may be extended to more complicated loadings and geometries...

  4. End-capping of amphiphilic nanotubes with phospholipid vesicles: impact of the phospholipid on the cap formation and vesicle loading under osmotic conditions.

    Science.gov (United States)

    Erne, Petra M; Štacko, Peter; van Dijken, Derk Jan; Chen, Jiawen; Stuart, Marc C A; Feringa, Ben L

    2016-09-22

    Soft amphiphilic nanotubes are capped with vesicles comprised of either overall neutral, zwitterionic phospholipids, or those that carry a net charge. The phase transition temperature of the zwitterionic phospholipids plays a crucial role in the phase separation that leads to the end-capped nanotubes. The cationic vesicle caps can be loaded into the nanotubes via osmosis whereas the anionic vesicle caps are stable under hyper-osmotic conditions. Furthermore, no additional salt needs to be added for the cationic vesicle caps to induce the loading of the vesicles into the nanotubes due to the presence of counterions.

  5. Demonstration that a mRNA Binding Protein is Responsible for GADD45 mRNA Destabilization

    National Research Council Canada - National Science Library

    Abcouwer, Steve

    2003-01-01

    ...) Using regions of the GADD45 mRNA 3'-untranslated region (UTR) in KNA gel shift assays, we have observed that glutamine causes distinct changes in RBP activities in cytoplasmic and nuclear protein extracts...

  6. Quantitative effect of suboptimal codon usage on translational efficiency of mRNA encoding HIV-1 gag in intact T cells.

    Directory of Open Access Journals (Sweden)

    Kholiswa C Ngumbela

    Full Text Available BACKGROUND: The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1 are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. METHODOLOGY AND PRINCIPAL FINDINGS: To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold. In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. CONCLUSIONS: We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.

  7. Quantitative effect of suboptimal codon usage on translational efficiency of mRNA encoding HIV-1 gag in intact T cells.

    Science.gov (United States)

    Ngumbela, Kholiswa C; Ryan, Kieran P; Sivamurthy, Rohini; Brockman, Mark A; Gandhi, Rajesh T; Bhardwaj, Nina; Kavanagh, Daniel G

    2008-06-04

    The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per microg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA.

  8. Impact Response Study on Covering Cap of Aircraft Big-Size Integral Fuel Tank

    Science.gov (United States)

    Wang, Fusheng; Jia, Senqing; Wang, Yi; Yue, Zhufeng

    2016-10-01

    In order to assess various design concepts and choose a kind of covering cap design scheme which can meet the requirements of airworthiness standard and ensure the safety of fuel tank. Using finite element software ANSYS/LS- DYNA, the impact process of covering cap of aircraft fuel tank by projectile were simulated, in which dynamical characteristics of simple single covering cap and gland double-layer covering cap impacted by titanium alloy projectile and rubber projectile were studied, as well as factor effects on simple single covering cap and gland double-layer covering cap under impact region, impact angle and impact energy were also studied. Though the comparison of critical damage velocity and element deleted number of the covering caps, it shows that the external covering cap has a good protection effect on internal covering cap. The regions close to boundary are vulnerable to appear impact damage with titanium alloy projectile while the regions close to center is vulnerable to occur damage with rubber projectile. Equivalent strain in covering cap is very little when impact angle is less than 15°. Element deleted number in covering cap reaches the maximum when impact angle is between 60°and 65°by titanium alloy projectile. While the bigger the impact angle and the more serious damage of the covering cap will be when rubber projectile impact composite covering cap. The energy needed for occurring damage on external covering cap and internal covering cap is less than and higher than that when single covering cap occur damage, respectively. The energy needed for complete breakdown of double-layer covering cap is much higher than that of single covering cap.

  9. Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus

    Energy Technology Data Exchange (ETDEWEB)

    Asojo, Oluwatoyin A., E-mail: oasojo@unmc.edu [Pathology and Microbiology Department, 986495 Nebraska Medical Center, Omaha, NE 68198-6495 (United States)

    2011-05-01

    The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite N. americanus refined to a resolution limit of 2.2 Å is presented. Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

  10. Integrated Modeling and Decision-Support System for Water Management in the Puget Sound Basin: Snow Caps to White Caps

    Energy Technology Data Exchange (ETDEWEB)

    Copping, Andrea E. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Yang, Zhaoqing [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Voisin, Nathalie [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Richey, Jeff [Univ. of Washington, Seattle, WA (United States); Wang, Taiping [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Taira, Randal Y. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Constans, Michael [Univ. of Washington, Seattle, WA (United States); Wigmosta, Mark S. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Van Cleve, Frances B. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Tesfa, Teklu K. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2013-12-01

    Final Report for the EPA-sponsored project Snow Caps to White Caps that provides data products and insight for water resource managers to support their predictions and management actions to address future changes in water resources (fresh and marine) in the Puget Sound basin. This report details the efforts of a team of scientists and engineers from Pacific Northwest National Laboratory (PNNL) and the University of Washington (UW) to examine the movement of water in the Snohomish Basin, within the watershed and the estuary, under present and future conditions, using a set of linked numerical models.

  11. The influence of connecting pile cap-column in the mechanisms of break in the two pile caps

    OpenAIRE

    MESQUITA, A. C.; ROCHA,A. S.; DELALIBERA,R. G.; W. A. DA SILVA

    2016-01-01

    Abstract The paper analyzes the two pile caps with partially embedded socket and subject a center load. Three models were experimentally tested, varying the type of conformation of the column and walls of the socket, with a smooth, the other rough, and a monolithic two pile cap, used for reference. The roughening of the column-socket interface was examined with the aim of verifying the difference of the distribution of compressive and tensile stresses in the strut an tie model used for design...

  12. An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication.

    Science.gov (United States)

    Kamel, Wael; Akusjärvi, Göran

    2017-11-01

    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.

  13. Optical Properties of ZnO Nanoparticles Capped with Polymers

    Directory of Open Access Journals (Sweden)

    Atsushi Noguchi

    2011-06-01

    Full Text Available Optical properties of ZnO nanoparticles capped with polymers were investigated. Polyethylene glycol (PEG and polyvinyl pyrrolidone (PVP were used as capping reagents. ZnO nanoparticles were synthesized by the sol-gel method. Fluorescence and absorption spectra were measured. When we varied the timing of the addition of the polymer to the ZnO nanoparticle solution, the optical properties were drastically changed. When PEG was added to the solution before the synthesis of ZnO nanoparticles, the fluorescence intensity increased. At the same time, the total particle size increased, which indicated that PEG molecules had capped the ZnO nanoparticles. The capping led to surface passivation, which increased fluorescence intensity. However, when PEG was added to the solution after the synthesis of ZnO nanoparticles, the fluorescence and particle size did not change. When PVP was added to the solution before the synthesis of ZnO nanoparticles, aggregation of nanoparticles occurred. When PVP was added to the solution after the synthesis of ZnO nanoparticles, fluorescence and particle size increased. This improvement of optical properties is advantageous to the practical usage of ZnO nanoparticles, such as bioimaging

  14. Risk premiums in cap rates of investment property

    NARCIS (Netherlands)

    Berkhout, T.M.; Wouwe, van M.; Tansens, P.R.

    2005-01-01

    Our research wants to establish links and patterns over time between the constituent components of the cap rates, and between real interest and risk premium in particular. We developed a statistical model to predict and explain the risk premium asked for by investors buying properties throughout

  15. Moving one of the ATLAS end-cap calorimeters

    CERN Multimedia

    Claudia Marcelloni

    2007-01-01

    One of the end-cap calorimeters for the ATLAS experiment is moved using a set of rails. This calorimeter will measure the energy of particles that are produced close to the axis of the beam when two protons collide. It is kept cool inside a cryostat to allow the detector to work at maximum efficiency.

  16. Effect of capping agents on optical and antibacterial properties of ...

    Indian Academy of Sciences (India)

    Administrator

    Abstract. Cadmium selenide quantum dots (CdSe QDs) were synthesized in aqueous phase by the freezing temperature injection technique using different capping agents (viz. thioglycolic acid, 1-thioglycerol,. L-cysteine). Absorption spectra of CdSe QDs exhibited a blue shift as compared to its bulk counterpart, which.

  17. Probability of pion absorption by. cap alpha. -cluster in nuclei

    Energy Technology Data Exchange (ETDEWEB)

    Hachenberg, F. (Heidelberg Univ. (Germany, F.R.). Inst. fuer Theoretische Physik; Max-Planck-Institut fuer Kernphysik, Heidelberg (Germany, F.R.))

    1982-07-08

    A multistep direct reaction model with pickup is extended to describe the formation of tritons and /sup 3/He-particles in nucleon- and pion-induced precompound reactions. In the latter ones, those tritons not accounted for by pickup can be explained as primary ones from absorption by ..cap alpha..-clusters, for which a 10% probability is deduced.

  18. Magnetohydrodynamic instability of polar caps on accreting neutron stars

    Science.gov (United States)

    Litwin, C.; Brown, E. F.; Rosner, R.

    2001-10-01

    We analyze equilibria and stability of the magnetically confined accreted matter in the polar cap on the surface of a neutron star. We find the equilibria by solving (both analytically and numerically) the Grad-Shafranov-like equation for the degenerate plasma in the neutron star ``ocean'' above the neutron star crust. Subsequently we employ the energy principle to analyze the stability of these equilibria to short-wavelength ballooning modes. We find that for strong magnetic fields (> 10^12 G) line-tying to the neutron star crust stabilizes these modes until the overpressure in the polar cap at the top of the neutron star crust exceeds the magnetic pressure by a factor ~ 8(a/h), where a and h are, respectively, the lateral extent of the polar cap and the density scale height. The most unstable modes are localized within a density scale height above the crust. We calculate the amount of mass that can be accumulated at the polar cap before the onset of instability. We also investigate the effect of incomplete degeneracy and nonuniform composition on stability.

  19. Lowering End-cap YE-1 in the CMS cavern

    CERN Document Server

    2008-01-01

    On Tuesday 22 January, the dance of the CMS end-caps came to an end with the lowering of YE-1, the heaviest of them all. After a spectacular lowering operation lsting ten hours, this mammoth component completed the 100-metre descent and was gently placed on the floor of the CMS cavern to the applause of the many onlookers.

  20. External caps: An approach to stress reduction in balloons

    Science.gov (United States)

    Hazlewood, K. H.

    Recent findings of the catastrophic balloon failures investigation in the U.S.A. indicate that very large gross inflations, in balloons using present design philosophy, over-stress currently available materials. External caps are proposed as an economic approach to reducting those stresses to an acceptable level.

  1. DRYING CHARACTERISTICS OF CAP AND STEM OF MUSHROOM

    African Journals Online (AJOL)

    Administrator

    Dehydrated mush- rooms are used as an important ingredient in several food formulations including instant soups, pasta salads, snack seasonings, stuffing, casseroles, and meat ... Cap and stem, of 40 mm length, were dried at air temperatures of 40, .... vegetables and agricultural products (Doymaz,. 2004 for green beans ...

  2. Philosophical enquiry as a pedagogical tool to implement the CAPS ...

    African Journals Online (AJOL)

    ... and constraints on implementation. Discussion focuses on the insights of participants, potential challenges, some limitations of the research and our plans to address them. Keywords: CAPS curriculum; citizenship education; cognitive education; community of enquiry pedagogy; critical thinking; Philosophy for Children; ...

  3. CAPS OpenACC Compilers: Performance and Portability

    CERN Multimedia

    CERN. Geneva

    2013-01-01

    The announcement late 2011 of the new OpenACC directive-based programming standard supported by CAPS, CRAY and PGI compilers has open up the door to more scientific applications that can be ported on many-core systems. Following a porting methodology, this talk will first review the principles of programming with OpenACC and then the advanced features available in the CAPS compilers to further optimize OpenACC applications: library integration, tuning directives with auto-tune mechanisms to build applications adaptive to different GPUs. CAPS compilers use hardware vendors' backends such as NVIDIA CUDA and OpenCL making them the only OpenACC compilers supporting various many-core architectures. About the speaker Stéphane Bihan is co-funder and currently Director of Sales and Marketing at CAPS enterprise. He has held several R&D positions in companies such as ARC international plc in London, Canon Research Center France, ACE compiler experts in Amsterdam and the INRIA r...

  4. CucCAP - Developing genomic resources for the cucurbit community

    Science.gov (United States)

    The U.S. cucurbit community has initiated a USDA-SCRI funded cucurbit genomics project, CucCAP: Leveraging applied genomics to increase disease resistance in cucurbit crops. Our primary objectives are: develop genomic and bioinformatic breeding tool kits for accelerated crop improvement across the...

  5. Working on an LHC dipole end-cap

    CERN Multimedia

    Laurent Guiraud

    1999-01-01

    A metal worker constructs an end-cap for an LHC dipole magnet. These magnets will be used to bend the proton beams around the LHC, which is due to start up in 2008. The handmade prototype seen here will be used to make a mold from which the final set of components will be made for the accelerator.

  6. Magnetic actuation and transition shapes of a bistable spherical cap

    Directory of Open Access Journals (Sweden)

    E.G. Loukaides

    2014-10-01

    Full Text Available Multistable shells have been proposed for a variety of applications; however, their actuation is almost exclusively addressed through embedded piezoelectric patches. Additional actuation techniques are needed for applications requiring high strains or where remote actuation is desirable. Part of the reason for the lack of research in this area is the absence of appropriate models describing the detailed deformation and energetics of such shells. This work presents a bistable spherical cap made of iron carbonyl-infused polydimethylsiloxane. The magnetizable structure can be actuated remotely through permanent magnets while the transition is recorded with a high-speed camera. Moreover, the experiment is reproduced in a finite element (FE dynamic model for comparison with the physical observations. High-speed footage of the physical cap inversion together with the FE modeling gives valuable insight on preferable intermediate geometries. Both methods return similar values for the magnetic field strength required for the snap-through. High-strain multistable spherical cap transformation is demonstrated, based on informed material selection. We discover that non-axisymmetric transition shapes are preferred in intermediate geometries by bistable spherical caps. We develop the methods for design and analysis of such actuators, including the feasibility of remote actuation methods for multistable shells.

  7. Reinforcement of silica aerogels using silane-end-capped polyurethanes.

    Science.gov (United States)

    Duan, Yannan; Jana, Sadhan C; Lama, Bimala; Espe, Matthew P

    2013-05-21

    Proper selection of silane precursors and polymer reinforcements yields more durable and stronger silica aerogels. This paper focuses on the use of silane-end-capped urethane prepolymer and chain-extended polyurethane for reinforcement of silica aerogels. The silane end groups were expected to participate in silica network formation and uniquely determine the amounts of urethanes incorporated into the aerogel network as reinforcement. The aerogels were prepared by one-step sol-gel process from mixed silane precursors tetraethoxysilane, aminopropyltriethoxysilane (APTES), and APTES-end-capped polyurethanes. The morphology and mechanical and surface properties of the resultant aerogels were investigated in addition to elucidation of chemical structures by solid-state (13)C and (29)Si nuclear magnetic resonance. Modification by 10 wt % APTES-end-capped chain-extended polyurethane yielded a 5-fold increase in compressive modulus and 60% increase in density. APTES-end-capped chain-extended polyurethane was found to be more effective in enhancement of mechanical properties and reduction of polarity.

  8. 1 | Page ARBITRATION AND CONCILIATION ACT CAP A18 LAWS ...

    African Journals Online (AJOL)

    Fr. Ikenga

    ARBITRATION AND CONCILIATION ACT CAP A18 LAWS OF THE. FEDERATION OF NIGERIA 2004 -CALL FOR AMENDMENT*. Abstract. The primary sources of the Nigerian Law of Arbitration are the English Common Law, the Nigerian. Customary law and Nigerian statues. The English common law and the doctrines of ...

