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Sample records for high levels nef

  1. Effector kinase coupling enables high-throughput screens for direct HIV-1 Nef antagonists with antiretroviral activity.

    PubMed

    Emert-Sedlak, Lori A; Narute, Purushottam; Shu, Sherry T; Poe, Jerrod A; Shi, Haibin; Yanamala, Naveena; Alvarado, John Jeff; Lazo, John S; Yeh, Joanne I; Johnston, Paul A; Smithgall, Thomas E

    2013-01-24

    HIV-1 Nef, a critical AIDS progression factor, represents an important target protein for antiretroviral drug discovery. Because Nef lacks intrinsic enzymatic activity, we developed an assay that couples Nef to the activation of Hck, a Src family member and Nef effector protein. Using this assay, we screened a large, diverse chemical library and identified small molecules that block Nef-dependent Hck activity with low micromolar potency. Of these, a diphenylpyrazolo compound demonstrated submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound binds directly to Nef via a pocket formed by the Nef dimerization interface and disrupts Nef dimerization in cells. Coupling of nonenzymatic viral accessory factors to host cell effector proteins amenable to high-throughput screening may represent a general strategy for the discovery of new antimicrobial agents. PMID:23352142

  2. Nef Stimulates Human Immunodeficiency Virus Type 1 Replication in Primary T Cells by Enhancing Virion-Associated gp120 Levels: Coreceptor-Dependent Requirement for Nef in Viral Replication†

    PubMed Central

    Lundquist, Christopher A.; Zhou, Jing; Aiken, Christopher

    2004-01-01

    The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) replication through an unknown mechanism. We and others have previously reported that efficient HIV-1 replication in activated primary CD4+ T cells depends on the ability of Nef to downregulate CD4 from the cell surface. Here we demonstrate that Nef greatly enhances the infectivity of HIV-1 particles produced in primary T cells. Nef-defective HIV-1 particles contained significantly reduced quantities of gp120 on their surface; however, Nef did not affect the levels of virion-associated gp41, indicating that Nef indirectly stabilizes the association of gp120 with gp41. Surprisingly, Nef was not required for efficient replication of viruses that use CCR5 for entry, nor did Nef influence the infectivity or gp120 content of these virions. Nef also inhibited the incorporation of CD4 into HIV-1 particles released from primary T cells. We propose that Nef, by downregulating cell surface CD4, enhances HIV-1 replication by inhibiting CD4-induced dissociation of gp120 from gp41. The preferential requirement for Nef in the replication of X4-tropic HIV-1 suggests that the ability of Nef to downregulate CD4 may be most important at later stages of disease when X4-tropic viruses emerge. PMID:15163722

  3. A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

    PubMed Central

    Lee, C H; Leung, B; Lemmon, M A; Zheng, J; Cowburn, D; Kuriyan, J; Saksela, K

    1995-01-01

    We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef. Images PMID:7588629

  4. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis.

    PubMed

    Cui, Huanhuan L; Ditiatkovski, Michael; Kesani, Rajitha; Bobryshev, Yuri V; Liu, Yingying; Geyer, Matthias; Mukhamedova, Nigora; Bukrinsky, Michael; Sviridov, Dmitri

    2014-07-01

    Patients with HIV are at an increased risk of cardiovascular disease. In this study we investigated the effect of Nef, a secreted HIV protein responsible for the impairment of cholesterol efflux, on the development of atherosclerosis in two animal models. ApoE(-/-) mice fed a high-fat diet and C57BL/6 mice fed a high-fat, high-cholesterol diet were injected with recombinant Nef (40 ng/injection) or vehicle, and the effects of Nef on development of atherosclerosis, inflammation, and dyslipidemia were assessed. In apoE(-/-) mice, Nef significantly increased the size of atherosclerotic lesions and caused vessel remodeling. Nef caused elevation of total cholesterol and triglyceride levels in the plasma while reducing high-density lipoprotein cholesterol levels. These changes were accompanied by a reduction of ABCA1 abundance in the liver, but not in the vessels. In C57BL/6 mice, Nef caused a significant number of lipid-laden macrophages presented in adventitia of the vessels; these cells were absent from the vessels of control mice. Nef caused sharp elevations of plasma triglyceride levels and body weight. Taken together, our findings suggest that Nef causes dyslipidemia and accumulation of cholesterol in macrophages within the vessel wall, supporting the role of Nef in pathogenesis of atherosclerosis in HIV-infected patients.-Cui, H. L., Ditiatkovski, M., Kesani, R., Bobryshev, Y. V., Liu, Y., Geyer, M., Mukhamedova, N., Bukrinsky, M., Sviridov, D. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis. PMID:24642731

  5. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis

    PubMed Central

    Cui, Huanhuan L.; Ditiatkovski, Michael; Kesani, Rajitha; Bobryshev, Yuri V.; Liu, Yingying; Geyer, Matthias; Mukhamedova, Nigora; Bukrinsky, Michael; Sviridov, Dmitri

    2014-01-01

    Patients with HIV are at an increased risk of cardiovascular disease. In this study we investigated the effect of Nef, a secreted HIV protein responsible for the impairment of cholesterol efflux, on the development of atherosclerosis in two animal models. ApoE−/− mice fed a high-fat diet and C57BL/6 mice fed a high-fat, high-cholesterol diet were injected with recombinant Nef (40 ng/injection) or vehicle, and the effects of Nef on development of atherosclerosis, inflammation, and dyslipidemia were assessed. In apoE−/− mice, Nef significantly increased the size of atherosclerotic lesions and caused vessel remodeling. Nef caused elevation of total cholesterol and triglyceride levels in the plasma while reducing high-density lipoprotein cholesterol levels. These changes were accompanied by a reduction of ABCA1 abundance in the liver, but not in the vessels. In C57BL/6 mice, Nef caused a significant number of lipid-laden macrophages presented in adventitia of the vessels; these cells were absent from the vessels of control mice. Nef caused sharp elevations of plasma triglyceride levels and body weight. Taken together, our findings suggest that Nef causes dyslipidemia and accumulation of cholesterol in macrophages within the vessel wall, supporting the role of Nef in pathogenesis of atherosclerosis in HIV-infected patients.—Cui, H. L., Ditiatkovski, M., Kesani, R., Bobryshev, Y. V., Liu, Y., Geyer, M., Mukhamedova, N., Bukrinsky, M., Sviridov, D. HIV protein Nef causes dyslipidemia and formation of foam cells in mouse models of atherosclerosis. PMID:24642731

  6. Requirements for the production of high-titre C3 nephritic factor (NEF) antibody in vitro.

    PubMed Central

    Marín, M A; Fontán, G; López-Trascasa, M

    1992-01-01

    C3 nephritic factor (NEF) is an IgG autoantibody directed against neoantigenic determinants of the alternative C3 convertase (C3b.Bb). Structural and functional studies require important amounts of this antibody, which are difficult to obtain from patients' sera. We have developed a method for increasing NEF production in vitro. Epstein-Barr virus is a herpes virus which transforms B lymphocytes. Some authors were able to induce the production of NEF in vitro after infection with Epstein-Barr virus (EBV). These works were preformed without any previous cellular selection of B cells. We have performed a method of preselecting antigen-binding cells prior to EBV transformation. Non-preselected cells yielded 0.16 U/million cells in culture (U/M) of NEF antibody, whereas enriched cells for NEF antibody in eliminated 8 U/M (sheep erythrocytes coated with anti-IgG, A, M). Specific NEF synthesis can be increased, in peripheral blood mononuclear cells (PBMC) from patients by in vitro stimulation with the antigens recognized by NEF [C3b.Bb, 21,000 MW protein from patients' E membranes and 26,000 MW protein from sheep E membranes (ShE)]. The highest stimulation is induced by the C3b.Bb and by 26,000 MW protein, 21,000 MW protein had lowest stimulatory effect. In this work also we have shown that patients having NEF antibody in sera have an increase of the CD5-CD19 subset, when compared with the controls. Images Figure 2 PMID:1321794

  7. Replication of HIV-1 deleted Nef mutants in chronically immune activated human T cells.

    PubMed

    Shapira-Nahor, Orit; Maayan, Shlomo; Peden, Keith W C; Rabinowitz, Ruth; Schlesinger, Michael; Alian, Akram; Panet, Amos

    2002-11-10

    Lymphocytes (PBMC) obtained from blood of HIV-sera negative Ethiopian immigrants (ETH) were highly susceptible to HIV-1 infection in vitro with no need for stimulation by mitogens. As the HIV nef gene product has been shown to enhance viral replication in stimulated primary lymphocytes, we investigated in this work the role of Nef in viral replication in the ETH cells. Lymphocytes obtained from ETH individuals supported high replication of wild-type HIV-1 and low but significant replication level of the two deleted Nef mutants (encode truncated Nef proteins consisting only of either the first 35 or the first 86 amino acids of Nef). In contrast, no replication was observed in nonactivated cells obtained from non-ETH individuals. After activation of the PBMC from ETH individuals with PHA, replication of both wild-type strains and the two deleted Nef mutant viruses further increased. The CD4(+) T cells of ETH individuals exhibited elevated levels of the surface activation markers CD45RO and HLA-DR, compared with T cells derived from non-ETH group. Likewise, expression of the chemokine receptors CCR5 and CXCR4 on these cells was higher in the ETH group than in the non-ETH group. Replication of HIV-1 wild-type and the isogenic-deleted Nef mutants was significantly correlated with the proportion of ETH cells expressing CD45RO and the chemokine receptors. This study suggests that HIV-1 may respond differently to several activation states characteristic of T cells. One activation state, defined by chronically activated lymphocytes from ETH individuals, is permissive to the wild-type HIV-1 and, to a lesser degree, to the Nef mutants. Further activation of these cells by exogenous stimuli enhances replication of the virus. Our results support the notion that Nef enhances the basal level of T cell activation and consequently, viral replication. PMID:12482665

  8. Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

    PubMed Central

    Brügger, Britta; Krautkrämer, Ellen; Tibroni, Nadine; Munte, Claudia E; Rauch, Susanne; Leibrecht, Iris; Glass, Bärbel; Breuer, Sebastian; Geyer, Matthias; Kräusslich, Hans-Georg; Kalbitzer, Hans Robert; Wieland, Felix T; Fackler, Oliver T

    2007-01-01

    Background The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. Results Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. Conclusion Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo. PMID:17908312

  9. Secretion Modification Region-Derived Peptide Disrupts HIV-1 Nef's Interaction with Mortalin and Blocks Virus and Nef Exosome Release

    PubMed Central

    Shelton, Martin N.; Huang, Ming-Bo; Ali, Syed A.; Powell, Michael D.

    2012-01-01

    Nef is secreted from infected cells in exosomes and is found in abundance in the sera of HIV-infected individuals. Secreted exosomal Nef (exNef) induces apoptosis in uninfected CD4+ T cells and may be a key component of HIV pathogenesis. The exosomal pathway has been implicated in HIV-1 virus release, suggesting a possible link between these two viral processes. However, the underlying mechanisms and cellular components of exNef secretion have not been elucidated. We have previously described a Nef motif, the secretion modification region (SMR; amino acids 66 to 70), that is required for exNef secretion. In silico modeling data suggest that this motif can form a putative binding pocket. We hypothesized that the Nef SMR binds a cellular protein involved in protein trafficking and that inhibition of this interaction would abrogate exNef secretion. By using tandem mass spectrometry and coimmunoprecipitation with a novel SMR-based peptide (SMRwt) that blocks exNef secretion and HIV-1 virus release, we identified mortalin as an SMR-specific cellular protein. A second set of coimmunoprecipitation experiments with full-length Nef confirmed that mortalin interacts with Nef via Nef's SMR motif and that this interaction is disrupted by the SMRwt peptide. Overexpression and microRNA knockdown of mortalin revealed a positive correlation between exNef secretion levels and mortalin protein expression. Using antibody inhibition we demonstrated that the Nef/mortalin interaction is necessary for exNef secretion. Taken together, this work constitutes a significant step in understanding the underlying mechanism of exNef secretion, identifies a novel host-pathogen interaction, and introduces an HIV-derived peptide with antiviral properties. PMID:22013042

  10. Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody

    PubMed Central

    2014-01-01

    Background The HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies. Results We have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication. Conclusions Together, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef. PMID:24620746

  11. HIV-1 Nef: Taking Control of Protein Trafficking.

    PubMed

    Pereira, Estela A; daSilva, Luis L P

    2016-09-01

    The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host-cell-interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV-1 virions. PMID:27161574

  12. Translation of HLA-HIV associations to the cellular level: HIV adapts to inflate CD8 T cell responses against Nef and HLA-adapted variant epitopes.

    PubMed

    Almeida, Coral-Ann M; Bronke, Corine; Roberts, Steven G; McKinnon, Elizabeth; Keane, Niamh M; Chopra, Abha; Kadie, Carl; Carlson, Jonathan; Haas, David W; Riddler, Sharon A; Haubrich, Richard; Heckerman, David; Mallal, Simon; John, Mina

    2011-09-01

    Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 individuals from the same cohort, which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA-HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design. PMID:21821798

  13. Analysis of human immunodeficiency virus type 1 nef gene sequences present in vivo.

    PubMed Central

    Shugars, D C; Smith, M S; Glueck, D H; Nantermet, P V; Seillier-Moiseiwitsch, F; Swanstrom, R

    1993-01-01

    The nef genes of the human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) and the related simian immunodeficiency viruses (SIVs) encode a protein (Nef) whose role in virus replication and cytopathicity remains uncertain. As an attempt to elucidate the function of nef, we characterized the nucleotide and corresponding protein sequences of naturally occurring nef genes obtained from several HIV-1-infected individuals. A consensus Nef sequence was derived and used to identify several features that were highly conserved among the Nef sequences. These features included a nearly invariant myristylation signal, regions of sequence polymorphism and variable duplication, a region with an acidic charge, a (Pxx)4 repeat sequence, and a potential protein kinase C phosphorylation site. Clustering of premature stop codons at position 124 was noted in 6 of the 54 Nef sequences. Further analysis revealed four stretches of residues that were highly conserved not only among the patient-derived HIV-1 Nef sequences, but also among the Nef sequences of HIV-2 and the SIVs, suggesting that Nef proteins expressed by these retroviruses are functionally equivalent. The "Nef-defining" sequences were used to evaluate the sequence alignments of known proteins reported to share sequence similarity with Nef sequences and to conduct additional computer-based searches for similar protein sequences. A gene encoding the consensus Nef sequence was also generated. This gene encodes a full-length Nef protein that should be a valuable tool in further studies of Nef function. Images PMID:8043040

  14. A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny

    PubMed Central

    Sabino Cunha, Marcela; Lima Sampaio, Thatiane; Peterlin, B. Matija; Jesus da Costa, Luciana

    2016-01-01

    Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. PMID:27399760

  15. A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny.

    PubMed

    Sabino Cunha, Marcela; Lima Sampaio, Thatiane; Peterlin, B Matija; Jesus da Costa, Luciana

    2016-01-01

    Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity. PMID:27399760

  16. One hundred years and still counting: the story of NEF--yesterday, today, and tomorrow.

    PubMed

    Bowar-Ferres, Susan L; Fitzpatrick, M Louise; McClure, Margaret L

    2014-01-01

    Nurses Educational Funds, Inc (NEF) is the oldest and largest professionally endorsed source of funds for advanced study in nursing, which celebrated its Centennial in 2012. This philanthropic nonprofit organization is notable for its roots; NEF was organized by nurses specifically for nurses. Its history dates back to 1912, when it began in memory of Isabel Hampton Robb at Teacher's College, where the first graduate nursing education programs began. The initial Robb Memorial Fund was incorporated as a nonprofit organization in 1941 and officially became Nursing Educational Funds, Inc, in 1954. The NEF's sole mission is to raise money and give it for graduate-level scholarships in nursing education, service, practice, and research. Since its origin, more than 1000 doctoral and master's students from a broad array of schools across the nation have been recipients of awards. The NEF Board is a totally volunteer, highly dedicated group of nursing, business, and other professional leaders, who are steadfastly committed to this critical effort. Scholarships for graduate nursing education are imperative to meet the need to grow the pipeline of faculty. As charged by the 2010 Institute of Medicine report, the goal to increase the number of baccalaureate nurses to 80% of the workforce and to double the number of nurses with doctoral degrees both by 2020 speak to the heart of NEF. Funds raised currently are largely from Board members, individual donors, modest foundational support, and a number of bequests. As the nursing population grows older, the potential for bequests or planned giving becomes a realistic goal. Former NEF scholars have not unfortunately been a financial source, although pay back is an expectation. Nurses are the best ones to tell this compelling story to corporations and foundations as NEF continues to persist in the commitment to support graduate nursing education. PMID:25208153

  17. Protein–protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein

    PubMed Central

    Betzi, Stéphane; Restouin, Audrey; Opi, Sandrine; Arold, Stefan T.; Parrot, Isabelle; Guerlesquin, Françoise; Morelli, Xavier; Collette, Yves

    2007-01-01

    Protein–protein recognition is the cornerstone of multiple cellular and pathological functions. Therefore, protein–protein interaction inhibition (2P2I) is endowed with great therapeutic potential despite the initial belief that 2P2I was refractory to small-molecule intervention. Improved knowledge of complex molecular binding surfaces has recently stimulated renewed interest for 2P2I, especially after identification of “hot spots” and first inhibitory compounds. However, the combination of target complexity and lack of starting compound has thwarted experimental results and created intellectual barriers. Here we combined virtual and experimental screening when no previously known inhibitors can be used as starting point in a structure-based research program that targets an SH3 binding surface of the HIV type I Nef protein. High-throughput docking and application of a pharmacophoric filter on one hand and search for analogy on the other hand identified drug-like compounds that were further confirmed to bind Nef in the micromolar range (isothermal titration calorimetry), to target the Nef SH3 binding surface (NMR experiments), and to efficiently compete for Nef–SH3 interactions (cell-based assay, GST pull-down). Initial identification of these compounds by virtual screening was validated by screening of the very same library of compounds in the cell-based assay, demonstrating that a significant enrichment factor was attained by the in silico screening. To our knowledge, our results identify the first set of drug-like compounds that functionally target the HIV-1 Nef SH3 binding surface and provide the basis for a powerful discovery process that should help to speed up 2P2I strategies and open avenues for new class of antiviral molecules. PMID:18042718

  18. Association of Nef with the Human Immunodeficiency Virus Type 1 Core

    PubMed Central

    Kotov, Alexander; Zhou, Jing; Flicker, Paula; Aiken, Christopher

    1999-01-01

    Highly conserved among primate lentiviruses, the human immunodeficiency virus type 1 (HIV-1) Nef protein enhances viral infectivity by an unknown mechanism. Nef-defective virions are blocked at a stage of the HIV-1 life cycle between entry and reverse transcription, possibly virus uncoating. Nef is present in purified HIV-1 particles; however, it has not been determined whether Nef is specifically recruited into HIV-1 particles or whether virion-associated Nef plays a functional role in HIV-1 replication. To address the specificity and potential functionality of virion-associated Nef, we determined the subviral localization of Nef. HIV-1 cores were isolated by detergent treatment of concentrated virions followed by equilibrium density gradient sedimentation. Relative to HIV-1 virions, HIV-1 cores contained equivalent amounts of reverse transcriptase and integrase, decreased amounts of the viral matrix protein, and trace quantities of the viral transmembrane glycoprotein gp41. Examination of the particles by electron microscopy revealed cone-shaped structures characteristic of lentiviral cores. Similar quantities of proteolytically processed Nef protein were detected in gradient fractions of HIV-1 cores and intact virions. In addition, detergent-resistant subviral complexes isolated from immature HIV-1 particles contained similar quantities of Nef as untreated virions. These results demonstrate that Nef stably associates with the HIV-1 core and suggest that virion-associated Nef plays a functional role in accelerating HIV-1 replication. PMID:10482638

  19. HIV-1 Nef Breaches Placental Barrier in Rat Model

    PubMed Central

    Singh, Poonam; Agnihotri, Saurabh Kumar; Tewari, Mahesh Chandra; Kumar, Sadan; Sachdev, Monika; Tripathi, Raj Kamal

    2012-01-01

    The vertical transmission of HIV-1 from the mother to fetus is known, but the molecular mechanism regulating this transmission is not fully characterized. The fetus is highly protected by the placenta, which does not permit microbial pathogens to cross the placental barrier. In the present study, a rat model was established to observe the effect of HIV-1 protein Nef on placental barrier. Evans blue dye was used to assay permeability of placental barrier and fourteen day pregnant Sprague Dawley rats were injected intravenously with 2% Evans blue dye along with various concentrations of recombinant Nef. After an hour, animals were sacrificed and dye migration was observed through the assimilation of peripheral blood into fetus. Interestingly, traces of recombinant Nef protein were detected in the embryo as well as amniotic fluid and amniotic membrane along with placenta and uterus. Our study indicates that recombinant HIV-1-Nef protein breaches the placental barrier and allows the migration of Evans blue dye to the growing fetus. Further the concentration of Nef protein in blood is directly proportional to the intensity of dye migration and to the amount of Nef protein detected in uterus, placenta, amniotic membrane, amniotic fluid and embryo. Based on this study, it can be concluded that the HIV-1 Nef protein has a direct effect on breaching of the placental barrier in the model we have established in this study. Our observations will be helpful to understand the molecular mechanisms related to this breach of placental barrier by Nef in humans and may be helpful to identify specific Nef inhibitors. PMID:23240037

  20. HIV-1 Nef mutations abrogating downregulation of CD4 affect other Nef functions and show reduced pathogenicity in transgenic mice

    SciTech Connect

    Hanna, Zaher . E-mail: Zaher.Hanna@ircm.qc.ca; Priceputu, Elena; Hu, Chunyan; Vincent, Patrick; Jolicoeur, Paul

    2006-03-01

    HIV-1 Nef has the ability to downmodulate CD4 cell surface expression. Several studies have shown that CD4 downregulation is required for efficient virus replication and high infectivity. However, the pathophysiological relevance of this phenomenon in vivo, independently of its role in sustaining high virus loads, remains unclear. We studied the impact of the CD4 downregulation function of Nef on its pathogenesis in vivo, in the absence of viral replication, in the CD4C/HIV transgenic (Tg) mouse model. Two independent Nef mutants (RD35/36AA and D174K), known to abrogate CD4 downregulation, were tested in Tg mice. Flow cytometry analysis showed that downregulation of murine CD4 was severely decreased or abrogated on Tg T cells expressing respectively Nef{sup RD35/36AA} and Nef{sup D174K}. Similarly, the severe depletion of double-positive CD4{sup +}CD8{sup +} and of single-positive CD4{sup +}CD8{sup -} thymocytes, usually observed with Nef{sup Wt}, was not detected in Nef{sup RD35/36AA} and Nef{sup D174K} Tg mice. However, both mutant Tg mice showed a partial depletion of peripheral CD4{sup +} T cells. This was accompanied, as previously reported for Net{sup Wt} Tg mice, by the presence of an activated/memory-like phenotype (CD69{sup +}, CD25{sup +}, CD44{sup +}, CD45RB{sup Low}, CD62{sup Low}) of CD4{sup +} T cells expressing Nef{sup RD35/36AA} and to a lesser extent Nef{sup D174K}. In addition, both mutants retained the ability to block CD4{sup +} T cell proliferation in vitro after anti-CD3 stimulation, but not to enhance apoptosis/death of CD4{sup +} T cells. Therefore, it appears that Nef-mediated CD4 downregulation is associated with thymic defects, but segregates independently of the activated/memory-like phenotype, of the partial depletion and of the impaired in vitro proliferation of peripheral CD4{sup +} T cells. Histopathological assessment revealed the total absence of or decrease severity and frequency of organ AIDS-like diseases (lung, heart and kidney

  1. HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

    NASA Astrophysics Data System (ADS)

    Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

    2014-03-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.

  2. HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

    PubMed Central

    Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

    2014-01-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes. PMID:24658403

  3. Genetic and Functional Diversity of Human Immunodeficiency Virus Type 1 Subtype B Nef Primary Isolates

    PubMed Central

    Foster, John L.; Molina, Rene P.; Luo, Tianci; Arora, Vivek K.; Huang, Yaoxing; Ho, David D.; Garcia, J. Victor

    2001-01-01

    We have characterized the functional integrity of seven primary Nef isolates: five from a long-term nonprogressing human immunodeficiency virus (HIV)-infected individual and one each from two patients with AIDS. One of the seven Nefs was defective for CD4 downregulation, two others were defective for PAK-2 activation, and one Nef was defective for PAK-2 activation and major histocompatibility complex (MHC) class I downregulation. Five of the Nefs were tested and found to be functional for the enhancement of virus particle infectivity. The structural basis for each of the functional defects has been analyzed by constructing a consensus nef, followed by mutational analysis of the variant amino acid residues. Mutations A29V and F193I were deleterious to CD4 downregulation and PAK-2 activation, respectively, while S189R rendered Nef defective for both MHC class I downregulation and PAK-2 activation. A search of the literature identified HIVs from five patients with Nefs predominantly mutated at F193 and from one patient with Nefs predominantly mutated at A29. A29 is highly conserved in all HIV subtypes except for subtype E. F193 is conserved in subtype B (and possibly in the closely related subtype D), but none of the other HIV group M subtypes. Our results suggest that functional distinctions may exist between HIV subtypes. PMID:11160665

  4. Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells

    PubMed Central

    Weiler, Andrea M.; Das, Arpita; Akinyosoye, Oluwasayo; Cui, Sherry; O'Connor, Shelby L.; Scheef, Elizabeth A.; Reed, Jason S.; Panganiban, Antonito T.; Sacha, Jonah B.; Rakasz, Eva G.; Friedrich, Thomas C.

    2015-01-01

    Nef-specific CD8+ T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef165–173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T170I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication. PMID:26637459

  5. Acute Viral Escape Selectively Impairs Nef-Mediated Major Histocompatibility Complex Class I Downmodulation and Increases Susceptibility to Antiviral T Cells.

