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Sample records for chimeras expressing nef

  1. Astrocytic expression of HIV-1 Nef impairs spatial and recognition memory.

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    Chompre, Gladys; Cruz, Emmanuel; Maldonado, Lucianette; Rivera-Amill, Vanessa; Porter, James T; Noel, Richard J

    2013-01-01

    Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. A Highly Conserved Residue in HIV-1 Nef Alpha Helix 2 Modulates Protein Expression.

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    Johnson, Aaron L; Dirk, Brennan S; Coutu, Mathieu; Haeryfar, S M Mansour; Arts, Eric J; Finzi, Andrés; Dikeakos, Jimmy D

    2016-01-01

    Extensive genetic diversity is a defining characteristic of human immunodeficiency virus type 1 (HIV-1) and poses a significant barrier to the development of an effective vaccine. To better understand the impact of this genetic diversity on the HIV-1 pathogenic factor Nef, we compiled a panel of reference strains from the NIH Los Alamos HIV Database. Initial sequence analysis identified point mutations at Nef residues 13, 84, and 92 in subtype C reference strain C.BR92025 from Brazil. Functional analysis revealed impaired major histocompatibility complex class I and CD4 downregulation of strain C.BR92025 Nef, which corresponded to decreased protein expression. Metabolic labeling demonstrated that strain C.BR92025 Nef has a greater rate of protein turnover than subtype B reference strain B.JRFL that, on the basis of mutational analysis, is related to Nef residue A84. An alanine-to-valine substitution at position 84, located in alpha helix 2 of Nef, was sufficient to alter the rate of turnover of an otherwise highly expressed Nef protein. In conclusion, these findings highlight HIV-1 Nef residue A84 as a major determinant of protein expression that may offer an additional avenue to disrupt or mediate the effects of this key HIV-1 pathogenic factor. IMPORTANCE The HIV-1 Nef protein has been established as a key pathogenic determinant of HIV/AIDS, but there is little knowledge of how the extensive genetic diversity of HIV-1 affects Nef function. Upon compiling a set of subtype-specific reference strains, we identified a subtype C reference strain, C.BR92025, that contained natural polymorphisms at otherwise highly conserved residues 13, 84, and 92. Interestingly, strain C.BR92025 Nef displayed impaired Nef function and had decreased protein expression. We have demonstrated that strain C.BR92025 Nef has a higher rate of protein turnover than highly expressed Nef proteins and that this higher rate of protein turnover is due to an alanine-to-valine substitution at Nef

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

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    Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex; Klaver, Bep; Siteur-van Rijnstra, Esther; Frankin, Esmay; Berkhout, Ben; Das, Atze T., E-mail: a.t.das@amc.uva.nl

    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. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.

  4. Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

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    Baschieri Selene

    2007-02-01

    Full Text Available Abstract Background Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa and a truncated form (p25, 25 kDa. Here we report the expression and purification of HIV Nef from transgenic tobacco. Results We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. Conclusion We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue.

  5. Biology of HIV-Nef in vivo

    NARCIS (Netherlands)

    Koenen, P.G.F.

    2007-01-01

    HIV-Nef is established as an important factor for pathogenicity during HIV infection. The exact role and underlying mechanisms however are still unclear. Several studies have shown that Nef expression in T cells results in increased T cell activation. This Nef-mediated T cell activation has been

  6. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

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    Bianco Linda

    2009-11-01

    Full Text Available Abstract Background In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein. In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. Results The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19 gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. Conclusion We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor

  7. High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus.

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    Lombardi, Raffaele; Circelli, Patrizia; Villani, Maria Elena; Buriani, Giampaolo; Nardi, Luca; Coppola, Valentina; Bianco, Linda; Benvenuto, Eugenio; Donini, Marcello; Marusic, Carla

    2009-11-20

    In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue. We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with

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

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    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.

  9. Nef protein of human immunodeficiency virus type 1: evidence against its role as a transcriptional inhibitor.

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    Hammes, S. R.; Dixon, E P; Malim, M H; Cullen, B R; Greene, W C

    1989-01-01

    The type 1 human immunodeficiency virus (HIV-1) encodes a 27-kDa protein termed Nef (negative factor). Nef has been reported to down-regulate viral gene transcription directed by the HIV-1 long terminal repeat. To assess the possible role of Nef in the initiation or maintenance of viral latency, we prepared two different nef expression vectors (pNEF from the HXB-3 proviral clone; pNEF-2/3 from HXB-2 and HXB-3) and a control vector containing a frameshift mutation in the HXB-3 nef coding seque...

  10. HIV-1 Nef is released in extracellular vesicles derived from astrocytes: evidence for Nef-mediated neurotoxicity

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    Sami Saribas, A; Cicalese, Stephanie; Ahooyi, Taha Mohseni; Khalili, Kamel; Amini, Shohreh; Sariyer, Ilker Kudret

    2017-01-01

    Human immunodeficiency virus-associated neurological disorders (HANDs) affect the majority of AIDS patients and are a significant problem among HIV-1-infected individuals who live longer because of combined anti-retroviral therapies. HIV-1 utilizes a number of viral proteins and subsequent cytokine inductions to unleash its toxicity on neurons. Among HIV-1 viral proteins, Nef is a small protein expressed abundantly in astrocytes of HIV-1-infected brains and has been suggested to have a role in the pathogenesis of HAND. In order to explore its effect in the central nervous system, HIV-1 Nef was expressed in primary human fetal astrocytes (PHFAs) using an adenovirus. Our results revealed that HIV-1 Nef is released in extracellular vesicles (EVs) derived from PHFA cells expressing the protein. Interestingly, HIV-1 Nef release in EVs was enriched significantly when the cells were treated with autophagy activators perifosine, tomaxifen, MG-132, and autophagy inhibitors LY294002 and wortmannin suggesting a novel role of autophagy signaling in HIV-1 Nef release from astrocytes. Next, Nef-carrying EVs were purified from astrocyte cultures and neurotoxic effects on neurons were analyzed. We observed that HIV-1 Nef-containing EVs were readily taken up by neurons as demonstrated by immunocytochemistry and immunoblotting. Furthermore, treatment of neurons with Nef-carrying EVs induced oxidative stress as evidenced by a decrease in glutathione levels. To further investigate its neurotoxic effects, we expressed HIV-1 Nef in primary neurons by adenoviral transduction. Intracellular expression of HIV-1 Nef caused axonal and neurite degeneration of neurons. Furthermore, expression of HIV-1 Nef decreased the levels of phospho-tau while enhancing total tau in primary neurons. In addition, treatment of primary neurons with Nef-carrying EVs suppressed functional neuronal action potential assessed by multielectrode array studies. Collectively, these data suggested that HIV-1 Nef can be

  11. Nef dimension of minimal models

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    Ambro, Florin

    2003-01-01

    We reduce the Abundance Conjecture in dimension 4 to the following numerical statement: if the canonical divisor K is nef and has maximal nef dimension, then K is big. From this point of view, we ``classify'' in dimension 2 nef divisors which have maximal nef dimension, but which are not big.

  12. Replication of different clones of human immunodeficiency virus type 1 in primary fetal human astrocytes: enhancement of viral gene expression by Nef.

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    Bencheikh, M; Bentsman, G; Sarkissian, N; Canki, M; Volsky, D J

    1999-04-01

    Dementia is a common complication of AIDS which is associated with human immunodeficiency virus type 1 (HIV-1) infection of brain macrophages and microglia. Recent studies have shown that astrocytes are also infected in the brain but HIV-1 replication in these cells is restricted. To determine virus specificity of this restriction we tested the expression of 15 HIV-1 molecular clones in primary human fetal astrocytes by infection and DNA transfection. Infection with cell-free viruses was poorly productive and revealed no clone-specific differences. In contrast, transfected cells produced transiently high levels of HIV-1 p24 core antigen, up to 50 nanograms per ml culture supernatant, and nanogram levels of p24 were detected 3-4 weeks after transfection of some viral clones. The average peak expression of HIV-1 in astrocytes varied as a function of viral clone used by a factor of 15 but the differences and the subsequent virus spread did not correlate with the tropism of the viral clones to T cells or macrophages. Functional vif, vpu, and vpr genes were dispensable for virus replication from transfected DNA, but intact nef provided a detectable enhancement of early viral gene expression and promoted maintenance of HIV-1 infection. We conclude that primary astrocytes present no fundamental barriers to moderate expression of different strains of HIV-1 and that the presence of functional Nef is advantageous to virus infection in these cells.

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

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    Marcela Sabino Cunha

    2016-07-01

    Full Text Available 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.

  14. Attenuation of multiple Nef functions in HIV-1 elite controllers

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    Mwimanzi Philip

    2013-01-01

    Full Text Available Abstract Background Impaired HIV-1 Gag, Pol, and Env function has been described in elite controllers (EC who spontaneously suppress plasma viremia to Results In general, EC Nef clones were functional; however, all five activities were significantly lower in EC compared to CP. Nef clones from HLA-B*57-expressing EC exhibited poorer CD4 down-regulation function compared to those from non-B*57 EC, and the number of EC-specific B*57-associated Nef polymorphisms correlated inversely with 4 of 5 Nef functions in these individuals. Conclusion Results indicate that decreased HIV-1 Nef function, due in part to host immune selection pressures, may be a hallmark of the EC phenotype.

  15. Heterologous expression of NaV1.9 chimeras in various cell systems.

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    Goral, R Oliver; Leipold, Enrico; Nematian-Ardestani, Ehsan; Heinemann, Stefan H

    2015-12-01

    SCN11A encodes the voltage-gated sodium channel NaV1.9, which deviates most strongly from the other eight NaV channels expressed in mammals. It is characterized by resistance to the prototypic NaV channel blocker tetrodotoxin and exhibits slow activation and inactivation gating. Its expression in dorsal root ganglia neurons suggests a role in motor or pain signaling functions as also recently demonstrated by the occurrence of various mutations in human SCN11A leading to altered pain sensation syndromes. The systematic investigation of human NaV1.9, however, is severely hampered because of very poor heterologous expression in host cells. Using patch-clamp and two-electrode voltage-clamp methods, we show that this limitation is caused by the C-terminal structure of NaV1.9. A chimera of NaV1.9 harboring the C terminus of NaV1.4 yields functional expression not only in neuronal cells but also in non-excitable cells, such as HEK 293T or Xenopus oocytes. The major functional difference of the chimeric channel with respect to NaV1.9 is an accelerated activation and inactivation. Since the entire transmembrane domain is preserved, it is suited for studying pharmacological properties of the channel and the functional impact of disease-causing mutations. Moreover, we demonstrate how mutation S360Y makes NaV1.9 channels sensitive to tetrodotoxin and saxitoxin and that the unusual slow open-state inactivation of NaV1.9 is also mediated by the IFM (isoleucine-phenylalanine-methionine) inactivation motif located in the linker connecting domains III and IV.

  16. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

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    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  17. HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

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    Pene-Dumitrescu Teodora

    2012-03-01

    Full Text Available Abstract Background Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency. Conclusions These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.

  18. Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

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    Marusic, Carla; Vitale, Allessandro; Pedrazzini, Emanuela; Donini, Marcello; Frigerio, Lorenzo; Bock, Ralph; Dix, Philip J.; McCabe, Matthew S.; Bellucci, Michele; Benvenuto, Eugenio

    2009-01-01

    The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10?years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to ...

  19. Immunogenicity of seven new recombinant yellow fever viruses 17D expressing fragments of SIVmac239 Gag, Nef, and Vif in Indian rhesus macaques.

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    Mauricio A Martins

    Full Text Available An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV. Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (rYF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIVmac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5 vectors resulted in robust expansion of SIV-specific CD8(+ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4(+ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D - a clinically relevant vaccine vector - can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D

  20. Proteomic analysis of HIV-1 Nef cellular binding partners reveals a role for exocyst complex proteins in mediating enhancement of intercellular nanotube formation

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    Mukerji Joya

    2012-06-01

    Full Text Available Abstract Background HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunneling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. Results To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3. We report that wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6, an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Conclusions Exocyst complex proteins are likely a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Linkages revealed between Nef and the exocyst complex suggest a new paradigm of

  1. Species-specific activity of SIV Nef and HIV-1 Vpu in overcoming restriction by tetherin/BST2.

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    Bin Jia

    2009-05-01

    Full Text Available Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host-cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIV(smm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIV(mac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIV(smm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts.

  2. HIV-1 Nef targets MHC-I and CD4 for degradation via a final common beta-COP-dependent pathway in T cells.

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    Malinda R Schaefer

    2008-08-01

    Full Text Available To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4 and molecules capable of presenting HIV antigens to the immune system (MHC-I. To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7(+ vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, beta-COP. Moreover, we demonstrate that Nef contains two separable beta-COP binding sites. One site, an arginine (RXR motif in the N-terminal alpha helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.

  3. HIV-1 Nef targets MHC-I and CD4 for degradation via a final common beta-COP-dependent pathway in T cells.

    Science.gov (United States)

    Schaefer, Malinda R; Wonderlich, Elizabeth R; Roeth, Jeremiah F; Leonard, Jolie A; Collins, Kathleen L

    2008-08-22

    To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7(+) vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, beta-COP. Moreover, we demonstrate that Nef contains two separable beta-COP binding sites. One site, an arginine (RXR) motif in the N-terminal alpha helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.

  4. Nef-M1, a peptide antagonist of CXCR4, inhibits tumor angiogenesis and epithelial-to-mesenchymal transition in colon and breast cancers

    Science.gov (United States)

    Katkoori, Venkat R.; Basson, Marc D.; Bond, Vincent C.; Manne, Upender; Bumpers, Harvey L.

    2015-01-01

    The Nef-M1 peptide competes effectively with the natural ligand of CXC chemokine receptor 4 (CXCR4), stromal cell-derived factor 1-alpha, to induce apoptosis and inhibit growth in colon cancer (CRC) and breast cancer (BC). Its role in tumor angiogenesis, and epithelial-to-mesenchymal transition (EMT) regulation, key steps involved in tumor growth and metastasis, are unknown. We evaluated the angioinhibitory effect of Nef-M1 peptide and examined its role in the inhibition of EMT in these cancers. Colon (HT29) and breast (MDA-MB231) cancer cells expressing CXCR4 were studied in vitro and in xenograft tumors propagated in severe combined immunodeficient mice. The mice were treated intraperitoneally with Nef-M1 or scrambled amino acid sequence of Nef-M1 (sNef-M1) peptide, a negative control, starting at the time of tumor implantation. Sections from tumors were evaluated for tumor angiogenesis, as measured by microvessel density (MVD) based on immunostaining of endothelial markers. In vitro tumor angiogenesis was assessed by treating human umbilical vein endothelial cells with conditioned media from the tumor cell lines. A BC cell line (MDA-MB 468) which does not express CXCR4 was used to study the actions of Nef-M1 peptide. Western blot and immunofluorescence analyses assessed the effect of Nef-M1 on tumor angiogenesis and EMT in both tumors and cancer cells. Metastatic lesions of CRC and BC expressed more CXCR4 than primary lesions. It was also found that tumors from mice treated with sNef-M1 had well established vascularity, while Nef-M1 treated tumors had very poor vascularization. Indeed, the mean MVD was lower in tumors from Nef-M1 treated mice than in sNef-M1 treated tumors. Nef-M1 treated tumor has poor morphology and loss of endothelial integrity. Although conditioned medium from CRC or BC cells supported HUVEC tube formation, the conditioned medium from Nef-M1 treated CRC or BC cells did not support tube formation. Western blot analyses revealed that Nef-M1

  5. Nef-M1, a CXCR4 Peptide Antagonist, Enhances Apoptosis and Inhibits Primary Tumor Growth and Metastasis in Breast Cancer.

    Science.gov (United States)

    Bumpers, Harvey; Huang, Ming-Bo; Katkoori, Venkat; Manne, Upender; Bond, Vincent

    2013-06-01

    Results from studies with animal models suggest that, in many cancers, CXCR4 is an important therapeutic target and that CXCR4 antagonists may be promising treatments for primary cancers and for metastases. The Nef protein effectively competes with CXCR4's natural ligand, SDF-1α, and induces apoptosis. As described in this report, the Nef-M1 peptide (Nef protein amino acids 50 - 60) inhibits primary tumor growth and metastasis of breast cancer (BC). Four BC cell lines (MDA-MB-231, MDA-MB-468, MCF 7, and DU4475) and primary human mammary epithelium (HME) cells were evaluated for their response to the Nef protein and to the Nef-M1 peptide. The presence of CXCR4 receptors in these cells was determined by RT-PCR, Western blot (WB), and immunohistochemical analyses. The apoptotic effect of Nef-M1 was assessed by terminal transferase dUTP nick-end labeling (TUNEL). WBs was used to assess caspase 3 activation. BC xenografts grown in SCID mice were evaluated for the presence of CXCR4 and for their metastatic potential. CXCR4 was presented in MDA-MB-231, MCF 7, and DU 4475 BC cells but not in MDA-MB-468 BC or HME cells. Cells expressing CXCR4 and treated with Nef-M1 peptide or the Nef protein had higher rates of apoptosis than untreated cells. Caspase-3 activation increased in MDA-MB 231 cells treated with the Nef protein, the Nef 41 - 60 peptide, or Nef-M1. Nef-M1, administered to mice starting at the time of xenograft implantation, inhibited growth of primary tumors and metastatic spread. Untreated mice developed diffuse intraperitoneal metastases. We conclude that, in BCs, Nef-M1, through interaction with CXCR4, inhibits primary tumor growth and metastasis by causing apoptosis.

  6. Ability of HIV-1 Nef to downregulate CD4 and HLA class I differs among viral subtypes.

    Science.gov (United States)

    Mann, Jaclyn K; Byakwaga, Helen; Kuang, Xiaomei T; Le, Anh Q; Brumme, Chanson J; Mwimanzi, Philip; Omarjee, Saleha; Martin, Eric; Lee, Guinevere Q; Baraki, Bemuluyigza; Danroth, Ryan; McCloskey, Rosemary; Muzoora, Conrad; Bangsberg, David R; Hunt, Peter W; Goulder, Philip J R; Walker, Bruce D; Harrigan, P Richard; Martin, Jeff N; Ndung'u, Thumbi; Brockman, Mark A; Brumme, Zabrina L

    2013-09-16

    The highly genetically diverse HIV-1 group M subtypes may differ in their biological properties. Nef is an important mediator of viral pathogenicity; however, to date, a comprehensive inter-subtype comparison of Nef in vitro function has not been undertaken. Here, we investigate two of Nef's most well-characterized activities, CD4 and HLA class I downregulation, for clones obtained from 360 chronic patients infected with HIV-1 subtypes A, B, C or D. Single HIV-1 plasma RNA Nef clones were obtained from N=360 antiretroviral-naïve, chronically infected patients from Africa and North America: 96 (subtype A), 93 (B), 85 (C), and 86 (D). Nef clones were expressed by transfection in an immortalized CD4+ T-cell line. CD4 and HLA class I surface levels were assessed by flow cytometry. Nef expression was verified by Western blot. Subset analyses and multivariable linear regression were used to adjust for differences in age, sex and clinical parameters between cohorts. Consensus HIV-1 subtype B and C Nef sequences were synthesized and functionally assessed. Exploratory sequence analyses were performed to identify potential genotypic correlates of Nef function. Subtype B Nef clones displayed marginally greater CD4 downregulation activity (p = 0.03) and markedly greater HLA class I downregulation activity (p class I downregulation remained statistically significant after controlling for differences in age, sex, and clinical parameters (p A/D > C for Nef-mediated CD4 and HLA class I downregulation. The mechanisms underlying these differences and their relevance to HIV-1 pathogenicity merit further investigation.

  7. DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli.

    Science.gov (United States)

    Liu, Yamei; Stepanov, Victor G; Strych, Ulrich; Willson, Richard C; Jackson, George W; Fox, George E

    2010-12-06

    Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA) was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use in clinical and biomedical research.

  8. The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4+ T cell apoptosis.

    Science.gov (United States)

    Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Dirk, Brennan S; Van Nynatten, Logan R; Haeryfar, S M Mansour; Dikeakos, Jimmy D

    2017-09-01

    Acquired Immune Deficiency Syndrome is characterized by a decline in CD4+ T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4+ T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (NefM20A or NefEEEE62-65AAAA) or Nef:AP-2 (NefLL164/165AA), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (NefH89A or NefF191A) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Gargoyles, Grotesques, & Chimeras

    Science.gov (United States)

    Wilbert, Nancy Corrigan

    2010-01-01

    Gargoyles, grotesques, and chimeras are scary, mythical (and sometimes humorous) creatures that have functional, decorative, and spiritual significance in medieval architecture. In this article, the author describes how her ceramics students created contemporary versions of gargoyles, chimeras, and grotesque faces. (Contains 1 online resource.)

  10. Exogenous HIV-1 Nef Upsets the IFN-γ-Induced Impairment of Human Intestinal Epithelial Integrity

    Science.gov (United States)

    Quaranta, Maria Giovanna; Vincentini, Olimpia; Felli, Cristina; Spadaro, Francesca; Silano, Marco; Moricoli, Diego; Giordani, Luciana; Viora, Marina

    2011-01-01

    Background The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line. Methodology/Principal Findings We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepitelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade. Conclusion/Significance Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions. PMID:21858117

  11. Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity.

    Directory of Open Access Journals (Sweden)

    Maria Giovanna Quaranta

    Full Text Available The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.

  12. Self-association of the Lentivirus protein, Nef

    Directory of Open Access Journals (Sweden)

    Garcia J Victor

    2010-09-01

    Full Text Available Abstract Background The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization. Results By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123. Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIVMAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface. Conclusions We have

  13. Macropinocytosis and TAK1 mediate anti-inflammatory to pro-inflammatory macrophage differentiation by HIV-1 Nef.

    Science.gov (United States)

    Hashimoto, M; Nasser, H; Chihara, T; Suzu, S

    2014-05-29

    Macrophages (MΦ) are functionally classified into two types, anti-inflammatory M2 and pro-inflammatory M1. Importantly, we recently revealed that soluble HIV-1 proteins, particularly the pathogenetic protein Nef, preferentially activate M2-MΦ and drive them towards an M1-like MΦ, which might explain the sustained immune activation seen in HIV-1-infected patients. Here, we show that the preferential effect of Nef on M2-MΦ is mediated by TAK1 (TGF-β-activated kinase 1) and macropinocytosis. As with MAP kinases and NF-κB pathway, Nef markedly activated TAK1 in M-CSF-derived M2-MΦ but not in GM-CSF-derived M1-MΦ. Two Nef mutants, which were unable to activate MAP kinases and NF-κB pathway, failed to activate TAK1. Indeed, the TAK1 inhibitor 5Z-7-oxozeaenol as well as the ectopic expression of a dominant-negative mutant of TAK1 or TRAF2, an upstream molecule of TAK1, inhibited Nef-induced signaling activation and M1-like phenotypic differentiation of M2-MΦ. Meanwhile, the preferential effect of Nef on M2-MΦ correlated with the fact the Nef entered M2-MΦ more efficiently than M1-MΦ. Importantly, the macropinosome formation inhibitor EIPA completely blocked the internalization of Nef into M2-MΦ. Because the macropinocytosis activity of M2-MΦ was higher than that of M1-MΦ, our findings indicate that Nef enters M2-MΦ efficiently by exploiting their higher macropinocytosis activity and drives them towards M1-like MΦ by activating TAK1.

  14. DNAzyme-mediated recovery of small recombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Willson Richard C

    2010-12-01

    Full Text Available Abstract Background Manufacturing large quantities of recombinant RNAs by overexpression in a bacterial host is hampered by their instability in intracellular environment. To overcome this problem, an RNA of interest can be fused into a stable bacterial RNA for the resulting chimeric construct to accumulate in the cytoplasm to a sufficiently high level. Being supplemented with cost-effective procedures for isolation of the chimera from cells and recovery of the recombinant RNA from stabilizing scaffold, this strategy might become a viable alternative to the existing methods of chemical or enzymatic RNA synthesis. Results Sequence encoding a 71-nucleotide recombinant RNA was inserted into a plasmid-borne deletion mutant of the Vibrio proteolyticus 5S rRNA gene in place of helix III - loop C segment of the original 5S rRNA. After transformation into Escherichia coli, the chimeric RNA (3×pen aRNA was expressed constitutively from E. coli rrnB P1 and P2 promoters. The RNA chimera accumulated to levels that exceeded those of the host's 5S rRNA. A novel method relying on liquid-solid partitioning of cellular constituents was developed for isolation of total RNA from bacterial cells. This protocol avoids toxic chemicals, and is therefore more suitable for large scale RNA purification than traditional methods. A pair of biotinylated 8-17 DNAzymes was used to bring about the quantitative excision of the 71-nt recombinant RNA from the chimera. The recombinant RNA was isolated by sequence-specific capture on beads with immobilized complementary deoxyoligonucleotide, while DNAzymes were recovered by biotin affinity chromatography for reuse. Conclusions The feasibility of a fermentation-based approach for manufacturing large quantities of small RNAs in vivo using a "5S rRNA scaffold" strategy is demonstrated. The approach provides a route towards an economical method for the large-scale production of small RNAs including shRNAs, siRNAs and aptamers for use

  15. A Conserved GPG-Motif in the HIV-1 Nef Core Is Required for Principal Nef-Activities.

    Directory of Open Access Journals (Sweden)

    Marta Martínez-Bonet

    Full Text Available To find out new determinants required for Nef activity we performed a functional alanine scanning analysis along a discrete but highly conserved region at the core of HIV-1 Nef. We identified the GPG-motif, located at the 121-137 region of HIV-1 NL4.3 Nef, as a novel protein signature strictly required for the p56Lck dependent Nef-induced CD4-downregulation in T-cells. Since the Nef-GPG motif was dispensable for CD4-downregulation in HeLa-CD4 cells, Nef/AP-1 interaction and Nef-dependent effects on Tf-R trafficking, the observed effects on CD4 downregulation cannot be attributed to structure constraints or to alterations on general protein trafficking. Besides, we found that the GPG-motif was also required for Nef-dependent inhibition of ring actin re-organization upon TCR triggering and MHCI downregulation, suggesting that the GPG-motif could actively cooperate with the Nef PxxP motif for these HIV-1 Nef-related effects. Finally, we observed that the Nef-GPG motif was required for optimal infectivity of those viruses produced in T-cells. According to these findings, we propose the conserved GPG-motif in HIV-1 Nef as functional region required for HIV-1 infectivity and therefore with a potential interest for the interference of Nef activity during HIV-1 infection.

  16. Characterization of the mammalian RNA exonuclease 5/NEF-sp as a testis-specific nuclear 3' → 5' exoribonuclease.

    Science.gov (United States)

    Silva, Sara; Homolka, David; Pillai, Ramesh S

    2017-09-01

    Ribonucleases catalyze maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp (RNA exonuclease 5 [REXO5] or LOC81691) as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3' → 5' exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length. The presence of a 2'-O-methyl modification at the 3' end of the RNA substrate is inhibitory. Ectopically expressed NEF-sp localizes to the nucleolar/nuclear compartment in mammalian cell cultures and this is dependent on an amino-terminal nuclear localization signal. Finally, mice lacking NEF-sp are viable and display normal fertility, likely indicating overlapping functions with other nucleases. Taken together, our study provides the first biochemical and genetic exploration of the role of the NEF-sp exoribonuclease in the mammalian genome. © 2017 Silva et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. A Discontinuous Galerkin Chimera Overset Solver

    Science.gov (United States)

    Galbraith, Marshall Christopher

    This work summarizes the development of an accurate, efficient, and flexible Computational Fluid Dynamics computer code that is an improvement relative to the state of the art. The improved accuracy and efficiency is obtained by using a high-order discontinuous Galerkin (DG) discretization scheme. In order to maximize the computational efficiency, quadrature-free integration and numerical integration optimized as matrix-vector multiplications is employed and implemented through a pre-processor (PyDG). Using the PyDG pre-processor, a C++ polynomial library has been developed that uses overloaded operators to design an efficient Domain Specific Language (DSL) that allows expressions involving polynomials to be written as if they are scalars. The DSL, which makes the syntax of computer code legible and intuitive, promotes maintainability of the software and simplifies the development of additional capabilities. The flexibility of the code is achieved by combining the DG scheme with the Chimera overset method. The Chimera overset method produces solutions on a set of overlapping grids that communicate through an exchange of data on grid boundaries (known as artificial boundaries). Finite volume and finite difference discretizations use fringe points, which are layers of points on the artificial boundaries, to maintain the interior stencil on artificial boundaries. The fringe points receive solution values interpolated from overset grids. Proper interpolation requires fringe points to be contained in overset grids. Insufficient overlap must be corrected by modifying the grid system. The Chimera scheme can also exclude regions of grids that lie outside the computational domain; a process commonly known as hole cutting. The Chimera overset method has traditionally enabled the use of high-order finite difference and finite volume approaches such as WENO and compact differencing schemes, which require structured meshes, for modeling fluid flow associated with complex

  18. PATENTABILITY OF HUMAN-ANIMAL CHIMERAS

    National Research Council Canada - National Science Library

    Ryan Hagglund

    2008-01-01

    .... The advent of human-animal chimera technology naturally raises the issue of whether development of human-animal chimeras should be encouraged by the issuance of patents to inventors of human-animal chimeras...

  19. Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: Study of immunological memory and long-term toxicology

    Directory of Open Access Journals (Sweden)

    Engström Gunnel

    2007-07-01

    Full Text Available Abstract Background The human immunodeficiency virus type 1 (HIV-1 regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types in vitro. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed. Results The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs. Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8+ T cell memory responses, whereas MVA-encoded nef induced CD4+ T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate

  20. HIV-1 Nef Signaling in Intestinal Mucosa Epithelium Suggests the Existence of an Active Inter-kingdom Crosstalk Mediated by Exosomes

    Directory of Open Access Journals (Sweden)

    Cristina Felli

    2017-06-01

    Full Text Available The human intestinal mucosal surface represents the first defense against pathogens and regulates the immune response through the combination of epithelial cell (EC functions and immunological factors. ECs act as sensors of luminal stimuli and interact with the immune cells through signal–transduction pathways, thus representing the first barrier that HIV-1 virus encounters during infection. In particular, the HIV-1 Nef protein plays a crucial role in viral invasion and replication. Nef is expressed early during viral infection and interacts with numerous cellular proteins as a scaffold/adaptor. Nef is localized primarily to cellular membranes and affects several signaling cascades in infected cells modulating the expression of cell surface receptors critical for HIV-1 infection and transmission, also accompanied by the production of specific cytokines and progressive depletion of CD4+ T cells. At the intestinal level, Nef contributes to affect the mucosal barrier by increasing epithelial permeability, that results in the translocation of microbial antigens and consequently in immune system activation. However, the pathological role of Nef in mucosal dysfunction has not been fully elucidated. Interestingly, Nef is secreted also within exosomes and contributes to regulate the intercellular communication exploiting the vesicular trafficking machinery of the host. This can be considered as a potential inter-kingdom communication pathway between virus and humans, where viral Nef contributes to modulate and post-transcriptionally regulate the host gene expression and immune response. In this mini-review we discuss the effects of HIV-1 Nef protein on intestinal epithelium and propose the existence of an inter-kingdom communication process mediated by exosomes.

  1. The HIV-1 Nef protein and phagocyte NADPH oxidase activation

    DEFF Research Database (Denmark)

    Vilhardt, Frederik; Plastre, Olivier; Sawada, Makoto

    2002-01-01

    Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia...

  2. Complex macromolecular chimeras.

    Science.gov (United States)

    Karatzas, Anastasis; Iatrou, Hermis; Hadjichristidis, Nikos; Inoue, Kyouichi; Sugiyama, Kenji; Hirao, Akira

    2008-07-01

    By combining two living polymerizations, anionic and ring opening (ROP), the following novel multiblock multicomponent linear and miktoarm star (micro-star) polymer/polypeptide hybrids (macromolecular chimeras) were synthesized: Linear, PBLL-b-PBLG-b-PS-b-PBLG-b-PBLL; 3micro-stars, (PS)2(PBLG or PBLL), (PS)(PI)(PBLG or PBLL); 4micro-stars, (PS)2[P(alpha-MeS)](PBLG or PBLL), (PS)2(PBLG or PBLL)2 [PS, polystyrene; PI, polyisoprene; P(alpha-MeS), poly(alpha-methylstyrene); PBLG, poly(gamma-benzyl-L-glutamate); and PBLL, poly(-tert-butyloxycarbonyl-L-lysine)]. The procedure involves (a) the synthesis of end- or in-chain amino-functionalized polymers, by anionic polymerization high vacuum techniques and appropriate linking chemistry and (b) the use of the amino groups for the ROP of alpha-amino acid carboxyanhydrides (NCAs). Molecular characterization revealed the high molecular weight and compositional homogeneity of the macromolecular chimeras prepared. The success of the synthesis was based mainly on the high vacuum techniques used for the ROP of NCAs, ensuring the avoidance of unwanted polymerization mechanisms and termination reactions.

  3. Recurrence quantification analysis of chimera states

    Energy Technology Data Exchange (ETDEWEB)

    Santos, M.S. [Pós-Graduação em Ciências/Física, Universidade Estadual de Ponta Grossa, 84030-900, Ponta Grossa, PR (Brazil); Szezech, J.D., E-mail: jdanilo@gmail.com [Departamento de Matemática e Estatística, Universidade Estadual de Ponta Grossa, 84030-900, Ponta Grossa, PR (Brazil); Batista, A.M., E-mail: antoniomarcosbatista@gmail.com [Departamento de Matemática e Estatística, Universidade Estadual de Ponta Grossa, 84030-900, Ponta Grossa, PR (Brazil); Caldas, I.L. [Instituto de Física, Universidade de São Paulo, 05315-970, São Paulo, SP (Brazil); Viana, R.L.; Lopes, S.R. [Departamento de Física, Universidade Federal do Paraná, 81531-990, Curitiba, PR (Brazil)

    2015-10-02

    Chimera states, characterised by coexistence of coherence and incoherence in coupled dynamical systems, have been found in various physical systems, such as mechanical oscillator networks and Josephson-junction arrays. We used recurrence plots to provide graphical representations of recurrent patterns and identify chimera states. Moreover, we show that recurrence plots can be used as a diagnostic of chimera states and also to identify the chimera collapse. - Highlights: • Chimera states have been found in various physical systems. • Recurrence plots is a graphical method useful to locate recurring patterns. • We used recurrence plots to identify the chimera states. • We show also the recurrence plots can identify the chimera collapse.

  4. Enstore with Chimera namespace provider

    Energy Technology Data Exchange (ETDEWEB)

    Litvintsev, Dmitry [Fermilab; Moibenko, Alexander [Fermilab; Oleynik, Gene [Fermilab; Zalokar, Michael [Fermilab

    2012-01-01

    Enstore is a mass storage system developed by Fermilab that provides distributed access and management of data stored on tapes. It uses a namespace service, PNFS, developed by DESY to provide a filesystem-like view of the stored data. PNFS is a legacy product and is being replaced by a new implementation, called Chimera, which is also developed by DESY. Chimera offers multiple advantages over PNFS in terms of performance and functionality. The Enstore client component, encp, has been modified to work with Chimera, as well as with any other namespace provider. We performed high load end-to-end acceptance test of Enstore with the Chimera namespace. This paper describes the modifications to Enstore, the test procedure and the results of the acceptance testing.

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

    Science.gov (United States)

    Korber, Bette T [Los Alamos, NM; Perkins, Simon [Los Alamos, NM; Bhattacharya, Tanmoy [Los Alamos, NM; Fischer, William M [Los Alamos, NM; Theiler, James [Los Alamos, NM; Letvin, Norman [Boston, MA; Haynes, Barton F [Durham, NC; Hahn, Beatrice H [Birmingham, AL; Yusim, Karina [Los Alamos, NM; Kuiken, Carla [Los Alamos, NM

    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.

  6. HIV Nef-M1 Effects on Colorectal Cancer Growth in Tumor-induced Spleens and Hepatic Metastasis.

    Science.gov (United States)

    Harrington, Willie; Bond, Vincent; Huang, Ming Bo; Powell, Michael; Lillard, James; Manne, Upender; Bumpers, Harvey

    2009-08-05

    CXCR4 receptors have been implicated in tumorigenesis and proliferation, making it a potential target for colorectal cancer therapy. Expression of this chemokine receptor on cellular surfaces appears to promote metastasis by directly stimulating tumor cell migration and invasion. The receptor/ligand, CXCR4/SDF-1alpha, pair are critically important to angiogenesis and vascular remodeling which supports cancer proliferation. Our work has shown that a novel apoptotic peptide of HIV-1, Nef-M1, can act as a CXCR4 antagonist, inducing apoptosis in CXCR4 containing cells. Four colorectal tumor cell lines (HT-29, LS174t, SW480, WiDr), were evaluated for their response to Nef-M1 peptide via in vivo and in vitro. The presence of CXCR4 receptors on tumor cells was determined using immunohistochemical and RT-PCR analyses. Solid xenografts derived from tumor cell lines grown in SCID mice, were evaluated for the persistence of the receptor. Xenografts propagated in SCID mice from each of the four cell lines demonstrated high levels of receptor expression as well. The effects of Nef-M1 in vivo via splenic injected mice and subsequent hepatic metastasis also demonstrated dramatic reduction of primary tumor growth in the spleen and secondary invasion of the liver. We concluded that Nef-M1 peptide, through physical interaction(s) with CXCR4, drives apoptotic reduction in in vivo primary tumor growth and metastasis.

  7. T-cell responses targeting HIV Nef uniquely correlate with infected cell frequencies after long-term antiretroviral therapy.

    Directory of Open Access Journals (Sweden)

    Allison S Thomas

    2017-09-01

    Full Text Available HIV-specific CD8+ T-cell responses limit viral replication in untreated infection. After the initiation of antiretroviral therapy (ART, these responses decay and the infected cell population that remains is commonly considered to be invisible to T-cells. We hypothesized that HIV antigen recognition may persist in ART-treated individuals due to low-level or episodic protein expression. We posited that if persistent recognition were occurring it would be preferentially directed against the early HIV gene products Nef, Tat, and Rev as compared to late gene products, such as Gag, Pol, and Env, which have higher barriers to expression. Using a primary cell model of latency, we observed that a Nef-specific CD8+ T-cell clone exhibited low-level recognition of infected cells prior to reactivation and robust recognition shortly thereafter. A Gag-specific CD8+ T-cell clone failed to recognized infected cells under these conditions, corresponding with a lack of detectable Gag expression. We measured HIV-specific T-cell responses in 96 individuals who had been suppressed on ART for a median of 7 years, and observed a significant, direct correlation between cell-associated HIV DNA levels and magnitudes of IFN-γ-producing Nef/Tat/Rev-specific T-cell responses. This correlation was confirmed in an independent cohort (n = 18. Correlations were not detected between measures of HIV persistence and T-cell responses to other HIV antigens. The correlation with Nef/Tat/Rev-specific T-cells was attributable to Nef-specific responses, the breadth of which also correlated with HIV DNA levels. These results suggest that ongoing Nef expression in ART-treated individuals drives preferential maintenance and/or expansion of T-cells reactive to this protein, implying sensing of infected cells by the immune system. The direct correlation, however, suggests that recognition does not result in efficient elimination of infected cells. These results raise the possibility that

  8. Cloning, production, and functional expression of the bacteriocin sakacin A (SakA) and two SakA-derived chimeras in lactic acid bacteria (LAB) and the yeasts Pichia pastoris and Kluyveromyces lactis.

    Science.gov (United States)

    Jiménez, Juan J; Borrero, Juan; Diep, Dzung B; Gútiez, Loreto; Nes, Ingolf F; Herranz, Carmen; Cintas, Luis M; Hernández, Pablo E

    2013-09-01

    Mature sakacin A (SakA, encoded by sapA) and its cognate immunity protein (SakI, encoded by sapiA), and two SakA-derived chimeras mimicking the N-terminal end of mature enterocin P (EntP/SakA) and mature enterocin A (EntA/SakA) together with SakI, were fused to different signal peptides (SP) and cloned into the protein expression vectors pNZ8048 and pMG36c for evaluation of their production and functional expression by different lactic acid bacteria. The amount, antimicrobial activity, and specific antimicrobial activity of SakA and its chimeras produced by Lactococcus lactis subsp. cremoris NZ9000 depended on the SP and the expression vector. Only L. lactis NZ9000 (pNUPS), producing EntP/SakA, showed higher bacteriocin production and antimicrobial activity than the natural SakA-producer Lactobacillus sakei Lb706. The lower antimicrobial activity of the SakA-producer L. lactis NZ9000 (pNUS) and that of the EntA/SakA-producer L. lactis NZ9000 (pNUAS) could be ascribed to secretion of truncated bacteriocins. On the other hand, of the Lb. sakei Lb706 cultures transformed with the pMG36c-derived vectors only Lb. sakei Lb706 (pGUS) overproducing SakA showed a higher antimicrobial activity than Lb. sakei Lb706. Finally, cloning of SakA and EntP/SakA into pPICZαA and pKLAC2 permitted the production of SakA and EntP/SakA by recombinant Pichia pastoris X-33 and Kluyveromyces lactis GG799 derivatives although their antimicrobial activity was lower than expected from their production.

  9. Inefficient Nef-mediated downmodulation of CD3 and MHC-I correlates with loss of CD4+T cells in natural SIV infection.

    Directory of Open Access Journals (Sweden)

    Michael Schindler

    2008-07-01

    Full Text Available Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500 and 15 animals with low (<500 CD4+ T-cells/microl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL-2 and programmed death (PD-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.

  10. HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation

    Science.gov (United States)

    Muthumani, Karuppiah; Choo, Andrew Y.; Hwang, Daniel S.; Premkumar, Arumugam; Dayes, Nathanael S.; Harris, Crafford; Green, Douglas R.; Wadsworth, Scott A.; Siekierka, John J.; Weiner, David B.

    2005-01-01

    The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro. (Blood. 2005;106:2059-2068) PMID:15928037

  11. Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3.

    Science.gov (United States)

    Manrique, Santiago; Sauter, Daniel; Horenkamp, Florian A; Lülf, Sebastian; Yu, Hangxing; Hotter, Dominik; Anand, Kanchan; Kirchhoff, Frank; Geyer, Matthias

    2017-09-05

    Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.HIV and simian immunodeficiency virus (SIV) Nef proteins both stimulate the clathrin-mediated endocytosis of CD4 but differ in downmodulation of the immune receptor CD3. Here, the authors present the structure of SIV Nef bound to the ExxxLM motif of another Nef molecule, which allows them to propose a model how Nef recognizes these motifs in CD3 and CD4.

  12. Impaired Downregulation of NKG2D Ligands by Nef Proteins from Elite Controllers Sensitizes HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Alsahafi, Nirmin; Richard, Jonathan; Prévost, Jérémie; Coutu, Mathieu; Brassard, Nathalie; Parsons, Matthew S; Kaufmann, Daniel E; Brockman, Mark; Finzi, Andrés

    2017-08-15

    HIV-1 Nef clones isolated from a rare subset of HIV-1-infected elite controllers (EC), with the ability to suppress viral load to undetectable levels in the absence of antiretroviral therapy, are unable to fully downregulate CD4 from the plasma membrane of CD4(+) T cells. Residual CD4 left at the plasma membrane allows Env-CD4 interaction, which leads to increased exposure of Env CD4-induced epitopes and increases susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). ADCC is mediated largely by natural killer (NK) cells, which control their activation status through the cumulative signals received through activating and inhibitory receptors. Recently, the activating NKG2D receptor was demonstrated to positively influence ADCC responses. Since HIV-1 Nef has been reported to reduce the expression of NKG2D ligands, we evaluated the relative abilities of Nef from EC and progressors to downmodulate NKG2D ligands. Furthermore, we assessed the impact of EC and progressor Nef on the ADCC susceptibility of HIV-1-infected cells. We observed a significantly increased expression of NKG2D ligands on cells infected with viruses coding for Nef from EC. Importantly, NKG2D ligand expression levels correlated with enhanced susceptibility of HIV-1-infected cells to ADCC. The biological significance of this correlation was corroborated by the demonstration that antibody-mediated blockade of NKG2D significantly reduced ADCC of cells infected with viruses carrying Nef from EC. These results suggest the involvement of NKG2D-NKG2D ligand interactions in the enhanced susceptibility of EC HIV-1-infected cells to ADCC responses.IMPORTANCE Attenuated Nef functions have been reported in HIV-1 isolated from EC. The inability of elite controller Nef to fully remove CD4 from the surface of infected cells enhanced their susceptibility to elimination by ADCC. We now show that downregulation of NKG2D ligands by HIV-1 Nef from EC is inefficient and leaves infected cells

  13. Identification of a novel binding site between HIV type 1 Nef C-terminal flexible loop and AP2 required for Nef-mediated CD4 downregulation.

    Science.gov (United States)

    Jin, Yong-Jiu; Cai, Catherine Yi; Mezei, Mihaly; Ohlmeyer, Michael; Sanchez, Roberto; Burakoff, Steven J

    2013-04-01

    HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

  14. Serological detection of attenuated HIV-1 variants with nef gene deletions.

    Science.gov (United States)

    Greenway, A L; Mills, J; Rhodes, D; Deacon, N J; McPhee, D A

    1998-04-16

    To investigate whether members of a transfusion-linked cohort (the Sydney Bloodbank Cohort) infected with a nef-deleted strain of HIV-1 could be differentiated from individuals infected with wild-type strains of HIV-1 by characterizing the Nef antibody response of cohort members. Retrospective and prospective analysis of the nef gene sequence and the antibody response to Nef peptides in HIV-infected subjects. Plasma was obtained from all individuals of the Sydney cohort, and from a variety of HIV-1-infected and uninfected controls. Antibodies recognizing full-length recombinant HIV-1NL43 Nef protein and synthetic peptide analogues were assessed by enzyme-linked immunosorbent assay. All 34 individuals infected with wild-type HIV-1 had antibodies reacting with full-length Nef protein as well as with a series of synthetic peptides (6-23-mers) spanning most of the Nef protein of HIV-1NL43. Although the HIV-1 quasispecies infecting the Sydney cohort had a consensus deletion of the nef gene corresponding to amino-acids 165-206, HIV-1 strains from individual members of the cohort had additional deletions comprising up to 80% of the nef gene. Members of the cohort had antibodies to peptides homologous to all regions of the Nef protein tested, except for a single peptide (amino-acids 162-177) that lies within the consensus nef deletion for the cohort quasispecies. These data show that nef-deleted strains of HIV-1 can be detected serologically. In the Sydney cohort, detection of antibodies to all regions of Nef tested, except that corresponding to amino-acids 162-177, suggests that observed deletions outside this domain occurred after this virus had infected these subjects and stimulated an immune response. A Nef peptide serological assay may be useful for identifying further examples of individuals infected with nef-deleted, attenuated HIV-1 quasispecies and for assessing the evolution of those variants in vivo.

  15. 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.

    Science.gov (United States)

    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; Salemi, Marco

    2015-08-01

    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. The SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate

  16. Heterologous Src Homology 4 Domains Support Membrane Anchoring and Biological Activity of HIV-1 Nef*

    Science.gov (United States)

    Geist, Miriam M.; Pan, Xiaoyu; Bender, Silke; Bartenschlager, Ralf; Nickel, Walter; Fackler, Oliver T.

    2014-01-01

    The HIV-1 pathogenicity factor Nef enhances viral replication by modulation of multiple host cell transport and signaling pathways. Nef associates with membranes via an N-terminal Src homology 4 (SH4) domain, and membrane association is believed to be essential for its biological functions. At which subcellular site(s) Nef exerts its different functions and how kinetics of membrane interactions contribute to its biological activity are unknown. To address how specific characteristics of Nef membrane association affect its biological properties, the SH4 domain of Nef was replaced by heterologous membrane targeting domains. The use of a panel of heterologous SH4 domains resulted in chimeric Nef proteins with distinct steady state subcellular localization, membrane association efficiency, and anterograde transport routes. Irrespective of these modifications, cardinal Nef functions affecting host cell vesicular transport and actin dynamics were fully preserved. In contrast, stable targeting of Nef to the surface of mitochondria, peroxisomes, or the Golgi apparatus, and thus prevention of plasma membrane delivery, caused potent and broad loss of Nef activity. These results support the concept that Nef adopts its active conformation in the membrane-associated state but exclude that membrane-associated Nef simply acts by recruiting soluble factors independently of its local microenvironment. Rather than its steady state subcellular localization or membrane affinity, the ability to undergo dynamic anterograde and internalization cycles appear to determine Nef function. These results reveal that functional membrane interactions of Nef underlie critical spatiotemporal regulation and suggest that delivery to distinct subcellular sites via such transport cycles provides the basis for the multifunctionality of Nef. PMID:24706755

  17. Recent results of CHIMERA activity

    Directory of Open Access Journals (Sweden)

    Pagano A.

    2012-07-01

    Full Text Available The experimental activity of CHIMERA in recent years has been characterized by a steady progress in the detection technique and data analysis. Since 2008 the detector system benefits of new implementations: a new reaction chamber, a new charged particle identification in silicon detector made by an extended pulse shape method and an efficient system for the identification of exotic beams produced by projectile-like fragmentation (In-flight method. These implementations appear to be promising tools in view of further exclusive experiments in the field of isospin physics. The coupling of CHIMERA with other equipments (such as interferometers and highly segmented arrays, magnetic elements, neutron detectors, etc. is also envisaged in order to extend the studies of the reaction mechanism in heavy ion physics.

  18. HIV-1 Group P is unable to antagonize human tetherin by Vpu, Env or Nef

    Directory of Open Access Journals (Sweden)

    Sauter Daniel

    2011-12-01

    Full Text Available Abstract Background A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317 suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans. Results Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression. Conclusions Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.

  19. Effect of promoter-leader sequences on transient expression of reporter gene chimeras biolistically transferred into sugarbeet (Beta vulgaris) suspension cells.

    Science.gov (United States)

    Ingersoll, J C; Heutte, T M; Owens, L D

    1996-08-01

    Chimeric constructs consisting of the gus coding region fused downstream of promoterun-translated leader sequences from the tobacco osmotin and PR-S genes, the potato proteinase inhibitor 2 gene (pin2), and the cauliflower mosaic virus (CaMV) 35S promoter were biolistically transferred into sugarbeet suspension cells. Each construct was expressed in recipient cells at 6 h after bombardment with maximum levels observed between 12 and 48 h. Expression of the PR-S construct mimicked the time-course expression of the constitutively expressed 35S construct but reached levels almost 50% higher. The pin2-promoter construct was ultimately expressed at levels similar to that of PR-S. Expression of the osmotin promoter-leader construct was highest, reaching levels approximately 2.5-fold higher than those of the 35S construct.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

    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 EEEE{sub 65} 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. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

  1. Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

    Science.gov (United States)

    Puckette, Michael; Burrage, Thomas; Neilan, John G; Rasmussen, Max

    2017-06-12

    The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

  2. HIV-1 Nef control of cell signalling molecules: multiple strategies to ...

    Indian Academy of Sciences (India)

    Unknown

    HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle.