  9. Traditional M e n Caps o f Kyrgyzstan

    Directory of Open Access Journals (Sweden)

    Serap TANRISEVER

    2015-07-01

    Full Text Available Kyrgyzstan has an extraordinarily rich mosaic of folk culture. There is a great importance of wear clothing, crafts and decorative arts in this mosaic. Traditional caps and clothes of Kyrgyzstan are the best elements that reflect the presence of culture, t raditions, geography inhabited, climatic conditions that they already live in. Kyrgyzstan has been trying to keep alive the traditions, styles of lives by using the required crafts and handmade products although the global fashion, time and technological development. According to research, it has still been seen that the caps and clothes of Kyrgyzstan are being used less in daily life. Caps, Kalpak , Tebetey, Malakay, are some of the best known extant that come to this age related with the traditions of ca p s of Kyrgyzstan’s men . In this research, it has been worked of decorations of Kirghiz men caps , specialties of color and design, techniques of sewing, characteristics of mold and fabrics, Moreover the place and the meaning of the titles has been trying to determine in the cultural life. The scan method has been used by doing regional research.

  10. Antioxidant properties of cap and stipe from Coprinus comatus.

    Science.gov (United States)

    Li, Bo; Lu, Fei; Suo, Xiaomin; Nan, Haijuan; Li, Bin

    2010-03-09

    Coprinus comatus, also called chicken drumstick mushroom, is currently commercially available in China. Hot water and ethanolic extracts were prepared from cap and stipe of C. comatus fruit bodies and their antioxidant properties were studied. Ethanolic extract from stipe showed high antioxidant activity (80.6%) at 1 mg/mL. Reducing power of hot water extracts from cap was 1.653 at 10 mg/mL. Extracts from cap showed better scavenging ability on DPPH (57.9% at 1 mg/mL) than stipe ones. Ethanolic extracts were more effective in scavenging ability on hydroxyl radicals (57.4-61.3% at 5 mg/mL) than hot water extracts. Ethanolic extracts showed moderate scavenging ability on superoxide radicals (46.3-47.0% at 20 mg/mL). Naturally occurring antioxidant components including total phenols (3.60-20.00 mg/g), tocopherols (0.58-11.93 mg/g), flavonoids (0.19-3.52 mg/g) and polysaccharides (58.52-547.86 mg/g) were found in the extracts. Overall, extracts from cap were more effective for the antioxidant properties assayed.

  11. Antioxidant Properties of Cap and Stipe from Coprinus comatus

    Directory of Open Access Journals (Sweden)

    Haijuan Nan

    2010-03-01

    Full Text Available Coprinus comatus, also called chicken drumstick mushroom, is currently commercially available in China. Hot water and ethanolic extracts were prepared from cap and stipe of C. comatus fruit bodies and their antioxidant properties were studied. Ethanolic extract from stipe showed high antioxidant activity (80.6% at 1 mg/mL. Reducing power of hot water extracts from cap was 1.653 at 10 mg/mL. Extracts from cap showed better scavenging ability on DPPH (57.9% at 1 mg/mL than stipe ones. Ethanolic extracts were more effective in scavenging ability on hydroxyl radicals (57.4–61.3% at 5 mg/mL than hot water extracts. Ethanolic extracts showed moderate scavenging ability on superoxide radicals (46.3–47.0% at 20 mg/mL. Naturally occurring antioxidant components including total phenols (3.60–20.00 mg/g, tocopherols (0.58–11.93 mg/g, flavonoids (0.19–3.52 mg/g and polysaccharides (58.52–547.86 mg/g were found in the extracts. Overall, extracts from cap were more effective for the antioxidant properties assayed.

  12. Green-capped Eremomela Eremomela scotops kikuyuensis in Nairobi

    African Journals Online (AJOL)

    The little known Kenya endemic race kikuyuensis of the Green-capped Eremomela was collected by van Someren in November 1916 (believed to have been from the western Nairobi suburbs), but was not described until 1931 (van Someren 1931). Today, it remains extremely localized in the Meru, Embu, Thika, Kiambu ...

  13. Crater Population and Resurfacing of the Martian North Polar Cap

    Science.gov (United States)

    Banks, M. E.; Galla, K. G.; Bryne, S.; Murray, B. C.; McEwen, A. S.; HiRISE Team

    2009-03-01

    Over 100 impact craters have been identified in CTX images of the north polar layered deposits (NPLD) and residual cap (NRC). Using NRC cratering data we will use landscape evolution modeling to investigate the recent mass-balance history of the NRC.

  14. Performance of a pulsed-cap microcolumn for protein extraction

    Directory of Open Access Journals (Sweden)

    Rabelo A.P.B.

    2003-01-01

    Full Text Available This work presents the results obtained using a microcolumn agitated by pulsed caps, using aqueous two-phase systems formed polyethylene glycol and salts (monobasic and dibasic potassium phosphate for protein extraction. Proteins used were extracted cytochrome b5 and the enzyme ascorbic oxidoreductase. It was observed that operation of this equipment was stable and high efficiency values were achieved.

  15. Shelf life of pie caps with biodegradable films as spacers

    Directory of Open Access Journals (Sweden)

    Daniela Verónica Escobar Gianni

    2013-01-01

    Full Text Available Commonly pie caps at market use polyethylene films as spacers between them. This paper studies the conventional spacers replacement with edible and biodegradable films made with whey protein isolate (WPI and potassium sorbate as a preservative. Besides facilitating the separation of pie caps, with this application is intended to increase their shelf life. The films made by the compression molding method were used as spacers in pie caps without preservative in their formula (A and with preservative (B and they were compared with conventional polyethylene spacers (C. During four months, monthly sensory, microbiological and physicochemical (humidity evaluations were done on the pie caps, together with humidity and solubility evaluations of the films. None of the samples showed microbiological or sensory deterioration. The sensory attributes showed no or slight difference in study time. Between samples the differences were minor: the best scores were for sample A in color, sample C in flavor, and samples B and C in texture and overall liking. The edible films have an interesting potential for this application, although studies in disguise the flavor of serum should be done.

  16. Mississippi CaP HBCU Undergraduate Research Training Program

    Science.gov (United States)

    2015-09-01

    TITLE AND SUBTITLE Mississippi CaP HBCU Undergraduate Research Training Program 5a. CONTRACT NUMBER W81XWH-14-1-0151 5b. GRANT NUMBER PC131783... Bacillus bacteria and pediatric brain malignancies. He has been listed as an author on papers from the lab of Dr. Bianca Garner of Tougaloo College

  17. Indirect pulp capping in primary molar using glass ionomer cements

    Directory of Open Access Journals (Sweden)

    Murtia Metalita

    2014-12-01

    Full Text Available Background: Indirect pulp capping in primary teeth, however, is more rarely conducted than permanent teeth, since it thought to have low impact and most suggestion is for taking caries lesion aggressively on primary teeth. Purpose: The study was aimed to evaluate the subjective complaint, clinical symptom, and radiographic appearance of indirect pulp capping treatment using glass ionomers cements in primary molar. Methods: Sixteen children in range of age 6 to 8 years old, who visited Clinic of Pediatric Dentistry Universitas Airlangga Dental Hospital, Surabaya Indonesia, were the subject of study. They had one occlusal dental caries on one side of maxillary or mandibular primary molar with the diagnose of pulpitis reversible. The experimental group, had indirect pulp capping treatment with glass ionomer cements (GC Fuji VII®, while the control group, had indirect pulp capping treatment with calcium hydroxide (Metapaste. Each group was filled with GC Fuji IX® as permanent restoration. After one week, one month, and three months later, the observations were made on subjective complaint, clinical symptom, and radiographic appearance. Results: The results showed no subjective complaint such as pain or problem on mastication; no negative clinical symptoms such as pain on palpation, gingivitis or periodontitis, and abnormal tooth mobility; no negative radiographic appearance such as pathological apical radioluscency, internal or external resorbtion, and change of ligament periodontal widthafter the treatment. Conclusion: The study suggested that indirect pulp capping treatment using glass ionomer cement materials on primary teeth might be considered to be the treatment choice.Latar belakang: Indirect pulp capping pada gigi sulung lebih jarang dilakukan dibandingkan gigi permanen, karena dianggap memiliki dampak yang rendah dan sebagian besar menyarankan untuk mengambil lesi karies secara agresif pada gigi sulung. Tujuan: Penelitian ini bertujuan

  18. Nebulisation of IVT mRNA Complexes for Intrapulmonary Administration.

    Directory of Open Access Journals (Sweden)

    Sarah M Johler

    Full Text Available During the last years the potential role of in vitro transcribed (IVT mRNA as a vehicle to deliver genetic information has come into focus. IVT mRNA could be used for anti-cancer therapies, vaccination purposes, generation of pluripotent stem cells and also for genome engineering or protein replacement. However, the administration of IVT mRNA into the target organ is still challenging. The lung with its large surface area is not only of interest for delivery of genetic information for treatment of e.g. for cystic fibrosis or alpha-1-antitrypsin deficiency, but also for vaccination purposes. Administration of IVT mRNA to the lung can be performed by direct intratracheal instillation or by aerosol inhalation/nebulisation. The latter approach shows a non-invasive tool, although it is not known, if IVT mRNA is resistant during the process of nebulisation. Therefore, we investigated the transfection efficiency of non-nebulised and nebulised IVT mRNA polyplexes and lipoplexes in human bronchial epithelial cells (16HBE. A slight reduction in transfection efficiency was observed for lipoplexes (Lipofectamine 2000 in the nebulised part compared to the non-nebulised which can be overcome by increasing the amount of Lipofectamine. However, Lipofectamine was more than three times more efficient in transfecting 16HBE than DMRIE and linear PEI performed almost 10 times better than its branched derivative. By contrast, the nebulisation process did not affect the cationic polymer complexes. Furthermore, aerosolisation of IVT mRNA complexes did neither affect the protein duration nor the toxicity of the cationic complexes. Taken together, these data show that aerosolisation of cationic IVT mRNA complexes constitute a potentially powerful means to transfect cells in the lung with the purpose of protein replacement for genetic diseases such as cystic fibrosis or alpha-1-antitrypsin deficiency or for infectious disease vaccines, while bringing along the advantages

  19. Protein targeting to subcellular organelles via MRNA localization.

    Science.gov (United States)

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William

    2013-02-01

    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  20. Polar Rain Gradients and Field-Aligned Polar Cap Potentials

    Science.gov (United States)

    Fairfield, D. H.; Wing, S.; Newell, P. T.; Ruohoniemi, J. M.; Gosling, J. T.; Skoug, R. M.

    2008-01-01

    ACE SWEPAM measurements of solar wind field-aligned electrons have been compared with simultaneous measurements of polar rain electrons precipitating over the polar cap and detected by DMSP spacecraft. Such comparisons allow investigation of cross-polarcap gradients in the intensity of otherwise-steady polar rain. The generally good agreement of the distribution functions, f, from the two data sources confirms that direct entry of solar electrons along open field lines is indeed the cause of polar rain. The agreement between the data sets is typically best on the side of the polar cap with most intense polar rain but the DMSP f's in less intense regions can be brought into agreement with ACE measurements by shifting all energies by a fixed amounts that range from tens to several hundred eV. In most cases these shifts are positive which implies that field-aligned potentials of these amounts exist on polar cap field lines which tend to retard the entry of electrons and produce the observed gradients. These retarding potentials undoubtedly appear in order to prevent the entry of low-energy electrons and maintain charge quasi-neutrality that would otherwise be violated since most tailward flowing magnetosheath ions are unable to follow polar rain electrons down to the polar cap. In more limited regions near the boundary of the polar cap there is sometimes evidence for field-aligned potentials of the opposite sign that accelerate polar rain electrons. A solar electron burst is also studied and it is concluded that electrons from such bursts can enter the magnetotail and precipitate in the same manner as polar rain.

  1. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites.

    Science.gov (United States)

    Schwartz, Schraga; Mumbach, Maxwell R; Jovanovic, Marko; Wang, Tim; Maciag, Karolina; Bushkin, G Guy; Mertins, Philipp; Ter-Ovanesyan, Dmitry; Habib, Naomi; Cacchiarelli, Davide; Sanjana, Neville E; Freinkman, Elizaveta; Pacold, Michael E; Satija, Rahul; Mikkelsen, Tarjei S; Hacohen, Nir; Zhang, Feng; Carr, Steven A; Lander, Eric S; Regev, Aviv

    2014-07-10

    N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing "basal" degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Translational control of human acetyl-CoA carboxylase 1 mRNA is mediated by an internal ribosome entry site in response to ER stress, serum deprivation or hypoxia mimetic CoCl2.

    Science.gov (United States)

    Damiano, Fabrizio; Testini, Mariangela; Tocci, Romina; Gnoni, Gabriele V; Siculella, Luisa

    2018-01-14

    Acetyl-CoA carboxylase 1 (ACC1) is a cytosolic enzyme catalyzing the rate limiting step in de novo fatty acid biosynthesis. There is mounting evidence showing that ACC1 is susceptible to dysregulation and that it is over-expressed in liver diseases associated with lipid accumulation and in several cancers. In the present study, ACC1 regulation at the translational level is reported. Using several experimental approaches, the presence of an internal ribosome entry site (IRES) has been established in the 5' untranslated region (5' UTR) of the ACC1 mRNA. Transfection experiments with the ACC1 5' UTR inserted in a dicistronic reporter vector show a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. The endoplasmic reticulum (ER) stress condition and the related unfolded protein response (UPR), triggered by treatment with thapsigargin and tunicamycin, cause an increase of the cap-independent translation of ACC1 mRNA in HepG2 cells, despite the overall reduction in global protein synthesis. Other stress conditions, such as serum starvation and incubation with hypoxia mimetic agent CoCl2, up-regulate ACC1 expression in HepG2 cells at the translational level. Overall, these findings indicate that the presence of an IRES in the ACC1 5' UTR allows ACC1 mRNA translation in conditions that are inhibitory to cap-dependent translation. A potential involvement of the cap-independent translation of ACC1 in several pathologies, such as obesity and cancer, has been discussed. Copyright © 2018. Published by Elsevier B.V.

  3. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B

    2009-01-01

    . In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to beta-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients. RESULTS: The alpha1(IV......). The level of alpha 6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p alpha 6(IV) mRNA coincides...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6...

  4. Recognition of nonsense mRNA: towards a unified model.

    Science.gov (United States)

    Mühlemann, Oliver

    2008-06-01

    Among the different cellular surveillance mechanisms that ensure accurate gene expression, nonsense-mediated mRNA decay rapidly degrades mRNAs harbouring PTCs (premature translation-termination codons) and thereby prevents the accumulation of potentially deleterious proteins with C-terminal truncations. In the present article, I review recent data from yeast, fluitflies, nematode worms and human cells and endeavour to merge these results into a unified model for recognition of nonsense mRNA. According to this model, the distinction between translation termination at PTCs and at 'normal' termination codons relies on the physical distance between the terminating ribosome and PABP [poly(A)-binding protein]. Correct translation termination is promoted by a PABP-mediated signal to the terminating ribosome, whereas the absence of this signal leads to the assembly of an mRNA decay-promoting protein complex including the conserved NMD factors UPF (up-frameshift) 1-3.

  5. Post-transcriptional gene regulation by mRNA modifications

    Science.gov (United States)

    Zhao, Boxuan Simen; Roundtree, Ian A.; He, Chuan

    2016-01-01

    The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis. PMID:27808276

  6. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  7. FDA Widens Access to 'Cooling Cap' to Stop Hair Loss in Cancer Patients

    Science.gov (United States)

    ... FDA Widens Access to 'Cooling Cap' to Stop Hair Loss in Cancer Patients Treatment now OK'd for ... cancer patients may be able to ward off hair loss during chemotherapy treatment. A cooling cap approved in ...

  8. Coastal Change Analysis Program (C-CAP) Regional Land Cover Data and Change Data

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  9. NOAA's Coastal Change Analysis Program (C-CAP) 2001 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  10. NOAA's Coastal Change Analysis Program (C-CAP) 1985 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  11. NOAA's Coastal Change Analysis Program (C-CAP) 2006 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  12. NOAA's Coastal Change Analysis Program (C-CAP) 1996 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  13. NOAA's Coastal Change Analysis Program (C-CAP) 2010 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  14. NOAA's Coastal Change Analysis Program (C-CAP) 1975 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  15. NOAA's Coastal Change Analysis Program (C-CAP) 1996 Forest Fragmentation Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  16. Barrier Methods of Birth Control: Spermicide, Condom, Sponge, Diaphragm, and Cervical Cap

    Science.gov (United States)

    ... ACOG Barrier Methods of Birth Control: Spermicide, Condom, Sponge, Diaphragm, and Cervical Cap Home For Patients Search ... 2016 Barrier Methods of Birth Control: Spermicide, Condom, Sponge, Diaphragm, and Cervical Cap This product is undergoing ...