    PubMed

    Weiler, Andrea M; Das, Arpita; Akinyosoye, Oluwasayo; Cui, Sherry; O'Connor, Shelby L; Scheef, Elizabeth A; Reed, Jason S; Panganiban, Antonito T; Sacha, Jonah B; Rakasz, Eva G; Friedrich, Thomas C; Maness, Nicholas J

    2016-02-01

    Nef-specific CD8(+) T lymphocytes (CD8TL) are associated with control of simian immunodeficiency virus (SIV) despite extensive nef variation between and within animals. Deep viral sequencing of the immunodominant Mamu-B*017:01-restricted Nef165-173IW9 epitope revealed highly restricted evolution. A common acute escape variant, T170I, unexpectedly and uniquely degraded Nef's major histocompatibility complex class I (MHC-I) downregulatory capacity, rendering the virus more vulnerable to CD8TL targeting other epitopes. These data aid in a mechanistic understanding of Nef functions and suggest means of immunity-mediated control of lentivirus replication. PMID:26637459

  6. Primate lentiviral Nef proteins deregulate T-cell development by multiple mechanisms

    PubMed Central

    2013-01-01

    Background A nef gene is present in all primate lentiviral genomes and is important for high viral loads and progression to AIDS in human or experimental macaque hosts of HIV or SIV, respectively. In these hosts, infection of the thymus results in a decreased output of naive T cells that may contribute to the development of immunodeficiency. We have previously shown that HIV-1 subtype B Nef proteins can block human T-cell development. However, the underlying mechanism(s) and the conservation of this Nef function between different groups of HIV and SIV remained to be determined. Results We investigated whether reduction of thymic output is a conserved function of highly divergent lentiviral Nef proteins including those from both types of human immunodeficiency viruses (HIV-1 and HIV-2), their direct simian counterparts (SIVcpz, SIVgor and SIVsmm, respectively), and some additional SIV strains. We found that expression of most of these nef alleles in thymocyte progenitors impaired T-cell development and reduced thymic output. For HIV-1 Nef, binding to active p21 protein (Cdc42/Rac)-activated kinase (PAK2) was a major determinant of this function. In contrast, selective disruption of PAK2 binding did not eliminate the effect on T-cell development of SIVmac239 Nef, as was shown by expressing mutants in a newly discovered PAK2 activating structural motif (PASM) constituted by residues I117, H121, T218 and Y221, as well as previously described mutants. Rather, down-modulation of cell surface CD3 was sufficient for reduced thymic output by SIVmac Nef, while other functions of SIV Nefs contributed. Conclusions Our results indicate that primate lentiviral Nef proteins impair development of thymocyte precursors into T cells in multiple ways. The interaction of HIV-1 Nef with active PAK2 by HIV-1 seem to be most detrimental, and downregulation of CD3 by HIV-2 and most SIV Nef proteins sufficient for reduced thymic output. Since the reduction of thymic output by Nef is a

  7. HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo

    PubMed Central

    Stolp, Bettina; Imle, Andrea; Coelho, Fernanda Matos; Hons, Miroslav; Gorina, Roser; Lyck, Ruth; Stein, Jens V.; Fackler, Oliver T.

    2012-01-01

    HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef’s influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef’s ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor. PMID:23093676

  8. A truncated form of Nef selected during pathogenic reversion of simian immunodeficiency virus SIVmac239Deltanef increases viral replication.

    PubMed

    Chakrabarti, Lisa A; Metzner, Karin J; Ivanovic, Tijana; Cheng, Hua; Louis-Virelizier, Jean; Connor, Ruth I; Cheng-Mayer, Cecilia

    2003-01-01

    The live, attenuated vaccine simian immunodeficiency virus SIVmac239Deltanef efficiently protects rhesus macaques against infection with wild-type SIVmac but occasionally causes CD4(+) T-cell depletion and progression to simian AIDS (SAIDS). Virus recovered from a vaccinated macaque (Rh1490) that progressed to SAIDS had acquired an additional deletion in the nef gene, resulting in a frameshift that restored the original nef open reading frame (R. I. Connor, D. C. Montefiori, J. M. Binley, J. P. Moore, S. Bonhoeffer, A. Gettie, E. A. Fenamore, K. E. Sheridan, D. D. Ho, P. J. Dailey, and P. A. Marx, J. Virol. 72:7501-7509, 1998). Intravenous inoculation of the Rh1490 viral isolate into four naive rhesus macaques induced CD4(+) T-cell depletion and disease in three out of four animals within 2 years, indicating a restoration of virulence. A DNA fragment encompassing the truncated nef gene amplified from the Rh1490 isolate was inserted into the genetic backbone of SIVmac239. The resulting clone, SIVmac239-Delta2nef, expressed a Nef protein of approximately 23 kDa, while the original SIVmac239Deltanef clone expressed a shorter protein of 8 kDa. The revertant form of Nef did not cause downregulation of CD4, CD3, or major histocompatibility complex class I. The infectivity of SIVmac239-Delta2nef was similar to that of SIVmac239Deltanef in single-cycle assays using indicator cell lines. In contrast, SIVmac239-Delta2nef replicated more efficiently than SIVmac239Deltanef in peripheral blood mononuclear cell (PBMC) cultures infected under unstimulated conditions. The p27 Gag antigen levels in SIVmac239-Delta2nef-infected cultures were still lower than those obtained with wild-type SIVmac239, consistent with a partial recovery of Nef function. The transcriptional activity of long terminal repeat (LTR)-luciferase constructs containing the nef deletions did not differ markedly from that of wild-type LTR. Introduction of a premature stop codon within Nef-Delta2 abolished the

  9. HIV-1 Nef and Vpu Are Functionally Redundant Broad-Spectrum Modulators of Cell Surface Receptors, Including Tetraspanins

    PubMed Central

    Haller, Claudia; Müller, Birthe; Fritz, Joëlle V.; Lamas-Murua, Miguel; Stolp, Bettina; Pujol, François M.; Keppler, Oliver T.

    2014-01-01

    ABSTRACT HIV-1 Nef and Vpu are thought to optimize virus replication in the infected host, at least in part via their ability to interfere with vesicular host cell trafficking. Despite the use of distinct molecular mechanisms, Nef and Vpu share specificity for some molecules such as CD4 and major histocompatibility complex class I (MHC-I), while disruption of intracellular transport of the host cell restriction factor CD317/tetherin represents a specialized activity of Vpu not exerted by HIV-1 Nef. To establish a profile of host cell receptors whose intracellular transport is affected by Nef, Vpu, or both, we comprehensively analyzed the effect of these accessory viral proteins on cell surface receptor levels on A3.01 T lymphocytes. Thirty-six out of 105 detectable receptors were significantly downregulated by HIV-1 Nef, revealing a previously unappreciated scope with which HIV-1 Nef remodels the cell surface of infected cells. Remarkably, the effects of HIV-1 Vpu on host cell receptor exposure largely matched those of HIV-1 Nef in breadth and specificity (32 of 105, all also targeted by Nef), even though the magnitude was generally less pronounced. Of particular note, cell surface exposure of all members of the tetraspanin (TSPAN) protein family analyzed was reduced by both Nef and Vpu, and the viral proteins triggered the enrichment of TSPANs in a perinuclear area of the cell. While Vpu displayed significant colocalization and physical association with TSPANs, interactions of Nef with TSPANs were less robust. TSPANs thus emerge as a major target of deregulation in host cell vesicular transport by HIV-1 Nef and Vpu. The conservation of this activity in two independent accessory proteins suggests its importance for the spread of HIV-1 in the infected host. IMPORTANCE In this paper, we define that HIV-1 Nef and Vpu display a surprising functional overlap and affect the cell surface exposure of a previously unexpected breadth of cellular receptors. Our analyses

  10. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef.

    PubMed

    Usami, Yoshiko; Wu, Yuanfei; Göttlinger, Heinrich G

    2015-10-01

    HIV-1 Nef and the unrelated mouse leukaemia virus glycosylated Gag (glycoGag) strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of both SERINC3 and SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivity. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4(+) T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS. PMID:26416733

  11. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef

    PubMed Central

    Usami, Yoshiko; Wu, Yuanfei; Göttlinger, Heinrich G.

    2015-01-01

    HIV-1 Nef and the unrelated murine leukemia virus glycoGag strongly enhance the infectivity of HIV-1 virions produced in certain cell types in a clathrin-dependent manner. Here we show that Nef and glycoGag prevent the incorporation of the multipass transmembrane proteins SERINC3 and SERINC5 into HIV-1 virions to an extent that correlates with infectivity enhancement. Silencing of SERINC3 together with SERINC5 precisely phenocopied the effects of Nef and glycoGag on HIV-1 infectivities. The infectivity of nef-deficient virions increased more than 100-fold when produced in double-knockout human CD4+ T cells that lack both SERINC3 and SERINC5, and re-expression experiments confirmed that the absence of SERINC3 and SERINC5 accounted for the infectivity enhancement. Furthermore, SERINC3 and SERINC5 together restricted HIV-1 replication, and this restriction was evaded by Nef. SERINC3 and SERINC5 are highly expressed in primary human HIV-1 target cells, and inhibiting their downregulation by Nef is a potential strategy to combat HIV/AIDS. PMID:26416733

  12. Expression of Nef Downregulates CXCR4, the Major Coreceptor of Human Immunodeficiency Virus, from the Surfaces of Target Cells and Thereby Enhances Resistance to Superinfection▿

    PubMed Central

    Venzke, Stephanie; Michel, Nico; Allespach, Ina; Fackler, Oliver T.; Keppler, Oliver T.

    2006-01-01

    Lentiviral Nef proteins are key factors for pathogenesis and are known to downregulate functionally important molecules, including CD4 and major histocompatibility complex class I (MHC-I), from the surfaces of infected cells. Recently, we demonstrated that Nef reduces cell surface levels of the human immunodeficiency virus type 1 (HIV-1) entry coreceptor CCR5 (N. Michel, I. Allespach, S. Venzke, O. T. Fackler, and O. T. Keppler, Curr. Biol. 15:714-723, 2005). Here, we report that Nef downregulates the second major HIV-1 coreceptor, CXCR4, from the surfaces of HIV-infected primary CD4 T lymphocytes with efficiencies comparable to those of the natural CXCR4 ligand, stromal cell-derived factor-1 alpha. Analysis of a panel of mutants of HIV-1SF2 Nef revealed that the viral protein utilized the same signature motifs for downmodulation of CXCR4 and MHC-I, including the proline-rich motif P73P76P79P82 and the acidic cluster motif E66E67E68E69. Expression of wild-type Nef, but not of specific Nef mutants, resulted in a perinuclear accumulation of the coreceptor. Remarkably, the carboxy terminus of CXCR4, which harbors the classical motifs critical for basal and ligand-induced receptor endocytosis, was dispensable for the Nef-mediated reduction of surface exposure. Functionally, the ability of Nef to simultaneously downmodulate CXCR4 and CD4 correlated with maximum-level protection of Nef-expressing target cells from fusion with cells exposing X4 HIV-1 envelopes. Furthermore, the Nef-mediated downregulation of CXCR4 alone on target T lymphocytes was sufficient to diminish cells' susceptibility to X4 HIV-1 virions at the entry step. The downregulation of chemokine coreceptors is a conserved activity of Nef to modulate infected cells, an important functional consequence of which is an enhanced resistance to HIV superinfection. PMID:16928758

  13. Nef Neutralizes the Ability of Exosomes from CD4+ T Cells to Act as Decoys during HIV-1 Infection

    PubMed Central

    da Silva, Elaine Z. M.; Silveira, Paola P.; da Silva-Januário, Mara E.; Arruda, Eurico; Jamur, Maria C.; Oliver, Constance; Aguiar, Renato S.; daSilva, Luis L. P.

    2014-01-01

    Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4− T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface. PMID:25423108

  14. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    PubMed Central

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  15. Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

    PubMed Central

    Greenway, A; Azad, A; Mills, J; McPhee, D

    1996-01-01

    It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis. PMID:8794306

  16. Pathogenesis of HIV-associated pulmonary hypertension: potential role of HIV-1 Nef.

    PubMed

    Almodovar, Sharilyn; Hsue, Priscilla Y; Morelli, Julie; Huang, Laurence; Flores, Sonia C

    2011-06-01

    Infection with HIV increases the risk for lung diseases, including noninfectious pulmonary hypertension (PH). HIV-associated PH (HIV-PH) is an important lung disease in HIV-infected persons who live longer with antiretrovirals. The early stages of HIV-PH may be overlooked by healthcare providers due to nonspecific symptoms, including progressive dyspnea and nonproductive cough. HIV-PH may be detected via chest radiographs, CT scans, or electrocardiograms, but Doppler echocardiography is the most useful screening test to identify candidates for right heart catheterization. HIV-PH has a poor prognosis with high mortality; improved biomarkers to identify earlier stages of PH would benefit clinical care. The HIV-PH mechanism remains unknown, but HIV proteins such as Tat and Nef may play a role. HIV-1 Nef is a broad-spectrum adaptor protein that may affect HIV-infected and uninfected pulmonary vascular cells. Studies in macaques suggest that Nef is important in HIV-PH pathogenesis because monkeys infected with a chimeric simian immunodeficiency virus (SIV) expressing HIV-nef (SHIVnef) alleles, but not monkeys infected with the native SIV, develop pulmonary vascular remodeling. Four consistent amino acid mutations arose spontaneously in Nef passaged in the monkeys. To translate these findings to humans, one research endeavor of the Lung HIV Study focuses on the identification of HIV nef mutations in HIV-infected individuals with PH compared with HIV-infected normotensive patients. We present some of the preliminary evidence. Ongoing longitudinal studies will establish the connection between Nef mutations and the propensity for HIV-PH. PMID:21653533

  17. CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef.

    PubMed

    Milicic, Anita; Price, David A; Zimbwa, Peter; Booth, Bruce L; Brown, Helen L; Easterbrook, Philippa J; Olsen, Kara; Robinson, Nicola; Gileadi, Uzi; Sewell, Andrew K; Cerundolo, Vincenzo; Phillips, Rodney E

    2005-10-01

    CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated. PMID:16177107

  18. The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner.

    PubMed

    Tarafdar, Sreya; Poe, Jerrod A; Smithgall, Thomas E

    2014-05-30

    The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle. PMID:24722985

  19. Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action.

    PubMed

    Dikeakos, Jimmy D; Atkins, Katelyn M; Thomas, Laurel; Emert-Sedlak, Lori; Byeon, In-Ja L; Jung, Jinwon; Ahn, Jinwoo; Wortman, Matthew D; Kukull, Ben; Saito, Masumichi; Koizumi, Hirokazu; Williamson, Danielle M; Hiyoshi, Masateru; Barklis, Eric; Takiguchi, Masafumi; Suzu, Shinya; Gronenborn, Angela M; Smithgall, Thomas E; Thomas, Gary

    2010-10-01

    HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1-mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2-dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1-dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action. PMID:20702582

  20. Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease

    PubMed Central

    2011-01-01

    Background The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). Results Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. Conclusions In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential. PMID:21366921

  1. Membrane-Associated Conformation of HIV-1 Nef Investigated with Hydrogen Exchange Mass Spectrometry at a Langmuir Monolayer.

    PubMed

    Pirrone, Gregory F; Emert-Sedlak, Lori A; Wales, Thomas E; Smithgall, Thomas E; Kent, Michael S; Engen, John R

    2015-07-21

    In the companion paper to this work, we described development of a new type of hydrogen exchange (HX) mass spectrometry (MS) measurement that integrates Langmuir monolayers. With Langmuir monolayers, the lipid packing density can be reproducibly controlled and changed as desired. Analysis of HX in proteins that may undergo conformational changes as a function of lipid packing (for example, conformational rearrangements after insertion into a lipid layer) are then possible. We previously used neutron reflection to characterize just such a conformational change in the myristoylated HIV-1 Nef protein (myrNef): at high lipid packing density, myrNef could not insert into the lipids and maintained a compact conformation adjacent to the monolayer, whereas at lower lipid packing density, myrNef was able to insert N-terminal arm residues, causing displacement of the core domain away from the monolayer. In order to locate where conformation may have been altered by lipid association, we applied the HX MS Langmuir monolayer method to myrNef associated with monolayers of packing densities identical to those used for the prior neutron reflection measurements. The results show that the N-terminal region and the C-terminal unstructured loop undergo conformational changes when associated with a low density lipid monolayer. The results are not consistent with the hypothesis of myrNef dimerization upon membrane association in the absence of other myrNef binding partners. The HX MS Langmuir monolayer method provides new and meaningful information for myrNef that helps explain necessary conformational changes required for function at the membrane. PMID:26133569

  2. The HIV-1 Nef Protein Binds Argonaute-2 and Functions as a Viral Suppressor of RNA Interference

    PubMed Central

    Aqil, Madeeha; Naqvi, Afsar Raza; Bano, Aalia Shahr; Jameel, Shahid

    2013-01-01

    The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR). PMID:24023945

  3. Human immunodeficiency virus type 1 Nef recruits the guanine exchange factor Vav1 via an unexpected interface into plasma membrane microdomains for association with p21-activated kinase 2 activity.

    PubMed

    Rauch, Susanne; Pulkkinen, Kati; Saksela, Kalle; Fackler, Oliver T

    2008-03-01

    Alterations of T-cell receptor signaling by human immunodeficiency virus type 1 (HIV-1) Nef involve its association with a highly active subpopulation of p21-activated kinase 2 (PAK2) within a dynamic signalosome assembled in detergent-insoluble membrane microdomains. Nef-PAK2 complexes contain the GTPases Rac and Cdc42 as well as a factor providing guanine nucleotide exchange factor (GEF) activity for Rac/Cdc42. However, the identity of this GEF has remained controversial. Previous studies suggested the association of Nef with at least three independent GEFs, Vav, DOCK2/ELMO1, and betaPix. Here we used a broad panel of approaches to address which of these GEFs is involved in the functional interaction of Nef with PAK2 activity. Biochemical fractionation and confocal microscopy revealed that Nef recruits Vav1, but not DOCK2/ELMO1 or betaPix, to membrane microdomains. Transient RNAi knockdown, analysis of cell lines defective for expression of Vav1 or DOCK2 as well as use of a betaPix binding-deficient PAK2 variant confirmed a role for Vav1 but not DOCK2 or betaPix in Nef's association with PAK2 activity. Nef-mediated microdomain recruitment of Vav1 occurred independently of the Src homology 3 domain binding PxxP motif, which is known to connect Nef to many cellular signaling processes. Instead, a recently described protein interaction surface surrounding Nef residue F195 was identified as critical for Nef-mediated raft recruitment of Vav1. These results identify Vav1 as a relevant component of the Nef-PAK2 signalosome and provide a molecular basis for the role of F195 in formation of a catalytically active Nef-PAK2 complex. PMID:18094167

  4. Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

    PubMed

    Alberti, Michael O; Jones, Jennifer J; Miglietta, Riccardo; Ding, Haitao; Bakshi, Rakesh K; Edmonds, Tara G; Kappes, John C; Ochsenbauer, Christina

    2015-12-01

    We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus

  5. Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

    PubMed Central

    Serena, Michela; Giorgetti, Alejandro; Busato, Mirko; Gasparini, Francesca; Diani, Erica; Romanelli, Maria Grazia; Zipeto, Donato

    2016-01-01

    HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction. PMID:26927806

  6. Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

    NASA Astrophysics Data System (ADS)

    Serena, Michela; Giorgetti, Alejandro; Busato, Mirko; Gasparini, Francesca; Diani, Erica; Romanelli, Maria Grazia; Zipeto, Donato

    2016-03-01

    HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.

  7. Downregulation of the T-Cell Receptor by Human Immunodeficiency Virus Type 2 Nef Does Not Protect against Disease Progression▿

    PubMed Central

    Feldmann, Jérôme; Leligdowicz, Aleksandra; Jaye, Assan; Dong, Tao; Whittle, Hilton; Rowland-Jones, Sarah L.

    2009-01-01

    Chronic immune activation is thought to play a major role in human immunodeficiency virus (HIV) pathogenesis, but the relative contributions of multiple factors to immune activation are not known. One proposed mechanism to protect against immune activation is the ability of Nef proteins from some HIV and simian immunodeficiency virus strains to downregulate the T-cell receptor (TCR)-CD3 complex of the infected cell, thereby reducing the potential for deleterious activation. HIV type 1 (HIV-1) Nef has lost this property. In contrast to HIV-1, HIV-2 infection is characterized by a marked disparity in the disease course, with most individuals maintaining a normal life span. In this study, we examined the relationship between the ability of HIV-2 Nef proteins to downregulate the TCR and immune activation, comparing progressors and nonprogressors. Representative Nef variants were isolated from 28 HIV-2-infected individuals. We assessed their abilities to downregulate the TCR from the surfaces of CD4 T cells. In the same individuals, the activation of peripheral lymphocytes was evaluated by measurement of the expression levels of HLA-DR and CD38. We observed a striking correlation of the TCR downregulation efficiency of HIV-2 Nef variants with immune activation in individuals with a low viral load. This strongly suggests that Nef expression can influence the activation state of the immune systems of infected individuals. However, the efficiency of TCR downregulation by Nef was not reduced in progressing individuals, showing that TCR downregulation does not protect against progression in HIV-2 infection. PMID:19812166

  8. Dynamic features of apo and bound HIV-Nef protein reveal the anti-HIV dimerization inhibition mechanism.

    PubMed

    Moonsamy, Suri; Bhakat, Soumendranath; Soliman, Mahmoud E S

    2015-01-01

    The first account on the dynamic features of Nef or negative factor, a small myristoylated protein located in the cytoplasm believes to increase HIV-1 viral titer level, is reported herein. Due to its major role in HIV-1 pathogenicity, Nef protein is considered an emerging target in anti-HIV drug design and discovery process. In this study, comparative long-range all-atom molecular dynamics simulations were employed for apo and bound protein to unveil molecular mechanism of HIV-Nef dimerization and inhibition. Results clearly revealed that B9, a newly discovered Nef inhibitor, binds at the dimeric interface of Nef protein and caused significant separation between orthogonally opposed residues, namely Asp108, Leu112 and Gln104. Large differences in magnitudes were observed in the radius of gyration (∼1.5 Å), per-residue fluctuation (∼2 Å), C-alpha deviations (∼2 Å) which confirm a comparatively more flexible nature of apo conformation due to rapid dimeric association. Compared to the bound conformer, a more globally correlated motion in case of apo structure of HIV-Nef confirms the process of dimeric association. This clearly highlights the process of inhibition as a result of ligand binding. The difference in principal component analysis (PCA) scatter plot and per-residue mobility plot across first two normal modes further justifies the same findings. The in-depth dynamic analyses of Nef protein presented in this report would serve crucial in understanding its function and inhibition mechanisms. Information on inhibitor binding mode would also assist in designing of potential inhibitors against this important HIV target. PMID:26355431

  9. Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination.

    PubMed

    Guerra-Pérez, Natalia; Aravantinou, Meropi; Veglia, Filippo; Goode, Diana; Truong, Rosaline; Derby, Nina; Blanchard, James; Grasperge, Brooke; Gettie, Agegnehu; Robbiani, Melissa; Martinelli, Elena

    2016-01-01

    Prevalent HSV-2 infection increases the risk of HIV acquisition both in men and women even in asymptomatic subjects. Understanding the impact of HSV-2 on the mucosal microenvironment may help to identify determinants of susceptibility to HIV. Vaginal HSV-2 infection increases the frequency of cells highly susceptible to HIV in the vaginal tissue of women and macaques and this correlates with increased susceptibility to vaginal SHIV infection in macaques. However, the effect of rectal HSV-2 infection on HIV acquisition remains understudied. We developed a model of rectal HSV-2 infection in macaques in combination with rectal SIVmac239Δnef (SIVΔnef) vaccination and our results suggest that rectal HSV-2 infection may increase the susceptibility of macaques to rectal SIVmac239 wild-type (wt) infection even in SIVΔnef-infected animals. Rectal SIVΔnef infection/vaccination protected 7 out of 7 SIVΔnef-infected macaques from SIVmac239wt rectal infection (vs 12 out of 16 SIVΔnef-negative macaques), while 1 out of 3 animals co-infected with SIVΔnef and HSV-2 acquired SIVmac239wt infection. HSV-2/SIVmac239wt co-infected animals had increased concentrations of inflammatory factors in their plasma and rectal fluids and a tendency toward higher acute SIVmac239wt plasma viral load. However, they had higher blood CD4 counts and reduced depletion of CCR5+ CD4+ T cells compared to SIVmac239wt-only infected animals. Thus, rectal HSV-2 infection generates a pro-inflammatory environment that may increase susceptibility to rectal SIV infection and may impact immunological and virological parameters during acute SIV infection. Studies with larger number of animals are needed to confirm these findings. PMID:26886938

  10. Rectal HSV-2 Infection May Increase Rectal SIV Acquisition Even in the Context of SIVΔnef Vaccination

    PubMed Central

    Veglia, Filippo; Goode, Diana; Truong, Rosaline; Derby, Nina; Blanchard, James; Grasperge, Brooke; Gettie, Agegnehu; Robbiani, Melissa; Martinelli, Elena

    2016-01-01

    Prevalent HSV-2 infection increases the risk of HIV acquisition both in men and women even in asymptomatic subjects. Understanding the impact of HSV-2 on the mucosal microenvironment may help to identify determinants of susceptibility to HIV. Vaginal HSV-2 infection increases the frequency of cells highly susceptible to HIV in the vaginal tissue of women and macaques and this correlates with increased susceptibility to vaginal SHIV infection in macaques. However, the effect of rectal HSV-2 infection on HIV acquisition remains understudied. We developed a model of rectal HSV-2 infection in macaques in combination with rectal SIVmac239Δnef (SIVΔnef) vaccination and our results suggest that rectal HSV-2 infection may increase the susceptibility of macaques to rectal SIVmac239 wild-type (wt) infection even in SIVΔnef-infected animals. Rectal SIVΔnef infection/vaccination protected 7 out of 7 SIVΔnef-infected macaques from SIVmac239wt rectal infection (vs 12 out of 16 SIVΔnef-negative macaques), while 1 out of 3 animals co-infected with SIVΔnef and HSV-2 acquired SIVmac239wt infection. HSV-2/SIVmac239wt co-infected animals had increased concentrations of inflammatory factors in their plasma and rectal fluids and a tendency toward higher acute SIVmac239wt plasma viral load. However, they had higher blood CD4 counts and reduced depletion of CCR5+ CD4+ T cells compared to SIVmac239wt-only infected animals. Thus, rectal HSV-2 infection generates a pro-inflammatory environment that may increase susceptibility to rectal SIV infection and may impact immunological and virological parameters during acute SIV infection. Studies with larger number of animals are needed to confirm these findings. PMID:26886938

  11. Structural constraints on human immunodeficiency virus type 1 Nef function

    SciTech Connect

    Raney, Alexa; Shaw, Alice Y.; Foster, John L.; Garcia, J. Victor

    2007-11-10

    HIV-1 Nef is a multifunctional protein that exerts its activities through interactions with multiple cellular partners. Nef uses different domains and mechanisms to exert its functions including cell surface down-modulation of CD4 and MHC-I receptors and activation of the serine/threonine kinase PAK-2. We inserted tags at the C-terminus and proximal to the N-terminus of Nef and the effects on Nef's structure/function relationships were examined. We discovered significant defects in MHC-I down-modulation with the insertion of HA/FLAG tags at either region. We also found impaired PAK-2 activation with a C-terminal fusion with GFP. Interestingly, Nef-GFP and Nef-GH{sub 7} induced MHC-I down-modulation, suggesting that the negative charge of the HA/FLAG tag could contribute to the observed defect. Together, these observations highlight elements of Nef's functional complexity and demonstrate previously unsuspected structural requirements for PAK-2 activation and MHC-1 down-modulation in Nef's flexible N- and C-terminal regions.

  12. High potassium level

    MedlinePlus

    High potassium level is a problem in which the amount of potassium in the blood is higher than normal. The medical ... There are often no symptoms with a high level of potassium. When symptoms do occur, they may ...