  3. Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix

    Directory of Open Access Journals (Sweden)

    Kuo Lillian S

    2012-05-01

    Full Text Available Abstract Background HIV-1 Nef is a multifunctional protein required for full pathogenicity of the virus. As Nef has no known enzymatic activity, it necessarily functions through protein-protein interaction interfaces. A critical Nef protein interaction interface is centered on its polyproline segment (P69VRPQVPLRP78 which contains the helical SH3 domain binding protein motif, PXXPXR. We hypothesized that any Nef-SH3 domain interactions would be lost upon mutation of the prolines or arginine of PXXPXR. Further, mutation of the non-motif “X” residues, (Q73, V74, and L75 would give altered patterns of inhibition for different Nef/SH3 domain protein interactions. Results We found that mutations of either of the prolines or the arginine of PXXPXR are defective for Nef-Hck binding, Nef/activated PAK2 complex formation and enhancement of virion infectivity (EVI. Mutation of the non-motif “X” residues (Q, V and L gave similar patterns of inhibition for Nef/activated PAK2 complex formation and EVI which were distinct from the pattern for Hck binding. These results implicate an SH3 domain containing protein other than Hck for Nef/activated PAK2 complex formation and EVI. We have also mutated Nef residues at the N-and C-terminal ends of the polyproline segment to explore interactions outside of PXXPXR. We discovered a new locus GFP/F (G67, F68, P69 and F90 that is required for Nef/activated PAK2 complex formation and EVI. MHC Class I (MHCI downregulation was only partially inhibited by mutating the PXXPXR motif residues, but was fully inhibited by mutating the C-terminal P78. Further, we observed that MHCI downregulation strictly requires G67 and F68. Our mutational analysis confirms the recently reported structure of the complex between Nef, AP-1 μ1 and the cytoplasmic tail of MHCI, but does not support involvement of an SH3 domain protein in MHCI downregulation. Conclusion Nef has evolved to be dependent on interactions with multiple SH3 domain

  4. Intermittent chaotic chimeras for coupled rotators

    DEFF Research Database (Denmark)

    Olmi, Simona; Martens, Erik Andreas; Thutupalli, Shashi

    2015-01-01

    Two symmetrically coupled populations of N oscillators with inertia m display chaotic solutions with broken symmetry similar to experimental observations with mechanical pendulums. In particular, we report evidence of intermittent chaotic chimeras, where one population is synchronized and the other...

  5. HIRENASD Chimera medium-size grid

    Data.gov (United States)

    National Aeronautics and Space Administration — Chimera medium-size grid for HIRENASD. Two files, fort.501 for grid for wing, fuselage, and 'world' zones, fort.503 for collar zone. File format is plot3d,...

  6. Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

    Science.gov (United States)

    Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Zhang, Kai; Batt, Micheal; Fitchett, Jonathan R; He, Dongmei; Rick, Heather L; Conner, Elaine M; Demarest, Stephen J

    2015-01-01

    Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing CH1 with Cα and CL with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases. PMID:25611120

  7. Enhancing UCSF Chimera through web services.

    Science.gov (United States)

    Huang, Conrad C; Meng, Elaine C; Morris, John H; Pettersen, Eric F; Ferrin, Thomas E

    2014-07-01

    Integrating access to web services with desktop applications allows for an expanded set of application features, including performing computationally intensive tasks and convenient searches of databases. We describe how we have enhanced UCSF Chimera (http://www.rbvi.ucsf.edu/chimera/), a program for the interactive visualization and analysis of molecular structures and related data, through the addition of several web services (http://www.rbvi.ucsf.edu/chimera/docs/webservices.html). By streamlining access to web services, including the entire job submission, monitoring and retrieval process, Chimera makes it simpler for users to focus on their science projects rather than data manipulation. Chimera uses Opal, a toolkit for wrapping scientific applications as web services, to provide scalable and transparent access to several popular software packages. We illustrate Chimera's use of web services with an example workflow that interleaves use of these services with interactive manipulation of molecular sequences and structures, and we provide an example Python program to demonstrate how easily Opal-based web services can be accessed from within an application. Web server availability: http://webservices.rbvi.ucsf.edu/opal2/dashboard?command=serviceList. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. Processing of ovine PrP(ARQ)C-EGFP chimeras containing Asn138 and Cys151 polymorphisms.

    Science.gov (United States)

    Bragason, Birkir Thor; Palsdottir, Astridur

    2005-10-21

    Polymorphisms in the prion protein, PrP(C), affect the susceptibility of sheep to scrapie. Three rare polymorphisms, M137T, S138N, and R151C, have been found in Icelandic sheep. Observations suggest that R151C may be associated with lower scrapie susceptibility, whereas S138N is neutral. The effects of the S138N and R151C polymorphisms on the cellular processing of PrP(C) were examined in a model system consisting of the expression of ovine PrP(C)-EGFP (green fluorescent protein) chimeras in the mouse neuroblastoma cell line N2a. Chimeras with the haplotypes A136R154Q171 (ARQ), AN138RQ, and AC151RQ were compared. The chimeras did not differ regarding their translocation into the secretory system, glycosylation, and transport to the cell surface. However, the AC151RQ chimera differed from the other chimeras regarding disulfide bonding characteristics; furthermore, a slight difference was detected between AC151RQ and the other chimeras by limited proteolysis. The processing of the ARQ and AN138RQ chimeras was identical in the experiments performed consistent with observations that it is neutral.

  9. Characterization of Three nef-Defective Human Immunodeficiency Virus Type 1 Strains Associated with Long-Term Nonprogression

    Science.gov (United States)

    Rhodes, David I.; Ashton, Lesley; Solomon, Ajantha; Carr, Andrew; Cooper, David; Kaldor, John; Deacon, Nicholas

    2000-01-01

    Long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection provide an opportunity to investigate both viral and host factors that influence the rate of disease progression. We have identified three HIV-1-infected individuals in Australia who have been infected for over 11 years with viruses that contain deletions in the nef and nef-long terminal repeat (nef/LTR) overlap regions. These viruses differ from each other and from other nef-defective strains of HIV-1 previously identified in Australia. One individual, LTS 3, is infected with a virus containing a nef gene with a deletion of 29 bp from the nef/LTR overlap region, resulting in a truncated Nef open reading frame. In addition to the Nef defect, only viruses containing truncated Vif open reading frames of 37 or 69 amino acids could be detected in peripheral blood mononuclear cells isolated from this patient. LTS 3 had a viral load of less than 20 copies of RNA/ml of plasma. The other two long-term survivors, LTS 9 and LTS 11, had loads of less than 200 copies of RNA/ml of plasma and are infected with viruses with larger deletions in both the nef alone and nef/LTR overlap regions. These viruses contain wild-type vif, vpu, and vpr accessory genes. All three strains of virus had envelope sequences characteristic of macrophagetropic viruses. These findings further indicate the reduced pathogenic potential of nef-defective viruses. PMID:11044102

  10. Human-animal chimeras for vaccine development: an endangered species or opportunity for the developing world?

    Directory of Open Access Journals (Sweden)

    Daar Abdallah S

    2010-05-01

    Full Text Available Abstract Background In recent years, the field of vaccines for diseases such as Human Immunodeficiency Virus (HIV which take a heavy toll in developing countries has faced major failures. This has led to a call for more basic science research, and development as well as evaluation of new vaccine candidates. Human-animal chimeras, developed with a 'humanized' immune system could be useful to study infectious diseases, including many neglected diseases. These would also serve as an important tool for the efficient testing of new vaccine candidates to streamline promising candidates for further trials in humans. However, developing human-animal chimeras has proved to be controversial. Discussion Development of human-animal chimeras for vaccine development has been slowed down because of opposition by some philosophers, ethicists and policy makers in the west-they question the moral status of such animals, and also express discomfort about transgression of species barriers. Such opposition often uses a contemporary western world view as a reference point. Human-animal chimeras are often being created for diseases which cause significantly higher morbidity and mortality in the developing world as compared to the developed world. We argue in our commentary that given this high disease burden, we should look at socio-cultural perspectives on human-animal chimera like beings in the developing world. On examination, it's clear that such beings have been part of mythology and cultural descriptions in many countries in the developing world. Summary To ensure that important research on diseases afflicting millions like malaria, HIV, Hepatitis-C and dengue continues to progress, we recommend supporting human-animal chimera research for vaccine development in developing countries (especially China and India which have growing technical expertise in the area. The negative perceptions in some parts of the west about human-animal chimeras can be used as an

  11. Human-animal chimeras for vaccine development: an endangered species or opportunity for the developing world?

    Science.gov (United States)

    Bhan, Anant; Singer, Peter A; Daar, Abdallah S

    2010-05-19

    In recent years, the field of vaccines for diseases such as Human Immunodeficiency Virus (HIV) which take a heavy toll in developing countries has faced major failures. This has led to a call for more basic science research, and development as well as evaluation of new vaccine candidates. Human-animal chimeras, developed with a 'humanized' immune system could be useful to study infectious diseases, including many neglected diseases. These would also serve as an important tool for the efficient testing of new vaccine candidates to streamline promising candidates for further trials in humans. However, developing human-animal chimeras has proved to be controversial. Development of human-animal chimeras for vaccine development has been slowed down because of opposition by some philosophers, ethicists and policy makers in the west-they question the moral status of such animals, and also express discomfort about transgression of species barriers. Such opposition often uses a contemporary western world view as a reference point. Human-animal chimeras are often being created for diseases which cause significantly higher morbidity and mortality in the developing world as compared to the developed world. We argue in our commentary that given this high disease burden, we should look at socio-cultural perspectives on human-animal chimera like beings in the developing world. On examination, it's clear that such beings have been part of mythology and cultural descriptions in many countries in the developing world. To ensure that important research on diseases afflicting millions like malaria, HIV, Hepatitis-C and dengue continues to progress, we recommend supporting human-animal chimera research for vaccine development in developing countries (especially China and India which have growing technical expertise in the area). The negative perceptions in some parts of the west about human-animal chimeras can be used as an opportunity for nurturing important vaccine development

  12. A novel dimer-tetramer transition captured by the crystal structure of the HIV-1 Nef.

    Directory of Open Access Journals (Sweden)

    Pankaj Singh

    Full Text Available HIV-1 Nef modulates disease progression through interactions with over 30 host proteins. Individual chains fold into membrane-interacting N-terminal and C-terminal core (Nef(core domains respectively. Nef exists as small oligomers near membranes and associates into higher oligomers such as tetramers or hexadecamers in the cytoplasm. Earlier structures of the Nef(core in apo and complexed forms with the Fyn-kinase SH3 domain revealed dimeric association details and the role of the conserved PXXP recognition motif (residues 72-78 of Nef in SH3-domain interactions. The crystal structure of the tetrameric Nef reported here corresponds to the elusive cytoplasmic stage. Comparative analyses show that subunits of Nef(core dimers (open conformation swing out with a relative displacement of ~22 Å and rotation of ~174° to form the 'closed' tetrameric structure. The changes to the association are around Asp125, a conserved residue important for viral replication and the important XR motif (residues 107-108. The tetramer associates through C4 symmetry instead of the 222 symmetry expected when two dimers associate together. This novel dimer-tetramer transition agrees with earlier solution studies including small angle X-ray scattering, analytical ultracentrifugation, dynamic laser light scattering and our glutaraldehyde cross-linking experiments. Comparisons with the Nef(core--Fyn-SH3 domain complexes reveal that the PXXP motif that interacts with the SH3-domain in the dimeric form is sterically occluded in the tetramer. However the 151-180 loop that is distal to the PXXP motif and contains several protein interaction motifs remains accessible. The results suggest how changes to the oligomeric state of Nef can help it distinguish between protein partners.

  13. Creation of mixed bone marrow chimeras with appropriate controls

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Jennifer Gommerman & Olga Rojas ### Abstract We report that IgA+ PC also can produce the anti-microbial mediators TNFα and iNOS which appears to arise in the unique environment of the gut, and may be critical to mount effective responses to microbial assault. To examine the functional relevance of TNFα/iNOS expression in IgA+ cells, we created mixed-bone marrow (BM) chimeras from TNFα/iNOS double knockout mice (dKO), in combination with B-cell deficient (JH-/-), using JH-...

  14. Chimera states in mechanical oscillator networks

    DEFF Research Database (Denmark)

    Martens, Erik Andreas; Thutupalli, Shashi; Fourrière, Antoine

    2013-01-01

    of whether chimeras are indeed characteristic of natural systems. This calls for a palpable realization of chimera states without any fine-tuning, from which physical mechanisms underlying their emergence can be uncovered. Here, we devise a simple experiment with mechanical oscillators coupled...... in our experiments is controlled by elementary dynamical equations from mechanics that are ubiquitous in many natural and technological systems. The symmetry-breaking mechanism revealed by our experiments may thus be prevalent in systems exhibiting collective behavior, such as power grids, optomechanical...

  15. Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41.

    Directory of Open Access Journals (Sweden)

    Rachel P J Lai

    2011-12-01

    Full Text Available Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs that target the membrane proximal external region (MPER of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly

  16. Lipidomic dataset of plasma from patients infected with wild type and nef-deficient HIV-1 strain

    Directory of Open Access Journals (Sweden)

    Peter Meikle

    2016-03-01

    Full Text Available Previous in vitro and in vivo studies demonstrated that HIV protein nef plays a key role in impairing cellular and systemic cholesterol metabolism in HIV disease, but clinical support for these findings is lacking. Here we present the data of comparative lipidomic analysis (330 lipid species of plasma samples from HIV-negative subjects, patients infected with WT HIV-1 strain and patients infected with nef-deficient strain of HIV-1. We determine which effects of HIV on plasma lipidome are explained by the presence of nef. The data can be used to evaluate cardiovascular risk in HIV disease and to assess the role of nef in HIV-induced disturbances in systemic lipid metabolism. The full impact of nef deficiency on lipid and lipoprotein metabolism in HIV-infected patients is presented in the accompanying study “Lipid Metabolism in Patients Infected with Nef-deficient HIV-1 Strain” [1].

  17. Basins of Attraction for Chimera States

    DEFF Research Database (Denmark)

    Martens, Erik Andreas; Panaggio, Mark; Abrams, Daniel

    2016-01-01

    evaluate asymptotic states and associated destination maps, and demonstrate that basins form a complex twisting structure in phase space. Understanding the basins' precise nature may help in the development of control methods to switch between chimera patterns, with possible technological and neural system...

  18. α-Peptide/ß-Peptoid Chimeras

    DEFF Research Database (Denmark)

    Olsen, Christian Adam; Bonke, Gitte; Vedel, Line

    2007-01-01

    We describe the synthesis and characterization of the first generation of oligomers consisting of alternating repeats of a-amino acids and chiral N-alkyl-ß-alanine (ß-peptoid) residues. These chimeras are stable toward proteolysis, non-hemolytic, and possess antibacterial activity comparable...

  19. A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts.

    Science.gov (United States)

    Ealba, Erin L; Schneider, Richard A

    2013-07-01

    Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level.

  20. Characterization of Three nef-Defective Human Immunodeficiency Virus Type 1 Strains Associated with Long-Term Nonprogression

    OpenAIRE

    David I. Rhodes; Ashton, Lesley; Solomon, Ajantha; Carr, Andrew; Cooper, David; Kaldor, John; Deacon, Nicholas

    2000-01-01

    Long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection provide an opportunity to investigate both viral and host factors that influence the rate of disease progression. We have identified three HIV-1-infected individuals in Australia who have been infected for over 11 years with viruses that contain deletions in the nef and nef-long terminal repeat (nef/LTR) overlap regions. These viruses differ from each other and from other nef-defective strains of HIV-1 previous...

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

    Science.gov (United States)

    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.

  2. Trans-cellular introduction of HIV-1 protein Nef induces pathogenic response in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Aamir Nazir

    Full Text Available BACKGROUND: Caenorhabditis elegans has emerged as a very powerful model for studying the host pathogen interactions. Despite the absence of a naturally occurring viral infection for C. elegans, the model is now being exploited experimentally to study the basic aspects of virus-host interplay. The data generated from recent studies suggests that the virus that infects mammalian cells does infect, replicate and accumulate in C. elegans. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of the easy-to-achieve protein introduction in C. elegans and employing the methodology, we administered HIV-1 protein Nef into live worms. Nef is known to be an important protein for exacerbating HIV-1 pathogenesis in host by enhancing viral replication. The deletion of nef from the viral genome has been reported to inhibit its replication in the host, thereby leading to delayed pathogenesis. Our studies, employing Nef introduction into C. elegans, led to creation of an in-vivo model that allowed us to study, whether or not, the protein induces effect in the whole organism. We observed a marked lipodystrophy, effect on neuromuscular function, impaired fertility and reduced longevity in the worms exposed to Nef. The observed effects resemble to those observed in Nef transgenic mice and most interestingly the effects also relate to some of the pathogenic aspects exhibited by human AIDS patients. CONCLUSIONS/SIGNIFICANCE: Our studies underline the importance of this in vivo model for studying the interactions of Nef with host proteins, which could further be used for identifying possible inhibitors of such interactions.

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

    Directory of Open Access Journals (Sweden)

    Geyer Matthias

    2007-10-01

    Full Text Available Abstract 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.

  4. Generation of Hprt-disrupted rat through mouse←rat ES chimeras

    OpenAIRE

    Ayako Isotani; Kazuo Yamagata; Masaru Okabe; Masahito Ikawa

    2016-01-01

    We established rat embryonic stem (ES) cell lines from a double transgenic rat line which harbours CAG-GFP for ubiquitous expression of GFP in somatic cells and Acr3-EGFP for expression in sperm (green body and green sperm: GBGS rat). By injecting the GBGS rat ES cells into mouse blastocysts and transplanting them into pseudopregnant mice, rat spermatozoa were produced in mouse?rat ES chimeras. Rat spermatozoa from the chimeric testis were able to fertilize eggs by testicular sperm extraction...

  5. Cell Surface Downregulation of NK Cell Ligands by Patient-Derived HIV-1 Vpu and Nef Alleles.

    Science.gov (United States)

    Galaski, Johanna; Ahmad, Fareed; Tibroni, Nadine; Pujol, Francois M; Müller, Birthe; Schmidt, Reinhold E; Fackler, Oliver T

    2016-05-01

    HIV-1 Vpu and Nef proteins downregulate cell surface levels of natural killer (NK) cell ligands but functional consequences of individual downregulation events are unclear. We tested how well-conserved NK cell ligand downregulation is among Vpu and Nef variants isolated from chronic HIV patients. Proviral vpu and nef sequences were amplified from 27 chronic HIV patients, subcloned, and tested for their ability to downregulate cell surface receptors. Cell surface downregulation of CD4, CD317/tetherin, and major histocompatibility complex class 1 that exert biological functions other than NK cell activation were well conserved among patient-derived Vpu and Nef variants. Among NK cell ligands, NK-T-B-antigen, poliovirus receptor, and UL16-binding protein were identified as main targets for Vpu and Nef, the downregulation of which by at least 1 viral protein was highly conserved. NK cell ligands displayed specific sensitivity to Vpu (NK-T-B-antigen) or Nef (poliovirus receptor), and downregulation of cell surface UL16-binding protein was identified as a novel and highly conserved activity of HIV-1 Vpu but not Nef. The conservation of downregulation of major NK cell ligands by either HIV-1 Vpu or Nef suggests an important pathophysiological role of this activity, which may impact the acute but not the chronic phase of HIV infection.

  6. Intrahost and interhost variability of the HIV type 1 nef gene in Brazilian children.

    Science.gov (United States)

    Cavalieri, Elizabeth; Florido, Camila; Leal, Elcio; Machado, Daisy Maria; Camargo, Michelle; Diaz, Ricardo S; Janini, Luiz Mario

    2009-11-01

    Many aspects of HIV-1 pathogenesis are affected by Nef protein activity, and efforts have been made to study variation in the nef gene and how that variation relates to disease outcome. We studied the genetic diversity of the nef gene in distinct clones obtained from the same patient (intrahost) and in sequences obtained from different hosts (interhost). The set of sequences analyzed was obtained from HIV-1-infected Brazilian children and contained 112 clones from 25 children (intrahost samples), as well as 55 sequences from epidemiologically unlinked children (interhost samples). We found extensive site polymorphisms and amino acid length variations, mainly in the amino terminal region of the nef gene, between the myristoylation motif (MGxxxS) and the MHC-1 downregulation motif (Rxx). Analysis of the sequences deposited in the Los Alamos HIV sequences database ( www.hiv.lanl.gov ) indicated that the most frequent motif at the MHC-1 downregulation site in the subtype B strain is R(86%)A(64%)E(82%) (n = 1040) and R(78%)T(74%)E(56%) in the subtype C strain (n = 549). Conversely, the Brazilian subtype B isolates presented the motif R(81%)T(62%)E(67%) at this site (n = 64). A detailed analysis of selective pressures identified a concentration of codons under strong positive selection in the amino terminal region of the nef gene. We also determined that different sites are under positive selection in the subtype B and subtype C viruses. The amino acid composition in the MHC-1 downregulation motif of the nef gene in our sequences may indicate a distinct adaptive pattern of HIV-1 subtype B to the Brazilian host population.

  7. Reevaluating αE-catenin monomer and homodimer functions by characterizing E-cadherin/αE-catenin chimeras.

    Science.gov (United States)

    Bianchini, Julie M; Kitt, Khameeka N; Gloerich, Martijn; Pokutta, Sabine; Weis, William I; Nelson, W James

    2015-09-28

    As part of the E-cadherin-β-catenin-αE-catenin complex (CCC), mammalian αE-catenin binds F-actin weakly in the absence of force, whereas cytosolic αE-catenin forms a homodimer that interacts more strongly with F-actin. It has been concluded that cytosolic αE-catenin homodimer is not important for intercellular adhesion because E-cadherin/αE-catenin chimeras thought to mimic the CCC are sufficient to induce cell-cell adhesion. We show that, unlike αE-catenin in the CCC, these chimeras homodimerize, bind F-actin strongly, and inhibit the Arp2/3 complex, all of which are properties of the αE-catenin homodimer. To more accurately mimic the junctional CCC, we designed a constitutively monomeric chimera, and show that E-cadherin-dependent cell adhesion is weaker in cells expressing this chimera compared with cells in which αE-catenin homodimers are present. Our results demonstrate that E-cadherin/αE-catenin chimeras used previously do not mimic αE-catenin in the native CCC, and imply that both CCC-bound monomer and cytosolic homodimer αE-catenin are required for strong cell-cell adhesion. © 2015 Bianchini et al.

  8. Morphology and cytology of flower chimeras in hybrids of Brassica ...

    African Journals Online (AJOL)

    Hybridization between white flowered Brassica carinata and yellow flowered B. rapa were made, and the flower chimeras were observed in a few hybrids. The simple single sequence molecular markers verified the hybridity of those hybrids. Chimeras were justified and totally classified based on the morphological ...

  9. Hemopoietic cell kinase (Hck) and p21-activated kinase 2 (PAK2) are involved in the down-regulation of CD1a lipid antigen presentation by HIV-1 Nef in dendritic cells.

    Science.gov (United States)

    Shinya, Eiji; Shimizu, Masumi; Owaki, Atsuko; Paoletti, Samantha; Mori, Lucia; De Libero, Gennaro; Takahashi, Hidemi

    2016-01-01

    Dendritic cells (DCs) play a major role in in vivo pathogenesis of HIV-1 infection. Therefore, DCs may provide a promising strategy to control and eventually overcome the fatal infection. Especially, immature DCs express all CD1s, the non-MHC lipid antigen -presenting molecules, and HIV-1 Nef down-regulates CD1 expression besides MHC. Moreover, CD1d-restricted CD4(+) NKT cells are infected by HIV-1, reducing the number of these cells in HIV-1-infected individuals. To understand the exact role of DCs and CD1-mediated immune response during HIV-1 infection, Nef down-regulation of CD1a-restricted lipid/glycolipid Ag presentation in iDCs was analyzed. We demonstrated the involvement of the association of Nef with hemopoietic cell kinase (Hck) and p21-activated kinase 2 (PAK2), and that Hck, which is expressed strongly in iDCs, augmented this mutual interaction. Hck might be another therapeutic target to preserve the function of HIV-1 infected DCs, which are potential reservoirs of HIV-1 even after antiretroviral therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Isotropy of Angular Frequencies and Weak Chimeras with Broken Symmetry

    Science.gov (United States)

    Bick, Christian

    2017-04-01

    The notion of a weak chimeras provides a tractable definition for chimera states in networks of finitely many phase oscillators. Here, we generalize the definition of a weak chimera to a more general class of equivariant dynamical systems by characterizing solutions in terms of the isotropy of their angular frequency vector—for coupled phase oscillators the angular frequency vector is given by the average of the vector field along a trajectory. Symmetries of solutions automatically imply angular frequency synchronization. We show that the presence of such symmetries is not necessary by giving a result for the existence of weak chimeras without instantaneous or setwise symmetries for coupled phase oscillators. Moreover, we construct a coupling function that gives rise to chaotic weak chimeras without symmetry in weakly coupled populations of phase oscillators with generalized coupling.

  11. HIV-1 Nef control of cell signalling molecules: multiple strategies to ...

    Indian Academy of Sciences (India)

    The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. This small protein of approximately 27 kDa is required for maximal virus replication and disease progression. The mechanisms by which it is able to act as a positive factor during virus replication is an area of intense research and although some ...

  12. HIV-1 nef protein structures associated with brain infection and dementia pathogenesis.

    Directory of Open Access Journals (Sweden)

    Susanna L Lamers

    2011-02-01

    Full Text Available The difference between regional rates of HIV-associated dementia (HAD in patients infected with different subtypes of HIV suggests that genetic determinants exist within HIV that influence the ability of the virus to replicate in the central nervous system (in Uganda, Africa, subtype D HAD rate is 89%, while subtype A HAD rate is 24%. HIV-1 nef is a multifunctional protein with known toxic effects in the brain compartment. The goal of the current study was to identify if specific three-dimensional nef structures may be linked to patients who developed HAD. HIV-1 nef structures were computationally derived for consensus brain and non-brain sequences from a panel of patients infected with subtype B who died due to varied disease pathologies and consensus subtype A and subtype D sequences from Uganda. Site directed mutation analysis identified signatures in brain structures that appear to change binding potentials and could affect folding conformations of brain-associated structures. Despite the large sequence variation between HIV subtypes, structural alignments confirmed that viral structures derived from patients with HAD were more similar to subtype D structures than to structures derived from patient sequences without HAD. Furthermore, structures derived from brain sequences of patients with HAD were more similar to subtype D structures than they were to their own non-brain structures. The potential finding of a brain-specific nef structure indicates that HAD may result from genetic alterations that alter the folding or binding potential of the protein.

  13. HIV-1 nef protein structures associated with brain infection and dementia pathogenesis.

    Science.gov (United States)

    Lamers, Susanna L; Poon, Art F Y; McGrath, Michael S

    2011-02-09

    The difference between regional rates of HIV-associated dementia (HAD) in patients infected with different subtypes of HIV suggests that genetic determinants exist within HIV that influence the ability of the virus to replicate in the central nervous system (in Uganda, Africa, subtype D HAD rate is 89%, while subtype A HAD rate is 24%). HIV-1 nef is a multifunctional protein with known toxic effects in the brain compartment. The goal of the current study was to identify if specific three-dimensional nef structures may be linked to patients who developed HAD. HIV-1 nef structures were computationally derived for consensus brain and non-brain sequences from a panel of patients infected with subtype B who died due to varied disease pathologies and consensus subtype A and subtype D sequences from Uganda. Site directed mutation analysis identified signatures in brain structures that appear to change binding potentials and could affect folding conformations of brain-associated structures. Despite the large sequence variation between HIV subtypes, structural alignments confirmed that viral structures derived from patients with HAD were more similar to subtype D structures than to structures derived from patient sequences without HAD. Furthermore, structures derived from brain sequences of patients with HAD were more similar to subtype D structures than they were to their own non-brain structures. The potential finding of a brain-specific nef structure indicates that HAD may result from genetic alterations that alter the folding or binding potential of the protein.

  14. Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor.

    Science.gov (United States)

    Amorim, Nathaly A; da Silva, Eulália M L; de Castro, Rodrigo O; da Silva-Januário, Mara E; Mendonça, Luiza M; Bonifacino, Juan S; da Costa, Luciana J; daSilva, Luis L P

    2014-10-03

    Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Interaction of HIV-1 Nef Protein with the Host Protein Alix Promotes Lysosomal Targeting of CD4 Receptor*

    Science.gov (United States)

    Amorim, Nathaly A.; da Silva, Eulália M. L.; de Castro, Rodrigo O.; da Silva-Januário, Mara E.; Mendonça, Luiza M.; Bonifacino, Juan S.; da Costa, Luciana J.; daSilva, Luis L. P.

    2014-01-01

    Nef is an accessory protein of human immunodeficiency viruses that promotes viral replication and progression to AIDS through interference with various host trafficking and signaling pathways. A key function of Nef is the down-regulation of the coreceptor CD4 from the surface of the host cells. Nef-induced CD4 down-regulation involves at least two independent steps as follows: acceleration of CD4 endocytosis by a clathrin/AP-2-dependent pathway and targeting of internalized CD4 to multivesicular bodies (MVBs) for eventual degradation in lysosomes. In a previous work, we found that CD4 targeting to the MVB pathway was independent of CD4 ubiquitination. Here, we report that this targeting depends on a direct interaction of Nef with Alix/AIP1, a protein associated with the endosomal sorting complexes required for transport (ESCRT) machinery that assists with cargo recruitment and intraluminal vesicle formation in MVBs. We show that Nef interacts with both the Bro1 and V domains of Alix. Depletion of Alix or overexpression of the Alix V domain impairs lysosomal degradation of CD4 induced by Nef. In contrast, the V domain overexpression does not prevent cell surface removal of CD4 by Nef or protein targeting to the canonical ubiquitination-dependent MVB pathway. We also show that the Nef-Alix interaction occurs in late endosomes that are enriched in internalized CD4. Together, our results indicate that Alix functions as an adaptor for the ESCRT-dependent, ubiquitin-independent targeting of CD4 to the MVB pathway induced by Nef. PMID:25118280

  16. Persistence of attenuated HIV-1 rev alleles in an epidemiologically linked cohort of long-term survivors infected with nef-deleted virus

    Directory of Open Access Journals (Sweden)

    Wesselingh Steven L

    2007-07-01

    Full Text Available Abstract Background The Sydney blood bank cohort (SBBC of long-term survivors consists of multiple individuals infected with nef-deleted, attenuated strains of human immunodeficiency virus type 1 (HIV-1. Although the cohort members have experienced differing clinical courses and now comprise slow progressors (SP as well as long-term nonprogressors (LTNP, longitudinal analysis of nef/long-terminal repeat (LTR sequences demonstrated convergent nef/LTR sequence evolution in SBBC SP and LTNP. Thus, the in vivo pathogenicity of attenuated HIV-1 strains harboured by SBBC members is dictated by factors other than nef/LTR. Therefore, to determine whether defects in other viral genes contribute to attenuation of these HIV-1 strains, we characterized dominant HIV-1 rev alleles that persisted in 4 SBBC subjects; C18, C64, C98 and D36. Results The ability of Rev derived from D36 and C64 to bind the Rev responsive element (RRE in RNA binding assays was reduced by approximately 90% compared to Rev derived from HIV-1NL4-3, C18 or C98. D36 Rev also had a 50–60% reduction in ability to express Rev-dependent reporter constructs in mammalian cells. In contrast, C64 Rev had only marginally decreased Rev function despite attenuated RRE binding. In D36 and C64, attenuated RRE binding was associated with rare amino acid changes at 3 highly conserved residues; Gln to Pro at position 74 immediately N-terminal to the Rev activation domain, and Val to Leu and Ser to Pro at positions 104 and 106 at the Rev C-terminus, respectively. In D36, reduced Rev function was mapped to an unusual 13 amino acid extension at the Rev C-terminus. Conclusion These findings provide new genetic and mechanistic insights important for Rev function, and suggest that Rev function, not Rev/RRE binding may be rate limiting for HIV-1 replication. In addition, attenuated rev alleles may contribute to viral attenuation and long-term survival of HIV-1 infection in a subset of SBBC members.

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

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    Martha J Lewis

    Full Text Available 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

  18. Brain transcriptome-wide screen for HIV-1 Nef protein interaction partners reveals various membrane-associated proteins.

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    Ellen C Kammula

    Full Text Available HIV-1 Nef protein contributes essentially to the pathology of AIDS by a variety of protein-protein-interactions within the host cell. The versatile functionality of Nef is partially attributed to different conformational states and posttranslational modifications, such as myristoylation. Up to now, many interaction partners of Nef have been identified using classical yeast two-hybrid screens. Such screens rely on transcriptional activation of reporter genes in the nucleus to detect interactions. Thus, the identification of Nef interaction partners that are integral membrane proteins, membrane-associated proteins or other proteins that do not translocate into the nucleus is hampered. In the present study, a split-ubiquitin based yeast two-hybrid screen was used to identify novel membrane-localized interaction partners of Nef. More than 80% of the hereby identified interaction partners of Nef are transmembrane proteins. The identified hits are GPM6B, GPM6A, BAP31, TSPAN7, CYB5B, CD320/TCblR, VSIG4, PMEPA1, OCIAD1, ITGB1, CHN1, PH4, CLDN10, HSPA9, APR-3, PEBP1 and B3GNT, which are involved in diverse cellular processes like signaling, apoptosis, neurogenesis, cell adhesion and protein trafficking or quality control. For a subfraction of the hereby identified proteins we present data supporting their direct interaction with HIV-1 Nef. We discuss the results with respect to many phenotypes observed in HIV infected cells and patients. The identified Nef interaction partners may help to further elucidate the molecular basis of HIV-related diseases.

  19. Is repulsion good for the health of chimeras?

    Science.gov (United States)

    Jalan, Sarika; Ghosh, Saptarshi; Patra, Bibhabasu

    2017-10-01

    Yes! Very much so. A chimera state refers to the coexistence of a coherent-incoherent dynamical evolution of identically coupled oscillators. We investigate the impact of multiplexing of a layer having repulsively coupled oscillators on the occurrence of chimeras in the layer having attractively coupled identical oscillators. We report that there exists an enhancement in the appearance of the chimera state in one layer of the multiplex network in the presence of repulsive coupling in the other layer. Furthermore, we show that a small amount of inhibition or repulsive coupling in one layer is sufficient to yield the chimera state in another layer by destroying its synchronized behavior. These results can be used to obtain insight into dynamical behaviors of those systems where both attractive and repulsive couplings exist among their constituents.

  20. Determinants in HIV-1 Nef for enhancement of virus replication and depletion of CD4+ T lymphocytes in human lymphoid tissue ex vivo

    Directory of Open Access Journals (Sweden)

    Sertel Serkan

    2009-01-01

    Full Text Available Abstract Background HIV-1 Nef critically contributes to AIDS in part by augmenting virus titers in infected individuals. Analyzing which of Nef's activities contribute to HIV pathogenesis has been hampered by the lack of a cell culture model in which Nef exerts pronounced effects on HIV replication. The human lymphoid aggregate culture (HLAC from tonsil maintains the cell populations and cytokine milieu found in vivo, supports a productive infection without exogenous stimulation, and Nef contributes to efficient HIV-1 replication as well as CD4+ T cell depletion in this experimental ex vivo-model. Results To identify determinants in Nef that mediate these activities, we infected HLAC with a panel of isogenic HIV-1NL4-3 strains that encode for well-characterized mutants of HIV-1SF2 Nef. Determination of HIV-1 replication revealed that enhancement of the virus spread by Nef is governed by a complex set of protein interaction surfaces. In contrast, increased CD4+ T lymphocyte depletion depended on only two protein interaction surfaces in Nef that mediate either downregulation of cell surface CD4 or interaction with the NAKC signalosome. Consistently, in HLAC from 9 out of 14 donors, Nef enhanced CD4+ T cell depletion in the absence of a significant effect on virus replication. Moreover, our results suggest that this Nef-dependent enhancement in depletion occurred predominately in uninfected bystander CD4+ T cells. Conclusion Our findings suggest that Nef facilitates depletion of CD4+ T lymphocytes in HIV-1-infected lymphoid tissue ex vivo by increasing the pool of productively infected cells and by sensitizing bystander cells for killing. This ability might contribute to Nef's pathogenic potential in vivo.

  1. Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

    Directory of Open Access Journals (Sweden)

    Meuwissen Pieter J

    2012-04-01

    Full Text Available Abstract Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2 and non-canonical (B2 and C1422 HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2, the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we

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

    Directory of Open Access Journals (Sweden)

    Grant R Campbell

    2015-06-01

    Full Text Available 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.

  3. Formyl peptide receptor chimeras define domains involved in ligand binding.

    Science.gov (United States)

    Perez, H D; Holmes, R; Vilander, L R; Adams, R R; Manzana, W; Jolley, D; Andrews, W H

    1993-02-05

    We have begun to study the structural requirements for the binding of formyl peptides to their specific receptors. As an initial approach, we constructed C5a-formyl peptide receptor chimeras. Unique (and identical) restriction sites were introduced within the transmembrane domains of these receptors that allowed for the exchange of specific areas. Four types of chimeric receptors were generated. 1) The C5a receptor was progressively substituted by the formyl peptide receptor. 2) The formyl peptide receptor was progressively substituted by the C5a receptor. 3) Specific domains of the C5a receptor were substituted by the corresponding domain of the formyl peptide receptor. 4) Specific domains of the formyl peptide receptor were replaced by the same corresponding domain of the C5a receptor. Wild type and chimeric receptors were transfected into COS 7 cells and their ability to bind formyl peptide determined, taking into account efficiency of transfection and expression of chimeric protein. Based on these results, a ligand binding model is presented in which the second, third, and fourth extracellular (and/or their transmembrane) domains together with the first transmembrane domain form a ligand binding pocket for formyl peptides. It is proposed that the amino-terminal domain plays a role by presumably providing a "lid" to the pocket. The carboxyl-terminal cytoplasmic tail appears to modulate ligand binding by regulating receptor affinity.

  4. Prototype and Chimera-Type Galectins in Placentas with Spontaneous and Recurrent Miscarriages

    Directory of Open Access Journals (Sweden)

    Laura Unverdorben

    2016-04-01

    Full Text Available Galectins are galactose binding proteins and, in addition, factors for a wide range of pathologies in pregnancy. We have analyzed the expression of prototype (gal-1, -2, -7, -10 and chimera-type (gal-3 galectins in the placenta in cases of spontaneous abortions (SPA and recurrent abortions (RA in the first trimester. Fifteen placental samples from healthy pregnancies were used as a control group. Nine placentas were examined for spontaneous abortions, and 12 placentas for recurrent abortions. For differentiation and evaluation of different cell types of galectin-expression in the decidua, immunofluorescence was used. For all investigated prototype galectins (gal-1, -2, -7, -10 in SPA and RA placenta trophoblast cells the expression is significantly decreased. In the decidua/extravillous trophoblast only gal-2 expression was significantly lowered, which could be connected to its role in angiogenesis. In trophoblasts in first-trimester placentas and in cases of SPA and RA, prototype galectins are altered in the same way. We suspect prototype galectins have a similar function in placental tissue because of their common biochemical structure. Expression of galectin 3 as a chimera type galectin was not found to be significantly altered in abortive placentas.

  5. Plant chimeras: The good, the bad, and the 'Bizzaria'.

    Science.gov (United States)

    Frank, Margaret H; Chitwood, Daniel H

    2016-11-01

    Chimeras - organisms that are composed of cells of more than one genotype - captured the human imagination long before they were formally described and used in the laboratory. These organisms owe their namesake to a fire-breathing monster from Greek mythology that has the head of a lion, the body of a goat, and the tail of a serpent. The first description of a non-fictional chimera dates back to the middle of the seventeenth century when the Florentine gardener Pietro Nati discovered an adventitious shoot growing from the graft junction between sour orange (Citrus aurantium) and citron (Citrus medica). This perplexing chimera that grows with sectors phenotypically resembling each of the citrus progenitors inspired discussion and wonder from the scientific community and was fittingly named the 'Bizzaria'. Initially, the 'Bizzaria' was believed to be an asexual hybrid that formed from a cellular fusion between the grafted parents; however, in-depth cellular analyses carried out centuries later demonstrated that the 'Bizzaria', along with other chimeras, owe their unique sectored appearance to a conglomeration of cells from the two donors. Since this pivotal discovery at the turn of the twentieth century, chimeras have served both as tools and as unique biological phenomena that have contributed to our understanding of plant development at the cellular, tissue, and organismal level. Rapid advancements in genome sequencing technologies have enabled the establishment of new model species with novel morphological and developmental features that enable the generation of chimeric organisms. In this review, we show that genetic mosaic and chimera studies provide a technologically simple way to delve into the organismal, genetic, and genomic inner workings underlying the development of diverse model organisms. Moreover, we discuss the unique opportunity that chimeras present to explore universal principles governing intercellular communication and the coordination of

  6. Tools for integrated sequence-structure analysis with UCSF Chimera

    Directory of Open Access Journals (Sweden)

    Huang Conrad C

    2006-07-01

    Full Text Available Abstract Background Comparing related structures and viewing the structures in the context of sequence alignments are important tasks in protein structure-function research. While many programs exist for individual aspects of such work, there is a need for interactive visualization tools that: (a provide a deep integration of sequence and structure, far beyond mapping where a sequence region falls in the structure and vice versa; (b facilitate changing data of one type based on the other (for example, using only sequence-conserved residues to match structures, or adjusting a sequence alignment based on spatial fit; (c can be used with a researcher's own data, including arbitrary sequence alignments and annotations, closely or distantly related sets of proteins, etc.; and (d interoperate with each other and with a full complement of molecular graphics features. We describe enhancements to UCSF Chimera to achieve these goals. Results The molecular graphics program UCSF Chimera includes a suite of tools for interactive analyses of sequences and structures. Structures automatically associate with sequences in imported alignments, allowing many kinds of crosstalk. A novel method is provided to superimpose structures in the absence of a pre-existing sequence alignment. The method uses both sequence and secondary structure, and can match even structures with very low sequence identity. Another tool constructs structure-based sequence alignments from superpositions of two or more proteins. Chimera is designed to be extensible, and mechanisms for incorporating user-specific data without Chimera code development are also provided. Conclusion The tools described here apply to many problems involving comparison and analysis of protein structures and their sequences. Chimera includes complete documentation and is intended for use by a wide range of scientists, not just those in the computational disciplines. UCSF Chimera is free for non-commercial use and is

  7. Tools for integrated sequence-structure analysis with UCSF Chimera.

    Science.gov (United States)

    Meng, Elaine C; Pettersen, Eric F; Couch, Gregory S; Huang, Conrad C; Ferrin, Thomas E

    2006-07-12

    Comparing related structures and viewing the structures in the context of sequence alignments are important tasks in protein structure-function research. While many programs exist for individual aspects of such work, there is a need for interactive visualization tools that: (a) provide a deep integration of sequence and structure, far beyond mapping where a sequence region falls in the structure and vice versa; (b) facilitate changing data of one type based on the other (for example, using only sequence-conserved residues to match structures, or adjusting a sequence alignment based on spatial fit); (c) can be used with a researcher's own data, including arbitrary sequence alignments and annotations, closely or distantly related sets of proteins, etc.; and (d) interoperate with each other and with a full complement of molecular graphics features. We describe enhancements to UCSF Chimera to achieve these goals. The molecular graphics program UCSF Chimera includes a suite of tools for interactive analyses of sequences and structures. Structures automatically associate with sequences in imported alignments, allowing many kinds of crosstalk. A novel method is provided to superimpose structures in the absence of a pre-existing sequence alignment. The method uses both sequence and secondary structure, and can match even structures with very low sequence identity. Another tool constructs structure-based sequence alignments from superpositions of two or more proteins. Chimera is designed to be extensible, and mechanisms for incorporating user-specific data without Chimera code development are also provided. The tools described here apply to many problems involving comparison and analysis of protein structures and their sequences. Chimera includes complete documentation and is intended for use by a wide range of scientists, not just those in the computational disciplines. UCSF Chimera is free for non-commercial use and is available for Microsoft Windows, Apple Mac OS X

  8. ChimPipe: accurate detection of fusion genes and transcription-induced chimeras from RNA-seq data.

    Science.gov (United States)

    Rodríguez-Martín, Bernardo; Palumbo, Emilio; Marco-Sola, Santiago; Griebel, Thasso; Ribeca, Paolo; Alonso, Graciela; Rastrojo, Alberto; Aguado, Begoña; Guigó, Roderic; Djebali, Sarah

    2017-01-03

    Chimeric transcripts are commonly defined as transcripts linking two or more different genes in the genome, and can be explained by various biological mechanisms such as genomic rearrangement, read-through or trans-splicing, but also by technical or biological artefacts. Several studies have shown their importance in cancer, cell pluripotency and motility. Many programs have recently been developed to identify chimeras from Illumina RNA-seq data (mostly fusion genes in cancer). However outputs of different programs on the same dataset can be widely inconsistent, and tend to include many false positives. Other issues relate to simulated datasets restricted to fusion genes, real datasets with limited numbers of validated cases, result inconsistencies between simulated and real datasets, and gene rather than junction level assessment. Here we present ChimPipe, a modular and easy-to-use method to reliably identify fusion genes and transcription-induced chimeras from paired-end Illumina RNA-seq data. We have also produced realistic simulated datasets for three different read lengths, and enhanced two gold-standard cancer datasets by associating exact junction points to validated gene fusions. Benchmarking ChimPipe together with four other state-of-the-art tools on this data showed ChimPipe to be the top program at identifying exact junction coordinates for both kinds of datasets, and the one showing the best trade-off between sensitivity and precision. Applied to 106 ENCODE human RNA-seq datasets, ChimPipe identified 137 high confidence chimeras connecting the protein coding sequence of their parent genes. In subsequent experiments, three out of four predicted chimeras, two of which recurrently expressed in a large majority of the samples, could be validated. Cloning and sequencing of the three cases revealed several new chimeric transcript structures, 3 of which with the potential to encode a chimeric protein for which we hypothesized a new role. Applying ChimPipe to

  9. Maintenance of AP-2 Dependent Functional Activities of Nef Restricts Pathways of Immune Escape from CD8 T Lymphocyte Responses.

    Science.gov (United States)

    Schouest, Blake; Weiler, Andrea M; Janaka, Sanath Kumar; Myers, T Alix; Das, Arpita; Wilder, Sarah C; Furlott, Jessica; Baddoo, Melody; Flemington, Erik K; Rakasz, Eva G; Evans, David T; Friedrich, Thomas C; Maness, Nicholas J

    2017-12-13

    Nef-specific CD8+ T lymphocytes (CD8TL) are linked to extraordinary control of primate lentiviral replication, but the mechanisms underlying their efficacy remain largely unknown. The immunodominant, Mamu-B*017:01+ restricted Nef195-203MW9 epitope in SIVmac239 partially overlaps a sorting motif important for interactions with host AP-2 proteins and, hence, downmodulation of several host proteins including Tetherin (CD317/BST-2), CD28, CD4, SERINC3, and SERINC5. We reasoned that CD8TL-driven evolution in this epitope might compromise Nef's ability to modulate these important molecules. Here we used deep sequencing of SIV from nine B*017:01+ macaques throughout infection with SIVmac239 to characterize the patterns of viral escape in this epitope and then assayed impacts of these variants on Nef-mediated modulation of multiple host molecules. Acute variation in multiple Nef195-203MW9 residues significantly compromised Nef's ability to downregulate surface Tetherin, CD4, and CD28, and reduced its ability to prevent SERINC5-mediated reduction in viral infectivity but did not impact downregulation of CD3 or MHC-I, suggesting selective disruption of immunomodulatory pathways involving Nef AP-2 interactions. Together, our data illuminate a pattern of viral escape dictated by a selective balance to maintain AP-2 mediated downregulation while evading epitope-specific CD8TL responses. These data could shed light on mechanisms of both CD8TL-driven viral control generally and on Mamu-B*017:01-mediated viral control specifically.ImportanceA rare subset of humans infected with HIV-1 and macaques infected with SIV can control the virus without aid of antiviral medications. A common feature of these individuals is the ability to mount unusually effective CD8 T lymphocyte responses against the virus. One of the most formidable aspects of HIV is its ability to evolve to evade immune responses, particularly CD8 T lymphocytes. We show that macaques that target a specific peptide in the

  10. Chimera states in coupled Kuramoto oscillators with inertia

    Energy Technology Data Exchange (ETDEWEB)

    Olmi, Simona, E-mail: simona.olmi@fi.isc.cnr.it [CNR - Consiglio Nazionale delle Ricerche - Istituto dei Sistemi Complessi, via Madonna del Piano 10, I-50019 Sesto Fiorentino (Italy); INFN Sez. Firenze, via Sansone, 1 - I-50019 Sesto Fiorentino (Italy)

    2015-12-15

    The dynamics of two symmetrically coupled populations of rotators is studied for different values of the inertia. The system is characterized by different types of solutions, which all coexist with the fully synchronized state. At small inertia, the system is no more chaotic and one observes mainly quasi-periodic chimeras, while the usual (stationary) chimera state is not anymore observable. At large inertia, one observes two different kind of chaotic solutions with broken symmetry: the intermittent chaotic chimera, characterized by a synchronized population and a population displaying a turbulent behaviour, and a second state where the two populations are both chaotic but whose dynamics adhere to two different macroscopic attractors. The intermittent chaotic chimeras are characterized by a finite life-time, whose duration increases as a power-law with the system size and the inertia value. Moreover, the chaotic population exhibits clear intermittent behavior, displaying a laminar phase where the two populations tend to synchronize, and a turbulent phase where the macroscopic motion of one population is definitely erratic. In the thermodynamic limit, these states survive for infinite time and the laminar regimes tends to disappear, thus giving rise to stationary chaotic solutions with broken symmetry contrary to what observed for chaotic chimeras on a ring geometry.

  11. Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef.

    Directory of Open Access Journals (Sweden)

    Mahdi Sarmady

    Full Text Available Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.

  12. Double-well chimeras in 2D lattice of chaotic bistable elements

    Science.gov (United States)

    Shepelev, I. A.; Bukh, A. V.; Vadivasova, T. E.; Anishchenko, V. S.; Zakharova, A.

    2018-01-01

    We investigate spatio-temporal dynamics of a 2D ensemble of nonlocally coupled chaotic cubic maps in a bistability regime. In particular, we perform a detailed study on the transition ;coherence - incoherence; for varying coupling strength for a fixed interaction radius. For the 2D ensemble we show the appearance of amplitude and phase chimera states previously reported for 1D ensembles of nonlocally coupled chaotic systems. Moreover, we uncover a novel type of chimera state, double-well chimera, which occurs due to the interplay of the bistability of the local dynamics and the 2D ensemble structure. Additionally, we find double-well chimera behavior for steady states which we call double-well chimera death. A distinguishing feature of chimera patterns observed in the lattice is that they mainly combine clusters of different chimera types: phase, amplitude and double-well chimeras.