  17. Integration of the End Cap TEC+ of the CMS Silicon Strip Tracker

    CERN Document Server

    Adler, Volker; Ageron, Michel; Agram, Jean-Laurent; Atz, Bernd; Barvich, Tobias; Baulieu, Guillaume; Beaumont, Willem; Beissel, Franz; Bergauer, Thomas; Berst, Jean-Daniel; Blüm, Peter; Bock, E; Bogelsbacher, F; de Boer, Wim; Bonnet, Jean-Luc; Bonnevaux, Alain; Boudoul, Gaelle; Bouhali, Othmane; Braunschweig, Wolfgang; Bremer, R; Brom, Jean-Marie; Butz, Erik; Chabanat, Eric; Chabert, Eric Christian; Clerbaux, Barbara; Contardo, Didier; De Callatay, Bernard; Dehm, Philip; Delaere, Christophe; Della Negra, Rodolphe; Dewulf, Jean-Paul; D'Hondt, Jorgen; Didierjean, Francois; Dierlamm, Alexander; Dirkes, Guido; Dragicevic, Marko; Drouhin, Frédéric; Ernenwein, Jean-Pierre; Esser, Hans; Estre, Nicolas; Fahrer, Manuel; Feld, Lutz; Fernández, J; Florins, Benoit; Flossdorf, Alexander; Flucke, Gero; Flügge, Günter; Fontaine, Jean-Charles; Freudenreich, Klaus; Frey, Martin; Friedl, Markus; Furgeri, Alexander; Giraud, Noël; Goerlach, Ulrich; Goorens, Robert; Graehling, Philippe; Grégoire, Ghislain; Gregoriev, E; Gross, Laurent; Hansel, S; Haroutunian, Roger; Hartmann, Frank; Heier, Stefan; Hermanns, Thomas; Heydhausen, Dirk; Heyninck, Jan; Hosselet, J; Hrubec, Josef; Jahn, Dieter; Juillot, Pierre; Kaminski, Jochen; Karpinski, Waclaw; Kaussen, Gordon; Keutgen, Thomas; Klanner, Robert; Klein, Katja; König, Stefan; Kosbow, M; Krammer, Manfred; Ledermann, Bernhard; Lemaître, Vincent; De Lentdecker, Gilles; Linn, Alexander; Lounis, Abdenour; Lübelsmeyer, Klaus; Lumb, Nicholas; Maazouzi, Chaker; Mahmoud, Tariq; Michotte, Daniel; Militaru, Otilia; Mirabito, Laurent; Müller, Thomas; Neukermans, Lionel; Ollivetto, C; Olzem, Jan; Ostapchuk, Andrey; Pandoulas, Demetrios; Pein, Uwe; Pernicka, Manfred; Perriès, Stephane; Piaseki, C; Pierschel, Gerhard; Piotrzkowski, Krzysztof; Poettgens, Michael; Pooth, Oliver; Rouby, Xavier; Sabellek, Andreas; Schael, Stefan; Schirm, Norbert; Schleper, Peter; Schmitz, Stefan Antonius; Schultz von Dratzig, Arndt; Siedling, Rolf; Simonis, Hans-Jürgen; Stahl, Achim; Steck, Pia; Steinbruck, G; Stoye, Markus; Strub, Roger; Tavernier, Stefaan; Teyssier, Daniel; Theel, Andreas; Trocmé, Benjamin; Udo, Fred; Van der Donckt, M; Van der Velde, C; Van Hove, Pierre; Vanlaer, Pascal; Van Lancker, Luc; Van Staa, Rolf; Vanzetto, Sylvain; Weber, Markus; Weiler, Thomas; Weseler, Siegfried; Wickens, John; Wittmer, Bruno; Wlochal, Michael; De Wolf, Eddi A; Zhukov, Valery; Zoeller, Marc Henning

    2009-01-01

    The silicon strip tracker of the CMS experiment has been completed and inserted into the CMS detector in late 2007. The largest sub-system of the tracker is its end cap system, comprising two large end caps (TEC) each containing 3200 silicon strip modules. To ease construction, the end caps feature a modular design: groups of about 20 silicon modules are placed on sub-assemblies called petals and these self-contained elements are then mounted into the TEC support structures. Each end cap consists of 144 petals, and the insertion of these petals into the end cap structure is referred to as TEC integration. The two end caps were integrated independently in Aachen (TEC+) and at CERN (TEC--). This note deals with the integration of TEC+, describing procedures for end cap integration and for quality control during testing of integrated sections of the end cap and presenting results from the testing.

  18. Innovative Capping Technology To Prevent The Migration of Toxic Chemicals From Contaminated Sediments

    Science.gov (United States)

    Capping is a common strategy for decreasing the risk associated with contaminated sediments in lakes and streams. Historically, caps have been designed to physically isolate contaminated sediments and prevent the transport of contaminants from sediments into the water above them...

  19. NOAA's Coastal Change Analysis Program (C-CAP) 2001 Forest Fragmentation Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  20. NOAA's Coastal Change Analysis Program (C-CAP) 2010 Forest Fragmentation Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  1. NOAA's Coastal Change Analysis Program (C-CAP) 1992 Forest Fragmentation Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  2. INFLUENCE OF A ROUND CAP ON THE BEARING CAPACITY OF A LATERALLY LOADED PILE

    Directory of Open Access Journals (Sweden)

    Buslov Anatoliy Semenovich

    2012-07-01

    The data has proven that cap-covered piles are substantially more economical (over 40 % in terms of materials consumption rate if compared to constant cross-section piles (cap-free or broadening piles, all other factors being equal.

  3. NOAA's Coastal Change Analysis Program (C-CAP) 2006 Forest Fragmentation Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  4. NOAA's Coastal Change Analysis Program (C-CAP) 1985 Forest Fragmentation Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  5. NOAA's Coastal Change Analysis Program (C-CAP) 2016 Regional Land Cover Data - Coastal United States

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Coastal Change Analysis Program (C-CAP) produces national standardized land cover and change products for the coastal regions of the U.S. C-CAP products...

  6. Seasonal Changes in Mars' North Polar Ice Cap

    Science.gov (United States)

    1997-01-01

    These images, which seem to have been taken while NASA's Hubble Space Telescope (HST) was looking directly down on the Martian North Pole, were actually created by assembling mosaics of three sets of images taken by HST in October, 1996 and in January and March, 1997 and projecting them to appear as they would if seen from above the pole. This first mosaic is a view which could not actually be seen in nature because at this season a portion of the pole would have actually been in shadow; the last view, taken near the summer solstice, would correspond to the Midnight Sun on Earth with the pole fully illuminated all day. The resulting polar maps begin at 50 degrees N latitude and are oriented with 0 degrees longitude at the 12 o'clock position. This series of pictures captures the seasonal retreat of Mars' north polar cap.October 1996 (early spring in the Northern hemisphere): In this map, assembled from images obtained between Oct. 8 and 15, the cap extends down to 60 degrees N latitude, nearly it's maximum winter extent. (The notches are areas where Hubble data were not available). A thin, comma-shaped cloud of dust can be seen as a salmon-colored crescent at the 7 o'clock position. The cap is actually fairly circular about the geographic pole at this season; the bluish 'knobs' where the cap seems to extend further are actually clouds that occurred near the edges of the three separate sets of images used to make the mosaic.January 1997 (mid-spring): Increased warming as spring progresses in the northern hemisphere has sublimated the carbon dioxide ice and frost below 70 degrees north latitude. The faint darker circle inside the cap boundary marks the location of circumpolar sand dunes (see March '97 map); these dark dunes are warmed more by solar heating than are the brighter surroundings, so the surface frost sublimates from the dunes earlier than from the neighboring areas. Particularly evident is the marked hexagonal shape of the polar cap at this season, noted

  7. Processing of a phosphoglycerate kinase reporter mRNA in Trypanosoma brucei is not coupled to transcription by RNA polymerase II.

    Science.gov (United States)

    Stewart, Mhairi; Haile, Simon; Jha, Bhaskar Anand; Cristodero, Marina; Li, Chi-Ho; Clayton, Christine

    2010-08-01

    Capping of mRNAs is strictly coupled to RNA polymerase II transcription and there is evidence, mainly from metazoans, that other steps in pre-mRNA processing show a similar linkage. In trypanosomes, however, the mRNA cap is supplied by a trans spliced leader sequence. Thus pre-mRNAs transcribed by RNA Polymerase I are capped by trans splicing, and translation-competent transgenic mRNAs can be produced by RNA Polymerase I and T7 RNA polymerase so long as the primary transcript has a splice acceptor signal. We quantified the efficiency of processing of trypanosome pre-mRNAs produced from a plasmid integrated either at the tubulin locus, or in an rRNA spacer, and transcribed by RNA polymerase II, RNA polymerase I or T7 RNA polymerase. The processing efficiencies were similar for primary transcripts from the tubulin locus, produced by RNA polymerase II, and for RNA from an rRNA spacer, transcribed by RNA polymerase I. Primary transcripts produced by T7 RNA polymerase from the tubulin locus were processed almost as well. There was therefore no evidence for recruitment of the 3'-splicing apparatus by the RNA polymerase. Abundant transcripts transcribed from the rRNA locus by T7 RNA polymerase were somewhat less efficiently processed.

  8. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interactions and gene expression profiling

    Directory of Open Access Journals (Sweden)

    Mo Min

    2008-05-01

    Full Text Available Abstract Background The polyadenylation of mRNA is one of the critical processing steps during expression of almost all eukaryotic genes. It is tightly integrated with transcription, particularly its termination, as well as other RNA processing events, i.e. capping and splicing. The poly(A tail protects the mRNA from unregulated degradation, and it is required for nuclear export and translation initiation. In recent years, it has been demonstrated that the polyadenylation process is also involved in the regulation of gene expression. The polyadenylation process requires two components, the cis-elements on the mRNA and a group of protein factors that recognize the cis-elements and produce the poly(A tail. Here we report a comprehensive pairwise protein-protein interaction mapping and gene expression profiling of the mRNA polyadenylation protein machinery in Arabidopsis. Results By protein sequence homology search using human and yeast polyadenylation factors, we identified 28 proteins that may be components of Arabidopsis polyadenylation machinery. To elucidate the protein network and their functions, we first tested their protein-protein interaction profiles. Out of 320 pair-wise protein-protein interaction assays done using the yeast two-hybrid system, 56 (~17% showed positive interactions. 15 of these interactions were further tested, and all were confirmed by co-immunoprecipitation and/or in vitro co-purification. These interactions organize into three distinct hubs involving the Arabidopsis polyadenylation factors. These hubs are centered around AtCPSF100, AtCLPS, and AtFIPS. The first two are similar to complexes seen in mammals, while the third one stands out as unique to plants. When comparing the gene expression profiles extracted from publicly available microarray datasets, some of the polyadenylation related genes showed tissue-specific expression, suggestive of potential different polyadenylation complex configurations. Conclusion An

  9. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  10. 38 CFR 8.33 - Cash value for term-capped policies.

    Science.gov (United States)

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2010-07-01 2010-07-01 false Cash value for term-capped policies. 8.33 Section 8.33 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF VETERANS AFFAIRS NATIONAL SERVICE LIFE INSURANCE Appeals § 8.33 Cash value for term-capped policies. (a) What is a term-capped policy? A term-capped policy is a...

  11. [Utilization of CAP Survey, Based on Questionnaire Results from Survey Participants].

    Science.gov (United States)

    Hirano, Akiko; Ohno, Hiroie

    2015-08-01

    The survey provided by the College of American Pathologists (CAP) is chosen as one of the proficiency testing programs in Japan, and, recently, the numbers of participating facilities have increased. CAP provides 754 programs, and more than 1,000 tests were provided in 2014. Materials are translated as the "CAP global inter-laboratory comparison program" under the instruction of the Japanese Society of Laboratory Medicine (JSLM) selected from CAP surveys in Japan, and 68 programs and 261 items are provided. The total number of participating facilities was 174. CAP itself and the other services CAP provides are not well-known, while recognition of "the CAP survey as the proficiency test" has increased. The question "What is CAP and the CAP survey" was analyzed as a result of the questionnaire surveys conducted in 2014, and the advantage of the CAP survey and how to utilize it were considered. A questionnaire survey was conducted about the CAP survey for Japanese participants in 2014. Fifty-three questions were asked about their satisfaction level, intended use, and improvement. Eighty replies were analyzed. As a result, most CAP survey participants are satisfied. They intend to mainly use the CAP survey for their quality control. Furthermore, they can continuously monitor their systems throughout all testing phases as the survey has numbers of shipments a year and several specimens per each mailing. This helps in laboratory performance improvement. The Evaluation and Participant Summary (PSR) also effectively improves the laboratories' performance. CAP-accredited laboratories are required to participate in all survey programs concerning the test menu which they provide. Therefore, they have become accustomed to reviewing the evaluation and performing self-evaluation with a high usage rate of the Evaluation and PSR of the CAP survey. The questionnaire proved that performing the CAP survey properly enhanced the laboratories' quality control, and this meets the

  12. On complete caps in the projective geometries over F_3. II

    DEFF Research Database (Denmark)

    Barat, Janos

    2004-01-01

    Hill, Landjev, Jones, Storme and Bar\\'at proved in a previous article on caps in $\\pg(5,3)$ and $\\pg(6,3)$ that every 53-cap in $\\pg(5,3)$ is contained in the 56-cap of Hill and that there exist complete 48-caps in $\\pg(5,3)$. The first result was used to lower the upper bound on $m_2(6,3)$ on th...

  13. Properties of electric turbulence in the polar cap ionosphere

    Science.gov (United States)

    Golovchanskaya, I. V.; Kozelov, B. V.

    2010-10-01

    Small-scale (scales of ˜0.5-256 km) electric fields in the polar cap ionosphere are studied on the basis of measurements of the Dynamics Explorer 2 (DE-2) low-altitude satellite with a polar orbit. Nineteen DE-2 passes through the high-latitude ionosphere from the morning side to the evening side are considered when the IMF z component was southward. A rather extensive polar cap, which could be identified using the ɛ- t spectrograms of precipitating particles with auroral energies, was formed during the analyzed events. It is shown that the logarithmic diagrams (LDs), constructed using the discrete wavelet transform of electric fields in the polar cap, are power law (μ ˜ s α). Here, μ is the variance of the detail coefficients of the signal discrete wavelet transform, s is the wavelet scale, and index α characterizes the LD slope. The probability density functions P(δ E, s) of the electric field fluctuations δ E observed on different scales s are non-Gaussian and have intensified wings. When the probability density functions are renormalized, that is constructed of δ E/ s γ, where γ is the scaling exponent, they lie near a single curve, which indicates that the studied fields are statistically self-similar. In spite of the fact that the amplitude of electric fluctuations in the polar cap is much smaller than in the auroral zone, the quantitative characteristics of field scaling in the two regions are similar. Two possible causes of the observed turbulent structure of the electric field in the polar cap are considered: (1) the structure is transferred from the solar wind, which is known to have turbulent properties, and (2) the structure is generated by convection velocity shears in the region of open magnetic field lines. The detected dependence of the characteristic distribution of turbulent electric fields over the polar cap region on IMF B y and the correlation of the rms amplitudes of δ E fluctuations with IMF B z and the solar wind transfer

  14. Design and implementation of the protective cap/biobarrier experiment at the Idaho National Engineering Laboratory

    Energy Technology Data Exchange (ETDEWEB)

    Limbach, W.E.; Ratzlaff, T.D.; Anderson, J.E.; Reynolds, T.D.; Laundre, J.W. [Idaho State Univ., Pocatello, ID (United States)]|[Environmental Science and Research Foundation, Idaho Falls, ID (United States)

    1994-12-31

    The Protective Cap/Biobarrier Experiment (PCBE), initiated in 1993 at the Idaho National Engineering Laboratory (INEL), is a strip-split plot experiment with three replications designed to rigorously test a 2.0-m loessal soil cap against a cap recommended by the US Environmental Protection Agency and two caps with biological intrusion barriers. Past research at INEL indicates that it should be possible to exclude water from buried wastes using natural materials and natural processes in arid environments rather than expensive materials (geotextiles) and highly engineered caps. The PCBE will also test the effects of two vegetal covers and three irrigation levels on cap performance. Drainage pans, located at the bottom of each plot, will monitor cap failure. Soil water profiles will be monitored biweekly by neutron probe and continuously by time domain reflectometry. The performance of each cap design will be monitored under a variety of conditions through 1998. From 1994 to 1996, the authors will assess plant establishment, rooting depths, patterns of moisture extraction and their interactions among caps, vegetal covers, and irrigation levels. In 1996, they will introduce ants and burrowing mammals to test the structural integrity of each cap design. In 1998, the authors will apply sufficient water to determine the failure limit for each cap design. The PCBE should provide reliable knowledge of the performances of the four cap designs under a variety of conditions and aid in making hazardous-waste management decisions at INEL and at disposal sites in similar environments.