  13. CD8 T Cell Response Maturation Defined by Anentropic Specificity and Repertoire Depth Correlates with SIVΔnef-induced Protection

    PubMed Central

    Adnan, Sama; Colantonio, Arnaud D.; Yu, Yi; Gillis, Jacqueline; Wong, Fay E.; Becker, Ericka A.; Reeves, R. Keith; Lifson, Jeffrey D.; O’Connor, Shelby L.; Johnson, R. Paul

    2015-01-01

    The live attenuated simian immunodeficiency virus (LASIV) vaccine SIVΔnef is one of the most effective vaccines in inducing protection against wild-type lentiviral challenge, yet little is known about the mechanisms underlying its remarkable protective efficacy. Here, we exploit deep sequencing technology and comprehensive CD8 T cell epitope mapping to deconstruct the CD8 T cell response, to identify the regions of immune pressure and viral escape, and to delineate the effect of epitope escape on the evolution of the CD8 T cell response in SIVΔnef-vaccinated animals. We demonstrate that the initial CD8 T cell response in the acute phase of SIVΔnef infection is mounted predominantly against more variable epitopes, followed by widespread sequence evolution and viral escape. Furthermore, we show that epitope escape expands the CD8 T cell repertoire that targets highly conserved epitopes, defined as anentropic specificity, and generates de novo responses to the escaped epitope variants during the vaccination period. These results correlate SIVΔnef-induced protection with expanded anentropic specificity and increased response depth. Importantly, these findings render SIVΔnef, long the gold standard in HIV/SIV vaccine research, as a proof-of-concept vaccine that highlights the significance of the twin principles of anentropic specificity and repertoire depth in successful vaccine design. PMID:25688559

  14. High blood cholesterol levels

    MedlinePlus

    ... gov/ency/article/000403.htm High blood cholesterol levels To use the sharing features on this page, ... called "bad" cholesterol For many people, abnormal cholesterol levels are partly due to an unhealthy lifestyle. This ...

  15. Activation pathways and human immunodeficiency virus type 1 replication are not altered in CD4+ T cells expressing the nef protein.

    PubMed

    Schwartz, O; Arenzana-Seisdedos, F; Heard, J M; Danos, O

    1992-05-01

    While recent studies in Rhesus monkeys have pointed out the importance of an intact nef gene for the development of acquired immunodeficiency syndrome (AIDS), no biological function has been so far unambiguously attributed to its product. Since Nef has been described to possess GTP-binding properties and to down-regulate CD4 cell surface expression, we looked for evidences of Nef interfering with the transduction of activating signals in human CD4+ T cells. We used a murine leukemia retroviral vector to express the HIV-1BRU nef gene in two permanent tumoral T-cell lines (CEM and Jurkat) and in two nonimmortalized, interleukin-2 (IL2)-dependent, T-cell clones. The single copy recombinant provirus integrated in the genome of these cells directed the synthesis of a 27-kD protein with a half-life greater than 5 h. The levels of expression of cell surface molecules involved in T-cell functions (CD4, CD3, CD28, CD29, IL-2 receptor) were not modified in cell populations expressing Nef. In immunocompetent T-cell clones, cell proliferation and lymphokine production in response to activating stimuli (IL-2, alloantigens, phorbol esters, or antibodies directed against CD2, CD3, CD4, CD28) remained unmodified. Moreover, the presence of Nef did not change the kinetics of human immunodeficiency virus (HIV) infection. PMID:1355346

  16. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin.

    PubMed

    de Virgilio, Maddalena; De Marchis, Francesca; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

    2008-01-01

    Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein gamma-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of gamma-zein). Protein blots and pulse-chase indicate that fusions between Nef and the same gamma-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin-Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its gamma-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

  17. The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

    PubMed Central

    de Virgilio, Maddalena; Bellucci, Michele; Mainieri, Davide; Rossi, Marika; Benvenuto, Eugenio; Arcioni, Sergio; Vitale, Alessandro

    2008-01-01

    Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants. PMID:18540021

  18. Immunogenic compositions comprising human immunodeficiency virus (HIV) mosaic Nef proteins

    DOEpatents

    Korber, Bette T.; Perkins, Simon; Bhattacharya, Tanmoy; Fischer, William M.; Theiler, James; Letvin, Norman; Haynes, Barton F.; Hahn, Beatrice H.; Yusim, Karina; Kuiken, Carla

    2012-02-21

    The present invention relates to mosaic clade M HIV-1 Nef polypeptides and to compositions comprising same. The polypeptides of the invention are suitable for use in inducing an immune response to HIV-1 in a human.

  19. Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration.

    PubMed

    Campbell, Grant R; Rawat, Pratima; Bruckman, Rachel S; Spector, Stephen A

    2015-06-01

    HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival. PMID:26115100

  20. Human Immunodeficiency Virus Type 1 Nef Inhibits Autophagy through Transcription Factor EB Sequestration

    PubMed Central

    Campbell, Grant R.; Rawat, Pratima; Bruckman, Rachel S.; Spector, Stephen A.

    2015-01-01

    HIV Nef acts as an anti-autophagic maturation factor through interaction with beclin-1 (BECN1). We report that exposure of macrophages to infectious or non-infectious purified HIV induces toll-like receptor 8 (TLR8) and BECN1 dependent dephosphorylation and nuclear translocation of TFEB and that this correlates with an increase in autophagy markers. RNA interference for ATG13, TFEB, TLR8, or BECN1 inhibits this HIV-induced autophagy. However, once HIV establishes a productive infection, TFEB phosphorylation and cytoplasmic sequestration are increased resulting in decreased autophagy markers. Moreover, by 7 d post-infection, autophagy levels are similar to mock infected controls. Conversely, although Nef deleted HIV similarly induces TFEB dephosphorylation and nuclear localization, and increases autophagy, these levels remain elevated during continued productive infection. Thus, the interaction between HIV and TLR8 serves as a signal for autophagy induction that is dependent upon the dephosphorylation and nuclear translocation of TFEB. During permissive infection, Nef binds BECN1 resulting in mammalian target of rapamycin (MTOR) activation, TFEB phosphorylation and cytosolic sequestration, and the inhibition of autophagy. To our knowledge, this is the first report of a virus modulating TFEB localization and helps to explain how HIV modulates autophagy to promote its own replication and cell survival. PMID:26115100

  1. High level nuclear waste

    SciTech Connect

    Crandall, J L

    1980-01-01

    The DOE Division of Waste Products through a lead office at Savannah River is developing a program to immobilize all US high-level nuclear waste for terminal disposal. DOE high-level wastes include those at the Hanford Plant, the Idaho Chemical Processing Plant, and the Savannah River Plant. Commercial high-level wastes, for which DOE is also developing immobilization technology, include those at the Nuclear Fuel Services Plant and any future commercial fuels reprocessing plants. The first immobilization plant is to be the Defense Waste Processing Facility at Savannah River, scheduled for 1983 project submission to Congress and 1989 operation. Waste forms are still being selected for this plant. Borosilicate glass is currently the reference form, but alternate candidates include concretes, calcines, other glasses, ceramics, and matrix forms.

  2. Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue.

    PubMed Central

    Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

    1992-01-01

    The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to HIV-1-tat stained solitary cells in the germinal centers and interfollicular zones, and vascular endothelium. Staining by an anti-nef monoclonal antibody was restricted to follicular dendritic cells, whereas anti-rev antibody bound to fibriohistiocytes and high endothelial venules. The antibodies used labeled several cell types in tissues from uninfected individuals. Anti-HIV-1-tat antibodies labeled blood vessels and Hassall's corpuscles in skin and thymus; goblet cells in intestinal tissue and trachea; neural cells in brain and spinal cord; and zymogen-producing cells in pancreas. Anti-rev antibody stained high endothelial venules, Hassall's corpuscles and histiocytes. One anti-nef antibody solely stained follicular dendritic cells in spleen, tonsil, lymph node and Peyer's patches, whereas two other anti-nef antibodies bound to astrocytes, solitary cells in the interfollicular zones of lymph nodes, and skin cells. The current results hamper the immunohistochemical study for pathogenetic and diagnostic use of HIV regulatory protein expression in infected tissue specimens or cells. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1279980

  3. Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex

    PubMed Central

    Imle, Andrea; Abraham, Libin; Tsopoulidis, Nikolaos; Hoflack, Bernard; Saksela, Kalle

    2015-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics. PMID:26350970

  4. Formation of a unique cluster of G-quadruplex structures in the HIV-1 Nef coding region: implications for antiviral activity.

    PubMed

    Perrone, Rosalba; Nadai, Matteo; Poe, Jerrod A; Frasson, Ilaria; Palumbo, Manlio; Palù, Giorgio; Smithgall, Thomas E; Richter, Sara N

    2013-01-01

    G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment. PMID:24015290

  5. Formation of a Unique Cluster of G-Quadruplex Structures in the HIV-1 nef Coding Region: Implications for Antiviral Activity

    PubMed Central

    Perrone, Rosalba; Nadai, Matteo; Poe, Jerrod A.; Frasson, Ilaria; Palumbo, Manlio; Palù, Giorgio; Smithgall, Thomas E.; Richter, Sara N.

    2013-01-01

    G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis invitro and invivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment. PMID:24015290

  6. A Genome-Wide Screen for Machinery Involved in Downregulation of MHC Class I by HIV-1 Nef

    PubMed Central

    Choma, Maja K.; Lumb, Jennifer; Kozik, Patrycja; Robinson, Margaret S.

    2015-01-01

    The HIV-1-encoded protein, Nef, plays a key role in the development of AIDS. One of Nef’s functions is to keep MHC class I off the surface of infected cells, a process that requires the host proteins clathrin and AP-1. To identify other proteins involved in this pathway, we carried out a genome-wide siRNA library screen on HeLa cells co-expressing HLA-A2 and an inducible form of Nef. Out of 21,121 siRNA pools, 100 were selected for further analysis, based on their ability to either inhibit or enhance downregulation of MHC-I by Nef. When cells were treated with the same siRNA pools as those used in the screen, 79% produced a similar phenotype. However, when the cells were treated with different siRNA reagents targeting the same genes, only 16% produced a similar phenotype. This indicates that most of the hits found in the original screen are likely to have been off-target, an important concern that is often not taken into account in siRNA screening studies. Nevertheless, we identified novel host factors involved in Nef-induced downregulation of MHC-I, including four genes, MIIP, CAMSAP3, SLC6A3, and KCTD19, where multiple reagents produced a strong inhibitory effect on Nef activity. Other hits slightly below our very high stringency cutoff point may also deserve further study. Thus, our dataset is a valuable resource for scientists investigating the pathogenesis of HIV. PMID:26466362

  7. Tracking the Emergence of Host-Specific Simian Immunodeficiency Virus env and nef Populations Reveals nef Early Adaptation and Convergent Evolution in Brain of Naturally Progressing Rhesus Macaques

    PubMed Central

    Lamers, Susanna L.; Nolan, David J.; Rife, Brittany D.; Fogel, Gary B.; McGrath, Michael S.; Burdo, Tricia H.; Autissier, Patrick; Williams, Kenneth C.; Goodenow, Maureen M.

    2015-01-01

    ABSTRACT While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (∼92 days) before they were detected in gp120 (∼182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains. IMPORTANCE The SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral

  8. Live Attenuated Rev-Independent Nef¯SIV Enhances Acquisition of Heterologous SIVsmE660 in Acutely Vaccinated Rhesus Macaques

    PubMed Central

    Byrareddy, Siddappa N.; Ayash-Rashkovsky, Mila; Kramer, Victor G.; Lee, Sandra J.; Correll, Mick; Novembre, Francis J.; Villinger, Francois; Johnson, Welkin E.; von Gegerfelt, Agneta; Felber, Barbara K.; Ruprecht, Ruth M.

    2013-01-01

    Background Rhesus macaques (RMs) inoculated with live-attenuated Rev-Independent Nef¯ simian immunodeficiency virus (Rev-Ind Nef¯SIV) as adults or neonates controlled viremia to undetectable levels and showed no signs of immunodeficiency over 6-8 years of follow-up. We tested the capacity of this live-attenuated virus to protect RMs against pathogenic, heterologous SIVsmE660 challenges. Methodology/Principal Findings Three groups of four RM were inoculated with Rev-Ind Nef¯SIV and compared. Group 1 was inoculated 8 years prior and again 15 months before low dose intrarectal challenges with SIVsmE660. Group 2 animals were inoculated with Rev-Ind Nef¯SIV at 15 months and Group 3 at 2 weeks prior to the SIVsmE660 challenges, respectively. Group 4 served as unvaccinated controls. All RMs underwent repeated weekly low-dose intrarectal challenges with SIVsmE660. Surprisingly, all RMs with acute live-attenuated virus infection (Group 3) became superinfected with the challenge virus, in contrast to the two other vaccine groups (Groups 1 and 2) (P=0.006 for each) and controls (Group 4) (P=0.022). Gene expression analysis showed significant upregulation of innate immune response-related chemokines and their receptors, most notably CCR5 in Group 3 animals during acute infection with Rev-Ind Nef¯SIV. Conclusions/Significance We conclude that although Rev-Ind Nef¯SIV remained apathogenic, acute replication of the vaccine strain was not protective but associated with increased acquisition of heterologous mucosal SIVsmE660 challenges. PMID:24098702

  9. Characterization of CD8+ T Cell Differentiation following SIVΔnef Vaccination by Transcription Factor Expression Profiling

    PubMed Central

    Billingsley, James M.; Rajakumar, Premeela A.; Connole, Michelle A.; Salisch, Nadine C.; Adnan, Sama; Kuzmichev, Yury V.; Hong, Henoch S.; Reeves, R. Keith; Kang, Hyung-joo; Li, Wenjun; Li, Qingsheng; Haase, Ashley T.; Johnson, R. Paul

    2015-01-01

    The onset of protective immunity against pathogenic SIV challenge in SIVΔnef-vaccinated macaques is delayed for 15-20 weeks, a process that is related to qualitative changes in CD8+ T cell responses induced by SIVΔnef. As a novel approach to characterize cell differentiation following vaccination, we used multi-target qPCR to measure transcription factor expression in naïve and memory subsets of CD8++ T cells, and in SIV-specific CD8+ T cells obtained from SIVΔnef-vaccinated or wild type SIVmac239-infected macaques. Unsupervised clustering of expression profiles organized naïve and memory CD8+ T cells into groups concordant with cell surface phenotype. Transcription factor expression patterns in SIV-specific CD8+ T cells in SIVΔnef-vaccinated animals were distinct from those observed in purified CD8+ T cell subsets obtained from naïve animals, and were intermediate to expression profiles of purified central memory and effector memory T cells. Expression of transcription factors elicited by SIVΔnef vaccination also varied over time: cells obtained at later time points, temporally associated with greater protection, appeared more central-memory like than cells obtained at earlier time points, which appeared more effector memory-like. Expression of transcription factors associated with effector differentiation, such as ID2 and RUNX3, were decreased over time, while expression of transcription factors associated with quiescence or memory differentiation, such as TCF7, BCOR and EOMES, increased. CD8+ T cells specific for a more conserved epitope expressed higher levels of TBX21 and BATF, and appeared more effector-like than cells specific for an escaped epitope, consistent with continued activation by replicating vaccine virus. These data suggest transcription factor expression profiling is a novel method that can provide additional data complementary to the analysis of memory cell differentiation based on classical phenotypic markers. Additionally, these data

  10. Nef gene evolution from a single transmitted strain in acute SIV infection

    PubMed Central

    Bimber, Benjamin N; Chugh, Pauline; Giorgi, Elena E; Kim, Baek; Almudevar, Anthony L; Dewhurst, Stephen; O'Connor, David H; Lee, Ha Youn

    2009-01-01

    Background The acute phase of immunodeficiency virus infection plays a crucial role in determining steady-state virus load and subsequent progression of disease in both humans and nonhuman primates. The acute period is also the time when vaccine-mediated effects on host immunity are likely to exert their major effects on virus infection. Recently we developed a Monte-Carlo (MC) simulation with mathematical analysis of viral evolution during primary HIV-1 infection that enables classification of new HIV-1 infections originating from multiple versus single transmitted viral strains and the estimation of time elapsed following infection. Results A total of 322 SIV nef SIV sequences, collected during the first 3 weeks following experimental infection of two rhesus macaques with the SIVmac239 clone, were analyzed and found to display a comparable level of genetic diversity, 0.015% to 0.052%, with that of env sequences from acute HIV-1 infection, 0.005% to 0.127%. We confirmed that the acute HIV-1 infection model correctly identified the experimental SIV infections in rhesus macaques as "homogenous" infections, initiated by a single founder strain. The consensus sequence of the sampled strains corresponded to the transmitted sequence as the model predicted. However, measured sequential decrease in diversity at day 7, 11, and 18 post infection violated the model assumption, neutral evolution without any selection. Conclusion While nef gene evolution over the first 3 weeks of SIV infection originating from a single transmitted strain showed a comparable rate of sequence evolution to that observed during acute HIV-1 infection, a purifying selection for the founder nef gene was observed during the early phase of experimental infection of a nonhuman primate. PMID:19505314

  11. Nef Enhances HIV-1 Infectivity via Association with the Virus Assembly Complex

    PubMed Central

    Qi, Mingli; Aiken, Christopher

    2008-01-01

    The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle. PMID:18191978

  12. Nef enhances HIV-1 infectivity via association with the virus assembly complex

    SciTech Connect

    Qi Mingli; Aiken, Christopher

    2008-04-10

    The HIV-1 accessory protein Nef enhances virus infectivity by facilitating an early post-entry step of infection. Nef acts in the virus producer cell, leading to a beneficial modification to HIV-1 particles. Nef itself is incorporated into HIV-1 particles, where it is cleaved by the viral protease during virion maturation. To probe the role of virion-associated Nef in HIV-1 infection, we generated a fusion protein consisting of the host protein cyclophilin A (CypA) linked to the amino terminus of Nef. The resulting CypA-Nef protein enhanced the infectivity of Nef-defective HIV-1 particles and was specifically incorporated into the virions via association with Gag during particle assembly. Pharmacologic or genetic inhibition of CypA-Nef binding to Gag prevented incorporation of CypA-Nef into virions and inhibited infectivity enhancement. Our results indicate that infectivity enhancement by Nef requires its association with a component of the assembling HIV-1 particle.

  13. High blood cholesterol levels

    MedlinePlus

    Steps you can take to improve their cholesterol levels, and help prevent heart disease and a heart attack include: Quit smoking. This is the single biggest change you can make to reduce your risk of heart attack and stroke. Eat foods ...

  14. Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants.

    PubMed

    Wales, Thomas E; Poe, Jerrod A; Emert-Sedlak, Lori; Morgan, Christopher R; Smithgall, Thomas E; Engen, John R

    2016-06-01

    Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure. If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time, and protease digestion patterns all complicate the data analysis. We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain. Regions with similar sequence could be compared between variants. Although much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same. These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation. The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS. Graphical Abstract ᅟ. PMID:27032648

  15. Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants

    NASA Astrophysics Data System (ADS)

    Wales, Thomas E.; Poe, Jerrod A.; Emert-Sedlak, Lori; Morgan, Christopher R.; Smithgall, Thomas E.; Engen, John R.

    2016-03-01

    Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure. If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time, and protease digestion patterns all complicate the data analysis. We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain. Regions with similar sequence could be compared between variants. Although much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same. These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation. The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS.

  16. Hydrogen Exchange Mass Spectrometry of Related Proteins with Divergent Sequences: A Comparative Study of HIV-1 Nef Allelic Variants

    NASA Astrophysics Data System (ADS)

    Wales, Thomas E.; Poe, Jerrod A.; Emert-Sedlak, Lori; Morgan, Christopher R.; Smithgall, Thomas E.; Engen, John R.

    2016-06-01

    Hydrogen exchange mass spectrometry can be used to compare the conformation and dynamics of proteins that are similar in tertiary structure. If relative deuterium levels are measured, differences in sequence, deuterium forward- and back-exchange, peptide retention time, and protease digestion patterns all complicate the data analysis. We illustrate what can be learned from such data sets by analyzing five variants (Consensus G2E, SF2, NL4-3, ELI, and LTNP4) of the HIV-1 Nef protein, both alone and when bound to the human Hck SH3 domain. Regions with similar sequence could be compared between variants. Although much of the hydrogen exchange features were preserved across the five proteins, the kinetics of Nef binding to Hck SH3 were not the same. These observations may be related to biological function, particularly for ELI Nef where we also observed an impaired ability to downregulate CD4 surface presentation. The data illustrate some of the caveats that must be considered for comparison experiments and provide a framework for investigations of other protein relatives, families, and superfamilies with HX MS.

  17. Relative Resistance of HLA-B to Downregulation by Naturally Occurring HIV-1 Nef Sequences

    PubMed Central

    Mahiti, Macdonald; Toyoda, Mako; Jia, Xiaofei; Kuang, Xiaomei T.; Mwimanzi, Francis; Mwimanzi, Philip; Walker, Bruce D.; Xiong, Yong; Brumme, Zabrina L.; Brockman, Mark A.

    2016-01-01

    ABSTRACT HIV-1 Nef binds to the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules from the surface of virus-infected cells, thus evading immune detection by CD8+ T cells. Polymorphic residues within the HLA cytoplasmic region may affect Nef’s downregulation activity. However, the effects of HLA polymorphisms on recognition by primary Nef isolates remain elusive, as do the specific Nef regions responsible for downregulation of HLA-A versus HLA-B. Here, we examined 46 Nef clones isolated from chronically HIV-1 subtype B-infected subjects for their ability to downregulate various HLA-A, HLA-B, and HLA-C molecules on the surface of virus-infected cells. Overall, HLA-B exhibited greater resistance to Nef-mediated downregulation than HLA-A, regardless of the cell type examined. As expected, no Nef clone downregulated HLA-C. Importantly, the differential abilities of patient-derived Nef clones to downregulate HLA-A and HLA-B correlated inversely with the sensitivities of HIV-infected target cells to recognition by effector cells expressing an HIV-1 Gag-specific T cell receptor. Nef codon function analysis implicated amino acid variation at position 202 (Nef-202) in differentially affecting the ability to downregulate HLA-A and HLA-B, an observation that was subsequently confirmed by experiments using Nef mutants constructed by site-directed mutagenesis. The in silico and mutagenesis analyses further suggested that Nef-202 may interact with the C-terminal Cys-Lys-Val residues of HLA-A, which are absent in HLA-B. Taken together, the results show that natural polymorphisms within Nef modulate its interaction with natural polymorphisms in the HLA cytoplasmic tails, thereby affecting the efficiency of HLA downregulation and consequent recognition by HIV-specific T cells. These results thus extend our understanding of this complex pathway of retroviral immune evasion. PMID:26787826

  18. Mechanistic Independence of Nef and Cyclophilin A Enhancement of Human Immunodeficiency Virus Type 1 Infectivity

    PubMed Central

    Aiken, Christopher

    2013-01-01

    Optimal HIV-1 infectivity requires the presence of both the viral factor Nef and the cellular protein cyclophilin A (CyPA) during virion assembly. These two proteins are integral components of HIV-1 particles. Both CyPA and Nef facilitate a step in the viral life cycle occurring between penetration and reverse transcription, suggesting a common mechanism of action. Experiments were performed to test the potential interplay of Nef- and CyPA-mediated enhancement of HIV-1 infectivity. In single-cycle infection assays, nef-defective virions were partially resistant to cyclosporin A (CsA), a drug which inhibits the binding of CyPA to the HIV-1 Gag precursor and CyPA incorporation into virions. Genetic dissection of the relative contributions of Nef and the cyclophilin A-Gag interaction to HIV-1 infectivity demonstrated the independence of these two effects. Nef was not required for incorporation of CyPA into HIV-1 virions, and vice-versa. Surprisingly, CyPA-deficient virions remained sensitive to inhibition by CsA, in a manner that depended strongly on the presence of a functional nef gene. These results demonstrate that Nef and CyPA act independently to render HIV-1 particles fully infectious. They further suggest that in addition to blocking the CyPA-Gag interaction, CsA can also inhibit HIV-1 replication through a novel mechanism involving suppression of Nef-directed enhancement of virus infectivity. PMID:9705263

  19. Gene expression profiling reveals Nef induced deregulation of lipid metabolism in HIV-1 infected T cells.

    PubMed

    Shrivastava, Surya; Trivedi, Jay; Mitra, Debashis

    2016-03-25

    Human Immunodeficiency Virus-1 (HIV-1) encodes a 27 kDa Negative Factor or Nef protein, which is increasingly proving to be a misnomer. Nef seems to be crucial for AIDS progression as individuals infected with nef-deleted strain of HIV were reported to become Long Term Non Progressors (LTNP). These findings necessitate tracing of Nef's footprint on landscape of cellular transcriptome favoring HIV-1 pathogenesis. We have tried to explore effect of Nef on cellular gene expression profile in conjunction with rest of HIV-1 proteins. Our results show that 237 genes are differentially regulated due to the presence of Nef during infection, which belong to several broad categories like "signaling", "apoptosis", "transcription" and "lipid metabolism" in gene ontology analysis. Furthermore, our results show that Nef causes disruption of lipid content in HIV-1 infected T cells. Molecular inhibitors of lipid metabolism like Atorvastatin and Ranolazine were found to have profound effect on wild type virus as compared to nef-deleted HIV-1. Thus our results suggest that interference in lipid metabolism is a potential mechanism through which Nef contributes in enhancing HIV-1 pathogenesis. PMID:26915805

  20. HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling.

    PubMed

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal

    2016-05-20

    HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE65 acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. PMID:27105913

  1. Neutron Reflectometry Study of the Conformation of HIV Nef Bound to Lipid Membranes

    PubMed Central

    Kent, Michael S.; Murton, Jaclyn K.; Sasaki, Darryl Y.; Satija, Sushil; Akgun, Bulent; Nanda, Hirsh; Curtis, Joseph E.; Majewski, Jaroslaw; Morgan, Christopher R.; Engen, John R.

    2010-01-01

    Nef is an HIV-1 accessory protein that directly contributes to AIDS progression. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its medical relevance, to our knowledge there is no direct information about the conformation of membrane-bound Nef. In this work, we used neutron reflection to reveal what we believe are the first details of the conformation of membrane-bound Nef. The conformation of Nef was probed upon binding to Langmuir monolayers through the interaction of an N-terminal His tag with a synthetic metal-chelating lipid, which models one of the possible limiting cases for myr-Nef. The data indicate that residues are inserted into the lipid headgroups during interaction, and that the core domain lies directly against the lipid headgroups, with a thickness of ∼40 Å. Binding of Nef through the N-terminal His tag apparently facilitates insertion of residues, as no insertion occurred upon binding of Nef through weak electrostatic interactions in the absence of the specific interaction through the His tag. PMID:20858440

  2. Structural basis of evasion of cellular adaptive immunity by HIV-1 Nef

    SciTech Connect

    Jia, Xiaofei; Singh, Rajendra; Homann, Stefanie; Yang, Haitao; Guatelli, John; Xiong, Yong

    2012-10-24

    The HIV-1 protein Nef inhibits antigen presentation by class I major histocompatibility complex (MHC-I). We determined the mechanism of this activity by solving the crystal structure of a protein complex comprising Nef, the MHC-I cytoplasmic domain (MHC-I CD) and the {mu}1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-{mu}1 interface, which encompasses the cargo-recognition site of {mu}1 and the proline-rich strand of Nef. The Nef C terminus induces a previously unobserved conformational change in {mu}1, whereas the N terminus binds the Nef core to position it optimally for complex formation. Positively charged patches on {mu}1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity.