  13. Nef does not contribute to replication differences between R5 pre-AIDS and AIDS HIV-1 clones from patient ACH142

    Directory of Open Access Journals (Sweden)

    Rekosh David

    2008-05-01

    Full Text Available Abstract AIDS-associated, CCR5-tropic (R5 HIV-1 clones, isolated from a patient that never developed CXCR4-tropic HIV-1, replicate to a greater extent and cause greater cytopathic effects than R5 HIV-1 clones isolated before the onset of AIDS. Previously, we showed that HIV-1 Env substantially contributed to the enhanced replication of an AIDS clone. In order to determine if Nef makes a similar contribution, we cloned and phenotypically analyzed nef genes from a series of patient ACH142 derived R5 HIV-1 clones. The AIDS-associated Nef contains a series of residues found in Nef proteins from progressors 1. In contrast to other reports 123, this AIDS-associated Nef downmodulated MHC-I to a greater extent and CD4 less than pre-AIDS Nef proteins. Additionally, all Nef proteins enhanced infectivity similarly in a single round of replication. Combined with our previous study, these data show that evolution of the HIV-1 env gene, but not the nef gene, within patient ACH142 significantly contributed to the enhanced replication and cytopathic effects of the AIDS-associated R5 HIV-1 clone.

  14. Chimera States in Continuous Media: Existence and Distinctness

    Science.gov (United States)

    Nicolaou, Zachary G.; Riecke, Hermann; Motter, Adilson E.

    2017-12-01

    The defining property of chimera states is the coexistence of coherent and incoherent domains in systems that are structurally and spatially homogeneous. The recent realization that such states might be common in oscillator networks raises the question of whether an analogous phenomenon can occur in continuous media. Here, we show that chimera states can exist in continuous systems even when the coupling is strictly local, as in many fluid and pattern forming media. Using the complex Ginzburg-Landau equation as a model system, we characterize chimera states consisting of a coherent domain of a frozen spiral structure and an incoherent domain of amplitude turbulence. We show that in this case, in contrast with discrete network systems, fluctuations in the local coupling field play a crucial role in limiting the coherent regions. We suggest these findings shed light on new possible forms of coexisting order and disorder in fluid systems.

  15. Mechanism of chimera formation during the Multiple Displacement Amplification reaction

    Directory of Open Access Journals (Sweden)

    Stockwell Timothy B

    2007-04-01

    Full Text Available Abstract Background Multiple Displacement Amplification (MDA is a method used for amplifying limiting DNA sources. The high molecular weight amplified DNA is ideal for DNA library construction. While this has enabled genomic sequencing from one or a few cells of unculturable microorganisms, the process is complicated by the tendency of MDA to generate chimeric DNA rearrangements in the amplified DNA. Determining the source of the DNA rearrangements would be an important step towards reducing or eliminating them. Results Here, we characterize the major types of chimeras formed by carrying out an MDA whole genome amplification from a single E. coli cell and sequencing by the 454 Life Sciences method. Analysis of 475 chimeras revealed the predominant reaction mechanisms that create the DNA rearrangements. The highly branched DNA synthesized in MDA can assume many alternative secondary structures. DNA strands extended on an initial template can be displaced becoming available to prime on a second template creating the chimeras. Evidence supports a model in which branch migration can displace 3'-ends freeing them to prime on the new templates. More than 85% of the resulting DNA rearrangements were inverted sequences with intervening deletions that the model predicts. Intramolecular rearrangements were favored, with displaced 3'-ends reannealing to single stranded 5'-strands contained within the same branched DNA molecule. In over 70% of the chimeric junctions, the 3' termini had initiated priming at complimentary sequences of 2–21 nucleotides (nts in the new templates. Conclusion Formation of chimeras is an important limitation to the MDA method, particularly for whole genome sequencing. Identification of the mechanism for chimera formation provides new insight into the MDA reaction and suggests methods to reduce chimeras. The 454 sequencing approach used here will provide a rapid method to assess the utility of reaction modifications.

  16. Chimera States in Two Populations with Heterogeneous Phase-lag

    DEFF Research Database (Denmark)

    Martens, Erik Andreas; Bick, Christian; Panaggio, Mark

    2016-01-01

    The simplest network of coupled phase-oscillators exhibiting chimera states is given by two populations with disparate intra- and inter-population coupling strengths. We explore the effects of heterogeneous coupling phase-lags between the two populations. Such heterogeneity arises naturally......-uniform synchronization, including in-phase and anti-phase synchrony, full incoherence (splay state), chimera states with phase separation of 0 or π between populations, and states where both populations remain desynchronized. These desynchronized states exhibit stable, oscillatory, and even chaotic dynamics. Moreover...

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

    Directory of Open Access Journals (Sweden)

    Bimber Benjamin N

    2009-06-01

    Full Text Available Abstract 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.

  18. Rituximab fails where eculizumab restores renal function in C3nef-related DDD.

    Science.gov (United States)

    Rousset-Rouvière, Caroline; Cailliez, Mathilde; Garaix, Florentine; Bruno, Daniele; Laurent, Daniel; Tsimaratos, Michel

    2014-06-01

    Dense deposit disease (DDD), a C3 glomerulopathy (C3G), is a rare disease with unfavorable progression towards end-stage kidney disease. The pathogenesis of DDD is due to cytotoxic effects related to acquired or genetic dysregulation of the complement alternative pathway, which is at times accompanied by the production of C3 nephritic factor (C3NeF), an auto-antibody directed against the alternative C3 convertase. Available treatments include plasma exchange, CD20-targeted antibodies, and a terminal complement blockade via the anti-C5 monoclonal antibody eculizumab. We report here the case of an 8-year-old child with C3NeF and refractory DDD who presented with a nephritic syndrome. She tested positive for C3NeF activity; C3 was undetectable. Genetic analyses of the alternative complement pathway were normal. Methylprednisolone pulses and mycophenolate mofetil treatment resulted in complete recovery of renal function and a reduction in proteinuria. Corticosteroids were tapered and then withdrawn. Four months after corticosteroid discontinuation, hematuria and proteinuria recurred, and a renal biopsy confirmed an active DDD with a majority of extracapillary crescents. Despite an increase in immunosuppressive drugs, including methylprednisolone pulses and rituximab therapy, the patient suffered acute renal failure within 3 weeks, requiring dialysis. Eculizumab treatment resulted in a quick and impressive response. Hematuria very quickly resolved, kidney function improved, and no further dialysis was required. The patient received bimonthly eculizumab injections of 600 mg, allowing for normalization of renal function and reduction of proteinuria to DDD prior to the development of glomerulosclerosis. Our data provide evidence supporting the pivotal role of complement alternative pathway abnormalities in C3G with DDD.

  19. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes from...... mice immunized with the synthetic peptide coupled to keyhole limpet hemocyanin (KLH). The hybridomas were screened and selected by ELISA with the peptide coupled to bovine serum albumin (BSA) immobilized to the polystyrene surface and specificity for the peptide was confirmed by competitive ELISA...

  20. A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of an Adjuvanted HIV-1 Gag-Pol-Nef Fusion Protein and Adenovirus 35 Gag-RT-Int-Nef Vaccine in Healthy HIV-Uninfected African Adults.

    Directory of Open Access Journals (Sweden)

    Gloria Omosa-Manyonyi

    Full Text Available Sequential prime-boost or co-administration of HIV vaccine candidates based on an adjuvanted clade B p24, RT, Nef, p17 fusion protein (F4/AS01 plus a non-replicating adenovirus 35 expressing clade A Gag, RT, Int and Nef (Ad35-GRIN may lead to a unique immune profile, inducing both strong T-cell and antibody responses.In a phase 1, double-blind, placebo-controlled trial, 146 healthy adult volunteers were randomized to one of four regimens: heterologous prime-boost with two doses of F4/AS01E or F4/AS01B followed by Ad35-GRIN; Ad35-GRIN followed by two doses of F4/AS01B; or three co-administrations of Ad35-GRIN and F4/AS01B. T cell and antibody responses were measured.The vaccines were generally well-tolerated, and did not cause serious adverse events. The response rate, by IFN-γ ELISPOT, was greater when Ad35-GRIN was the priming vaccine and in the co-administration groups. F4/AS01 induced CD4+ T-cells expressing primarily CD40L and IL2 +/- TNF-α, while Ad35-GRIN induced predominantly CD8+ T-cells expressing IFN-γ +/- IL2 or TNF-α. Viral inhibition was induced after Ad35-GRIN vaccination, regardless of the regimen. Strong F4-specific antibody responses were induced. Immune responses persisted at least a year after the last vaccination. The complementary response profiles, characteristic of each vaccine, were both expressed after co-administration.Co-administration of an adjuvanted protein and an adenovirus vector showed an acceptable safety and reactogenicity profile and resulted in strong, multifunctional and complementary HIV-specific immune responses.ClinicalTrials.gov NCT01264445.

  1. Book Review: Evolution's Chimera: Bats and the Marvel of ...

    African Journals Online (AJOL)

    Book Title: Evolution's Chimera: Bats and the Marvel of Evolutionary Adaptation. Book Author: David Jacobs. 2016, University of Cape Town Press, Juta and Company (Pty) Ltd, PO Box 14373, Lansdowne 7779, Cape Town, South Africa Softcover, 204 pages. ISBN 978-1-77582-212-7. Price R299 ...

  2. Use of fragmentation beams at LNS with CHIMERA detector

    Directory of Open Access Journals (Sweden)

    Gianí R.

    2012-07-01

    Full Text Available The recent intensity upgrade of the LNS fragmentation beam is discussed. The available beams, the tagging procedures and details on the beam quality are reported. The experimental program started with the CHIMERA detector using such beams is also discussed with preliminary results and future perspectives.

  3. CHIMERA CBRN protective suit. Advanced embodiment design. Final report

    NARCIS (Netherlands)

    Bogerd, C.P.; Smit, B. de; Olarte, C.; Kane, G.; Bie, M. de; Megen, X. van; Schenk, J.; Hooop, J. de

    2015-01-01

    The Chimera project started of with the following design challenge: Designing a switchable CBRN (chemical, biological, radiological, nuclear) protective suit for soldiers, one phase being a regular work state and the other phase being a protective state to enable the soldier to get away from the

  4. Chimera states in a population of identical oscillators under planar ...

    Indian Academy of Sciences (India)

    2015-01-30

    Jan 30, 2015 ... We report the existence of chimera states in an assembly of identical nonlinear oscillators that are globally linked to each other in a simple planar cross-coupled form. The rotational symmetry breaking of the coupling term appears to be responsible for the emergence of these collective states that display a ...

  5. HIV-1 Infection of T Cells and Macrophages Are Differentially Modulated by Virion-Associated Hck: A Nef-Dependent Phenomenon

    Directory of Open Access Journals (Sweden)

    Gilda Tachedjian

    2013-09-01

    Full Text Available 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.

  6. Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ thymocytes

    Directory of Open Access Journals (Sweden)

    Zou Wei

    2012-05-01

    Full Text Available Abstract Background The outcome of untreated HIV-1 infection is progression to AIDS and death in nearly all cases. Some important exceptions are the small number of patients infected with HIV-1 deleted for the accessory gene, nef. With these infections, disease progression is entirely suppressed or greatly delayed. Whether Nef is critical for high levels of replication or is directly cytotoxic remains controversial. The major problem in determining the role of Nef in HIV/AIDS has been the lack of tractable in vivo models where Nef’s complex pathogenic phenotype can be recapitulated. Results Intravenous inoculation (3000 to 600,000 TCIU of BLT humanized mice with HIV-1LAI reproducibly establishes a systemic infection. HIV-1LAI (LAI replicates to high levels (peak viral load in blood 8,200,000 ± 1,800,000 copies of viral RNA/ml, range 3,600,000 to 20,400,000; n = 9 and exhaustively depletes CD4+ T cells in blood and tissues. CD4+CD8+ thymocytes were also efficiently depleted but CD4+CD8- thymocytes were partially resistant to cell killing by LAI. Infection with a nef-deleted LAI (LAINefdd gave lower peak viral loads (1,220,000 ± 330,000, range 27,000 to 4,240,000; n = 17. For fourteen of seventeen LAINefdd-infected mice, there was little to no loss of either CD4+ T cells or thymocytes. Both LAI- and LAINefdd-infected mice had about 8% of total peripheral blood CD8+ T cells that were CD38+HLA-DR+ compared dd-infected mice that lost CD4+ T cells received 600,000 TCIU. All three exhibited peak viral loads over 3,000,000 copies of LAINefdd RNA/ml. Over an extended time course, substantial systemic CD4+ T cell loss was observed for the three mice, but there was no loss of CD4+CD8+ or CD4+CD8- thymocytes. Conclusion We conclude Nef is necessary for elevated viral replication and as a result indirectly contributes to CD4+ T cell killing. Further, Nef was not necessary for the activation of peripheral blood CD8+ T cells following

  7. Proteomic Analysis Reveals the Leaf Color Regulation Mechanism in Chimera Hosta “Gold Standard” Leaves

    Science.gov (United States)

    Yu, Juanjuan; Zhang, Jinzheng; Zhao, Qi; Liu, Yuelu; Chen, Sixue; Guo, Hongliang; Shi, Lei; Dai, Shaojun

    2016-01-01

    Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta “Gold Standard” is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta “Gold Standard”, as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta “Gold Standard”. For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves. PMID:27005614

  8. Using CHIMERA detector at LNS for gamma-particle coincidences

    Directory of Open Access Journals (Sweden)

    Cardella G.

    2016-01-01

    Full Text Available We have recently evaluated the quality of γ-ray angular distributions that can be extracted in particle-gamma coincidence measurements using the CHIMERA detector at LNS. γ-rays have been detected using the CsI(Tl detectors of the spherical part of the CHIMERA array. Very clean γ-rays angular distributions were extracted in reactions induced by different stable beams impinging on 12C thin targets. The results evidenced an effect of projectile spin flip on the γ-rays angular distributions. γ-particle coincidence measurements were also performed in reactions induced by neutron rich exotic beams produced through in-flight fragmentation at LNS. In recent experiments also the Farcos array was used to improve energy and angular resolution measurements of the detected charged particles. Results obtained with both stable and radioactive beams are reported.

  9. No evidence for selection of HIV-1 with enhanced gag-protease or Nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.

    Directory of Open Access Journals (Sweden)

    Denis R Chopera

    Full Text Available Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial.We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein downregulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis.Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.

  10. Multiclustered chimeras in large semiconductor laser arrays with nonlocal interactions

    Science.gov (United States)

    Shena, J.; Hizanidis, J.; Hövel, P.; Tsironis, G. P.

    2017-09-01

    The dynamics of a large array of coupled semiconductor lasers is studied numerically for a nonlocal coupling scheme. Our focus is on chimera states, a self-organized spatiotemporal pattern of coexisting coherence and incoherence. In laser systems, such states have been previously found for global and nearest-neighbor coupling, mainly in small networks. The technological advantage of large arrays has motivated us to study a system of 200 nonlocally coupled lasers with respect to the emerging collective dynamics. Moreover, the nonlocal nature of the coupling allows us to obtain robust chimera states with multiple (in)coherent domains. The crucial parameters are the coupling strength, the coupling phase and the range of the nonlocal interaction. We find that multiclustered chimera states exist in a wide region of the parameter space and we provide quantitative characterization for the obtained spatiotemporal patterns. By proposing two different experimental setups for the realization of the nonlocal coupling scheme, we are confident that our results can be confirmed in the laboratory.

  11. Insights into the Activity and Substrate Binding of Xylella fastidiosa Polygalacturonase by Modification of a Unique QMK Amino Acid Motif Using Protein Chimeras.

    Science.gov (United States)

    Warren, Jeremy G; Lincoln, James E; Kirkpatrick, Bruce C

    2015-01-01

    Polygalacturonases (EC 3.2.1.15) catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa possesses a single polygalacturonase gene, pglA (PD1485), and X. fastidiosa mutants deficient in the production of polygalacturonase are non-pathogenic and show a compromised ability to systemically infect grapevines. These results suggested that grapevines expressing sufficient amounts of an inhibitor of X. fastidiosa polygalacturonase might be protected from disease. Previous work in our laboratory and others have tried without success to produce soluble active X. fastidiosa polygalacturonase for use in inhibition assays. In this study, we created two enzymatically active X. fastidiosa / A. vitis polygalacturonase chimeras, AX1A and AX2A to explore the functionality of X. fastidiosa polygalacturonase in vitro. The AX1A chimera was constructed to specifically test if recombinant chimeric protein, produced in Escherichia coli, is soluble and if the X. fastidiosa polygalacturonase catalytic amino acids are able to hydrolyze polygalacturonic acid. The AX2A chimera was constructed to evaluate the ability of a unique QMK motif of X. fastidiosa polygalacturonase, most polygalacturonases have a R(I/L)K motif, to bind to and allow the hydrolysis of polygalacturonic acid. Furthermore, the AX2A chimera was also used to explore what effect modification of the QMK motif of X. fastidiosa polygalacturonase to a conserved RIK motif has on enzymatic activity. These experiments showed that both the AX1A and AX2A polygalacturonase chimeras were soluble and able to hydrolyze the polygalacturonic acid substrate. Additionally, the modification of the QMK motif to the conserved RIK motif eliminated hydrolytic activity, suggesting that the QMK motif is important for the activity of X. fastidiosa polygalacturonase. This result suggests X. fastidiosa polygalacturonase may preferentially hydrolyze a different pectic substrate or

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

    Science.gov (United States)

    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.

  13. Characteristic distribution of finite-time Lyapunov exponents for chimera states

    Science.gov (United States)

    Botha, André E.

    2016-01-01

    Our fascination with chimera states stems partially from the somewhat paradoxical, yet fundamental trait of identical, and identically coupled, oscillators to split into spatially separated, coherently and incoherently oscillating groups. While the list of systems for which various types of chimeras have already been detected continues to grow, there is a corresponding increase in the number of mathematical analyses aimed at elucidating the fundamental reasons for this surprising behaviour. Based on the model systems, there are strong indications that chimera states may generally be ubiquitous in naturally occurring systems containing large numbers of coupled oscillators – certain biological systems and high-Tc superconducting materials, for example. In this work we suggest a new way of detecting and characterising chimera states. Specifically, it is shown that the probability densities of finite-time Lyapunov exponents, corresponding to chimera states, have a definite characteristic shape. Such distributions could be used as signatures of chimera states, particularly in systems for which the phases of all the oscillators cannot be measured directly. For such cases, we suggest that chimera states could perhaps be detected by reconstructing the characteristic distribution via standard embedding techniques, thus making it possible to detect chimera states in systems where they could otherwise exist unnoticed. PMID:27374473

  14. Virtual chimera states for delayed-feedback systems.

    Science.gov (United States)

    Larger, Laurent; Penkovsky, Bogdan; Maistrenko, Yuri

    2013-08-02

    Time-delayed systems are found to display remarkable temporal patterns the dynamics of which split into regular and chaotic components repeating at the interval of a delay. This novel long-term behavior for delay dynamics results from strongly asymmetric nonlinear delayed feedback driving a highly damped harmonic oscillator dynamics. In the corresponding virtual space-time representation, the behavior is found to develop as a chimeralike state, a new paradigmatic object from the network theory characterized by the coexistence of synchronous and incoherent oscillations. Numerous virtual chimera states are obtained and analyzed, through experiment, theory, and simulations.

  15. Ethical aspects of creating human-nonhuman chimeras capable of human gamete production and human pregnancy.

    Science.gov (United States)

    Palacios-González, César

    2015-01-01

    In this paper I explore some of the moral issues that could emerge from the creation of human-nonhuman chimeras (HNH-chimeras) capable of human gamete production and human pregnancy. First I explore whether there is a cogent argument against the creation of HNH-chimeras that could produce human gametes. I conclude that so far there is none, and that in fact there is at least one good moral reason for producing such types of creatures. Afterwards I explore some of the moral problems that could emerge from the fact that a HNH-chimera could become pregnant with a human conceptus. I focus on two sets of problems: problems that would arise by virtue of the fact that a human is gestated by a nonhuman creature, and problems that would emerge from the fact that such pregnancies could affect the health of the HNH-chimera.

  16. Targeted chimera delivery to ovarian cancer cells by heterogeneous gold magnetic nanoparticle

    Science.gov (United States)

    Chen, Yao; Xu, Mengjiao; Guo, Yi; Tu, Keyao; Wu, Weimin; Wang, Jianjun; Tong, Xiaowen; Wu, Wenjuan; Qi, Lifeng; Shi, Donglu

    2017-01-01

    Efficient delivery of small interfering RNAs (siRNAs) to the targeted cells has remained a significant challenge in clinical applications. In the present study, we developed a novel aptamer-siRNA chimera delivery system mediated by cationic Au-Fe3O4 nanoparticles (NPs). The chimera constructed by VEGF RNA aptamer and Notch3 siRNA was bonded with heterogeneous Au-Fe3O4 nanoparticles by electrostatic interaction. The obtained complex exhibited much higher silencing efficiency against Notch3 gene compared with chimera alone and lipofectamine-siRNA complex, and improved the antitumor effects of the loaded chimera. Moreover, the efficient delivery of the chimera by Au-Fe3O4 NPs could reverse multi-drug resistance (MDR) of ovarian cancer cells against the chemotherapeutic drug cisplatin, indicating its potential capability for future targeted cancer therapy while overcoming MDR.

  17. Experimental observation of chimera and cluster states in a minimal globally coupled network

    Energy Technology Data Exchange (ETDEWEB)

    Hart, Joseph D. [Institute for Research in Electronics and Applied Physics, University of Maryland, College Park, Maryland 20742 (United States); Department of Physics, University of Maryland, College Park, Maryland 20742 (United States); Bansal, Kanika [Department of Mathematics, University at Buffalo, SUNY Buffalo, New York 14260 (United States); US Army Research Laboratory, Aberdeen Proving Ground, Maryland 21005 (United States); Murphy, Thomas E. [Institute for Research in Electronics and Applied Physics, University of Maryland, College Park, Maryland 20742 (United States); Department of Electrical and Computer Engineering, University of Maryland, College Park, Maryland 20742 (United States); Roy, Rajarshi [Institute for Research in Electronics and Applied Physics, University of Maryland, College Park, Maryland 20742 (United States); Department of Physics, University of Maryland, College Park, Maryland 20742 (United States); Institute for Physical Science and Technology, University of Maryland, College Park, Maryland 20742 (United States)

    2016-09-15

    A “chimera state” is a dynamical pattern that occurs in a network of coupled identical oscillators when the symmetry of the oscillator population is broken into synchronous and asynchronous parts. We report the experimental observation of chimera and cluster states in a network of four globally coupled chaotic opto-electronic oscillators. This is the minimal network that can support chimera states, and our study provides new insight into the fundamental mechanisms underlying their formation. We use a unified approach to determine the stability of all the observed partially synchronous patterns, highlighting the close relationship between chimera and cluster states as belonging to the broader phenomenon of partial synchronization. Our approach is general in terms of network size and connectivity. We also find that chimera states often appear in regions of multistability between global, cluster, and desynchronized states.

  18. Laser chimeras as a paradigm for multistable patterns in complex systems.

    Science.gov (United States)

    Larger, Laurent; Penkovsky, Bogdan; Maistrenko, Yuri

    2015-07-14

    A chimera state is a rich and fascinating class of self-organized solutions developed in high-dimensional networks. Necessary features of the network for the emergence of such complex but structured motions are non-local and symmetry breaking coupling. An accurate understanding of chimera states is expected to bring important insights on deterministic mechanism occurring in many structurally similar high-dimensional dynamics such as living systems, brain operation principles and even turbulence in hydrodynamics. Here we report on a powerful and highly controllable experiment based on an optoelectronic delayed feedback applied to a wavelength tuneable semiconductor laser, with which a wide variety of chimera patterns can be accurately investigated and interpreted. We uncover a cascade of higher-order chimeras as a pattern transition from N to N+1 clusters of chaoticity. Finally, we follow visually, as the gain increases, how chimera state is gradually destroyed on the way to apparent turbulence-like system behaviour.

  19. Chimera states in a Hodgkin-Huxley model of thermally sensitive neurons

    Science.gov (United States)

    Glaze, Tera A.; Lewis, Scott; Bahar, Sonya

    2016-08-01

    Chimera states occur when identically coupled groups of nonlinear oscillators exhibit radically different dynamics, with one group exhibiting synchronized oscillations and the other desynchronized behavior. This dynamical phenomenon has recently been studied in computational models and demonstrated experimentally in mechanical, optical, and chemical systems. The theoretical basis of these states is currently under active investigation. Chimera behavior is of particular relevance in the context of neural synchronization, given the phenomenon of unihemispheric sleep and the recent observation of asymmetric sleep in human patients with sleep apnea. The similarity of neural chimera states to neural "bump" states, which have been suggested as a model for working memory and visual orientation tuning in the cortex, adds to their interest as objects of study. Chimera states have been demonstrated in the FitzHugh-Nagumo model of excitable cells and in the Hindmarsh-Rose neural model. Here, we demonstrate chimera states and chimera-like behaviors in a Hodgkin-Huxley-type model of thermally sensitive neurons both in a system with Abrams-Strogatz (mean field) coupling and in a system with Kuramoto (distance-dependent) coupling. We map the regions of parameter space for which chimera behavior occurs in each of the two coupling schemes.

  20. MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

    Directory of Open Access Journals (Sweden)

    STANCA CLAUDIA

    2007-01-01

    Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

  1. Functional characterization of two prokaryotic pentameric ligand-gated ion channel chimeras - role of the GLIC transmembrane domain in proton sensing.

    Science.gov (United States)

    Hénault, Camille M; Baenziger, John E

    2017-02-01

    With the long-term goal of using a chimeric approach to dissect the distinct lipid sensitivities and thermal stabilities of the pentameric ligand-gated ion channels (pLGIC), GLIC and ELIC, we constructed chimeras by cross-combining their extracellular (ECD) and transmembrane (TMD) domains. As expected, the chimera formed between GLIC-ECD and ELIC-TMD (GE) responded to protons, the agonist for GLIC, but not cysteamine, the agonist for ELIC, although GE exhibited a 25-fold decrease in proton-sensitivity relative to wild type. The chimera formed between ELIC-ECD and the GLIC-TMD (EG) was usually toxic, unless it contained a pore-lining Ile9'Ala gain-of-function mutation. No significant improvements in expression/toxicity were observed with extensive loop substitutions at the ECD/TMD interface. Surprisingly, oocytes expressing EG-I9'A responded to both the ELIC agonist, cysteamine and the GLIC agonist, protons - the latter at pH values ≤4.0. The cysteamine- and proton-induced currents in EG-I9'A were inhibited by the GLIC TMD pore blocker, amantadine. The cysteamine-induced response of EG-I9'A was also inhibited by protons at pH values down to 4.5, but potentiated at lower pH values. Proton-induced gating at low pH was not abolished by mutation of an intramembrane histidine residue previously implicated in GLIC TMD function. We show that the TMD plays a major role governing the thermal stability of a pLGIC, and identify three distinct mechanisms by which agonists and protons influence the gating of the EG chimera. A structural basis for the impaired function of GE is suggested. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Chimeras of Bet v 1 and Api g 1 reveal heterogeneous IgE responses in patients with birch pollen allergy.

    Science.gov (United States)

    Gepp, Barbara; Lengger, Nina; Bublin, Merima; Hemmer, Wolfgang; Breiteneder, Heimo; Radauer, Christian

    2014-07-01

    Characterization of IgE-binding epitopes of allergens and determination of their patient-specific relevance is crucial for the diagnosis and treatment of allergy. We sought to assess the contribution of specific surface areas of the major birch pollen allergen Bet v 1.0101 to binding IgE of individual patients. Four distinct areas of Bet v 1 representing in total 81% of its surface were grafted onto the scaffold of its homolog, Api g 1.0101, to yield the chimeras Api-Bet-1 to Api-Bet-4. The chimeras were expressed in Escherichia coli and purified. IgE binding of 64 sera from Bet v 1-sensitized subjects with birch pollen allergy was determined by using direct ELISA. Specificity was assessed by means of inhibition ELISA. rApi g 1.0101, Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4 bound IgE from 44%, 89%, 80%, 78%, and 48% of the patients, respectively. By comparing the amount of IgE binding to the chimeras and to rApi g 1.0101, 81%, 70%, 75%, and 45% of the patients showed significantly enhanced IgE binding to Api-Bet-1, Api-Bet-2, Api-Bet-3, and Api-Bet-4, respectively. The minority (8%) of the sera revealed enhanced IgE binding exclusively to a single chimera, whereas 31% showed increased IgE binding to all 4 chimeras compared with rApi g 1.0101. The chimeras inhibited up to 70% of IgE binding to rBet v 1.0101, confirming the specific IgE recognition of the grafted regions. The Bet v 1-specific IgE response is polyclonal, and epitopes are spread across the entire Bet v 1 surface. Furthermore, the IgE recognition profile of Bet v 1 is highly patient specific. Copyright © 2014 The Authors. Published by Mosby, Inc. All rights reserved.

  3. Robust Weak Chimeras in Oscillator Networks with Delayed Linear and Quadratic Interactions

    Science.gov (United States)

    Bick, Christian; Sebek, Michael; Kiss, István Z.

    2017-10-01

    We present an approach to generate chimera dynamics (localized frequency synchrony) in oscillator networks with two populations of (at least) two elements using a general method based on a delayed interaction with linear and quadratic terms. The coupling design yields robust chimeras through a phase-model-based design of the delay and the ratio of linear and quadratic components of the interactions. We demonstrate the method in the Brusselator model and experiments with electrochemical oscillators. The technique opens the way to directly bridge chimera dynamics in phase models and real-world oscillator networks.

  4. Phenotype and envelope gene diversity of nef-deleted HIV-1 isolated from long-term survivors infected from a single source

    Directory of Open Access Journals (Sweden)

    Sullivan John S

    2007-07-01

    Full Text Available Abstract Background The Sydney blood bank cohort (SBBC of long-term survivors consists of multiple individuals infected with attenuated, nef-deleted variants of human immunodeficiency virus type 1 (HIV-1 acquired from a single source. Long-term prospective studies have demonstrated that the SBBC now comprises slow progressors (SP as well as long-term nonprogressors (LTNP. Convergent evolution of nef sequences in SBBC SP and LTNP indicates the in vivo pathogenicity of HIV-1 in SBBC members is dictated by factors other than nef. To better understand mechanisms underlying the pathogenicity of nef-deleted HIV-1, we examined the phenotype and env sequence diversity of sequentially isolated viruses (n = 2 from 3 SBBC members. Results The viruses characterized here were isolated from two SP spanning a three or six year period during progressive HIV-1 infection (subjects D36 and C98, respectively and from a LTNP spanning a two year period during asymptomatic, nonprogressive infection (subject C18. Both isolates from D36 were R5X4 phenotype and, compared to control HIV-1 strains, replicated to low levels in peripheral blood mononuclear cells (PBMC. In contrast, both isolates from C98 and C18 were CCR5-restricted. Both viruses isolated from C98 replicated to barely detectable levels in PBMC, whereas both viruses isolated from C18 replicated to low levels, similar to those isolated from D36. Analysis of env by V1V2 and V3 heteroduplex tracking assay, V1V2 length polymorphisms, sequencing and phylogenetic analysis showed distinct intra- and inter-patient env evolution. Conclusion Independent evolution of env despite convergent evolution of nef may contribute to the in vivo pathogenicity of nef-deleted HIV-1 in SBBC members, which may not necessarily be associated with changes in replication capacity or viral coreceptor specificity.

  5. A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer.

    Directory of Open Access Journals (Sweden)

    Mohammad Razuanul Hoque

    Full Text Available With the progress of optogenetics, the activities of genetically identified neurons can be optically silenced to determine whether the neurons in question are necessary for the network performance of the behavioral expression. This logical induction is expected to be improved by the application of the Na+ pump rhodopsins (NaRs, which hyperpolarize the membrane potential with negligible influence on the ionic/pH balance. Here, we made several chimeric NaRs between two NaRs, KR2 and IaNaR from Krokinobacter eikastus and Indibacter alkaliphilus, respectively. We found that one of these chimeras, named I1K6NaR, exhibited some improvements in the membrane targeting and photocurrent properties over native NaRs. The I1K6NaR-expressing cortical neurons were stably silenced by green light irradiation for a certain long duration. With its rapid kinetics and voltage dependency, the photoactivation of I1K6NaR would specifically counteract the generation of action potentials with less hyperpolarization of the neuronal membrane potential than KR2.

  6. A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer.

    Science.gov (United States)

    Hoque, Mohammad Razuanul; Ishizuka, Toru; Inoue, Keiichi; Abe-Yoshizumi, Rei; Igarashi, Hiroyuki; Mishima, Takaaki; Kandori, Hideki; Yawo, Hiromu

    2016-01-01

    With the progress of optogenetics, the activities of genetically identified neurons can be optically silenced to determine whether the neurons in question are necessary for the network performance of the behavioral expression. This logical induction is expected to be improved by the application of the Na+ pump rhodopsins (NaRs), which hyperpolarize the membrane potential with negligible influence on the ionic/pH balance. Here, we made several chimeric NaRs between two NaRs, KR2 and IaNaR from Krokinobacter eikastus and Indibacter alkaliphilus, respectively. We found that one of these chimeras, named I1K6NaR, exhibited some improvements in the membrane targeting and photocurrent properties over native NaRs. The I1K6NaR-expressing cortical neurons were stably silenced by green light irradiation for a certain long duration. With its rapid kinetics and voltage dependency, the photoactivation of I1K6NaR would specifically counteract the generation of action potentials with less hyperpolarization of the neuronal membrane potential than KR2.

  7. Greengenes: Chimera-checked 16S rRNA gene database and workbenchcompatible in ARB

    Energy Technology Data Exchange (ETDEWEB)

    DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie,E.L; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L.

    2006-02-01

    A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera-screening, standard alignments and taxonomic classification using multiple published taxonomies. It was revealed that incongruent taxonomic nomenclature exists among curators even at the phylum-level. Putative chimeras were identified in 3% of environmental sequences and 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages within the Archaea and Bacteria.

  8. Neuronal nitric-oxide synthase interaction with calmodulin-troponin C chimeras.

    Science.gov (United States)

    Gachhui, R; Abu-Soud, H M; Ghosha, D K; Presta, A; Blazing, M A; Mayer, B; George, S E; Stuehr, D J

    1998-03-06

    Calmodulin (CaM) binding activates neuronal nitric-oxide synthase (nNOS) catalytic functions and also up-regulates electron transfer into its flavin and heme centers. Here, we utilized seven tight binding CaM-troponin C chimeras, which variably activate nNOS NO synthesis to examine the relationship between CaM domain structure, activation of catalytic functions, and control of internal electron transfer at two points within nNOS. Chimeras that were singly substituted with troponin C domains 4, 3, 2, or 1 were increasingly unable to activate NO synthesis, but all caused some activation of cytochrome c reduction compared with CaM-free nNOS. The magnitude by which each chimera activated NO synthesis was approximately proportional to the rate of heme iron reduction supported by each chimera, which varied from 0% to approximately 80% compared with native CaM and remained coupled to NO synthesis in all cases. In contrast, chimera activation of cytochrome c reduction was not always associated with accelerated reduction of nNOS flavins, and certain chimeras activated cytochrome c reduction without triggering heme iron reduction. We conclude: 1) CaM effects on electron transfer at two points within nNOS can be functionally separated. 2) CaM controls NO synthesis by governing heme iron reduction, but enhances reductase activity by two mechanisms, only one of which is associated with an increased rate of flavin reduction.

  9. UCSF Chimera--a visualization system for exploratory research and analysis.

    Science.gov (United States)

    Pettersen, Eric F; Goddard, Thomas D; Huang, Conrad C; Couch, Gregory S; Greenblatt, Daniel M; Meng, Elaine C; Ferrin, Thomas E

    2004-10-01

    The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/. Copyright 2004 Wiley Periodicals, Inc.

  10. CHIMERAS WITH MOSAIC PATTERN IN ARCHEOSPORE GERMLINGS OF PYROPIA YEZOENSIS UEDA (BANGIALES, RHODOPHYTA)(1).

    Science.gov (United States)

    Niwa, Kyosuke; Abe, Tomoko

    2012-06-01

    In the marine crop Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi, it is known that conchospores from heterozygous conchocelis develop into sectored gametophytic blades (chimeras), but archeospores asexually released from haploid blades do not usually grow into chimeric blades. In this study, chimeras with mosaic pattern consisting of the green and wildtype colors were developed from archeospores that were released from a blade piece containing a cell cluster of green color induced by heavy-ion beam irradiation. To make clear whether these archeospores were produced from the green-colored cells or the wildtype-colored cells, cell clusters of the green mutant, wildtype, and mosaic pattern were cut out from the grown chimera, and archeospores were released from each of the three blade pieces. Archeospores from the green-mutant blade piece and from the wildtype blade piece developed into only green-mutant blades and wildtype blades, respectively. In contrast, archeospores from the blade piece with mosaic pattern developed into green-mutant blades, wildtype blades, and chimeric blades with mosaic pattern of the two colors, although the frequency of the chimeras was low. Because each gametophytic cell possesses a single plastid, it is difficult to explain the occurrence of the new chimeras as a mutation of the plastid DNA. Thus, the new chimeras are considered to be due to transposable elements in Pyropia. © 2012 Phycological Society of America.

  11. Human Leukocyte Antigen (HLA Class I Down-Regulation by Human Immunodeficiency Virus Type 1 Negative Factor (HIV-1 Nef: What Might We Learn From Natural Sequence Variants?

    Directory of Open Access Journals (Sweden)

    Philip Mwimanzi

    2012-09-01

    Full Text Available HIV-1 causes a chronic infection in humans that is characterized by high plasma viremia, progressive loss of CD4+ T lymphocytes, and severe immunodeficiency resulting in opportunistic disease and AIDS. Viral persistence is mediated in part by the ability of the Nef protein to down-regulate HLA molecules on the infected cell surface, thereby allowing HIV-1 to evade recognition by antiviral CD8+ T lymphocytes. Extensive research has been conducted on Nef to determine protein domains that are required for its immune evasion activities and to identify critical cellular co-factors, and our mechanistic understanding of this process is becoming more complete. This review highlights our current knowledge of Nef-mediated HLA class I down-regulation and places this work in the context of naturally occurring sequence variation in this protein. We argue that efforts to fully understand the critical role of Nef for HIV-1 pathogenesis will require greater analysis of patient-derived sequences to elucidate subtle differences in immune evasion activity that may alter clinical outcome.

  12. Persistent Low-Level Replication of SIVΔnef Drives Maturation of Antibody and CD8 T Cell Responses to Induce Protective Immunity against Vaginal SIV Infection.

    Directory of Open Access Journals (Sweden)

    Sama Adnan

    2016-12-01

    Full Text Available Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination.

  13. Present status of the Chimera-Isospin experiment

    Energy Technology Data Exchange (ETDEWEB)

    Politi, G.; Arena, N.; Cardella, G.; DeFilippo, E.; Lanzano, G.; Nigro, S.L.; Pagano, A.; Papa, M.; Pirrone, S.; Russotto, P. [Catania Univ., INFN (Italy); Alderighi, M.; Sechi, G.; Sperduto, M.L. [Milano Univ., INFN, CNR (Italy); Amorini, F.; Anzalone, A.; Baran, V.; Bonasera, A.; Cavallaro, S.L.; Colonna, M.; Di Toro, M.; LaGuidara, E.; Lanzalone, G.; IaconoManno, M.; Giustolisi, F.; Maiolino, C.; Porto, F.; Rizzo, F.; Trifiro, A.; Trimarchi, M. [Catania Univ., INFN, Lab. Nazionali del Sud (Italy); Auditore, L.; Barna, R.; DePasquale, D. [Messina Univ., INFN (Italy); Berceanu, I.; Petrovici, M.; Pop, A. [Inst. for Physics and Nuclear Engineering, Bucharest (Romania); Blicharska, J.; Grzeszczuk, A.; Kowalski, S.; Zipper, W. [Univ. of Silesia, Inst. of Physics, Katowice (Poland); Borderie, B.; LeNeindre, N.; Rivet, M.F. [Paris-11 Univ., IPN-IN2P3-CNRS, 91 - Orsay (France); Bougault, R. [Caen Univ., LPC-ISMRA (France); Briczycnski, J.; Gawlikowicz, W.; Majka, Z.; Planeta, R. [M. Smoluchowski Inst. of Physics, Jagellonian Univ., Cracow (Poland); Bruno, M.; D' Agostino, M.; Fuschini, E.; Geraci, E.; Vannini, G. [Bologna Univ., INFN (Italy); Chatterjee, M.B. [Saha Inst. of Nuclear Physics, NIS Div., Kolkata (India); Chbihi, A.; Wieleczko, J.P. [GANIL -CEA-IN2P3-CNRS, 14 - Caen (France); Cibor, J. [H.Niewodniczanski Inst. of Nuclear Physics, Cracow (Poland); Dayras, R. [CEA Saclay, Dept. d' Astrophysique, de Physique des Particules, de Physique Nucleaire et de l' Instrumentation Associee, SPhN, 91- Gif sur Yvette (France); Guazzoni, P.; Russo, S.; Sassi, M.; Zetta, L. [Milano Univ., INFN (Italy); Guinet, D. [Univ. Claude Bernard, IPN-IN2P3-CNRS, 69 - Lyon (France); Li, S.; Wu, H.; Xiao, Z. [Inst. of Modern Physics, Lanzhou (China); Nicolis, N.G. [Ioannina Univ., Dept. of Physics (Greece); Piasecki, E.; Swiderski, L.; Siwek-Wilczynska, K.; Skwira, I. [Warsaw Univ., Inst. for Experimental Physics (Poland); Rosato, E.; Vigilante, M.; Wilczynski, J.

    2003-07-01

    The CHIMERA detector was designed to significantly contribute to multifragmentation studies in the field of heavy ion collisions at intermediate energies. The device has been used at 'Laboratori Nazionali del Sud' (LNS) in Catania (Italy) to study different aspects of the relevant nuclear reaction mechanism, in two different campaigns: the first one in 2000, by using the forward part (1 - 30 degrees) of the device, and the second one in 2003, by using the 4{pi} geometry. The experimental results have confirmed the capability of the apparatus for good isotopic identification of light charged particles and light fragments (3

  14. Kinetic characterization of the interaction of biotinylated human interleukin 5 with an Fc chimera of its receptor alpha subunit and development of an ELISA screening assay using real-time interaction biosensor analysis.

    Science.gov (United States)

    Bennett, D; Morton, T; Breen, A; Hertzberg, R; Cusimano, D; Appelbaum, E; McDonnell, P; Young, P; Matico, R; Chaiken, I

    1995-01-01

    The interaction of biotinylated human interleukin 5 ([BT]hIL5) with immobilized receptor was measured with a real-time biosensor, and these results were used as a basis for configuring an ELISA for screening antagonists of hIL5-receptor binding. The recombinant proteins used, hIL5 and shIL5R alpha-Fc (chimeric fusion receptor constructed by linking the soluble component of the hIL5 receptor alpha subunit to the constant domain (Fc) of immunoglobulin G), were produced by the expression of cloned vectors in Drosophila schneider (S2) cells. Initial attempts to develop a screening assay by direct immobilization of soluble IL5 receptor to microtiter plates proved unsatisfactory and led to use of the Fc chimera attached by oriented immobilization via protein A. Hence, shIL5R alpha-Fc was bound to protein A covalently immobilized on a carboxymethyl dextran (CM-5) biosensor chip. Specific binding was demonstrated of [BT]hIL5 to protein A/shIL5R alpha-Fc receptor complex. The binding was high affinity (Kdapp = 6 nM), reversible and saturable. The affinity of [BT]hIL5 was similar to that determined with the biosensor assay for unmodified hIL5. The observed kinetics of the interactions of Fc chimera with protein A (slow dissociation) and of [BT]hIL5 with immobilized Fc chimera (faster dissociation) were favorable for subsequently establishing a microtiter plate based ELISA assay. In the latter, Fc chimera was immobilized to the plate via protein A as in the biosensor experiment. Binding of [BT]hIL5 to immobilized Fc chimera in the ELISA was concentration dependent and was competed by both hIL5 and shIL5R alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Efficient Agrobacterium-based transient expression system for the production of biopharmaceuticals in plants.

    Science.gov (United States)

    Circelli, Patrizia; Donini, Marcello; Villani, Maria Elena; Benvenuto, Eugenio; Marusic, Carla

    2010-01-01

    We have recently described an efficient transient expression system mediated by Agrobacterium tumefaciens for the production of HIV-1 Nef protein in Nicotiana benthamiana plants. In order to enhance the yield of recombinant protein we assayed the effect of three gene-silencing viral suppressor proteins (P25 of Potato Virus X, P19 of Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef expression levels. Results demonstrated that AMCV-P19 gave the highest Nef yield (1.3% of total soluble protein) and that this effect was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms. Here we report additional data on the production of different heterologous proteins including human immunoglobulin heavy and light chains and a virus coat protein that demonstrate the robustness of this co-agroinfiltration expression system boosted by the AMCV-P19 gene-silencing suppressor. © 2010 Landes Bioscience

  16. A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras.

    Science.gov (United States)

    Maekawa, Fumihiko; Sakurai, Miyano; Yamashita, Yuki; Tanaka, Kohichi; Haraguchi, Shogo; Yamamoto, Kazutoshi; Tsutsui, Kazuyoshi; Yoshioka, Hidefumi; Murakami, Shizuko; Tadano, Ryo; Goto, Tatsuhiko; Shiraishi, Jun-ichi; Tomonari, Kohei; Oka, Takao; Ohara, Ken; Maeda, Teruo; Bungo, Takashi; Tsudzuki, Masaoki; Ohki-Hamazaki, Hiroko

    2013-01-01

    Sexual differentiation leads to structural and behavioural differences between males and females. Here we investigate the intrinsic sex identity of the brain by constructing chicken chimeras in which the brain primordium is switched between male and female identities before gonadal development. We find that the female chimeras with male brains display delayed sexual maturation and irregular oviposition cycles, although their behaviour, plasma concentrations of sex steroids and luteinizing hormone levels are normal. The male chimeras with female brains show phenotypes similar to typical cocks. In the perinatal period, oestrogen concentrations in the genetically male brain are higher than those in the genetically female brain. Our study demonstrates that male brain cells retain male sex identity and do not differentiate into female cells to drive the normal oestrous cycle, even when situated in the female hormonal milieu. This is clear evidence for a sex-specific feature that develops independent of gonadal steroids.

  17. Chimera states in a multilayer network of coupled and uncoupled neurons

    Science.gov (United States)

    Majhi, Soumen; Perc, Matjaž; Ghosh, Dibakar

    2017-07-01

    We study the emergence of chimera states in a multilayer neuronal network, where one layer is composed of coupled and the other layer of uncoupled neurons. Through the multilayer structure, the layer with coupled neurons acts as the medium by means of which neurons in the uncoupled layer share information in spite of the absence of physical connections among them. Neurons in the coupled layer are connected with electrical synapses, while across the two layers, neurons are connected through chemical synapses. In both layers, the dynamics of each neuron is described by the Hindmarsh-Rose square wave bursting dynamics. We show that the presence of two different types of connecting synapses within and between the two layers, together with the multilayer network structure, plays a key role in the emergence of between-layer synchronous chimera states and patterns of synchronous clusters. In particular, we find that these chimera states can emerge in the coupled layer regardless of the range of electrical synapses. Even in all-to-all and nearest-neighbor coupling within the coupled layer, we observe qualitatively identical between-layer chimera states. Moreover, we show that the role of information transmission delay between the two layers must not be neglected, and we obtain precise parameter bounds at which chimera states can be observed. The expansion of the chimera region and annihilation of cluster and fully coherent states in the parameter plane for increasing values of inter-layer chemical synaptic time delay are illustrated using effective range measurements. These results are discussed in the light of neuronal evolution, where the coexistence of coherent and incoherent dynamics during the developmental stage is particularly likely.

  18. Glassy Chimeras Could Be Blind to Quantum Speedup: Designing Better Benchmarks for Quantum Annealing Machines

    Science.gov (United States)

    Katzgraber, Helmut G.; Hamze, Firas; Andrist, Ruben S.

    2014-04-01

    Recently, a programmable quantum annealing machine has been built that minimizes the cost function of hard optimization problems by, in principle, adiabatically quenching quantum fluctuations. Tests performed by different research teams have shown that, indeed, the machine seems to exploit quantum effects. However, experiments on a class of random-bond instances have not yet demonstrated an advantage over classical optimization algorithms on traditional computer hardware. Here, we present evidence as to why this might be the case. These engineered quantum annealing machines effectively operate coupled to a decohering thermal bath. Therefore, we study the finite-temperature critical behavior of the standard benchmark problem used to assess the computational capabilities of these complex machines. We simulate both random-bond Ising models and spin glasses with bimodal and Gaussian disorder on the D-Wave Chimera topology. Our results show that while the worst-case complexity of finding a ground state of an Ising spin glass on the Chimera graph is not polynomial, the finite-temperature phase space is likely rather simple because spin glasses on Chimera have only a zero-temperature transition. This means that benchmarking optimization methods using spin glasses on the Chimera graph might not be the best benchmark problems to test quantum speedup. We propose alternative benchmarks by embedding potentially harder problems on the Chimera topology. Finally, we also study the (reentrant) disorder-temperature phase diagram of the random-bond Ising model on the Chimera graph and show that a finite-temperature ferromagnetic phase is stable up to 19.85(15)% antiferromagnetic bonds. Beyond this threshold, the system only displays a zero-temperature spin-glass phase. Our results therefore show that a careful design of the hardware architecture and benchmark problems is key when building quantum annealing machines.

  19. Glassy Chimeras Could Be Blind to Quantum Speedup: Designing Better Benchmarks for Quantum Annealing Machines

    Directory of Open Access Journals (Sweden)

    Helmut G. Katzgraber

    2014-04-01

    Full Text Available Recently, a programmable quantum annealing machine has been built that minimizes the cost function of hard optimization problems by, in principle, adiabatically quenching quantum fluctuations. Tests performed by different research teams have shown that, indeed, the machine seems to exploit quantum effects. However, experiments on a class of random-bond instances have not yet demonstrated an advantage over classical optimization algorithms on traditional computer hardware. Here, we present evidence as to why this might be the case. These engineered quantum annealing machines effectively operate coupled to a decohering thermal bath. Therefore, we study the finite-temperature critical behavior of the standard benchmark problem used to assess the computational capabilities of these complex machines. We simulate both random-bond Ising models and spin glasses with bimodal and Gaussian disorder on the D-Wave Chimera topology. Our results show that while the worst-case complexity of finding a ground state of an Ising spin glass on the Chimera graph is not polynomial, the finite-temperature phase space is likely rather simple because spin glasses on Chimera have only a zero-temperature transition. This means that benchmarking optimization methods using spin glasses on the Chimera graph might not be the best benchmark problems to test quantum speedup. We propose alternative benchmarks by embedding potentially harder problems on the Chimera topology. Finally, we also study the (reentrant disorder-temperature phase diagram of the random-bond Ising model on the Chimera graph and show that a finite-temperature ferromagnetic phase is stable up to 19.85(15% antiferromagnetic bonds. Beyond this threshold, the system only displays a zero-temperature spin-glass phase. Our results therefore show that a careful design of the hardware architecture and benchmark problems is key when building quantum annealing machines.

  20. Chronic Exposure to Androgenic-Anabolic Steroids Exacerbates Axonal Injury and Microgliosis in the CHIMERA Mouse Model of Repetitive Concussion.