  15. Green wet chemical route to synthesize capped CdSe quantum dots

    Indian Academy of Sciences (India)

    In the present work, we report green synthesis of tartaric acid (TA) and triethanolamine (TEA) capped cadmium selenide quantum dots (CdSe QDs) employing chemical bath deposition (CBD) method. The mechanism of capping using non-toxic binary capping agents is also discussed. Stable QDs of various sizes were ...

  16. An ATLAS inner detector end-cap is placed in its cryostat

    CERN Multimedia

    2007-01-01

    The instrumentation housed inside the inner end-cap must be kept cool to avoid thermal noise. This cooling is achieved on ATLAS by placing the end-cap inside a liquid argon cryostat. The end-cap measures particles that are produced close to the direction of the beam pipe and would otherwise be missed.

  17. 50 CFR 679.92 - Amendment 80 Program use caps and sideboard limits.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Amendment 80 Program use caps and... ECONOMIC ZONE OFF ALASKA Amendment 80 Program § 679.92 Amendment 80 Program use caps and sideboard limits. (a) Use caps—(1) General. Use caps limit the amount of Amendment 80 QS units and Amendment 80 species...

  18. 40 CFR 1060.521 - How do I test fuel caps for permeation emissions?

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false How do I test fuel caps for permeation... EQUIPMENT Test Procedures § 1060.521 How do I test fuel caps for permeation emissions? If you measure a fuel tank's permeation emissions with a nonpermeable covering in place of the fuel cap under § 1060.520(b)(5...

  19. 77 FR 57197 - Proposed Collection; Comment Request for Form 1099-CAP

    Science.gov (United States)

    2012-09-17

    ... Internal Revenue Service Proposed Collection; Comment Request for Form 1099-CAP AGENCY: Internal Revenue...(c)(2)(A)). Currently, the IRS is soliciting comments concerning Form 1099-CAP, Changes in Corporate...: Changes in Corporate Control and Capital Structure. OMB Number: 1545-1814. Form Number: 1099-CAP. Abstract...

  20. File list: Oth.Dig.10.MethylCap.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Dig.10.MethylCap.AllCell hg19 TFs and others MethylCap Digestive tract SRX10526...1,SRX105262,SRX105260 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Dig.10.MethylCap.AllCell.bed ...

  1. Construction of quantum caps in projective space PG( r, 4) and quantum codes of distance 4

    Science.gov (United States)

    Li, Ruihu; Fu, Qiang; Guo, Luobin; Li, Xueliang

    2016-02-01

    Constructions of quantum caps in projective space PG( r, 4) by recursive methods and computer search are discussed. For each even n satisfying n≥282 and each odd z satisfying z≥275, a quantum n-cap and a quantum z-cap in PG(k-1, 4) with suitable k are constructed, and [[n,n-2k,4

  2. Meteorological observations in support of a hill cap cloud experiment

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, Morten

    1998-06-01

    Humid air flows form a hill cap cloud over the Agana mountain ridge in the north-east of Tenerife. The HILLCLOUD project utilised this cloud formation to investigate the chemical and physical properties of cloud aerosols by land based observations. The project was part of the second Aerosol characterisation Experiment (ACE-2) of the International Global Atmospheric chemistry project (IGAC). The present report describes meteorological observations in support of the hill cap cloud experiment. Time-series of wind speed, wind direction, temperature and humidity were collected at ground-based meteorological stations during a period starting one year in advance of the main campaign. A series of radiosonde detecting the upstream stability and wind profile were launched during the main campaign. (au) 5 tabs., 32 ills., 6 refs.

  3. Simple MoCap System for Home Usage

    Directory of Open Access Journals (Sweden)

    Martin Magdin

    2017-08-01

    Full Text Available Nowadays many MoCap systems exist. Generating 3D facial animation of characters is currently realized by using the motion capture data (MoCap data, which is obtained by tracking the facial markers from an actor/actress. In general it is a professional solution that is sophisticated and costly. This paper presents a solution with a system that is inexpensive. We propose a new easy-to-use system for home usage, through which we are making character animation. In its implementation we paid attention to the elimination of errors from the previous solutions. In this paper the authors describe the method how motion capture characters on a treadmill and as well as an own Java application that processes the video for its further use in Cinema 4D. This paper describes the implementation of this technology of sensing in a way so that the animated character authentically imitated human movement on a treadmill.

  4. Nanoscale gadolinium oxide capping layers on compositionally variant gate dielectrics

    KAUST Repository

    Alshareef, Husam N.

    2010-11-19

    Metal gate work function enhancement using nanoscale (1.0 nm) Gd2O3 interfacial layers has been evaluated as a function of silicon oxide content in the HfxSiyOz gate dielectric and process thermal budget. It is found that the effective work function tuning by the Gd2O3 capping layer varied by nearly 400 mV as the composition of the underlying dielectric changed from 0% to 100% SiO2, and by nearly 300 mV as the maximum process temperature increased from ambient to 1000 °C. A qualitative model is proposed to explain these results, expanding the existing models for the lanthanide capping layer effect.

  5. Polar cap F layer patches: structure and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Weber, E.J.; Klobuchar, J.A.; Buchau, J.; Carlson H.C. Jr.; Livingston, R.C.; De La Beaujardiere, O.; McCready, M.; Moore, J.G.; Bishop, G.J.

    1986-11-01

    Coordinated measurements of F region plasma patches were conducted on February 3/4, 1984, from Thule and Sondrestrom, Greenland. Optical, ionosonde, amplitude scintillation, total electron content (TEC), and incoherent scatter radar measurements were combined to reveal several new aspects of the structure and transport of these localized regions of enhanced F region ionization. For the first time these patches were directly tracked flowing in the antisunward direction over distances of 3000 km from the center of the polar cap to the poleward edge of the auroral oval. Quantitative measurements of TEC show increases of 10--15 TEC units within the patches, above a background polar cap value of 5 TEC units. Amplitude scintillation measurements show the presence of ionospheric irregularities through the entire patch, with a weak indication of stronger scintillation on the trailing (or E x B unstable) edge.

  6. Polar cap F layer patches: structure and dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Weber, E.J.; Klobuchar, J.A.; Buchau, J.; Carlson, H.C.; Livingston, R.C.

    1986-11-01

    Coordinated measurements of F-region plasma patches were conducted on February 3/4, 1984, from Thule and Sondrestrom, Greenland. Optical, ionsonde, amplitude scintillation, total electron content (TEC), and incoherent scatter radar measurements were combined to reveal several new aspects of the structure and transport of these localized regions of enhanced F region ionization. For the first time, these patches were directly tracked flowing in the antisunward direction over distances of 3000 km from the center of the polar cap to the poleward edge of the auroral oval. Quantative measurements of TEC show increases of 10-15 TEC units within the patches, above a background polar cap value of 5 TEC units. Amplitude scintillation measurements show the presence of ionospheric irregularities through the entire patch, with a weak indication of stronger scintillation on the trailing (or E x B unstable) edge.

  7. Thermal analysis of the airflow around ATLAS muon end cap

    CERN Document Server

    Gasser, D

    2003-01-01

    A thermal analysis of the airflow inside the UX15 cavern and through the ATLAS detector is presented. This study is done using a CFD (Computational Fluid Dynamics) model. This model includes a simplified geometry of the detector and the experimental cavern, the ventilation flow rate and the released heat dissipation figures are taken into account. This analysis aims at estimate the temperature gradients that develop in the muons end cap area. Indeed, light rays seen by CCD camera will be used in this area in order to align the muon chambers. The rays should not be too much distorted by temperature difference, which would hinder the chamber alignment. The simulation results show that a light ray projected through the whole end cap area should not encounter a gradient higher than 5 K. Nevertheless, the results of this analysis are valid if and only if the spaces represented as empty in the model are allowed to remain empty in ATLAS.

  8. Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages

    National Research Council Canada - National Science Library

    Van Arsdell, S W; Murphy, K P; Pazmany, C; Erickson, D; Burns, C; Moody, M D

    2000-01-01

    Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA...

  9. Coronavirus Nonstructural Protein 16 Is a Cap-0 Binding Enzyme Possessing (Nucleoside-2′O)-Methyltransferase Activity▿

    Science.gov (United States)

    Decroly, Etienne; Imbert, Isabelle; Coutard, Bruno; Bouvet, Mickaël; Selisko, Barbara; Alvarez, Karine; Gorbalenya, Alexander E.; Snijder, Eric J.; Canard, Bruno

    2008-01-01

    The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-l-methionine (AdoMet)-dependent RNA (nucleoside-2′O)-methyltransferase (2′O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap (7MeGpppAC3-6) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2′O position of the first transcribed nucleotide, thus converting 7MeGpppAC3-6 into 7MeGpppA2′OMeC3-6. The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2′O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2′O methylation follows an order of events in which 2′O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase. PMID:18417574

  10. Coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'O)-methyltransferase activity.

    Science.gov (United States)

    Decroly, Etienne; Imbert, Isabelle; Coutard, Bruno; Bouvet, Mickaël; Selisko, Barbara; Alvarez, Karine; Gorbalenya, Alexander E; Snijder, Eric J; Canard, Bruno

    2008-08-01

    The coronavirus family of positive-strand RNA viruses includes important pathogens of livestock, companion animals, and humans, including the severe acute respiratory syndrome coronavirus that was responsible for a worldwide outbreak in 2003. The unusually complex coronavirus replicase/transcriptase is comprised of 15 or 16 virus-specific subunits that are autoproteolytically derived from two large polyproteins. In line with bioinformatics predictions, we now show that feline coronavirus (FCoV) nonstructural protein 16 (nsp16) possesses an S-adenosyl-L-methionine (AdoMet)-dependent RNA (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is capable of cap-1 formation. Purified recombinant FCoV nsp16 selectively binds to short capped RNAs. Remarkably, an N7-methyl guanosine cap ((7Me)GpppAC(3-6)) is a prerequisite for binding. High-performance liquid chromatography analysis demonstrated that nsp16 mediates methyl transfer from AdoMet to the 2'O position of the first transcribed nucleotide, thus converting (7Me)GpppAC(3-6) into (7Me)GpppA(2')(O)(Me)C(3-6). The characterization of 11 nsp16 mutants supported the previous identification of residues K45, D129, K169, and E202 as the putative K-D-K-E catalytic tetrad of the enzyme. Furthermore, residues Y29 and F173 of FCoV nsp16, which may be the functional counterparts of aromatic residues involved in substrate recognition by the vaccinia virus MTase VP39, were found to be essential for both substrate binding and 2'O-MTase activity. Finally, the weak inhibition profile of different AdoMet analogues indicates that nsp16 has evolved an atypical AdoMet binding site. Our results suggest that coronavirus mRNA carries a cap-1, onto which 2'O methylation follows an order of events in which 2'O-methyl transfer must be preceded by guanine N7 methylation, with the latter step being performed by a yet-unknown N7-specific MTase.

  11. Analysis of mRNA recognition by human thymidylate synthase.

    Science.gov (United States)

    Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B; Ghassemian, Majid; Hermann, Thomas

    2014-12-23

    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site.

  12. Association between VDAC1 mRNA expression and intracellular ...

    African Journals Online (AJOL)

    One way in which xenobiotics induce apoptotic cell death is to alter the selective permeability of the intracellular voltage-dependent anion channel (VDAC1) in the mitochondrial membrane. In this study, we explored the association between VDAC1 mRNA expression and mitochondrial function during hexavalent chromium ...

  13. Cytokine mRNA expression during experimental corneal allograft rejection

    NARCIS (Netherlands)

    Torres, P. F.; de Vos, A. F.; van der Gaag, R.; Martins, B.; Kijlstra, A.

    1996-01-01

    Allograft rejection is the main cause of corneal graft failure. T lymphocytes and macrophages have been implied to be involved in corneal rejection, but little is known about the molecular mechanism in this process. In this study, cytokine mRNA expression in the cornea was analysed during

  14. Human mRNA response to exercise and temperature.

    Science.gov (United States)

    Slivka, D R; Dumke, C L; Tucker, T J; Cuddy, J S; Ruby, B

    2012-02-01

    The purpose of this research was to determine the mRNA response to exercise in different environmental temperatures. 9 recreationally active males (27±1 years, 77.4±2.7  kg, 13.5±1.5% fat, 4.49±0.15  L · min (-1) VO2 max) completed 3 trials consisting of 1 h cycling exercise at 60% Wmax followed by a 3 h recovery in the cold (7°C), room temperature (20°C), and hot (33°C) environments. Muscle biopsies were obtained pre, post, and 3 h post exercise for the analysis of glycogen and mRNA. Expired gases were collected to calculate substrate use. PGC-1α increased to a greater degree in the cold trial than in the room temperature trial (p=0.036) and the hot trial (p=0.006). PGC1-α mRNA was also higher after the room temperature trial than the hot trial (p=0.050). UCP3 and MFN2 mRNA increased with exercise (pcold than exercise in the heat. However, VO2 was higher during recovery in the cold trial than in the room temperature and hot trials (p<0.05). This study presents evidence of PGC-1α temperature sensitivity in human skeletal muscle. © Georg Thieme Verlag KG Stuttgart · New York.

  15. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results indicated that the ...

  16. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    Jane

    2011-07-13

    Jul 13, 2011 ... This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye. (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results.

  17. Regulation of mRNA Trafficking by Nuclear Pore Complexes

    Directory of Open Access Journals (Sweden)

    Amandine Bonnet

    2014-09-01

    Full Text Available Over the last two decades, multiple studies have explored the mechanisms governing mRNA export out of the nucleus, a crucial step in eukaryotic gene expression. During transcription and processing, mRNAs are assembled into messenger ribonucleoparticles (mRNPs. mRNPs are then exported through nuclear pore complexes (NPCs, which are large multiprotein assemblies made of several copies of a limited number of nucleoporins. A considerable effort has been put into the dissection of mRNA export through NPCs at both cellular and molecular levels, revealing the conserved contributions of a subset of nucleoporins in this process, from yeast to vertebrates. Several reports have also demonstrated the ability of NPCs to sort out properly-processed mRNPs for entry into the nuclear export pathway. Importantly, changes in mRNA export have been associated with post-translational modifications of nucleoporins or changes in NPC composition, depending on cell cycle progression, development or exposure to stress. How NPC modifications also impact on cellular mRNA export in disease situations, notably upon viral infection, is discussed.

  18. MRNA-based skin identification for forensic applications

    NARCIS (Netherlands)

    M. Visser (Mijke); D. Zubakov (Dmitry); K. Ballantyne (Kaye); M.H. Kayser (Manfred)

    2011-01-01

    textabstractAlthough the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in

  19. Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

    Science.gov (United States)

    Hou, Guichuan; Mohamalawari, Deepti R.; Blancaflor, Elison B.

    2003-01-01

    The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.

  20. Polar cap flow channel events: spontaneous and driven responses

    Directory of Open Access Journals (Sweden)

    P. E. Sandholt

    2010-11-01

    Full Text Available We present two case studies of specific flow channel events appearing at the dusk and/or dawn polar cap boundary during passage at Earth of interplanetary (IP coronal mass ejections (ICMEs on 10 January and 25 July 2004. The channels of enhanced (>1 km/s antisunward convection are documented by SuperDARN radars and dawn-dusk crossings of the polar cap by the DMSP F13 satellite. The relationship with Birkeland currents (C1–C2 located poleward of the traditional R1–R2 currents is demonstrated. The convection events are manifest in ground magnetic deflections obtained from the IMAGE (International Monitor for Auroral Geomagnetic Effects Svalbard chain of ground magnetometer stations located within 71–76° MLAT. By combining the ionospheric convection data and the ground magnetograms we are able to study the temporal behaviour of the convection events. In the two ICME case studies the convection events belong to two different categories, i.e., directly driven and spontaneous events. In the 10 January case two sharp southward turnings of the ICME magnetic field excited corresponding convection events as detected by IMAGE and SuperDARN. We use this case to determine the ground magnetic signature of enhanced flow channel events (the NH-dusk/By<0 variant. In the 25 July case a several-hour-long interval of steady southwest ICME field (Bz<0; By<0 gave rise to a long series of spontaneous convection events as detected by IMAGE when the ground stations swept through the 12:00–18:00 MLT sector. From the ground-satellite conjunction on 25 July we infer the pulsed nature of the polar cap ionospheric flow channel events in this case. The typical duration of these convection enhancements in the polar cap is 10 min.