  3. Structural Basis of Evasion of Cellular Adaptive Immunity by HIV-1 Nef

    PubMed Central

    Jia, Xiaofei; Singh, Rajendra; Homann, Stefanie; Yang, Haitao; Guatelli, John; Xiong, Yong

    2012-01-01

    The HIV-1 protein Nef inhibits antigen presentation by class I MHC (MHC-I). Here the mechanism of this activity is revealed by the crystal structure of a protein complex consisting of Nef, the MHC-I cytoplasmic domain (MHC-I CD), and the μ1 subunit of the clathrin adaptor protein complex 1. A ternary, cooperative interaction clamps the MHC-I CD into a narrow binding groove at the Nef-μ1 interface encompassing the cargo-recognition site of μ1 and the proline rich strand of Nef. The Nef C-terminus induces a novel conformational change in μ1, while the N-terminus binds the Nef core to position it optimally for complex formation. Positively charged patches on μ1 recognize acidic clusters in Nef and MHC-I. The structure shows how Nef functions as a clathrin-associated sorting protein to alter the specificity of host membrane trafficking and enable viral evasion of adaptive immunity. PMID:22705789

  4. Nef Does Not Affect the Efficiency of Human Immunodeficiency Virus Type 1 Fusion with Target Cells

    PubMed Central

    Tobiume, Minoru; Lineberger, Janet E.; Lundquist, Christopher A.; Miller, Michael D.; Aiken, Christopher

    2003-01-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by an unknown mechanism. Recent studies have suggested that Nef may act by regulating the efficiency of virus entry into cells. Here we provide evidence to the contrary. Using a quantitative assay of HIV-1 virus-cell fusion, we observed equivalent rates and extents of fusion of wild-type and Nef-defective HIV-1 particles with MT-4 cells and CD4-expressing HeLa cells. In studies using soluble CD4 (sCD4) to inhibit infection, wild-type and Nef-defective HIV-1 escaped the sCD4 block with similar kinetics. We conclude that Nef acts at a postentry step in infection, probably by facilitating intracellular transport of the HIV-1 ribonucleoprotein complex. PMID:12970449

  5. High-level-waste immobilization

    SciTech Connect

    Crandall, J L

    1982-01-01

    Analysis of risks, environmental effects, process feasibility, and costs for disposal of immobilized high-level wastes in geologic repositories indicates that the disposal system safety has a low sensitivity to the choice of the waste disposal form.

  6. Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements.

    PubMed

    van der Velden, Yme U; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T

    2016-01-15

    Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. PMID:26615334

  7. HIV-1 Nef Inhibits Ruffles, Induces Filopodia, and Modulates Migration of Infected Lymphocytes▿

    PubMed Central

    Nobile, Cinzia; Rudnicka, Dominika; Hasan, Milena; Aulner, Nathalie; Porrot, Françoise; Machu, Christophe; Renaud, Olivier; Prévost, Marie-Christine; Hivroz, Claire; Schwartz, Olivier; Sol-Foulon, Nathalie

    2010-01-01

    The HIV-1 Nef protein is a pathogenic factor modulating the behavior of infected cells. Nef induces actin cytoskeleton changes and impairs cell migration toward chemokines. We further characterized the morphology, cytoskeleton dynamics, and motility of HIV-1-infected lymphocytes. By using scanning electron microscopy, confocal immunofluorescence microscopy, and ImageStream technology, which combines flow cytometry and automated imaging, we report that HIV-1 induces a characteristic remodeling of the actin cytoskeleton. In infected lymphocytes, ruffle formation is inhibited, whereas long, thin filopodium-like protrusions are induced. Cells infected with HIV with nef deleted display a normal phenotype, and Nef expression alone, in the absence of other viral proteins, induces morphological changes. We also used an innovative imaging system to immobilize and visualize living individual cells in suspension. When combined with confocal “axial tomography,” this technique greatly enhances three-dimensional optical resolution. With this technique, we confirmed the induction of long filopodium-like structures in unfixed Nef-expressing lymphocytes. The cytoskeleton reorganization induced by Nef is associated with an important impairment of cell movements. The adhesion and spreading of infected cells to fibronectin, their spontaneous motility, and their migration toward chemokines (CXCL12, CCL3, and CCL19) were all significantly decreased. Therefore, Nef induces complex effects on the lymphocyte actin cytoskeleton and cellular morphology, which likely impacts the capacity of infected cells to circulate and to encounter and communicate with bystander cells. PMID:20015995

  8. Mutational analysis of the human immunodeficiency virus type 1 Eli Nef function.

    PubMed Central

    Zazopoulos, E; Haseltine, W A

    1992-01-01

    The studies presented here define an internally consistent experimental system that permits systematic analysis of the effect of nef on the rate of the human immunodeficiency virus type 1 (HIV-1) replication in a CD4+ tumor T-cell line and in primary peripheral blood mononuclear cells. The parental full-length Nef protein, derived from the Eli strain of HIV-1, accelerates virus replication in both cell types. Mutations that destabilize or alter the intracellular location of the protein affect the ability of the Nef protein to accelerate virus replication. A set of mutants was made in amino acids proposed to be required for Nef function, including threonine and serine residues proposed to be targets for phosphorylation, and in sequences thought to resemble the G-1, G-3, and G-4 sites of the family of G proteins. In most cases alterations of the critical amino acids yield stable Nef proteins of parental phenotype. These results challenge the existing theories for the mechanism of Nef function. The results also identify two residues in the carboxyl half of the protein that are important for Nef function. Images PMID:1631166

  9. High pressure liquid level monitor

    DOEpatents

    Bean, Vern E.; Long, Frederick G.

    1984-01-01

    A liquid level monitor for tracking the level of a coal slurry in a high-pressure vessel including a toroidal-shaped float with magnetically permeable bands thereon disposed within the vessel, two pairs of magnetic field generators and detectors disposed outside the vessel adjacent the top and bottom thereof and magnetically coupled to the magnetically permeable bands on the float, and signal processing circuitry for combining signals from the top and bottom detectors for generating a monotonically increasing analog control signal which is a function of liquid level. The control signal may be utilized to operate high-pressure control valves associated with processes in which the high-pressure vessel is used.

  10. Nef Enhances Human Immunodeficiency Virus Type 1 Infectivity Resulting from Intervirion Fusion: Evidence Supporting a Role for Nef at the Virion Envelope

    PubMed Central

    Zhou, Jing; Aiken, Christopher

    2001-01-01

    The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by facilitating an early event in the HIV-1 life cycle. Although no structural or biochemical defects in Nef-defective HIV-1 particles have been demonstrated, the Nef protein is incorporated into HIV-1 particles. To localize the function of Nef within the virus particle, we developed a novel technology involving fusion of enveloped donor HIV-1 particles bearing core defects with envelope-defective target virions bearing HIV-1 receptors. Although neither virus alone was capable of infecting CD4+ target cells, the incubation of donor and target virions prior to addition to target cells resulted in infection. This effect, termed “virion transcomplementation,” required a functional Env protein on the donor virus and CD4 and an appropriate coreceptor on target virions. To provide evidence for intervirion fusion as the mechanism of complementation, experiments were performed using dual-enveloped HIV-1 particles bearing both HIV-1 and ecotropic murine leukemia virus (E-MLV) Env proteins as donor virions. Infection of CD4-negative target cells bearing E-MLV receptors was prevented by HIV-1 entry inhibitors when added before, but not after, incubation of donor and target virions prior to the addition to cells. When we used Nef+ and Nef− donor and target virions, Nef enhanced infection when present in donor virions. In contrast, no effect of Nef was detected when present in the target virus. These results reveal a potential mechanism for enhancing HIV-1 diversity in vivo through the rescue of defective viral genomes and provide a novel genetic system for the functional analysis of virion-associated proteins in HIV-1 infection. PMID:11390586

  11. Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8+ T-Cell Responses Focused on a Single Nef Epitope Select for Escape Variants Shortly after Infection

    PubMed Central

    Tully, Damien C.; Cruz, Michael A.; Power, Karen A.; Veloso de Santana, Marlon G.; Bean, David J.; Ogilvie, Colin B.; Gadgil, Rujuta; Lima, Noemia S.; Magnani, Diogo M.; Ejima, Keisuke; Allison, David B.; Piatak, Michael; Altman, John D.; Parks, Christopher L.; Rakasz, Eva G.; Capuano, Saverio; Galler, Ricardo; Bonaldo, Myrna C.; Lifson, Jeffrey D.; Allen, Todd M.; Watkins, David I.

    2015-01-01

    ABSTRACT Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed “elite controllers” [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08+ animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08+ macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8+ T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8+ T-cell response would facilitate the development of elite control in Mamu-B*08+ animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08+ animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8+ T cells. These vaccine-induced effector memory CD8+ T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8+ T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08+ macaques. IMPORTANCE Since elite control of chronic-phase viremia is a classic

  12. High-Level Radioactive Waste.

    ERIC Educational Resources Information Center

    Hayden, Howard C.

    1995-01-01

    Presents a method to calculate the amount of high-level radioactive waste by taking into consideration the following factors: the fission process that yields the waste, identification of the waste, the energy required to run a 1-GWe plant for one year, and the uranium mass required to produce that energy. Briefly discusses waste disposal and…

  13. The CMS high level trigger

    NASA Astrophysics Data System (ADS)

    Gori, Valentina

    2014-05-01

    The CMS experiment has been designed with a 2-level trigger system: the Level 1 Trigger, implemented on custom-designed electronics, and the High Level Trigger (HLT), a streamlined version of the CMS offline reconstruction software running on a computer farm. A software trigger system requires a tradeoff between the complexity of the algorithms running on the available computing power, the sustainable output rate, and the selection efficiency. Here we will present the performance of the main triggers used during the 2012 data taking, ranging from simpler single-object selections to more complex algorithms combining different objects, and applying analysis-level reconstruction and selection. We will discuss the optimisation of the triggers and the specific techniques to cope with the increasing LHC pile-up, reducing its impact on the physics performance.

  14. The CMS High Level Trigger

    NASA Astrophysics Data System (ADS)

    Trocino, Daniele

    2014-06-01

    The CMS experiment has been designed with a two-level trigger system: the Level-1 Trigger, implemented in custom-designed electronics, and the High-Level Trigger (HLT), a streamlined version of the CMS offline reconstruction software running on a computer farm. A software trigger system requires a tradeoff between the complexity of the algorithms running with the available computing power, the sustainable output rate, and the selection efficiency. We present the performance of the main triggers used during the 2012 data taking, ranging from simple single-object selections to more complex algorithms combining different objects, and applying analysis-level reconstruction and selection. We discuss the optimisation of the trigger and the specific techniques to cope with the increasing LHC pile-up, reducing its impact on the physics performance.

  15. Disassembly of Human Immunodeficiency Virus Type 1 Cores In Vitro Reveals Association of Nef with the Subviral Ribonucleoprotein Complex

    PubMed Central

    Forshey, Brett M.; Aiken, Christopher

    2003-01-01

    The human immunodeficiency virus type 1 (HIV-1) virulence factor Nef enhances viral infectivity in single-cycle infection assays and accelerates HIV-1 replication in vitro. It has been reported that the effects of Nef are mediated early after viral entry and before the completion of reverse transcription, as viral DNA synthesis is strongly attenuated during infection by Nef-defective virions. Our previous work has demonstrated that Nef is associated with mature HIV-1 cores, implicating Nef in the regulation of HIV-1 core stability. Here we report a comparative analysis of HIV-1 cores isolated from wild-type and Nef-defective particles. We observed no effect of Nef on HIV-1 core structure or stability; however, Nef cosedimented with a subviral ribonucleoprotein complex following dissociation of CA. These results indicate that Nef interacts tightly with an internal component of the HIV-1 core. They further suggest that virion-associated Nef may facilitate an early step in HIV-1 infection following dissociation of the viral capsid in the target cell. PMID:12634398

  16. Human immunodeficiency virus (HIV) nef-specific cytotoxic T lymphocytes in noninfected heterosexual contact of HIV-infected patients.

    PubMed Central

    Langlade-Demoyen, P; Ngo-Giang-Huong, N; Ferchal, F; Oksenhendler, E

    1994-01-01

    We report on the detection of HIV-specific cytotoxic T lymphocytes (CTL) among 23 regular partners of HIV-infected individuals. 15 of the 46 individuals enrolled in the study were positive for HLA-A2.1 typing. Among the 23 contacts studied, 7 were seropositive and 16 were seronegative on repeated tests. None of the 16 seronegative contacts were positive for p24 antigenemia nor were they positive by the lymphocytes coculture assay, although, in two instances HIV-1 DNA could be detected by PCR (in one case using a gag SK 38/39 primer, and in the other using a primer for the pol P3/P4 primer). These two individuals remained seronegative for 18 and 36 mo, respectively. HIV-specific cytotoxicity was performed in the 15 HLA-A2.1 subjects (7 indexes, 2 seropositive contacts, and 6 seronegative contacts) and in 4 HLA-matched HIV negative donors. CTL specific for env, gag, or nef proteins could not be detected in unstimulated bulk cultures of peripheral blood lymphocytes in any of the six seronegative contacts. However, using a limiting dilution assay we found an usually high frequency of HIV nef-specific CTL precursors (CTLp) for HIV env and gag was very similar to that observed in seronegative HLA-matched healthy donors. Because no presence of HIV could be demonstrated in these individuals, these findings argue against the possibility of a silent HIV infection and suggest that a CTL response against nef may be involved in a rapid and effective clearance of the virus after sexual exposure. PMID:8132769

  17. Conformational transition of membrane-associated terminally-acylated HIV-1 Nef

    PubMed Central

    Akgun, Bulent; Satija, Sushil; Nanda, Hirsh; Pirrone, Gregory F.; Shi, Xiaomeng; Engen, John R.; Kent, Michael S.

    2013-01-01

    Many proteins are post-translationally modified by acylation targetting them to lipid membranes. While methods such as X-ray crystallography and NMR are available to determine the structure of folded proteins in solution, the precise position of folded domains relative to a membrane remains largely unknown. We used neutron and X-ray reflection methods to measure the displacement of the core domain of HIV Nef from lipid membranes upon insertion of the N-terminal myristate group. Nef is one of several HIV-1 accessory proteins and an essential factor in AIDS progression. Upon insertion of the myristate and residues from the N-terminal arm, Nef transitions from a closed to open conformation that positions the core domain 70 Å from the lipid headgroups. This work rules out speculation that the Nef core remains closely associated with the membrane to optimize interactions with the cytoplasmic domain of MHC-1. PMID:24035710

  18. p21-Activated Kinase 1 Plays a Critical Role in Cellular Activation by Nef

    PubMed Central

    Fackler, Oliver T.; Lu, Xiaobin; Frost, Jeffrey A.; Geyer, Matthias; Jiang, Bing; Luo, Wen; Abo, Arie; Alberts, Arthur S.; Peterlin, B. Matija

    2000-01-01

    The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef. PMID:10713183

  19. HIV-Nef and ADAM17-Containing Plasma Extracellular Vesicles Induce and Correlate with Immune Pathogenesis in Chronic HIV Infection

    PubMed Central

    Lee, Jung-Hyun; Schierer, Stephan; Blume, Katja; Dindorf, Jochen; Wittki, Sebastian; Xiang, Wei; Ostalecki, Christian; Koliha, Nina; Wild, Stefan; Schuler, Gerold; Fackler, Oliver T.; Saksela, Kalle; Harrer, Thomas; Baur, Andreas S.

    2016-01-01

    Antiretroviral therapy (ART) efficiently suppresses HIV replication but immune activation and low CD4 T cell counts often persist. The underlying mechanism of this ART-resistant pathogenesis is not clear. We observed that levels of plasma extracellular vesicles (pEV) are strongly elevated in HIV infection and do not decline during ART. Surprisingly, these vesicles contained the viral accessory proteins Nef and Vpu, which are assumed to be not expressed under efficient ART, as well as pro-inflammatory effectors, including activated ADAM17. HIV pEV were characterized by the presence of activated αvβ3 and absence of CD81 and Tsg101. Correlating with immune activation, peripheral monocytes ingested large amounts of pEV, giving rise to an increased population of CD1c+ CD14+ cells that secreted inflammatory cytokines. Importantly, the pro-inflammatory content, particularly ADAM17 activity, correlated with low T cell counts. Preliminary evidence suggested that HIV pEV derived from peripheral mononuclear cells and from an unknown myeloid cell population. In summary we propose an important role of pro-inflammatory pEV in chronic HIV infection due to ongoing viral Nef activity. PMID:27211553

  20. Selective Restriction of Nef-Defective Human Immunodeficiency Virus Type 1 by a Proteasome-Dependent Mechanism▿

    PubMed Central

    Qi, Mingli; Aiken, Christopher

    2007-01-01

    The Nef protein enhances human immunodeficiency virus type 1 (HIV-1) infectivity by facilitating an early postentry step in the virus life cycle. We report here that the addition of MG132 or lactacystin, each a specific inhibitor of cellular proteasome activity, preferentially enhances cellular permissiveness to infection by Nef-defective versus wild-type HIV-1. Pseudotyping by the glycoprotein of vesicular stomatitis virus rendered Nef-defective HIV-1 particles minimally responsive to the enhancing effects of proteasome inhibitors. These results suggest that Nef enhances the infectivity of HIV-1 particles by reducing their susceptibility to proteasomal degradation in target cells. PMID:17108041

  1. Human immunodeficiency virus type 1 Nef associates with a member of the p21-activated kinase family.

    PubMed Central

    Nunn, M F; Marsh, J W

    1996-01-01

    Although human immunodeficiency virus (HIV) Nef is essential for the induction of AIDS, its biochemical function has remained an enigma. In this study, HIV Nef protein is shown to associate with a serine-threonine kinase that recognizes histone H4 as a substrate, is serologically related to rat p21-activated kinase (PAK), and is specifically activated by Rac and Cdc42. These characteristics define the Nef-associated kinase as belonging to the PAK family. PAKs initiate kinase cascades in response to environmental stimuli, and their identification as a target of Nef implicates these signaling molecules in HIV pathogenesis and provides a novel target for clinical intervention. PMID:8709241

  2. A Bidirectional SF2/ASF- and SRp40-Dependent Splicing Enhancer Regulates Human Immunodeficiency Virus Type 1 rev, env, vpu, and nef Gene Expression

    PubMed Central

    Caputi, Massimo; Freund, Marcel; Kammler, Susanne; Asang, Corinna; Schaal, Heiner

    2004-01-01

    The integrated human immunodeficiency virus type 1 (HIV-1) genome is transcribed in a single pre-mRNA that is alternatively spliced into more than 40 mRNAs. We characterized a novel bidirectional exonic splicing enhancer (ESE) that regulates the expression of the HIV-1 env, vpu, rev, and nef mRNAs. The ESE is localized downstream of the vpu-, env-, and nef-specific 3′ splice site no. 5. SF2/ASF and SRp40 activate the ESE and are required for efficient 3′ splice site usage and binding of the U1 snRNP to the downstream 5′ splice site no. 4. U1 snRNP binding to the 5′ splice site no. 4 is required for splicing of the rev and nef mRNAs and to increase expression of the partially spliced env mRNA. Finally, our results indicate that this ESE is necessary for the recruitment of the U1 snRNP to the 5′ splice site no. 4, even when the 5′ splice site and the U1 snRNA have been mutated to obtain a perfect complementary match. The ESE characterized here is highly conserved in most viral subtypes. PMID:15163745

  3. Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

    PubMed Central

    Saxena, Reshu; Gupta, Sudipti; Singh, Kavita; Mitra, Kalyan; Tripathi, Anil Kumar; Tripathi, Raj Kamal

    2015-01-01

    Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 (55AAAAAAA61) and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T

  4. High level white noise generator

    DOEpatents

    Borkowski, Casimer J.; Blalock, Theron V.

    1979-01-01

    A wide band, stable, random noise source with a high and well-defined output power spectral density is provided which may be used for accurate calibration of Johnson Noise Power Thermometers (JNPT) and other applications requiring a stable, wide band, well-defined noise power spectral density. The noise source is based on the fact that the open-circuit thermal noise voltage of a feedback resistor, connecting the output to the input of a special inverting amplifier, is available at the amplifier output from an equivalent low output impedance caused by the feedback mechanism. The noise power spectral density level at the noise source output is equivalent to the density of the open-circuit thermal noise or a 100 ohm resistor at a temperature of approximately 64,000 Kelvins. The noise source has an output power spectral density that is flat to within 0.1% (0.0043 db) in the frequency range of from 1 KHz to 100 KHz which brackets typical passbands of the signal-processing channels of JNPT's. Two embodiments, one of higher accuracy that is suitable for use as a standards instrument and another that is particularly adapted for ambient temperature operation, are illustrated in this application.

  5. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation

    PubMed Central

    Rosa, Annachiara; Chande, Ajit; Ziglio, Serena; Sanctis, Veronica De; Bertorelli, Roberto; Goh, Shih Lin; McCauley, Sean M.; Nowosielska, Anetta; Antonarakis, Stylianos E.; Luban, Jeremy; Santoni, Federico Andrea; Pizzato, Massimo

    2016-01-01

    HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor. PMID:26416734

  6. Optimizing High Level Waste Disposal

    SciTech Connect

    Dirk Gombert

    2005-09-01

    If society is ever to reap the potential benefits of nuclear energy, technologists must close the fuel-cycle completely. A closed cycle equates to a continued supply of fuel and safe reactors, but also reliable and comprehensive closure of waste issues. High level waste (HLW) disposal in borosilicate glass (BSG) is based on 1970s era evaluations. This host matrix is very adaptable to sequestering a wide variety of radionuclides found in raffinates from spent fuel reprocessing. However, it is now known that the current system is far from optimal for disposal of the diverse HLW streams, and proven alternatives are available to reduce costs by billions of dollars. The basis for HLW disposal should be reassessed to consider extensive waste form and process technology research and development efforts, which have been conducted by the United States Department of Energy (USDOE), international agencies and the private sector. Matching the waste form to the waste chemistry and using currently available technology could increase the waste content in waste forms to 50% or more and double processing rates. Optimization of the HLW disposal system would accelerate HLW disposition and increase repository capacity. This does not necessarily require developing new waste forms, the emphasis should be on qualifying existing matrices to demonstrate protection equal to or better than the baseline glass performance. Also, this proposed effort does not necessarily require developing new technology concepts. The emphasis is on demonstrating existing technology that is clearly better (reliability, productivity, cost) than current technology, and justifying its use in future facilities or retrofitted facilities. Higher waste processing and disposal efficiency can be realized by performing the engineering analyses and trade-studies necessary to select the most efficient methods for processing the full spectrum of wastes across the nuclear complex. This paper will describe technologies being

  7. Nef-Mediated Downregulation of CD4 Enhances Human Immunodeficiency Virus Type 1 Replication in Primary T Lymphocytes

    PubMed Central

    Lundquist, Christopher A.; Tobiume, Minoru; Zhou, Jing; Unutmaz, Derya; Aiken, Christopher

    2002-01-01

    The accessory protein Nef plays a crucial role in primate lentivirus pathogenesis. Nef enhances human immunodeficiency virus type 1 (HIV-1) infectivity in culture and stimulates viral replication in primary T cells. In this study, we investigated the relationship between HIV-1 replication efficiency in CD4+ T cells purified from human blood and two various known activities of Nef, CD4 downregulation and single-cycle infectivity enhancement. Using a battery of reporter viruses containing point mutations in nef, we observed a strong genetic correlation between CD4 downregulation by Nef during acute HIV-1 infection of activated T cells and HIV-1 replication efficiency in T cells. In contrast, HIV-1 replication ability was not significantly correlated with the ability of Nef to enhance single-cycle virion infectivity, as determined by using viruses produced in cells lacking CD4. These results demonstrate that CD4 downregulation by Nef plays a crucial role in HIV-1 replication in activated T cells and underscore the potential for the development of therapies targeting this conserved activity of Nef. PMID:11932428

  8. Potential impact of viral load and genetic makeup of HIV type 1 on mother-to-child transmission: characterization of env-C2V3C3 and nef sequences.

    PubMed

    Pádua, Elizabeth; Parreira, Ricardo; Tendeiro, Rita; Nunes, Baltazar; Castela, João; Soares, Isabel; Mouzinho, Ana; Reis, Eduarda; Paixão, Maria Teresa

    2009-11-01

    HIV-1 mother-to-child transmission (MTCT) was evaluated in terms of the molecular characterization of the env and nef genomic regions and quantification of maternal RNA viral loads. Assignment of viral subtype was achieved by direct sequencing of PCR 1172 products amplified from proviral DNA in 45 HIV-1-nontransmitting mothers (NTM), along with 13 pairs of HIV-1-transmitting mothers (TM) and their infected children (C). Analysis of the env C2V3C3 and nef sequences revealed that subtypes G and B, and their genetic combinations (AG, BG), accounted for over 84.5% of all viruses identified. The genetic structure form envA-nefG was the most commonly observed, with a lower frequency in the NTM (13.3%) compared to the TM (23.1%) group. A greater number of genetic forms was observed among NTM, namely the presence of sequences assigned to subtypes D and F, as well as the intergenetic A/J, and C/U, recombinant forms, along with a mosaic provirus with a complex putative envA-nefEGE genetic structure. No significant differences were found when RNA viral loads were evaluated as a function of the viral subtypes. Nevertheless, a relatively high quantification of HIV-1 RNA was obtained in the NTM group, emphasizing the importance of the compliance and effectiveness of therapeutic schemes to control viral replication and reduce the risk of HIV vertical transmission. V3 sequences displaying features associated with the R5 phenotype dominated in both groups. Both C2V3C3 and Nef's functional domains were conserved during HIV-1 vertical transmission. PMID:19886833

  9. High temperature liquid level sensor

    DOEpatents

    Tokarz, Richard D.

    1983-01-01

    A length of metal sheathed metal oxide cable is perforated to permit liquid access to the insulation about a pair of conductors spaced close to one another. Changes in resistance across the conductors will be a function of liquid level, since the wetted insulation will have greater electrical conductivity than that of the dry insulation above the liquid elevation.

  10. High-Level Data Races

    NASA Technical Reports Server (NTRS)

    Artho, Cyrille; Havelund, Klaus; Biere, Armin; Koga, Dennis (Technical Monitor)

    2003-01-01

    Data races are a common problem in concurrent and multi-threaded programming. They are hard to detect without proper tool support. Despite the successful application of these tools, experience shows that the notion of data race is not powerful enough to capture certain types of inconsistencies occurring in practice. In this paper we investigate data races on a higher abstraction layer. This enables us to detect inconsistent uses of shared variables, even if no classical race condition occurs. For example, a data structure representing a coordinate pair may have to be treated atomically. By lifting the meaning of a data race to a higher level, such problems can now be covered. The paper defines the concepts view and view consistency to give a notation for this novel kind of property. It describes what kinds of errors can be detected with this new definition, and where its limitations are. It also gives a formal guideline for using data structures in a multi-threading environment.

  11. HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

    PubMed Central

    Park, In-Woo; Fan, Yan; Luo, Xiaoyu; Ryou, Myoung-Gwi; Liu, Jinfeng; Green, Linden; He, Johnny J.