    Science.gov (United States)

    Namjoshi, Dhananjay R; Cheng, Wai Hang; Carr, Michael; Martens, Kris M; Zareyan, Shahab; Wilkinson, Anna; McInnes, Kurt A; Cripton, Peter A; Wellington, Cheryl L

    2016-01-01

    Concussion is a serious health concern. Concussion in athletes is of particular interest with respect to the relationship of concussion exposure to risk of chronic traumatic encephalopathy (CTE), a neurodegenerative condition associated with altered cognitive and psychiatric functions and profound tauopathy. However, much remains to be learned about factors other than cumulative exposure that could influence concussion pathogenesis. Approximately 20% of CTE cases report a history of substance use including androgenic-anabolic steroids (AAS). How acute, chronic, or historical AAS use may affect the vulnerability of the brain to concussion is unknown. We therefore tested whether antecedent AAS exposure in young, male C57Bl/6 mice affects acute behavioral and neuropathological responses to mild traumatic brain injury (TBI) induced with the CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) platform. Male C57Bl/6 mice received either vehicle or a cocktail of three AAS (testosterone, nandrolone and 17α-methyltestosterone) from 8-16 weeks of age. At the end of the 7th week of treatment, mice underwent two closed-head TBI or sham procedures spaced 24 h apart using CHIMERA. Post-repetitive TBI (rTBI) behavior was assessed for 7 d followed by tissue collection. AAS treatment induced the expected physiological changes including increased body weight, testicular atrophy, aggression and downregulation of brain 5-HT1B receptor expression. rTBI induced behavioral deficits, widespread axonal injury and white matter microgliosis. While AAS treatment did not worsen post-rTBI behavioral changes, AAS-treated mice exhibited significantly exacerbated axonal injury and microgliosis, indicating that AAS exposure can alter neuronal and innate immune responses to concussive TBI.

  1. Chronic Exposure to Androgenic-Anabolic Steroids Exacerbates Axonal Injury and Microgliosis in the CHIMERA Mouse Model of Repetitive Concussion.

    Directory of Open Access Journals (Sweden)

    Dhananjay R Namjoshi

    Full Text Available Concussion is a serious health concern. Concussion in athletes is of particular interest with respect to the relationship of concussion exposure to risk of chronic traumatic encephalopathy (CTE, a neurodegenerative condition associated with altered cognitive and psychiatric functions and profound tauopathy. However, much remains to be learned about factors other than cumulative exposure that could influence concussion pathogenesis. Approximately 20% of CTE cases report a history of substance use including androgenic-anabolic steroids (AAS. How acute, chronic, or historical AAS use may affect the vulnerability of the brain to concussion is unknown. We therefore tested whether antecedent AAS exposure in young, male C57Bl/6 mice affects acute behavioral and neuropathological responses to mild traumatic brain injury (TBI induced with the CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration platform. Male C57Bl/6 mice received either vehicle or a cocktail of three AAS (testosterone, nandrolone and 17α-methyltestosterone from 8-16 weeks of age. At the end of the 7th week of treatment, mice underwent two closed-head TBI or sham procedures spaced 24 h apart using CHIMERA. Post-repetitive TBI (rTBI behavior was assessed for 7 d followed by tissue collection. AAS treatment induced the expected physiological changes including increased body weight, testicular atrophy, aggression and downregulation of brain 5-HT1B receptor expression. rTBI induced behavioral deficits, widespread axonal injury and white matter microgliosis. While AAS treatment did not worsen post-rTBI behavioral changes, AAS-treated mice exhibited significantly exacerbated axonal injury and microgliosis, indicating that AAS exposure can alter neuronal and innate immune responses to concussive TBI.

  2. The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior.

    Science.gov (United States)

    An, Bo; DesRochers, Teresa M; Qin, Guokui; Xia, Xiaoxia; Thiagarajan, Geetha; Brodsky, Barbara; Kaplan, David L

    2013-01-01

    Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Polariton Chimeras: Bose-Einstein Condensates with Intrinsic Chaoticity and Spontaneous Long-Range Ordering

    Science.gov (United States)

    Gavrilov, S. S.

    2018-01-01

    The system of cavity polaritons driven by a plane electromagnetic wave is found to undergo the spontaneous breaking of spatial symmetry, which results in a lifted phase locking with respect to the driving field and, consequently, in the possibility of internal ordering. In particular, periodic spin and intensity patterns arise in polariton wires; they exhibit strong long-range order and can serve as media for signal transmission. Such patterns have the properties of dynamical chimeras: they are formed spontaneously in perfectly homogeneous media and can be partially chaotic. The reported new mechanism of chimera formation requires neither time-delayed feedback loops nor nonlocal interactions.

  4. Thoughts on the chimera method of simulation of three-dimensional viscous flow

    Science.gov (United States)

    Steger, Joseph L.

    1991-01-01

    The chimera overset grid is reviewed and discussed relative to other procedures for simulating flow about complex configurations. It is argued that while more refinement of the technique is needed, current schemes are competitive to unstructured grid schemes and should ultimately prove more useful.

  5. "American Chimera: The Ever-Present Domination of Whiteness, Patriarchy, and Capitalism…A Parable"

    Science.gov (United States)

    Montoya, Roberto; Matias, Cheryl E.; Nishi, Naomi W. M.; Sarcedo, Geneva L.

    2016-01-01

    In Greek mythology, the Chimera is a fire-breathing monster with three heads: one of a lion, one of a horned goat, and one of a powerful dragon. Of similar construction is the presence of three structures in US society, whiteness, patriarchy, and capitalism, which are overwhelmingly represented, valued, and espoused when examining areas of…

  6. Growth control of genetically modified cells using an antibody/c-Kit chimera.

    Science.gov (United States)

    Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

    2012-05-01

    Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. A Phase 1 Study of 4 Live, Recombinant Human Cytomegalovirus Towne/Toledo Chimera Vaccines in Cytomegalovirus-Seronegative Men.

    Science.gov (United States)

    Adler, Stuart P; Manganello, Anne-Marie; Lee, Ronzo; McVoy, Michael A; Nixon, Daniel E; Plotkin, Stanley; Mocarski, Edward; Cox, Josephine H; Fast, Patricia E; Nesterenko, Pavlo A; Murray, Susan E; Hill, Ann B; Kemble, George

    2016-11-01

     Human cytomegalovirus (HCMV) infection causes disease in newborns and transplant recipients. A HCMV vaccine (Towne) protects transplant recipients.  The genomes of Towne and the nonattenuated Toledo strain were recombined, yielding 4 Towne/Toledo chimera vaccines. Each of 36 HCMV-seronegative men received 1 subcutaneous dose of 10, 100, or 1000 plaque-forming units (PFU) in cohorts of 3. Safety and immunogenicity were evaluated over 12 weeks after immunization and for 52 weeks for those who seroconverted.  There were no serious local or systemic reactions. No subject had HCMV in urine or saliva. For chimera 3, none of 9 subjects seroconverted. For chimera 1, 1 of 9 seroconverted (the seroconverter received 100 PFU). For chimera 2, 3 subjects seroconverted (1 received 100 PFU, and 2 received 1000 PFU). For chimera 4, 7 subjects seroconverted (1 received 10 PFU, 3 received 100 PFU, and 3 received 1000 PFU). All 11 seroconverters developed low but detectable levels of neutralizing activity. CD4+ T-cell responses were detectable in 1 subject (who received 100 PFU of chimera 4). Seven subjects receiving chimera 2 or 4 had detectable CD8+ T-cell responses to IE1; 3 responded to 1-2 additional antigens.  The Towne/Toledo chimera vaccine candidates were well tolerated and were not excreted. Additional human trials of chimeras 2 and 4 are appropriate.  NCT01195571. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  8. Chimera states in multi-strain epidemic models with temporary immunity

    Science.gov (United States)

    Bauer, Larissa; Bassett, Jason; Hövel, Philipp; Kyrychko, Yuliya N.; Blyuss, Konstantin B.

    2017-11-01

    We investigate a time-delayed epidemic model for multi-strain diseases with temporary immunity. In the absence of cross-immunity between strains, dynamics of each individual strain exhibit emergence and annihilation of limit cycles due to a Hopf bifurcation of the endemic equilibrium, and a saddle-node bifurcation of limit cycles depending on the time delay associated with duration of temporary immunity. Effects of all-to-all and non-local coupling topologies are systematically investigated by means of numerical simulations, and they suggest that cross-immunity is able to induce a diverse range of complex dynamical behaviors and synchronization patterns, including discrete traveling waves, solitary states, and amplitude chimeras. Interestingly, chimera states are observed for narrower cross-immunity kernels, which can have profound implications for understanding the dynamics of multi-strain diseases.

  9. UCSF ChimeraX: Meeting modern challenges in visualization and analysis.

    Science.gov (United States)

    Goddard, Thomas D; Huang, Conrad C; Meng, Elaine C; Pettersen, Eric F; Couch, Gregory S; Morris, John H; Ferrin, Thomas E

    2018-01-01

    UCSF ChimeraX is next-generation software for the visualization and analysis of molecular structures, density maps, 3D microscopy, and associated data. It addresses challenges in the size, scope, and disparate types of data attendant with cutting-edge experimental methods, while providing advanced options for high-quality rendering (interactive ambient occlusion, reliable molecular surface calculations, etc.) and professional approaches to software design and distribution. This article highlights some specific advances in the areas of visualization and usability, performance, and extensibility. ChimeraX is free for noncommercial use and is available from http://www.rbvi.ucsf.edu/chimerax/ for Windows, Mac, and Linux. © 2017 The Protein Society.

  10. The contribution of human/non-human animal chimeras to stem cell research

    Directory of Open Access Journals (Sweden)

    Sonya Levine

    2017-10-01

    Full Text Available Chimeric animals are made up of cells from two separate zygotes. Human/non-human animal chimeras have been used for a number of research purposes, including human disease modeling. Pluripotent stem cell (PSC research has relied upon the chimera approach to examine the developmental potential of stem cells, to determine the efficacy of cell replacement therapies, and to establish a means of producing human organs. Based on ethical issues, this work has faced pushback from various sources including funding agencies. We discuss here the essential role these studies have played, from gaining a better understanding of human biology to providing a stepping stone to human disease treatments. We also consider the major ethical issues, as well as the current status of support for this work in the United States.

  11. Chimera states and collective chaos in pulse-coupled neural networks

    OpenAIRE

    Olmi Simona; Politi Antonio; Torcini Alessandro

    2011-01-01

    Understanding the collective motion of networks of oscillators is crucial in many contexts, starting from neuronal circuits [1]. So far, most of the efforts have been devoted to the characterization of strong forms of synchronization. However, more subtle phenomena, like the onset of coherent oscillations in an ensemble of neurons can also play a relevant role for information coding. A peculiar coherent state, termed Chimera, appears in two symmetrically coupled populations of oscillators, wh...

  12. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

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    CÂRSTEA V. B

    2007-01-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  13. A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae.

    OpenAIRE

    Sherwood, P. W.; Katic, I.; Sanz, P; Carlson, M.

    2000-01-01

    A family of glucose transporters mediates glucose uptake in Saccharomyces cerevisiae. We show that the dominant mutation GSF4-1, which impairs glucose repression of SUC2, results in a nonfunctional chimera of the transporters Hxt1p and Hxt4p. Hxt1/4p inhibits the function of wild-type glucose transporters. Similar mutations may facilitate analysis of the major facilitator superfamily.

  14. The History of Modern Psyycology in "İlmü'n-Nefs" writed by İzmirli İsmail Hakkı

    OpenAIRE

    KIZILGEÇİT, Muhammed

    2013-01-01

    Human being who many-sided has been in a struggle for knowing and under-standing himself since he took part in the possible existence scene. Both that revelation started with the first man and the knowledge about self (nefs) has been inherited from generation to generation are an evidence for it. When psy-chology is talked about, a modern science gaining acceleration in the 1960s comes to mind. The connection of this field with Islam tradition, especially with the Works in the Ottoman period...

  15. Serine integrase chimeras with activity in E. coli and HeLa cells

    Directory of Open Access Journals (Sweden)

    Alfonso P. Farruggio

    2014-09-01

    Full Text Available In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC and phiC31-TG1 (CT hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

  16. A Universal Protein Tag for Delivery of SiRNA-Aptamer Chimeras

    Science.gov (United States)

    Liu, Hong Yan; Gao, Xiaohu

    2013-11-01

    siRNA-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of siRNA due to the modularity of their diblock RNA structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. However, additional challenges must be addressed for efficient RNA interference (RNAi), in particular, endosomal escape. Currently, vast majority of siRNA delivery vehicles are based on cationic materials, which form complexes with negatively charged siRNA. Unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. The protein selectively tags along the siRNA block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition. More interestingly, we found that extending the c-terminal polyhistidine segment in the protein tag to 18 amino acids completely abolishes the RNA binding function of dsRBD.

  17. TLR4 Expression by Liver Resident Cells Mediates the Development of Glucose Intolerance and Insulin Resistance in Experimental Periodontitis

    Science.gov (United States)

    Ilievski, Vladimir; Cho, Yale; Katwala, Priya; Rodriguez, Heriberto; Tulowiecka, Margaret; Kurian, David; Leoni, Lara; Christman, John W.; Unterman, Terry G.; Watanabe, Keiko

    2015-01-01

    Background Results from epidemiological studies indicate a close association between periodontitis and type 2 diabetes mellitus. However, the mechanism linking periodontitis to glucose intolerance (GI) and insulin resistance (IR) is unknown. We therefore tested the hypothesis that periodontitis induces the development of GI/IR through a liver Toll-like receptor 4 (TLR4) dependent mechanism. Methods TLR4 chimeric mice were developed by bone marrow transplantation using green fluorescent protein expressing TLR4WT mouse (GFPWT) as donor and TLR4 WT or TLR4-/- as recipient mice (GFPWT:WT and GFPWT:KO chimeras respectively). These chimeras were subjected to experimental chronic periodontitis induced by repeated applications of LPS to the gingival sulci for 18 weeks. The levels of GI/IR were monitored and plasma cytokines and LPS were determined at 18 weeks when differences in glucose tolerance were most apparent. Cytokine gene expression was measured in liver tissue by qPCR. Results Alveolar bone loss was significantly greater in GFPWT:WT chimeras treated with LPS compared with chimeras treated with PBS or GFPWT:KO chimeras. However, the degree of gingival inflammation was similar between GFPWT:WT and GFPWT:KO mice with LPS application. Severe GI/IR occurred in GFPWT:WT chimeras but not in the GFPWT:KO chimeras that were subjected to 18 weeks of LPS. Serum LPS was detected only in animals to which LPS was applied and the level was similar in GFPWT:WT and GFPWT:KO mice at the 18 week time point. Surprisingly, there was no significant difference in the plasma levels of IL1β, IL6 and TNFα at 18 weeks in spite of the severe GI/IR in the GFPWT:WT chimeras with LPS application. Also, no difference in the expression of TNFα or IL6 mRNA was detected in the liver of GFPWT:WT vs GFPWT:KO mice. In contrast, liver IL1β expression was significantly greater in GFPWT:WT chimeras compared to GFPWT:KO chimeras treated with LPS. Conclusion We observed that GFPWT:WT, but not GFPWT

  18. Anti-tetherin activities in Vpu-expressing primate lentiviruses

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    Haworth Kevin G

    2010-02-01

    Full Text Available Abstract Background The anti-viral activity of the cellular restriction factor, BST-2/tetherin, was first observed as an ability to block the release of Vpu-minus HIV-1 from the surface of infected cells. However, tetherin restriction is also counteracted by primate lentiviruses that do not express a Vpu protein, where anti-tetherin functions are provided by either the Env protein (HIV-2, SIVtan or the Nef protein (SIVsm/mac and SIVagm. Within the primate lentiviruses, Vpu is also present in the genomes of SIVcpz and certain SIVsyk viruses. We asked whether, in these viruses, anti-tetherin activity was always a property of Vpu, or if it had selectively evolved in HIV-1 to perform this function. Results We found that despite the close relatedness of HIV-1 and SIVcpz, the chimpanzee viruses use Nef instead of Vpu to counteract tetherin. Furthermore, SIVcpz Nef proteins had activity against chimpanzee but not human tetherin. This specificity mapped to a short sequence that is present in the cytoplasmic tail of primate but not human tetherins, and this also accounts for the specificity of SIVsm/mac Nef for primate but not human tetherins. In contrast, Vpu proteins from four diverse members of the SIVsyk lineage all displayed an anti-tetherin activity that was active against macaque tetherin. Interestingly, Vpu from a SIVgsn isolate was also found to have activity against human tetherin. Conclusions Primate lentiviruses show a high degree of flexibility in their use of anti-tetherin factors, indicating a strong selective pressure to counteract tetherin restriction. The identification of an activity against human tetherin in SIVgsn Vpu suggests that the presence of Vpu in the ancestral SIVmus/mon/gsn virus believed to have contributed the 3' half of the HIV-1 genome may have played a role in the evolution of viruses that could counteract human tetherin and infect humans.

  19. Systematic Characteristic Exploration of the Chimeras Generated in Multiple Displacement Amplification through Next Generation Sequencing Data Reanalysis.

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    Jing Tu

    Full Text Available The chimeric sequences produced by phi29 DNA polymerase, which are named as chimeras, influence the performance of the multiple displacement amplification (MDA and also increase the difficulty of sequence data process. Despite several articles have reported the existence of chimeric sequence, there was only one research focusing on the structure and generation mechanism of chimeras, and it was merely based on hundreds of chimeras found in the sequence data of E. coli genome.We finished data mining towards a series of Next Generation Sequencing (NGS reads which were used for whole genome haplotype assembling in a primary study. We established a bioinformatics pipeline based on subsection alignment strategy to discover all the chimeras inside and achieve their structural visualization. Then, we artificially defined two statistical indexes (the chimeric distance and the overlap length, and their regular abundance distribution helped illustrate of the structural characteristics of the chimeras. Finally we analyzed the relationship between the chimera type and the average insertion size, so that illustrate a method to decrease the proportion of wasted data in the procedure of DNA library construction.131.4 Gb pair-end (PE sequence data was reanalyzed for the chimeras. Totally, 40,259,438 read pairs (6.19% with chimerism were discovered among 650,430,811 read pairs. The chimeric sequences are consisted of two or more parts which locate inconsecutively but adjacently on the chromosome. The chimeric distance between the locations of adjacent parts on the chromosome followed an approximate bimodal distribution ranging from 0 to over 5,000 nt, whose peak was at about 250 to 300 nt. The overlap length of adjacent parts followed an approximate Poisson distribution and revealed a peak at 6 nt. Moreover, unmapped chimeras, which were classified as the wasted data, could be reduced by properly increasing the length of the insertion segment size through a

  20. Arne Jönsson, Valborg Lindgärde, Elisabet Göransson (eds., Wår Lärda Skalde-Fru Sophia Elisabeth Brenner och hennes tid (Ängelholm: Skåneförlaget, 2011

    Directory of Open Access Journals (Sweden)

    Camilla Storskog

    2011-03-01

    Full Text Available Book review of Wår Lärda Skalde-Fru Sophia Elisabeth Brenner och hennes tid, edited by Arne Jönsson, Valborg Lindgärde and, Elisabet Göransson, published by Skåneförlaget, 2011, pp. 536. ISBN: 978-91-87976-33-9

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

    Science.gov (United States)

    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.

  2. Explosive synchronization in clustered scale-free networks: Revealing the existence of chimera state

    Science.gov (United States)

    Berec, V.

    2016-02-01

    The collective dynamics of Kuramoto oscillators with a positive correlation between the incoherent and fully coherent domains in clustered scale-free networks is studied. Emergence of chimera states for the onsets of explosive synchronization transition is observed during an intermediate coupling regime when degree-frequency correlation is established for the hubs with the highest degrees. Diagnostic of the abrupt synchronization is revealed by the intrinsic spectral properties of the network graph Laplacian encoded in the heterogeneous phase space manifold, through extensive analytical investigation, presenting realistic MC simulations of nonlocal interactions in discrete time dynamics evolving on the network.

  3. Multitasking for flows about multiple body configurations using the chimera grid scheme

    Science.gov (United States)

    Dougherty, F. C.; Morgan, R. L.

    1987-01-01

    The multitasking of a finite-difference scheme using multiple overset meshes is described. In this chimera, or multiple overset mesh approach, a multiple body configuration is mapped using a major grid about the main component of the configuration, with minor overset meshes used to map each additional component. This type of code is well suited to multitasking. Both steady and unsteady two dimensional computations are run on parallel processors on a CRAY-X/MP 48, usually with one mesh per processor. Flow field results are compared with single processor results to demonstrate the feasibility of running multiple mesh codes on parallel processors and to show the increase in efficiency.

  4. Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras.

    Science.gov (United States)

    Kuo, Lili; Hurst-Hess, Kelley R; Koetzner, Cheri A; Masters, Paul S

    2016-05-01

    The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a

  5. Acute serum amyloid A induces migration, angiogenesis, and inflammation in synovial cells in vitro and in a human rheumatoid arthritis/SCID mouse chimera model.

    LENUS (Irish Health Repository)

    Connolly, Mary

    2010-06-01

    Serum amyloid A (A-SAA), an acute-phase protein with cytokine-like properties, is expressed at sites of inflammation. This study investigated the effects of A-SAA on chemokine-regulated migration and angiogenesis using rheumatoid arthritis (RA) cells and whole-tissue explants in vitro, ex vivo, and in vivo. A-SAA levels were measured by real-time PCR and ELISA. IL-8 and MCP-1 expression was examined in RA synovial fibroblasts, human microvascular endothelial cells, and RA synovial explants by ELISA. Neutrophil transendothelial cell migration, cell adhesion, invasion, and migration were examined using transwell leukocyte\\/monocyte migration assays, invasion assays, and adhesion assays with or without anti-MCP-1\\/anti-IL-8. NF-kappaB was examined using a specific inhibitor and Western blotting. An RA synovial\\/SCID mouse chimera model was used to examine the effects of A-SAA on cell migration, proliferation, and angiogenesis in vivo. High expression of A-SAA was demonstrated in RA patients (p < 0.05). A-SAA induced chemokine expression in a time- and dose-dependent manner (p < 0.05). Blockade with anti-scavenger receptor class B member 1 and lipoxin A4 (A-SAA receptors) significantly reduced chemokine expression in RA synovial tissue explants (p < 0.05). A-SAA induced cell invasion, neutrophil-transendothelial cell migration, monocyte migration, and adhesion (all p < 0.05), effects that were blocked by anti-IL-8 or anti-MCP-1. A-SAA-induced chemokine expression was mediated through NF-kappaB in RA explants (p < 0.05). Finally, in the RA synovial\\/SCID mouse chimera model, we demonstrated for the first time in vivo that A-SAA directly induces monocyte migration from the murine circulation into RA synovial grafts, synovial cell proliferation, and angiogenesis (p < 0.05). A-SAA promotes cell migrational mechanisms and angiogenesis critical to RA pathogenesis.

  6. Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

    Science.gov (United States)

    Yao, Hongwei; Lee, Myungwoon; Liao, Shu-Yu; Hong, Mei

    2016-12-13

    The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13 C- 13 C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may

  7. Artificial modulation of the gating behavior of a K+ channel in a KvAP-DNA chimera.

    Science.gov (United States)

    Wang, Andrew; Zocchi, Giovanni

    2011-04-19

    We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel.

  8. A Biochemical/Biophysical Assay Dyad for HTS-Compatible Triaging of Inhibitors of the HIV-1 Nef/Hck SH3 Interaction

    KAUST Repository

    Breuer, Sebastian

    2013-07-26

    The current treatment regimens for HIV include over 20 anti-retrovirals. However, adverse drug effects and the emergence of drug resistance necessitates the continued improvement of the existing drug classes as well as the development of novel drugs that target as yet therapeutically unexploited viral and cellular pathways. Here we demonstrate a strategy for the discovery of protein-protein interaction inhibitors of the viral pathogenicity factor HIV-1 Nef and its interaction with the host factor SH3. A combination of a time-resolved fluorescence resonance energy resonance energy transfer-based assay and a label-free resonant waveguide grating-based assay was optimized for high-throughput screening formats.

  9. A Comprehensive, Automatically Updated Fungal ITS Sequence Dataset for Reference-Based Chimera Control in Environmental Sequencing Efforts.

    Science.gov (United States)

    Nilsson, R Henrik; Tedersoo, Leho; Ryberg, Martin; Kristiansson, Erik; Hartmann, Martin; Unterseher, Martin; Porter, Teresita M; Bengtsson-Palme, Johan; Walker, Donald M; de Sousa, Filipe; Gamper, Hannes Andres; Larsson, Ellen; Larsson, Karl-Henrik; Kõljalg, Urmas; Edgar, Robert C; Abarenkov, Kessy

    2015-01-01

    The nuclear ribosomal internal transcribed spacer (ITS) region is the most commonly chosen genetic marker for the molecular identification of fungi in environmental sequencing and molecular ecology studies. Several analytical issues complicate such efforts, one of which is the formation of chimeric-artificially joined-DNA sequences during PCR amplification or sequence assembly. Several software tools are currently available for chimera detection, but rely to various degrees on the presence of a chimera-free reference dataset for optimal performance. However, no such dataset is available for use with the fungal ITS region. This study introduces a comprehensive, automatically updated reference dataset for fungal ITS sequences based on the UNITE database for the molecular identification of fungi. This dataset supports chimera detection throughout the fungal kingdom and for full-length ITS sequences as well as partial (ITS1 or ITS2 only) datasets. The performance of the dataset on a large set of artificial chimeras was above 99.5%, and we subsequently used the dataset to remove nearly 1,000 compromised fungal ITS sequences from public circulation. The dataset is available at http://unite.ut.ee/repository.php and is subject to web-based third-party curation.

  10. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  11. Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence.

    Science.gov (United States)

    McAuley, Alexander J; Torres, Maricela; Plante, Jessica A; Huang, Claire Y-H; Bente, Dennis A; Beasley, David W C

    2016-05-01

    Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence. The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a

  12. Immunizing adult female mice with a TcpA-A2-CTB chimera provides a high level of protection for their pups in the infant mouse model of cholera.

    Directory of Open Access Journals (Sweden)

    Gregory A Price

    2014-12-01

    Full Text Available Vibrio cholerae expresses two primary virulence factors, cholera toxin (CT and the toxin-coregulated pilus (TCP. CT causes profuse watery diarrhea, and TCP (composed of repeating copies of the major pilin TcpA is required for intestinal colonization by V. cholerae. Antibodies to CT or TcpA can protect against cholera in animal models. We developed a TcpA holotoxin-like chimera (TcpA-A2-CTB to elicit both anti-TcpA and anti-CTB antibodies and evaluated its immunogenicity and protective efficacy in the infant mouse model of cholera. Adult female CD-1 mice were immunized intraperitoneally three times with the TcpA-A2-CTB chimera and compared with similar groups immunized with a TcpA+CTB mixture, TcpA alone, TcpA with Salmonella typhimurium flagellin subunit FliC as adjuvant, or CTB alone. Blood and fecal samples were analyzed for antigen-specific IgG or IgA, respectively, using quantitative ELISA. Immunized females were mated; their reared offspring were challenged orogastrically with 10 or 20 LD50 of V. cholerae El Tor N16961; and vaccine efficacy was assessed by survival of the challenged pups at 48 hrs. All pups from dams immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture survived at both challenge doses. In contrast, no pups from dams immunized with TcpA+FliC or CTB alone survived at the 20 LD50 challenge dose, although the anti-TcpA or anti-CTB antibody level elicited by these immunizations was comparable to the corresponding antibody level achieved by immunization with TcpA-A2-CTB or TcpA+CTB. Taken together, these findings comprise strong preliminary evidence for synergistic action between anti-TcpA and anti-CTB antibodies in protecting mice against cholera. Weight loss analysis showed that only immunization of dams with TcpA-A2-CTB chimera or TcpA+CTB mixture protected their pups against excess weight loss from severe diarrhea. These data support the concept of including both TcpA and CTB as immunogens in development of an

  13. Immunizing adult female mice with a TcpA-A2-CTB chimera provides a high level of protection for their pups in the infant mouse model of cholera.

    Science.gov (United States)

    Price, Gregory A; Holmes, Randall K

    2014-12-01

    Vibrio cholerae expresses two primary virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP). CT causes profuse watery diarrhea, and TCP (composed of repeating copies of the major pilin TcpA) is required for intestinal colonization by V. cholerae. Antibodies to CT or TcpA can protect against cholera in animal models. We developed a TcpA holotoxin-like chimera (TcpA-A2-CTB) to elicit both anti-TcpA and anti-CTB antibodies and evaluated its immunogenicity and protective efficacy in the infant mouse model of cholera. Adult female CD-1 mice were immunized intraperitoneally three times with the TcpA-A2-CTB chimera and compared with similar groups immunized with a TcpA+CTB mixture, TcpA alone, TcpA with Salmonella typhimurium flagellin subunit FliC as adjuvant, or CTB alone. Blood and fecal samples were analyzed for antigen-specific IgG or IgA, respectively, using quantitative ELISA. Immunized females were mated; their reared offspring were challenged orogastrically with 10 or 20 LD50 of V. cholerae El Tor N16961; and vaccine efficacy was assessed by survival of the challenged pups at 48 hrs. All pups from dams immunized with the TcpA-A2-CTB chimera or the TcpA+CTB mixture survived at both challenge doses. In contrast, no pups from dams immunized with TcpA+FliC or CTB alone survived at the 20 LD50 challenge dose, although the anti-TcpA or anti-CTB antibody level elicited by these immunizations was comparable to the corresponding antibody level achieved by immunization with TcpA-A2-CTB or TcpA+CTB. Taken together, these findings comprise strong preliminary evidence for synergistic action between anti-TcpA and anti-CTB antibodies in protecting mice against cholera. Weight loss analysis showed that only immunization of dams with TcpA-A2-CTB chimera or TcpA+CTB mixture protected their pups against excess weight loss from severe diarrhea. These data support the concept of including both TcpA and CTB as immunogens in development of an effective multivalent

  14. Requirement for Tumor Necrosis Factor Receptor 2 Expression on Vascular Cells To Induce Experimental Cerebral Malaria

    OpenAIRE

    Stoelcker, Benjamin; Hehlgans, Thomas; Weigl, Karin; Bluethmann, Horst; Grau, Georges E.; Männel, Daniela N.

    2002-01-01

    Using tumor necrosis factor receptor type 2 (TNFR2)-deficient mice and generating bone marrow chimeras which express TNFR2 on either hematopoietic or nonhematopoietic cells, we demonstrated the requirement for TNFR2 expression on tissue cells to induce lethal cerebral malaria. Thus, TNFR2 on the brain vasculature mediates tumor necrosis factor-induced neurovascular lesions in experimental cerebral malaria.

  15. T cell receptor zeta allows stable expression of receptors containing the CD3gamma leucine-based receptor-sorting motif

    DEFF Research Database (Denmark)

    Dietrich, J; Geisler, C

    1998-01-01

    The leucine-based motif in the T cell receptor (TCR) subunit CD3gamma constitutes a strong internalization signal. In fully assembled TCR this motif is inactive unless phosphorylated. In contrast, the motif is constitutively active in CD4/CD3gamma and Tac/CD3gamma chimeras independently...... to mask the CD3gamma leucine-based motif. By studying CD4/CD3gamma and CD16/CD3gamma chimeras, we found that CD16/CD3gamma chimeras associated with TCRzeta. The CD16/CD3gamma-TCRzeta complexes were stably expressed at the cell surface and had a low spontaneous internalization rate, indicating...... that the leucine-based motif in these complexes was inactive. In contrast, the CD4/CD3gamma chimeras did not associate with TCRzeta, and the leucine-based motif in these chimeras was constitutively active resulting in a high spontaneous internalization rate and low expression of the chimeras at the cell surface...

  16. Nef Annliese (dir., Les territoires de la Méditerranée, XIe-XVIe siècle, Rennes, Presses universitaires de Rennes, 2013, 238 p.

    Directory of Open Access Journals (Sweden)

    Pascal Buresi

    2014-02-01

    Full Text Available Cet ouvrage est le fruit d’un séminaire collectif annuel, consacré à l’espace économique méditerranéen au Moyen Âge et animé par D. Coulon, A. Nef, C. Picard et D. Valérian. Après une courte introduction d’A. Nef, il comporte quatorze articles, classés sous trois rubriques : « Le contrôle des espaces maritimes » (p. 13-71, « Stratégies économiques » (p. 73-157 et « L’encadrement institutionnel, juridique et fiscal de la production et des échanges » (p. 159-231. Dans « L’Adriatique, une pro...

  17. Monsters, dreams and madness: Commentary on 'The arms of the chimeras'.

    Science.gov (United States)

    Reis, Bruce

    2016-04-01

    Considering Freudian and Post-Freudian approaches to the intersubjective Beatrice Ithier puts the work of Michel de M'Uzan and Thomas Ogden in comparison. To this comparison I add a consideration of the work of Christopher Bollas. The highly creative clinical approaches these three theorists take is shown to be informed by their elaborations of the Freudian notion of unconscious communication and by new approaches to the issue of identity. Attention is paid to differentiating traumatic from fanciful chimeras; and to the experience of the analyst undergoing the sorts of transformations requisite to entering this psychic space marked by fluid exchanges of being and becoming, wherein analyst becomes patient, new subjects are created through shared dreams, and through which monsters appear. Copyright © 2016 Institute of Psychoanalysis.

  18. EBARDenovo: highly accurate de novo assembly of RNA-Seq with efficient chimera-detection.

    Science.gov (United States)

    Chu, Hsueh-Ting; Hsiao, William W L; Chen, Jen-Chih; Yeh, Tze-Jung; Tsai, Mong-Hsun; Lin, Han; Liu, Yen-Wenn; Lee, Sheng-An; Chen, Chaur-Chin; Tsao, Theresa T H; Kao, Cheng-Yan

    2013-04-15

    High-accuracy de novo assembly of the short sequencing reads from RNA-Seq technology is very challenging. We introduce a de novo assembly algorithm, EBARDenovo, which stands for Extension, Bridging And Repeat-sensing Denovo. This algorithm uses an efficient chimera-detection function to abrogate the effect of aberrant chimeric reads in RNA-Seq data. EBARDenovo resolves the complications of RNA-Seq assembly arising from sequencing errors, repetitive sequences and aberrant chimeric amplicons. In a series of assembly experiments, our algorithm is the most accurate among the examined programs, including de Bruijn graph assemblers, Trinity and Oases. EBARDenovo is available at http://ebardenovo.sourceforge.net/. This software package (with patent pending) is free of charge for academic use only. Supplementary data are available at Bioinformatics online.

  19. Test of GET Electronics for the CHIMERA and FARCOS multi-detectors

    Science.gov (United States)

    De Luca, S.; Acosta, L.; Auditore, L.; Boiano, C.; Cardella, G.; Castoldi, A.; D'Andrea, M.; De Filippo, E.; Dell'Aquila, D.; Fichera, F.; Gnoffo, B.; Guazzoni, C.; Lanzalone, G.; Lombardo, I.; Martorana, N. S.; Minniti, T.; Norella, S.; Pagano, A.; Pagano, E. V.; Papa, M.; Pirrone, S.; Politi, G.; Quattrocchi, L.; Rizzo, F.; Russotto, P.; Saccà, G.; Trifirò, A.; Trimarchi, M.; Verde, G.; Vigilante, M.

    2017-11-01

    In this paper we present the results of the tests on the new digital electronics GET (General Electronics for Tpc), which will be used for the readout of the CsI(Tl) detectors of CHIMERA (Charged Heavy Ion Mass and Energy Resolving Array) and for the new correlator FARCOS (Femtoscope ARray for COrrelations and Spectroscopy). The new electronics allows us to digitize the full waveform of the signals produced by the detector. Among its features it is worth noticing the compactness and low power consumption (5W for 256 channels). Tests have been performed with pulsers, radioactive sources and ion beams. With such electronics very good results in energy resolution and isotope separation of the detected fragments were obtained, by using both hardware and software filters.

  20. Chimera and modulated drift states in a ring of nonlocally coupled oscillators with heterogeneous phase lags

    Science.gov (United States)

    Choe, Chol-Ung; Kim, Ryong-Son; Ri, Ji-Song

    2017-09-01

    We consider a ring of phase oscillators with nonlocal coupling strength and heterogeneous phase lags. We analyze the effects of heterogeneity in the phase lags on the existence and stability of a variety of steady states. A nonlocal coupling with heterogeneous phase lags that allows the system to be solved analytically is suggested and the stability of solutions along the Ott-Antonsen invariant manifold is explored. We present a complete bifurcation diagram for stationary patterns including the uniform drift and modulated drift states as well as chimera state, which reveals that the stable modulated drift state and a continuum of metastable drift states could occur due to the heterogeneity of the phase lags. We verify our theoretical results using the direct numerical simulations of the model system.

  1. Musicianship boosts perceptual learning of pseudoword-chimeras: an electrophysiological approach.

    Science.gov (United States)

    Kühnis, Jürg; Elmer, Stefan; Meyer, Martin; Jäncke, Lutz

    2013-01-01

    A vast amount of previous work has consistently revealed that professional music training is associated with functional and structural alterations of auditory-related brain regions. Meanwhile, there is also an increasing array of evidence, which shows that musicianship facilitates segmental, as well as supra-segmental aspects of speech processing. Based on this evidence, we addressed a novel research question, namely whether professional music training has an influence on the perceptual learning of speech sounds. In the context of an EEG experiment, we presented auditory pseudoword-chimeras, manipulated in terms of spectral- or envelope-related acoustic information, to a group of professional musicians and non-musicians. During EEG measurements, participants were requested to assign the auditory-presented pseudoword-chimeras to one out of four visually presented templates. As expected, both groups showed behavioural learning effects during the time course of the experiment. These learning effects were associated with an increase in accuracy, a decrease in reaction time, as well as a decrease in the P2-like microstate duration in both groups. Notably, the musicians showed an increased learning performance compared to the controls during the first two runs of the spectral condition. This perceptual learning effect, which varies as a function of musical expertise, was reflected by a reduction of the P2-like microstate duration. Results may mirror transfer effects from musical training to the processing of spectral information in speech sounds. Hence, this study provides first evidence for a relationship between changes in microstates, musical expertise, and perceptual verbal learning mechanisms.

  2. Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera.

    Science.gov (United States)

    Errico Provenzano, Alfredo; Posteri, Riccardo; Giansanti, Francesco; Angelucci, Francesco; Flavell, Sopsamorn U; Flavell, David J; Fabbrini, Maria Serena; Porro, Danilo; Ippoliti, Rodolfo; Ceriotti, Aldo; Branduardi, Paola; Vago, Riccardo

    2016-11-14

    The big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types. To selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity. Our results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.

  3. La charpente de la nef de la cathédrale de Bourges The structure of the nave in Bourges cathedral

    Directory of Open Access Journals (Sweden)

    Frédéric Epaud

    2012-03-01

    Full Text Available La cathédrale gothique de Bourges a été édifiée en deux campagnes avec le chœur, de 1195 à 1214, puis la nef, de 1225 à 1255, après 10 ans d’interruption du chantier. Ses charpentes d’origine ont été en partie reconstruites sur l’ensemble des collatéraux, suite à un incendie en 1559, et sur la partie médiane du grand vaisseau, entre 1747 et 1754, du fait de la suppression du faux transept et de sa flèche. Le relevé archéologique de la charpente gothique subsistante sur la nef, et l’analyse dendrochronologique de ses bois démontrent que les abattages se sont étalés de 1230 à 1257, avec deux grandes campagnes de coupes de bois entre 1240 et 1244 et en automne-hiver 1254-1255, juste avant la mise en œuvre et le levage de la charpente en 1256 ou peu après. Ces coupes successives supposent un approvisionnement du chantier par des donations de bois ou de parcelles forestières exploitées aussitôt, obligeant à un stockage des bois dans l’attente du chantier. La charpente à chevrons-formant-fermes a été exécutée selon un premier projet qui a été modifié en cours de réalisation pour y insérer un dispositif de contreventement longitudinal, avec une nouvelle structure des fermes principales permettant de le recevoir. Ce contreventement axial est doublé par un second situé dans le plan des chevrons, attesté pour la première fois par la dendrochronologie du milieu du xiiie s. En 1262, les travées orientales de la charpente de la nef sont reprises pour permettre le rajout d’une flèche en bois et d’un faux-transept à l’aplomb de la quatrième travée de voûtes.The Gothic cathedral of Bourges was built in two phases, with the chancel from 1195 to 1214 then the nave from 1225 to 1255, after a 10-year interruption to the building work. The original roof structure was in part reconstructed on all the sides, following a fire in 1559, and on the middle part of the great nave, between 1747 and 1754, due to

  4. Study on optimized upwind schemes for computational areoacoustics and extension of a fully conservative Chimera to finite difference schemes

    Science.gov (United States)

    Chen, Rangfu

    A computational methodology has been developed in the first part of the thesis for the simulations of acoustic radiation, propagation and reflection. The developed methodology is high order accurate, uses less grid points per wave length comparing to standard high order accurate numerical methods, and automatically damps out spurious short waves. Furthermore, the methodology can be applied to acoustic problems in the presence of objects with curved geometries. To achieve these results, high order accurate optimized upwind schemes, which are applied to discretize spatial derivatives on interior grid points, have been developed. High order accurate optimized one- side biased schemes, which are only applied to discretize the spatial derivatives on grid points near computational boundaries, have also been constructed. The developed schemes are combined with a time difference scheme to fully discretize acoustic field equations in multi- dimension in arbitrary curvilinear coordinates. Numerical boundary conditions are investigated and intuitively illustrated. Applications of the developed methodology to a sequence of one-dimensional and multi-dimensional acoustic problems are performed. The numerical results have validated the developed methodology and demonstrated advantages of the methodology over central-difference Dispersion-Relation-Preserving method. Numerical results have also shown that the optimized upwind schemes minimize not only the dissipation error but also the dissipation error, while retaining the numerical stability. The second part of the thesis deals with a fully conservative Chimera methodology. The fully conservative Chimera was originally developed based on a finite volume approach. A finite difference scheme is shown to be identical to a finite volume scheme with proper definition of control volumes and metrics. The fully conservative Chimera has been successfully extended to finite difference schemes for viscous flows including turbulence models

  5. A Navier-Strokes Chimera Code on the Connection Machine CM-5: Design and Performance

    Science.gov (United States)

    Jespersen, Dennis C.; Levit, Creon; Kwak, Dochan (Technical Monitor)

    1994-01-01

    We have implemented a three-dimensional compressible Navier-Stokes code on the Connection Machine CM-5. The code is set up for implicit time-stepping on single or multiple structured grids. For multiple grids and geometrically complex problems, we follow the 'chimera' approach, where flow data on one zone is interpolated onto another in the region of overlap. We will describe our design philosophy and give some timing results for the current code. A parallel machine like the CM-5 is well-suited for finite-difference methods on structured grids. The regular pattern of connections of a structured mesh maps well onto the architecture of the machine. So the first design choice, finite differences on a structured mesh, is natural. We use centered differences in space, with added artificial dissipation terms. When numerically solving the Navier-Stokes equations, there are liable to be some mesh cells near a solid body that are small in at least one direction. This mesh cell geometry can impose a very severe CFL (Courant-Friedrichs-Lewy) condition on the time step for explicit time-stepping methods. Thus, though explicit time-stepping is well-suited to the architecture of the machine, we have adopted implicit time-stepping. We have further taken the approximate factorization approach. This creates the need to solve large banded linear systems and creates the first possible barrier to an efficient algorithm. To overcome this first possible barrier we have considered two options. The first is just to solve the banded linear systems with data spread over the whole machine, using whatever fast method is available. This option is adequate for solving scalar tridiagonal systems, but for scalar pentadiagonal or block tridiagonal systems it is somewhat slower than desired. The second option is to 'transpose' the flow and geometry variables as part of the time-stepping process: Start with x-lines of data in-processor. Form explicit terms in x, then transpose so y-lines of data are

  6. A Direct Mapping of Max k-SAT and High Order Parity Checks to a Chimera Graph.

    Science.gov (United States)

    Chancellor, N; Zohren, S; Warburton, P A; Benjamin, S C; Roberts, S

    2016-11-18

    We demonstrate a direct mapping of max k-SAT problems (and weighted max k-SAT) to a Chimera graph, which is the non-planar hardware graph of the devices built by D-Wave Systems Inc. We further show that this mapping can be used to map a similar class of maximum satisfiability problems where the clauses are replaced by parity checks over potentially large numbers of bits. The latter is of specific interest for applications in decoding for communication. We discuss an example in which the decoding of a turbo code, which has been demonstrated to perform near the Shannon limit, can be mapped to a Chimera graph. The weighted max k-SAT problem is the most general class of satisfiability problems, so our result effectively demonstrates how any satisfiability problem may be directly mapped to a Chimera graph. Our methods faithfully reproduce the low energy spectrum of the target problems, so therefore may also be used for maximum entropy inference.

  7. A Direct Mapping of Max k-SAT and High Order Parity Checks to a Chimera Graph

    Science.gov (United States)

    Chancellor, N.; Zohren, S.; Warburton, P. A.; Benjamin, S. C.; Roberts, S.

    2016-11-01

    We demonstrate a direct mapping of max k-SAT problems (and weighted max k-SAT) to a Chimera graph, which is the non-planar hardware graph of the devices built by D-Wave Systems Inc. We further show that this mapping can be used to map a similar class of maximum satisfiability problems where the clauses are replaced by parity checks over potentially large numbers of bits. The latter is of specific interest for applications in decoding for communication. We discuss an example in which the decoding of a turbo code, which has been demonstrated to perform near the Shannon limit, can be mapped to a Chimera graph. The weighted max k-SAT problem is the most general class of satisfiability problems, so our result effectively demonstrates how any satisfiability problem may be directly mapped to a Chimera graph. Our methods faithfully reproduce the low energy spectrum of the target problems, so therefore may also be used for maximum entropy inference.

  8. Epitope Mapping of Neutralizing Monoclonal Antibodies to Human Interferon-γ Using Human-Bovine Interferon-γ Chimeras

    Science.gov (United States)

    Zuber, Bartek; Rudström, Karin; Ehrnfelt, Cecilia

    2016-01-01

    Our aim was to identify conformational epitopes, recognized by monoclonal antibodies (mAbs) made against human (h) interferon (IFN)-γ. Based on the mAbs' (n = 12) ability to simultaneously bind hIFN-γ in ELISA, 2 epitope clusters with 5 mAbs in each were defined; 2 mAbs recognized unique epitopes. Utilizing the mAbs' lack of reactivity with bovine (b) IFN-γ, epitopes were identified using 7 h/bIFN-γ chimeras where the helical regions (A-F) or the C terminus were substituted with bIFN-γ residues. Chimeras had a N-terminal peptide tag enabling the analysis of mAb recognition of chimeras in ELISA. The 2 mAb clusters mapped to region A and E, respectively; the epitopes of several mAbs also involved additional regions. MAbs in cluster A neutralized, to various degrees, IFN-γ-mediated activation of human cells, in line with the involvement of region A in the IFN-γ receptor interaction. MAbs mapping to region E displayed a stronger neutralizing capacity although this region has not been directly implicated in the receptor interaction. The results corroborate earlier studies and provide a detailed picture of the link between the epitope specificity and neutralizing capacity of mAbs. They further demonstrate the general use of peptide-tagged chimeric proteins as a powerful and straightforward method for efficient mapping of conformational epitopes. PMID:27336613

  9. All together now: Analogies between chimera state collapses and epileptic seizures

    Science.gov (United States)

    Andrzejak, Ralph G.; Rummel, Christian; Mormann, Florian; Schindler, Kaspar

    2016-03-01

    Conceptually and structurally simple mathematical models of coupled oscillator networks can show a rich variety of complex dynamics, providing fundamental insights into many real-world phenomena. A recent and not yet fully understood example is the collapse of coexisting synchronous and asynchronous oscillations into a globally synchronous motion found in networks of identical oscillators. Here we show that this sudden collapse is promoted by a further decrease of synchronization, rather than by critically high synchronization. This strikingly counterintuitive mechanism can be found also in nature, as we demonstrate on epileptic seizures in humans. Analyzing spatiotemporal correlation profiles derived from intracranial electroencephalographic recordings (EEG) of seizures in epilepsy patients, we found a pronounced decrease of correlation at the seizure onsets. Applying our findings in a closed-loop control scheme to models of coupled oscillators in chimera states, we succeed in both provoking and preventing outbreaks of global synchronization. Our findings not only advance the understanding of networks of coupled dynamics but can open new ways to control them, thus offering a vast range of potential new applications.

  10. Aerodynamic study of sounding rocket flows using Chimera and patched multiblock meshes

    Directory of Open Access Journals (Sweden)

    João Alves de Oliveira Neto

    2011-01-01

    Full Text Available Aerodynamic flow simulations over a typical sounding rocket are presented in this paper. The work is inserted in the effort of developing computational tools necessary to simulate aerodynamic flows over configurations of interest for Instituto de Aeronáutica e Espaço of Departamento de Ciência e Tecnologia Aeroespacial. Sounding rocket configurations usually require fairly large fins and, quite frequently, have more than one set of fins. In order to be able to handle such configurations, the present paper presents a novel methodology which combines both Chimera and patched multiblock grids in the discretization of the computational domain. The flows of interest are modeled using the 3-D Euler equations and the work describes the details of discretization procedure, which uses a finite difference approach for structure, body-conforming, multiblock grids. The method is used to calculate the aerodynamics of a sounding rocket vehicle. The results indicate that the present approach can be a powerful aerodynamic analysis and design tool.

  11. Generation of Efficient Germ-Line Chimeras Using Embryonic Stem Cell Injection.

    Science.gov (United States)

    Ritchie, William A

    2015-01-01

    There are many different reasons for producing germ-line chimeras, so a method for producing these is very important both for the testing of stem cells (SC) and for the production of an animal which may be genetically modified (Voncken, Methods Mol Biol 693:11-36, 2011). As with many scientific procedures the theory behind the process is very simple: in this case injection of cells into the blastocoel cavity of an embryo which has developed to the blastocyst stage so as the injected cells can contribute to the inner cell mass (ICM) and hopefully contribute to the germ line of the animal produced (Schneider et al., Stem Cell Rev 5(4):369-377, 2009). Incorporation of the cells into the gonads of the animal produced will allow the testing of those cells and the resulting animal which may be derived from the injected cells (Bradley et al., Nature 309(5965):255-256, 1984). The problems arise because of the size of the cells and the challenge of injection into the blastocoel cavity of a developing embryo.

  12. An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario; Cavaleiro, Mafalda; Christensen, Ulla

    2017-01-01

    tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed...

  13. Magnetorotational dynamo chimeras. The missing link to turbulent accretion disk dynamo models?

    Science.gov (United States)

    Riols, A.; Rincon, F.; Cossu, C.; Lesur, G.; Ogilvie, G. I.; Longaretti, P.-Y.