  1. Cavity Attenuated Phase Shift (CAPS) Monitor Instrument Handbook

    Energy Technology Data Exchange (ETDEWEB)

    Sedlacek, Arthur J. [Brookhaven National Lab. (BNL), Upton, NY (United States)

    2016-04-01

    The CAPS PMex monitor is a cavity attenuated phase shift extinction instrument. It operates as an optical extinction spectrometer, using a visible-light-emitting diode (LED) as the light source, a sample cell incorporating two high-reflectivity mirrors centered at the wavelength of the LED, and a vacuum photodiode detector. Its efficacy is based on the fact that aerosols are broadband scatterers and absorbers of light.

  2. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism.

    Directory of Open Access Journals (Sweden)

    Christina R Muratore

    Full Text Available The folate and vitamin B12-dependent enzyme methionine synthase (MS is highly sensitive to cellular oxidative status, and lower MS activity increases production of the antioxidant glutathione, while simultaneously decreasing more than 200 methylation reactions, broadly affecting metabolic activity. MS mRNA levels in postmortem human cortex from subjects across the lifespan were measured and a dramatic progressive biphasic decrease of more than 400-fold from 28 weeks of gestation to 84 years was observed. Further analysis revealed alternative splicing of MS mRNA, including deletion of folate-binding domain exons and age-dependent deletion of exons from the cap domain, which protects vitamin B12 (cobalamin from oxidation. Although three species of MS were evident at the protein level, corresponding to full-length and alternatively spliced mRNA transcripts, decreasing mRNA levels across the lifespan were not associated with significant changes in MS protein or methionine levels. MS mRNA levels were significantly lower in autistic subjects, especially at younger ages, and this decrease was replicated in cultured human neuronal cells by treatment with TNF-α, whose CSF levels are elevated in autism. These novel findings suggest that rather than serving as a housekeeping enzyme, MS has a broad and dynamic role in coordinating metabolism in the brain during development and aging. Factors adversely affecting MS activity, such as oxidative stress, can be a source of risk for neurological disorders across the lifespan via their impact on methylation reactions, including epigenetic regulation of gene expression.

  3. Activin-treated Urodele Animal Caps : II. Inductive Interactions in Newt Animal Caps After Treatment with Activin A(Developmental Biology)

    OpenAIRE

    Takashi, Ariizumi; Shinji, Komazaki; Makoto, Asashima; CREST, Japan Science and Technology Corporation; The 2nd Department of Anatomy, Saitama Medical School

    1999-01-01

    The inductive interactions between activin-induced and non-induced cells were investigated in newt animal cap explants. A wide range of concentrations of activin A (0.1-100 ng/ml) induced mesodermal tissues in the animal caps, but at generally low frequencies. Animal caps treated with 100 ng/ml of activin A, on the other hand, differentiated solely into nonspecific endoderm. At this concentration, various mesodermal tissues were induced in addition to endoderm as the animal caps increased in ...

  4. Structural characterisation of alkyl amine-capped zinc sulphide nanoparticles.

    Science.gov (United States)

    Kremser, Gabriele; Rath, Thomas; Kunert, Birgit; Edler, Michael; Fritz-Popovski, Gerhard; Resel, Roland; Letofsky-Papst, Ilse; Grogger, Werner; Trimmel, Gregor

    2012-03-01

    Nanoparticles capped with amine ligands with different steric properties, dodecylamine and oleylamine, respectively, are investigated in the solid state as well as in solution. A combined X-ray diffraction, small angle X-ray scattering and electron microscopy investigation showed that the nanoparticles exhibit the sphalerite modification of ZnS as crystal phase with a diameter of 3-5 nm. A close packing of the monocrystalline nanoparticles in the solid state is observed. However, in the dodecylamine sample, besides spherical particles, a fraction of the nanoparticles is elongated. The nanoparticles are readily resoluble in apolar solvents like hexane. Dynamic light scattering (DLS) and SAXS investigations of the solutions reveal that the nanoparticles are dissolved as singular particles. In the case of oleylamine-capped ZnS, a defined core-shell structure with a ZnS core with a diameter of 4 nm and an organic shell with a thickness of approximately 2 nm have been found. Dodecylamine-capped nanoparticles slightly tend to form agglomerates with a diameter of approximately 40 nm. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Results from the First Validation Phase of CAP code

    Energy Technology Data Exchange (ETDEWEB)

    Choo, Yeon Joon; Hong, Soon Joon; Hwang, Su Hyun; Kim, Min Ki; Lee, Byung Chul [FNC Tech., SNU, Seoul (Korea, Republic of); Ha, Sang Jun; Choi, Hoon [Korea Electric Power Research Institute, Daejeon (Korea, Republic of)

    2010-10-15

    The second stage of Safety Analysis Code Development for Nuclear Power Plants was lunched on Apirl, 2010 and is scheduled to be through 2012, of which the scope of work shall cover from code validation to licensing preparation. As a part of this project, CAP(Containment Analysis Package) will follow the same procedures. CAP's validation works are organized hieratically into four validation steps using; 1) Fundamental phenomena. 2) Principal phenomena (mixing and transport) and components in containment. 3) Demonstration test by small, middle, large facilities and International Standard Problems. 4) Comparison with other containment codes such as GOTHIC or COMTEMPT. In addition, collecting the experimental data related to containment phenomena and then constructing the database is one of the major works during the second stage as a part of this project. From the validation process of fundamental phenomenon, it could be expected that the current capability and the future improvements of CAP code will be revealed. For this purpose, simple but significant problems, which have the exact analytical solution, were selected and calculated for validation of fundamental phenomena. In this paper, some results of validation problems for the selected fundamental phenomena will be summarized and discussed briefly

  6. Structural basis for capping protein sequestration by myotrophin (V-1).

    Science.gov (United States)

    Zwolak, Adam; Fujiwara, Ikuko; Hammer, John A; Tjandra, Nico

    2010-08-13

    Capping protein (CP) is a ubiquitously expressed, heterodimeric 62-kDa protein that binds the barbed end of the actin filament with high affinity to block further filament elongation. Myotrophin (V-1) is a 13-kDa ankyrin repeat-containing protein that binds CP tightly, sequestering it in a totally inactive complex in vitro. Here, we elucidate the molecular interaction between CP and V-1 by NMR. Specifically, chemical shift mapping and intermolecular paramagnetic relaxation enhancement experiments reveal that the ankyrin loops of V-1, which are essential for V-1/CP interaction, bind the basic patch near the joint of the alpha tentacle of CP shown previously to drive most of the association of CP with and affinity for the barbed end. Consistently, site-directed mutagenesis of CP shows that V-1 and the strong electrostatic binding site for CP on the barbed end compete for this basic patch on CP. These results can explain how V-1 inactivates barbed end capping by CP and why V-1 is incapable of uncapping CP-capped actin filaments, the two signature biochemical activities of V-1.

  7. Resonance Raman spectra of. cap alpha. -copper phthalocyanine

    Energy Technology Data Exchange (ETDEWEB)

    Bovill, A.J.; McConnell, A.A.; Nimmo, J.A.; Smith, W.E.

    1986-02-13

    Raman spectra of ..cap alpha..-copper phthalocyanine (..cap alpha..-CuPc) were recorded at room temperature and at 10 K with excitation wavelengths between 457 and 714 nm. Resonance enhancement was greatest for modes for which the largest displacements were on either the inner five-membered ring of the isoindole groups or the inner macrocycle and consequently assignment of the bands to modes of the entire molecule was possible by comparison with nickel octaethylporphyrin. Four out of five bands resonant in the Q band region and preresonant near the B band absorption region are totally symmetric modes. B band preresonance occurs more strongly with high-frequency modes. At low temperatures, multimode interactions are reduced and profiles were obtained which can be compared with solution profiles of porphyrins. Both Q/sub x/ and Q/sub y/ 0-0 scattering can be identified and a helper mode is evident. A term enhancement predominates, with B/sub 1g/ and B/sub 2g/ modes enhanced because of a Jahn-Teller distortion of the excited state. The resonance studies, together with electronic absorption spectra and published theoretical studies, confirm that the Q band in ..cap alpha..-CuPc is largely due to an allowed ..pi..-..pi..* transition associated mainly with the macrocycle and inner five-membered rings of the isoindole groups. 25 references, 5 figures, 2 tables.

  8. Completion of the first TRT End-cap

    CERN Multimedia

    Catinaccio, A; Rohne, O

    On July 1, the first end-cap of the ATLAS Transition Radiation Tracker (TRT) was successfully completed in terms of the integration of the wheels assembled in Russia with their front-end electronics. The two groups of the detector, fully assembled and equipped with front-end electronics, were rotated from their horizontal position during stacking to their nominal vertical position, in which they will be integrated with the corresponding end-cap silicon-strip (SCT) detector towards the end of 2005, before installation into ATLAS in spring 2006. After starting the assembly in the SR building one year ago, the TRT team reached this important milestone, which marks the final realization and validation of the engineering concept developed by the CERN DT1 (ex-TA1) and ATT teams. A TRT end-cap consists of two sets of identical and independent wheels. The first type of wheels (type A, 12 wheels, positioned closest to the primary interaction point) contains 6144 radial straws positioned in eight successive layers s...

  9. First Wheel of the Hadronic EndCap Calorimeter Completed

    CERN Document Server

    Oram, C.J.

    2002-01-01

    With the LAr calorimeters well advanced in module production, the attention is turning to Batiment 180 where the calorimeter modules are formed into complete detectors and inserted into their respective cryostats. For the Hadronic End Cap (HEC) Group the task in B180 is to assemble the wheels, rotate them into their final orientation, and put them onto the cradle in front of the End Cap Cryostat. These tasks have been completed for the first HEC wheel in the B180 End Cap Clean Room. Given that this wheel weighs 70 tons the group is very relieved to have established that these gymnastics with the wheel proceed in a routine fashion. To assemble a wheel we take modules that have already been cold tested, do the final electrical testing and locate them onto the HEC wheel assembly table. Four wheels are required in total, each consisting of 32 modules. Wheel assembly is done in the horizontal position, creating a doughnut-like object sitting on the HEC table. The first picture shows the last module being added ...

  10. Capping Repels Oxygen and Selectively Adsorbs Hydrogen via Kubas Interaction

    Science.gov (United States)

    Ahmad, Rafia; Singh, Abhishek K.

    Hydrogen bound via Kubas interaction on metal decorated light weight materials forebodes a much efficient alternative for the fast depleting fossil fuels. O2 interference in hydrogen storage on metal decorated carbonaceous systems remains one of the major stumbling blocks in successful realization of the theoretically promised high storage of Kubas bound H2. The interference is a consequence of preferred O2 binding at the metal site, thereby blocking it for H2 adsorption. Here, we report that arene capping of a Sc-metallacarborane (MCB) efficiently reverses the preferential adsorption of O2 over H2. The capped Sc-MCB completely repels O2 off the Sc-site allowing solely Kubas binding of H2, consequent of the down-shift of the d-band center of Sc from 1.29 eV in the uncapped to 5.67 eV in the capped MCB, below the Fermi-level, respectively. This optimised d-band center position enables the empty Sc antibonding states to only be available for hydrogen adsorption via Kubas interaction, thereby, to completely avoid oxygen binding. This result provides an approach by tuning a cluster electronically to enhance gas adsorption selectivity, which can be efficiently ulitized in various catalytic, sensing and gas storage systems.

  11. Commissioning Test of ATLAS End-Cap Toroidal Magnets

    CERN Document Server

    Dudarev, A; Foussat, A; Benoit, P; Jeckel, M; Olyunin, A; Kopeykin, N; Stepanov, V; Deront, L; Olesen, G; Ponts, X; Ravat, S; Sbrissa, K; Barth, J; Bremer, J; Delruelle, J; Metselaar, J; Pengo, R; Pirotte, O; Buskop, J; Baynham, D E; Carr, F S; Holtom, E

    2009-01-01

    The system of superconducting toroids in the ATLAS experiment at CERN consists of three magnets. The Barrel Toroid was assembled and successfully tested in 2006. Next, two End-Cap Toroids have been tested on surface at 77 K and installed in the cavern, 100-m underground. The End Cap Toroids are based on Al stabilized Nb-Ti/Cu Rutherford cables, arranged in double pancake coils and conduction cooled at 4.6 K. The nominal current is 20.5 kA at 4.1 T peak field in the windings and the stored energy is 250 MJ per toroid. Prior to final testing of the entire ATLAS Toroidal system, each End Cap Toroid passed a commissioning test up to 21 kA to guarantee a reliable performance in the final assembly. In this paper the test results are described. It includes the stages of test preparation, isolation vacuum pumping and leak testing, cooling down, step-by-step charging to full current, training quenches and quench recovery. By fast discharges the quench detection and protection system was checked to demonstrate a safe e...

  12. Damage detection in prestressed pile to bent cap connections

    Science.gov (United States)

    Larosche, Aaron; Mangual, Jese; Ziehl, Paul

    2011-04-01

    South Carolina is one of the most seismically active states in the eastern U.S. Due to this high level of seismic activity, structural health monitoring is important to ensure a high level of confidence in the state's infrastructure. The University of South Carolina (U.SC) is currently studying the behavior of prestressed pile to bent-cap connections that are typical of construction used in the state. Bent caps are generally constructed with multiple piles. In these tests single pile specimens were created for both interior and exterior piles. Interior specimens were subjected to a constant compressive load while exterior specimens experienced both compressive and tensile loads. Acoustic Emission (AE) sensing was utilized on fullscale test specimens to investigate the feasibility of detecting and characterizing damage in these connections during a seismic event. Seven full-scale prestressed concrete piles have been embedded into cast-in-place (CIP) reinforced concrete bent caps and tested under reverse cyclic loading. AE data has been gathered with eight strategically placed AE sensors. Preliminary analysis of the data indicates that AE is promising method with respect to the detection of damage prior to detection by visual observation. AE activity is used to detect both the onset and location of cracking and to characterize the extent of damage at later stages of degradation. One focus of the work is to minimize the amount of AE data recorded for the development of wireless systems having low power consumption.

  13. Panther cap Amanita pantherina poisoning case report and review.

    Science.gov (United States)

    Satora, Leszek; Pach, Dorota; Ciszowski, Krzysztof; Winnik, Lidia

    2006-04-01

    An analysis of patients with mushroom poisoning hospitalized in the Clinic of Toxicology in Cracow revealed that only a small percentage of cases had been caused by the death cap Amanita phalloides (Vaill. ex Fr.) Secr. The most important factors contributing to intoxication are confusion of toxic mushrooms with edible species, and non-specific mushroom poisoning. The genus Amanita has a global distribution and is one of the most well-known genera of macrofungi. Active toxins present in the panther cap (A. pantherina) (DC ex Fr.) Secr are ibotenic acid and muscimol, which are rapidly absorbed from the gastrointestinal tract. It is likely that other substances also participate in the psychotropic effects. Five frayed panther cap fruiting bodies were eaten by mistake by two persons (27 and 47 years of age). Symptoms onset occurred after 120 min with central nervous system (CNS) depression, ataxia, waxing and waning obtundation, religious hallucinations and hyperkinetic behaviour. In the present case, successful general symptomatic treatment was administered, which consisted of controlling the nervous symptoms and stabilizing the electrolyte balance. The poisoning regressed with no organ complications.