    2014-01-01

    HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. PMID:24911518

  12. High fertility level in Tibet.

    PubMed

    Zhang, T

    1997-08-01

    This article presents a profile of fertility patterns in Tibet Autonomous Region for 1989. Data were obtained from the 1982 and 1990 China Censuses and from a 1988 fertility and contraception survey. Findings indicate that the total fertility rate (TFR) was 4.36 children/woman in 1989. TFR in Tibet is higher than in other provinces with a Tibetan population. Tibetan TFR was 4.07 among Tibetan women in Qinghai, 3.30 in Sichuan, 3.0 in Yunnan, and 2.8 in Gansu. TFR was 2.16 in Tibet's towns and 1.82 in Lhasa city, compared to 4.57 in counties. In Tibet, the birth rate rose from 31.05/1000 in 1981, to 31.14 in 1989. Ngari prefecture had an even higher birth rate of 35.67 in 1989. Other Tibetan prefectures had lower birth rates. During 1982-89, the birth rate declined to 26.19 in Lhasa City, to 29.36 in Shannan prefecture, and to 27.09 in Nagqu prefecture. Tibet's TFR fluctuated during 1950-57 between 3.20 and 3.80. It rose after 1958, from 4.0 to over 5.0, and remained high at 4.22 in 1989. TFR increased from older to younger women. The average number of live births also increased from older to younger women. This unusual feature is linked with high infertility and the number of women remaining celibate. Infertility was 17.7% among women aged 60-64 years, 14.8% among women aged 55-59, and 12.2% among women aged 50-54. 45.29% of women had parities of 1-2 children, ranging from 85.21% in the city, 77.92% in towns, and 42.20% in rural areas. The percentage of Tibetan women having 4 or more children was 5.82% in the city, 10.14% in towns, and 42.91% in rural areas. TFR was reversely correlated with educational status. TFR was highest among illiterates and semiliterates (4.59) and lowest for college educated women (1.28). Illiterate and semi-literate women comprised 13% of women with 4 children. PMID:12321529

  13. HIV-1 Nef promotes the localization of Gag to the cell membrane and facilitates viral cell-to-cell transfer

    PubMed Central

    2013-01-01

    Background Newly synthesized HIV-1 particles assemble at the plasma membrane of infected cells, before being released as free virions or being transferred through direct cell-to-cell contacts to neighboring cells. Localization of HIV-1 Gag precursor at the cell membrane is necessary and sufficient to trigger viral assembly, whereas the GagPol precursor is additionally required to generate a fully matured virion. HIV-1 Nef is an accessory protein that optimizes viral replication through partly defined mechanisms. Whether Nef modulates Gag and/or GagPol localization and assembly at the membrane and facilitates viral cell-to-cell transfer has not been extensively characterized so far. Results We report that Nef increases the total amount of Gag proteins present in infected cells, and promotes Gag localization at the cell membrane. Moreover, the processing of p55 into p24 is improved in the presence of Nef. We also examined the effect of Nef during HIV-1 cell-to-cell transfer. We show that without Nef, viral transfer through direct contacts between infected cells and target cells is impaired. With a nef-deleted virus, the number of HIV-1 positive target cells after a short 2h co-culture is reduced, and viral material transferred to uninfected cells is less matured. At later time points, this defect is associated with a reduction in the productive infection of new target cells. Conclusions Our results highlight a previously unappreciated role of Nef during the viral replication cycle. Nef promotes HIV-1 Gag membrane localization and processing, and facilitates viral cell-to-cell transfer. PMID:23899341

  14. Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor

    PubMed Central

    Fackler, Oliver T.

    2015-01-01

    The Nef protein is an accessory gene product encoded by human immunodeficiency virus types 1 and 2 (HIV-1/-2) and simian immunodeficiency virus (SIV) that boosts virus replication in the infected host and accelerates disease progression. Unlike the HIV-1 accessory proteins Vif, Vpr and Vpu, Nef was, until recently, not known to antagonize the antiviral activity of a host cell restriction factor. Two recent reports now describe the host cell proteins serine incorporator 3 and 5 (SERINC3 and SERINC5) as potent inhibitors of HIV-1 particle infectivity and demonstrate that Nef counteracts these effects. These findings establish SERINC3/5 as restrictions to HIV replication in human cells and define a novel activity for the HIV pathogenesis factor Nef. PMID:26703715

  15. HIV-1 Nef sequence and functional compartmentalization in the gut is not due to differential cytotoxic T lymphocyte selective pressure.

    PubMed

    Lewis, Martha J; Frohnen, Patricia; Ibarrondo, F Javier; Reed, Diane; Iyer, Varun; Ng, Hwee L; Elliott, Julie; Yang, Otto O; Anton, Peter

    2013-01-01

    The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef's function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef's sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary nef quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env(-) Vpu(-) pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of nef sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in

  16. Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases.

    PubMed Central

    Schorr, J; Kellner, R; Fackler, O; Freund, J; Konvalinka, J; Kienzle, N; Kräusslich, H G; Mueller-Lantzsch, N; Kalbitzer, H R

    1996-01-01

    Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1. PMID:8971042

  17. Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef

    SciTech Connect

    Bouzar, Baya Amel; Rea, Angela; Hoc-Villet, Stephanie; Garnier, Celine; Guiguen, Francois; Jin Yuhuai; Narayan, Opendra; Chebloune, Yahia . E-mail: ychebloune@kumc.edu

    2007-08-01

    Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes.

  18. Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

    PubMed

    Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

    2016-03-01

    The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection. PMID:26950141

  19. Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence

    PubMed Central

    Sugden, Scott M.; Bego, Mariana G.; Pham, Tram N.Q.; Cohen, Éric A.

    2016-01-01

    The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection. PMID:26950141

  20. Parallel Processing at the High School Level.

    ERIC Educational Resources Information Center

    Sheary, Kathryn Anne

    This study investigated the ability of high school students to cognitively understand and implement parallel processing. Data indicates that most parallel processing is being taught at the university level. Instructional modules on C, Linux, and the parallel processing language, P4, were designed to show that high school students are highly…

  1. High-Level Application Framework for LCLS

    SciTech Connect

    Chu, P; Chevtsov, S.; Fairley, D.; Larrieu, C.; Rock, J.; Rogind, D.; White, G.; Zalazny, M.; /SLAC

    2008-04-22

    A framework for high level accelerator application software is being developed for the Linac Coherent Light Source (LCLS). The framework is based on plug-in technology developed by an open source project, Eclipse. Many existing functionalities provided by Eclipse are available to high-level applications written within this framework. The framework also contains static data storage configuration and dynamic data connectivity. Because the framework is Eclipse-based, it is highly compatible with any other Eclipse plug-ins. The entire infrastructure of the software framework will be presented. Planned applications and plug-ins based on the framework are also presented.

  2. Future high sea levels in south Sweden

    SciTech Connect

    Blomgren, S.H.; Hanson, H.

    1997-12-31

    An estimation of future mean high water levels in Oeresund and the southwest Baltic Sea is presented together with a discussion of probable consequences for Falsterbo Peninsula, a trumpet-shaped sandy formation of some 25 km{sup 2} size situated in the very southwest corner of Sweden. A literature review coupled with sea-level measurements and observations made in the area every four hours since October 1945 are given and comprise the base for the present analysis.

  3. A Software Architecture for High Level Applications

    SciTech Connect

    Shen,G.

    2009-05-04

    A modular software platform for high level applications is under development at the National Synchrotron Light Source II project. This platform is based on client-server architecture, and the components of high level applications on this platform will be modular and distributed, and therefore reusable. An online model server is indispensable for model based control. Different accelerator facilities have different requirements for the online simulation. To supply various accelerator simulators, a set of narrow and general application programming interfaces is developed based on Tracy-3 and Elegant. This paper describes the system architecture for the modular high level applications, the design of narrow and general application programming interface for an online model server, and the prototype of online model server.

  4. High-level waste processing and disposal

    NASA Astrophysics Data System (ADS)

    Crandall, J. L.; Drause, H.; Sombret, C.; Uematsu, K.

    The national high level waste disposal plans for France, the Federal Republic of Germany, Japan, and the United States are covered. Three conclusions are reached. The first conclusion is that an excellent technology already exists for high level waste disposal. With appropriate packaging, spent fuel seems to be an acceptable waste form. Borosilicate glass reprocessing waste forms are well understood, in production in France, and scheduled for production in the next few years in a number of other countries. For final disposal, a number of candidate geological repository sites have been identified and several demonstration sites opened. The second conclusion is that adequate financing and a legal basis for waste disposal are in place in most countries. Costs of high level waste disposal will probably and about 5 to 10% to the costs of nuclear electric power. Third conclusion is less optimistic.

  5. PAIRWISE BLENDING OF HIGH LEVEL WASTE (HLW)

    SciTech Connect

    CERTA, P.J.

    2006-02-22

    The primary objective of this study is to demonstrate a mission scenario that uses pairwise and incidental blending of high level waste (HLW) to reduce the total mass of HLW glass. Secondary objectives include understanding how recent refinements to the tank waste inventory and solubility assumptions affect the mass of HLW glass and how logistical constraints may affect the efficacy of HLW blending.

  6. High-level radioactive wastes. Supplement 1

    SciTech Connect

    McLaren, L.H.

    1984-09-01

    This bibliography contains information on high-level radioactive wastes included in the Department of Energy's Energy Data Base from August 1982 through December 1983. These citations are to research reports, journal articles, books, patents, theses, and conference papers from worldwide sources. Five indexes, each preceded by a brief description, are provided: Corporate Author, Personal Author, Subject, Contract Number, and Report Number. 1452 citations.

  7. Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef.

    PubMed

    Matheson, Nicholas J; Sumner, Jonathan; Wals, Kim; Rapiteanu, Radu; Weekes, Michael P; Vigan, Raphael; Weinelt, Julia; Schindler, Michael; Antrobus, Robin; Costa, Ana S H; Frezza, Christian; Clish, Clary B; Neil, Stuart J D; Lehner, Paul J

    2015-10-14

    Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism. PMID:26439863

  8. Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef

    PubMed Central

    Matheson, Nicholas J.; Sumner, Jonathan; Wals, Kim; Rapiteanu, Radu; Weekes, Michael P.; Vigan, Raphael; Weinelt, Julia; Schindler, Michael; Antrobus, Robin; Costa, Ana S.H.; Frezza, Christian; Clish, Clary B.; Neil, Stuart J.D.; Lehner, Paul J.

    2015-01-01

    Summary Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism. PMID:26439863

  9. The CMS High-Level Trigger

    SciTech Connect

    Covarelli, R.

    2009-12-17

    At the startup of the LHC, the CMS data acquisition is expected to be able to sustain an event readout rate of up to 100 kHz from the Level-1 trigger. These events will be read into a large processor farm which will run the 'High-Level Trigger'(HLT) selection algorithms and will output a rate of about 150 Hz for permanent data storage. In this report HLT performances are shown for selections based on muons, electrons, photons, jets, missing transverse energy, {tau} leptons and b quarks: expected efficiencies, background rates and CPU time consumption are reported as well as relaxation criteria foreseen for a LHC startup instantaneous luminosity.

  10. Plants tolerant of high boron levels.

    PubMed

    Miwa, Kyoko; Takano, Junpei; Omori, Hiroyuki; Seki, Motoaki; Shinozaki, Kazuo; Fujiwara, Toru

    2007-11-30

    Reduced crop productivity due to soils containing toxic levels of boron (B) is a worldwide problem in food production. It is estimated that up to 17% of the barley yield losses in southern Australia are caused by B toxicity. We found that the expression of AtBOR4, an Arabidopsis paralog of BOR1, the first identified boron transporter gene, generates plants that are tolerant of high B levels. BOR4 is a polarly localized borate exporter that enhances B efflux from roots. The present study is a foundation for the improvement of crop productivity in soils containing excess B, which are distributed in arid areas of the world. PMID:18048682

  11. High-Level Waste Melter Study Report

    SciTech Connect

    Perez Jr, Joseph M; Bickford, Dennis F; Day, Delbert E; Kim, Dong-Sang; Lambert, Steven L; Marra, Sharon L; Peeler, David K; Strachan, Denis M; Triplett, Mark B; Vienna, John D; Wittman, Richard S

    2001-07-13

    At the Hanford Site in Richland, Washington, the path to site cleanup involves vitrification of the majority of the wastes that currently reside in large underground tanks. A Joule-heated glass melter is the equipment of choice for vitrifying the high-level fraction of these wastes. Even though this technology has general national and international acceptance, opportunities may exist to improve or change the technology to reduce the enormous cost of accomplishing the mission of site cleanup. Consequently, the U.S. Department of Energy requested the staff of the Tanks Focus Area to review immobilization technologies, waste forms, and modifications to requirements for solidification of the high-level waste fraction at Hanford to determine what aspects could affect cost reductions with reasonable long-term risk. The results of this study are summarized in this report.

  12. High level intelligent control of telerobotics systems

    NASA Technical Reports Server (NTRS)

    Mckee, James

    1988-01-01

    A high level robot command language is proposed for the autonomous mode of an advanced telerobotics system and a predictive display mechanism for the teleoperational model. It is believed that any such system will involve some mixture of these two modes, since, although artificial intelligence can facilitate significant autonomy, a system that can resort to teleoperation will always have the advantage. The high level command language will allow humans to give the robot instructions in a very natural manner. The robot will then analyze these instructions to infer meaning so that is can translate the task into lower level executable primitives. If, however, the robot is unable to perform the task autonomously, it will switch to the teleoperational mode. The time delay between control movement and actual robot movement has always been a problem in teleoperations. The remote operator may not actually see (via a monitor) the results of high actions for several seconds. A computer generated predictive display system is proposed whereby the operator can see a real-time model of the robot's environment and the delayed video picture on the monitor at the same time.

  13. Performance of the CMS High Level Trigger

    NASA Astrophysics Data System (ADS)

    Perrotta, Andrea

    2015-12-01

    The CMS experiment has been designed with a 2-level trigger system. The first level is implemented using custom-designed electronics. The second level is the so-called High Level Trigger (HLT), a streamlined version of the CMS offline reconstruction software running on a computer farm. For Run II of the Large Hadron Collider, the increases in center-of-mass energy and luminosity will raise the event rate to a level challenging for the HLT algorithms. The increase in the number of interactions per bunch crossing, on average 25 in 2012, and expected to be around 40 in Run II, will be an additional complication. We present here the expected performance of the main triggers that will be used during the 2015 data taking campaign, paying particular attention to the new approaches that have been developed to cope with the challenges of the new run. This includes improvements in HLT electron and photon reconstruction as well as better performing muon triggers. We will also present the performance of the improved tracking and vertexing algorithms, discussing their impact on the b-tagging performance as well as on the jet and missing energy reconstruction.

  14. Highly Pathogenic Simian Immunodeficiency Virus mne Variants That Emerge during the Course of Infection Evolve Enhanced Infectivity and the Ability To Downregulate CD4 but Not Class I Major Histocompatibility Complex Antigens

    PubMed Central

    Patel, Parul G.; Yu Kimata, Monica T.; Biggins, Julia E.; Wilson, Joelle M.; Kimata, Jason T.

    2002-01-01

    The replicative, cytopathic, and antigenic properties of simian immunodeficiency virus (SIV) variants influence its replication efficiency in vivo. To further define the viral properties and determinants that may be important for high-level replication in vivo and progression to AIDS, we compared a minimally pathogenic SIVmne molecular clone with two highly pathogenic variants cloned from late stages of infection. Both variants had evolved greater infectivity than the parental clone due to mutations in nef. Interestingly, a pol determinant in one of the highly pathogenic variants also contributed to its increased infectivity. Furthermore, because replication in vivo may also be influenced by the ability of a virus to evade the cellular immune response of the host, we examined whether the variants were more capable of downregulating surface expression of class I major histocompatibility complex (MHC). Decreased MHC class I expression was not observed in cells infected with any of the viruses. Furthermore, the Nef proteins of the highly pathogenic variants only slightly reduced surface MHC class I expression in transfected cells, although they efficiently downregulated CD4. Together, these data demonstrate that mutations which can enhance viral infectivity, as well as CD4 downregulation, may be important for efficient replication of SIV in the host. However, Nef-mediated reduction of MHC class I expression does not appear to be critical for the increased in vivo replicative ability of highly pathogenic late variants. PMID:12050354

  15. The high-level trigger of ALICE

    NASA Astrophysics Data System (ADS)

    Tilsner, H.; Alt, T.; Aurbakken, K.; Grastveit, G.; Helstrup, H.; Lindenstruth, V.; Loizides, C.; Nystrand, J.; Roehrich, D.; Skaali, B.; Steinbeck, T.; Ullaland, K.; Vestbo, A.; Vik, T.

    One of the main tracking detectors of the forthcoming ALICE Experiment at the LHC is a cylindrical Time Projection Chamber (TPC) with an expected data volume of about 75 MByte per event. This data volume, in combination with the presumed maximum bandwidth of 1.2 GByte/s to the mass storage system, would limit the maximum event rate to 20 Hz. In order to achieve higher event rates, online data processing has to be applied. This implies either the detection and read-out of only those events which contain interesting physical signatures or an efficient compression of the data by modeling techniques. In order to cope with the anticipated data rate, massive parallel computing power is required. It will be provided in form of a clustered farm of SMP-nodes, based on off-the-shelf PCs, which are connected with a high bandwidth low overhead network. This High-Level Trigger (HLT) will be able to process a data rate of 25 GByte/s online. The front-end electronics of the individual sub-detectors is connected to the HLT via an optical link and a custom PCI card which is mounted in the clustered PCs. The PCI card is equipped with an FPGA necessary for the implementation of the PCI-bus protocol. Therefore, this FPGA can also be used to assist the host processor with first-level processing. The first-level processing done on the FPGA includes conventional cluster-finding for low multiplicity events and local track finding based on the Hough Transformation of the raw data for high multiplicity events. PACS: 07.05.-t Computers in experimental physics - 07.05.Hd Data acquisition: hardware and software - 29.85.+c Computer data analysis

  16. EAP high-level product architecture

    NASA Astrophysics Data System (ADS)

    Gudlaugsson, T. V.; Mortensen, N. H.; Sarban, R.

    2013-04-01

    EAP technology has the potential to be used in a wide range of applications. This poses the challenge to the EAP component manufacturers to develop components for a wide variety of products. Danfoss Polypower A/S is developing an EAP technology platform, which can form the basis for a variety of EAP technology products while keeping complexity under control. High level product architecture has been developed for the mechanical part of EAP transducers, as the foundation for platform development. A generic description of an EAP transducer forms the core of the high level product architecture. This description breaks down the EAP transducer into organs that perform the functions that may be present in an EAP transducer. A physical instance of an EAP transducer contains a combination of the organs needed to fulfill the task of actuator, sensor, and generation. Alternative principles for each organ allow the function of the EAP transducers to be changed, by basing the EAP transducers on a different combination of organ alternatives. A model providing an overview of the high level product architecture has been developed to support daily development and cooperation across development teams. The platform approach has resulted in the first version of an EAP technology platform, on which multiple EAP products can be based. The contents of the platform have been the result of multi-disciplinary development work at Danfoss PolyPower, as well as collaboration with potential customers and research institutions. Initial results from applying the platform on demonstrator design for potential applications are promising. The scope of the article does not include technical details.

  17. Regulation of high density lipoprotein levels

    SciTech Connect

    Krauss, R.M.

    1982-03-01

    An increasing awareness of the physiologic and pathologic importance of serum high density lipoproteins (HDL) has led to a large number of observations regarding factors which influence their concentrations. HDL consists of a heterogeneous collection of macromolecules with diverse physical properties and chemical constituents. While laboratory techniques have made it possible to measure HDL and their individual components, there are as yet large gaps in our knowledge of the biochemical mechanisms and clinical significance of changes in these laboratory parameters. In this review, current concepts of the structure and metabolism of HDL will be briefly summarized, and the factors influencing their levels in humans will be surveyed. 313 references.

  18. The effects of high level infrasound

    SciTech Connect

    Johnson, D.L.

    1980-02-01

    This paper will attempt to survey the current knowledge on the effects of relative high levels of infrasound on humans. While this conference is concerned mainly about hearing, some discussion of other physiological effects is appropriate. Such discussion also serves to highlight a basic question, 'Is hearing the main concern of infrasound and low frequency exposure, or is there a more sensitive mechanism'. It would be comforting to know that the focal point of this conference is indeed the most important concern. Therefore, besides hearing loss and auditory threshold of infrasonic and low frequency exposure, four other effects will be provided. These are performance, respiration, annoyance, and vibration.

  19. Service Oriented Architecture for High Level Applications

    SciTech Connect

    Chu, Chungming; Chevtsov, Sergei; Wu, Juhao; Shen, Guobao; /Brookhaven

    2012-06-28

    Standalone high level applications often suffer from poor performance and reliability due to lengthy initialization, heavy computation and rapid graphical update. Service-oriented architecture (SOA) is trying to separate the initialization and computation from applications and to distribute such work to various service providers. Heavy computation such as beam tracking will be done periodically on a dedicated server and data will be available to client applications at all time. Industrial standard service architecture can help to improve the performance, reliability and maintainability of the service. Robustness will also be improved by reducing the complexity of individual client applications.

  20. High-level waste qualification: Managing uncertainty

    SciTech Connect

    Pulsipher, B.A.

    1993-09-01

    A vitrification facility is being developed by the U.S. Department of Energy (DOE) at the West Valley Demonstration Plant (WVDP) near Buffalo, New York, where approximately 300 canisters of high-level nuclear waste glass will be produced. To assure that the produced waste form is acceptable, uncertainty must be managed. Statistical issues arise due to sampling, waste variations, processing uncertainties, and analytical variations. This paper presents elements of a strategy to characterize and manage the uncertainties associated with demonstrating that an acceptable waste form product is achieved. Specific examples are provided within the context of statistical work performed by Pacific Northwest Laboratory (PNL).

  1. The High Level Data Reduction Library

    NASA Astrophysics Data System (ADS)

    Ballester, P.; Gabasch, A.; Jung, Y.; Modigliani, A.; Taylor, J.; Coccato, L.; Freudling, W.; Neeser, M.; Marchetti, E.

    2015-09-01

    The European Southern Observatory (ESO) provides pipelines to reduce data for most of the instruments at its Very Large telescope (VLT). These pipelines are written as part of the development of VLT instruments, and are used both in the ESO's operational environment and by science users who receive VLT data. All the pipelines are highly specific geared toward instruments. However, experience showed that the independently developed pipelines include significant overlap, duplication and slight variations of similar algorithms. In order to reduce the cost of development, verification and maintenance of ESO pipelines, and at the same time improve the scientific quality of pipelines data products, ESO decided to develop a limited set of versatile high-level scientific functions that are to be used in all future pipelines. The routines are provided by the High-level Data Reduction Library (HDRL). To reach this goal, we first compare several candidate algorithms and verify them during a prototype phase using data sets from several instruments. Once the best algorithm and error model have been chosen, we start a design and implementation phase. The coding of HDRL is done in plain C and using the Common Pipeline Library (CPL) functionality. HDRL adopts consistent function naming conventions and a well defined API to minimise future maintenance costs, implements error propagation, uses pixel quality information, employs OpenMP to take advantage of multi-core processors, and is verified with extensive unit and regression tests. This poster describes the status of the project and the lesson learned during the development of reusable code implementing algorithms of high scientific quality.

  2. HIV-1 infection of T cells and macrophages are differentially modulated by virion-associated Hck: a Nef-dependent phenomenon.

    PubMed

    Cornall, Alyssa; Mak, Johnson; Greenway, Alison; Tachedjian, Gilda

    2013-09-01

    The proline repeat motif (PxxP) of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell. PMID:24051604

  3. HIV-1 Nef and KSHV oncogene K1 synergistically promote angiogenesis by inducing cellular miR-718 to regulate the PTEN/AKT/mTOR signaling pathway

    PubMed Central

    Xue, Min; Yao, Shuihong; Hu, Minmin; Li, Wan; Hao, Tingting; Zhou, Feng; Zhu, Xiaofei; Lu, Hongmei; Qin, Di; Yan, Qin; Zhu, Jianzhong; Gao, Shou-Jiang; Lu, Chun

    2014-01-01

    Kaposi's sarcoma (KS) is an AIDS-defining cancer with aberrant neovascularization caused by KS-associated herpesvirus (KSHV). Although the interaction between HIV-1 and KSHV plays a pivotal role in promoting the aggressive manifestations of KS, the pathogenesis underlying AIDS-KS remains largely unknown. Here we examined HIV-1 Nef protein promotion of KSHV oncoprotein K1-induced angiogenesis. We showed that both internalized and ectopic expression of Nef in endothelial cells synergized with K1 to facilitate vascular tube formation and cell proliferation, and enhance angiogenesis in a chicken CAM model. In vivo experiments further indicated that Nef accelerated K1-induced angiogenesis and tumorigenesis in athymic nu/nu mice. Mechanistic studies revealed that Nef and K1 synergistically activated PI3K/AKT/mTOR signaling by downregulating PTEN. Furthermore, Nef and K1 induced cellular miR-718, which inhibited PTEN expression by directly targeting a seed sequence in the 3′ UTR of its mRNA. Inhibition of miR-718 expression increased PTEN synthesis and suppressed the synergistic effect of Nef- and K1-induced angiogenesis and tumorigenesis. These results indicate that, by targeting PTEN, miR-718 mediates Nef- and K1-induced angiogenesis via activation of AKT/mTOR signaling. Our results demonstrate an essential role of miR-718/AKT/mTOR axis in AIDS-KS and thus may represent an attractive therapeutic target. PMID:25104021

  4. Oral immunization with recombinant Mycobacterium bovis BCG simian immunodeficiency virus nef induces local and systemic cytotoxic T-lymphocyte responses in mice.

    PubMed Central

    Lagranderie, M; Balazuc, A M; Gicquel, B; Gheorghiu, M

    1997-01-01

    Recombinant live Mycobacterium bovis BCG vectors (rBCG) induce strong cellular and humoral immune responses against various antigens after either systemic or oral immunization of mice. Cytotoxic T-lymphocyte (CTL) responses may contribute to the control of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infections whose portal of entry is the gastrointestinal or genital mucosa. In this study, we immunized BALB/c mice with a recombinant BCG SIV nef and observed its behavior in oropharyngeal and target organ lymphoid tissues. The cellular immune responses, particularly the intestinal intraepithelial and systemic CTL responses, were investigated. The results showed that rBCG SIV nef translocated the oropharyngeal mucosa and intestinal epithelium. It diffused to and persisted in target lymphoid organs. Specific SIV Nef peptide proliferative responses and cytokine production were observed. Strong systemic and mucosal CTL responses were induced. In particular, we demonstrated direct specific anti-Nef CTL in intestinal intraepithelial CD8beta+ T cells. These findings provide evidence that orally administered rBCG SIV nef may contribute to local defenses against viral invasion. Therefore, rBCG SIV nef could be a candidate vaccine to protect against SIV infection and may be used to develop an oral rBCG HIV nef vaccine. PMID:9032366

  5. Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag–pol–nef antigen

    PubMed Central

    Herath, S.; Le Heron, A.; Colloca, S.; Bergin, P.; Patterson, S.; Weber, J.; Tatoud, R.; Dickson, G.