    2017-02-01

    In Keplerian accretion disks, turbulence and magnetic fields may be jointly excited through a subcritical dynamo mechanisminvolving magnetorotational instability (MRI). This dynamo may notably contribute to explaining the time-variability of various accreting systems, as high-resolution simulations of MRI dynamo turbulence exhibit statistical self-organization into large-scale cyclic dynamics. However, understanding the physics underlying these statistical states and assessing their exact astrophysical relevance is theoretically challenging. The study of simple periodic nonlinear MRI dynamo solutions has recently proven useful in this respect, and has highlighted the role of turbulent magnetic diffusion in the seeming impossibility of a dynamo at low magnetic Prandtl number (Pm), a common regime in disks. Arguably though, these simple laminar structures may not be fully representative of the complex, statistically self-organized states expected in astrophysical regimes. Here, we aim at closing this seeming discrepancy by reporting the numerical discovery of exactly periodic, yet semi-statistical "chimeral MRI dynamo states" which are the organized outcome of a succession of MRI-unstable, non-axisymmetric dynamical stages of different forms and amplitudes. Interestingly, these states, while reminiscent of the statistical complexity of turbulent simulations, involve the same physical principles as simpler laminar cycles, and their analysis further confirms the theory that subcritical turbulent magnetic diffusion impedes the sustainment of an MRI dynamo at low Pm. Overall, chimera dynamo cycles therefore offer an unprecedented dual physical and statistical perspective on dynamos in rotating shear flows, which may prove useful in devising more accurate, yet intuitive mean-field models of time-dependent turbulent disk dynamos. Movies associated to Fig. 1 are available at http://www.aanda.org

  14. Plasmid Replicons from Pseudomonas Are Natural Chimeras of Functional, Exchangeable Modules

    Science.gov (United States)

    Bardaji, Leire; Añorga, Maite; Ruiz-Masó, José A.; del Solar, Gloria; Murillo, Jesús

    2017-01-01

    Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas, including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities. PMID:28243228

  15. Novel pre-mRNA splicing of intronically integrated HBV generates oncogenic chimera in hepatocellular carcinoma.

    Science.gov (United States)

    Chiu, Yung-Tuen; Wong, John K L; Choi, Shing-Wan; Sze, Karen M F; Ho, Daniel W H; Chan, Lo-Kong; Lee, Joyce M F; Man, Kwan; Cherny, Stacey; Yang, Wan-Ling; Wong, Chun-Ming; Sham, Pak-Chung; Ng, Irene O L

    2016-06-01

    Hepatitis B virus (HBV) integration is common in HBV-associated hepatocellular carcinoma (HCC) and may play an important pathogenic role through the production of chimeric HBV-human transcripts. We aimed to screen the transcriptome for HBV integrations in HCCs. Transcriptome sequencing was performed on paired HBV-associated HCCs and corresponding non-tumorous liver tissues to identify viral-human chimeric sites. Validation was further performed in an expanded cohort of human HCCs. Here we report the discovery of a novel pre-mRNA splicing mechanism in generating HBV-human chimeric protein. This mechanism was exemplified by the formation of a recurrent HBV-cyclin A2 (CCNA2) chimeric transcript (A2S), as detected in 12.5% (6 of 48) of HCC patients, but in none of the 22 non-HCC HBV-associated cirrhotic liver samples examined. Upon the integration of HBV into the intron of the CCNA2 gene, the mammalian splicing machinery utilized the foreign splice sites at 282nt. and 458nt. of the HBV genome to generate a pseudo-exon, forming an in-frame chimeric fusion with CCNA2. The A2S chimeric protein gained a non-degradable property and promoted cell cycle progression, demonstrating its potential oncogenic functions. A pre-mRNA splicing mechanism is involved in the formation of HBV-human chimeric proteins. This represents a novel and possibly common mechanism underlying the formation of HBV-human chimeric transcripts from intronically integrated HBV genome with functional impact. HBV is involved in the mammalian pre-mRNA splicing machinery in the generation of potential tumorigenic HBV-human chimeras. This study also provided insight on the impact of intronic HBV integration with the gain of splice sites in the development of HBV-associated HCC. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  16. The Crystal Structure of a Maxi/Mini-Ferritin Chimera Reveals Guiding Principles for the Assembly of Protein Cages

    Energy Technology Data Exchange (ETDEWEB)

    Cornell, Thomas A. [Department; Division; Srivastava, Yogesh [Genome; Jauch, Ralf [Genome Institute of Singapore, Singapore; Genome; Fan, Rongli [Division; Orner, Brendan P. [Department; Division

    2017-07-19

    Cage proteins assemble into nanoscale structures with large central cavities. They play roles, including those as virus capsids and chaperones, and have been applied to drug delivery and nanomaterials. Furthermore, protein cages have been used as model systems to understand and design protein quaternary structure. Ferritins are ubiquitous protein cages that manage iron homeostasis and oxidative damage. Two ferritin subfamilies have strongly similar tertiary structure yet distinct quaternary structure: maxi-ferritins normally assemble into 24-meric, octahedral cages with C-terminal E-helices centered around 4-fold symmetry axes, and mini-ferritins are 12-meric, tetrahedral cages with 3-fold axes defined by C-termini lacking E-domains. To understand the role E-domains play in ferritin quaternary structure, we previously designed a chimera of a maxi-ferritin E-domain fused to the C-terminus of a mini-ferritin. The chimera is a 12-mer cage midway in size between those of the maxi- and mini-ferritin. The research described herein sets out to understand (a) whether the increase in size over a typical mini-ferritin is due to a frozen state where the E-domain is flipped out of the cage and (b) whether the symmetrical preference of the E-domain in the maxi-ferritin (4-fold axis) overrules the C-terminal preference in the mini-ferritin (3-fold axis). With a 1.99 Å resolution crystal structure, we determined that the chimera assembles into a tetrahedral cage that can be nearly superimposed with the parent mini-ferritin, and that the E-domains are flipped external to the cage at the 3-fold symmetry axes.

  17. Immunogenicity of a West Nile Virus DIII-Cholera Toxin A2/B Chimera after Intranasal Delivery

    Directory of Open Access Journals (Sweden)

    Juliette K. Tinker

    2014-04-01

    Full Text Available West Nile virus (WNV causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.

  18. Particle identification method in the CsI(Tl) scintillator used for the CHIMERA 4 pi detector

    CERN Document Server

    Alderighi, M; Basssini, R; Berceanu, I; Blicharska, J; Boiano, C; Borderie, B; Bougault, R; Bruno, M; Cali, C; Cardella, G; Cavallaro, S; D'Agostino, M; D'andrea, M; Dayras, R; De Filippo, E; Fichera, F; Geraci, E; Giustolisi, F; Grzeszczuk, A; Guardone, N; Guazzoni, P; Guinet, D; Iacono-Manno, M; Kowalski, S; La Guidara, E; Lanchais, A L; Lanzalone, G; Lanzanò, G; Le Neindre, N; Li, S; Maiolino, C; Majka, Z; Manfredi, G; Nicotra, D; Paduszynski, T; Pagano, A; Papa, M; Petrovici, C M; Piasecki, E; Pirrone, S; Politi, G; Pop, A; Porto, F; Rivet, M F; Rosato, E; Sacca, G; Sechi, G; Simion, V; Sperduto, M L; Steckmeyer, J C; Trifiró, A; Trimarchi, M; Urso, S; Vannini, G; Vigilante, M; Wilczynski, J; Wu, H; Xiao, Z; Zetta, L; Zipper, W

    2002-01-01

    The charged particle identification obtained by the analysis of signals coming from the CsI(Tl) detectors of the CHIMERA 4 pi heavy-ion detector is presented. A simple double-gate integration method, with the use of the cyclotron radiofrequency as reference time, results in low thresholds for isotopic particle identification. The dependence of the identification quality on the gate generation timing is discussed. Isotopic identification of light ions up to Beryllium is clearly seen. For the first time also the identification of Z=5 particles is observed. The identification of neutrons interacting with CsI(Tl) by (n,alpha) and (n,gamma) reactions is also discussed.

  19. Newspaper coverage of human-pig chimera research: A qualitative study on select media coverage of scientific breakthrough.

    Science.gov (United States)

    Hagan-Brown, Abena; Favaretto, Maddalena; Borry, Pascal

    2017-07-01

    A recently published article in the journal Cell by scientists from the Salk Institute highlighted the successful integration of stem cells from humans in pig embryos. This marks the first step toward the goal of growing human organs in animals for transplantation. There has, to date, been no research performed on the presentation of this breakthrough in the media. We thus assessed early newspaper coverage of the chimera study, looking into the descriptions as well as the benefits and concerns raised by the study mentioned by newspaper sources. We looked at newspaper coverage of the human-pig chimera study in the two weeks after the publication of the article describing the breakthrough in Cell. This time period spanned from January 26 to February 9, 2017. We used the LexisNexis Academic database and identified articles using the search string "hybrid OR chimera AND pig OR human OR embryo." The relevant articles were analyzed using qualitative content analysis. Two researchers openly coded the articles independently using themes that emerged from the raw texts. Our search yielded 31 unique articles, after extensive screening for relevance and duplicates. Through our analysis, we were able to identify several themes in a majority of the texts. Almost every article gave descriptive information about the chimera experiment with details about the study findings. All of the articles mentioned the benefits of the study, citing both immediate- and long-term goals, which included creating transplantable human organs, disease and drug development, and personalized medicine, among others. Some of the articles highlighted some ethical, social, and health concerns that the study and its future implications pose. Many of the articles also offered reassurances over the concerns brought up by the experiment. Our results appeared to align with similar research performed on the media representation of sensitive scientific news coverage. We also explored the inconsistency between

  20. Gene Amplification by PCR and Subcloning into a GFP-Fusion Plasmid Expression Vector as a Molecular Biology Laboratory Course

    Science.gov (United States)

    Bornhorst, Joshua A.; Deibel, Michael A.; Mulnix, Amy B.

    2004-01-01

    A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the…

  1. An Activin A/BMP2 chimera displays bone healing properties superior to those of BMP2

    Science.gov (United States)

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C.; Ye, Sang-kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-01-01

    Recombinant Bone Morphogenetic Protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue which when mutated to the corresponding BMP2 residue resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures. PMID:24692083

  2. An activin A/BMP2 chimera, AB204, displays bone-healing properties superior to those of BMP2.

    Science.gov (United States)

    Yoon, Byung-Hak; Esquivies, Luis; Ahn, Chihoon; Gray, Peter C; Ye, Sang-Kyu; Kwiatkowski, Witek; Choe, Senyon

    2014-09-01

    Recombinant bone morphogenetic protein 2 (rhBMP2) has been used clinically to treat bone fractures in human patients. However, the high doses of rhBMP2 required for a therapeutic response can cause undesirable side effects. Here, we demonstrate that a novel Activin A/BMP2 (AB2) chimera, AB204, promotes osteogenesis and bone healing much more potently and effectively than rhBMP2. Remarkably, 1 month of AB204 treatment completely heals tibial and calvarial defects of critical size in mice at a concentration 10-fold lower than a dose of rhBMP2 that only partially heals the defect. We determine the structure of AB204 to 2.3 Å that reveals a distinct BMP2-like fold in which the Activin A sequence segments confer insensitivity to the BMP2 antagonist Noggin and an affinity for the Activin/BMP type II receptor ActRII that is 100-fold greater than that of BMP2. The structure also led to our identification of a single Activin A-derived amino acid residue, which, when mutated to the corresponding BMP2 residue, resulted in a significant increase in the affinity of AB204 for its type I receptor BMPRIa and a further enhancement in AB204's osteogenic potency. Together, these findings demonstrate that rationally designed AB2 chimeras can provide BMP2 substitutes with enhanced potency for treating non-union bone fractures. © 2014 American Society for Bone and Mineral Research.

  3. A testis-mediated germline chimera production based on transfer of chicken testicular cells directly into heterologous testes.

    Science.gov (United States)

    Lee, Young Mok; Jung, Jin Gyoung; Kim, Jin Nam; Park, Tae Sub; Kim, Tae Min; Shin, Sang Su; Kang, Dae Kyung; Lim, Jeong Mook; Han, Jae Yong

    2006-09-01

    In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 x 10(7) cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 x 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.

  4. SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

    Science.gov (United States)

    Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

    2013-12-20

    Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

  5. Rational management of hunting and gathering: An aristotelian entelechy or a chimera?

    Directory of Open Access Journals (Sweden)

    Juan Mario Vargas Yáñez

    2018-01-01

    Full Text Available Rational management of hunting and gathering: An Aristotelian entelechy or a chimera? Since the dawn of time, consumption of animal protein has been crucially important to the evolution of humans. Cooperative hunting is an Evolutionarily Stable Strategy from the time when Palaeolithic hunters specialized in the pursuit of large animals. The entire process involves making decisions related to game management, some of which are still used by contemporary hunter-gatherers. In these societies, which still practice subsistence hunting, the composition of their diets and the exploitation techniques employed are explained by the theoretical underpinnings contained in the Optimal Foraging Theory (OFT. In order to test this theory, we have taken into account four decisions involving the adoption of different management strategies by hunter-gatherers: I what to eat, II where and how to look for food, III the most appropriate size and composition of the group of hunter-gatherers and IV the most efficient guidelines for the handling and distribution of prey. The OFT has been tested several times, and in a high percentage of cases studied, its predictions fit observations made in various hunter-gatherer societies. This means that maximizing catch yields in the short term is their main concern, rather than behaving as ‘cautious predators’ or passively selecting prey according to their size, accessibility and abundance. Such behaviour does not contradict the possibility that hunter-gatherers can act as conservationists, but discards the idea that this is the main focus of their way of life. There is no doubt that indigenous peoples have a wide knowledge of the environment, but it is unclear this knowledge is used to maintain a balance with nature or to be more efficient hunters. It more likely that the presence of sustainable hunting of game is due to low hunting pressure in areas where animal prey is abundant, and is thus an epiphenomenon rather than a

  6. Stability and Conformation of a Chemoreceptor HAMP Domain Chimera Correlates with Signaling Properties.

    Science.gov (United States)

    Sukomon, Nattakan; Widom, Joanne; Borbat, Peter P; Freed, Jack H; Crane, Brian R

    2017-04-11

    HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two

  7. Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

    Science.gov (United States)

    Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

    2015-08-01

    A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

  8. Pharmacological characterisation of α6β4* nicotinic acetylcholine receptors assembled from three different α6/α3 subunit chimeras in tsA201 cells

    DEFF Research Database (Denmark)

    Jensen, Anne Bjørnskov; Hoestgaard-Jensen, Kirsten; Jensen, Anders A.

    2014-01-01

    should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these receptors....

  9. Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

    Science.gov (United States)

    Freer, E; Moreno, E; Moriyón, I; Pizarro-Cerdá, J; Weintraub, A; Gorvel, J P

    1996-01-01

    A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or

  10. A GTPase chimera illustrates an uncoupled nucleotide affinity and release rate, Providing insight into the activation mechanism

    DEFF Research Database (Denmark)

    Guilfoyle, Amy P.; Deshpande, Chandrika N.; Font Sadurni, Josep

    2014-01-01

    The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example...... for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains...... GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped...

  11. Optimization of three-dimensional imaging on in vitro produced porcine blastocysts and chimeras for stem cell testing

    DEFF Research Database (Denmark)

    Secher, Jan Ole Bertelsen; Freude, Kristine; Li, Rong

    2015-01-01

    of the ICM they are likely to contribute to the fetus and if they are located in the area of the TE they are likely to contribute to the fetal membranes. In this article, we optimize on methods for embryo staining and mounting so that the exact location of injected stem cells within preimplantation porcine......Differential staining is an immunocytochemical staining that visualizes trophectoderm (TE) and the inner cell mass (ICM) of the blastocysts. It is used to determine the blastocyst quality, but could also be a useful tool to assess the integration site of injected cells into the early embryo....... This is relevant for testing of presumed pluripotent stem cells. The gold standard for pluripotent stem cells is to test if the cells are capable of contributing to germline chimeras. Differential staining can be used to evaluate the possibility of chimeric contribution; if the cells are located in the area...

  12. Neratinib Plus Paclitaxel vs Trastuzumab Plus Paclitaxel in Previously Untreated Metastatic ERBB2-Positive Breast Cancer: The NEfERT-T Randomized Clinical Trial.

    Science.gov (United States)

    Awada, Ahmad; Colomer, Ramon; Inoue, Kenichi; Bondarenko, Igor; Badwe, Rajendra A; Demetriou, Georgia; Lee, Soo-Chin; Mehta, Ajay O; Kim, Sung-Bae; Bachelot, Thomas; Goswami, Chanchal; Deo, Suryanarayan; Bose, Ron; Wong, Alvin; Xu, Feng; Yao, Bin; Bryce, Richard; Carey, Lisa A

    2016-12-01

    Efficacious ERBB2 (formerly HER2 or HER2/neu)-directed treatments, in addition to trastuzumab and lapatinib, are needed. To determine whether neratinib, an irreversible pan-ERBB tyrosine kinase inhibitor, plus paclitaxel improves progression-free survival compared with trastuzumab plus paclitaxel in the first-line treatment of recurrent and/or metastatic ERBB2-positive breast cancer. In the randomized, controlled, open-label NEfERT-T trial conducted from August 2009 to December 2014 at 188 centers in 34 countries in Europe, Asia, Africa, and North America, 479 women with previously untreated recurrent and/or metastatic ERBB2-positive breast cancer were randomized to 1 of 2 treatment arms (neratinib-paclitaxel [n = 242] or trastuzumab-paclitaxel [n = 237]). Women with asymptomatic central nervous system metastases were eligible, and randomization was stratified by prior trastuzumab and lapatinib exposure, hormone-receptor status, and region. Women received neratinib (240 mg/d orally) or trastuzumab (4 mg/kg then 2 mg/kg weekly), each combined with paclitaxel (80 mg/m2 on days 1, 8, and 15 every 28 days). Primary prophylaxis for diarrhea was not mandatory. The primary outcome was progression-free survival. Secondary end points were response rate, clinical benefit rate, duration of response, frequency, and time to symptomatic and/or progressive central nervous system lesions, and safety. The intent-to-treat population comprised 479 women 18 years or older (neratinib-paclitaxel, n = 242; trastuzumab-paclitaxel, n = 237) randomized and stratified in their respective treatment arms by prior trastuzumab and lapatinib exposure, hormone-receptor status, and region. Median progression-free survival was 12.9 months (95% CI, 11.1-14.9) with neratinib-paclitaxel and 12.9 months (95% CI, 11.1-14.8) with trastuzumab-paclitaxel (hazard ratio [HR], 1.02; 95% CI, 0.81-1.27; P =.89). With neratinib-paclitaxel, the incidence of central nervous system recurrences was

  13. Three Dimensional Visualization and Fractal Analysis of Mosaic Patches in Rat Chimeras: Cell Assortment in Liver, Adrenal Cortex and Cornea

    Science.gov (United States)

    Iannaccone, Stephen; Zhou, Yue; Walterhouse, David; Taborn, Greg; Landini, Gabriel; Iannaccone, Philip

    2012-01-01

    The production of organ parenchyma in a rapid and reproducible manner is critical to normal development. In chimeras produced by the combination of genetically distinguishable tissues, mosaic patterns of cells derived from the combined genotypes can be visualized. These patterns comprise patches of contiguously similar genotypes and are different in different organs but similar in a given organ from individual to individual. Thus, the processes that produce the patterns are regulated and conserved. We have previously established that mosaic patches in multiple tissues are fractal, consistent with an iterative, recursive growth model with simple stereotypical division rules. Fractal dimensions of various tissues are consistent with algorithmic models in which changing a single variable (e.g. daughter cell placement after division) switches the mosaic pattern from islands to stripes of cells. Here we show that the spiral pattern previously observed in mouse cornea can also be visualized in rat chimeras. While it is generally held that the pattern is induced by stem cell division dynamics, there is an unexplained discrepancy in the speed of cellular migration and the emergence of the pattern. We demonstrate in chimeric rat corneas both island and striped patterns exist depending on the age of the animal. The patches that comprise the pattern are fractal, and the fractal dimension changes with the age of the animal and indicates the constraint in patch complexity as the spiral pattern emerges. The spiral patterns are consistent with a loxodrome. Such data are likely to be relevant to growth and cell division in organ systems and will help in understanding how organ parenchyma are generated and maintained from multipotent stem cell populations located in specific topographical locations within the organ. Ultimately, understanding algorithmic growth is likely to be essential in achieving organ regeneration in vivo or in vitro from stem cell populations. PMID:22347498

  14. Tick-Borne Langat/Mosquito-Borne Dengue Flavivirus Chimera, a Candidate Live Attenuated Vaccine for Protection against Disease Caused by Members of the Tick-Borne Encephalitis Virus Complex: Evaluation in Rhesus Monkeys and in Mosquitoes

    Science.gov (United States)

    Pletnev, Alexander G.; Bray, Michael; Hanley, Kathryn A.; Speicher, Jim; Elkins, Randy

    2001-01-01

    Langat virus (LGT), strain TP21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained LGT genes for premembrane (preM) and envelope (E) glycoprotein and all other sequences derived from dengue type 4 virus (DEN4). The live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective E epitopes among themselves and with LGT. Toward that end the chimera, initially recovered in mosquito cells, was adapted to grow to high titer in qualified simian Vero cells. When inoculated intraperitoneally (i.p.), the Vero cell-adapted LGT TP21/DEN4 chimera remained completely attenuated for SCID mice. Significantly, the chimera protected immunocompetent mice against the most virulent tick-borne encephalitis virus (TBEV). Subsequently, rhesus monkeys were immunized in groups of 4 with 105 or 107 PFU of LGT strain TP21, with 105 PFU of DEN4, or with 103, 105, or 107 PFU of the chimera. Each of the monkeys inoculated with DEN4 or LGT TP21 became viremic, and the duration of viremia ranged from 1 to 5 days. In contrast, viremia was detected in only 1 of 12 monkeys inoculated with the LGT TP21/DEN4 chimera; in this instance the level of viremia was at the limit of detection. All monkeys immunized with the chimera or LGT TP21 virus developed a moderate to high level of neutralizing antibodies against LGT TP21 as well as TBEV and were completely protected against subsequent LGT TP21 challenge, whereas monkeys previously immunized with DEN4 virus became viremic when challenged with LGT TP21. These observations suggest that the chimera is attenuated, immunogenic, and able to induce a protective immune response. Furthermore, passive transfer of serum from monkeys immunized with chimera conferred significant protection to mice subsequently challenged with 100 i.p. 50% lethal doses of the highly virulent TBEV. The issue of transmissibility of the chimera

  15. Bid chimeras indicate that most BH3-only proteins can directly activate Bak and Bax, and show no preference for Bak versus Bax.

    Science.gov (United States)

    Hockings, C; Anwari, K; Ninnis, R L; Brouwer, J; O'Hely, M; Evangelista, M; Hinds, M G; Czabotar, P E; Lee, E F; Fairlie, W D; Dewson, G; Kluck, R M

    2015-04-23

    The mitochondrial pathway of apoptosis is initiated by Bcl-2 homology region 3 (BH3)-only members of the Bcl-2 protein family. On upregulation or activation, certain BH3-only proteins can directly bind and activate Bak and Bax to induce conformation change, oligomerization and pore formation in mitochondria. BH3-only proteins, with the exception of Bid, are intrinsically disordered and therefore, functional studies often utilize peptides based on just their BH3 domains. However, these reagents do not possess the hydrophobic membrane targeting domains found on the native BH3-only molecule. To generate each BH3-only protein as a recombinant protein that could efficiently target mitochondria, we developed recombinant Bid chimeras in which the BH3 domain was replaced with that of other BH3-only proteins (Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk). The chimeras were stable following purification, and each immunoprecipitated with full-length Bcl-xL according to the specificity reported for the related BH3 peptide. When tested for activation of Bak and Bax in mitochondrial permeabilization assays, Bid chimeras were ~1000-fold more effective than the related BH3 peptides. BH3 sequences from Bid and Bim were the strongest activators, followed by Puma, Hrk, Bmf and Bik, while Bad and Noxa were not activators. Notably, chimeras and peptides showed no apparent preference for activating Bak or Bax. In addition, within the BH3 domain, the h0 position recently found to be important for Bax activation, was important also for Bak activation. Together, our data with full-length proteins indicate that most BH3-only proteins can directly activate both Bak and Bax.

  16. Microbicidal effect of the lactoferrin peptides lactoferricin17-30, lactoferrampin265-284, and lactoferrin chimera on the parasite Entamoeba histolytica.

    Science.gov (United States)

    López-Soto, Fernando; León-Sicairos, Nidia; Nazmi, Kamran; Bolscher, Jan G; de la Garza, Mireya

    2010-06-01

    Entamoeba histolytica is a parasitic protozoan that produces amoebiasis, an intestinal disease characterized by ulcerative colitis and dysentery. In some cases, trophozoites can travel to the liver leading to hepatic abscesses and death. Recently, lactoferrin and lactoferricin B have been shown to be amoebicidal in axenic cultures. The aim of this work was to determine whether the lactoferrin-peptides lactoferricin amino acids 17-30, lactoferrampin amino acids 265-284, and lactoferrin chimera which is a fusion product of the two peptides, are capable of producing a microbicidal effect to trophozoites of E. histolytica. We evaluated the killing effect of these peptides in growth kinetics carried out in axenic culture medium to which different concentrations of peptides were added. At 50 muM of peptide concentration, lactoferricin and lactoferrampin had a moderate amoebicidal effect, since a 45-50% of trophozoites remained viable at 24 h culture. However, at 50 microM of the lactoferrin chimera 75% amoeba were killed whereas at 100 microM all cells died. These data indicate that of lactoferrin-peptides mainly the chimera have amoebicidal activity in a time- and concentration-dependent manner. The lactoferrin-peptides might be useful as therapeutic agents against amoebiasis and thereby diminish the use of metronidazole, which is extremely toxic for the host.

  17. α-Peptide-Oligourea Chimeras: Stabilization of Short α-Helices by Non-Peptide Helical Foldamers.

    Science.gov (United States)

    Fremaux, Juliette; Mauran, Laura; Pulka-Ziach, Karolina; Kauffmann, Brice; Odaert, Benoit; Guichard, Gilles

    2015-08-17

    Short α-peptides with less than 10 residues generally display a low propensity to nucleate stable helical conformations. While various strategies to stabilize peptide helices have been previously reported, the ability of non-peptide helical foldamers to stabilize α-helices when fused to short α-peptide segments has not been investigated. Towards this end, structural investigations into a series of chimeric oligomers obtained by joining aliphatic oligoureas to the C- or N-termini of α-peptides are described. All chimeras were found to be fully helical, with as few as 2 (or 3) urea units sufficient to propagate an α-helical conformation in the fused peptide segment. The remarkable compatibility of α-peptides with oligoureas described here, along with the simplicity of the approach, highlights the potential of interfacing natural and non-peptide backbones as a means to further control the behavior of α-peptides. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Localization of the CAPRICE-ENHANCER OF TRY AND CPC1 chimera protein in Arabidopsis root epidermis.

    Science.gov (United States)

    Tominaga-Wada, Rumi; Kurata, Tetsuya; Wada, Takuji

    2017-09-01

    The CAPRICE (CPC) encodes an R3-type MYB transcription factor, which promotes root-hair differentiation. Previously, we showed that the CPC protein moves from the non-hair cell to the neighboring cell and induces root-hair differentiation in Arabidopsis. In addition, we proposed two cell-to-cell movement signal sequences, S1 and S2, in CPC. However, an S1:2xGFP:S2 chimera protein did not move between root epidermal cells. Here, we show that the S1 and S2 sequences do not confer cell-to-cell movement or nuclear localization ability to a GFP protein. The ENHANCER OF TRY AND CPC1 (ETC1) gene encodes the CPC homolog R3 MYB; this protein does not possess cell-to-cell movement ability or the S1 sequence. To elucidate whether the S1 sequence can induce cell-to-cell movement ability in ETC1, CPCp:S1:ETC1:2xGFP was constructed and introduced into Arabidopsis. Our results indicate that the addition of the S1 sequence was not sufficient for ETC1 to acquire cell-to-cell movement ability.

  19. Particle gamma correlations in {sup 12}C measured with the CsI(Tl) based detector array CHIMERA

    Energy Technology Data Exchange (ETDEWEB)

    Cardella, G., E-mail: cardella@ct.infn.it [INFN - Sezione di Catania, Via S. Sofia, 95123 Catania (Italy); Acosta, L. [INFN - Sezione di Catania, Via S. Sofia, 95123 Catania (Italy); Amorini, F. [INFN - Laboratori Nazionali del Sud, Via S. Sofia, Catania (Italy); Auditore, L. [INFN Gruppo collegato di Messina and Dip. di Fisica e Scienze della Terra, Università di Messina (Italy); Berceanu, I. [Institute for Physics and Nuclear Engineering, Bucharest (Romania); Castoldi, A. [INFN Sezione di Milano e Politecnico Milano (Italy); De Filippo, E. [INFN - Sezione di Catania, Via S. Sofia, 95123 Catania (Italy); Dell' Aquila, D. [Dipartimento di scienze Fisiche, Università Federico II and INFN Sezione di Napoli (Italy); Francalanza, L. [INFN - Laboratori Nazionali del Sud, Via S. Sofia, Catania (Italy); Dip. di Fisica e Astronomia, Università di Catania, Via S. Sofia, Catania (Italy); Gnoffo, B. [INFN - Sezione di Catania, Via S. Sofia, 95123 Catania (Italy); Guazzoni, C. [INFN Sezione di Milano e Politecnico Milano (Italy); Lanzalone, G. [INFN - Laboratori Nazionali del Sud, Via S. Sofia, Catania (Italy); Facoltà di Ingegneria e Architettura, Università Kore, Enna (Italy); Lombardo, I. [Dipartimento di scienze Fisiche, Università Federico II and INFN Sezione di Napoli (Italy); Minniti, T.; Morgana, E.; Norella, S. [INFN Gruppo collegato di Messina and Dip. di Fisica e Scienze della Terra, Università di Messina (Italy); Pagano, A. [INFN - Sezione di Catania, Via S. Sofia, 95123 Catania (Italy); Pagano, E.V. [INFN - Laboratori Nazionali del Sud, Via S. Sofia, Catania (Italy); Dip. di Fisica e Astronomia, Università di Catania, Via S. Sofia, Catania (Italy); Papa, M.; Pirrone, S. [INFN - Sezione di Catania, Via S. Sofia, 95123 Catania (Italy); and others

    2015-11-01

    The gamma decay of the first excited 4.44 MeV 2+level of {sup 12}C, populated by inelastic scattering of proton and {sup 16}O beams at various energies was studied in order to test γ-ray detection efficiency and the quality of angular distribution information given by the CsI(Tl) detectors of the 4π CHIMERA array. The γ-decay was measured in coincidence with ejectile scattered particles in an approximately 4π geometry allowing to extract the angular distribution in the reference frame of recoiling {sup 12}C target. The typical sin{sup 2} (2θ) behavior of angular distribution was observed in the case of {sup 16}O beam. Besides that, for the proton beam, in order to explain the observed distribution, the addition of an incoherent flat contribution was required. This latter is the effect of proton spin flip events allowing the population of M=±1 magnetic substates, that is not possible in reactions induced by {sup 16}O beam. A comparison with previously collected data, obtained measuring only in and out of plane proton-γ-ray coincidences, confirms the good quality of the angular distribution information given by the apparatus. Possible applications with radioactive beams are outlined.

  20. Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing.

    Science.gov (United States)

    Aronoff-Spencer, Eliah; Venkatesh, A G; Sun, Alex; Brickner, Howard; Looney, David; Hall, Drew A

    2016-12-15

    Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a "renewable" capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Chimeras Reveal a Single Lipid-Interface Residue that Controls MscL Channel Kinetics as well as Mechanosensitivity

    Directory of Open Access Journals (Sweden)

    Li-Min Yang

    2013-02-01

    Full Text Available MscL, the highly conserved bacterial mechanosensitive channel of large conductance, serves as an osmotic “emergency release valve,” is among the best-studied mechanosensors, and is a paradigm of how a channel senses and responds to membrane tension. Although all homologs tested thus far encode channel activity, many show functional differences. We tested Escherichia coli and Staphylococcus aureus chimeras and found that the periplasmic region of the protein, particularly E. coli I49 and the equivalent S. aureus F47 at the periplasmic lipid-aqueous interface of the first transmembrane domain, drastically influences both the open dwell time and the threshold of channel opening. One mutant shows a severe hysteresis, confirming the importance of this residue in determining the energy barriers for channel gating. We propose that this site acts similarly to a spring for a clasp knife, adjusting the resistance for obtaining and stabilizing an open or closed channel structure.

  2. CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response

    Directory of Open Access Journals (Sweden)

    Birx Deborah L

    2006-04-01

    Full Text Available Abstract Background Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A. Results We have used the IFN-γ ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296–304; YL9 was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa, revealed that peptide sets based upon an homologous subtype (either isolate or consensus only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein, each set was capable of detecting unique responses not identified with the other peptide sets. Conclusion Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in

  3. Efficient expression of functional (α6β22β3 AChRs in Xenopus oocytes from free subunits using slightly modified α6 subunits.

    Directory of Open Access Journals (Sweden)

    Carson Kai-Kwong Ley

    Full Text Available Human (α6β2(α4β2β3 nicotinic acetylcholine receptors (AChRs are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β22β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β22β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells.

  4. [Leaf anatomy of the mosaic ficus benjamina cv. Starlight and interaction of source and sink chimera components].

    Science.gov (United States)

    Labunskaia, E A; Zhigalova, T V; Chub, V V

    2007-01-01

    Leaf anatomy was studied in the mosaic Ficus benjamina cv. Starlight and non-chimeric Ficus benjamina cv. Daniel. The number of chloroplasts in a white, chlorophyll-deficient tissue declines as compared to the green tissue. However, their functional activity is retained. The leaf of the mosaic F. benjamina contains two or, sometimes, three subepidermal layers. Mesophyll forms one layer in the green and white parts of leaf palisade and one white and one green layer in the transitional zone (edge). In the transitional zone, green spongy mesophyll is located between two white spongy layers and the proportion of photosynthesizing cells varies. In cv. Daniel, there are two subepidermal layers and one layer of columnar mesophyll cells. According to the morphometry data, the proportion of white zone in the leaf correlates with the leaf position in the whole shoot: the higher the branch order, the larger the proportion of white zone. The total leaf area depends also on its position in the shoot. No such correlation was found in non-chimeric F. benjamina cv. Daniel. In the mosaic chimera, the source-sink status appears to depend on the leaf position in the shoot. Experiments with individual shoots of the same order and elimination of all lateral shoots have shown that the proportion of white zone in new leaves on the shoot increases with the total area of green zone. Thus, the area of assimilating shoot surface affects the formation of leaves in the meristem. A hypothesis was put forward that the source-sink state affects the ratio of green and white parts in the leaf primordium. Products of photosynthesis (carbohydrates) are a possible metabolic signal affecting the meristem. It cannot be excluded as well that the hormonal state undergoes changes in the chimeric plant.

  5. Chimera-free, high copy number YAC libraries and efficient methods of analysis. Final technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-10-01

    The first experiment involved a low chimera YAC library in recombination deficient host strains. To determine if the genetic background of the yeast host strain contributes to the formation of chimeric YACs the same YAC ligation mixture was introduced into three isogenic yeast hosts differing only in their recombination abilities. To prepare YACs, human genomic DNA was partially digested with EcoR1 and then ligated to YAC vector pCGS966 arms. DNA was size fractionated before and after ligation by preparative pulsed field gel electrophoresis (CHEF), selecting for fragments greater than 400 kb, and introduced into competent spheroplasts. CHEF gel Southern blots of resulting colony-purified YACs were probed with human DNA to determine if multiple YACs or YAC fragments were present in the same cell. The frequency of chimeric YACs was measured by fluorescence in situ hybridization (FISH) of YACs to human prometaphase spreads. YACs that hybridized to more than one location were assumed to be chimeric. In the second experiment new YAC vectors featuring tags for capture of YACs and YAC inserts were constructed. Yeast Artificial Chromosomes (YACs) have been of tremendous value in the physical mapping of the human genome. Because they can carry very large inserts, YACs are likely not only to contain entire genes but also their control elements. However, the only mode of purification of YAC DNA from current commonly used YAC libraries such as the CEPH library is by pulsed field gel electrophoresis. This is an inefficient, time consuming process and due to the single copy nature of these YACs, often result in poor yields. The vector pCGS1000 was designed to test new efficient ways of YAC DNA purification.

  6. CHIMERA: Top-down model for hierarchical, overlapping and directed cluster structures in directed and weighted complex networks

    Science.gov (United States)

    Franke, R.

    2016-11-01

    In many networks discovered in biology, medicine, neuroscience and other disciplines special properties like a certain degree distribution and hierarchical cluster structure (also called communities) can be observed as general organizing principles. Detecting the cluster structure of an unknown network promises to identify functional subdivisions, hierarchy and interactions on a mesoscale. It is not trivial choosing an appropriate detection algorithm because there are multiple network, cluster and algorithmic properties to be considered. Edges can be weighted and/or directed, clusters overlap or build a hierarchy in several ways. Algorithms differ not only in runtime, memory requirements but also in allowed network and cluster properties. They are based on a specific definition of what a cluster is, too. On the one hand, a comprehensive network creation model is needed to build a large variety of benchmark networks with different reasonable structures to compare algorithms. On the other hand, if a cluster structure is already known, it is desirable to separate effects of this structure from other network properties. This can be done with null model networks that mimic an observed cluster structure to improve statistics on other network features. A third important application is the general study of properties in networks with different cluster structures, possibly evolving over time. Currently there are good benchmark and creation models available. But what is left is a precise sandbox model to build hierarchical, overlapping and directed clusters for undirected or directed, binary or weighted complex random networks on basis of a sophisticated blueprint. This gap shall be closed by the model CHIMERA (Cluster Hierarchy Interconnection Model for Evaluation, Research and Analysis) which will be introduced and described here for the first time.

  7. Screening of biotechnical parameters for production of bovine inter-subspecies embryonic chimeras by the aggregation of tetraploid Bos indicus and diploid crossbred Bos taurus embryos.

    Science.gov (United States)

    Razza, Eduardo M; Satrapa, Rafael A; Emanuelli, Isabele P; Barros, Ciro M; Nogueira, Marcelo F G

    2016-03-01

    The aggregation of a tetraploid zebu embryo (Bos indicus, a thermotolerant breed) with a diploid taurine embryo (Bos taurus, a thermosensitive breed) should create a complete taurine fetus, whose extra-embryonic components, e.g., the chorion, is derived mainly from the zebu embryo. These zebu-derived extra-embryonic components may interact positively with the taurine embryo/fetus during pregnancy in a tropical environment. We tested different parameters for the production of tetraploid Nelore (Bos indicus) embryos to be combined via aggregation with crossbred Bos taurus (diploid) embryos in order to produce viable chimeric blastocysts. Bovine (Bos indicus or crossbred Bos taurus) embryos were produced in vitro according to standard procedures. Two-cell Bos indicus embryos were submitted to electrofusion with varying numbers of pulses (1 or 2), voltages (0.4, 0.5, 0.75, 1.0, 1.4 and 5.0 kV/cm) and time (20, 25, 50 and 60 μs) to produce tetraploid embryos. Electrofused embryos were cultured with crossbred non-fused embryos to form chimeras that developed until the blastocyst stage. The best fusion parameter was 0.75 kV/cm for 60 μs. Four chimeric blastocysts (tetraploid Nelore with diploid crossbred Holstein) were formed after 31 attempts in 4 replicates (13%). We established an optimal procedure for the production of tetraploid Bos indicus (4n) embryos and embryonic chimeras by aggregation of crossbred Bos taurus (2n) with Bos indicus (4n) embryos. This technique would be valid in applied research, by producing exclusively taurine calves, but with placental elements from the Bos indicus breed, following transfer of these chimeras into recipient cows. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  8. RNA/aTNA Chimeras: RNAi Effects and Nucleases Resistance of Single and Double Stranded RNAs

    Directory of Open Access Journals (Sweden)

    Adele Alagia

    2014-11-01

    Full Text Available The RNA interference pathway (RNAi is a specific and powerful biological process, triggered by small non-coding RNA molecules and involved in gene expression regulation. In this work, we explored the possibility of increasing the biological stability of these RNA molecules by replacing their natural ribose ring with an acyclic L-threoninol backbone. In particular, this modification has been incorporated at certain positions of the oligonucleotide strands and its effects on the biological properties of the siRNA have been evaluated. In vitro cellular RNAi assays have demonstrated that the L-threoninol backbone is well tolerated by the RNAi machinery in both double and single-stranded fashion, with activities significantly higher than those evinced by the unmodified RNAs and comparable to the well-known phosphorothioate modification. Additionally, this modification conferred extremely strong resistance to serum and 3′/5′-exonucleases. In view of these results, we applied this modification to the knockdown of a therapeutically relevant human gene such as apolipoprotein B (ApoB. Further studies on the activation of the innate immune system showed that L-threoninol-modified RNAs are slightly less stimulatory than unmodified RNAs.

  9. Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera.

    Science.gov (United States)

    Cabrera-Mora, Monica; Fonseca, Jairo Andres; Singh, Balwan; Oliveira-Ferreira, Joseli; Lima-Junior, Josué da Costa; Calvo-Calle, J Mauricio; Moreno, Alberto

    2015-09-01

    Plasmodium vivax is the most widespread species of Plasmodium, causing up to 50% of the malaria cases occurring outside sub-Saharan Africa. An effective vaccine is essential for successful control and potential eradication. A well-characterized vaccine candidate is the circumsporozoite protein (CSP). Preclinical and clinical trials have shown that both antibodies and cellular immune responses have been correlated with protection induced by immunization with CSP. On the basis of our reported approach of developing chimeric Plasmodium yoelii proteins to enhance protective efficacy, we designed PvRMC-CSP, a recombinant chimeric protein based on the P. vivax CSP (PvCSP). In this engineered protein, regions of the PvCSP predicted to contain human T cell epitopes were genetically fused to an immunodominant B cell epitope derived from the N-terminal region I and to repeat sequences representing the two types of PvCSP repeats. The chimeric protein was expressed in soluble form with high yield. As the immune response to PvCSP has been reported to be genetically restricted in the murine model, we tested the immunogenicity of PvRMC-CSP in groups of six inbred strains of mice. PvRMC-CSP was able to induce robust antibody responses in all the mouse strains tested. Synthetic peptides representing the allelic forms of the P. vivax CSP were also recognized to a similar extent regardless of the mouse strain. Furthermore, the immunization regimen induced high frequencies of multifunctional CD4(+) and CD8(+) PvRMC-CSP-specific T cells. The depth and breadth of the immune responses elicited suggest that immunization with PvRMC-CSP can circumvent the genetic restriction of the immune response to P. vivax CSP. Interestingly, PvRMC-CSP was also recognized by naturally acquired antibodies from individuals living in areas where malaria is endemic. These features make PvRMC-CSP a promising vaccine candidate for further development. Copyright © 2015, American Society for Microbiology. All

  10. Bacterial membrane activity of a-peptide/b-peptoid chimeras: Influence of amino acid composition and chain length on the activity against different bacterial strains

    DEFF Research Database (Denmark)

    Hein-Kristensen, Line; Knapp, Kolja M; Franzyk, Henrik

    2011-01-01

    BACKGROUND: Characterization and use of antimicrobial peptides (AMPs) requires that their mode of action is determined. The interaction of membrane-active peptides with their target is often established using model membranes, however, the actual permeabilization of live bacterial cells and subseq...... of the bacterial cell envelope, and the outer membrane may act as a barrier in Gram-negative bacteria. The tolerance of S. marcescens to chimeras may be due to differences in the composition of the lipopolysaccharide layer also responsible for its resistance to polymyxin B.......BACKGROUND: Characterization and use of antimicrobial peptides (AMPs) requires that their mode of action is determined. The interaction of membrane-active peptides with their target is often established using model membranes, however, the actual permeabilization of live bacterial cells...... and subsequent killing is usually not tested. In this report, six α-peptide/β-peptoid chimeras were examined for the effect of amino acid/peptoid substitutions and chain length on the membrane perturbation and subsequent killing of food-borne and clinical bacterial isolates. RESULTS: All six AMP analogues...

  11. Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

    Energy Technology Data Exchange (ETDEWEB)

    van Bruggen, M.C.; van den Broek, M.F.; van den Berg, W.B. (University Hospital Nijmegen, (Netherlands))

    1991-09-01

    Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication the authors describe their studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 rats reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.

  12. Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

    Directory of Open Access Journals (Sweden)

    Qi Zhao

    Full Text Available Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

  13. Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction.

    Science.gov (United States)

    Molina, H; Brenner, C; Jacobi, S; Gorka, J; Carel, J C; Kinoshita, T; Holers, V M

    1991-07-05

    The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7.

  14. Chimeras: an ethical consideration

    Directory of Open Access Journals (Sweden)

    H. J.G. Zandman

    2011-07-01

    Full Text Available Scientists have started with experimentation that raises difficult ethical questions. It comprises taking material from the human blueprint (DNA and inserting this in various test animals. The purpose of such research is noble, namely the alleviation of hu- man suffering. Yet the ethical ramifications of blending the hu- man and animal genome are significant, especially for Chris- tians. The creation of all living entities after their kind and the image-bearing dignity attributed to man both come under se- vere ethical stress for those who presuppose divine order in God’s ecology.  For non-Christians the philosophical dilemma ought not to exist in the ethical sense if applied at the purest level. If the human is merely a kind of animal, along with and ontologically not diffe- rent from other animals, there is little logical reason to object to chimeric research apart from a concern about what such re- search and application might do to the order of life pragmati- cally. However, many non-Christian do object. Man is made in God’s image and the concept of human dignity and a universal sense of right and wrong still binds Christians and non-Chris- tians when considering ethics in the field of chimeric research. As the mixing of human stem cells with embryonic animals takes place, certain non-Christian authors protest that human dignity is being diminished and the animal essence is being vio- lated.

  15. Chimeras: an ethical consideration

    Directory of Open Access Journals (Sweden)

    H.J.G. Zandman

    2011-07-01

      For non-Christians the philosophical dilemma ought not to exist in the ethical sense if applied at the purest level. If the human is merely a kind of animal, along with and ontologically not diffe- rent from other animals, there is little logical reason to object to chimeric research apart from a concern about what such re- search and application might do to the order of life pragmati- cally. However, many non-Christian do object. Man is made in God’s image and the concept of human dignity and a universal sense of right and wrong still binds Christians and non-Chris- tians when considering ethics in the field of chimeric research. As the mixing of human stem cells with embryonic animals takes place, certain non-Christian authors protest that human dignity is being diminished and the animal essence is being vio- lated.

  16. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields.......Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  17. Continua or Chimera?

    Science.gov (United States)

    Booth, Tony

    1994-01-01

    This article looks at two concepts in the British 1993 draft Code of Practice concerning students with special needs: the concepts of a "continuum of needs" and a "continuum of provision." Issues involved in connecting the two continua are addressed, including whether service delivery decisions should be based on severity of…

  18. Experimental effects of dynamics and thermodynamics in nuclear reactions on the symmetry energy as seen by the CHIMERA 4 π detector

    Energy Technology Data Exchange (ETDEWEB)

    De Filippo, E.; Pagano, A. [INFN, Catania (Italy)

    2014-02-15

    Heavy-ion collisions have been widely used in the last decade to constrain the parameterizations of the symmetry energy term of the nuclear equation of state (EOS) for asymmetric nuclear matter as a function of baryonic density. In the Fermi energy domain one is faced with variations of the density within a narrow range of values around the saturation density ρ{sub 0}=0.16 fm{sup -3} down towards sub-saturation densities. The experimental observables which are sensitive to the symmetry energy are constructed starting from the detected light particles, clusters and heavy fragments that, in heavy-ion collisions, are generally produced by different emission mechanisms at different stages and time scales of the reaction. In this review the effects of dynamics and thermodynamics on the symmetry energy in nuclear reactions are discussed and characterized using an overview of the data taken so far with the CHIMERA multi detector array. (orig.)

  19. Primordial germ cell-mediated chimera technology produces viable pure-line Houbara bustard offspring: potential for repopulating an endangered species.

    Directory of Open Access Journals (Sweden)

    Ulrich Wernery

    2010-12-01

    Full Text Available The Houbara bustard (Chlamydotis undulata is a wild seasonal breeding bird populating arid sandy semi-desert habitats in North Africa and the Middle East. Its population has declined drastically during the last two decades and it is classified as vulnerable. Captive breeding programmes have, hitherto, been unsuccessful in reviving population numbers and thus radical technological solutions are essential for the long term survival of this species. The purpose of this study was to investigate the use of primordial germ cell-mediated chimera technology to produce viable Houbara bustard offspring.Embryonic gonadal tissue was dissected from Houbara bustard embryos at eight days post-incubation. Subsequently, Houbara tissue containing gonadal primordial germ cells (gPGCs was injected into White Leghorn chicken (Gallus gallus domesticus embryos, producing 83/138 surviving male chimeric embryos, of which 35 chimeric roosters reached sexual maturity after 5 months. The incorporation and differentiation of Houbara gPGCs in chimeric chicken testis were assessed by PCR with Houbara-specific primers and 31.3% (5/16 gonads collected from the injected chicken embryos showed the presence of donor Houbara cells. A total of 302 semen samples from 34 chimeric roosters were analyzed and eight were confirmed as germline chimeras. Semen samples from these eight roosters were used to artificially inseminate three female Houbara bustards. Subsequently, 45 Houbara eggs were obtained and incubated, two of which were fertile. One egg hatched as a male live born Houbara; the other was female but died before hatching. Genotyping confirmed that the male chick was a pure-line Houbara derived from a chimeric rooster.This study demonstrates for the first time that Houbara gPGCs can migrate, differentiate and eventually give rise to functional sperm in the chimeric chicken testis. This approach may provide a promising tool for propagation and conservation of endangered avian

  20. Recombinant adenovirus type 5 HIV gag/pol/nef vaccine in South Africa: unblinded, long-term follow-up of the phase 2b HVTN 503/Phambili study.

    Science.gov (United States)

    Gray, Glenda E; Moodie, Zoe; Metch, Barbara; Gilbert, Peter B; Bekker, Linda-Gail; Churchyard, Gavin; Nchabeleng, Maphoshane; Mlisana, Koleka; Laher, Fatima; Roux, Surita; Mngadi, Kathryn; Innes, Craig; Mathebula, Matsontso; Allen, Mary; McElrath, M Julie; Robertson, Michael; Kublin, James; Corey, Lawrence

    2014-05-01

    The HVTN 503/Phambili study, which assessed the efficacy of the Merck Ad5 gag/pol/nef subtype B HIV-1 preventive vaccine in South Africa, was stopped when futility criteria in the Step study (assessing the same vaccine in the Americas, Caribbean, and Australia) were met. Here we report long-term follow-up data. HVTN 503/Phambili was a double-blind, placebo-controlled, randomised trial that recruited HIV-1 uninfected, sexually active adults aged 18-35 years from five sites in South Africa. Eligible participants were randomly assigned (1:1) by computer-generated random numbers to either vaccine or placebo, stratified by site and sex. Cox proportional hazards models were used to estimate HIV-1 infection in the modified intention-to-treat cohort, all of whom were unmasked early in follow-up. The trial is registered with ClinicalTrials.gov, number NCT00413725 and the South African National Health Research Database, number DOH-27-0207-1539. Between Jan 24, 2007, and Sept 19, 2007, 801 participants (26·7%) of a planned 3000 were randomly assigned (400 to vaccine, 401 to placebo); 216 (27%) received only one injection, 529 (66%) received only two injections, and 56 (7%) received three injections. At a median follow-up of 42 months (IQR 31-42), 63 vaccine recipients (16%) had HIV-1 infection compared with 37 placebo recipients (9%; adjusted HR 1·70, 95% CI 1·13-2·55; p=0·01). Risk for HIV-1 infection did not differ according to the number of vaccinations received, sex, circumcision, or adenovirus type 5 (Ad5) serostatus. Differences in risk behaviour at baseline or during the study, or annualised dropout rate (7·7% [95% CI 6·2-9·5] for vaccine recipients vs 8·8% [7·1-10·7] for placebo recipients; p=0·40) are unlikely explanations for the increased rate of HIV-1 infections seen in vaccine recipients. The increased risk of HIV-1 acquisition in vaccine recipients, irrespective of number of doses received, warrants further investigation to understand the biological

  1. Bactericidal effect of bovine lactoferrin and synthetic peptide lactoferrin chimera in Streptococcus pneumoniae and the decrease in luxS gene expression by lactoferrin

    NARCIS (Netherlands)

    León-Sicairos, N.; Angulo-Zamudio, U.A.; Vidal, J.E.; López-Torres, C.A.; Bolscher, J.G.M.; Nazmi, K.; Reyes-Cortes, R.; Reyes-López, M.; de la Garza, M.; Canizalez-Román, A.