  14. The thermospheric effects of a rapid polar cap expansion

    Directory of Open Access Journals (Sweden)

    D. W. Idenden

    Full Text Available In a previous publication we used results from a coupled thermosphere-ionosphere-plasmasphere model to illustrate a new mechanism for the formation of a large-scale patch of ionisation arising from a rapid polar cap expansion. Here we describe the thermospheric response to that polar cap expansion, and to the ionospheric structure produced. The response is dominated by the energy and momentum input at the dayside throat during the expansion phase itself. These inputs give rise to a large-scale travelling atmospheric disturbance (TAD that propagates both antisunward across the polar cap and equatorward at speeds much greater than both the ion drifts and the neutral winds. We concentrate only on the initially poleward travelling disturbance. The disturbance is manifested in the neutral temperature and wind fields, the height of the pressure level surfaces and in the neutral density at fixed heights. The thermospheric effects caused by the ionospheric structure produced during the expansion are hard to discern due to the dominating effects of the TAD.

    Key words. Ionosphere (ionosphere · atmosphere interaction; modeling and forecasting; plasma convection.

  15. The thermospheric effects of a rapid polar cap expansion

    Directory of Open Access Journals (Sweden)

    D. W. Idenden

    1998-10-01

    Full Text Available In a previous publication we used results from a coupled thermosphere-ionosphere-plasmasphere model to illustrate a new mechanism for the formation of a large-scale patch of ionisation arising from a rapid polar cap expansion. Here we describe the thermospheric response to that polar cap expansion, and to the ionospheric structure produced. The response is dominated by the energy and momentum input at the dayside throat during the expansion phase itself. These inputs give rise to a large-scale travelling atmospheric disturbance (TAD that propagates both antisunward across the polar cap and equatorward at speeds much greater than both the ion drifts and the neutral winds. We concentrate only on the initially poleward travelling disturbance. The disturbance is manifested in the neutral temperature and wind fields, the height of the pressure level surfaces and in the neutral density at fixed heights. The thermospheric effects caused by the ionospheric structure produced during the expansion are hard to discern due to the dominating effects of the TAD.Key words. Ionosphere (ionosphere · atmosphere interaction; modeling and forecasting; plasma convection.

  16. Last End Cap Toroid installation : The Pharaonic enterprise

    CERN Document Server

    Arnaud Foussat

    After the successful and impressive transport feat from Building 191 to Point 1 was carried out by the Friderici crew on 28th June, the second and last Toroid End Cap, ECT-C, was transferred into the surface building, SX1, on 2nd July. The ECT-C was installed in the ATLAS cavern on the C-side on 12th July. As the person responsible for the project, in my opinion, one of the crucial points of this project was to design all the tooling and installation sequences taking into account the building infrastructure dimensional constraints. View of the ECT installation tooling and preparation for the ECT-C descent into the ATLAS 80m-shaft by the ATLAS magnet group and DBS teams. The movement of the 240-ton magnet and 12-m diameter toroid end-cap was achieved in collaboration with SCALES, a subcontractor company, using a hydraulic gantry able to lower the ECT inside the shaft by 5m below the floor level . This allowed the DBS team to attach the end-cap with the 2 x 140 tons overhead crane and lower it onto the c...

  17. The role of the poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants

    Science.gov (United States)

    Gallie, Daniel R

    2014-01-01

    Translation initiation in eukaryotes requires the involvement of multiple initiation factors (eIFs) that facilitate the binding of the 40 S ribosomal subunit to an mRNA and assemble the 80 S ribosome at the correct initiation codon. eIF4F, composed of eIF4E, eIF4A, and eIF4G, binds to the 5′-cap structure of an mRNA and prepares an mRNA for recruitment of a 40 S subunit. eIF4B promotes the ATP-dependent RNA helicase activity of eIF4A and eIF4F needed to unwind secondary structure present in a 5′-leader that would otherwise impede scanning of the 40 S subunit during initiation. The poly(A) binding protein (PABP), which binds the poly(A) tail, interacts with eIF4G and eIF4B to promote circularization of an mRNA and stimulates translation by promoting 40 S subunit recruitment. Thus, these factors serve essential functions in the early steps of protein synthesis. Their assembly and function requires multiple interactions that are competitive in nature and determine the nature of interactions between the termini of an mRNA. In this review, the domain organization and partner protein interactions are presented for the factors in plants which share similarities with those in animals and yeast but differ in several important respects. The functional consequences of their interactions on factor activity are also discussed. PMID:26779409

  18. What Should You Pay to Cap your ARM?—A Note on Capped Adjustable Rate Mortgages

    Directory of Open Access Journals (Sweden)

    Maj-Britt Nordfang

    2017-03-01

    Full Text Available In this paper, an Adjustable Rate Mortgage (ARM and a Fixed Rate Mortgage (FRM are formalized and studied in a simple continuous-time setting under the assumption of a simple one-factor Affine Term Structure (ATS. Through an application of existing results from ATS theory, it is shown that when the short rate reaches a certain pre-determined boundary, the constant payment stream on a new FRM equals the payments on an existing ARM. Hereby, this paper provides a theoretical build-in cap on the formalized ARM. The finite boundary for the short-rate suggests that certain caps on ARMs should (in theory be offered free of charge.

  19. Dynamic Interaction between Cap & Trade and Electricity Markets

    Science.gov (United States)

    Jeev, Kumar

    Greenhouse Gases (GHG), such as Carbon-Dioxide (CO2), which is released in the atmosphere due to anthropogenic activities like power production, are now accepted as the main culprits for global warming. The Regional Greenhouse Gas Initiative (RGGI), an initiative of the North East and Mid-Atlantic States of the United States (US) for limiting the emission of GHG, has developed a regional cap-and-trade program for CO2 emissions for power plants. Existing cap-and-trade programs in US and Europe for Greenhouse Gases have recently been plagued by over-allocation. Carbon prices recently collapsed in all these markets during the global recession. Since then, there have been significant policy changes, which have resulted in the adoption of aggressive emission cap targets by most major carbon emission markets. This is expected to make carbon emissions availability more restrictive, raising the prices of these credits. These emissions markets are expected to have a major impact on the wholesale electricity markets. Two models to study the interaction of these two markets are presented. These models assess the impact of the emissions market on wholesale electricity prices. The first model characterizes the competition between two types of power plants (coal and gas) in both the electricity and emissions markets as a dynamic game using the Cournot approximation. Under this approximation, we find that in the Nash equilibrium the plants increase their permit allocation to high-demand periods and the marginal value of each credit for a plant is identical in all periods under their optimal equilibrium strategy. The second numerical model allows us to explicitly evaluate the closed loop equilibrium of the dynamic interaction of two competitors in these markets. We find that plants often try to corner the market and push prices all the way to the price cap. Power plants derive most of their profits from these extreme price regimes. In the experiments where trading is allowed

  20. Evaluation of MerCAP for Power Plant Mercury Control

    Energy Technology Data Exchange (ETDEWEB)

    Carl Richardson

    2008-09-30

    This report is submitted to the U.S. Department of Energy National Energy Technology Laboratory (DOE-NETL) as part of Cooperative Agreement DE-FC26-03NT41993, 'Evaluation of EPRI's MerCAP{trademark} Technology for Power Plant Mercury Control'. This project has investigated the mercury removal performance of EPRI's Mercury Capture by Amalgamation Process (MerCAP{trademark}) technology. Test programs were conducted to evaluate gold-based MerCAP{trademark} at Great River Energy's Stanton Station Unit 10 (Site 1), which fired both North Dakota lignite (NDL) and Power River Basin (PRB) coal during the testing period, and at Georgia Power's Plant Yates Unit 1 (Site 2) [Georgia Power is a subsidiary of The Southern Company] which fires a low sulfur Eastern bituminous coal. Additional tests were carried out at Alabama Power's Plant Miller, which fires Powder River Basin Coal, to evaluate a carbon-based MerCAP{trademark} process for removing mercury from flue gas downstream of an electrostatic precipitator [Alabama Power is a subsidiary of The Southern Company]. A full-scale gold-based sorbent array was installed in the clean-air plenum of a single baghouse compartment at GRE's Stanton Station Unit 10, thereby treating 1/10th of the unit's exhaust gas flow. The substrates that were installed were electroplated gold screens oriented parallel to the flue gas flow. The sorbent array was initially installed in late August of 2004, operating continuously until its removal in July 2006, after nearly 23 months. The initial 4 months of operation were conducted while the host unit was burning North Dakota lignite (NDL). In November 2004, the host unit switched fuel to burn Powder River Basin (PRB) subbituminous coal and continued to burn the PRB fuel for the final 19 months of this program. Tests were conducted at Site 1 to evaluate the impacts of flue gas flow rate, sorbent plate spacing, sorbent pre-cleaning and regeneration, and spray

  1. ACTIVE CAPPING TECHNOLOGY - NEW APPROACHES FOR IN SITU REMEDIATION OF CONTAMINATED SEDIMENTS

    Energy Technology Data Exchange (ETDEWEB)

    Knox, A.; Paller, M.; Roberts, J.

    2012-02-13

    This study evaluated pilot-scale active caps composed of apatite, organoclay, biopolymers, and sand for the remediation of metal-contaminated sediments. The active caps were constructed in Steel Creek, at the Savannah River Site near Aiken, South Carolina. Monitoring was conducted for 12 months. Effectiveness of the caps was based on an evaluation of contaminant bioavailability, resistance to erosion, and impacts on benthic organisms. Active caps lowered metal bioavailability in the sediment during the one-year test period. Biopolymers reduced sediment suspension during cap construction, increased the pool of carbon, and lowered the release of metals. This field validation showed that active caps can effectively treat contaminants by changing their speciation, and that caps can be constructed to include more than one type of amendment to achieve multiple goals.

  2. Hfq affects mRNA levels independently of degradation

    Directory of Open Access Journals (Sweden)

    Hajnsdorf Eliane

    2010-02-01

    Full Text Available Abstract Background The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. Results The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the β-galactosidase yield remained about the same as in the parent wild-type strain. Conclusions The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript

  3. The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

    Directory of Open Access Journals (Sweden)

    Stephanie L Timmerman

    2007-09-01

    Full Text Available A single internal ribosomal entry site (IRES in conjunction with IRES transactivating factors (ITAFs is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt, both of which include the common 3' exon (Ex2, 344 nt. Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1. Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

  4. Controlled attenuation parameter (CAP) for the diagnosis of steatosis: a prospective study of 5323 examinations.

    Science.gov (United States)

    de Lédinghen, Victor; Vergniol, Julien; Capdepont, Maylis; Chermak, Faiza; Hiriart, Jean-Baptiste; Cassinotto, Christophe; Merrouche, Wassil; Foucher, Juliette; Brigitte, Le Bail

    2014-05-01

    Controlled attenuation parameter (CAP) evaluated with transient elastography (FibroScan®) is a recent method for non-invasive assessment of steatosis. Its usefulness in clinical practice is unknown. We prospectively investigated the determinants of CAP failure and the relationships between CAP and clinical or biological parameters in a large cohort of consecutive patients. All CAP examinations performed in adult patients with suspected chronic liver disease were included. CAP failure was defined as zero valid shot. The following factors were analyzed for their influence on CAP value and the relationships between CAP and clinico-biological parameters: age, gender, body mass index, waist circumference, hypertension, diabetes, metabolic syndrome, alcohol use, liver stiffness measurement, indication, and different biological parameters. CAP failure occurred in 7.7% of 5323 examinations. By multivariate analysis, factors independently associated with CAP measurement failure were female gender, BMI, and metabolic syndrome. By multivariate analysis, factors significantly associated with elevated CAP were BMI [25-30]kg/m(2), BMI >30kg/m(2), metabolic syndrome, alcohol >14 drink/week and liver stiffness >6kPa. CAP increased with the number of parameters of metabolic syndrome, BMI, waist circumference, the presence of diabetes or hypertension, and the cause of the disease. In the 440 patients with liver biopsy, for the diagnosis of steatosis >10%, steatosis >33%, and steatosis >66%, AUROCs of CAP were 0.79 (95% CI 0.74-0.84, pCAP provides an immediate assessment of steatosis simultaneously with liver stiffness measurement. The strong association of CAP with the metabolic syndrome and alcohol use could be of interest for the follow-up of NAFLD or alcoholic patients. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  5. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Daugaard, Tina Fuglsang; Holm, Ida E

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapa is the most predominant isoform. The Gfapd isoform is expressed in proliferating......RNA localization patterns were dependent on the different 39-exon sequences included in Gfapd and Gfapa mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential...

  6. Induction of rat cytochrome P-450 3 and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl (HCB): comparison to the induction of P-450c in liver and extrahepatic tissues

    Energy Technology Data Exchange (ETDEWEB)

    Yeowell, H.N.; Waxman, D.J.; Linko, P.; Goldstein, J.A.

    1987-05-01

    The present study assesses the effects of HCB on P-450 3 (P-450a/P-450 UT-F) and its translatable mRNA in adult male rats. Although a 50% increase in hepatic polysomal P-450 3 mRNA could be detected within 24 hours following a single dose of HCB, maximal induction was not observed until 5 days. In contrast, P-450c mRNA peaked 2-3 days after a single dose of HCB. The increase in P-450 3 and its associated steroid hormone 7..cap alpha..-hydroxylase activity also occurred later than the increase in P-450c. Neither P-450 3 nor 7..cap alpha..-OHase activity was detected in lung, kidney or prostate microsomes isolated from control or HCB-treated rats, whereas P-450c was induced in all three extrahepatic tissues (prostate > lung = kidney). These findings establish that P-450 3, which is relatively resistant to induction by many xenobiotics, can be induced several-fold by HCB. P-450 3 induction proceeds more slowly and to a significantly lower extent than P-450c induction, and in contrast to P-450c, does not occur in extrahepatic tissues. Finally, increased enzyme synthesis appears to be the major mechanism for the induction of P-450 3 by HCB.

  7. High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions

    Science.gov (United States)

    Peyrane, F.; Selisko, B.; Decroly, E.; Vasseur, J. J.; Benarroch, D.; Canard, B.; Alvarez, K.

    2007-01-01

    Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, 7MeG5′-ppp5′-A2′OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and 7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppACn and 7MeGpppACn (1 ≤ n ≤ 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 ≤ n ≤ 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases. PMID:17259217

  8. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...... response. Thus, our data suggest a role for IL-21 in the early stages of adaptive immune response against virus infections....

  9. Translation with frameshifting of ribosome along mRNA transcript

    CERN Document Server

    Li, Jingwei

    2015-01-01

    Translation is an important process for prokaryotic and eukaryotic cells to produce necessary proteins for cell growth. Numerious experiments have been performed to explore the translational properties. Diverse models have also been developed to determine the biochemical mechanism of translation. However, to simplify the majority of the existing models, the frameshifting of ribosome along the mRNA transcript is neglected, which actually occurs in real cells and has been extensively experimentally studied. The frameshifting of ribosome evidently influences the efficiency and speed of translation, considering that the peptide chains synthesized by shifted ribosomes will not fold into functional proteins and will degrade rapidly. In this study, a theoretical model is presented to describe the translational process based on the model for totally asymmetric simple exclusion process. In this model, the frameshifting of the ribosome along the mRNA transcript and the attachment/detachment of the ribosome to/from the ...

  10. Translatability of rat kidney mRNA after mercury administration.

    Science.gov (United States)

    Samji, S; Kuliszewski, M J; Girgis, G R; Nicholls, D M

    1985-09-01

    Young male rats received an intraperitoneal injection of 0.5 mg HgCl2/kg body weight and 16 h later the kidneys were removed and homogenized to prepare the polysomal fraction from which the poly(A)+ RNA was obtained. The activity of this fraction was assessed by translating the poly(A)+ RNA in a mRNA-dependent rabbit reticulocyte lysate and the activity was markedly elevated relative to preparations from control rat kidneys. The incorporation of labelled leucine and cysteine, but not phenylalanine, into a low molecular weight protein (approximately 10 000 as judged by denaturing polyacrylamide gel electrophoresis) accounted for the increased mRNA activity. The mobility during electrophoresis of the denatured labelled product and carboxymethylated product, as well as their acidic isoelectric points, provided evidence that it is metallothionein mRNA which exhibits increased translatability in preparations derived from mercury-treated rats.