    2015-01-01

    Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene. PMID:26546736

  6. Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag-pol-nef antigen.

    PubMed

    Herath, S; Le Heron, A; Colloca, S; Bergin, P; Patterson, S; Weber, J; Tatoud, R; Dickson, G

    2015-12-16

    Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene. PMID:26546736

  7. CMS High Level Trigger Timing Measurements

    NASA Astrophysics Data System (ADS)

    Richardson, Clint

    2015-12-01

    The two-level trigger system employed by CMS consists of the Level 1 (L1) Trigger, which is implemented using custom-built electronics, and the High Level Trigger (HLT), a farm of commercial CPUs running a streamlined version of the offline CMS reconstruction software. The operational L1 output rate of 100 kHz, together with the number of CPUs in the HLT farm, imposes a fundamental constraint on the amount of time available for the HLT to process events. Exceeding this limit impacts the experiment's ability to collect data efficiently. Hence, there is a critical need to characterize the performance of the HLT farm as well as the algorithms run prior to start up in order to ensure optimal data taking. Additional complications arise from the fact that the HLT farm consists of multiple generations of hardware and there can be subtleties in machine performance. We present our methods of measuring the timing performance of the CMS HLT, including the challenges of making such measurements. Results for the performance of various Intel Xeon architectures from 2009-2014 and different data taking scenarios are also presented.

  8. Technetium Chemistry in High-Level Waste

    SciTech Connect

    Hess, Nancy J.

    2006-06-01

    Tc contamination is found within the DOE complex at those sites whose mission involved extraction of plutonium from irradiated uranium fuel or isotopic enrichment of uranium. At the Hanford Site, chemical separations and extraction processes generated large amounts of high level and transuranic wastes that are currently stored in underground tanks. The waste from these extraction processes is currently stored in underground High Level Waste (HLW) tanks. However, the chemistry of the HLW in any given tank is greatly complicated by repeated efforts to reduce volume and recover isotopes. These processes ultimately resulted in mixing of waste streams from different processes. As a result, the chemistry and the fate of Tc in HLW tanks are not well understood. This lack of understanding has been made evident in the failed efforts to leach Tc from sludge and to remove Tc from supernatants prior to immobilization. Although recent interest in Tc chemistry has shifted from pretreatment chemistry to waste residuals, both needs are served by a fundamental understanding of Tc chemistry.

  9. High accuracy electronic material level sensor

    DOEpatents

    McEwan, Thomas E.

    1997-01-01

    The High Accuracy Electronic Material Level Sensor (electronic dipstick) is a sensor based on time domain reflectometry (TDR) of very short electrical pulses. Pulses are propagated along a transmission line or guide wire that is partially immersed in the material being measured; a launcher plate is positioned at the beginning of the guide wire. Reflected pulses are produced at the material interface due to the change in dielectric constant. The time difference of the reflections at the launcher plate and at the material interface are used to determine the material level. Improved performance is obtained by the incorporation of: 1) a high accuracy time base that is referenced to a quartz crystal, 2) an ultrawideband directional sampler to allow operation without an interconnect cable between the electronics module and the guide wire, 3) constant fraction discriminators (CFDs) that allow accurate measurements regardless of material dielectric constants, and reduce or eliminate errors induced by triple-transit or "ghost" reflections on the interconnect cable. These improvements make the dipstick accurate to better than 0.1%.

  10. High accuracy electronic material level sensor

    DOEpatents

    McEwan, T.E.

    1997-03-11

    The High Accuracy Electronic Material Level Sensor (electronic dipstick) is a sensor based on time domain reflectometry (TDR) of very short electrical pulses. Pulses are propagated along a transmission line or guide wire that is partially immersed in the material being measured; a launcher plate is positioned at the beginning of the guide wire. Reflected pulses are produced at the material interface due to the change in dielectric constant. The time difference of the reflections at the launcher plate and at the material interface are used to determine the material level. Improved performance is obtained by the incorporation of: (1) a high accuracy time base that is referenced to a quartz crystal, (2) an ultrawideband directional sampler to allow operation without an interconnect cable between the electronics module and the guide wire, (3) constant fraction discriminators (CFDs) that allow accurate measurements regardless of material dielectric constants, and reduce or eliminate errors induced by triple-transit or ``ghost`` reflections on the interconnect cable. These improvements make the dipstick accurate to better than 0.1%. 4 figs.

  11. Genetic drift can dominate short-term human immunodeficiency virus type 1 nef quasispecies evolution in vivo.

    PubMed Central

    Plikat, U; Nieselt-Struwe, K; Meyerhans, A

    1997-01-01

    The evolution of human immunodeficiency virus (HIV) type 1 nef quasispecies in a patient clonally infected with a contaminated batch of blood clotting factor IX was monitored. nef sequences were derived at 11, 25, and 41 months postinfection from infected peripheral blood mononuclear cells after molecular cloning of PCR-amplified proviral DNA. The phylogenetic relationships among a total of 41 informative sequences were established by split decomposition analysis and used as a basis to establish a substitution matrix and to score synonymous (s) and nonsynonymous (ns) substitutions. The number of observed in-phase stop codons within the nef sequences was comparable to that expected on a random basis. Similarly, the numbers of observed s and ns substitutions did not differ significantly from expected values. No codon position was preferentially mutated. The maximum sequence divergence increased in a linear manner, with approximately 4.4 nucleotide and approximately 3.2 amino acid changes per year. It appears that stochastic processes strongly influence short-term HIV nef quasispecies evolution in vivo. PMID:9151810

  12. Intra-host competition between nef-defective escape mutants and wild-type human immunodeficiency virus type 1.

    PubMed Central

    Altes, H K; Jansen, V A

    2000-01-01

    Various forms of nef genes with deletions at conserved positions along the sequence have been reported to persist in human immunodeficiency virus type 1 infected patients. We investigate the forces maintaining such variants in the proviral population. The main selection pressures are preservation of function and host immune response. The crippled Nef protein might have fewer epitopes, and as such be less visible to the specific immune response, but it will lose some function. Does a trade-off between avoidance of the immune response and loss of function explain the dynamics of the crippled virus found in the patients? To answer this question, we formulated a deterministic model of the virus-host interactions. We found that when the crippled protein presents few epitopes and suffers little loss of function, the two viral types can coexist. Otherwise, the wild-type comes to prevail. The mutant form might initially dominate, but as the selective pressure by the CD84+ T cells decreases over the course of infection, the advantage for the crippled form of losing epitopes disappears. Hence, we go from a situation of coexistence of wild-type and mutant, to a situation of only full-length nef. The results are discussed in the context of the suggested use of live attenuated vaccines having deletions in nef. PMID:10687825

  13. Visual high-level regions respond to high-level stimulus content in the absence of low-level confounds.

    PubMed

    Schindler, Andreas; Bartels, Andreas

    2016-05-15

    High-level regions of the ventral stream exhibit strong category selectivity to stimuli such as faces, houses, or objects. However, recent studies suggest that at least part of this selectivity stems from low-level differences inherent to images of the different categories. For example, visual outdoor and indoor scenes as well as houses differ in spatial frequency, rectilinearity and obliqueness when compared to face or object images. Correspondingly, scene responsive para-hippocampal place area (PPA) showed strong preference to low-level properties of visual scenes also in the absence of high-level scene content. This raises the question whether all high-level responses in PPA, the fusiform face area (FFA), or the object-responsive lateral occipital compex (LOC) may actually be explained by systematic differences in low-level features. In the present study we contrasted two classes of simple stimuli consisting of ten rectangles each. While both were matched in visual low-level features only one class of rectangle arrangements gave rise to a percept compatible with a high-level 3D layout such as a scene or an object. We found that areas PPA, transverse occipital sulcus (TOS, also referred to as occipital place area, OPA), as well as FFA and LOC showed robust responses to the visual scene class compared to the low-level matched control. Our results suggest that visual category responsive regions are not purely driven by low-level visual features but also by the high-level perceptual stimulus interpretation. PMID:26975552

  14. Beam size measurement at high radiation levels

    SciTech Connect

    Decker, F.J.

    1991-05-01

    At the end of the Stanford Linear Accelerator the high energy electron and positron beams are quite small. Beam sizes below 100 {mu}m ({sigma}) as well as the transverse distribution, especially tails, have to be determined. Fluorescent screens observed by TV cameras provide a quick two-dimensional picture, which can be analyzed by digitization. For running the SLAC Linear Collider (SLC) with low backgrounds at the interaction point, collimators are installed at the end of the linac. This causes a high radiation level so that the nearby cameras die within two weeks and so-called radiation hard'' cameras within two months. Therefore an optical system has been built, which guides a 5 mm wide picture with a resolution of about 30 {mu}m over a distance of 12 m to an accessible region. The overall resolution is limited by the screen thickness, optical diffraction and the line resolution of the camera. Vibration, chromatic effects or air fluctuations play a much less important role. The pictures are colored to get fast information about the beam current, size and tails. Beside the emittance, more information about the tail size and betatron phase is obtained by using four screens. This will help to develop tail compensation schemes to decrease the emittance growth in the linac at high currents. 4 refs., 2 figs.

  15. Reusable and Extensible High Level Data Distributions

    NASA Technical Reports Server (NTRS)

    Diaconescu, Roxana E.; Chamberlain, Bradford; James, Mark L.; Zima, Hans P.

    2005-01-01

    This paper presents a reusable design of a data distribution framework for data parallel high performance applications. We are implementing the design in the context of the Chapel high productivity programming language. Distributions in Chapel are a means to express locality in systems composed of large numbers of processor and memory components connected by a network. Since distributions have a great effect on,the performance of applications, it is important that the distribution strategy can be chosen by a user. At the same time, high productivity concerns require that the user is shielded from error-prone, tedious details such as communication and synchronization. We propose an approach to distributions that enables the user to refine a language-provided distribution type and adjust it to optimize the performance of the application. Additionally, we conceal from the user low-level communication and synchronization details to increase productivity. To emphasize the generality of our distribution machinery, we present its abstract design in the form of a design pattern, which is independent of a concrete implementation. To illustrate the applicability of our distribution framework design, we outline the implementation of data distributions in terms of the Chapel language.

  16. Umbra's High Level Architecture (HLA) Interface

    SciTech Connect

    GOTTLIEB, ERIC JOSEPH; MCDONALD, MICHAEL J.; OPPEL III, FRED J.

    2002-04-01

    This report describes Umbra's High Level Architecture HLA library. This library serves as an interface to the Defense Simulation and Modeling Office's (DMSO) Run Time Infrastructure Next Generation Version 1.3 (RTI NG1.3) software library and enables Umbra-based models to be federated into HLA environments. The Umbra library was built to enable the modeling of robots for military and security system concept evaluation. A first application provides component technologies that ideally fit the US Army JPSD's Joint Virtual Battlespace (JVB) simulation framework for Objective Force concept analysis. In addition to describing the Umbra HLA library, the report describes general issues of integrating Umbra with RTI code and outlines ways of building models to support particular HLA simulation frameworks like the JVB.

  17. High-level connectionist models. Semiannual report

    SciTech Connect

    Pollack, J.B.

    1989-08-01

    The major achievement of this semiannum was the significant revision and extension of the Recursive Auto-Associative Memory (RAAM) work for publication in the journal Artificial Intelligence. Included as an appendix to this report, the article includes several new elements: (1) Background - The work was more clearly set into the area of recursive distributed representations, machine learning, and the adequacy of the connectionist approach for high-level cognitive modeling; (2) New Experiment - RAAM was applied to finding compact representations for sequences of letters; (3) Analysis - The developed representations were analyzed as features which range from categorical to distinctive. Categorical features distinguish between conceptual categories while distinctive features vary within categories and discriminate or label the members. The representations were also analyzed geometrically; and (4) Applications - Feasibility studies were performed and described on inference by association, and on using RAAM-generated patterns along with cascaded networks for natural language parsing. Both of these remain long-term goals of the project.

  18. Airway injury during high-level exercise.

    PubMed

    Kippelen, Pascale; Anderson, Sandra D

    2012-05-01

    Airway epithelial cells act as a physical barrier against environmental toxins and injury, and modulate inflammation and the immune response. As such, maintenance of their integrity is critical. Evidence is accumulating to suggest that exercise can cause injury to the airway epithelium. This seems the case particularly for competitive athletes performing high-level exercise, or when exercise takes place in extreme environmental conditions such as in cold dry air or in polluted air. Dehydration of the small airways and increased forces exerted on to the airway surface during severe hyperpnoea are thought to be key factors in determining the occurrence of injury of the airway epithelium. The injury-repair process of the airway epithelium may contribute to the development of the bronchial hyper-responsiveness that is documented in many elite athletes. PMID:22247295

  19. HIGH LEVEL RF FOR THE SNS RING.

    SciTech Connect

    ZALTSMAN,A.; BLASKIEWICZ,M.; BRENNAN,J.; BRODOWSKI,J.; METH,M.; SPITZ,R.; SEVERINO,F.

    2002-06-03

    A high level RF system (HLRF) consisting of power amplifiers (PA's) and ferrite loaded cavities is being designed and built by Brookhaven National Laboratory (BNL) for the Spallation Neutron Source (SNS) project. It is a fixed frequency, two harmonic system whose main function is to maintain a gap for the kicker rise time. Three cavities running at the fundamental harmonic (h=l) will provide 40 kV and one cavity at the second harmonic (h=2) will provide 20 kV. Each cavity has two gaps with a design voltage of 10 kV per gap and will be driven by a power amplifier (PA) directly adjacent to it. The PA uses a 600kW tetrode to provide the necessary drive current. The anode of the tetrode is magnetically coupled to the downstream cell of the cavity. Drive to the PA will be provided by a wide band, solid state amplifier located remotely. A dynamic tuning scheme will be implemented to help compensate for the effect of beam loading.

  20. Decontamination of high-level waste canisters

    SciTech Connect

    Nesbitt, J.F.; Slate, S.C.; Fetrow, L.K.

    1980-12-01

    This report presents evaluations of several methods for the in-process decontamination of metallic canisters containing any one of a number of solidified high-level waste (HLW) forms. The use of steam-water, steam, abrasive blasting, electropolishing, liquid honing, vibratory finishing and soaking have been tested or evaluated as potential techniques to decontaminate the outer surfaces of HLW canisters. Either these techniques have been tested or available literature has been examined to assess their applicability to the decontamination of HLW canisters. Electropolishing has been found to be the most thorough method to remove radionuclides and other foreign material that may be deposited on or in the outer surface of a canister during any of the HLW processes. Steam or steam-water spraying techniques may be adequate for some applications but fail to remove all contaminated forms that could be present in some of the HLW processes. Liquid honing and abrasive blasting remove contamination and foreign material very quickly and effectively from small areas and components although these blasting techniques tend to disperse the material removed from the cleaned surfaces. Vibratory finishing is very capable of removing the bulk of contamination and foreign matter from a variety of materials. However, special vibratory finishing equipment would have to be designed and adapted for a remote process. Soaking techniques take long periods of time and may not remove all of the smearable contamination. If soaking involves pickling baths that use corrosive agents, these agents may cause erosion of grain boundaries that results in rough surfaces.

  1. One Protein to Rule them All: Modulation of Cell Surface Receptors and Molecules by HIV Nef

    PubMed Central

    Landi, Alessia; Iannucci, Veronica; Nuffel, Anouk Van; Meuwissen, Pieter; Verhasselt, Bruno

    2011-01-01

    The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic. PMID:22103833

  2. HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

    PubMed Central

    Fiorucci, Gianna; Vaccari, Gabriele; Acconcia, Filippo; Chiarabelli, Cristiano; Leone, Stefano; Noto, Alessia; Horenkamp, Florian A.; Manrique, Santiago; Romeo, Giovanna; Polticelli, Fabio; Geyer, Matthias; Affabris, Elisabetta

    2011-01-01

    Background HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3. Methodology/Principal Findings Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. Conclusions Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ. PMID:21886773

  3. DEFENSE HIGH LEVEL WASTE GLASS DEGRADATION

    SciTech Connect

    W. Ebert

    2001-09-20

    The purpose of this Analysis/Model Report (AMR) is to document the analyses that were done to develop models for radionuclide release from high-level waste (HLW) glass dissolution that can be integrated into performance assessment (PA) calculations conducted to support site recommendation and license application for the Yucca Mountain site. This report was developed in accordance with the ''Technical Work Plan for Waste Form Degradation Process Model Report for SR'' (CRWMS M&O 2000a). It specifically addresses the item, ''Defense High Level Waste Glass Degradation'', of the product technical work plan. The AP-3.15Q Attachment 1 screening criteria determines the importance for its intended use of the HLW glass model derived herein to be in the category ''Other Factors for the Postclosure Safety Case-Waste Form Performance'', and thus indicates that this factor does not contribute significantly to the postclosure safety strategy. Because the release of radionuclides from the glass will depend on the prior dissolution of the glass, the dissolution rate of the glass imposes an upper bound on the radionuclide release rate. The approach taken to provide a bound for the radionuclide release is to develop models that can be used to calculate the dissolution rate of waste glass when contacted by water in the disposal site. The release rate of a particular radionuclide can then be calculated by multiplying the glass dissolution rate by the mass fraction of that radionuclide in the glass and by the surface area of glass contacted by water. The scope includes consideration of the three modes by which water may contact waste glass in the disposal system: contact by humid air, dripping water, and immersion. The models for glass dissolution under these contact modes are all based on the rate expression for aqueous dissolution of borosilicate glasses. The mechanism and rate expression for aqueous dissolution are adequately understood; the analyses in this AMR were conducted to

  4. Fermilab tevatron high level RF accelerating systems

    SciTech Connect

    Kerns, Q.; Kerns, C.; Miller, H.; Reid, J.; Tawzer, S.; Webber, R.; Wildman, D.

    1985-10-01

    Eight tuned rf cavities have been installed and operated in the F0 straight section of the Tevatron. Their mechanical placement along the beam line enables them to be operated for colliding beams as two independent groups of four cavities, group 1-4 accelerating antiprotons and group 5-8 accelerating protons. The only difference is that the spacing between cavities 4 and 5 was increased to stay clear of the F0 colliding point. The cavities can easily be rephased by switching cables in a low-level distribution system (fan-out) so that the full accelerating capability of all eight cavities can be used during fixed target operations. Likewise, the cables from capacitive probes on each cavity gap can be switched to proper lengths and summed in a fan-back system to give an rf signal representing the amplitude and phase as ''seen by the beam,'' separately for protons and antiprotons. Such signals have been used to phase lock the Tevatron to the Main Ring for synchronous transfer. A cavity consists of two quarter-wave resonators placed back to back with a coaxial drift tube separating the two accelerating gaps by ..pi.. radians. The cavities are very similar to the prototype which has been previously described/sup 3/ and is operating as Station 8 in the Tevatron. Only additional water cooling around the high current region of the drift tube supports and a double loop used to monitor the unbalance current through the Hipernom mode damping resistor have been added. Each cavity has a Q of about7100, a shunt impedance of 1.2 M..cap omega.., and is capable of running cw with a peak accelerating voltage of 360

  5. Human Immunodeficiency Virus Type 1 Particles Pseudotyped with Envelope Proteins That Fuse at Low pH No Longer Require Nef for Optimal Infectivity

    PubMed Central

    Chazal, Nathalie; Singer, Gregory; Aiken, Christopher; Hammarskjöld, Marie-Louise; Rekosh, David

    2001-01-01

    We have investigated the effects of Nef on infectivity in the context of various viral envelope proteins. These experiments were performed with a minimal vector system where Nef is the only accessory protein present. Our results support the hypothesis that the route of entry influences the ability of Nef to enhance human immunodeficiency virus (HIV) infectivity. We show that HIV particles pseudotyped with Ebola virus glycoprotein or vesicular stomatitis virus glycoprotein (VSV-G), which fuse at low pH, do not require Nef for optimal infectivity. In contrast, Nef significantly enhances the infectivity of virus particles that contain envelope proteins that fuse at neutral pH (CCR5-dependent HIV Env, CXCR4-dependent HIV Env, or amphotropic murine leukemia virus Env). In addition, our results demonstrate that virus particles containing mixed CXCR4-dependent HIV and VSV-G envelope proteins show a conditional requirement for Nef for optimal infectivity, depending on which protein is allowed to facilitate entry. PMID:11264394

  6. High bicarbonate levels in narcoleptic children.

    PubMed

    Franco, Patricia; Junqua, Aurelie; Guignard-Perret, Anne; Raoux, Aude; Perier, Magali; Raverot, Veronique; Claustrat, Bruno; Gustin, Marie-Paule; Inocente, Clara Odilia; Lin, Jian-Sheng

    2016-04-01

    The objective of this study was to evaluate the levels of plasma bicarbonate levels in narcoleptic children. Clinical, electrophysiological data and bicarbonate levels were evaluated retrospectively in children seen in our paediatric national reference centre for hypersomnia. The cohort included 23 control subjects (11.5 ± 4 years, 43% boys) and 51 patients presenting de-novo narcolepsy (N) (12.7 ± 3.7 years, 47% boys). In narcoleptic children, cataplexy was present in 78% and DQB1*0602 was positive in 96%. The control children were less obese (2 versus 47%, P = 0.001). Compared with control subjects, narcoleptic children had higher bicarbonate levels (P = 0.02) as well as higher PCO2 (P < 0.01) and lower venous pH gas (P < 0.01). Bicarbonate levels higher than 27 mmol L(-1) were found in 41.2% of the narcoleptic children and 4.2% of the controls (P = 0.001). Bicarbonate levels were correlated with the Adapted Epworth Sleepiness Scale (P = 0.01). Narcoleptic patients without obesity often had bicarbonate levels higher than 27 mmol L (-1) (55 versus 25%, P = 0.025). No differences were found between children with and without cataplexy. In conclusion, narcoleptic patients had higher bicarbonate plasma levels compared to control children. This result could be a marker of hypoventilation in this pathology, provoking an increase in PCO2 and therefore a respiratory acidosis, compensated by an increase in plasma bicarbonates. This simple screening tool could be useful for prioritizing children for sleep laboratory evaluation in practice. PMID:26574184

  7. Statistics of high-level scene context

    PubMed Central

    Greene, Michelle R.

    2013-01-01

    Context is critical for recognizing environments and for searching for objects within them: contextual associations have been shown to modulate reaction time and object recognition accuracy, as well as influence the distribution of eye movements and patterns of brain activations. However, we have not yet systematically quantified the relationships between objects and their scene environments. Here I seek to fill this gap by providing descriptive statistics of object-scene relationships. A total of 48, 167 objects were hand-labeled in 3499 scenes using the LabelMe tool (Russell et al., 2008). From these data, I computed a variety of descriptive statistics at three different levels of analysis: the ensemble statistics that describe the density and spatial distribution of unnamed “things” in the scene; the bag of words level where scenes are described by the list of objects contained within them; and the structural level where the spatial distribution and relationships between the objects are measured. The utility of each level of description for scene categorization was assessed through the use of linear classifiers, and the plausibility of each level for modeling human scene categorization is discussed. Of the three levels, ensemble statistics were found to be the most informative (per feature), and also best explained human patterns of categorization errors. Although a bag of words classifier had similar performance to human observers, it had a markedly different pattern of errors. However, certain objects are more useful than others, and ceiling classification performance could be achieved using only the 64 most informative objects. As object location tends not to vary as a function of category, structural information provided little additional information. Additionally, these data provide valuable information on natural scene redundancy that can be exploited for machine vision, and can help the visual cognition community to design experiments guided by

  8. High School Accounting at Two Instructional Levels.

    ERIC Educational Resources Information Center

    Helme, Rebecca E.

    1980-01-01

    Offering two levels of accounting results in a broader base of student enrollment. Reiterating theory, giving practical examples, and stressing "why" as well as "how" enable the instructor to foster the environment the students need. The teacher must also instill self-confidence in the students. (JOW)

  9. Period analysis at high noise level

    NASA Astrophysics Data System (ADS)

    Kovacs, G.

    1980-05-01

    Analytical expressions are derived for the variances of three types of periodograms due to normal-distributed noise present in the data: the conventional Fourier spectrum, the method of Warner and Robinson (1972), and Jurkevich's method (1971). The equivalence of the Jurkevich method and that of Warner and Robinson is proved. The optimum phase cell number of the Warner-Robinson method is given; this number depends on the data length, signal form, and noise level. Results are illustrated by the analysis of two typical forms of light curves: an eclipsing type light curve and an RRa curve.

  10. High Power Amplifier Harmonic Output Level Measurement

    NASA Technical Reports Server (NTRS)

    Perez, R. M.; Hoppe, D. J.; Khan, A. R.

    1995-01-01

    A method is presented for the measurement of the harmonic output power of high power klystron amplifiers, involving coherent hemispherical radiation pattern measurements of the radiated klystron output. Results are discussed for the operation in saturated and unsaturated conditions, and with a waveguide harmonic filter included.

  11. How High Glucose Levels Affect Tendon Homeostasis.

    PubMed

    Snedeker, Jess G

    2016-01-01

    Among the many factors playing a role in tendon disease, unregulated biochemical reactions between glucose and the collagen extracellular matrix are coming increasingly into focus. We have shown that formation of advanced glycation end-products that cross-link the collagen extracellular matrix can drastically affect cellular level mechanical properties of the matrix, and in turn affect cell-level biomechanical stimuli during physiological loading of the tissue. We suggest that these may adversely affect tendon cell response to matrix damage, as well as the quality of the consequent repair. If such mechanical feedback loops are altered, the ability of tendon cells to maintain tissue in a functional, healthy state may be compromised. Although key foundational elements of biochemical, biomechanical, and biological understanding are now in place, the full extent of how these aspects interact, including the precise mechanisms by which advanced glycation end-products pathologically disrupt connective tissue homeostasis and damage repair, are only beginning to be adequately appreciated. PMID:27535261

  12. 46 CFR 182.530 - Bilge high level alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Bilge high level alarms. 182.530 Section 182.530... TONS) MACHINERY INSTALLATION Bilge and Ballast Systems § 182.530 Bilge high level alarms. (a) On a... operating station to indicate a high water level in each of the following normally unmanned spaces: (1)...

  13. 46 CFR 182.530 - Bilge high level alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Bilge high level alarms. 182.530 Section 182.530... TONS) MACHINERY INSTALLATION Bilge and Ballast Systems § 182.530 Bilge high level alarms. (a) On a... operating station to indicate a high water level in each of the following normally unmanned spaces: (1)...

  14. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  15. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  16. 46 CFR 182.530 - Bilge high level alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Bilge high level alarms. 182.530 Section 182.530... TONS) MACHINERY INSTALLATION Bilge and Ballast Systems § 182.530 Bilge high level alarms. (a) On a... operating station to indicate a high water level in each of the following normally unmanned spaces: (1)...

  17. 46 CFR 182.530 - Bilge high level alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Bilge high level alarms. 182.530 Section 182.530... TONS) MACHINERY INSTALLATION Bilge and Ballast Systems § 182.530 Bilge high level alarms. (a) On a... operating station to indicate a high water level in each of the following normally unmanned spaces: (1)...