    2014-01-01

    Streptococcus pneumoniae (pneumococcus) is responsible for nearly one million child deaths annually. Pneumococcus causes infections such as pneumonia, otitis media, meningitis, and sepsis. The human immune system includes antibacterial peptides and proteins such as lactoferrin (LF), but its activity

  2. Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein {alpha}-subunit

    Energy Technology Data Exchange (ETDEWEB)

    Abdulaev, Najmoutin G. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Zhang Cheng; Dinh, Andy [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States); Ngo, Tony; Bryan, Philip N. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States); Brabazon, Danielle M. [Loyola College in Maryland, Department of Chemistry (United States); Marino, John P. [University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology (United States)], E-mail: marino@carb.nist.gov; Ridge, Kevin D. [University of Texas Health Science Center, Center for Membrane Biology, Department of Biochemistry and Molecular Biology (United States)

    2005-05-15

    Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein {alpha}-subunit (G{sub {alpha}}) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G{sub {alpha}} chimera ({approx}40 kDa polypeptide) has been tested. The results show that a prodomain fused G{sub {alpha}} chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G{sub {alpha}} isolated from natural sources. To assay for the functional integrity of the purified G{sub {alpha}} chimera at NMR concentrations and probe for changes in the structure and dynamics of G{sub {alpha}} that result from activation, {sup 15}N-HSQC spectra of the GDP/Mg{sup 2+} bound form of G{sub {alpha}} obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the {sup 15}N-HSQC spectra reveals a number of changes in chemical shifts of the {sup 1}HN, {sup 15}N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G{sub {alpha}} activation.

  3. Functional Expression of Aquaporin-2 Tagged with Photoconvertible Fluorescent Protein in mpkCCD Cells

    Directory of Open Access Journals (Sweden)

    Kay-Pong Yip

    2015-05-01

    Full Text Available Background: Vasopressin induced trafficking of aquaporin-2 (AQP2 containing vesicles has been studied in kidney cell lines using conventional fluorescent proteins as tags. However, trafficking of fluorescent tagged AQP2, which resembles the vectorial translocation of native AQP2 from cytoplasm to apical membrane has not been demonstrated at real time. Using a photoconvertible fluorescent protein tag on AQP2 might allow the simultaneous tracking of two separate populations of AQP2 vesicle after subcellular local photoconversion. Methods: A spacer was used to link a photoconvertible fluorescent protein (mEos2 to the amino-terminus of AQP2. The DNA constructs were expressed in mpkCCD cells. The trafficking of chimeric protein was visualized with high speed confocal microscopy in 4 dimensions. Results: Chimeric AQP2 expressed in mpkCCD cell conferred osmotic water permeability to the cells. Subcellular photoconversion with a 405 nm laser pulse converted green chimeras to red chimeras locally. Forskolin stimulation triggered chimeric AQP2 to translocate from acidic organelles to apical plasma membrane. By serendipity, the rate of apical accumulation was found to increase when mEos2 was tagged to the carboxyl-terminus in at least one of the AQP2 molecules within the tetramer. Conclusion: Functional photoconvertible chimeric AQP2 was successfully expressed in mpkCCD cells, in which forskolin induced apical trafficking and accumulation of chimeric AQP2. The proof-of-concept to monitor two populations of AQP2 vesicle simultaneously was demonstrated.

  4. Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

    KAUST Repository

    Nomme, Julian

    2010-08-01

    We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

  5. Preliminary X-ray crystallographic analysis of an engineered variant of human chimera-type galectin-3 with a shortened N-terminal domain.

    Science.gov (United States)

    Flores-Ibarra, Andrea; Ruiz, Federico M; Vértesy, Sabine; André, Sabine; Gabius, Hans-Joachim; Romero, Antonio

    2015-02-01

    How lectins translate sugar-encoded information into cellular effects not only depends on glycan recognition. Other domains of the protein can contribute to the functional profile of a lectin. Human galectin-3 (Gal-3), an adhesion/growth-regulatory galectin, is composed of three different domains and is thus called a chimera-type protein. In addition to the carbohydrate-recognition domain, this lectin encompasses an N-terminal domain consisting of a peptide harbouring two phosphorylation sites and nine non-triple-helical collagen-like repeats. This region plays an as yet structurally undefined role in Gal-3 aggregation and ligand recognition. To date, crystallization of full-length Gal-3 has not been achieved. With the aim of providing structural insights into this modular organization, a Gal-3 variant was crystallized maintaining the terminal peptide and three of the nine collagen-like repeats. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 94.04, b = 97.96, c = 236.20 Å, and diffracted to a resolution of 3.3 Å.

  6. HIV-1 induces DCIR expression in CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Alexandra A Lambert

    2010-11-01

    Full Text Available The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4(+ T cells found in the synovial tissue from rheumatoid arthritis (RA patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4(+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4(+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons and cells acutely infected in vitro (seen in both virus-infected and uninfected cells. Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4(+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals and -independent intrinsic apoptotic pathways (involving the death effector AIF. Finally, we demonstrate that the higher surface expression of DCIR in CD4(+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4(+ T cells, a process that might promote virus dissemination throughout the infected organism.

  7. HIV-1 Induces DCIR Expression in CD4+ T Cells

    Science.gov (United States)

    Lambert, Alexandra A.; Imbeault, Michaël; Gilbert, Caroline; Tremblay, Michel J.

    2010-01-01

    The C-type lectin receptor DCIR, which has been shown very recently to act as an attachment factor for HIV-1 in dendritic cells, is expressed predominantly on antigen-presenting cells. However, this concept was recently challenged by the discovery that DCIR can also be detected in CD4+ T cells found in the synovial tissue from rheumatoid arthritis (RA) patients. Given that RA and HIV-1 infections share common features such as a chronic inflammatory condition and polyclonal immune hyperactivation status, we hypothesized that HIV-1 could promote DCIR expression in CD4+ T cells. We report here that HIV-1 drives DCIR expression in human primary CD4+ T cells isolated from patients (from both aviremic/treated and viremic/treatment naive persons) and cells acutely infected in vitro (seen in both virus-infected and uninfected cells). Soluble factors produced by virus-infected cells are responsible for the noticed DCIR up-regulation on uninfected cells. Infection studies with Vpr- or Nef-deleted viruses revealed that these two viral genes are not contributing to the mechanism of DCIR induction that is seen following acute infection of CD4+ T cells with HIV-1. Moreover, we report that DCIR is linked to caspase-dependent (induced by a mitochondria-mediated generation of free radicals) and -independent intrinsic apoptotic pathways (involving the death effector AIF). Finally, we demonstrate that the higher surface expression of DCIR in CD4+ T cells is accompanied by an enhancement of virus attachment/entry, replication and transfer. This study shows for the first time that HIV-1 induces DCIR membrane expression in CD4+ T cells, a process that might promote virus dissemination throughout the infected organism. PMID:21085612

  8. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum

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    Mihail Kantor

    2013-01-01

    Full Text Available The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9 in transgenic tomato plants from the T0 and T1 generations.

  9. Dynamics of Somatic Cell-Lineage Competition in Chimeras of Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa Dinámica de competencia entre líneas celulares somáticas en quimeras de Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa

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    Schwarz Ryan S.

    2007-12-01

    Full Text Available Sessile colonial invertebrates often fuse with conspecifics to form chimeras. Chimerism represents an unequivocal instance of withinindividual selection where genetically different celllineages compete for representation in the somatic and gametic pools. We analyzed temporal and spatial variations in somatic celllineage composition of laboratoryestablished chimeras of the hydroid Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa. Using three clones with different allotypic specificities (i.e., two rejecting one another but fusing with a third one, we established two classes of twoway chimeras, a single threeway chimera class, and an incompatible interaction as control. Chimeras were sampled at five time intervals for a year. Celllineages in samples were identified by polyp fusibility assays against tester colonies of known fusibility. The cell lineages composing the chimeras showed a differential competitive ability, with one of them representing close to 80% by the end of the study. Rare celllineages stabilized at low frequencies but preserved their ability to gain somatic representation and to colonize distant parts of the chimera. This behavior characterizes cell parasites. As a consequence of the reproductive plasticity of most colonial invertebrates, celllineage variability may be transmitted to the offspring both sexually and asexually. Successful somatic competitors are expected to be preferentially transmitted asexually, whereas cell parasites would be preferentially transmitted sexually.Los invertebrados coloniales y sésiles con frecuencia se fusionan con conespecíficos para formar quimeras. Estas quimeras son un ejemplo de selección natural actuando al interior del individuo en donde células genéticamente distintas compiten por acceso tanto a la línea somática como a la germinal. En este estudio se analizaron las variaciones temporal y espacial de linajes celulares somáticos en quimeras establecidas en el

  10. DYNAMICS OF SOMATIC CELLLINEAGE COMPETITION IN CHIMERAS OF Hydractinia symbiolongicarpus (CNIDARIA: HYDROZOA Dinámica de competencia entre líneas celulares somáticas en quimeras de Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa

    Directory of Open Access Journals (Sweden)

    RYAN S SCHWARZ

    Full Text Available Sessile colonial invertebrates often fuse with conspecifics to form chimeras. Chimerism represents an unequivocal instance of withinindividual selection where genetically different celllineages compete for representation in the somatic and gametic pools. We analyzed temporal and spatial variations in somatic celllineage composition of laboratoryestablished chimeras of the hydroid Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa. Using three clones with different allotypic specificities (i.e., two rejecting one another but fusing with a third one, we established two classes of twoway chimeras, a single threeway chimera class, and an incompatible interaction as control. Chimeras were sampled at five time intervals for a year. Celllineages in samples were identified by polyp fusibility assays against tester colonies of known fusibility. The cell lineages composing the chimeras showed a differential competitive ability, with one of them representing close to 80% by the end of the study. Rare celllineages stabilized at low frequencies but preserved their ability to gain somatic representation and to colonize distant parts of the chimera. This behavior characterizes cell parasites. As a consequence of the reproductive plasticity of most colonial invertebrates, celllineage variability may be transmitted to the offspring both sexually and asexually. Successful somatic competitors are expected to be preferentially transmitted asexually, whereas cell parasites would be preferentially transmitted sexuallyLos invertebrados coloniales y sésiles con frecuencia se fusionan con conespecíficos para formar quimeras. Estas quimeras son un ejemplo de selección natural actuando al interior del individuo en donde células genéticamente distintas compiten por acceso tanto a la línea somática como a la germinal. En este estudio se analizaron las variaciones temporal y espacial de linajes celulares somáticos en quimeras establecidas en el laboratorio del

  11. A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo.

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    Angela M Jansen

    2011-07-01

    Full Text Available The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+, harbouring the Salmonella pathogenicity island (SPI-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+ colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+ and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+ resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(- infected animals. C5.507 Vi(+ infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-. The modulating effect associated with Vi was not observed in MyD88(-/- and was reduced in TLR4(-/- mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.

  12. Human-derived IgG level as an indicator for EBV-associated lymphoma model in Hu-PBL/SCID chimeras.

    Science.gov (United States)

    Tang, Yunlian; He, Rongfang; Zhang, Yang; Liu, Fang; Cheng, Ailan; Wu, Yimou; Gan, Runliang

    2011-05-09

    Epstein-Barr virus (EBV) has a close association with various types of human lymphomas. Animal models are essential to elucidate the pathogenesis of human EBV-associated lymphomas. The aim of the present study is to evaluate the association between human IgG concentration and EBV-associated lymphoma development in huPBL/SCID mice. Human peripheral blood lymphocytes (hu-PBL) from EBV-seropositive donors were inoculated intraperitoneally into SCID mouse. Immunohistochemical staining was used to examine differentiated antigens of tumor cells. EBV infection of the induced tumors was detected by in situ hybridization. IgG concentrations in the serums of 12 SCID mice were measured by unidirectional immunodiffusion assay. 21 out of 29 mice developed tumors in their body. Immunohistochemical staining showed that all induced tumors were LCA (leukocyte common antigen) positive, B-cell markers (CD20, CD79a) positive, and T-cell markers (both CD3 and CD45RO) negative. The tumors can be diagnosed as human B-cell lymphomas by these morphological and immunohistochemical features. In situ hybridization exhibited resultant tumor cells had EBV encoded small RNA-1 (EBER-1). Human-derived IgG could be found in the serum from SCID mice on the 15th day following hu-PBL transplantation, and IgG levels increased with the tumor development in 6 hu-PBL/SCID chimeras. Intraperitoneal transfer of hu-PBLs from EBV+ donors to SCID mice leads to high human IgG levels in mouse serum and B cell lymphomas. Our findings suggest that increasing levels of human-derived IgG in peripheral blood from hu-PBL/SCID mice could be used to monitor EBV-related human B-cell lymphoma development in experimental animals.

  13. Human-derived IgG level as an indicator for EBV-associated lymphoma model in Hu-PBL/SCID chimeras

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    Cheng Ailan

    2011-05-01

    Full Text Available Abstract Background Epstein-Barr virus (EBV has a close association with various types of human lymphomas. Animal models are essential to elucidate the pathogenesis of human EBV-associated lymphomas. The aim of the present study is to evaluate the association between human IgG concentration and EBV-associated lymphoma development in huPBL/SCID mice. Methods Human peripheral blood lymphocytes (hu-PBL from EBV-seropositive donors were inoculated intraperitoneally into SCID mouse. Immunohistochemical staining was used to examine differentiated antigens of tumor cells. EBV infection of the induced tumors was detected by in situ hybridization. IgG concentrations in the serums of 12 SCID mice were measured by unidirectional immunodiffusion assay. Results 21 out of 29 mice developed tumors in their body. Immunohistochemical staining showed that all induced tumors were LCA (leukocyte common antigen positive, B-cell markers (CD20, CD79a positive, and T-cell markers (both CD3 and CD45RO negative. The tumors can be diagnosed as human B-cell lymphomas by these morphological and immunohistochemical features. In situ hybridization exhibited resultant tumor cells had EBV encoded small RNA-1 (EBER-1. Human-derived IgG could be found in the serum from SCID mice on the 15th day following hu-PBL transplantation, and IgG levels increased with the tumor development in 6 hu-PBL/SCID chimeras. Conclusions Intraperitoneal transfer of hu-PBLs from EBV+ donors to SCID mice leads to high human IgG levels in mouse serum and B cell lymphomas. Our findings suggest that increasing levels of human-derived IgG in peripheral blood from hu-PBL/SCID mice could be used to monitor EBV-related human B-cell lymphoma development in experimental animals.

  14. A prenylated p47phox-p67phox-Rac1 chimera is a Quintessential NADPH oxidase activator: membrane association and functional capacity.

    Science.gov (United States)

    Mizrahi, Ariel; Berdichevsky, Yevgeny; Casey, Patrick J; Pick, Edgar

    2010-08-13

    The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b(559), a membrane-associated heterodimer composed of two subunits (Nox2 and p22(phox)), and four cytosolic proteins (p47(phox), p67(phox), Rac, and p40(phox)). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b(559) and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47(phox), p67(phox), and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122-22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47(phox)-p67(phox)-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47(phox) phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp(193) in p47(phox) persists. Prenylated GFP-p47(phox)-p67(phox)-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors.

  15. Investigation of nuclear architecture with a domain-presenting expression system.

    Science.gov (United States)

    Dreger, Christine K; König, Alexandra R; Spring, Herbert; Lichter, Peter; Herrmann, Harald

    2002-01-01

    We have investigated the topogenic properties of the nucleus by ectopic expression of chimeric proteins consisting of a NLS-modified cytoplasmic filament-forming protein, Xenopus laevis vimentin, and domains of inner nuclear membrane proteins. Whereas the "carrier" without cargo, the NLS-vimentin alone, is deposited in a few nuclear body-type structures (J.M. Bridger, H. Herrmann, C. Münkel, P. Lichter, J. Cell Sci., 111, 1241-1253), the distribution is entirely changed upon coupling with the evolutionarily conserved domain of the lamin B tail, the entire lamin B tail, the amino-terminal nucleoplasmic segment of the lamin B receptor (LBR), and the LEM domain of emerin, respectively. Remarkably, every individual chimeric protein exhibits a completely different distribution. Therefore, we assume that the chimeric parts are specifically recognized by factors engaged in nucleus-specific topogenesis. Thus, the conserved domain of the lamin B tail results in the formation of many small accumulations spread all over the nucleus. The chimera with the complete lamin B tail is deposited in short fibrillar aggregates within the nucleus. It does not mediate the integration of the chimeric protein into the nuclear membrane in cultured cells, indicating that the lamin tail alone is not sufficient to direct the integration of a protein into the lamina in vivo. In contrast, in the nuclear assembly system of Xenopus laevis the recombinant NLS-vimentin-lamin tail protein is concentrated at the nuclear membrane. The LBR chimera is arranged in a "beaded-chain"-type fashion, quite different from the more random deposition of NLS-vimentin alone. To our surprise, the LEM domain of emerin induces the retention of most of the chimeric proteins within the cytoplasm. Hence, it appears to be engaged in a strong cytoplasmic interaction that overrides the nuclear localization signal. Finally, the lamin chimera with the conserved part of the lamin B tail is shown to recruit LBR to the nuclear

  16. Comparison of the osteogenesis and fusion rates between activin A/BMP-2 chimera (AB204) and rhBMP-2 in a beagle's posterolateral lumbar spine model.

    Science.gov (United States)

    Zheng, Guang Bin; Yoon, Byung-Hak; Lee, Jae Hyup

    2017-10-01

    Activin A/BMP-2 chimera (AB204) could promote bone healing more effectively than recombinant bone morphogenetic protein 2 (rhBMP-2) with much lower dose in a rodent model, but there is no report about the effectiveness of AB204 in a large animal model. The purpose of this study was to compare the osteogenesis and fusion rate between AB204 and rhBMP-2 using biphasic calcium phosphate (BCP) as a carrier in a beagle's posterolateral lumbar fusion model. This is a randomized control animal study. Seventeen male beagle dogs were included. Bilateral posterolateral fusion was performed at the L1-L2 and L4-L5 levels. Biphasic calcium phosphate (2 cc), rhBMP-2 (50 µg)+BCP (2 cc), or AB204 (50 µg)+BCP (2 cc) were implanted into the intertransverse space randomly. X-ray was performed at 4 and 8 weeks. After 8 weeks, the animals were sacrificed, and new bone formation and fusion rate were evaluated by manual palpation, computed tomography (CT), and undecalcified histology. The AB204 group showed significantly higher fusion rate (90%) than the rhBMP-2 group (15%) or the Osteon group (6.3%) by manual palpation. On x-ray and CT assessment, fusion rate and the volume of newly formed bone were also significantly higher in AB204 group than other groups. In contrast, more osteolysis was found in rhBMP-2 group (40%) than in AB204 group (10%) on CT study. In histologic results, new bone formation was sufficient between transverse processes in AB204 group, and obvious trabeculation and bone remodeling were observed. But in rhBMP-2 group, new bone formation was less than AB204 group and osteolysis was observed between the intertransverse spaces. A low dose of AB204 with BCP as a carrier significantly promotes the fusion rate in a large animal model when compared with the rhBMP-2. These findings demonstrate that AB204 could be an alternative to rhBMP-2 to improve fusion rate. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Replication competent HIV-1 viruses that express intragenomic microRNA reveal discrete RNA-interference mechanisms that affect viral replication.

    Science.gov (United States)

    Klase, Zachary; Houzet, Laurent; Jeang, Kuan-Teh

    2011-11-23

    It remains unclear whether retroviruses can encode and express an intragenomic microRNA (miRNA). Some have suggested that processing by the Drosha and Dicer enzymes might preclude the viability of a replicating retroviral RNA genome that contains a cis-embedded miRNA. To date, while many studies have shown that lentiviral vectors containing miRNAs can transduce mammalian cells and express the inserted miRNA efficiently, no study has examined the impact on the replication of a lentivirus such as HIV-1 after the deliberate intragenomic insertion of a bona fide miRNA. We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication. This is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA.

  18. Impaired virus control and severe CD8+ T-cell-mediated immunopathology in chimeric mice deficient in gamma interferon receptor expression on both parenchymal and hematopoietic cells

    DEFF Research Database (Denmark)

    Henrichsen, Pernille; Bartholdy, Christina; Christensen, Jan Pravsgaard

    2005-01-01

    Bone marrow chimeras were used to determine the cellular target(s) for the antiviral activity of gamma interferon (IFN-gamma). By transfusing such mice with high numbers of naive virus-specific CD8(+) T cells, a system was created in which the majority of virus-specific CD8(+) T cells would...... be capable of responding to IFN-gamma, but expression of the relevant receptor on non-T cells could be experimentally controlled. Only when the IFN-gamma receptor is absent on both radioresistant parenchymal and bone marrow-derived cells will chimeric mice challenged with a highly invasive, noncytolytic...

  19. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

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    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  20. Fascaplysin sensitizes cells to TRAIL-induced apoptosis through upregulating DR5 expression

    Science.gov (United States)

    Wang, Feng; Chen, Haimin; Yan, Xiaojun; Zheng, Yanling

    2013-05-01

    This study investigated the molecular mechanism of anti-tumor effect of fascaplysin, a nitrogenous red pigment firstly isolated from a marine sponge. Microarray analysis show that the TNF and TNF receptor superfamily in human umbilical vein endothelial cells (HUVEC) and human hepatocarcinoma cells (BEL-7402) were significantly regulated by fascaplysin. Western Blot results reveal that fascaplysin increased the expression of cleaved caspase-9, active caspase-3, and decreased the level of procaspase-8 and Bid. Flow cytometry and cytotoxicity tests indicate that fascaplysin sensitized cells to tumor necrosis-related apoptosisinducing ligand-(TRAIL) induced apoptosis, which was markedly blocked by TRAIL R2/Fc chimera, a dominant negative form of TRAIL receptor DR5. Therefore, our results demonstrate that fascaplysin promotes apoptosis through the activation of TRAIL signaling pathway by upregulating DR5 expression.

  1. Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing

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    Dorothee Pflueger

    2013-11-01

    Full Text Available TFE3 translocation renal cell carcinoma (tRCC is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study,we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNAread-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2 point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs. The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P<.001 and 22 papillaryRCCs (P<.05-.07. Immunohistochemical analysis of selected genes from the tRCC gene signature showed significantly higher eukaryotic translation elongation factor 1 alpha 2 (EEF1A2 and Contactin 3 (CNTN3 expression in 16 TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in over 200 ccRCCs (P < .0001, both.

  2. The search for the chimera

    CERN Document Server

    Randi, J

    1991-01-01

    Session 1 Mr. Randi will give an update of his lecture to the American Physical Society on the occasion of his award of the 1989 Forum Prize. The citation said: "for his unique defense of Science and the scientific method in many disciplines, including physics, against pseudoscience, frauds and charlatans. His use of scientific techniques has contributed to refuting suspicious and fraudulent claims of paranormal results. He has contributed significantly to public understanding of important issues where science and society interact". He is a professional magician and author of many books. He worked with John Maddox, the Editor of Nature to investigate the claims of "water with memory".

  3. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

    Directory of Open Access Journals (Sweden)

    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  4. A revisit of high collision energy effects on collision-induced dissociation spectra using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF): application to the sequencing of RNA/DNA chimeras.

    Science.gov (United States)

    Mauger, Florence; Tabet, Jean-Claude; Gut, Ivo G

    2014-07-15

    High-energy collision-induced dissociation (CID) spectra of isomeric RNA/DNA chimeras using matrix-assisted laser desorption/ionization time-of-flight LIFT mass spectrometry (MALDI-LIFT-TOF/TOF) can potentially be applied for an exhaustive fragment characterization in a nucleic acid sequencing scheme. These chimeras contain deoxynucleotides and at the 3'-end a ribonucleotide with a 3'-phosphate group. Deprotonated RNA/DNA chimeras of 4-, 5-, 7- and 10-mers are analyzed by CID. This enhances consecutive dissociations from both the precursor and prompt product anions generated by MALDI and metastable fragmentations prior to entering the LIFT cell. Gas-phase fragmentations of 4- and 5-mers produced many fragment ions, from base release prior to consecutive cleavage of the nucleotide phosphate bond linkage phosphate. The unusual a4(-) product ion is a specific and diagnostic dissociation of the 4-mer if the ribonucleotide contains cytosine. As the size of RNA/DNA chimeras increase, several abundant product ions are generated mainly from zwitterionic forms (deprotonated phosphate ester and protonated base sites): [(M-H)-BiH](-), [ai-BiH](-), wj(-), [wj, (ai-BiH)](-) (if Bi  ≠ T) as internal product ion, and more rarely [wj-BiH](-). The absence of the majority of the [ai-BiH](-) series although the wj (-) series suggested that the higher critical energy processes with a loose transition state are favored yielding the wj(-) series. A large number of abundant fragment ions are detected which enable each isomer to be sequenced. This sequencing method is high-throughput, accurate and could be used to sequence isomers of up to 10-mers and also oligonucleotides of unknown sequence. However, RNA/DNA chimeras without thymine must be sufficiently concentrated to reach desorption of deprotonated molecular species to be selected in LIFT to produce all fragment ions within measurable abundances. Copyright © 2014 John Wiley & Sons, Ltd.

  5. Study on the isospin equilibration phenomenon in nuclear reactions 40Ca + 40Ca , 40Ca + 46Ti , 40Ca + 48Ca , 48Ca + 48Ca at 25 MeV/nucleon by using the CHIMERA multidetector

    Science.gov (United States)

    Martorana, N. S.; Auditore, L.; Berceanu, I.; Cardella, G.; Chatterjee, M. B.; De Luca, S.; De Filippo, E.; Dell'Aquila, D.; Gnoffo, B.; Lanzalone, G.; Lombardo, I.; Maiolino, C.; Norella, S.; Pagano, A.; Pagano, E. V.; Papa, M.; Pirrone, S.; Politi, G.; Porto, F.; Quattrocchi, L.; Rizzo, F.; Russotto, P.; Trifirò, A.; Trimarchi, M.; Verde, G.; Vigilante, M.

    2017-11-01

    We report on the results obtained by studying nuclear reactions between isotopes of Ca and Ti at 25 MeV/nucleon. We used the multidetector CHIMERA to detect charged reaction products. In particular, we studied two main effects: the isospin diffusion and the isospin drift. In order to study these processes we performed a moving-source analysis on kinetic energy spectra of the isobar nuclei ^{3H} and ^{3He} . This method allows to isolate the emission from the typical sources produced in reactions at Fermi energy: projectile like fragment (PLF), target like fragment (TLF), and mid-velocity (MV) emission. The obtained results are compared to previous experimental investigations and to simulations obtained with CoMD-II model.

  6. Human chimera-type galectin-3: defining the critical tail length for high-affinity glycoprotein/cell surface binding and functional competition with galectin-1 in neuroblastoma cell growth regulation.

    Science.gov (United States)

    Kopitz, Jürgen; Vértesy, Sabine; André, Sabine; Fiedler, Sabine; Schnölzer, Martina; Gabius, Hans-Joachim

    2014-09-01

    Many human proteins have a modular design with receptor and structural domains. Using adhesion/growth-regulatory galectin-3 as model, we describe an interdisciplinary strategy to define the functional significance of its tail established by nine non-triple helical collagen-like repeats (I-IX) and the N-terminal peptide. Genetic engineering with sophisticated mass spectrometric product analysis provided the tools for biotesting, i.e. eight protein variants with different degrees of tail truncation. Evidently,various aspects of galectin-3 activity (cis binding and cell bridging) are affected by tail shortening in a different manner. Thus, this combined approach reveals an unsuspected complexity of structure-function relationship, encouraging further application beyond this chimera-type galectin. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  7. Amino acid residues important for CMP-sialic acid recognition by the CMP-sialic acid transporter: analysis of the substrate specificity of UDP-galactose/CMP-sialic acid transporter chimeras.

    Science.gov (United States)

    Takeshima-Futagami, Taro; Sakaguchi, Masayoshi; Uehara, Eriko; Aoki, Kazuhisa; Ishida, Nobuhiro; Sanai, Yutaka; Sugahara, Yasusato; Kawakita, Masao

    2012-12-01

    In our previous studies, we demonstrated that chimeric molecules of the CMP-sialic acid (CMP-Sia) transporter (CST) and the UDP-galactose (Gal) transporter (UGT) in which the seventh transmembrane helix-containing segment was derived from the CST could transport both CMP-Sia and UDP-Gal and that the CST-derived seventh transmembrane helix segment was sufficient for the chimera to recognize CMP-Sia in the otherwise UGT context. In this study, we continued to more precisely define the submolecular region that is necessary for CMP-Sia recognition, and we demonstrated that the N-terminal half of the seventh transmembrane helix of CST is essential for the CMP-Sia transport mediated by the chimeric transporters. We further showed that Tyr214Gly and Ser216Phe mutations of a chimeric transporter that was capable of transporting both CMP-Sia and UDP-Gal led to the selective loss of CMP-Sia transport activity without affecting UDP-Gal transport activity. Conversely, when a residue in a chimeric transporter that was active for UDP-Gal transport but not CMP-Sia transport was replaced by Tyr, so that Tyr occupied the same position as in the CMP-Sia transporter, the resulting mutant chimera acquired the ability to transport CMP-Sia. These results demonstrated that Tyr214 and Ser216, located in the seventh transmembrane helix of the human CST, are critically important for the recognition of CMP-Sia as a transport substrate. Identification of determinants critical for the discrimination between relevant and irrelevant substrates will advance our understanding of the mechanisms of substrate recognition by nucleotide sugar transporters.

  8. HIV-1 expression induces cyclin D1 expression and pRb phosphorylation in infected podocytes: cell-cycle mechanisms contributing to the proliferative phenotype in HIV-associated nephropathy

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    Husain Mohammad

    2002-09-01

    Full Text Available Abstract Background The aberrant cell-cycle progression of HIV-1-infected kidney cells plays a major role in the pathogenesis of HIV-associated nephropathy, however the mechanisms whereby HIV-1 induces infected glomerular podocytes or infected tubular epithelium to exit quiescence are largely unknown. Here, we ask whether the expression of HIV-1 genes in infected podocytes induces cyclin D1 and phospho-pRb (Ser780 expression, hallmarks of cyclin D1-mediated G1 → S phase progression. Results We assessed cyclin D1 and phospho-pRb (Ser780 expression in two well-characterized models of HIV-associated nephropathy pathogenesis: HIV-1 infection of cultured podocytes and HIV-1 transgenic mice (Tg26. Compared to controls, cultured podocytes expressing HIV-1 genes, and podocytes and tubular epithelium from hyperplastic nephrons in Tg26 kidneys, had increased levels of phospho-pRb (Ser780, a target of active cyclin D1/cyclin-dependent kinase-4/6 known to promote G1 → S phase progression. HIV-1-infected podocytes showed markedly elevated cyclin D1 mRNA and cyclin D1 protein, the latter of which did not down-regulate during cell-cell contact or differentiation, suggesting post-transcriptional stabilization of cyclin D1 protein levels by HIV-1. The selective suppression of HIV-1 transcription by the cyclin-dependent kinase inhibitor, flavopiridol, abrogated cyclin D1 expression, underlying the requirement for HIV-1 encoded products to induce cyclin D1. Indeed, HIV-1 virus deleted of nef failed to induce cyclin D1 mRNA to the level of other single gene mutant viruses. Conclusions HIV-1 expression induces cyclin D1 and phospho-pRb (Ser780 expression in infected podocytes, suggesting that HIV-1 activates cyclin D1-dependent cell-cycle mechanisms to promote proliferation of infected renal epithelium.

  9. Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

    DEFF Research Database (Denmark)

    Gustafsson, E; Brakebusch, C; Hietanen, K

    2001-01-01

    Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid...... the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate...... germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth...

  10. Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal

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    Macmaster Suzanne

    2002-06-01

    Full Text Available Abstract Background Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes 1. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP in embryonic stem (ES cells and mice 2. Results In this study we have used embryonic stem (ES cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP and enhanced yellow fluorescent protein (EYFP, two mutant and spectrally distinct color variants of wild type (wt GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid diploid and polarized (tetraploid diploid chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. Conclusions GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or

  11. Oral vaccination with salmonella simultaneously expressing Yersinia pestis F1 and V antigens protects against bubonic and pneumonic plague.

    Science.gov (United States)

    Yang, Xinghong; Hinnebusch, B Joseph; Trunkle, Theresa; Bosio, Catharine M; Suo, Zhiyong; Tighe, Mike; Harmsen, Ann; Becker, Todd; Crist, Kathryn; Walters, Nancy; Avci, Recep; Pascual, David W

    2007-01-15

    The gut provides a large area for immunization enabling the development of mucosal and systemic Ab responses. To test whether the protective Ags to Yersinia pestis can be orally delivered, the Y. pestis caf1 operon, encoding the F1-Ag and virulence Ag (V-Ag) were cloned into attenuated Salmonella vaccine vectors. F1-Ag expression was controlled under a promoter from the caf1 operon; two different promoters (P), PtetA in pV3, PphoP in pV4, as well as a chimera of the two in pV55 were tested. F1-Ag was amply expressed; the chimera in the pV55 showed the best V-Ag expression. Oral immunization with Salmonella-F1 elicited elevated secretory (S)-IgA and serum IgG titers, and Salmonella-V-Ag(pV55) elicited much greater S-IgA and serum IgG Ab titers than Salmonella-V-Ag(pV3) or Salmonella-V-Ag(pV4). Hence, a new Salmonella vaccine, Salmonella-(F1+V)Ags, made with a single plasmid containing the caf1 operon and the chimeric promoter for V-Ag allowed the simultaneous expression of F1 capsule and V-Ag. Salmonella-(F1+V)Ags elicited elevated Ab titers similar to their monotypic derivatives. For bubonic plague, mice dosed with Salmonella-(F1+V)Ags and Salmonella-F1-Ag showed similar efficacy (>83% survival) against approximately 1000 LD(50) Y. pestis. For pneumonic plague, immunized mice required immunity to both F1- and V-Ags because the mice vaccinated with Salmonella-(F1+V)Ags protected against 100 LD(50) Y. pestis. These results show that a single Salmonella vaccine can deliver both F1- and V-Ags to effect both systemic and mucosal immune protection against Y. pestis.

  12. Computational analysis of HIV-1 resistance based on gene expression profiles and the virus-host interaction network.

    Directory of Open Access Journals (Sweden)

    Tao Huang

    Full Text Available A very small proportion of people remain negative for HIV infection after repeated HIV-1 viral exposure, which is called HIV-1 resistance. Understanding the mechanism of HIV-1 resistance is important for the development of HIV-1 vaccines and Acquired Immune Deficiency Syndrome (AIDS therapies. In this study, we analyzed the gene expression profiles of CD4+ T cells from HIV-1-resistant individuals and HIV-susceptible individuals. One hundred eighty-five discriminative HIV-1 resistance genes were identified using the Minimum Redundancy-Maximum Relevance (mRMR and Incremental Feature Selection (IFS methods. The virus protein target enrichment analysis of the 185 HIV-1 resistance genes suggested that the HIV-1 protein nef might play an important role in HIV-1 infection. Moreover, we identified 29 infection information exchanger genes from the 185 HIV-1 resistance genes based on a virus-host interaction network analysis. The infection information exchanger genes are located on the shortest paths between virus-targeted proteins and are important for the coordination of virus infection. These proteins may be useful targets for AIDS prevention or therapy, as intervention in these pathways could disrupt communication with virus-targeted proteins and HIV-1 infection.

  13. Machine learning to design integral membrane channelrhodopsins for efficient eukaryotic expression and plasma membrane localization.

    Science.gov (United States)

    Bedbrook, Claire N; Yang, Kevin K; Rice, Austin J; Gradinaru, Viviana; Arnold, Frances H

    2017-10-01

    There is growing interest in studying and engineering integral membrane proteins (MPs) that play key roles in sensing and regulating cellular response to diverse external signals. A MP must be expressed, correctly inserted and folded in a lipid bilayer, and trafficked to the proper cellular location in order to function. The sequence and structural determinants of these processes are complex and highly constrained. Here we describe a predictive, machine-learning approach that captures this complexity to facilitate successful MP engineering and design. Machine learning on carefully-chosen training sequences made by structure-guided SCHEMA recombination has enabled us to accurately predict the rare sequences in a diverse library of channelrhodopsins (ChRs) that express and localize to the plasma membrane of mammalian cells. These light-gated channel proteins of microbial origin are of interest for neuroscience applications, where expression and localization to the plasma membrane is a prerequisite for function. We trained Gaussian process (GP) classification and regression models with expression and localization data from 218 ChR chimeras chosen from a 118,098-variant library designed by SCHEMA recombination of three parent ChRs. We use these GP models to identify ChRs that express and localize well and show that our models can elucidate sequence and structure elements important for these processes. We also used the predictive models to convert a naturally occurring ChR incapable of mammalian localization into one that localizes well.

  14. Regulation of expression of two LY-6 family genes by intron retention and transcription induced chimerism

    Directory of Open Access Journals (Sweden)

    Mallya Meera

    2008-09-01

    Full Text Available Abstract Background Regulation of the expression of particular genes can rely on mechanisms that are different from classical transcriptional and translational control. The LY6G5B and LY6G6D genes encode LY-6 domain proteins, whose expression seems to be regulated in an original fashion, consisting of an intron retention event which generates, through an early premature stop codon, a non-coding transcript, preventing expression in most cell lines and tissues. Results The MHC LY-6 non-coding transcripts have shown to be stable and very abundant in the cell, and not subject to Nonsense Mediated Decay (NMD. This retention event appears not to be solely dependent on intron features, because in the case of LY6G5B, when the intron is inserted in the artificial context of a luciferase expression plasmid, it is fully spliced but strongly stabilises the resulting luciferase transcript. In addition, by quantitative PCR we found that the retained and spliced forms are differentially expressed in tissues indicating an active regulation of the non-coding transcript. EST database analysis revealed that these genes have an alternative expression pathway with the formation of Transcription Induced Chimeras (TIC. This data was confirmed by RT-PCR, revealing the presence of different transcripts that would encode the chimeric proteins CSNKβ-LY6G5B and G6F-LY6G6D, in which the LY-6 domain would join to a kinase domain and an Ig-like domain, respectively. Conclusion In conclusion, the LY6G5B and LY6G6D intron-retained transcripts are not subjected to NMD and are more abundant than the properly spliced forms. In addition, these genes form chimeric transcripts with their neighbouring same orientation 5' genes. Of interest is the fact that the 5' genes (CSNKβ or G6F undergo differential splicing only in the context of the chimera (CSNKβ-LY6G5B or G6F-LY6G6C and not on their own.

  15. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  16. Directed engineering of a high-expression chimeric transgene as a strategy for gene therapy of hemophilia A.

    Science.gov (United States)

    Doering, Christopher B; Denning, Gabriela; Dooriss, Kerry; Gangadharan, Bagirath; Johnston, Jennifer M; Kerstann, Keith W; McCarty, David A; Spencer, H Trent

    2009-07-01

    Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

  17. Cu(II) Binding to the Peptide Ala-His-His, a Chimera of the Canonical Cu(II)-Binding Motifs Xxx-His and Xxx-Zzz-His.

    Science.gov (United States)

    Gonzalez, Paulina; Vileno, Bertrand; Bossak, Karolina; El Khoury, Youssef; Hellwig, Petra; Bal, Wojciech; Hureau, Christelle; Faller, Peter

    2017-12-18

    Peptides and proteins with the N-terminal motifs NH 2 -Xxx-His and NH 2 -Xxx-Zzz-His form well-established Cu(II) complexes. The canonical peptides are Gly-His-Lys and Asp-Ala-His-Lys (from the wound healing factor and human serum albumin, respectively). Cu(II) is bound to NH 2 -Xxx-His via three nitrogens from the peptide and an external ligand in the equatorial plane (called 3N form here). In contrast, Cu(II) is bound to NH 2 -Xxx-Zzz-His via four nitrogens from the peptide in the equatorial plane (called 4N form here). These two motifs are not mutually exclusive, as the peptides with the sequence NH 2 -Xxx-His-His contain both of them. However, this chimera has never been fully explored. In this work, we use a multispectroscopic approach to analyze the Cu(II) binding to the chimeric peptide Ala-His-His (AHH). AHH is capable of forming the 3N- and 4N-type complexes in a pH dependent manner. The 3N form predominates at pH ∼ 4-6.5 and the 4N form at ∼ pH 6.5-10. NMR experiments showed that at pH 8.5, where Cu(II) is almost exclusively bound in the 4N form, the Cu(II)-exchange between AHH or the amidated AHH-NH 2 is fast, in comparison to the nonchimeric 4N form (AAH). Together, the results show that the chimeric AHH can access both Cu(II) coordination types, that minor changes in the second (or further) coordination sphere can impact considerably the equilibrium between the forms, and that Cu kinetic exchange is fast even when Cu-AHH is mainly in the 4N form.

  18. CD40/CD154 blockade inhibits dendritic cell expression of inflammatory cytokines but not costimulatory molecules.

    Science.gov (United States)

    Ferrer, Ivana R; Liu, Danya; Pinelli, David F; Koehn, Brent H; Stempora, Linda L; Ford, Mandy L

    2012-11-01

    Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonism on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor Ag (OVA). Adoptive transfer of OVA-specific CD4(+) and CD8(+) T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c(+) DCs compared with untreated controls. However, DCs isolated from anti-CD154-treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of Ag-specific CD4(+) and CD8(+) T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival.

  19. Placental Expression Patterns of Galectin-1, Galectin-2, Galectin-3 and Galectin-13 in Cases of Intrauterine Growth Restriction (IUGR

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    Stefan Hutter

    2016-04-01

    Full Text Available Galectins (gal are members of the mammalian β-galactoside-binding proteins and recognize Galβ1-4GlcNAc and Galβ1-4GalNac (Thomsen-Friedenreich antigen (TF sequences of several cell surface oligosaccharides. In this study, gal-1, -2, -3 and -13 were investigated systematically in the trophoblast and decidua compartment of intrauterine growth restriction (IUGR placentas and normal third trimester control placentas and stratified by fetal gender and gestational age. Within this study, 29 third trimester placentas after delivery were analyzed. Fetal gender was equally divided within both groups, and immunohistochemical staining was analyzed according to fetal gender and gestational age. Double immune-fluorescence with trophoblast-specific markers was used to identify galectin-expressing cells at the feto-maternal interface in the decidua. Gal-3 was significantly downregulated only in the extravillous trophoblast of IUGR placentas. In contrast, expressions of gal-2 and gal-13 were downregulated in both villous and extravillous trophoblast cells of IUGR placentas. In addition, gal-2 and gal-13 showed a highly correlated expression scheme in the placenta. There are significant gender-specific expression patterns for single prototype galectins with downregulation of gal-2 and gal-13 of male gender placentas in cases of IUGR. Gal-3 as the chimera type galectin shows only little gender-specific differences in expression, which disappear in IUGR cases.

  20. Placental Expression Patterns of Galectin-1, Galectin-2, Galectin-3 and Galectin-13 in Cases of Intrauterine Growth Restriction (IUGR).

    Science.gov (United States)

    Hutter, Stefan; Knabl, Julia; Andergassen, Ulrich; Hofmann, Simone; Kuhn, Christina; Mahner, Sven; Arck, Petra; Jeschke, Udo

    2016-04-07

    Galectins (gal) are members of the mammalian β-galactoside-binding proteins and recognize Galβ1-4GlcNAc and Galβ1-4GalNac (Thomsen-Friedenreich antigen (TF)) sequences of several cell surface oligosaccharides. In this study, gal-1, -2, -3 and -13 were investigated systematically in the trophoblast and decidua compartment of intrauterine growth restriction (IUGR) placentas and normal third trimester control placentas and stratified by fetal gender and gestational age. Within this study, 29 third trimester placentas after delivery were analyzed. Fetal gender was equally divided within both groups, and immunohistochemical staining was analyzed according to fetal gender and gestational age. Double immune-fluorescence with trophoblast-specific markers was used to identify galectin-expressing cells at the feto-maternal interface in the decidua. Gal-3 was significantly downregulated only in the extravillous trophoblast of IUGR placentas. In contrast, expressions of gal-2 and gal-13 were downregulated in both villous and extravillous trophoblast cells of IUGR placentas. In addition, gal-2 and gal-13 showed a highly correlated expression scheme in the placenta. There are significant gender-specific expression patterns for single prototype galectins with downregulation of gal-2 and gal-13 of male gender placentas in cases of IUGR. Gal-3 as the chimera type galectin shows only little gender-specific differences in expression, which disappear in IUGR cases.

  1. Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

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    Guidot David M

    2008-04-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 infection and the consequent acquired immunodeficiency syndrome (AIDS has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef. Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1 and Transforming Growth Factor-β1 (TGFβ1, three factors associated with muscle catabolism. Results Consistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss, the oxidized form of the anti-oxidant cysteine (Cys, and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold and TGFβ1 (~5-fold and ~3-fold in heart and

  2. Birbeck granule-like "organized smooth endoplasmic reticulum" resulting from the expression of a cytoplasmic YFP-tagged langerin.

    Directory of Open Access Journals (Sweden)

    Cédric Lenormand

    Full Text Available Langerin is required for the biogenesis of Birbeck granules (BGs, the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of "Organized Smooth Endoplasmic Reticulum" (OSER, with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a "double-lock" mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These

  3. Bone Marrow Chimeras and c-fms Conditional Ablation (Mafia) Mice Reveal an Essential Role for Resident Myeloid Cells in Lipopolysaccharide/TLR4-Induced Corneal Inflammation1

    Science.gov (United States)

    Chinnery, Holly R.; Carlson, Eric C.; Sun, Yan; Lin, Michelle; Burnett, Sandra H.; Perez, Victor L.; McMenamin, Paul G.; Pearlman, Eric

    2012-01-01

    The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation. PMID:19234168

  4. Bone marrow chimeras and c-fms conditional ablation (Mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation.

    Science.gov (United States)

    Chinnery, Holly R; Carlson, Eric C; Sun, Yan; Lin, Michelle; Burnett, Sandra H; Perez, Victor L; McMenamin, Paul G; Pearlman, Eric

    2009-03-01

    The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4(-/-) mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)(+)/C57BL/6 or eGFP(+)/TLR4(-/-) mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4(-/-) mice reconstituted with C57BL/6, but not TLR4(-/-) bone marrow cells donor cells were found to cause infiltration of eGFP(+) cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP(+) cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1alpha production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.

  5. Expression of galectin-3 in nephrotic syndrome glomerulopaties in children.

    Directory of Open Access Journals (Sweden)

    Dariusz Samulak

    2009-12-01

    Full Text Available BACKGROUND
    Galectins are a family of ancient animal carbohydrate binding proteins; the name is from their description as beta-galactoside-specific lectins. They have been strongly implicated in inflammation and cancer. Studies of the association of galectins with various aspects of kidney disease in humans are still at an early stage. In line with the above, the aim of the present report was to analyse the immunohistochemical expression of galectin-3 (the only chimera galectin currently identified in renal biopsy specimens of children with idiopathic nephrotic syndrome (INS.

    PATIENTS AND METHODS
    Eighteen children with minimal change disease (MCD, 30 with diffuse mesangial proliferation (DMP and 11 with focal segmental glomerulosclerosis (FSGS treated between 2003 and 2006 in the Department of Paediatric Cardiology and Nephrology, Poznan University of Medical Sciences. An indirect immunohistochemical protocol using a polyclonal rabbit antibody against human galectin-3 was employed.

    RESULTS
    In the control, MCD and DMP children who responded to steroid therapy anti-galectin-3 reactivity was present both in renal cortex and medulla. It was the strongest within cortical collecting ducts and subjectively less expressed in distal tubules. The total number of galectin-3 positive cortical and medullary segments of collecting ducts was significantly higher in the subjects who did not respond to steroid therapy These patients revealed also immunohistochemical reactivity of galectin-3 within nuclei of individual glomerular mesangial cells (p<0,001.

    CONCLUSIONS
    A suggested galectin-3 authority in mature human glomeruli during proteinuric glomerulopathies may indicate, on the one hand, its anti-inflammatory effect, but on the other can prognosticate a further glomerular reconstruction leading to FSGS. Taken together, both glomerular and

  6. Duct- and Acinar-Derived Pancreatic Ductal Adenocarcinomas Show Distinct Tumor Progression and Marker Expression

    Directory of Open Access Journals (Sweden)

    Rute M.M. Ferreira

    2017-10-01

    Full Text Available The cell of origin of pancreatic ductal adenocarcinoma (PDAC has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs, duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development.

  7. CD40/CD154 Blockade Inhibits Dendritic Cell Expression of Inflammatory Cytokines but not Costimulatory Molecules1

    Science.gov (United States)

    Ferrer, Ivana R.; Liu, Danya; Pinelli, David F.; Koehn, Brent H.; Stempora, Linda L.; Ford, Mandy L.

    2012-01-01

    Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonsim on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor antigen (OVA). Adoptive transfer of OVA-specific CD4+ and CD8+ T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c+ DC compared to untreated controls. However, DCs isolated from anti-CD154 treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of antigen-specific CD4+ and CD8+ T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival. PMID:23002440

  8. Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells.

    Science.gov (United States)

    Wu, C-M; Wu, T-Y; Kao, S-S; Ko, J-L; Jinn, T-R

    2008-05-01

    To develop an efficient and facile expression system supply of high purity and stable activity of rFip-fve for oral administration, medicinal study and applications. A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence, was constructed. The rFip-fve was purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified by Western blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip-fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip-fve was evaluated by measuring the amount of interleukin-2 released from murine splenocytes. A reliable scheme to express and purify active rFip-fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip-fve protein. The rFip-fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip-fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli.