  11. Size-related cytotoxicological aspects of polyvinylpyrrolidone-capped platinum nanoparticles.

    Science.gov (United States)

    Buchtelova, Hana; Dostalova, Simona; Michalek, Petr; Krizkova, Sona; Strmiska, Vladislav; Kopel, Pavel; Hynek, David; Richtera, Lukas; Ridoskova, Andrea; Adam, Pavlina; Kynicky, Jindrich; Brtnicky, Martin; Heger, Zbynek; Adam, Vojtech

    2017-07-01

    The nanotechnological concept is based on size-dependent properties of particles in the 1-100 nm range. Nevertheless, the connection between their size and effect is still not clear. Thus, we focused on reductive colloidal synthesis, characterization and biological testing of Pt nanoparticles (PtNPs) capped with biocompatible polymer polyvinylpyrrolidone (PVP). Synthesized PtNPs were of 3 different primary sizes (approx. ∼10; ∼14 and > 20 nm) and demonstrated exceptional haemocompatibility. In vitro treatment of three different types of malignant cells (prostate - LNCaP, breast - MDA-MB-231 and neuroblastoma - GI-ME-N) revealed that even marginal differences in PtNPs diameter resulted in changes in their cytotoxicity. The highest cytotoxicity was observed using the smallest PtNPs-10, where 24IC50 was lower (3.1-6.2 μg/mL) than for cisplatin (8.1-19.8 μg/mL). In contrast to MDA-MB-231 and LNCaP cells, in GI-ME-N cells PtNPs caused noticeable changes in their cellular structure without influencing their viability. Post-exposure analyses revealed that PtNPs-29 and PtNPs-40 were capable of forming considerably higher amount of reactive oxygen species with consequent stimulation of expression of metallothionein (MT1/2 and MT3), at both mRNA and protein level. Overall, our pilot study demonstrates that in the nanoscaled world even the smallest differences can have crucial biological effect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Stabilization of HIF-2α through redox regulation of mTORC2 activation and initiation of mRNA translation.

    Science.gov (United States)

    Nayak, B K; Feliers, D; Sudarshan, S; Friedrichs, W E; Day, R T; New, D D; Fitzgerald, J P; Eid, A; Denapoli, T; Parekh, D J; Gorin, Y; Block, K

    2013-06-27

    Hypoxia inducible factor-2α (HIF-2α) has a critical role in renal tumorigenesis. HIF-2α is stabilized in von Hippel-Lindau (VHL)-deficient renal cell carcinoma through mechanisms that require ongoing mRNA translation. Mammalian target of rapamycin (mTOR) functions in two distinct complexes: Raptor-associated mTORC1 and Rictor-associated mTORC2. Rictor-associated mTORC2 complex has been linked to maintaining HIF-2α protein in the absence of VHL; however, the mechanisms remain to be elucidated. Although Raptor-associated mTORC1 is a known key upstream regulator of mRNA translation, initiation and elongation, the role of mTORC2 in regulating mRNA translation is not clear. Complex assembly of the mRNA cap protein, eukaryotic translation initiation factor 4 (eIF4)E, with activators (eIF4 gamma (eIF4G)) and inhibitors (eIF4E-binding protein 1 (4E-BP1)) are rate-limiting determinants of mRNA translation. Our laboratory has previously demonstrated that reactive oxygen species, mediated by p22(phox)-based Nox oxidases, are enhanced in VHL-deficient cells and have a role in the activation of Akt on S473, a site phosphorylated by the mTORC2 complex. In this study, we examined the role of Rictor-dependent regulation of HIF-2α through eIF4E-dependent mRNA translation and examined the effects of p22(phox)-based Nox oxidases on TORC2 regulation. We demonstrate for the first time that mTORC2 complex stability and activation is redox sensitive, and further defined a novel role for p22(phox)-based Nox oxidases in eIF4E-dependent mRNA translation through mTORC2. Furthermore, we provide the first evidence that silencing of p22(phox) reduces HIF-2α-dependent gene targeting in vitro and tumor formation in vivo. The clinical relevance of these studies is demonstrated.

  13. Polysome Fractionation to Analyze mRNA Distribution Profiles.

    Science.gov (United States)

    Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

    2017-02-05

    Eukaryotic cells adapt to changes in external or internal signals by precisely modulating the expression of specific gene products. The expression of protein-coding genes is controlled at the transcriptional and post-transcriptional levels. Among the latter steps, the regulation of translation is particularly important in cellular processes that require rapid changes in protein expression patterns. The translational efficiency of mRNAs is altered by RNA-binding proteins (RBPs) and noncoding (nc)RNAs such as microRNAs (Panda et al., 2014a and 2014b; Abdelmohsen et al., 2014). The impact of factors that regulate selective mRNA translation is a critical question in RNA biology. Polyribosome (polysome) fractionation analysis is a powerful method to assess the association of ribosomes with a given mRNA. It provides valuable information about the translational status of that mRNA, depending on the number of ribosomes with which they are associated, and identifies mRNAs that are not translated (Panda et al., 2016). mRNAs associated with many ribosomes form large polysomes that are predicted to be actively translated, while mRNAs associated with few or no ribosomes are expected to be translated poorly if at all. In sum, polysome fractionation analysis allows the direct determination of translation efficiencies at the level of the whole transcriptome as well as individual mRNAs.

  14. Cup regulates oskar mRNA stability during oogenesis.

    Science.gov (United States)

    Broyer, Risa M; Monfort, Elena; Wilhelm, James E

    2017-01-01

    The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

  15. 77 FR 22061 - FTA Section 5307 Urbanized Area Formula Program: Allocation of Funding Caps for Treating Fuel and...

    Science.gov (United States)

    2012-04-12

    ... Federal Transit Administration FTA Section 5307 Urbanized Area Formula Program: Allocation of Funding Caps.... Based on the $100,000,000 cap on use of this provision, FTA has allocated funding caps to program... allocate the available resources, FTA has determined funding caps for all requesting UZAs and States (see...

  16. A Nucleolar Protein, Ribosomal RNA Processing 1 Homolog B (RRP1B), Enhances the Recruitment of Cellular mRNA in Influenza Virus Transcription.

    Science.gov (United States)

    Su, Wen-Chi; Hsu, Shih-Feng; Lee, Yi-Yuan; Jeng, King-Song; Lai, Michael M C

    2015-11-01

    Influenza A virus (IAV) undergoes RNA transcription by a unique capped-mRNA-dependent transcription, which is carried out by the viral RNA-dependent RNA polymerase (RdRp), consisting of the viral PA, PB1, and PB2 proteins. However, how the viral RdRp utilizes cellular factors for virus transcription is not clear. Previously, we conducted a genome-wide pooled short hairpin RNA (shRNA) screen to identify host factors important for influenza A virus replication. Ribosomal RNA processing 1 homolog B (RRP1B) was identified as one of the candidates. RRP1B is a nucleolar protein involved in ribosomal biogenesis. Upon IAV infection, part of RRP1B was translocated from the nucleolus to the nucleoplasm, where viral RNA synthesis likely takes place. The depletion of RRP1B significantly reduced IAV mRNA transcription in a minireplicon assay and in virus-infected cells. Furthermore, we showed that RRP1B interacted with PB1 and PB2 of the RdRp and formed a coimmunoprecipitable complex with RdRp. The depletion of RRP1B reduced the amount of capped mRNA in the RdRp complex. Taken together, these findings indicate that RRP1B is a host factor essential for IAV transcription and provide a target for new antivirals. Influenza virus is an important human pathogen that causes significant morbidity and mortality and threatens the human population with epidemics and pandemics every year. Due to the high mutation rate of the virus, antiviral drugs targeting viral proteins might ultimately lose their effectiveness. An alternative strategy that explores the genetic stability of host factors indispensable for influenza virus replication would thus be desirable. Here, we characterized the rRNA processing 1 homolog B (RRP1B) protein as an important cellular factor for influenza A virus transcription. We showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity, which is responsible for virus transcription and replication. Furthermore, we reported that RRP1B is

  17. Metal-silicon reaction rates - The effects of capping

    Science.gov (United States)

    Weizer, Victor G.; Fatemi, Navid S.

    1989-01-01

    Evidence is presented showing that the presence of the commonly used anti-reflection coating material Ta2O5 on the free surface of contact metallization can either suppress or enhance, depending on the system, the interaction that takes place at elevated temperatures between the metallization and the underlying Si. The cap layer is shown to suppress both the generation and annihilation of vacancies at the free surface of the metal which are necessary to support metal-Si interactons. Evidence is also presented indicating that the mechanical condition of the free metal surface has a significant effect on the metal-silicon reaction rate.

  18. Enhanced thermal stability of Ag nanorods through capping

    Energy Technology Data Exchange (ETDEWEB)

    Bachenheimer, Lou; Elliott, Paul [Department of Mechanical Engineering, University of Connecticut, Storrs, Connecticut 06269 (United States); Stagon, Stephen [Department of Mechanical Engineering, University of North Florida, Jacksonville, Florida 32224 (United States); Huang, Hanchen, E-mail: h.huang@neu.edu [Department of Mechanical and Industrial Engineering, Northeastern University, Boston, Massachusetts 02115 (United States)

    2014-11-24

    Ag nanorods may serve as sensors in the detection of trace amounts of chemical agents, even single molecules, through surface enhanced Raman spectroscopy (SERS). However, thermal coarsening of Ag nanorods near room temperature limits their applications. This letter proposes the use of a thin oxide capping layer to enhance the thermal stability of Ag nanorods beyond 100 °C. Using electron microscopy characterization and SERS tests, the authors show that the proposed method is effective in stabilizing both morphology and sensitivity of Ag nanorods. The results of this work extend the applicability of Ag nanorods as chemical sensors to higher temperatures.

  19. Autoinflammatory diseases in dermatology: CAPS, TRAPS, HIDS, FMF, Blau, CANDLE.

    Science.gov (United States)

    Tripathi, Shivani V; Leslie, Kieron S

    2013-07-01

    Autoinflammatory diseases, including CAPS, TRAPS, HIDS, FMF, Blau, and CANDLE, have unique dermatologic presentations that can be a clue to diagnosis. Although these conditions are rare, the morbidity and mortality can be severe, and well-informed physicians can place these conditions in their differential diagnosis when familiar with the dermatologic manifestations. This review article presents a brief overview of each condition, clues to diagnosis that focus of dermatologic manifestations and clinical images, basic laboratory tests and follow-up, a brief review of treatments, and concludes with an overview for these autoinflammatory conditions and their differential diagnoses. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Anomalous Structure of Palladium-Capped Magnesium Thin Films

    Directory of Open Access Journals (Sweden)

    Kazuki Yoshimura

    2012-07-01

    Full Text Available Pd capped pure Mg thin film (50 nm thick was prepared by magnetron sputtering and its hydrogenation at room temperature has been investigated. After exposure to 4% hydrogen gas diluted by argon, the Pd/Mg thin films show drastic optical changes from the metallic state to the transparent state within five seconds by hydrogenation. Transmission electron microscope observation reveals that this sample has an anomalous structure; Mg grain is surrounded by Pd. This structure may be the reason why Pd/Mg films can be hydrogenated so quickly at room temperature.

  1. License - RGP caps | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us RGP caps License License to Use This Database Last updated : 2015/05/12 You may use this database...license terms regarding the use of this database and the requirements you must follow in using this database...Share Alike 2.1 Japan . If you use data from this database, please be sure attribute this database as follow...ommons Attribution-Share Alike 2.1 Japan is found here . With regard to this database, you are licensed to: ...freely access part or whole of this database, and acquire data; freely redistribute part or whole of the data from this database

  2. Effectiveness evaluation of three RCRA caps at the Y-12 Plant, Oak Ridge, Tennessee

    Energy Technology Data Exchange (ETDEWEB)

    Shevenell, L.A. [Nevada Bureau of Mines and Geology, Reno, NV (United States); Goldstrand, P.M. [Univ. of Tennessee, Knoxville, TN (United States). Dept. of Geological Sciences

    1994-01-01

    Because installation of Resource Conservation and Recovery Act (RCRA)- engineered caps is costly, it is prudent to evaluate the effectiveness of this procedure for hydrologically isolating contaminants. The objective for installation of five-part engineered caps at the Y-12 Plant was to (1) satisfy the regulatory compliance issues, (2) minimize the risk of direct contact with the wastes, and (3) reduce rainfall infiltration. Although the original objectives of installing the caps were not to alter groundwater flow, a potential effect of reducing infiltration is to minimize leaching, thus retarding groundwater contaminant migration from the site. Hence, cap effectiveness with respect to reduced groundwater contaminant migration is evaluated using groundwater data in this report. Based on the available data at the Y-12 capped areas, evaluation of cap effectiveness includes studying water level and chemical variability in nearby monitoring wells. Three caps installed during 1989 are selected for evaluation in this report. These caps are located in three significantly different hydrogeologic settings: overlying a karst aquifer (Chestnut Ridge Security Pits [CRSP]), overlying shales located on a hill slope (Oil Landfarm Waste Management Area [OLWMA]), and overlying shales in a valley floor which is a site of convergent groundwater flow (New Hope Pond [NHP]). Presumably, the caps have been effective in minimizing risk of direct contact with the wastes and halting direct rainfall infiltration into the sites over the extent of the capped areas, but no evidence is presented in this report to directly demonstrate this. The caps installed over the three sites appear to have had a minimal effect on groundwater contaminant migration from the respective sites. Following cap construction, no changes in the configuration of the water table were observed. Migration of contaminant plumes occurred at all three sites, apparently without regard to the timing of cap installation.

  3. Evaluation of in situ capping with clean soils to control phosphate release from sediments

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Di [State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008 (China); Ding, Shiming, E-mail: smding@niglas.ac.cn [State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008 (China); Sun, Qin [College of Environmental Science and Engineering, Hohai University, Nanjing (China); Zhong, Jicheng [State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008 (China); Wu, Wei; Jia, Fei [College of Environmental Science and Engineering, Hohai University, Nanjing (China)

    2012-11-01

    Evaluation of in situ capping with clean soils to control phosphate release from the sediments of a eutrophic bay in Lake Taihu was performed after 18 months of capping. The concentrations of dissolved reactive phosphate (DRP) in pore waters and DRP resupply from native sediments and capped sediments were determined using high-resolution dialysis (HR-Peeper) and a Zr-oxide diffusive gradients in thin films (Zr-oxide DGT) technique. The adsorption isotherm of these sediments was further investigated using a modified Langmuir model. The results showed low concentrations of DRP in pore waters with a low resupply from the sediments for sustaining pore water DRP concentration after capping. The calculated flux to the overlying water following the capping treatment was approximately half of that for the native sediments, implying that the capping reduced the release of phosphate from the sediments. The low resupply of the sediments after capping was further demonstrated by larger partitioning coefficient (K{sub p}) values and greater adsorption capacity (Q{sub max}) values, while zero equilibrium concentrations (EPC{sub 0}s) were similar to those in native sediments. The larger K{sub p} and Q{sub max} were attributed to higher active Fe and Al introduced by the capping, indicating that the binding of phosphate onto the active Fe and Al played a critical role in reducing the internal loading of phosphorous. Highlights: Black-Right-Pointing-Pointer Evaluation of capping with soils was performed through high-resolution sampling. Black-Right-Pointing-Pointer Capping decreased the concentrations of DRP in pore waters and its release to waters. Black-Right-Pointing-Pointer Capping decreased the resupply of pore water DRP from the sediments. Black-Right-Pointing-Pointer Capped sediments had stronger abilities to adsorb and retain P. Black-Right-Pointing-Pointer Active Fe and Al introduced by capping played a critical role.

  4. Analytical and experimental studies of wakes behind circularly capped bubbles

    Science.gov (United States)

    Bessler, W. F.

    The wakes behind circularly capped bubbles are examined by means of an experimental and analytical study. A single two-inch diameter bubble is injected into a six by three foot fluid column, one half inch thick, producing an essentially two-dimensional flow. Aspirin powder placed in the fluid column just prior to bubble release highlights the structure of the flow field before dissolving. High speed film and sequenced photographs are taken to document the observed results. Pressure-time measurements are made with sensitive capacitive transducers mounted in the rear column wall and are synchronized with photographs using a high speed clock. Experimental fluids, prepared by mixing water and glycerine in varying proportions, are used to study the effects of viscosity on bubble shape and wake structure. Testing is performed over a range of Reynolds numbers from 14 to 29,655 which includes the transition from circularly capped to ellipsoidal bubble shape. Experimental data is reduced and summarized in convenient dimensionless form to permit comparison with analytical predictions.