  18. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  19. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  20. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  1. Fermilab Tevatron high level rf accelerating systems

    SciTech Connect

    Kerns, Q.; Kerns, C.; Miller, H.; Tawser, S.; Reid, J.; Webber, R.; Wildman, D.

    1985-06-01

    Eight tuned rf cavities have been installed and operated in the F0 straight section of the Tevatron. Their mechanical placement along the beam line enables them to be operated for colliding beams as two independent groups of four cavities, group 1-4 accelerating antiprotons and group 5-8 accelerating protons. The only difference is that the spacing between cavities 4 and 5 was increased to stay clear of the F0 colliding point. The cavities can easily be rephased by switching cables in a low-level distribution system (fan-out) so that the full accelerating capability of all eight cavities can be used during fixed target operations. Likewise, the cables from capacitive probes on each cavity gap can be switched to proper lengths and summed in a fan-back system to give an rf signal representing the amplitude and phase as ''seen by the beam,'' separately for protons and antiprotons. Such signals have been used to phase lock the Tevatron to the Main Ring for synchronous transfer.

  2. Bumblebee Pupae Contain High Levels of Aluminium

    PubMed Central

    Exley, Christopher; Rotheray, Ellen; Goulson, David

    2015-01-01

    The causes of declines in bees and other pollinators remains an on-going debate. While recent attention has focussed upon pesticides, other environmental pollutants have largely been ignored. Aluminium is the most significant environmental contaminant of recent times and we speculated that it could be a factor in pollinator decline. Herein we have measured the content of aluminium in bumblebee pupae taken from naturally foraging colonies in the UK. Individual pupae were acid-digested in a microwave oven and their aluminium content determined using transversely heated graphite furnace atomic absorption spectrometry. Pupae were heavily contaminated with aluminium giving values between 13.4 and 193.4 μg/g dry wt. and a mean (SD) value of 51.0 (33.0) μg/g dry wt. for the 72 pupae tested. Mean aluminium content was shown to be a significant negative predictor of average pupal weight in colonies. While no other statistically significant relationships were found relating aluminium to bee or colony health, the actual content of aluminium in pupae are extremely high and demonstrate significant exposure to aluminium. Bees rely heavily on cognitive function and aluminium is a known neurotoxin with links, for example, to Alzheimer’s disease in humans. The significant contamination of bumblebee pupae by aluminium raises the intriguing spectre of cognitive dysfunction playing a role in their population decline. PMID:26042788

  3. Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line

    PubMed Central

    Kumar, Amit; Abbas, Wasim; Colin, Laurence; Khan, Kashif Aziz; Bouchat, Sophie; Varin, Audrey; Larbi, Anis; Gatot, Jean-Stéphane; Kabeya, Kabamba; Vanhulle, Caroline; Delacourt, Nadège; Pasquereau, Sébastien; Coquard, Laurie; Borch, Alexandra; König, Renate; Clumeck, Nathan; De Wit, Stephane; Rohr, Olivier; Rouzioux, Christine; Fulop, Tamas; Van Lint, Carine; Herbein, Georges

    2016-01-01

    Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine473 and threonine308. In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients. PMID:27076174

  4. Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line.

    PubMed

    Kumar, Amit; Abbas, Wasim; Colin, Laurence; Khan, Kashif Aziz; Bouchat, Sophie; Varin, Audrey; Larbi, Anis; Gatot, Jean-Stéphane; Kabeya, Kabamba; Vanhulle, Caroline; Delacourt, Nadège; Pasquereau, Sébastien; Coquard, Laurie; Borch, Alexandra; König, Renate; Clumeck, Nathan; De Wit, Stephane; Rohr, Olivier; Rouzioux, Christine; Fulop, Tamas; Van Lint, Carine; Herbein, Georges

    2016-01-01

    Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients. PMID:27076174

  5. 40 CFR 227.30 - High-level radioactive waste.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false High-level radioactive waste. 227.30 Section 227.30 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING...-level radioactive waste. High-level radioactive waste means the aqueous waste resulting from...

  6. 40 CFR 227.30 - High-level radioactive waste.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false High-level radioactive waste. 227.30 Section 227.30 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING...-level radioactive waste. High-level radioactive waste means the aqueous waste resulting from...

  7. 40 CFR 227.30 - High-level radioactive waste.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true High-level radioactive waste. 227.30 Section 227.30 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING...-level radioactive waste. High-level radioactive waste means the aqueous waste resulting from...

  8. 40 CFR 227.30 - High-level radioactive waste.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false High-level radioactive waste. 227.30 Section 227.30 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING...-level radioactive waste. High-level radioactive waste means the aqueous waste resulting from...

  9. Analysis of Cyberbullying Sensitivity Levels of High School Students and Their Perceived Social Support Levels

    ERIC Educational Resources Information Center

    Akturk, Ahmet Oguz

    2015-01-01

    Purpose: The purpose of this paper is to determine the cyberbullying sensitivity levels of high school students and their perceived social supports levels, and analyze the variables that predict cyberbullying sensitivity. In addition, whether cyberbullying sensitivity levels and social support levels differed according to gender was also…

  10. A Framework for Translating a High Level Security Policy into Low Level Security Mechanisms

    NASA Astrophysics Data System (ADS)

    Hassan, Ahmed A.; Bahgat, Waleed M.

    2010-01-01

    Security policies have different components; firewall, active directory, and IDS are some examples of these components. Enforcement of network security policies to low level security mechanisms faces some essential difficulties. Consistency, verification, and maintenance are the major ones of these difficulties. One approach to overcome these difficulties is to automate the process of translation of high level security policy into low level security mechanisms. This paper introduces a framework of an automation process that translates a high level security policy into low level security mechanisms. The framework is described in terms of three phases; in the first phase all network assets are categorized according to their roles in the network security and relations between them are identified to constitute the network security model. This proposed model is based on organization based access control (OrBAC). However, the proposed model extend the OrBAC model to include not only access control policy but also some other administrative security policies like auditing policy. Besides, the proposed model enables matching of each rule of the high level security policy with the corresponding ones of the low level security policy. Through the second phase of the proposed framework, the high level security policy is mapped into the network security model. The second phase could be considered as a translation of the high level security policy into an intermediate model level. Finally, the intermediate model level is translated automatically into low level security mechanism. The paper illustrates the applicability of proposed approach through an application example.

  11. Low-level awareness accompanies "unconscious" high-level processing during continuous flash suppression.

    PubMed

    Gelbard-Sagiv, Hagar; Faivre, Nathan; Mudrik, Liad; Koch, Christof

    2016-01-01

    The scope and limits of unconscious processing are a matter of ongoing debate. Lately, continuous flash suppression (CFS), a technique for suppressing visual stimuli, has been widely used to demonstrate surprisingly high-level processing of invisible stimuli. Yet, recent studies showed that CFS might actually allow low-level features of the stimulus to escape suppression and be consciously perceived. The influence of such low-level awareness on high-level processing might easily go unnoticed, as studies usually only probe the visibility of the feature of interest, and not that of lower-level features. For instance, face identity is held to be processed unconsciously since subjects who fail to judge the identity of suppressed faces still show identity priming effects. Here we challenge these results, showing that such high-level priming effects are indeed induced by faces whose identity is invisible, but critically, only when a lower-level feature, such as color or location, is visible. No evidence for identity processing was found when subjects had no conscious access to any feature of the suppressed face. These results suggest that high-level processing of an image might be enabled by-or co-occur with-conscious access to some of its low-level features, even when these features are not relevant to the processed dimension. Accordingly, they call for further investigation of lower-level awareness during CFS, and reevaluation of other unconscious high-level processing findings. PMID:26756173

  12. Physical Activity Levels in Portuguese High School Physical Education

    ERIC Educational Resources Information Center

    Marmeleira, Jose Francisco Filipe; Aldeias, Nuno Micael Carrasqueira; da Graca, Pedro Miguel dos Santos Medeira

    2012-01-01

    The main aim of this study was to evaluate the physical activity (PA) levels of high school Portuguese students during physical education (PE) and investigate the association of PA levels with students' goal orientation and intrinsic motivation. Forty-six students from three high schools participated. Heart rate telemetry and pedometry were used…

  13. Process for solidifying high-level nuclear waste

    DOEpatents

    Ross, Wayne A.

    1978-01-01

    The addition of a small amount of reducing agent to a mixture of a high-level radioactive waste calcine and glass frit before the mixture is melted will produce a more homogeneous glass which is leach-resistant and suitable for long-term storage of high-level radioactive waste products.

  14. 46 CFR 153.409 - High level alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false High level alarms. 153.409 Section 153.409 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SHIPS CARRYING BULK LIQUID, LIQUEFIED GAS, OR COMPRESSED GAS HAZARDOUS MATERIALS Design and Equipment Cargo Gauging Systems § 153.409 High level alarms. When Table...

  15. Mucosal Humoral Immune Response to SIVmac239∆nef Vaccination and Vaginal Challenge.

    PubMed

    Zeng, Ming; Smith, Anthony J; Shang, Liang; Wietgrefe, Stephen W; Voss, James E; Carlis, John V; Li, Qingsheng; Piatak, Michael; Lifson, Jeffrey D; Johnson, R Paul; Haase, Ashley T

    2016-03-15

    Live attenuated vaccines such as SIV with a deleted nef gene have provided the most robust protection against subsequent vaginal challenge with wild-type (WT) SIV in the SIV-rhesus macaque model of HIV-1 transmission to women. Hence, identifying correlates of this protection could enable design of an effective HIV-1 vaccine. One such prechallenge correlate of protection from vaginal challenge has recently been identified as a system with three components: 1) IgG Abs reacting with the viral envelope glycoprotein trimeric gp41; 2) produced by plasma cells in the submucosa and ectopic tertiary lymphoid follicles in the ectocervix and vagina; and 3) concentrated on the path of virus entry by the neonatal FcR in the overlying epithelium. We now examine the mucosal production of the Ab component of this system after vaginal challenge. We show that vaginal challenge immediately elicits striking increases in plasma cells not only in the female reproductive tract but also at other mucosal sites, and that these increases correlate with low but persistent replication at mucosal sites. We describe vaginal ectopic follicles that are structurally and functionally organized similar to follicles in secondary lymphoid organs, and we provide inferential evidence for a key role of the female reproductive tract epithelium in facilitating Ab production, affinity maturation, and class switch recombination. Vaccination thus accesses an epithelial-immune system axis in the female reproductive tract to respond to exposure to mucosal pathogens. Designing strategies to mimic this system could advance development of an effective HIV-1 vaccine. PMID:26864031

  16. Predictors of Placement in Lower Level versus Higher Level High School Mathematics

    ERIC Educational Resources Information Center

    Archbald, Doug; Farley-Ripple, Elizabeth N.

    2012-01-01

    Educators and researchers have long been interested in determinants of access to honors level and college prep courses in high school. Factors influencing access to upper level mathematics courses are particularly important because of the hierarchical and sequential nature of this subject and because students who finish high school with only lower…

  17. Stable superconducting magnet. [high current levels below critical temperature

    NASA Technical Reports Server (NTRS)

    Boom, R. W. (Inventor)

    1967-01-01

    Operation of a superconducting magnet is considered. A method is described for; (1) obtaining a relatively high current in a superconducting magnet positioned in a bath of a gas refrigerant; (2) operating a superconducting magnet at a relatively high current level without training; and (3) operating a superconducting magnet containing a plurality of turns of a niobium zirconium wire at a relatively high current level without training.

  18. Reference commercial high-level waste glass and canister definition.

    SciTech Connect

    Slate, S.C.; Ross, W.A.; Partain, W.L.

    1981-09-01

    This report presents technical data and performance characteristics of a high-level waste glass and canister intended for use in the design of a complete waste encapsulation package suitable for disposal in a geologic repository. The borosilicate glass contained in the stainless steel canister represents the probable type of high-level waste product that will be produced in a commercial nuclear-fuel reprocessing plant. Development history is summarized for high-level liquid waste compositions, waste glass composition and characteristics, and canister design. The decay histories of the fission products and actinides (plus daughters) calculated by the ORIGEN-II code are presented.

  19. Human immunodeficiency virus type 1 Nef does not alter T-cell sensitivity to antigen-specific stimulation.

    PubMed Central

    Page, K A; van Schooten, W C; Feinberg, M B

    1997-01-01

    We have developed an in vitro model to study the influence that human immunodeficiency virus type 1 (HIV-1) may have on the ability of T cells to respond to antigenic challenge. We have examined consequences of HIV-1 gene expression on T-cell activation in antigen-dependent T cells that have stably integrated copies of replication-defective proviral HIV-1. Virus production by HIV-infected, antigen-dependent T cells was induced in response to antigenic stimulation and then decreased as infected cells returned to a state of quiescence. Contrary to the predictions of models proposing that Nef alters signal transduction pathways in T lymphocytes and thereby alters cellular activation, Nef expression in antigen-dependent T-cell clones did not influence their proliferative responses to low or intermediate concentrations of antigen and did not affect other measures of T-cell activation, such as induction of interleukin 2 receptor alpha-chain expression and cytokine production. In addition, we found no evidence for alteration of T-cell responsiveness to antigen by the gag, pol, vif, tat, or rev gene of HIV-1. PMID:9094653

  20. Production of recombinant HIV-1 nef protein using different expression host systems: a techno-economical comparison.

    PubMed

    Vermasvuori, Raisa; Koskinen, Jani; Salonen, Katri; Sirén, Noora; Weegar, Jan; Dahlbacka, John; Kalkkinen, Nisse; von Weymarn, Niklas

    2009-01-01

    Three popular expression host systems Escherichia coli, Pichia pastoris and Drosophila S2 were analyzed techno-economically using HIV-1 Nef protein as the model product. On scale of 100 mg protein, the labor costs corresponded to 52-83% of the manufacturing costs. When analyzing the cost impact of the different phases (strain/cell line construction, bioreactor production, and primary purification), we found that with the microbial host systems the strain construction phase was most significant generating 56% (E. coli) and 72% (P. pastoris) of the manufacturing costs, whereas with the Drosophila S2 system the cell line construction and bioreactor production phases were equally significant (46 and 47% of the total costs, respectively). With different titers and production goal of 100 mg of Nef protein, the costs of P. pastoris and Drosophila S2 systems were about two and four times higher than the respective costs of the E. coli system. When equal titers and bioreactor working volumes (10 L) were assumed for all three systems, the manufacturing costs of the bioreactor production of the P. pastoris and Drosophila S2 systems were about two and 2.5 times higher than the respective costs of the E. coli system. PMID:19224559

  1. Neptunium estimation in dissolver and high-level-waste solutions

    SciTech Connect

    Pathak, P.N.; Prabhu, D.R.; Kanekar, A.S.; Manchanda, V.K.

    2008-07-01

    This papers deals with the optimization of the experimental conditions for the estimation of {sup 237}Np in spent-fuel dissolver/high-level waste solutions using thenoyltrifluoroacetone as the extractant. (authors)

  2. HIGH-LEVEL OZONE DISINFECTION OF MUNICIPAL WASTEWATER EFFLUENTS

    EPA Science Inventory

    A 20 month operating experimental program was conducted at Marlborough, Massachusetts to evaluate the feasibility, engineering, and economic aspects of achieving high levels of effluent disinfection with ozone. The ozone research pilot facility was designed to operate at a consta...

  3. Field Trips as Cognitive Motivators for High Level Science Learning

    ERIC Educational Resources Information Center

    Hurley, Marlene M.

    2006-01-01

    Using a composite example of field trips from several years of traveling to Yellowstone with high school biology students, the author illustrates how to raise the cognitive level of science instruction and student learning through science field trips. The author examines what teachers can do to raise the level of both teaching and learning in all…

  4. High-Level Waste System Process Interface Description

    SciTech Connect

    d'Entremont, P.D.

    1999-01-14

    The High-Level Waste System is a set of six different processes interconnected by pipelines. These processes function as one large treatment plant that receives, stores, and treats high-level wastes from various generators at SRS and converts them into forms suitable for final disposal. The three major forms are borosilicate glass, which will be eventually disposed of in a Federal Repository, Saltstone to be buried on site, and treated water effluent that is released to the environment.

  5. Decision Document for Heat Removal from High Level Waste Tanks

    SciTech Connect

    WILLIS, W.L.

    2000-07-31

    This document establishes the combination of design and operational configurations that will be used to provide heat removal from high-level waste tanks during Phase 1 waste feed delivery to prevent the waste temperature from exceeding tank safety requirement limits. The chosen method--to use the primary and annulus ventilation systems to remove heat from the high-level waste tanks--is documented herein.

  6. 46 CFR 182.530 - Bilge high level alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Bilge high level alarms. 182.530 Section 182.530 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) MACHINERY INSTALLATION Bilge and Ballast Systems § 182.530 Bilge high level alarms. (a) On a vessel of at least 7.9 meters (26 feet) in...

  7. Effect of high fluoride and high fat on serum lipid levels and oxidative stress in rabbits.

    PubMed

    Sun, Liyan; Gao, Yanhui; Zhang, Wei; Liu, Hui; Sun, Dianjun

    2014-11-01

    The purpose of this study was to explore the effects of high fluoride and high fat on triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total antioxidant capacity (T-AOC), lipid peroxide (LPO) and malondialdehyde (MDA) in rabbits. A factorial experimental design was used, with two factors (fluoride and fat) and three levels. Seventy-two male rabbits were randomly assigned into nine groups according to initial weight and serum lipid levels. The rabbits were fed with basic feed, moderate fat feed or high fat feed and drank tap water, fluoridated water at levels of 50 and 100mgfluorion/L freely. Biological materials were collected after 5 months, and serum lipid, T-AOC, LPO, and MDA levels were then measured. Using these data, the separate and interactive effects of high fluoride and high fat were analyzed. High fluoride and high fat both increased serum levels of TC, HDL-C and LDL-C significantly (P<0.05), and there was also a synergistic effect between high fluoride and high fat (P<0.05). High fluoride and high fat had different effects on TG levels: high fat significantly increased TG levels (P<0.01) whereas high fluoride had nothing to do with TG levels (P>0.05). High fat significantly elevated LPO and MDA levels and lowered T-AOC levels in serum (P<0.05). Similarly, high fluoride significantly increased LPO and MDA levels in serum (P<0.05). However, there was no interactive effect between high fat and high fluoride on these indexes. In summary, high fluoride and high fat increased serum TC and LDL-C levels individually and synergistically, and this would cause and aggravate hypercholesterolemia in rabbits. At the same time, high fluoride and high fat both made the accumulation of product of oxidative stress in experimental animals. PMID:25461561

  8. Comparison of HIV-1 nef and gag Variations and Host HLA Characteristics as Determinants of Disease Progression among HIV-1 Vertically Infected Kenyan Children

    PubMed Central

    Saina, Matilda Chelimo; Bi, Xiuqiong; Lihana, Raphael; Lwembe, Raphael; Ishizaki, Azumi; Panikulam, Annie; Palakudy, Tresa; Musoke, Rachel; Owens, Mary; Songok, Elijah Maritim; Ichimura, Hiroshi

    2015-01-01

    Objectives Disease progression varies among HIV-1-infected individuals. The present study aimed to explore possible viral and host factors affecting disease progression in HIV-1-infected children. Methods Since 2000, 102 HIV-1 vertically-infected children have been followed-up in Kenya. Here we studied 29 children (15 male/14 female) who started antiretroviral treatment at <5 years of age (rapid progressors; RP), and 32 (17 male/15 female) who started at >10 years of age (slow progressors; SP). Sequence variations in the HIV-1 gag and nef genes and the HLA class I-related epitopes were compared between the two groups. Results Based on nef sequences, HIV-1 subtypes A1/D were detected in 62.5%/12.5% of RP and 66.7%/20% of SP, with no significant difference in subtype distribution between groups (p = 0.8). In the ten Nef functional domains, only the PxxP3 region showed significantly greater variation in RP (33.3%) than SP (7.7%, p = 0.048). Gag sequences did not significantly differ between groups. The reportedly protective HLA-A alleles, A*74:01, A*32:01 and A*26, were more commonly observed in SP (50.0%) than RP (11.1%, p = 0.010), whereas the reportedly disease-susceptible HLA-B*45:01 was more common in RP (33.3%) than SP (7.4%, p = 0.045). Compared to RP, SP showed a significantly higher median number of predicted HLA-B-related 12-mer epitopes in Nef (3 vs. 2, p = 0.037), HLA-B-related 11-mer epitopes in Gag (2 vs. 1, p = 0.029), and HLA-A-related 9-mer epitopes in Gag (4 vs. 1, p = 0.051). SP also had fewer HLA-C-related epitopes in Nef (median 4 vs. 5, p = 0.046) and HLA-C-related 11-mer epitopes in Gag (median 1 vs. 1.5, p = 0.044) than RP. Conclusions Compared to rapid progressors, slow progressors had more protective HLA-A alleles and more HLA-B-related epitopes in both the Nef and Gag proteins. These results suggest that the host factor HLA plays a stronger role in disease progression than the Nef and Gag sequence variations in HIV-1-infected Kenyan children

  9. The effect of high altitude on nasal nitric oxide levels.

    PubMed

    Altundag, Aytug; Salihoglu, Murat; Cayonu, Melih; Cingi, Cemal; Tekeli, Hakan; Hummel, Thomas

    2014-09-01

    The aim of the present study was to investigate whether nasal nitric oxide (nNO) levels change in relation to high altitude in a natural setting where the weather conditions were favorable. The present study included 41 healthy volunteers without a history of acute rhinosinusitis within 3 weeks and nasal polyposis. The study group consisted of 31 males (76 %) and 10 females (24 %) and the mean age of the study population was 38 ± 10 years. The volunteers encamped for 2 days in a mountain village at an altitude of 1,500 m above sea level (masl) and proceeded to highlands at an altitude of 2,200 masl throughout the day. The measurements of nNO were done randomly, either first at the mountain village or at sea level. Each participant had nNO values both at sea level and at high altitude at the end of the study. The nNO values of sea level and high altitude were compared to investigate the effect of high altitude on nNO levels. The mean of average nNO measurements at the high altitude was 74.2 ± 41 parts-per-billion (ppb) and the mean of the measurements at sea level was 93.4 ± 45 ppb. The change in nNO depending on the altitude level was statistically significant (p < 0.001). The current investigation showed that nNO levels were decreased at high altitude even if the weather conditions were favorable, such as temperature, humidity, and wind. PMID:24972544

  10. Does high serum uric acid level cause aspirin resistance?

    PubMed

    Yildiz, Bekir S; Ozkan, Emel; Esin, Fatma; Alihanoglu, Yusuf I; Ozkan, Hayrettin; Bilgin, Murat; Kilic, Ismail D; Ergin, Ahmet; Kaftan, Havane A; Evrengul, Harun

    2016-06-01

    In patients with coronary artery disease (CAD), though aspirin inhibits platelet activation and reduces atherothrombotic complications, it does not always sufficiently inhibit platelet function, thereby causing a clinical situation known as aspirin resistance. As hyperuricemia activates platelet turnover, aspirin resistance may be specifically induced by increased serum uric acid (SUA) levels. In this study, we thus investigated the association between SUA level and aspirin resistance in patients with CAD. We analyzed 245 consecutive patients with stable angina pectoris (SAP) who in coronary angiography showed more than 50% occlusion in a major coronary artery. According to aspirin resistance, two groups were formed: the aspirin resistance group (Group 1) and the aspirin-sensitive group (Group 2). Compared with those of Group 2, patients with aspirin resistance exhibited significantly higher white blood cell counts, neutrophil counts, neutrophil-to-lymphocyte ratios, SUA levels, high-sensitivity C-reactive protein levels, and fasting blood glucose levels. After multivariate analysis, a high level of SUA emerged as an independent predictor of aspirin resistance. The receiver-operating characteristic analysis provided a cutoff value of 6.45 mg/dl for SUA to predict aspirin resistance with 79% sensitivity and 65% specificity. Hyperuricemia may cause aspirin resistance in patients with CAD and high SUA levels may indicate aspirin-resistant patients. Such levels should thus recommend avoiding heart attack and stroke by adjusting aspirin dosage. PMID:26656902

  11. An overview of very high level software design methods

    NASA Technical Reports Server (NTRS)

    Asdjodi, Maryam; Hooper, James W.

    1988-01-01

    Very High Level design methods emphasize automatic transfer of requirements to formal design specifications, and/or may concentrate on automatic transformation of formal design specifications that include some semantic information of the system into machine executable form. Very high level design methods range from general domain independent methods to approaches implementable for specific applications or domains. Applying AI techniques, abstract programming methods, domain heuristics, software engineering tools, library-based programming and other methods different approaches for higher level software design are being developed. Though one finds that a given approach does not always fall exactly in any specific class, this paper provides a classification for very high level design methods including examples for each class. These methods are analyzed and compared based on their basic approaches, strengths and feasibility for future expansion toward automatic development of software systems.

  12. Student Achievement Levels Raised at Chandler High School.

    ERIC Educational Resources Information Center

    Profiles, Programs & Products, 1983

    1983-01-01

    Chandler (Arizona) High School has reported a dramatic increase in student achievement levels since implementing a number of management, curriculum, and discipline policy changes. Among the program changes that contributed to these gains are: (1) creation of a positive school environment, with high expectations for teachers, emphasis on high…

  13. A Testing Instrument for High School Arabic, Level III.

    ERIC Educational Resources Information Center

    Wolowelsky, Joel B.

    The Arabic language examination was designed for Jewish immigrants from Syria wishing to satisfy New York State language requirements for high school graduation by indicating their proficiency in Arabic. The test is essentially a translation of a state test of Hebrew, and is intended to test Arabic at the third-year high school level. The…

  14. Determination of the nuclear level density at high excitation energy

    SciTech Connect

    Chbihi, A.; Sobotka, L.G.; Nicolis, N.G.; Sarantites, D.G.; Stracener, D.W.; Majka, Z. ); Hensley, D.C.; Beene, J.R.; Halbert, M.L. )

    1991-02-01

    Evaporation simulations are presented to illustrate the problems associated with the determination of the nuclear level density constant at high excitation energy from evaporation spectra. The methods of using either the total (whole chain) spectra or the difference (from two different initial excitation energies) spectra are discussed. Data from the study of the reaction 701 MeV {sup 28}Si+{sup 100}Mo are presented and both methods are used to extract the level density constant. We find that in order to reproduce the slopes of the light particle spectra the level density constant must have a value near 1/10{ital A}-- 1 / 11 {ital A} for excited nuclei with statistical temperatures in the range of 3.5 to 5.5 MeV. This presumes that the only parameter adjustment required to treat the decay of highly exited nuclei is the level density constant. If this is so, the shapes of the evaporation spectra imply a reduction in the level density constant from the value required to explain the decay of less highly excited nuclei, a conclusion reached by others. However, the reduced level density constant leads to an overproduction of deuterons and tritons. This suggests that a more complicated set of parameter adjustments may be required to treat the decay of highly excited nuclei.

  15. An Investigation of Source and Receiver Apprehension at the Junior High, Senior High and College Levels.

    ERIC Educational Resources Information Center

    McDowell, Earl E.; McDowell, Carlene E.