  9. James Parkinson's Chimera: syndrome or disease?

    Science.gov (United States)

    Kempster, P A; Hurwitz, B; Lees, A J

    2017-06-01

    It is 200 years since James Parkinson published An Essay on the Shaking Palsy. While his monograph continues to be acclaimed for its precedence and clarity of description, what is often overlooked is the originality of Parkinson's ideas. Here we show that he appreciated the weakness of the systematic 18th century nosologies, which presupposed that medical species, the building blocks of these Linnaean taxonomic schemes, were as distinct as plant and animal species; and that Parkinson made a conceptual leap about combinations of clinical phenomena in recurring patterns, now recognised to be one of the germs of neurological thinking about syndromes. The Essay's written style underpins another aspect of significance to contemporary neurological practice - an inherent intellectual humility. In this commemorative year we locate the continuing importance of the related notions of syndrome and disease in successive frameworks of knowledge about the shaking palsy. Syndrome and disease are interpreted as dual character concepts, one clinically-based and the other restricted to pathophysiological causation. They both remain fundamental to understanding Parkinson's syndrome-disease today.

  10. Birbeck Granule-Like “Organized Smooth Endoplasmic Reticulum” Resulting from the Expression of a Cytoplasmic YFP-Tagged Langerin

    Science.gov (United States)

    Lenormand, Cédric; Spiegelhalter, Coralie; Cinquin, Bertrand; Bardin, Sabine; Bausinger, Huguette; Angénieux, Catherine; Eckly, Anita; Proamer, Fabienne; Wall, David; Lich, Ben; Tourne, Sylvie; Hanau, Daniel; Schwab, Yannick; Salamero, Jean; de la Salle, Henri

    2013-01-01

    Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of “Organized Smooth Endoplasmic Reticulum” (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a “double-lock” mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations

  11. Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation.

    Science.gov (United States)

    Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook; Kim, Sung-Gun; Hwang, Jihwan

    2016-09-01

    The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg(2+) concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study

  12. Interaction of the Hereditary Hemochromatosis Protein, HFE, with Transferrin Receptor 2 Is Required for Transferrin-Induced Hepcidin Expression

    Science.gov (United States)

    Gao, Junwei; Chen, Juxing; Kramer, Maxwell; Tsukamoto, Hidekazu; Zhang, An-Sheng; Enns, Caroline A.

    2009-01-01

    SUMMARY The mechanisms that allow the body to sense iron levels in order to maintain iron homeostasis are unknown. Patients with the most common form of hereditary iron overload have mutations in the hereditary hemochromatosis protein, HFE. They have lower levels of hepcidin, than unaffected individuals. Hepcidin, a hepatic peptide hormone, negatively regulates iron efflux from the intestines into the blood. We report two hepatic cell lines, WIF-B cells and HepG2 cells transfected with HFE, where hepcidin expression responded to iron-loaded transferrin. The response was abolished when endogenous transferrin receptor 2 (TfR2) was suppressed or in primary hepatocytes lacking either functional TfR2 or HFE. Furthermore, transferrin-treated HepG2 cells transfected with HFE chimeras containing only the α3 and cytoplasmic domains could upregulate hepcidin expression. Since the HFE α3 domain interacts with TfR2, these results supported our finding that TfR2/HFE complex is required for transcriptional regulation of hepcidin by holo-Tf. PMID:19254567

  13. Alternaria induces STAT-6 dependent acute airway eosinophila and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness

    Science.gov (United States)

    Doherty, Taylor A.; Khorram, Naseem; Sugimoto, Kotaro; Sheppard, Dean; Rosenthal, Peter; Youn Cho, Jae; Pham, Alexa; Miller, Marina; Croft, Michael; Broide, David H.

    2012-01-01

    The fungal allergen, Alternaria, is specifically associated with severe asthma, including life-threatening exacerbations. To better understand the acute innate airway response to Alternaria, naïve WT mice were challenged once intranasally with Alternaria. Naïve WT mice developed significant BAL eosinophila following Alternaria challenge when analyzed 24 hours later. In contrast to Alternaria, neither Aspergillus nor Candida induced BAL eosinophilia. Gene microarray analysis of airway epithelial cell brushings demonstrated that Alternaria-challenged naïve WT mice had an over 20 fold increase level of expression of “Found in Inflammatory Zone 1” (FIZZ1/Retnla), a resistin-like molecule. Lung immunostaining confirmed strong airway epithelial FIZZ1 expression present as early as 3 hours after a single Alternaria challenge that persisted for at least 5 days and was significantly reduced in STAT6-deficient, but not PAR-2-deficient mice. Bone marrow chimera studies revealed that STAT6 expressed in lung cells was required for epithelial FIZZ1 expression, while in contrast, STAT6 present in bone marrow derived cells contributed to airway eosinophilia. Studies investigating which cells in the non-challenged lung bind FIZZ1 demonstrated that CD45+CD11c+ (macrophages and dendritic cells) as well as collagen-1 producing CD45 negative cells (fibroblasts) can bind to FIZZ1. Importantly, direct administration of recombinant FIZZ1 to naïve WT mice led to airway eosinophilia, peribronchial fibrosis, and increased thickness of the airway epithelium. Thus, Alternaria induces STAT-6 dependent acute airway eosinophila and epithelial FIZZ1 expression that promotes airway fibrosis and epithelial thickness. This may provide some insight into the uniquely pathogenic aspects of Alternaria-associated asthma. PMID:22327070

  14. HIV-1 Adapts To Replicate in Cells Expressing Common Marmoset APOBEC3G and BST2

    Science.gov (United States)

    Fernández-Oliva, Alberto; Finzi, Andrés; Haim, Hillel; Menéndez-Arias, Luis; Sodroski, Joseph

    2015-01-01

    ABSTRACT Previous studies have shown that a major block to HIV-1 replication in common marmosets operates at the level of viral entry and that this block can be overcome by adaptation of the virus in tissue-cultured cells. However, our current studies indicate that HIV-1 encounters additional postentry blocks in common marmoset peripheral blood mononuclear cells. Here, we show that the common marmoset APOBEC3G (A3G) and BST2 proteins block HIV-1 in cell cultures. Using a directed-evolution method that takes advantage of the natural ability of HIV-1 to mutate during replication, we have been able to overcome these blocks in tissue-cultured cells. In the adapted viruses, specific changes were observed in gag, vif, env, and nef. The contribution of these changes to virus replication in the presence of the A3G and BST2 restriction factors was studied. We found that certain amino acid changes in Vif and Env that arise during adaptation to marmoset A3G and BST2 allow the virus to replicate in the presence of these restriction factors. The changes in Vif reduce expression levels and encapsidation of marmoset APOBEC3G, while the changes in Env increase viral fitness and discretely favor cell-to-cell transmission of the virus, allowing viral escape from these restriction factors. IMPORTANCE HIV-1 can infect only humans and chimpanzees. The main reason for this narrow tropism is the presence in many species of dominant-acting factors, known as restriction factors, that block viral replication in a species-specific way. We have been exploring the blocks to HIV-1 in common marmosets, with the ultimate goal of developing a new animal model of HIV-1 infection in these monkeys. In this study, we observed that common marmoset APOBEC3G and BST2, two known restriction factors, are able to block HIV-1 in cell cultures. We have adapted HIV-1 to replicate in the presence of these restriction factors and have characterized the mechanisms of escape. These studies can help in the

  15. HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splicing and virus production in macrophages

    Directory of Open Access Journals (Sweden)

    Purcell Damian FJ

    2008-02-01

    Full Text Available Abstract Background Macrophages are important targets and long-lived reservoirs of HIV-1, which are not cleared of infection by currently available treatments. In the primary monocyte-derived macrophage model of infection, replication is initially productive followed by a decline in virion output over ensuing weeks, coincident with a decrease in the levels of the essential viral transactivator protein Tat. We investigated two possible mechanisms in macrophages for regulation of viral replication, which appears to be primarily regulated at the level of tat mRNA: 1 differential mRNA stability, used by cells and some viruses for the rapid regulation of gene expression and 2 control of HIV-1 alternative splicing, which is essential for optimal viral replication. Results Following termination of transcription at increasing times after infection in macrophages, we found that tat mRNA did indeed decay more rapidly than rev or nef mRNA, but with similar kinetics throughout infection. In addition, tat mRNA decayed at least as rapidly in peripheral blood lymphocytes. Expression of cellular splicing factors in uninfected and infected macrophage cultures from the same donor showed an inverse pattern over time between enhancing factors (members of the SR family of RNA binding proteins and inhibitory factors (members of the hnRNP family. While levels of the SR protein SC35 were greatly up-regulated in the first week or two after infection, hnRNPs of the A/B and H groups were down-regulated. Around the peak of virus production in each culture, SC35 expression declined to levels in uninfected cells or lower, while the hnRNPs increased to control levels or above. We also found evidence for increased cytoplasmic expression of SC35 following long-term infection. Conclusion While no evidence of differential regulation of tat mRNA decay was found in macrophages following HIV-1 infection, changes in the balance of cellular splicing factors which regulate alternative

  16. Express web application development

    CERN Document Server

    Yaapa, Hage

    2013-01-01

    Express Web Application Development is a practical introduction to learning about Express. Each chapter introduces you to a different area of Express, using screenshots and examples to get you up and running as quickly as possible.If you are looking to use Express to build your next web application, ""Express Web Application Development"" will help you get started and take you right through to Express' advanced features. You will need to have an intermediate knowledge of JavaScript to get the most out of this book.

  17. The heme oxygenase-1 inducer THI-56 negatively regulates iNOS expression and HMGB1 release in LPS-activated RAW 264.7 cells and CLP-induced septic mice.

    Science.gov (United States)

    Park, Eun Jung; Jang, Hwa Jin; Tsoyi, Konstantin; Kim, Young Min; Park, Sang Won; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl

    2013-01-01

    The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1.

  18. High expression Zymomonas promoters

    Science.gov (United States)

    Viitanen, Paul V [West Chester, PA; Tao, Luan [Havertown, PA; Zhang, Yuying [New Hope, PA; Caimi, Perry G [Kennett Square, PA; McCole, Laura : Zhang, Min; Chou, Yat-Chen [Lakewood, CO; McCutchen, Carol M [Wilmington, DE; Franden, Mary Ann [Centennial, CO

    2011-08-02

    Identified are mutants of the promoter of the Z. mobilis glyceraldehyde-3-phosphate dehydrogenase gene, which direct improved expression levels of operably linked heterologous nucleic acids. These are high expression promoters useful for expression of chimeric genes in Zymomonas, Zymobacter, and other related bacteria.

  19. Extrachromosomal inducible expression

    NARCIS (Netherlands)

    Veltman, Douwe M; Van Haastert, Peter J M

    2013-01-01

    Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled

  20. Genetic control of eosinophilia in mice: gene(s) expressed in bone marrow-derived cells control high responsiveness

    Energy Technology Data Exchange (ETDEWEB)

    Vadas, M.A.

    1982-02-01

    A heterogeneity in the capacity of strains of mice to mount eosinophilia is described. BALB/c and C3H are eosinophil high responder strains (EO-HR) and CBA and A/J are eosinophil low responder strains (EO-LR), judged by the response of blood eosinophils to Ascaris suum, and the response of blood, bone marrow, and spleen eosinophils to keyhole limpet hemocyanin given 2 days after 150 mg/kg cyclophosphamide. Some of the gene(s) for high responsiveness appear to be dominant because (EO-HR x EO-LR)F/sub 1/ mice were intermediate to high responders. This gene is expressed in bone marrow-derived cells because radiation chimeras of the type EO-HR..-->..F/sub 1/ were high responders and EO-LR..-->..F/sub 1/ were low responders. This description of a genetic control of eosinophilia in mice may be useful in understanding the role of this cell in parasite immunity and allergy.

  1. Protein expression-yeast.

    Science.gov (United States)

    Nielsen, Klaus H

    2014-01-01

    Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. Many eukaryotic proteins contain posttranslational modifications that can be performed in yeast but not in bacterial expression systems. In comparison with mammalian cell culture expression systems, growing yeast is both faster and less expensive, and large-scale cultures can be performed using fermentation. While several different yeast expression systems exist, this chapter focuses on the budding yeast Saccharomyces cerevisiae and will briefly describe some options to consider when selecting vectors and tags to be used for protein expression. Throughout this chapter, the expression and purification of yeast eIF3 is shown as an example alongside a general scheme outline. © 2014 Elsevier Inc. All rights reserved.

  2. Expressiveness in musical emotions.

    Science.gov (United States)

    Vieillard, Sandrine; Roy, Mathieu; Peretz, Isabelle

    2012-09-01

    This study was designed to investigate how emotion category, characterized by distinct musical structures (happiness, sadness, threat) and expressiveness (mechanical, expressive) may influence overt and covert behavioral judgments and physiological responses in musically trained and untrained listeners. Mechanical and expressive versions of happy, sad and scary excerpts were presented while physiological measures were recorded. Participants rated the intensity of the emotion they felt. In addition, they monitored excerpts for the presence of brief breaths. Results showed that the emotion categories were rated higher in the expressive than in the mechanical versions and that this effect was larger in musicians. Moreover, expressive excerpts were found to increase skin conductance level more than the mechanical ones, independently of their arousal value, and to slow down response times in the breath detection task relative to the mechanical versions, suggesting enhanced capture of attention by expressiveness. Altogether, the results support the key role of the performer's expression in the listener's emotional response to music.

  3. Neuroglobin over expressing mice

    DEFF Research Database (Denmark)

    Raida, Zindy; Hundahl, Christian Ansgar; Nyengaard, Jens R

    2013-01-01

    thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain...... genetic background on ischemic damage was investigated. PRINCIPAL FINDINGS: A four to five fold increase in Neuroglobin mRNA and protein expression was seen in the brain of transgenic mice. A β-actin promoter was used to drive Neuroglobin over expression, but immunohistochemistry and in situ hybridization...... showed over expression to be confined to primarily the cortex, hippocampus, cerebellum, and only in neurons. The level and expression pattern of endogenous Neuroglobin was unaffected by insertion of the over expressing Ngb transgene. Neuroglobin over expression resulted in a significant reduction...

  4. Inhibition of HIV-1 gene expression by Sam68ΔC: multiple targets but a common mechanism?

    Directory of Open Access Journals (Sweden)

    Cochrane Alan

    2009-03-01

    Full Text Available Abstract Two recent publications have explored the mechanisms by which a mutant of the host protein Sam68 blocks HIV-1 structural protein synthesis and expands its activity to encompass Nef. Although the two studies propose different mechanisms for the responses observed, it is possible that a common activity is responsible. Understanding how this Sam68 mutant discriminates among the multiple viral mRNAs promises to reveal unique properties of HIV-1 RNA metabolism.

  5. EphA2 Expression Regulates Inflammation and Fibroproliferative Remodeling in Atherosclerosis.

    Science.gov (United States)

    Finney, Alexandra C; Funk, Steven D; Green, Jonette M; Yurdagul, Arif; Rana, Mohammad Atif; Pistorius, Rebecca; Henry, Miriam; Yurochko, Andrew; Pattillo, Christopher B; Traylor, James G; Chen, Jin; Woolard, Matthew D; Kevil, Christopher G; Orr, A Wayne

    2017-08-08

    Atherosclerotic plaque formation results from chronic inflammation and fibroproliferative remodeling in the vascular wall. We previously demonstrated that both human and mouse atherosclerotic plaques show elevated expression of EphA2, a guidance molecule involved in cell-cell interactions and tumorigenesis. Here, we assessed the role of EphA2 in atherosclerosis by deleting EphA2 in a mouse model of atherosclerosis (Apoe - /- ) and by assessing EphA2 function in multiple vascular cell culture models. After 8 to 16 weeks on a Western diet, male and female mice were assessed for atherosclerotic burden in the large vessels, and plasma lipid levels were analyzed. Despite enhanced weight gain and plasma lipid levels compared with Apoe -/- controls, EphA2 -/- Apoe -/- knockout mice show diminished atherosclerotic plaque formation, characterized by reduced proinflammatory gene expression and plaque macrophage content. Although plaque macrophages express EphA2, EphA2 deletion does not affect macrophage phenotype, inflammatory responses, and lipid uptake, and bone marrow chimeras suggest that hematopoietic EphA2 deletion does not affect plaque formation. In contrast, endothelial EphA2 knockdown significantly reduces monocyte firm adhesion under flow. In addition, EphA2 -/- Apoe -/- mice show reduced progression to advanced atherosclerotic plaques with diminished smooth muscle and collagen content. Consistent with this phenotype, EphA2 shows enhanced expression after smooth muscle transition to a synthetic phenotype, and EphA2 depletion reduces smooth muscle proliferation, mitogenic signaling, and extracellular matrix deposition both in atherosclerotic plaques and in vascular smooth muscle cells in culture. Together, these data identify a novel role for EphA2 in atherosclerosis, regulating both plaque inflammation and progression to advanced atherosclerotic lesions. Cell culture studies suggest that endothelial EphA2 contributes to atherosclerotic inflammation by promoting

  6. B cells require Type 1 interferon to produce alloantibodies to transfused KEL-expressing red blood cells in mice.

    Science.gov (United States)

    Gibb, David R; Liu, Jingchun; Santhanakrishnan, Manjula; Natarajan, Prabitha; Madrid, David J; Patel, Seema; Eisenbarth, Stephanie C; Tormey, Christopher A; Stowell, Sean R; Iwasaki, Akiko; Hendrickson, Jeanne E

    2017-11-01

    Alloantibodies to red blood cell (RBC) antigens can cause significant hemolytic events. Prior studies have demonstrated that inflammatory stimuli in animal models and inflammatory states in humans, including autoimmunity and viremia, promote alloimmunization. However, molecular mechanisms underlying these findings are poorly understood. Given that Type 1 interferons (IFN-α/β) regulate antiviral immunity and autoimmune pathology, the hypothesis that IFN-α/β regulates RBC alloimmunization was tested in a murine model. Leukoreduced murine RBCs expressing the human KEL glycoprotein were transfused into control mice (WT), mice lacking the unique IFN-α/β receptor (IFNAR1-/- ), or bone marrow chimeric mice lacking IFNAR1 on specific cell populations. Anti-KEL IgG production, expressed as mean fluorescence intensity (MFI), and B-cell differentiation were examined. Transfused WT mice produced anti-KEL IgG alloantibodies (peak response MFI, 50.4). However, the alloimmune response of IFNAR1-/- mice was almost completely abrogated (MFI, 4.2; p IFNAR1 expression in all hematopoietic cells or specifically in B cells was also diminished (MFI, 3.8 and 5.4, respectively, compared to control chimeras, MFI, 65.6; p IFNAR1-/- B cells into germinal center B cells and plasma cells was significantly reduced, compared to WT B cells. This study demonstrates that B cells require signaling from IFN-α/β to produce alloantibodies to the human KEL glycoprotein in mice. These findings provide a potential mechanistic basis for inflammation-induced alloimmunization. If these findings extend to human studies, patients with IFN-α/β-associated conditions may have an elevated risk of alloimmunization and benefit from personalized transfusion protocols. © 2017 AABB.

  7. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication

    Directory of Open Access Journals (Sweden)

    Balart Luis A

    2010-10-01

    Full Text Available Abstract Interferon alpha (IFN-α binds to a cell surface receptor that activates the Jak-Stat signaling pathway. A critical component of this pathway is the translocation of interferon stimulated gene factor 3 (a complex of three proteins Stat1, Stat2 and IRF9 to the nucleus to activate antiviral genes. A stable sub-genomic replicon cell line resistant to IFN-α was developed in which the nuclear translocation of Stat1 and Stat2 proteins was prevented due to the lack of phosphorylation; whereas the nuclear translocation of IRF9 protein was not affected. In this study, we sought to overcome defective Jak-Stat signaling and to induce an antiviral state in the IFN-α resistant replicon cell line by developing a chimera IRF9 protein fused with the trans activating domain (TAD of either a Stat1 (IRF9-S1C or Stat2 (IRF9-S2C protein. We show here that intracellular expression of fusion proteins using the plasmid constructs of either IRF9-S1C or IRF9-S2C, in the IFN-α resistant cells, resulted in an increase in Interferon Stimulated Response Element (ISRE luciferase promoter activity and significantly induced HLA-1 surface expression. Moreover, we show that transient transfection of IRF9-S1C or IRF9-S2C plasmid constructs into IFN-α resistant replicon cells containing sub-genomic HCV1b and HCV2a viruses resulted in an inhibition of viral replication and viral protein expression independent of IFN-α treatment. The results of this study indicate that the recombinant fusion proteins of IRF9-S1C, IRF9-S2C alone, or in combination, have potent antiviral properties against the HCV in an IFN-α resistant cell line with a defective Jak-Stat signaling.

  8. Molecular Pathogenesis of EBV Susceptibility in XLP as Revealed by Analysis of Female Carriers with Heterozygous Expression of SAP

    Science.gov (United States)

    Palendira, Umaimainthan; Low, Carol; Chan, Anna; Hislop, Andrew D.; Ho, Edwin; Phan, Tri Giang; Deenick, Elissa; Cook, Matthew C.; Riminton, D. Sean; Choo, Sharon; Loh, Richard; Alvaro, Frank; Booth, Claire; Gaspar, H. Bobby; Moretta, Alessandro; Khanna, Rajiv; Rickinson, Alan B.; Tangye, Stuart G.

    2011-01-01

    X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP+ and SAP− cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8+ T cells specific for CMV and influenza were distributed across SAP+ and SAP− populations, EBV-specific cells were exclusively SAP+. The preferential recruitment of SAP+ cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8+ T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP− clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP− CD8+ T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited. PMID:22069374

  9. Molecular pathogenesis of EBV susceptibility in XLP as revealed by analysis of female carriers with heterozygous expression of SAP.

    Directory of Open Access Journals (Sweden)

    Umaimainthan Palendira

    2011-11-01

    Full Text Available X-linked lymphoproliferative disease (XLP is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP(+ and SAP(- cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8(+ T cells specific for CMV and influenza were distributed across SAP(+ and SAP(- populations, EBV-specific cells were exclusively SAP(+. The preferential recruitment of SAP(+ cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8(+ T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP(- clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP(- CD8(+ T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited.

  10. Molecular pathogenesis of EBV susceptibility in XLP as revealed by analysis of female carriers with heterozygous expression of SAP.

    Science.gov (United States)

    Palendira, Umaimainthan; Low, Carol; Chan, Anna; Hislop, Andrew D; Ho, Edwin; Phan, Tri Giang; Deenick, Elissa; Cook, Matthew C; Riminton, D Sean; Choo, Sharon; Loh, Richard; Alvaro, Frank; Booth, Claire; Gaspar, H Bobby; Moretta, Alessandro; Khanna, Rajiv; Rickinson, Alan B; Tangye, Stuart G

    2011-11-01

    X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP(+) and SAP(-) cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8(+) T cells specific for CMV and influenza were distributed across SAP(+) and SAP(-) populations, EBV-specific cells were exclusively SAP(+). The preferential recruitment of SAP(+) cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8(+) T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP(-) clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP(-) CD8(+) T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited.

  11. Re-evaluation of the PBAN receptor (PBANR molecule: characterization of PBANR variants expressed in the pheromone glands of moths

    Directory of Open Access Journals (Sweden)

    Jae Min Lee

    2012-01-01

    Full Text Available Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR activation. PBANR was initially cloned from pheromone glands (PGs of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Multiple PBANRs have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to examine the prevalence of multiple PBANRs in the PGs of three moths and to ascertain their potential functional relevance. Multiple PBANR variants (As, A, B, and C were cloned from the PGs of all species examined with PBANR-C the most highly expressed. Alternative splicing of the C-terminal coding sequence of the PBAN gene gives rise to the variants, which are distinguishable only by the length and composition of their respective C-terminal tails. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous preferential amplification of PBANR-A like receptors from other species.

  12. Regular expressions cookbook

    CERN Document Server

    Goyvaerts, Jan

    2009-01-01

    This cookbook provides more than 100 recipes to help you crunch data and manipulate text with regular expressions. Every programmer can find uses for regular expressions, but their power doesn't come worry-free. Even seasoned users often suffer from poor performance, false positives, false negatives, or perplexing bugs. Regular Expressions Cookbook offers step-by-step instructions for some of the most common tasks involving this tool, with recipes for C#, Java, JavaScript, Perl, PHP, Python, Ruby, and VB.NET. With this book, you will: Understand the basics of regular expressions through a

  13. Regular expression containment

    DEFF Research Database (Denmark)

    Henglein, Fritz; Nielsen, Lasse

    2011-01-01

    * for Kleene-star, and a general coin- duction rule as the only additional rule. Our axiomatization gives rise to a natural computational inter- pretation of regular expressions as simple types that represent parse trees, and of containment proofs as coercions. This gives the axiom- atization a Curry...... to be undecidable. We discuss application of regular expressions as types to bit coding of strings and hint at other applications to the wide-spread use of regular expressions for substring matching, where classical automata-theoretic techniques are a priori inapplicable. Neither regular expressions as types nor...

  14. Regular Expression Pocket Reference

    CERN Document Server

    Stubblebine, Tony

    2007-01-01

    This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

  15. Genotype-Associated Differential NKG2D Expression on CD56+CD3+ Lymphocytes Predicts Response to Pegylated-Interferon/Ribavirin Therapy in Chronic Hepatitis C.

    Science.gov (United States)

    Chu, Po-sung; Ebinuma, Hirotoshi; Nakamoto, Nobuhiro; Sugiyama, Kazuo; Usui, Shingo; Wakayama, Yuko; Taniki, Nobuhito; Yamaguchi, Akihiro; Shiba, Shunsuke; Yamagishi, Yoshiyuki; Wakita, Takaji; Hibi, Toshifumi; Saito, Hidetsugu; Kanai, Takanori

    2015-01-01

    Hepatitis C virus (HCV) genotype 1 infections are significantly more difficult to eradicate with PEG-IFN/ribavirin therapy, compared to HCV genotype 2. The aim of this work is to investigate the difference of immunological impairments underlying this phenomenon. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes and CD56+CD3- NK cells from cases of chronic hepatitis C were analyzed and assessed by treatment effect. Two strains of HCV were used to co-incubate with immune cells in vitro. NKG2D expression on peripheral CD56+CD3+ lymphocytes, but not NK cells, was significantly impaired in genotype 1 infection, compared to genotype 2. When peripheral blood mononuclear cells from healthy donors were co-incubated with TNS2J1, a genotype 1b/2a chimera strain, or with JFH1, a genotype 2a strain, genotype-specific decrease of NKG2D on CD56+CD3+ lymphocytes, but not NK cells, was observed. Pre-treatment NKG2D expression on peripheral CD56+CD3+ lymphocytes significantly correlated with reduction in serum HCV RNA levels from week 0 to week 4, and predicted treatment response. Ex vivo stimulation of peripheral CD56+CD3+ lymphocytes showed NKG2D expression-correlated IFN-γ production. In conclusion, Decreased NKG2D expression on CD56+CD3+ lymphocytes in chronic HCV genotype 1 infection predicts inferior treatment response to PEG-IFN/ribavirin therapy compared to genotype 2.

  16. BST-2 Expression Modulates Small CD4-Mimetic Sensitization of HIV-1-Infected Cells to Antibody-Dependent Cellular Cytotoxicity.

    Science.gov (United States)

    Richard, Jonathan; Prévost, Jérémie; von Bredow, Benjamin; Ding, Shilei; Brassard, Nathalie; Medjahed, Halima; Coutu, Mathieu; Melillo, Bruno; Bibollet-Ruche, Frédéric; Hahn, Beatrice H; Kaufmann, Daniel E; Smith, Amos B; Sodroski, Joseph; Sauter, Daniel; Kirchhoff, Frank; Gee, Katrina; Neil, Stuart J; Evans, David T; Finzi, Andrés

    2017-06-01

    Antibodies recognizing conserved CD4-induced (CD4i) epitopes on human immunodeficiency virus type 1 (HIV-1) Env and able to mediate antibody-dependent cellular cytotoxicity (ADCC) have been shown to be present in sera from most HIV-1-infected individuals. These antibodies preferentially recognize Env in its CD4-bound conformation. CD4 downregulation by Nef and Vpu dramatically reduces exposure of CD4i HIV-1 Env epitopes and therefore reduce the susceptibility of HIV-1-infected cells to ADCC mediated by HIV-positive (HIV+) sera. Importantly, this mechanism of immune evasion can be circumvented with small-molecule CD4 mimetics (CD4mc) that are able to transition Env into the CD4-bound conformation and sensitize HIV-1-infected cells to ADCC mediated by HIV+ sera. However, HIV-1 developed additional mechanisms to avoid ADCC, including Vpu-mediated BST-2 antagonism, which decreases the overall amount of Env present at the cell surface. Accordingly, BST-2 upregulation in response to alpha interferon (IFN-α) was shown to increase the susceptibility of HIV-1-infected cells to ADCC despite the activity of Vpu. Here we show that BST-2 upregulation by IFN-β and interleukin-27 (IL-27) also increases the surface expression of Env and thus boosts the ability of CD4mc to sensitize HIV-1-infected cells to ADCC by sera from HIV-1-infected individuals.IMPORTANCE HIV-1 evolved sophisticated strategies to conceal Env epitopes from ADCC-mediating antibodies present in HIV+ sera. Vpu-mediated BST-2 downregulation was shown to decrease ADCC responses by limiting the amount of Env present at the cell surface. This effect of Vpu was shown to be attenuated by IFN-α treatment. Here we show that in addition to IFN-α, IFN-β and IL-27 also affect Vpu-mediated BST-2 downregulation and greatly enhance ADCC responses against HIV-1-infected cells in the presence of CD4mc. These findings may inform strategies aimed at HIV prevention and eradication. Copyright © 2017 American Society for

  17. Mechanisms of Cytotoxicity of the Aids Virus

    Science.gov (United States)

    1992-10-10

    the above experiments was a significant pcoblem in data interpretation. To mare clearly define the cellular localizaticn of vpr by imarflurrescence, we...Department of Pathology , George Washington University, Washington D.C. 200372; and Henry M. Jackson Foundation for the Advancement of Military Medicine and the...cellular transcriptional factors. (mouse mammary tumour virus) LTR-linked nef Specific targets of PKC, PKA, cAMP-dependent transgenic lines expressed Nef

  18. Darwin and Emotion Expression

    Science.gov (United States)

    Hess, Ursula; Thibault, Pascal

    2009-01-01

    In his book "The Expression of the Emotions in Man and Animals," Charles Darwin (1872/1965) defended the argument that emotion expressions are evolved and adaptive (at least at some point in the past) and serve an important communicative function. The ideas he developed in his book had an important impact on the field and spawned rich domains of…

  19. Pleiotrophin expression during odontogenesis.

    Science.gov (United States)

    Erlandsen, Heidi; Ames, Jennifer E; Tamkenath, Amena; Mamaeva, Olga; Stidham, Katherine; Wilson, Mary E; Perez-Pinera, Pablo; Deuel, Thomas F; Macdougall, Mary

    2012-05-01

    Pleiotrophin (PTN) is an extracellular matrix-associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix-secreting ameloblasts and odontoblasts of the adult mouse incisors and molars.

  20. Facial expression and sarcasm.

    Science.gov (United States)

    Rockwell, P

    2001-08-01

    This study examined facial expression in the presentation of sarcasm. 60 responses (sarcastic responses = 30, nonsarcastic responses = 30) from 40 different speakers were coded by two trained coders. Expressions in three facial areas--eyebrow, eyes, and mouth--were evaluated. Only movement in the mouth area significantly differentiated ratings of sarcasm from nonsarcasm.

  1. Expressive Faces Confuse Identity.

    Science.gov (United States)

    Redfern, Annabelle S; Benton, Christopher P

    2017-01-01

    We used highly variable, so-called 'ambient' images to test whether expressions affect the identity recognition of real-world facial images. Using movie segments of two actors unknown to our participants, we created image pairs - each image within a pair being captured from the same film segment. This ensured that, within pairs, variables such as lighting were constant whilst expressiveness differed. We created two packs of cards, one containing neutral face images, the other, their expressive counterparts. Participants sorted the card packs into piles, one for each perceived identity. As with previous studies, the perceived number of identities was higher than the veridical number of two. Interestingly, when looking within piles, we found a strong difference between the expressive and neutral sorting tasks. With expressive faces, identity piles were significantly more likely to contain cards of both identities. This finding demonstrates that, over and above other image variables, expressiveness variability can cause identity confusion; evidently, expression is not disregarded or factored out when we classify facial identity in real-world images. Our results provide clear support for a face processing architecture in which both invariant and changeable facial information may be drawn upon to drive our decisions of identity.

  2. [Prokaryotic expression systems].

    Science.gov (United States)

    Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał

    2013-03-01

    For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

  3. Expert Oracle application express

    CERN Document Server

    Scott, John Edward

    2011-01-01

    Expert Oracle Application Express brings you groundbreaking insights into developing with Oracle's enterprise-level, rapid-development tool from some of the best practitioners in the field today. Oracle Application Express (APEX) is an entirely web-based development framework that is built into every edition of Oracle Database. The framework rests upon Oracle's powerful PL/SQL language, enabling power users and developers to rapidly develop applications that easily scale to hundreds, even thousands of concurrent users. The 13 authors of Expert Oracle Application Express build their careers aro

  4. The Expressive Organization

    DEFF Research Database (Denmark)

    not only understand their distinct identity and their brands, but are also able to express these externally and internally. In order to thrive in an era of transparency and customer choice, the authors argue, organizations will have to be expressive. This book is intended for undergraduate and postgraduate...... branding on organizational structures and processes? How do organizations discover their identities? These are some of the vexing problems addressed in this book by a diverse international team of contributors. According to the authors, the future lies with "the expressive organization". Such organizations...... students of management, business strategy, accounting, marketing, and communication studies; MBA students; Managers and consultants....

  5. CD73 expression identifies a subset of IgM+ antigen-experienced cells with memory attributes that is T cell and CD40 signalling dependent.

    Science.gov (United States)

    D'Souza, Lucas; Gupta, Sneh Lata; Bal, Vineeta; Rath, Satyajit; George, Anna

    2017-12-01

    B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM+ cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73+ IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory. © 2017 John Wiley & Sons Ltd.

  6. Expression of the H+-ATPase AHA10 proton pump is associated with citric acid accumulation in lemon juice sac cells.

    Science.gov (United States)

    Aprile, Alessio; Federici, Claire; Close, Timothy J; De Bellis, Luigi; Cattivelli, Luigi; Roose, Mikeal L

    2011-12-01

    The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a "sweet" lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit (Faris acid; FA) on different branches; it is apparently a graft chimera with layer L1 derived from Millsweet limetta and layer L2 from a standard lemon. The transcription profiles of Faris sweet lemon were compared with Faris acid lemon and Frost Lisbon (L), which is a standard sour lemon genetically indistinguishable from Faris in prior work with SSR markers. Analysis of microarray data revealed that the transcriptomes of the two sour lemon genotypes were nearly identical. In contrast, the transcriptome of Faris sweet lemon was very different from those of both sour lemons. Among about 1,000 FNA-specific, presumably pH-related genes, the homolog of Arabidopsis H(+)-ATPase proton pump AHA10 was not expressed in FNA, but highly expressed in FA and L. Since Arabidopsis AHA10 is involved in biosynthesis and acidification of vacuoles, the lack of expression of the AHA10 citrus homolog represents a very conspicuous molecular feature of the FNA sweet phenotype. In addition, high expression of several 2-oxoglutarate degradation-related genes in FNA suggests activation of the GABA shunt and degradation of valine and tyrosine as components of the mechanism that reduces the level of citric acid in sweet lemon.

  7. Express.js blueprints

    CERN Document Server

    Augarten, Ben; Lin, Eric; Shaikh, Aidha; Soriani, Fabiano Pereira; Tisserand, Geoffrey; Zhang, Chiqing; Zhang, Kan

    2015-01-01

    This book is for beginners to Node.js and also for those who are technically advanced. By the end of this book, every competent developer will have achieved expertise in building web applications with Express.js.

  8. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  9. Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response.

    Science.gov (United States)

    Kielczewska, Agnieszka; Pyzik, Michal; Sun, Tianhe; Krmpotic, Astrid; Lodoen, Melissa B; Munks, Michael W; Babic, Marina; Hill, Ann B; Koszinowski, Ulrich H; Jonjic, Stipan; Lanier, Lewis L; Vidal, Silvia M

    2009-03-16

    Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatibility complex class I-restricted recognition of virally infected cells by an activating NK cell receptor.

  10. Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns.

    Science.gov (United States)

    Ben-Yehudah, Ahmi; Easley, Charles A; Hermann, Brian P; Castro, Carlos; Simerly, Calvin; Orwig, Kyle E; Mitalipov, Shoukhrat; Schatten, Gerald

    2010-08-05

    The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells.

  11. Potential roles of CCR5(+ CCR6(+ dendritic cells induced by nasal ovalbumin plus Flt3 ligand expressing adenovirus for mucosal IgA responses.

    Directory of Open Access Journals (Sweden)

    Yoshiko Fukuyama

    Full Text Available We assessed the role of CCR5(+/CCR6(+/CD11b(+/CD11c(+ dendritic cells (DCs for induction of ovalbumin (OVA-specific antibody (Ab responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL showed early expansion of CCR5(+/CCR6(+/CD11b(+/CD11c(+ DCs in nasopharyngeal-associated lymphoid tissue (NALT and cervical lymph nodes (CLNs. Subsequently, this DC subset became resident in submandibular glands (SMGs and nasal passages (NPs in response to high levels of CCR-ligands produced in these tissues. CD11b(+/CD11c(+ DCs were markedly decreased in both CCR5(-/- and CCR6(-/- mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR and CCR5(-/- or CD11c-DTR and CCR6(-/- mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA Ab responses in saliva and nasal washes. These results suggest that CCR5(+CCR6(+ DCs play an important role in the induction of Ag-specific SIgA Ab responses.

  12. Human formyl peptide receptor ligand binding domain(s). Studies using an improved mutagenesis/expression vector reveal a novel mechanism for the regulation of receptor occupancy.

    Science.gov (United States)

    Perez, H D; Vilander, L; Andrews, W H; Holmes, R

    1994-09-09

    Recently, we reported the domain requirements for the binding of formyl peptide to its specific receptor. Based on experiments using receptor chimeras, we also postulated an importance for the amino-terminal domain of the receptor in ligand binding (Perez, H. D., Holmes, R., Vilander, L., Adams, R., Manzana, W., Jolley, D., and Andrews, W. H. (1993) J. Biol. Chem. 268, 2292-2295). We have begun to perform a detailed analysis of the regions within the formyl peptide receptor involved in ligand binding. To address the importance of the receptor amino-terminal domain, we substituted (or inserted) hydrophilic sequences within the amino-terminal domain, expressed the receptors, and determined their ability to bind ligand. A stretch of nine amino acids next to the initial methionine was identified as crucial for receptor occupancy. A peptide containing such a sequence specifically completed binding of the ligand to the receptor. Alanine screen mutagenesis of the second extracellular domain also identified amino acids involved in ligand binding as well as a disulfide bond (Cys98 to Cys176) crucial for maintaining the binding pocket. These studies provide evidence for a novel mechanism involved in regulation of receptor occupancy. Binding of the ligand induces conformational changes in the receptor that result in the apposition of the amino-terminal domain over the ligand, providing a lid to the binding pocket.

  13. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation.

    Science.gov (United States)

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-Il; Uemura, Taku; Hirai, Shizuka; Kawada, Teruo

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPARgamma by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPARgamma target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPARgamma and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  14. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  15. HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates

    Science.gov (United States)

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan L.; Seaman, Michael S.; Montefiori, David C.; Yates, Nicole L.; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; Roederer, Mario; Self, Steven G.; Borate, Bhavesh; Gottardo, Raphael; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony D.; Weiss, Deborah E.; Lee, Carter; Kibler, Karen V.; Jacobs, Bertram L.; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe

    2017-01-01

    ABSTRACT The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions. IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1

  16. HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates.

    Science.gov (United States)

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan L; Seaman, Michael S; Montefiori, David C; Yates, Nicole L; Tomaras, Georgia D; Ferrari, Guido; Foulds, Kathryn E; Roederer, Mario; Self, Steven G; Borate, Bhavesh; Gottardo, Raphael; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W; Burke, Brian; Cristillo, Anthony D; Weiss, Deborah E; Lee, Carter; Kibler, Karen V; Jacobs, Bertram L; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe; Esteban, Mariano

    2017-05-01

    The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection

  17. Academic Freedom and Artistic Expression.

    Science.gov (United States)

    Stern, Carol Simpson

    1994-01-01

    Recent attempts to curtail artistic expression in colleges and universities, particularly in relation to federal funding, are examined and it is concluded that artistic expression merits protections of academic freedom similar to those accorded to intellectual expression. (Author/MSE)

  18. Cues and expressions

    Directory of Open Access Journals (Sweden)

    Thorbjörg Hróarsdóttir

    2005-02-01

    Full Text Available A number of European languages have undergone a change from object-verb to verb-object order. We focus on the change in English and Icelandic, showing that while the structural change was the same, it took place at different times and different ways in the two languages, triggered by different E-language changes. As seen from the English viewpoint, low-level facts of inflection morphology may express the relevant cue for parameters, and so the loss of inflection may lead to a grammar change. This analysis does not carry over to Icelandic, as the loss of OV there took place despite rich case morphology. We aim to show how this can be explained within a cue-style approach, arguing for a universal set of cues. However, the relevant cue may be expressed differently among languages: While it may have been expressed through morphology in English, it as expressed through information structure in Icelandic. In both cases, external effects led to fewer expressions of the relevant (universal cue and a grammar change took place.

  19. Lack of CD47 on nonhematopoietic cells induces split macrophage tolerance to CD47null cells

    OpenAIRE

    Wang, Hui; Madariaga, Maria Lucia; Wang, Shumei; Van Rooijen, Nico; Oldenborg, Per-Arne; Yang, Yong-Guang

    2007-01-01

    Macrophages recognize CD47 as a marker of “self” and phagocytose CD47null hematopoietic cells. Using CD47 chimera models, here, we show that the phagocytic activity of macrophages against CD47null hematopoietic cells is conferred by CD47 expression on nonhematopoietic cells, and this “education” process is hematopoietic cell-independent. Macrophages in the chimeras where nonhematopoietic cells express CD47 phagocytose CD47null cells, whereas those in the chimeras lacking CD47 on nonhematopoie...

  20. Antitumor HPV E7-specific CTL activity elicited by in vivo engineered exosomes produced through DNA inoculation.

    Science.gov (United States)

    Di Bonito, Paola; Chiozzini, Chiara; Arenaccio, Claudia; Anticoli, Simona; Manfredi, Francesco; Olivetta, Eleonora; Ferrantelli, Flavia; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio

    2017-01-01

    We recently proved that exosomes engineered in vitro to deliver high amounts of HPV E7 upon fusion with the Nef(mut) exosome-anchoring protein elicit an efficient anti-E7 cytotoxic T lymphocyte immune response. However, in view of a potential clinic application of this finding, our exosome-based immunization strategy was faced with possible technical difficulties including industrial manufacturing, cost of production, and storage. To overcome these hurdles, we designed an as yet unproven exosome-based immunization strategy relying on delivery by intramuscular inoculation of a DNA vector expressing Nef(mut) fused with HPV E7. In this way, we predicted that the expression of the Nef(mut)/E7 vector in muscle cells would result in a continuous source of endogenous (ie, produced by the inoculated host) engineered exosomes able to induce an E7-specific immune response. To assess this hypothesis, we first demonstrated that the injection of a Nef(mut)/green fluorescent protein-expressing vector led to the release of fluorescent exosomes, as detected in plasma of inoculated mice. Then, we observed that mice inoculated intramuscularly with a vector expressing Nef(mut)/E7 developed a CD8(+) T-cell immune response against both Nef and E7. Conversely, no CD8(+) T-cell responses were detected upon injection of vectors expressing either the wild-type Nef isoform of E7 alone, most likely a consequence of their inefficient exosome incorporation. The production of immunogenic exosomes in the DNA-injected mice was formally demonstrated by the E7-specific CD8(+) T-cell immune response we detected in mice inoculated with exosomes isolated from plasma of mice inoculated with the Nef(mut)/E7 vector. Finally, we provide evidence that the injection of Nef(mut)/E7 DNA led to the generation of effective antigen-specific cytotoxic T lymphocytes whose activity was likely part of the potent, therapeutic antitumor effect we observed in mice implanted with TC-1 tumor cells. In summary, we

  1. The Expressive Organization

    DEFF Research Database (Denmark)

    branding on organizational structures and processes? How do organizations discover their identities? These are some of the vexing problems addressed in this book by a diverse international team of contributors. According to the authors, the future lies with "the expressive organization". Such organizations...... not only understand their distinct identity and their brands, but are also able to express these externally and internally. In order to thrive in an era of transparency and customer choice, the authors argue, organizations will have to be expressive. This book is intended for undergraduate and postgraduate......This text challenges beliefs about organizational identity, reputation, and branding. It contains a wealth of new ideas for finding the elusive answers to questions troubling contemporary organizations. How does an organization create a strong reputation? What are the implications of corporate...

  2. The Expressive Organization

    DEFF Research Database (Denmark)

    This text challenges beliefs about organizational identity, reputation, and branding. It contains a wealth of new ideas for finding the elusive answers to questions troubling contemporary organizations. How does an organization create a strong reputation? What are the implications of corporate...... branding on organizational structures and processes? How do organizations discover their identities? These are some of the vexing problems addressed in this book by a diverse international team of contributors. According to the authors, the future lies with "the expressive organization". Such organizations...... not only understand their distinct identity and their brands, but are also able to express these externally and internally. In order to thrive in an era of transparency and customer choice, the authors argue, organizations will have to be expressive. This book is intended for undergraduate and postgraduate...

  3. The Expressive Organization

    DEFF Research Database (Denmark)

    not only understand their distinct identity and their brands, but are also able to express these externally and internally. In order to thrive in an era of transparency and customer choice, the authors argue, organizations will have to be expressive. This book is intended for undergraduate and postgraduate......This text challenges beliefs about organizational identity, reputation, and branding. It contains a wealth of new ideas for finding the elusive answers to questions troubling contemporary organizations. How does an organization create a strong reputation? What are the implications of corporate...... branding on organizational structures and processes? How do organizations discover their identities? These are some of the vexing problems addressed in this book by a diverse international team of contributors. According to the authors, the future lies with "the expressive organization". Such organizations...

  4. Materiality for Musical Expressions

    DEFF Research Database (Denmark)

    Lindell, Rikard; Tahiroğlu, Koray; Riis, Morten S.

    2016-01-01

    technology and possible musical expression with a strong connection to culture and place. The emphasis on performance provided closure and motivated teams to move forward in their design and artistic processes. On the basis of the course we discuss an interdisciplinary NIME course syllabus, and we infer...

  5. Freedom of expression

    Directory of Open Access Journals (Sweden)

    Oljana HOXHAJ

    2013-12-01

    Full Text Available In this paper "Freedom of expression" I’ ve tried to explain the close relationship between freedom right and other constitutional freedoms, which have a direct impact on values consolidation in a democratic society and giving possibilities for the public to be active in the decision making process. The researches are based in three directions: the doctrine of international low, in Albanian literature; in native and foreign legislation and also in jurisprudence of Albanians courts and the European Court of Human Rights. The theme dedicates a wide space freedom of expression in the context of public debate, thereby guaranteeing the public's right to know. Many cases of interference in freedom of expression, has been given special importance in legal terms. This intervention must have a legitimate purpose to protect more than one of the public interests. All of this work focuses on sharing the idea that everyone has the right to freedom of opinion and expression; this right includes freedom to hold opinions without interference, receive and impart information and ideas through any media and regardless of frontiers.

  6. Experience and Expression

    Science.gov (United States)

    Hanes, Jay Michael; Weisman, Eleanor

    2016-01-01

    Two artist-educators analyzed their creative process informed by John Dewey's concepts regarding the act of expression. The essay interweaves a description of their performance piece with a discussion of conceptual processes, including intermediality and collaboration as crucial in art making, learning, and pedagogical efficacy. Both the creation…

  7. Encouraging Self-Expression

    Science.gov (United States)

    Barnes, Peter

    2006-01-01

    In this article, the author discusses the importance of encouraging self-expression among students. He contends that allowing students to share their personal interests can be of benefit to all. The students' true personalities come out and they become more comfortable with one another as the year goes on.

  8. Virtual Self-Expression

    Science.gov (United States)

    Black, David V.

    2008-01-01

    This article introduces the art of three-dimensional modelling. Three-dimensional modelling is gaining acceptance as a new medium for self-expression. Students must first master the software programs, learn the tools and functions, the menu choices and settings, and use them to create realistic objects. (Contains 4 online resources.)

  9. Encircling Creative Expression

    Science.gov (United States)

    Brown, Susannah

    2007-01-01

    Artworks that are circular in nature are often referred to as mandalas. "Mandala" means center, circle, or circumference. Mandalas are created in many cultures for a variety of reasons, most of which are related to self-expression, ritual, and religion. In this article, the author describes how her students created mandalas. She also provides…

  10. Facial Expression Recognition

    NARCIS (Netherlands)

    Pantic, Maja; Li, S.; Jain, A.

    2009-01-01

    Facial expression recognition is a process performed by humans or computers, which consists of: 1. Locating faces in the scene (e.g., in an image; this step is also referred to as face detection), 2. Extracting facial features from the detected face region (e.g., detecting the shape of facial

  11. The Expressive Organization

    DEFF Research Database (Denmark)

    branding on organizational structures and processes? How do organizations discover their identities? These are some of the vexing problems addressed in this book by a diverse international team of contributors. According to the authors, the future lies with "the expressive organization". Such organizations...

  12. Facial expressions recognition with an emotion expressive robotic head

    Science.gov (United States)

    Doroftei, I.; Adascalitei, F.; Lefeber, D.; Vanderborght, B.; Doroftei, I. A.

    2016-08-01

    The purpose of this study is to present the preliminary steps in facial expressions recognition with a new version of an expressive social robotic head. So, in a first phase, our main goal was to reach a minimum level of emotional expressiveness in order to obtain nonverbal communication between the robot and human by building six basic facial expressions. To evaluate the facial expressions, the robot was used in some preliminary user studies, among children and adults.

  13. Assessing Pain by Facial Expression: Facial Expression as Nexus

    OpenAIRE

    Kenneth M Prkachin

    2009-01-01

    The experience of pain is often represented by changes in facial expression. Evidence of pain that is available from facial expression has been the subject of considerable scientific investigation. The present paper reviews the history of pain assessment via facial expression in the context of a model of pain expression as a nexus connecting internal experience with social influence. Evidence about the structure of facial expressions of pain across the lifespan is reviewed. Applications of fa...

  14. Immunogenic profiling in mice of a HIV/AIDS vaccine candidate (MVA-B expressing four HIV-1 antigens and potentiation by specific gene deletions.