  5. Photoaffinity labeling of the messenger RNA cap-binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Rhoads, R.; Patzelt, E.; Blaas, D.; Kuechler, E.

    1986-05-01

    A photoaffinity derivative of a cap analogue, ..gamma..(/sup 32/P)-(4-(benzoylphenyl)methylamido)-7-methylguanosine 5'-triphosphate, was reacted with the 28-kDa messenger RNA cap-binding protein (CBP), purified by affinity chromatography from rabbit erythrocyte lysate. The predominant labeling was of the 28-kDa species. No labeling occurred without photoillumination or in the presence of 250 ..mu..M m/sup 7/GTP. In addition CBP, two polypeptides of 19 and 17kDa, present in the affinity-purified preparation, were specifically labeled. Another sample tested was the eIF-3 region of a sucrose gradient fractionation, in 100 mM KCl, of rabbit reticulocyte high salt ribosomal wash. The only polypeptide labeled was the 28-kDa CBP. Labeling was more intense than with an equivalent amount of affinity-purified CBP, suggesting the presence of stabilizing or stimulatory factors in the eIF-3 preparation.

  6. Polar cap electron densities from DE 1 plasma wave observations

    Science.gov (United States)

    Persoon, A. M.; Gurnett, D. A.; Shawhan, S. D.

    1983-01-01

    Electric-field-spectum measurements from the plasma-wave instrument on the Dynamics Explorer 1 spacecraft are used to study the local electron density at high altitudes in the northern polar-cap region. The electron density is determined from the upper cutoff of whistler-mode radiation at the electron plasma frequency. Median density values over the polar cap at L greater than 10 are found to vary from 35.2 + or - 8.5 cu cm at 2.1 earth radii to 0.99 + or - 0.51 cu cm at 4.66 earth radii. The steady-state radial-outflow model is examined for consistency with the observed density profile. A power-law fit to the radial variation of the electron density yields an exponent of - 3.85 + or - 0.32, which for the radial-outflow model implies a flow velocity increasing nearly linearly with incresing radial distance. Comparison of the observed electron densities with theoretical polar-wind densities yields consistent results up to 2.8 earth radii. A comparison of the observed electron densities with low-altitude density profiles from the Alouette II and ISIS 1 spacecraft illustrates transitions in the slope of the profile at 1.16 earth radii and between 1.55 and 2.0 earth radii. The changes in the density profile suggest that changes occur in the basic radial-transport processes at these altitudes.

  7. Gravity Waves Near 300 km Over the Polar Caps

    Science.gov (United States)

    Johnson, F. S.; Hanson, W. B.; Hodges, R. R.; Coley, W. R.; Carignan, G. R.; Spencer, N. W.

    1995-01-01

    Distinctive wave forms in the distributions of vertical velocity and temperature of both neutral particles and ions are frequently observed from Dynamics Explorer 2 at altitudes above 250 km over the polar caps. These are interpreted as being due to internal gravity waves propagating in the neutral atmosphere. The disturbances characterized by vertical velocity perturbations of the order of 100 m/s and horizontal wave lengths along the satellite path of about 500 km. They often extend across the entire polar cap. The associated temperature perturbations indicate that the horizontal phase progression is from the nightside to the dayside. Vertical displacements are inferred to be of the order of 10 km and the periods to be of the order of 10(exp 3) s. The waves must propagate in the neutral atmosphere, but they usually are most clearly recognizable in the observations of ion vertical velocity and ion temperature. By combining the neutral pressure calculated from the observed neutral concentration and temperature with the vertical component of the neutral velocity, an upward energy flux of the order of 0.04 erg/sq cm-s at 250 km has been calculated, which is about equal to the maximum total solar ultraviolet heat input above that altitude. Upward energy fluxes calculated from observations on orbital passes at altitudes from 250 to 560 km indicate relatively little attenuation with altitude.

  8. The use of bottle caps as submerged aerated filter medium.

    Science.gov (United States)

    Damasceno de Oliveira, Laurence; Motlagh, Amir Mohaghegh; Goel, Ramesh; de Souza Missagia, Beatriz; Alves de Abreu Filho, Benício; Lautenschlager, Sandro Rogério

    2014-01-01

    In this study, a submerged aerated filter (SAF) using bottle caps as a support medium was evaluated. The system was fed with effluent from an upflow anaerobic sludge blanket system at ETE 2-South wastewater treatment plant, under different volumetric organic load rates (VOLRs). The population of a particular nitrifying microbial community was assessed by fluorescent in situ hybridization with specific oligonucleotide probes. The system showed an average removal of chemical oxygen demand (COD) equal to 76% for VOLRs between 2.6 and 13.6 kg COD m(-3)_media.day(-1). The process of nitrification in conjunction with the removal of organic matter was observed from applying VOLRs lower than 5.5 kg COD m(-3)_media.day(-1) resulting in 78% conversion of NH4(+)-N. As the applied organic load was reduced, an increase in the nitrifying bacteria population was observed compared with total 4'-6-diamidino-2-phenylindole (DAPI) stained cells. Generally, SAF using bottle caps as a biological aerated filter medium treating wastewater from an anaerobic system showed promising removal of chemical oxygen demand (COD) and conversion of NH4(+)-N.

  9. Self-assembly of colloids with magnetic caps

    Energy Technology Data Exchange (ETDEWEB)

    Novak, E.V., E-mail: ekaterina.novak@urfu.ru [Ural Federal University, Lenin Av. 51, Ekaterinburg (Russian Federation); Kantorovich, S.S. [Ural Federal University, Lenin Av. 51, Ekaterinburg (Russian Federation); University of Vienna, Sensengasse 8, Vienna (Austria)

    2017-06-01

    In our earlier work (Steinbach et al., 2016 ) we investigated a homogeneous system of magnetically capped colloidal particles that self-assembled via two structural patterns of different symmetry. The particles could form a compact, equilateral triangle with a three-fold rotational symmetry and zero dipole moment and a staggered chain with mirror symmetry with a net magnetisation perpendicular to the chain. The system exhibited a bistability already in clusters of three particles. Based on observations of a real magnetic particles system, analytical calculations and molecular dynamics simulations, it has been shown that the bistability is a result of an anisotropic magnetisation distribution with rotational symmetry inside the particles. The present study is a logical extension of the above research and forms a preparatory stage for the study of a self-assembly of such magnetic particles under the influence of an external magnetic field. Since the magnetic field is only an additive contribution to the total ground state energy, we can study the interparticle interaction energies of candidate ground state structures based on the field-free terms. - Highlights: • Analytical calculations of the energies of ground state candidates for colloids with magnetic caps. • Computer simulations confirmed the theoretical model. • The structural transition between ground states was found.

  10. The backward end-cap for the PANDA electromagnetic calorimeter

    Energy Technology Data Exchange (ETDEWEB)

    Capozza, Luigi; Maas, Frank; Rodriguez Pineiro, David; Valente, Roserio [Helmholtz-Institut Mainz - Johannes Gutenberg-Universitaet Mainz (Germany); GSI Helmholtzzentrum fuer Schwerionenforschung GmbH (Germany); Lin, Dexu; Noll, Oliver [Helmholtz-Institut Mainz - Johannes Gutenberg-Universitaet Mainz (Germany)

    2014-07-01

    The PANDA experiment at the new FAIR facility will cover a broad experimental programme in hadron structure and spectroscopy. As a multipurpose detector, the PANDA spectrometer needs to ensure almost 4π coverage of the scattering solid angle, full and accurate multiple-particle event reconstruction and very good particle identification capabilities. % The electromagnetic calorimeter (EMC) will be a key item for many of these aspects. Particle energies ranging from some MeVs to several GeVs have to be measured with a relative resolution of 1% + 2%/√(E/ GeV). % It will be a homogeneous calorimeter made of PbWO{sub 4} crystals and will be operated at -25 {sup circle} C, in order to improve the scintillation light yield. With the exception of the very forward section, the light will be detected by large area avalanche photodiodes. % The whole calorimeter has been designed in three sections: a forward end-cap, a central barrel and a backward end-cap (BWEC). % In this contribution, a status report on the development of the BWEC is given.

  11. The antimicrobial propeptide hCAP-18 plasma levels in neutropenia of various aetiologies

    DEFF Research Database (Denmark)

    Ye, Ying; Carlsson, Göran; Karlsson-Sjöberg, Jenny M T

    2015-01-01

    The underlying cause of neutropenia may be difficult to determine due to similar clinical presentation in many neutropenic conditions. The neutrophil protein hCAP-18 (pro-LL-37) is a major component of neutrophil secondary granules and in this prospective study we assessed the use of hCAP-18 levels...... in blood plasma for differential diagnosis of neutropenic patients (n = 133) of various aetiologies. Plasma levels of hCAP-18 were determined using immunoblot and ELISA. Patients with severe congenital neutropenia (n = 23) presented with the lowest levels of plasma hCAP-18 and differential diagnostic...... diagnostic value in differential diagnosis of chronic neutropenia. Neutropenic patients with Shwachman-Diamond syndrome, Barth syndrome, Cohen syndrome, acute myeloid leukaemia and specific granule deficiency presented with reduced plasma hCAP-18 levels as well. The blood plasma level of hCAP-18 was thus low...

  12. Role of Surface Capping Molecule Polarity on the Optical Properties of Solution Synthesized Germanium Nanocrystals.

    Science.gov (United States)

    McVey, B F P; O'Mara, P B; McGrath, A J; Faramus, A; Yasarapudi, V B; Gonçales, V R; Tan, V T G; Schmidt, T W; Gooding, J J; Tilley, R D

    2017-09-05

    The role surface capping molecules play in dictating the optical properties of semiconductor nanocrystals (NCs) is becoming increasingly evident. In this paper the role of surface capping molecule polarity on the optical properties of germanium NCs (Ge NCs) is explored. Capping molecules are split into two groups: nonpolar and polar. The NCs are fully characterized structurally and optically to establish the link between observed optical properties and surface capping molecules. Ge NC optical properties altered by surface capping molecule polarity include emission maximum, emission lifetime, quantum yield, and Stokes shift. For Ge NCs, this work also allows rational tuning of their optical properties through changes to surface capping molecule polarity, leading to improvements in emerging Ge based bioimaging and optoelectronic devices.

  13. Effects of intima stiffness and plaque morphology on peak cap stress

    Directory of Open Access Journals (Sweden)

    Virmani Renu

    2011-04-01

    Full Text Available Abstract Background Rupture of the cap of a vulnerable plaque present in a coronary vessel may cause myocardial infarction and death. Cap rupture occurs when the peak cap stress exceeds the cap strength. The mechanical stress within a cap depends on the plaque morphology and the material characteristics of the plaque components. A parametric study was conducted to assess the effect of intima stiffness and plaque morphology on peak cap stress. Methods Models with idealized geometries based on histology images of human coronary arteries were generated by varying geometric plaque features. The constructed multi-layer models contained adventitia, media, intima, and necrotic core sections. For adventitia and media layers, anisotropic hyperelastic material models were used. For necrotic core and intima sections, isotropic hyperelastic material models were employed. Three different intima stiffness values were used to cover the wide range reported in literature. According to the intima stiffness, the models were classified as stiff, intermediate and soft intima models. Finite element method was used to compute peak cap stress. Results The intima stiffness was an essential determinant of cap stresses. The computed peak cap stresses for the soft intima models were much lower than for stiff and intermediate intima models. Intima stiffness also affected the influence of morphological parameters on cap stresses. For the stiff and intermediate intima models, the cap thickness and necrotic core thickness were the most important determinants of cap stresses. The peak cap stress increased three-fold when the cap thickness was reduced from 0.25 mm to 0.05 mm for both stiff and intermediate intima models. Doubling the thickness of the necrotic core elevated the peak cap stress by 60% for the stiff intima models and by 90% for the intermediate intima models. Two-fold increase in the intima thickness behind the necrotic core reduced the peak cap stress by

  14. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-09-10

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

  15. Organic–Inorganic Nanostructure Architecture via Directly Capping Fullerenes onto Quantum Dots

    Directory of Open Access Journals (Sweden)

    Kim Jonggi

    2011-01-01

    Full Text Available Abstract A new form of fullerene-capped CdSe nanoparticles (PCBA-capped CdSe NPs, using carboxylate ligands with [60]fullerene capping groups that provides an effective synthetic methodology to attach fullerenes noncovalently to CdSe, is presented for usage in nanotechnology and photoelectric fields. Interestingly, either the internal charge transfer or the energy transfer in the hybrid material contributes to photoluminescence (PL quenching of the CdSe moieties.

  16. Organic-Inorganic Nanostructure Architecture via Directly Capping Fullerenes onto Quantum Dots.

    Science.gov (United States)

    Lee, Jae Kwan; Kim, Jonggi; Yang, Changduk

    2011-12-01

    A new form of fullerene-capped CdSe nanoparticles (PCBA-capped CdSe NPs), using carboxylate ligands with [60]fullerene capping groups that provides an effective synthetic methodology to attach fullerenes noncovalently to CdSe, is presented for usage in nanotechnology and photoelectric fields. Interestingly, either the internal charge transfer or the energy transfer in the hybrid material contributes to photoluminescence (PL) quenching of the CdSe moieties.

  17. Live Imaging of mRNA Synthesis in Drosophila.

    Science.gov (United States)

    Garcia, Hernan G; Gregor, Thomas

    2018-01-01

    mRNA synthesis is one of the earliest readouts of the activity of a transcribed gene, which is of particular interest in the context of metazoan cell fate specification. These processes are intrinsically dynamic and stochastic, which makes in vivo single-cell measurements inevitable. Here, we present the application of a technology that has been widely used in single celled organisms to measure transcriptional activity in developing embryos of the fruit fly Drosophila melanogaster. The method allows for quantification of instantaneous polymerase occupancy of active gene loci and thereby enables the development and testing of models of gene regulation in development.

  18. Update History of This Database - RGP caps | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available search(/contents-en/) != -1 || url.search(/index-e.html/) != -1 ) { document.getElementById(lang).innerHTML=... '[ Japanese | English ]'; } else if ( url.search(//(dbmeta|datameta)-(list|search)-e.html.../) != -1 ) { url = url.replace(-e.html,.html); document.getElementById(lang).innerHTML=[ Japanese |...chive site is opened. 2013/04/01 The URL of RGP caps is changed to ... http://rgp.dna.affrc.go.jp/E/publicdata/caps/index.html... . 2002/02/10 RGP caps (http://rgp.dna.affrc.go.jp/publicdata/caps/index.html) is opened. Abo

  19. Retention Strength of Conical Welding Caps for Fixed Implant-Supported Prostheses.

    Science.gov (United States)

    Nardi, Diego; Degidi, Marco; Sighinolfi, Gianluca; Tebbel, Florian; Marchetti, Claudio

    This study evaluated the retention strength of welding caps for Ankylos standard abutments using a pull-out test. Each sample consisted of an implant abutment and its welding cap. The tests were performed with a Zwick Roell testing machine with a 1-kN load cell. The retention strength of the welding caps increased with higher abutment diameters and higher head heights and was comparable or superior to the values reported in the literature for the temporary cements used in implant dentistry. Welding caps provide a reliable connection between an abutment and a fixed prosthesis without the use of cement.

  20. [A comparative study of MAST and CAP RAST with 90 patients with bronchial asthma].

    Science.gov (United States)

    Mita, S; Oda, N; Adachi, M

    1995-12-01

    We measured serum IgE antibodies by the MAST and the CAP RAST in 90 patients refered to our asthma clinic and compared their results. Furthermore the patients with CAP positive/MAST negative were investigated by bronchial provocation test with allergen, skin test and CAP RAST inhibition test. Significant correlations were obtained between the results of the MAST and those of the CAP RAST for house dust 2 (r = 0.617), for Dermatophagoides farinae (r = 0.776) and for Japanese cedar (r = 0.609), but not for all 3 mold allergens. CAP positive/MAST negative results were found in 1.4-27.8% and MAST positive/CAP negative results were found in 0-2.7%. The presences of specific IgE antibodies were confirmed by a positive bronchial provocation test with allergen, skin test and CAP RAST inhibition test in CAP positive/MAST negative results. Those results indicate that the CAP RAST is more sensitive than the MAST.