    1978-01-01

    The Personal Report of Communication Apprehension and the Receiver Apprehension Test are used to determine the relationship between speech and listening apprehension at each educational level (junior high, senior high, and college); differences among educational levels and between sexes; and the interaction effect of educational level and sex. (JF)

  16. Oxidative Stress Levels in Aqueous Humor from High Myopic Patients

    PubMed Central

    Kim, Eun Bi; Kim, Ha Kyoung; Hyon, Joon Young; Wee, Won Ryang

    2016-01-01

    Purpose To compare oxidative stress status in the aqueous humor of highly myopic eyes and control eyes. Methods Aqueous humor samples were collected from 15 highly myopic eyes (high myopia group) and 23 cataractous eyes (control group) during cataract surgery. Central corneal thickness, corneal endothelial cell density, hexagonality of corneal endothelial cells, and cell area of corneal endothelial cells were measured using specular microscopy. Axial length was measured using ultrasound biometry. 8-Hydroxydeoxyguanosine (8-OHdG) and malondialdehyde levels were measured using enzyme-linked immunosorbent assay. Results 8-OHdG level was lower in the aqueous humor of myopic patients than in that of control group (p = 0.014) and was positively correlated with central corneal thickness and negatively correlated with axial length (r = 0.511, p = 0.02; r = -0.382, p < 0.001). There was no correlation between 8-OHdG level and corneal endothelial cell density, hexagonality, or cell area. Malondialdehyde level did not show any correlation with any parameters evaluated. Conclusions 8-OHdG might be a sensitive biomarker for evaluating oxidative stress status in the eye. Oxidative stress level was lower in the aqueous humor of highly myopic eyes compared to that in control eyes, which indicates lower metabolic activity in these eyes. PMID:27247516

  17. Photovoltaic (PV) Impact Assessment for Very High Penetration Levels

    SciTech Connect

    Cheng, Danling; Mather, Barry A.; Seguin, Richard; Hambrick, Joshua; Broadwater, Robert P.

    2016-01-01

    This paper describes a granular approach for investigating the impacts of very high photovoltaic (PV) generation penetration. Studies on two real-world distribution feeders connected to PV plants are presented. The studies include both steady-state and time-series power flow analyses, which include the effects of solar variability. The goal of the study is to predict the effects of increasing levels of PV generation as it reaches very high penetration levels. The loss and return of generation with and without regulation is simulated to capture short-term problems such as voltage fluctuations. Impact results from the analyses are described along with potential mitigations.

  18. Disposal of high-level nuclear waste in space

    NASA Astrophysics Data System (ADS)

    Coopersmith, Jonathan

    1992-08-01

    A solution of launching high-level nuclear waste into space is suggested. Disposal in space includes solidifying the wastes, embedding them in an explosion-proof vehicle, and launching it into earth orbit, and then into a solar orbit. The benefits of such a system include not only the safe disposal of high-level waste but also the establishment of an infrastructure for large-scale space exploration and development. Particular attention is given to the wide range of technical choices along with the societal, economic, and political factors needed for success.

  19. High Level Waste (HLW) Feed Process Control Strategy

    SciTech Connect

    STAEHR, T.W.

    2000-06-14

    The primary purpose of this document is to describe the overall process control strategy for monitoring and controlling the functions associated with the Phase 1B high-level waste feed delivery. This document provides the basis for process monitoring and control functions and requirements needed throughput the double-shell tank system during Phase 1 high-level waste feed delivery. This document is intended to be used by (1) the developers of the future Process Control Plan and (2) the developers of the monitoring and control system.

  20. Final report on cermet high-level waste forms

    SciTech Connect

    Kobisk, E.H.; Quinby, T.C.; Aaron, W.S.

    1981-08-01

    Cermets are being developed as an alternate method for the fixation of defense and commercial high level radioactive waste in a terminal disposal form. Following initial feasibility assessments of this waste form, consisting of ceramic particles dispersed in an iron-nickel base alloy, significantly improved processing methods were developed. The characterization of cermets has continued through property determinations on samples prepared by various methods from a variety of simulated and actual high-level wastes. This report describes the status of development of the cermet waste form as it has evolved since 1977. 6 tables, 18 figures.

  1. The Use of ARTEMIS with High-Level Applications

    SciTech Connect

    B. A. Bowling; H. Shoaee; S. Witherspoon

    1995-10-01

    ARTEMIS is an online accelerator modeling server developed at CEBAF. One of the design goals of ARTEMIS was to provide an integrated modeling environment for high- level accelerator diagnostic and control applications such as automated beam steering, Linac Energy management (LEM) and the fast feedback system. This report illustrates the use of ARTEMIS in these applications as well as the application interface using the EPICS cdev device support API. Concentration is placed on the design and implementation aspects of high- level applications which utilize the ARTEMIS server for information on beam dynamics. Performance benchmarks for various model operations provided by ARTEMIS are also discussed.

  2. Sterilization, high-level disinfection, and environmental cleaning.

    PubMed

    Rutala, William A; Weber, David J

    2011-03-01

    Failure to perform proper disinfection and sterilization of medical devices may lead to introduction of pathogens, resulting in infection. New techniques have been developed for achieving high-level disinfection and adequate environmental cleanliness. This article examines new technologies for sterilization and high-level disinfection of critical and semicritical items, respectively, and because semicritical items carry the greatest risk of infection, the authors discuss reprocessing semicritical items such as endoscopes and automated endoscope reprocessors, endocavitary probes, prostate biopsy probes, tonometers, laryngoscopes, and infrared coagulation devices. In addition, current issues and practices associated with environmental cleaning are reviewed. PMID:21315994

  3. High level radioactive waste management facility design criteria

    SciTech Connect

    Sheikh, N.A.; Salaymeh, S.R.

    1993-10-01

    This paper discusses the engineering systems for the structural design of the Defense Waste Processing Facility (DWPF) at the Savannah River Site (SRS). At the DWPF, high level radioactive liquids will be mixed with glass particles and heated in a melter. This molten glass will then be poured into stainless steel canisters where it will harden. This process will transform the high level waste into a more stable, manageable substance. This paper discuss the structural design requirements for this unique one of a kind facility. A special emphasis will be concentrated on the design criteria pertaining to earthquake, wind and tornado, and flooding.

  4. Low voltage to high voltage level shifter and related methods

    NASA Technical Reports Server (NTRS)

    Mentze, Erik J. (Inventor); Hess, Herbert L. (Inventor); Buck, Kevin M. (Inventor); Cox, David F. (Inventor)

    2006-01-01

    A shifter circuit comprises a high and low voltage buffer stages and an output buffer stage. The high voltage buffer stage comprises multiple transistors arranged in a transistor stack having a plurality of intermediate nodes connecting individual transistors along the stack. The transistor stack is connected between a voltage level being shifted to and an input voltage. An inverter of this stage comprises multiple inputs and an output. Inverter inputs are connected to a respective intermediate node of the transistor stack. The low voltage buffer stage has an input connected to the input voltage and an output, and is operably connected to the high voltage buffer stage. The low voltage buffer stage is connected between a voltage level being shifted away from and a lower voltage. The output buffer stage is driven by the outputs of the high voltage buffer stage inverter and the low voltage buffer stage.

  5. Modeling future high-resolution dynamic sea level change

    NASA Astrophysics Data System (ADS)

    Brunnabend, Sandra-Esther; Dijkstra, Henk A.; Kliphuis, Michael A.; van Werkhoven, Ben; Bal, Henri E.; van Meersbergen, Maarten; Seinstra, Frank; Maassen, Jason

    2015-04-01

    Different studies have shown that resolving ocean eddies and representing boundary currents are of major importance when simulating changes in dynamic sea level on regional scale. Therefore, we use the strongly eddying global model version of the Parallel Ocean Program to simulate high-resolution future (up to the year 2100) sea surface height variations (SSH) under the SRES-A1B atmospheric forcing scenario. Results show dynamic sea level changes in the Southern Ocean that are caused by the southward shift in the westerly winds. The warming ocean (global mean sea surface temperature rises by about 2°C over the period 2000-2100) leads to a strong reduction of the Atlantic Meridional Overturning Circulation (AMOC). The magnitude of this reduction is affected by a feedback involving the heat transport to the sub-polar gyre region and evaporation over the North Atlantic region. The ocean circulation changes cause regional deviations from global mean sea level change in the North Atlantic. At coastal regions of eastern North America, dynamic sea level change leads to a positive deviation from global mean sea level change in the order of several decimeters. In the sub-polar gyre region a negative deviation from global mean sea level occurs. In the western North Atlantic, not only mean regional sea level is changed but also its variability, caused by shifted eddy pathways. This leads to a change in the frequency distribution of SSH anomalies, which has important consequences for regional sea level extremes.

  6. MMP for moduli of sheaves on K3s via wall-crossing: nef and movable cones, Lagrangian fibrations

    NASA Astrophysics Data System (ADS)

    Bayer, Arend; Macrì, Emanuele

    2014-12-01

    We use wall-crossing with respect to Bridgeland stability conditions to systematically study the birational geometry of a moduli space M of stable sheaves on a K3 surface X: 1. We describe the nef cone, the movable cone, and the effective cone of M in terms of the Mukai lattice of X. 2. We establish a long-standing conjecture that predicts the existence of a birational Lagrangian fibration on M whenever M admits an integral divisor class D of square zero (with respect to the Beauville-Bogomolov form). These results are proved using a natural map from the space of Bridgeland stability conditions Stab(X) to the cone Mov(X) of movable divisors on M; this map relates wall-crossing in Stab(X) to birational transformations of M. In particular, every minimal model of M appears as a moduli space of Bridgeland-stable objects on X.

  7. The tracking of high level waste shipments-TRANSCOM system

    SciTech Connect

    Johnson, P.E.; Joy, D.S.; Pope, R.B.

    1995-12-31

    The TRANSCOM (transportation tracking and communication) system is the U.S. Department of Energy`s (DOE`s) real-time system for tracking shipments of spent fuel, high-level wastes, and other high-visibility shipments of radioactive material. The TRANSCOM system has been operational since 1988. The system was used during FY1993 to track almost 100 shipments within the US.DOE complex, and it is accessed weekly by 10 to 20 users.

  8. Satellites to Delta n = 1 transitions between high-lying levels of multiply ionized atoms

    NASA Astrophysics Data System (ADS)

    Koenig, R.; Kolk, K.-H.; Koshelev, K. N.; Kunze, H.-J.

    1989-04-01

    In a theta pinch discharge satellites to Delta n = 1 transitions between high-lying levels are observed for the ions Si IX, Si X, and Si XI, but not for Si XII. They are identified as Delta n = 1 transitions between the corresponding levels of doubly excited systems. At high densities, the series of Rydberg levels above their respective thermal limit are collisionally coupled to their ionization limit. The intensity ratio of a transition to that of its satellite thus offers the unique possibility of measuring the ratio of the population density in the ground energy level of the next ionization stage to that in the lowest excited levels of this ion.

  9. The Estuary Guide. Level 3: High School. Draft.

    ERIC Educational Resources Information Center

    Alexander, Glen; And Others

    Estuaries are marine systems that serve as nurseries for animals, links in the migratory pathways, and habitat for a complex community of organisms. This curriculum guide intended for use at the high school level seeks to teach what estuaries are; provide opportunities to practice decision-making that affects estuaries; and encourage students to…

  10. 40 CFR 227.30 - High-level radioactive waste.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false High-level radioactive waste. 227.30 Section 227.30 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) OCEAN DUMPING CRITERIA FOR THE EVALUATION OF PERMIT APPLICATIONS FOR OCEAN DUMPING OF MATERIALS Definitions § 227.30...

  11. A Preview of High School Level Economic Software.

    ERIC Educational Resources Information Center

    Kincade, Jeannine H.

    The purpose of this study was to locate and evaluate high-school-level economic software compatible with an Apple II or IIe computer. To identify software, computer searches were run, bibliographies were scanned, and reviews were collected. Only software that was recommended by some outside source was included in the evaluation. The following…

  12. High level cognitive information processing in neural networks

    NASA Technical Reports Server (NTRS)

    Barnden, John A.; Fields, Christopher A.

    1992-01-01

    Two related research efforts were addressed: (1) high-level connectionist cognitive modeling; and (2) local neural circuit modeling. The goals of the first effort were to develop connectionist models of high-level cognitive processes such as problem solving or natural language understanding, and to understand the computational requirements of such models. The goals of the second effort were to develop biologically-realistic model of local neural circuits, and to understand the computational behavior of such models. In keeping with the nature of NASA's Innovative Research Program, all the work conducted under the grant was highly innovative. For instance, the following ideas, all summarized, are contributions to the study of connectionist/neural networks: (1) the temporal-winner-take-all, relative-position encoding, and pattern-similarity association techniques; (2) the importation of logical combinators into connection; (3) the use of analogy-based reasoning as a bridge across the gap between the traditional symbolic paradigm and the connectionist paradigm; and (4) the application of connectionism to the domain of belief representation/reasoning. The work on local neural circuit modeling also departs significantly from the work of related researchers. In particular, its concentration on low-level neural phenomena that could support high-level cognitive processing is unusual within the area of biological local circuit modeling, and also serves to expand the horizons of the artificial neural net field.

  13. The ATLAS Data Acquisition and High Level Trigger system

    NASA Astrophysics Data System (ADS)

    The ATLAS TDAQ Collaboration

    2016-06-01

    This paper describes the data acquisition and high level trigger system of the ATLAS experiment at the Large Hadron Collider at CERN, as deployed during Run 1. Data flow as well as control, configuration and monitoring aspects are addressed. An overview of the functionality of the system and of its performance is presented and design choices are discussed.

  14. Student Achievement Levels Climb at Ribault Senior High School.

    ERIC Educational Resources Information Center

    Profiles, Programs & Products, 1983

    1983-01-01

    Ribault (Florida) Senior High School has reported a dramatic increase in student achievement levels since implementing a comprehensive college preparation curriculum. Among the program changes that contributed to those gains are: (1) the establishment and consistent enforcement of a strong discipline program, including rules for student conduct, a…

  15. A comparison of high-level waste form characteristics

    SciTech Connect

    Salmon, R.; Notz, K.J.

    1991-01-01

    There are currently about 1055 million curies of high-level waste with a thermal output of about 2950 kilowatts (KW) at four sites in the United States: West Valley Demonstration Project (WVDP), Savannah River Site (SRS), Hanford Site (HANF), and Idaho National Engineering Laboratory (INEL). These quantities are expected to increase to about 1200 million curies and 3570 kw by the end of year 2020. Under the Nuclear Waste Policy Act, this high-level waste must ultimately be disposed of in a geologic repository. Accordingly, canisters of high-level waste immobilized in borosilicate glass or glass-ceramic mixtures are to be produced at the four sites and stored there until a repository becomes available. Data on the estimated production schedules and on the physical, chemical, and radiological characteristics of the canisters of immobilized high-level waste have been collected in OCRWM's Waste Characteristics Data Base, including recent updates an revisions. Comparisons of some of these data for the four sites are presented in this report. 14 refs., 3 tabs.

  16. Rice lines with high leaf mineral nutrient levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphorus (P) and Potassium (K), and sometimes other mineral nutrients are often applied as fertilizer, in addition to Nitrogen, to help achieve high yields in Texas rice production. For some mineral nutrients, total levels in soil would be sufficient to support the desired rice crop growth, but th...

  17. Typewriter Modifications for Persons Who Are High-Level Quadriplegics.

    ERIC Educational Resources Information Center

    O'Reagan, James R.; And Others

    Standard, common electric typewriters are not completely suited to the needs of a high-level quadriplegic typing with a mouthstick. Experiences show that for complete control of a typewriter a mouthstick user needs the combined features of one-button correction, electric forward and reverse indexing, and easy character viewing. To modify a…

  18. THE XAL INFRASTRUCTURE FOR HIGH LEVEL CONTROL ROOM APPLICATIONS

    SciTech Connect

    Shishlo, Andrei P; Allen, Christopher K; Chu, Paul; Galambos, John D; Pelaia II, Tom

    2009-01-01

    XAL is a Java programming framework for building high-level control applications related to accelerator physics. The structure, details of implementation, and interaction between components, auxiliary XAL packages, and the latest modifications are discussed. A general overview of XAL applications created for the SNS project is presented.

  19. High-Level waste process and product data annotated bibliography

    SciTech Connect

    Stegen, G.E.

    1996-02-13

    The objective of this document is to provide information on available issued documents that will assist interested parties in finding available data on high-level waste and transuranic waste feed compositions, properties, behavior in candidate processing operations, and behavior on candidate product glasses made from those wastes. This initial compilation is only a partial list of available references.

  20. The Politics of High-Level Manpower Supply in Tanzania

    ERIC Educational Resources Information Center

    Brooke-Smith, Robin

    1978-01-01

    In its policies related to high-level manpower, the Tanzanian Government attaches great importance to the university, viewing it as a key institution in its policies for national development. Describes the difficulties the administration of President Nyerere has had in using the university as a political tool and analyzes various instances of…

  1. MIXING PROCESSES IN HIGH-LEVEL WASTE TANKS

    EPA Science Inventory

    Flammable gases can be generated in DOE high-level waste tanks, including radiolytic hydrogen, and during cesium precipitation from salt solutions, benzene. Under normal operating conditions the potential for deflagration or detonation from these gases is precluded by purging and...

  2. Effective Communication: High-Level Management Receptive to Low-Level Management Ideas.

    ERIC Educational Resources Information Center

    Chressanthis, June D.; Janousek, Kelly

    Based on the premise that senior staff or high-level managers have the knowledge and experience required to make daily decisions and also have a degree of power and respect, this document proposes that these managers are in a position to be effective leaders who can and should foster an atmosphere of receptiveness for innovation and positive…

  3. High levels of plasma protein C in nephrotic syndrome.

    PubMed

    Pabinger-Fasching, I; Lechner, K; Niessner, H; Schmidt, P; Balzar, E; Mannhalter, C

    1985-02-18

    In patients with severe nephrotic syndrome determinations of plasma protein C: Ag levels (8 patients: 5 adults, 3 children) and protein C activity (3 out of 8 patients) revealed significantly elevated plasma protein C concentrations. Furthermore we observed a significant inverse correlation of protein C: Ag to AT III: Ag levels. No protein C: Ag could be detected in the urine of two patients studied. We conclude from our data, that changes of plasma protein C do not contribute to the high thrombotic tendency in nephrotic syndrome. PMID:3838827

  4. Overview of high-level waste management accomplishments

    SciTech Connect

    Lawroski, H; Berreth, J R; Freeby, W A

    1980-01-01

    Storage of power reactor spent fuel is necessary at present because of the lack of reprocessing operations particularly in the U.S. By considering the above solidification and storage scenario, there is more than reasonable assurance that acceptable, stable, low heat generation rate, solidified waste can be produced, and safely disposed. The public perception of no waste disposal solutions is being exploited by detractors of nuclear power application. The inability to even point to one overall system demonstration lends credibility to the negative assertions. By delaying the gathering of on-line information to qualify repository sites, and to implement a demonstration, the actions of the nuclear power detractors are self serving in that they can continue to point out there is no demonstration of satisfactory high-level waste disposal. By maintaining the liquid and solidified high-level waste in secure above ground storage until acceptable decay heat generation rates are achieved, by producing a compatible, high integrity, solid waste form, by providing a second or even third barrier as a compound container and by inserting the enclosed waste form in a qualified repository with spacing to assure moderately low temperature disposal conditions, there appears to be no technical reason for not progressing further with the disposal of high-level wastes and needed implementation of the complete nuclear power fuel cycle.

  5. High levels of molecular chlorine in the Arctic atmosphere

    NASA Astrophysics Data System (ADS)

    Liao, Jin; Huey, L. Gregory; Liu, Zhen; Tanner, David J.; Cantrell, Chris A.; Orlando, John J.; Flocke, Frank M.; Shepson, Paul B.; Weinheimer, Andrew J.; Hall, Samuel R.; Ullmann, Kirk; Beine, Harry J.; Wang, Yuhang; Ingall, Ellery D.; Stephens, Chelsea R.; Hornbrook, Rebecca S.; Apel, Eric C.; Riemer, Daniel; Fried, Alan; Mauldin, Roy L.; Smith, James N.; Staebler, Ralf M.; Neuman, J. Andrew; Nowak, John B.

    2014-02-01

    Chlorine radicals can function as a strong atmospheric oxidant, particularly in polar regions, where levels of hydroxyl radicals are low. In the atmosphere, chlorine radicals expedite the degradation of methane and tropospheric ozone, and the oxidation of mercury to more toxic forms. Here we present direct measurements of molecular chlorine levels in the Arctic marine boundary layer in Barrow, Alaska, collected in the spring of 2009 over a six-week period using chemical ionization mass spectrometry. We report high levels of molecular chlorine, of up to 400 pptv. Concentrations peaked in the early morning and late afternoon, and fell to near-zero levels at night. Average daytime molecular chlorine levels were correlated with ozone concentrations, suggesting that sunlight and ozone are required for molecular chlorine formation. Using a time-dependent box model, we estimate that the chlorine radicals produced from the photolysis of molecular chlorine oxidized more methane than hydroxyl radicals, on average, and enhanced the abundance of short-lived peroxy radicals. Elevated hydroperoxyl radical levels, in turn, promoted the formation of hypobromous acid, which catalyses mercury oxidation and the breakdown of tropospheric ozone. We therefore suggest that molecular chlorine exerts a significant effect on the atmospheric chemistry of the Arctic.

  6. Measuring the effects of high CO₂ levels in Caenorhabditis elegans.

    PubMed

    Zuela, Noam; Friedman, Nurit; Zaslaver, Alon; Gruenbaum, Yosef

    2014-08-01

    Carbon dioxide (CO2) is an important molecule in cell metabolism. It is also a byproduct of many physiological processes. In humans, impaired lung function and lung diseases disrupt the body's ability to dispose of CO2 and elevate its levels in the body (hypercapnia). Animal models allow further understanding of how CO2 is sensed in the body and what are the physiological responses to high CO2 levels. This information can provide new strategies in the battle against the detrimental effects of CO2 accumulation in lung diseases. The nematode Caenorhabditis elegans provides us with such a model animal due to its natural ability to sense and navigate through varying concentrations of CO2, as well as the fact that it can be genetically manipulated with ease. Here we describe the different methods used to measure the effects elevated levels of CO2 have on the molecular sensing mechanism and physiology of C. elegans. PMID:24650565

  7. High-level waste management technology program plan

    SciTech Connect

    Harmon, H.D.

    1995-01-01

    The purpose of this plan is to document the integrated technology program plan for the Savannah River Site (SRS) High-Level Waste (HLW) Management System. The mission of the SRS HLW System is to receive and store SRS high-level wastes in a see and environmentally sound, and to convert these wastes into forms suitable for final disposal. These final disposal forms are borosilicate glass to be sent to the Federal Repository, Saltstone grout to be disposed of on site, and treated waste water to be released to the environment via a permitted outfall. Thus, the technology development activities described herein are those activities required to enable successful accomplishment of this mission. The technology program is based on specific needs of the SRS HLW System and organized following the systems engineering level 3 functions. Technology needs for each level 3 function are listed as reference, enhancements, and alternatives. Finally, FY-95 funding, deliverables, and schedules are s in Chapter IV with details on the specific tasks that are funded in FY-95 provided in Appendix A. The information in this report represents the vision of activities as defined at the beginning of the fiscal year. Depending on emergent issues, funding changes, and other factors, programs and milestones may be adjusted during the fiscal year. The FY-95 SRS HLW technology program strongly emphasizes startup support for the Defense Waste Processing Facility and In-Tank Precipitation. Closure of technical issues associated with these operations has been given highest priority. Consequently, efforts on longer term enhancements and alternatives are receiving minimal funding. However, High-Level Waste Management is committed to participation in the national Radioactive Waste Tank Remediation Technology Focus Area. 4 refs., 5 figs., 9 tabs.

  8. A high resolution water level forecast for the German Bight

    NASA Astrophysics Data System (ADS)

    Niehüser, Sebastian; Dangendorf, Sönke; Arns, Arne; Jensen, Jürgen

    2016-04-01

    Many coastal regions worldwide are potentially endangered by storm surges which can cause disastrous damages and loss of life. Due to climate change induced sea level rise, an accumulation of such events is expected by the end of the 21th century. Therefore, advanced storm surge warnings are needed to be prepared when another storm surge hits the coast. In the shallow southeastern North Sea these storm surge warnings are nowadays routinely provided for selected tide gauge locations along a coastline through state-of-the-art forecast systems, which are based on a coupled system of empirical tidal predictions and numerical storm surge forecasts. Along the German North Sea coastline, the Federal Maritime and Hydrographic Agency in cooperation with the German Weather Service is responsible for the storm surge warnings. They provide accurate, high frequency and real-time water level forecasts for up to six days ahead at selected tide gauge sites via internet, telephone and broadcast. Since water levels along the German North Sea coastline are dominated by shallow water effects and a very complex bathymetric structure of the seabed, the pointwise forecast is not necessarily transferable to un-gauged areas between the tide gauges. Here we aim to close this existing gap and develop water level forecasts with a high spatial (continuously with a resolution of at least 1 kilometer) as well as a high temporal (at least 15-minute values) resolution along the entire German North Sea coastline. We introduce a new methodology for water level forecasts which combines empirical or statistical and numerical models. While the tidal forecast is performed by non-parametric interpolation techniques between un-gauged and gauged sites, storm surges are estimated on the basis of statistical/empirical storm surge formulas taken from a numerical model hindcast. The procedure will be implemented in the operational mode forced with numerical weather forecasts.

  9. Evaluation and selection of candidate high-level waste forms

    SciTech Connect

    Bernadzikowski, T. A.; Allender, J. S.; Butler, J. L.; Gordon, D. E.; Gould, Jr., T. H.; Stone, J. A.

    1982-03-01

    Seven candidate waste forms being developed under the direction of the Department of Energy's National High-Level Waste (HLW) Technology Program, were evaluated as potential media for the immobilization and geologic disposal of high-level nuclear wastes. The evaluation combined preliminary waste form evaluations conducted at DOE defense waste-sites and independent laboratories, peer review assessments, a product performance evaluation, and a processability analysis. Based on the combined results of these four inputs, two of the seven forms, borosilicate glass and a titanate based ceramic, SYNROC, were selected as the reference and alternative forms for continued development and evaluation in the National HLW Program. Both the glass and ceramic forms are viable candidates for use at each of the DOE defense waste-sites; they are also potential candidates for immobilization of commercial reprocessing wastes. This report describes the waste form screening process, and discusses each of the four major inputs considered in the selection of the two forms.

  10. Management of data quality of high level waste characterization

    SciTech Connect

    Winters, W.I., Westinghouse Hanford

    1996-06-12

    Over the past 10 years, the Hanford Site has been transitioning from nuclear materials production to Site cleanup operations. High-level waste characterization at the Hanford Site provides data to support present waste processing operations, tank safety programs, and future waste disposal programs. Quality elements in the high-level waste characterization program will be presented by following a sample through the data quality objective, sampling, laboratory analysis and data review process. Transition from production to cleanup has resulted in changes in quality systems and program; the changes, as well as other issues in these quality programs, will be described. Laboratory assessment through quality control and performance evaluation programs will be described, and data assessments in the laboratory and final reporting in the tank characterization reports will be discussed.