    Directory of Open Access Journals (Sweden)

    Juan García-Arriaza

    Full Text Available BACKGROUND: The immune parameters of HIV/AIDS vaccine candidates that might be relevant in protection against HIV-1 infection are still undefined. The highly attenuated poxvirus strain MVA is one of the most promising vectors to be use as HIV-1 vaccine. We have previously described a recombinant MVA expressing HIV-1 Env, Gag, Pol and Nef antigens from clade B (referred as MVA-B, that induced HIV-1-specific immune responses in different animal models and gene signatures in human dendritic cells (DCs with immunoregulatory function. METHODOLOGY/PRINCIPAL FINDINGS: In an effort to characterize in more detail the immunogenic profile of MVA-B and to improve its immunogenicity we have generated a new vector lacking two genes (A41L and B16R, known to counteract host immune responses by blocking the action of CC-chemokines and of interleukin 1beta, respectively (referred as MVA-B DeltaA41L/DeltaB16R. A DNA prime/MVA boost immunization protocol was used to compare the adaptive and memory HIV-1 specific immune responses induced in mice by the parental MVA-B and by the double deletion mutant MVA-B DeltaA41L/DeltaB16R. Flow cytometry analysis revealed that both vectors triggered HIV-1-specific CD4(+ and CD8(+ T cells, with the CD8(+ T-cell compartment responsible for >91.9% of the total HIV-1 responses in both immunization groups. However, MVA-B DeltaA41L/DeltaB16R enhanced the magnitude and polyfunctionality of the HIV-1-specific CD4(+ and CD8(+ T-cell immune responses. HIV-1-specific CD4(+ T-cell responses were polyfunctional and preferentially Env-specific in both immunization groups. Significantly, while MVA-B induced preferentially Env-specific CD8(+ T-cell responses, MVA-B DeltaA41L/DeltaB16R induced more GPN-specific CD8(+ T-cell responses, with an enhanced polyfunctional pattern. Both vectors were capable of producing similar levels of antibodies against Env. CONCLUSIONS/SIGNIFICANCE: These findings revealed that MVA-B and MVA-B DeltaA41L/DeltaB16R

  15. Kuiper Express: a sciencecraft

    Science.gov (United States)

    Rodgers, David H.; Alkalai, Leon; Beauchamp, Patricia M.; Chen, Gun-Shing; Crisp, Michael P.; Brown, Robert H.; Davidson, J. M.; Huxtable, Douglas D.; Penzo, P. A.; Petrick, Stanley W.; Soderblom, Laurance A.; Stewart, A.; Vane, Gregg; Yelle, Roger V.

    1996-10-01

    The Kuiper Express is a mission to achieve the first reconnaissance of one of the primitive objects that reside in the Kuiper Belt. The objects in the Kuiper Belt are the remnants of the planetesimal swarm that formed the four giant planets of the outer Solar System. These objects, because they are far from the Sun, have not been processed by solar heating and are essentially in their primordial state. This makes them unique objects and their study will provide information on the composition of the solar nebula that cannot be extracted from a study of other objects in the Solar System. The Kuiper Express is a sciencecraft mission. A sciencecraft is an integrated unit that combines into a single system the essential elements (but no more) necessary to achieve the science objectives of the mission, including science instruments, electronics, telecommunications, power, and propulsion. The design of a sciencecraft begins with the definition of mission science objectives and cost constraint. An observational sequence and sensor subsystem are then designed. This sensor subsystem in turn becomes the design driver for the sciencecraft architecture and hardware subsystems needed to deliver the sensor to its target and return the science data to the earth. Throughout the design process, shared functionality, shared redundancy, and reduced cost are strongly emphasized. The Kuiper Express will be launched using a Delta vehicle and will use solar electric propulsion to add velocity and shape its trajectory in the inner Solar System, executing two earth gravity-assist flybys. It will also execute flybys of main belt asteroids, Mars, Uranus, and Neptune/Triton en route to its target in the Kuiper belt, where it will arrive about ten years after launch. It will use no nuclear power. The surface constituents and morphology of the objects visited will be measured and their atmospheres will be characterized. The cost of the detailed design, fabrication, and launch of the Kuiper

  16. odo/pAe Vt'nef, 1791-1847. Uit

    African Journals Online (AJOL)

    Test

    Evangelie soos suurdeeg die deeg laat rys. Dis egter nie alleen KuYPER wat op die punt van die verhou ding van Kerk en Staat 'n volgeling van ViNET was nie, maar ook DE. SAVORNiN LOHMAN en VAN DER BRUGGHEN. ViNET het groot invloed uitgeoefen tot ver buite Switserland. Dr. KEijZER wys daarop dat hy 'n ...

  17. Data Mining for Expressivity of Recombinant Protein Expression

    Science.gov (United States)

    Kira, Satoshi; Isoai, Atsushi; Yamamura, Masayuki

    We analyzed the expressivity of recombinant proteins by using data mining methods. The expression technique of recombinant protein is a key step towards elucidating the functions of genes discovered through genomic sequence projects. We have studied the productive efficiency of recombinant proteins in fission yeast, Schizosaccharomyces pombe (S.pombe), by mining the expression results. We gathered 57 proteins whose expression levels were known roughly in the host. Correlation analysis, principal component analysis and decision tree analysis were applied to these expression data. Analysis featuring codon usage and amino acid composition clarified that the amino acid composition affected to the expression levels of a recombinant protein strongly than the effect of codon usage. Furthermore, analysis of amino acid composition showed that protein solubility and the metabolism cost of amino acids correlated with a protein expressivity. Codon usage was often interesting in the field of recombinant expressions. However, our analysis found the weak correlation codon features with expressivities. These results indicated that ready-made indices of codon bias were irrelevant ones for modeling the expressivities of recombinant proteins. Our data driven approach was an easy and powerful method to improve recombinant protein expression, and this approach should be concentrated attention with the huge amount of expression data accumulating through the post-genome era.

  18. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    Science.gov (United States)

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  19. Boolean Expression Diagrams

    DEFF Research Database (Denmark)

    Andersen, Henrik Reif; Hulgaard, Henrik

    2002-01-01

    a BED into a reduced ordered BDD. One is a generalized version of the BDD apply-operator while the other can exploit the structural information of the Boolean expression. This ability is demonstrated by verifying that two different circuit implementations of a 16-bit multiplier implement the same...... Boolean function. Using BEDs, this verification problem is solved efficiently, while using standard BDD techniques this problem is infeasible. Generally, BEDs are useful in applications, for example tautology checking, where the end-result as a reduced ordered BDD is small. Moreover, using operators...... for substitution and existential quantification they allow for the verification of large hierarchical circuits....

  20. Boolean Expression Diagrams

    DEFF Research Database (Denmark)

    Andersen, Henrik Reif; Hulgaard, Henrik

    1997-01-01

    properties of BDDs. Two algorithms are described for transforming a BED into a reduced ordered BDD. One closely mimics the BDD apply-operator while the other can exploit the structural information of the Boolean expression. The efficacy of the BED representation is demonstrated by verifying...... that the redundant and non-redundant versions of the ISCAS 85 benchmark circuits are identical. In particular, it is verified that the two 16-bit multiplication circuits (c6288 and c6288nr) implement the same Boolean functions. Using BEDs, this verification problem is solved in less than a second, while using...

  1. Expression-Invariant Age Estimation

    NARCIS (Netherlands)

    Alnajar, F.; Lou, Z.; Alvarez, J.; Gevers, T.; Valstar, M.; French, A.; Pridmore, T.

    2014-01-01

    In this paper, we investigate and exploit the influence of facial expressions on automatic age estimation. Different from existing approaches, our method jointly learns the age and expression by introducing a new graphical model with a latent layer between the age/expression labels and the features.

  2. The expressions of emotions

    DEFF Research Database (Denmark)

    Vishnivetz, Berta

    and of expressions of emotions and movement notation that provided the sources for a careful research plan for the empirical process of this study. On this basis I chose to record onto video the four previously choreographed movements that I considered to correspond each of the following emotions: joy, fear, sadness......, anger. The selection of these four emotions demanded previously to clear up the problems the above named survey ensued. When researchers want to describe a certain movement in the field of psychology and non-verbal communication, it may result in disagreements and misunderstandings which sometimes lead...... to methodological errors. I carried a judgement study and a component study to validate the empirical data: the recorded emotions. For the judgement study I asked 71 voluntary observers from four different countries to watch the video and made an identification of what emotion it was being displayed...

  3. Natural Art, False Expressions

    Directory of Open Access Journals (Sweden)

    Oscar Hernando Nossa García

    2011-05-01

    Full Text Available In the documentary My Kid Could Paint That, directed by Bar-Lev, which deals with Marla Olmstead, the child prodigy of painting, several interviews with persons in the art world are conducted, among them an artist who uses a magnifying glass and the thinnest brushes to do his work. This man, although happy for the success of the child’s abstract paintings, saw in the whole spectacle a mockery of art, and stood firmly by her work. The girl’s father, also an artist, was accused of plagiarism. Cameras entered the child’s studio in order to prove that Marla was the real artist. Why should such relevance be given to authorship? What is the cause of the dispute between the expressive and the rational?

  4. Expressing emotions in blogs

    DEFF Research Database (Denmark)

    Rodriguez-Hidalgo, Carmina Rodriguez-Hidalgo; Tan, Ed S.; Verlegh, Peeter

    2017-01-01

    Textual paralanguage cues (TPC) have been signaled as effective emotion transmitters online. Though several studies have investigated their properties and occurrence, there remains a gap concerning their communicative impact within specific psychological processes, such as the social sharing...... of emotion (SSE, Rimé, 2009). This study content-analyzed Live Journal blogposts for the occurrence of TPC in three phases of online SSE: initiation, feedback and repost. We compared these to TPC on a second type of emotional expression, emotional venting. Based on Social Information processing theory (SIP......, Walther, 1992), and on the Emotional Mimicry in Context (EMC, Hess & Fischer, 2013) framework, we study predictive relationships in TPC usage in our phased model of online SSE. Results showed that TPC prevailed in SSE blogposts and strongly dominated in emotional venting posts. TPC was more common...

  5. The Symbiotic Anthozoan: A Physiological Chimera between Alga and Animal.

    Science.gov (United States)

    Furla, Paola; Allemand, Denis; Shick, J Malcolm; Ferrier-Pagès, Christine; Richier, Sophie; Plantivaux, Amandine; Merle, Pierre-Laurent; Tambutté, Sylvie

    2005-08-01

    The symbiotic life style involves mutual ecological, physiological, structural, and molecular adaptations between the partners. In the symbiotic association between anthozoans and photosynthetic dinoflagellates (Symbiodinium spp., also called zooxanthellae), the presence of the endosymbiont in the animal cells has constrained the host in several ways. It adopts behaviors that optimize photosynthesis of the zooxanthellae. The animal partner has had to evolve the ability to absorb and concentrate dissolved inorganic carbon from seawater in order to supply the symbiont's photosynthesis. Exposing itself to sunlight to illuminate its symbionts sufficiently also subjects the host to damaging solar ultraviolet radiation. Protection against this is provided by biochemical sunscreens, including mycosporine-like amino acids, themselves produced by the symbiont and translocated to the host. Moreover, to protect itself against oxygen produced during algal photosynthesis, the cnidarian host has developed certain antioxidant defenses that are unique among animals. Finally, living in nutrient-poor waters, the animal partner has developed several mechanisms for nitrogen assimilation and conservation such as the ability to absorb inorganic nitrogen, highly unusual for a metazoan. These facts suggest a parallel evolution of symbiotic cnidarians and plants, in which the animal host has adopted characteristics usually associated with phototrophic organisms.

  6. A streptavidin-SOG chimera for all-optical immunoassays.

    Science.gov (United States)

    Wurtzler, Elizabeth M; Wendell, David

    2014-01-13

    Immunological detection has been developed into a sensitive and versatile technique and is based on two requisite elements: targeting antibodies and an indicator, usually in the form of horseradish peroxidase or alkaline phosphatase. The specificity and turnover rate of these enzymes provide an efficient means of signal amplification, but both require a stopping agent to prevent overdevelopment, which limits scalability to mechanized fluidic systems. As an alternative, we present a fully optical detection system based on a streptavidin-singlet oxygen generating chimeric protein that produces singlet oxygen from blue light. When used with trans-1-(2'-methoxyvinyl)pyrene, the photosynthetic streptavidin combines indicator development and reporting in sufficiently distinct visible wavelengths while retaining the sensitivity and scale of enzymatic systems that use horseradish peroxidase. By combining photosensitive development and detection into one system, we can enable future, highly parallel immunological testing to be controlled with the spatial and temporal precision of light.

  7. Aptamer-siRNA Chimeras: Discovery, Progress, and Future Prospects

    Science.gov (United States)

    Kruspe, Sven; Giangrande, Paloma H.

    2017-01-01

    Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery tools for many therapeutic oligonucleotide-based drugs, including small interfering RNAs (siRNAs). In this review, we summarize recent progress in the aptamer selection technology that has made possible the identification of cell-specific, cell-internalizing aptamers for the cell-targeted delivery of therapeutic oligonucleotides. In addition, we review the original, proof-of-concept aptamer-siRNA delivery studies and discuss recent advances in aptamer-siRNA conjugate designs for applications ranging from cancer therapy to the development of targeted antivirals. Challenges and prospects of aptamer-targeted siRNA drugs for clinical development are further highlighted. PMID:28792479

  8. RNA Chimeras as a Gene Signature of Breast Cancer

    Science.gov (United States)

    2013-05-01

    www.plosone.org 11 August 2012 | Volume 7 | Issue 8 | e41659 Human genes Human ACTB mRNA: >gi|168480144|ref|NM_001101.3| Homo sapiens actin, beta...TCCCCCTTTTTTGTCCCCCAACTTGAGATGTATGAAGGCTTTTGGTCTCCCTGGGAGTGGGTGGAGGCAGCCAGGGCTTACCTGTACACTGACTTGAGACCAGTTGAATAAA AGTGCACACCTTAAAAATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Human GAPDH mRNA: >gi|83641890|ref|NM_002046.4| Homo sapiens ... Homo sapiens hypoxanthine phosphoribosyltransferase 1 (HPRT1), mRNA

  9. Donor chimera model for tolerance induction in transplantation

    NARCIS (Netherlands)

    Rezaee, F.; Peppelenbosch, M.; Dashty, M.

    Tolerance induction is the basis of a successful transplantation with the goal being the re-establishment of homeostasis after transplantation. Non-autograft transplantation disrupts this maintenance drastically which would be avoided by administration of a novel procedure. At present, the blood

  10. A healthy, female chimera with 46,XX/46,XY karyotype.

    NARCIS (Netherlands)

    Binkhorst, M.; Leeuw, N. de; Otten, B.J.

    2009-01-01

    We report a healthy and unambiguously female newborn, whose phenotypic sex contradicted the expected male sex based on previously performed prenatal cytogenetic analysis. Both 46,XX and 46,XY cells were detected in a villus sample, the former having been attributed to maternal cell contamination.

  11. Resurrecting the chimera: Progressions in parenting and peer processes.

    Science.gov (United States)

    Forgatch, Marion S; Snyder, James J; Patterson, Gerald R; Pauldine, Michael R; Chaw, Yvonne; Elish, Katie; Harris, Jasmine B; Richardson, Eric B

    2016-08-01

    This report uses 6-year outcomes of the Oregon Divorce Study to examine the processes by which parenting practices affect deviant peer association during two developmental stages: early to middle childhood and late childhood to early adolescence. The participants were 238 newly divorced mothers and their 5- to 8-year-old sons who were randomly assigned to Parent Management Training-Oregon Model (PMTO®) or to a no-treatment control group. Parenting practices, child delinquent behavior, and deviant peer association were repeatedly assessed from baseline to 6 years after baseline using multiple methods and informants. PMTO had a beneficial effect on parenting practices relative to the control group. Two stage models linking changes in parenting generated by PMTO to children's growth in deviant peer association were supported. During the early to middle childhood stage, the relationship of improved parenting practices on deviant peer association was moderated by family socioeconomic status (SES); effective parenting was particularly important in mitigating deviant peer association for lower SES families whose children experience higher densities of deviant peers in schools and neighborhoods. During late childhood and early adolescence, the relationship of improved parenting to youths' growth in deviant peer association was mediated by reductions in the growth of delinquency during childhood; higher levels of early delinquency are likely to promote deviant peer association through processes of selective affiliation and reciprocal deviancy training. The results are discussed in terms of multilevel developmental progressions of diminished parenting, child involvement in deviancy producing processes in peer groups, and increased variety and severity of antisocial behavior, all exacerbated by ecological risks associated with low family SES.

  12. Plasma haptoglobin levels and radiation chimeras in mice. [Gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Hayakawa, J.; Tsuchiya, T.; Dei, T.

    1976-05-01

    When irradiated mice were grafted with allogeneic bone marrow, their plasma haptoglobin(Hp) significantly increased 2 weeks after grafting. To elucidate the generation of the increase of Hp in allogeneic bone marrow recipients, the relationship between the Hp levels and allograft reactions was assessed. The Hp levels of irradiated recipients, which were inoculated with allogeneic spleen cells, increased with the increase of alloantigen-sensitive units in donor cells. The positive correlation between the Hp levels and graft-versus-host reactions evaluated by spleen weight assay was also observed in unirradiated F/sub 1/ hybrid mice which received parental spleen cells. It is, therefore, suspected that the elevated Hp levels found in allogeneic-bone-marrow recipients are associated with lymphoid cell proliferation initiated by alloantigenic stimulations, and it was concluded that Hp levels in allogeneic-bone-marrow recipients provide an indicator of the degree of allograft reactions.

  13. Genes encoding chimeras of Neurospora crassa erg-3 and human ...

    Indian Academy of Sciences (India)

    Unknown

    In the yeast Saccharomyces cerevisiae, sterol. C-14 reductase is encoded by the ERG24 gene and erg24 null mutants are not viable on rich medium but they are viable on synthetic medium (Crowley et al 1996). Both the Neurospora and the yeast mutants have been used previously to test for sterol C-14 reductase function ...

  14. Genes encoding chimeras of Neurospora crassa erg-3 and human ...

    Indian Academy of Sciences (India)

    http://www.ias.ac.in/article/fulltext/jbsc/027/02/0105-0112. Keywords. Lamin B receptor; sterol reductase. Abstract. The human gene TM7SF2 encodes a polypeptide (SR-1) with high sequence similarity to sterol C-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. In Neurospora and yeast this ...

  15. Chemically tunable mucin chimeras assembled on living cells

    Science.gov (United States)

    Kramer, Jessica R.; Onoa, Bibiana; Bustamante, Carlos; Bertozzi, Carolyn R.

    2015-01-01

    Mucins are a family of secreted and transmembrane glycoproteins characterized by a massive domain of dense O-glycosylation on serine and threonine residues. Mucins are intimately involved in immunity and cancer, yet elucidation of the biological roles of their glycodomains has been complicated by their massive size, domain polymorphisms, and variable glycosylation patterns. Here we developed a synthetic route to a library of compositionally defined, high-molecular weight, dual end-functionalized mucin glycodomain constructs via N-carboxyanhydride polymerization. These glycopolypeptides are the first synthetic analogs to our knowledge to feature the native α-GalNAc linkage to serine with molecular weights similar to native mucins, solving a nearly 50-year synthetic challenge. Physical characterization of the mimics revealed insights into the structure and properties of mucins. The synthetic glycodomains were end-functionalized with an optical probe and a tetrazine moiety, which allowed site-specific bioorthogonal conjugation to an engineered membrane protein on live mammalian cells. This strategy in protein engineering will open avenues to explore the biological roles of cell surface mucins. PMID:26420872

  16. Chimera states in a population of identical oscillators under planar ...

    Indian Academy of Sciences (India)

    [20] F Sorrentino, New J. Phys. 14, 033035 (2012). [21] S Bilal and R Ramaswamy, Phys. Rev. E 89, 062923 (2014). [22] R FitzHugh, Bull. Math. Biophys. 17, 257 (1955). [23] V Icke, Force of symmetry (Cambridge University Press, Cambridge, 1995) p. 247. [24] H Daido and K Nakanishi, Phys. Rev. Lett. 96, 054101 (2006).

  17. Hunting down the chimera of multiple disciplinarity in conservation science.

    Science.gov (United States)

    Pooley, Simon P; Mendelsohn, J Andrew; Milner-Gulland, E J

    2014-02-01

    The consensus is that both ecological and social factors are essential dimensions of conservation research and practice. However, much of the literature on multiple disciplinary collaboration focuses on the difficulties of undertaking it. This review of the challenges of conducting multiple disciplinary collaboration offers a framework for thinking about the diversity and complexity of this endeavor. We focused on conceptual challenges, of which 5 main categories emerged: methodological challenges, value judgments, theories of knowledge, disciplinary prejudices, and interdisciplinary communication. The major problems identified in these areas have proved remarkably persistent in the literature surveyed (c.1960-2012). Reasons for these failures to learn from past experience include the pressure to produce positive outcomes and gloss over disagreements, the ephemeral nature of many such projects and resulting lack of institutional memory, and the apparent complexity and incoherence of the endeavor. We suggest that multiple disciplinary collaboration requires conceptual integration among carefully selected multiple disciplinary team members united in investigating a shared problem or question. We outline a 9-point sequence of steps for setting up a successful multiple disciplinary project. This encompasses points on recruitment, involving stakeholders, developing research questions, negotiating power dynamics and hidden values and conceptual differences, explaining and choosing appropriate methods, developing a shared language, facilitating on-going communications, and discussing data integration and project outcomes. Although numerous solutions to the challenges of multiple disciplinary research have been proposed, lessons learned are often lost when projects end or experienced individuals move on. We urge multiple disciplinary teams to capture the challenges recognized, and solutions proposed, by their researchers while projects are in process. A database of well-documented case studies would showcase theories and methods from a variety of disciplines and their interactions, enable better comparative study and evaluation, and provide a useful resource for developing future projects and training multiple disciplinary researchers. © 2013 The Authors. Conservation Biology published by Wiley Periodicals, Inc. on behalf of Society for Conservation Biology.

  18. Attention modulates emotional expression processing.

    Science.gov (United States)

    Wronka, Eligiusz; Walentowska, Wioleta

    2011-08-01

    To investigate the time course of emotional expression processing, we recorded ERPs to facial stimuli. The first task was to discriminate emotional expressions. Enhanced negativity of the face-specific N170 was elicited by emotional as opposed to neutral faces, followed by the occipital negativity (240-340 ms poststimulus). The second task was to classify face gender. Here, N170 was unaffected by the emotional expression. However, emotional expression effect was expressed in the anterior positivity (160-250 ms poststimulus) and subsequent occipital negativity (240-340 ms poststimulus). Results support the thesis that structural encoding relevant to gender recognition and simultaneous expression analysis are independent processes. Attention modulates facial emotion processing 140-185 ms poststimulus. Involuntary differentiation of facial expression was observed later (160-340 ms poststimulus), suggesting unintentional attention capture. Copyright © 2011 Society for Psychophysiological Research.

  19. Towards a semantics for mass expressions derived from gradable expressions

    OpenAIRE

    Nicolas, David

    2010-01-01

    What semantics should we attribute to mass expressions like wisdom and love, which are derived from gradable expressions (wise, to love)? We first examine how these expressions are used, then how they are interpreted in their various uses. We show in particular that, just like with ordinary concrete mass nouns (wine, furniture), sentences where they appear are liable to distributive, collective, and intermediate construals. We then propose a model to account for these data, in which derived m...

  20. Animal Cell Expression Systems.

    Science.gov (United States)

    Butler, M; Reichl, Ing U

    2017-10-03

    The glycan profile of therapeutic recombinant proteins such as monoclonal antibodies is a critical quality attribute, which affects the efficacy of the final product. The cellular glycosylation process during protein expression is dependent upon a number of factors such as the availability of substrates in the media, the intracellular content of nucleotide sugars, and the enzyme repertoire of the host cells. In order to control the variability of glycosylation it is important to understand the critical process parameters and their acceptable range of values to enable reproducible production of proteins with a predetermined glycan profile providing the desired biological function or therapeutic effect. The depletion of critical nutrients such as glucose or galactose, which may occur toward the end of a culture process, can lead to truncated glycans. Terminal galactosylation and sialyation are particularly variable but may be controlled by the presence of some key media components. Ammonia accumulation, pH, and dissolved oxygen levels are also known to be key bioprocess parameters that affect the glycosylation of recombinant proteins. Specific enzyme inhibitors can be added to the media to drive the formation of selected and predetermined glycan profiles. Various attempts have been made to predict the glycan profiles of cellular expressed proteins and have led to metabolic models based upon knowledge of metabolic flux and the kinetics of individual glycosylation reactions.In contrast to single recombinant proteins, the glycan profiles of viral vaccines are far more complex and difficult to predict. The example of influenza A virus shows that hemagglutinin, the major antigenic determinant, has three to nine N-glycans, which may influence the antigenicity and efficacy of the vaccine. Glycosylation of the influenza A virus has been largely unmonitored in the past as production has been from eggs, where glycan profiles of antigens are difficult if not impossible to

  1. Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

    Science.gov (United States)

    Fahrenkrog, Birthe; Martinelli, Valérie; Nilles, Nadine; Fruhmann, Gernot; Chatel, Guillaume; Juge, Sabine; Sauder, Ursula; Di Giacomo, Danika; Mecucci, Cristina; Schwaller, Jürg

    2016-01-01

    Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

  2. Expression of Concern.

    Science.gov (United States)

    2017-11-01

    The Journal Editors hereby issue this note of an expression of concern for the following publication: Benjamin, A. J., Jr., Kepes, S., & Bushman, B. J. (2017). Effects of weapons on aggressive thoughts, angry feelings, hostile appraisals, and aggressive behavior: A meta-analytic review of the weapons effect literature. Personality and Social Psychology Review. Advance online publication. doi:10.1177/1088868317725419 The authors of this manuscript contacted the editors indicating they had discovered some errors in the computation of effect sizes in their meta-analysis. Initial reanalysis suggested that at least one of the substantive conclusions of the manuscript was affected by the error; the authors now urge greater caution in interpreting the effect size of weapons on aggressive behavioral outcomes. The authors will undertake a thorough reanalysis and will modify the results and interpretations accordingly, and this notice will be updated upon their completion of the modifications. The editorial staff appreciates the proactive efforts of the authors in this matter. The Editors and SAGE strive to uphold the very highest standards of publication ethics and are committed to supporting the high standards of integrity of Personality and Social Psychology Review. Authors, reviewers, editors, and interested readers should consult the ethics section of SAGE and the Committee on Publication Ethics (COPE) website for guidelines on publication ethics.

  3. Emotional Expression in Reality TV

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    are being treated as ordinary people. My article will discuss different presentations of selves and especially the emotional verbal and nonverbal expressions in reality TV communication. Aspects of the intimate self and its emotional expressions seem to be strategically managed in reality TV and even......, that information ‘given off’ in lying behavior will must often be found in non-verbal mikro expressions and mikro gestures. I will end by discussing the strategic emotional expressions as production of floating identities in a broader framework on the basis of among others Gergen, Lipovetsky and Baumann...

  4. Oracle Application Express 4 Recipes

    CERN Document Server

    Zehoo, Edmund

    2011-01-01

    Oracle Application Express 4 Recipes provides an example-based approach to learning Application Express - the ground-breaking, rapid application development platform included with every Oracle Database license. The recipes format is ideal for the quick-study who just wants a good example or two to kick start their thinking and get pointed in the right direction. The recipes cover the gamut of Application Express development. Author and Application Express expert Edmund Zehoo shows how to create data entry screens, visualize data in the form of reports and charts, implement validation and back-

  5. Expression modeling for expression-invariant face recognition

    NARCIS (Netherlands)

    Haar, F.B. Ter; Veltkamp, R.C.

    2010-01-01

    Morphable face models have proven to be an effective tool for 3D face modeling and face recognition, but the extension to 3D face scans with expressions is still a challenge. The two main difficulties are (1) how to build a new morphable face model that deals with expressions, and (2) how to fit

  6. Creating an Expressive Performance Mindset

    Science.gov (United States)

    Broomhead, Paul; Skidmore, Jon B.

    2014-01-01

    Students in performance situations sometimes experience physiological symptoms that inhibit their ability to perform as expressively as they otherwise might possess the understanding and ability to do. As students set out to perform with an expressive mindset, the brain's limbic system may detect some perceived danger in the situation and…

  7. Pain expression as a biometric

    DEFF Research Database (Denmark)

    Haque, Mohammad A.; Nasrollahi, Kamal; Moeslund, Thomas B.

    2017-01-01

    Developing a vision-based efficient and automatic pain intensity measurement system requires the understanding of the relationship between self-reported pain intensity and pain expression in the facial videos. In this paper, we first demonstrate how pain expression in facial video frames may not ...

  8. Leishmania-based expression systems.

    Science.gov (United States)

    Taheri, Tahereh; Seyed, Negar; Mizbani, Amir; Rafati, Sima

    2016-09-01

    Production of therapeutic or medical recombinant proteins, such as monoclonal antibodies, proteins, or active enzymes, requires a highly efficient system allowing natural folding and perfect post-translation modifications of the expressed protein. These requirements lead to the generation of a variety of gene expression systems from bacteria to eukaryotes. To achieve the best form of eukaryotic proteins, two factors need to be taken into consideration: choosing a suitable organism to express the protein of interest, and selecting an efficient delivery system. For this reason, the expression of recombinant proteins in eukaryotic nonpathogenic Leishmania parasites is an interesting approach which meets both criteria. Here, new Leishmania-based expression systems are compared with current systems that have long histories in research and industry.

  9. Measuring facial expression of emotion.

    Science.gov (United States)

    Wolf, Karsten

    2015-12-01

    Research into emotions has increased in recent decades, especially on the subject of recognition of emotions. However, studies of the facial expressions of emotion were compromised by technical problems with visible video analysis and electromyography in experimental settings. These have only recently been overcome. There have been new developments in the field of automated computerized facial recognition; allowing real-time identification of facial expression in social environments. This review addresses three approaches to measuring facial expression of emotion and describes their specific contributions to understanding emotion in the healthy population and in persons with mental illness. Despite recent progress, studies on human emotions have been hindered by the lack of consensus on an emotion theory suited to examining the dynamic aspects of emotion and its expression. Studying expression of emotion in patients with mental health conditions for diagnostic and therapeutic purposes will profit from theoretical and methodological progress.

  10. Expression, sorting and transport studies for the orphan carrier SLC10A4 in neuronal and non-neuronal cell lines and in Xenopus laevis oocytes.

    Science.gov (United States)

    Schmidt, Stephanie; Moncada, Marcela; Burger, Simone; Geyer, Joachim

    2015-06-19

    SLC10A4 belongs to the solute carrier family SLC10 whose founding members are the Na(+)/taurocholate co-transporting polypeptide (NTCP, SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT, SLC10A2). These carriers maintain the enterohepatic circulation of bile acids between the liver and the gut. SLC10A4 was identified as a novel member of the SLC10 carrier family with the highest phylogenetic relationship to NTCP. The SLC10A4 protein was detected in synaptic vesicles of cholinergic and monoaminergic neurons of the peripheral and central nervous system, suggesting a transport function for any kind of neurotransmitter. Therefore, in the present study, we performed systematic transport screenings for SLC10A4 and also aimed to identify the vesicular sorting domain of the SLC10A4 protein. We detected a vesicle-like expression pattern of the SLC10A4 protein in the neuronal cell lines SH-SY5Y and CAD. Differentiation of these cells to the neuronal phenotype altered neither SLC10A4 gene expression nor its vesicular expression pattern. Functional transport studies with different neurotransmitters, bile acids and steroid sulfates were performed in SLC10A4-transfected HEK293 cells, SLC10A4-transfected CAD cells and in Xenopus laevis oocytes. For these studies, transport by the dopamine transporter DAT, the serotonin transporter SERT, the choline transporter CHT1, the vesicular monoamine transporter VMAT2, the organic cation transporter Oct1, and NTCP were used as positive control. SLC10A4 failed to show transport activity for dopamine, serotonin, norepinephrine, histamine, acetylcholine, choline, acetate, aspartate, glutamate, gamma-aminobutyric acid, pregnenolone sulfate, dehydroepiandrosterone sulfate, estrone-3-sulfate, and adenosine triphosphate, at least in the transport assays used. When the C-terminus of SLC10A4 was replaced by the homologous sequence of NTCP, the SLC10A4-NTCP chimeric protein revealed clear plasma membrane expression in CAD and HEK

  11. Transgenic tomato plants expressing the antigen gene PfCP-2.9 of Plasmodium falciparum Plantas transgênicas de tomate expressando o gene do antígeno PfCP-2.9 de Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Mihail Kantor

    2013-01-01

    Full Text Available The objective of this work was to obtain transgenic tomato plants expressing the PfCP-2.9 protein (a chimera of the antigens MSP1 and AMA1 of Plasmodium falciparum. Cotyledons of seven-day-old tomatoes, cultivar Summers, were transformed via Agrobacterium tumefaciens. Transgenic expression in the T0 plants was verified in the DNA extracted from fruits. PCR analysis was used to test the presence of the gene of interest in the T1 generation. Reverse transcriptase PCR provided evidence of gene expression at the RNA level, and Western blot analysis confirmed the presence of the protein of interest in the T1 plants. This is the first report of successful transformation with the expression of a malaria antigen (PfCP-2.9 in transgenic tomato plants from the T0 and T1 generations.O objetivo deste trabalho foi obter plantas transgênicas de tomate que expressem a proteína PfCP-2.9 (uma quimera dos antígenos MSP1 e AMA1 de Plasmodium falciparum. Cotilédones de tomate, cultivar Summers, com sete dias de idade, foram transformados via Agrobacterium tumefaciens. A expressão transgênica nas plantas T0 foi verificada no DNA extraído dos frutos. A análise por PCR foi utilizada para testar a presença do gene de interesse na geração T1. A evidência da expressão do gene no RNA foi constatada por meio da PCR de transcriptase reversa, e a análise "Western blot" confirmou a presença da proteína de interesse nas plantas T1. Este é o primeiro relato de transformação bem sucedida com a expressão de um antígeno da malária (PfCP-2,9 em plantas transgênicas de tomate da geração T0 e T1.

  12. Expression of amelogenin in odontoblasts.

    Science.gov (United States)

    Papagerakis, P; MacDougall, M; Hotton, D; Bailleul-Forestier, I; Oboeuf, M; Berdal, A

    2003-03-01

    Amelogenin is the major enamel protein produced by ameloblasts. Its expression has been shown to be down-regulated in ameloblasts of vitamin-D-deficient (-D) rats. The potential expression and localization of amelogenin in odontoblasts and its regulation by vitamin D were investigated in this study. RT-PCR and semi-quantitative Northern blot analyses were performed using the odontoblast cell line MO6-G3 and microdissected dental pulp mesenchyme. Both in vitro and in vivo odontoblasts expressed various alternatively spliced amelogenin transcripts. In situ hybridization studies showed that amelogenin expression was restricted to young odontoblasts during mantle dentin deposition. Electron microscopy studies localized the amelogenin protein in the odontoblast cell process cytoplasm and mantle dentin. Amelogenin immunolabeling was stronger in -D rats, suggesting an inverse regulation by vitamin D in odontoblasts. Furthermore, amelogenin mRNA steady-state levels were significantly increased in -D dental pulp mesenchyme. In addition, a temporal-spatial lengthening of the mantle dentin stage was observed in -D animals, suggesting that developmental perturbations occur in relation to the vitamin D status and/or amelogenin expression. These data show that amelogenin is expressed by odontoblasts selectively during mantle dentin deposition. This developmental regulated expression pattern is enhanced under vitamin-D-deficiency status and in a broader context may play an important role during ameloblast and odontoblast differentiation and function.

  13. Cortical control of facial expression.

    Science.gov (United States)

    Müri, René M

    2016-06-01

    The present Review deals with the motor control of facial expressions in humans. Facial expressions are a central part of human communication. Emotional face expressions have a crucial role in human nonverbal behavior, allowing a rapid transfer of information between individuals. Facial expressions can be either voluntarily or emotionally controlled. Recent studies in nonhuman primates and humans have revealed that the motor control of facial expressions has a distributed neural representation. At least five cortical regions on the medial and lateral aspects of each hemisphere are involved: the primary motor cortex, the ventral lateral premotor cortex, the supplementary motor area on the medial wall, and the rostral and caudal cingulate cortex. The results of studies in humans and nonhuman primates suggest that the innervation of the face is bilaterally controlled for the upper part and mainly contralaterally controlled for the lower part. Furthermore, the primary motor cortex, the ventral lateral premotor cortex, and the supplementary motor area are essential for the voluntary control of facial expressions. In contrast, the cingulate cortical areas are important for emotional expression, because they receive input from different structures of the limbic system. © 2015 Wiley Periodicals, Inc.

  14.  Prokaryotic expression systems

    Directory of Open Access Journals (Sweden)

    Dorota Porowińska

    2013-03-01

    Full Text Available For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host.The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized.Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities. 

  15. TempoExpress: An Expressivity-Preserving Musical Tempo Transformation

    OpenAIRE

    Grachten, Maarten; Arcos, Josep Ll.; Lopez de Mantaras, Ramon

    2006-01-01

    The research described in this paper focuses on global tempo transformations of monophonic audio recordings of saxophone jazz performances. More concretely, we have investigated the problem of how a performance played at a particular tempo can be automatically rendered at another tempo while preserving its expressivity. To do so we have developed a case-based reasoning system called TempoExpress. The results we have obtained have been extensively compared against a sta...

  16. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...... intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely...

  17. [Prosopagnosia and facial expression recognition].

    Science.gov (United States)

    Koyama, Shinichi

    2014-04-01

    This paper reviews clinical neuropsychological studies that have indicated that the recognition of a person's identity and the recognition of facial expressions are processed by different cortical and subcortical areas of the brain. The fusiform gyrus, especially the right fusiform gyrus, plays an important role in the recognition of identity. The superior temporal sulcus, amygdala, and medial frontal cortex play important roles in facial-expression recognition. Both facial recognition and facial-expression recognition are highly intellectual processes that involve several regions of the brain.

  18. Analysis of Facial Expression by Taste Stimulation

    Science.gov (United States)

    Tobitani, Kensuke; Kato, Kunihito; Yamamoto, Kazuhiko

    In this study, we focused on the basic taste stimulation for the analysis of real facial expressions. We considered that the expressions caused by taste stimulation were unaffected by individuality or emotion, that is, such expressions were involuntary. We analyzed the movement of facial muscles by taste stimulation and compared real expressions with artificial expressions. From the result, we identified an obvious difference between real and artificial expressions. Thus, our method would be a new approach for facial expression recognition.

  19. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa....... Not is a homeobox containing gene that regulates the formation of the notochord in chordates, while Cdx (caudal) is a ParaHox gene involved in the formation of posterior tissues of various animal phyla. The T. transversa homolog, TtrNot, is expressed in the ectoderm from the beginning of gastrulation until...... formation. TtrNot expression is absent in unfertilized eggs, in embryos prior to gastrulation, and in settled individuals during and after metamorphosis. Comparison with the expression patterns of Not genes in other metazoan phyla suggests an ancestral role for this gene in gastrulation and germ layer...

  20. DHL Express U.S

    National Research Council Canada - National Science Library

    Anonymous

    2016-01-01

      Comprised of commercial managers and support specialists located in every region of our global network, the DHL Express Public Sector Sales and Support Team provides risk management and contingency plan triggers...

  1. A Tattoo Is Expression, Too.

    Science.gov (United States)

    Dowling-Sendor, Benjamin

    1997-01-01

    In "Stephenson v. Davenport Community School District," the U.S. Eighth Circuit Court of Appeals ruled that schools cannot adopt unduly vague policies to regulate student expression, in this case, a cross-shaped tattoo. (LMI)

  2. Expressive communication in multimedia environments

    Science.gov (United States)

    Carey, John

    1993-01-01

    Narrow bandwidth networks have provided limited capabilities for the transmission of expressive communication that is present in face-to-face communication, e.g., facial expressions and gestures, as well as affective information that is present in audio/visual media, e.g., animated graphics and stereophonic music. An understanding of the structure and functions of expressive communication in face-to-face communication and audio/visual media can inform the development of new multi-media applications in broadband networks. At the same time, a review of existing knowledge suggests that there is a need for considerable research if the rich potential of expressive communication in these new settings is to be fully developed.

  3. Precise Analysis of String Expressions

    DEFF Research Database (Denmark)

    Christensen, Aske Simon; Møller, Anders; Schwartzbach, Michael Ignatieff

    2003-01-01

    We perform static analysis of Java programs to answer a simple question: which values may occur as results of string expressions? The answers are summarized for each expression by a regular language that is guaranteed to contain all possible values. We present several applications of this analysis......, including statically checking the syntax of dynamically generated expressions, such as SQL queries. Our analysis constructs flow graphs from class files and generates a context-free grammar with a nonterminal for each string expression. The language of this grammar is then widened into a regular language...... through a variant of an algorithm previously used for speech recognition. The collection of resulting regular languages is compactly represented as a special kind of multi-level automaton from which individual answers may be extracted. If a program error is detected, examples of invalid strings...

  4. Compound facial expressions of emotion

    National Research Council Canada - National Science Library

    Shichuan Du; Yong Tao; Aleix M. Martinez

    2014-01-01

    Understanding the different categories of facial expressions of emotion regularly used by us is essential to gain insights into human cognition and affect as well as for the design of computational...

  5. Leptospira Protein Expression During Infection

    Science.gov (United States)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  6. Expression homeostasis during DNA replication.

    Science.gov (United States)

    Voichek, Yoav; Bar-Ziv, Raz; Barkai, Naama

    2016-03-04

    Genome replication introduces a stepwise increase in the DNA template available for transcription. Genes replicated early in S phase experience this increase before late-replicating genes, raising the question of how expression levels are affected by DNA replication. We show that in budding yeast, messenger RNA (mRNA) synthesis rate is buffered against changes in gene dosage during S phase. This expression homeostasis depends on acetylation of H3 on its internal K56 site by Rtt109/Asf1. Deleting these factors, mutating H3K56 or up-regulating its deacetylation, increases gene expression in S phase in proportion to gene replication timing. Therefore, H3K56 acetylation on newly deposited histones reduces transcription efficiency from replicated DNA, complementing its role in guarding genome stability. Our study provides molecular insight into the mechanism maintaining expression homeostasis during DNA replication. Copyright © 2016, American Association for the Advancement of Science.

  7. Facial Asymmetry and Emotional Expression

    OpenAIRE

    Pickin, Andrew

    2011-01-01

    This report is about facial asymmetry, its connection to emotional expression, and methods of measuring facial asymmetry in videos of faces. The research was motivated by two factors: firstly, there was a real opportunity to develop a novel measure of asymmetry that required minimal human involvement and that improved on earlier measures in the literature; and secondly, the study of the relationship between facial asymmetry and emotional expression is both interesting in its own right, and im...

  8. The motivation to express prejudice

    Science.gov (United States)

    Forscher, Patrick S.; Cox, William T. L.; Graetz, Nicholas; Devine, Patricia G.

    2015-01-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001; 2009) suggest that some expressions of prejudice are intentional. These phenomena and findings are difficult to explain solely from the motivations to respond without prejudice. We argue that some people are motivated to express prejudice, and we develop the motivation to express prejudice (MP) scale to measure this motivation. In seven studies involving more than 6,000 participants, we demonstrate that, across scale versions targeted at Black people and gay men, the MP scale has good reliability and convergent, discriminant, and predictive validity. In normative climates that prohibit prejudice, the internal and external motivations to express prejudice are functionally non-independent, but they become more independent when normative climates permit more prejudice toward a target group. People high in the motivation to express prejudice are relatively likely to resist pressure to support programs promoting intergroup contact and vote for political candidates who support oppressive policies. The motivation to express prejudice predicted these outcomes even when controlling for attitudes and the motivations to respond without prejudice. This work encourages contemporary prejudice researchers to broaden the range of samples, target groups, and phenomena that they study, and more generally to consider the intentional aspects of negative intergroup behavior. PMID:26479365

  9. Human Lacrimal Gland Gene Expression.

    Directory of Open Access Journals (Sweden)

    Vinay Kumar Aakalu

    Full Text Available The study of human lacrimal gland biology and development is limited. Lacrimal gland tissue is damaged or poorly functional in a number of disease states including dry eye disease. Development of cell based therapies for lacrimal gland diseases requires a better understanding of the gene expression and signaling pathways in lacrimal gland. Differential gene expression analysis between lacrimal gland and other embryologically similar tissues may be helpful in furthering our understanding of lacrimal gland development.We performed global gene expression analysis of human lacrimal gland tissue using Affymetrix ® gene expression arrays. Primary data from our laboratory was compared with datasets available in the NLM GEO database for other surface ectodermal tissues including salivary gland, skin, conjunctiva and corneal epithelium.The analysis revealed statistically significant difference in the gene expression of lacrimal gland tissue compared to other ectodermal tissues. The lacrimal gland specific, cell surface secretory protein encoding genes and critical signaling pathways which distinguish lacrimal gland from other ectodermal tissues are described.Differential gene expression in human lacrimal gland compared with other ectodermal tissue types revealed interesting patterns which may serve as the basis for future studies in directed differentiation among other areas.

  10. Epimorphin expression in interstitial pneumonia

    Directory of Open Access Journals (Sweden)

    Suga Moritaka

    2005-01-01

    Full Text Available Abstract Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP, and lungs with usual interstitial pneumonia (UIP; we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling.

  11. Extracting expression modules from perturbational gene expression compendia

    Directory of Open Access Journals (Sweden)

    Van Dijck Patrick

    2008-04-01

    Full Text Available Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclustering methods on the other hand are specifically designed to capture such partial coexpression patterns, but they show a variety of other drawbacks. For instance, some biclustering methods are less suited to identify overlapping biclusters, while others generate highly redundant biclusters. Also, none of the existing biclustering tools takes advantage of the staple of perturbational expression data analysis: the identification of differentially expressed genes. Results We introduce a novel method, called ENIGMA, that addresses some of these issues. ENIGMA leverages differential expression analysis results to extract expression modules from perturbational gene expression data. The core parameters of the ENIGMA clustering procedure are automatically optimized to reduce the redundancy between modules. In contrast to the biclusters produced by most other methods, ENIGMA modules may show internal substructure, i.e. subsets of genes with distinct but significantly related expression patterns. The grouping of these (often functionally related patterns in one module greatly aids in the biological interpretation of the data. We show that ENIGMA outperforms other methods on artificial datasets, using a quality criterion that, unlike other criteria, can be used for algorithms that generate overlapping clusters and that can be modified to take redundancy between clusters into account. Finally, we apply ENIGMA to the Rosetta compendium of expression profiles for

  12. Comparative gene expression signature of pig, human and mouse induced pluripotent stem cell lines reveals insight into pig pluripotency gene networks.

    Science.gov (United States)

    Liu, Yajun; Ma, Yangyang; Yang, Jeong-Yeh; Cheng, De; Liu, Xiaopeng; Ma, Xiaoling; West, Franklin D; Wang, Huayan

    2014-04-01

    Reported pig induced pluripotent stem cells (piPSCs) have shown either a bFGF-dependent state with human embryonic stem cell (ESC) and mouse epiblast stem cell (EpiSC) morphology and molecular features or piPSCs exist in a LIF-dependent state and resemble fully reprogrammed mouse iPSCs. The features of authentic piPSCs and molecular events during the reprogramming are largely unknown. In this study, we assessed the transcriptome profile of multiple piPSC lines derived from different laboratories worldwide and compared to mouse and human iPSCs to determine the molecular signaling pathways that might play a central role in authentic piPSCs. The results demonstrated that the up-regulation of endogenous epithelial cells adhesion molecule (EpCAM) was correlated with the pluripotent state of pig pluripotent cells, which could be utilized as a marker for evaluating pig cell reprogramming. Comparison of key signaling pathways JAK-STAT, NOTCH, TGFB1, WNT and VEGF in pig, mouse and human iPSCs showed that the core transcriptional network to maintain pluripotency and self-renewal in pig were different from that in mouse, but had significant similarities to human. Pig iPSCs, which lacked expression of specific naïve state markers KLF2/4/5 and TBX3, but expressed the primed state markers of Otx2 and Fabp7, share defining features with human ESCs and mouse EpiSCs. The cluster of imprinted genes delineated by the delta-like homolog 1 gene and the type III iodothyronine deiodinase gene (DLK1-DIO3) were silenced in piPSCs as previously seen in mouse iPSCs that have limited ability to contribute to chimaeras. These key differences in naïve state gene and imprinting gene expression suggests that so far known piPSC lines may be more similar to primed state cells. The primed state of these cells may potentially explain the rare ability of piPSCS to generate chimeras and cloned offspring.

  13. Toll-like receptor 2-expressing macrophages are required and sufficient for rhinovirus-induced airway inflammation.

    Science.gov (United States)

    Han, Mingyuan; Chung, Yutein; Young Hong, Jun; Rajput, Charu; Lei, Jing; Hinde, Joanna L; Chen, Qiang; Weng, Steven P; Bentley, J Kelley; Hershenson, Marc B

    2016-12-01

    We have shown that rhinovirus, a cause of asthma exacerbation, colocalizes with CD68(+) and CD11b(+) airway macrophages after experimental infection in human subjects. We have also shown that rhinovirus-induced cytokine expression is abolished in Toll-like receptor (TLR2)(-/-) bone marrow-derived macrophages. We hypothesize that TLR2(+) macrophages are required and sufficient for rhinovirus-induced airway inflammation in vivo. Naive and ovalbumin (OVA)-sensitized and challenged C57BL/6 wild-type and TLR2(-/-) mice were infected with RV1B, followed by IgG or anti-TLR2, to determine the requirement and sufficiency of TLR2 for rhinovirus-induced airway responses. Bone marrow chimera experiments using OVA-treated C57BL/6 and TLR2(-/-) mice were also performed. Finally, naive TLR2(-/-) mice underwent intranasal transfer of bone marrow-derived wild-type macrophages. RV1B infection of naive wild-type mice induced an influx of airway neutrophils and CD11b(+) exudative macrophages, which was reduced in TLR2(-/-) mice. After allergen exposure, rhinovirus-induced neutrophilic and eosinophilic airway inflammation and hyperresponsiveness were reduced in TLR2(-/-) and anti-TLR2-treated mice. Transfer of TLR2(-/-) bone marrow into wild-type, OVA-treated C57BL/6 mice blocked rhinovirus-induced airway responses, whereas transfer of wild-type marrow to TLR2(-/-) mice restored them. Finally, transfer of wild-type macrophages to naive TLR2(-/-) mice was sufficient for neutrophilic inflammation after rhinovirus infection, whereas macrophages treated with IL-4 (to induce M2 polarization) were sufficient for eosinophilic inflammation, mucous metaplasia, and airways hyperresponsiveness. TLR2 is required for early inflammatory responses induced by rhinovirus, and TLR2(+) macrophages are sufficient to confer airway inflammation to TLR2(-/-) mice, with the pattern of inflammation depending on the macrophage activation state. Copyright © 2016 American Academy of Allergy, Asthma

  14. Phylogenetic analysis of gene expression.

    Science.gov (United States)

    Dunn, Casey W; Luo, Xi; Wu, Zhijin

    2013-11-01

    Phylogenetic analyses of gene expression have great potential for addressing a wide range of questions. These analyses will, for example, identify genes that have evolutionary shifts in expression that are correlated with evolutionary changes in morphological, physiological, and developmental characters of interest. This will provide entirely new opportunities to identify genes related to particular phenotypes. There are, however, 3 key challenges that must be addressed for such studies to realize their potential. First, data on gene expression must be measured from multiple species, some of which may be field-collected, and parameterized in such a way that they can be compared across species. Second, it will be necessary to develop comparative phylogenetic methods suitable for large multidimensional datasets. In most phylogenetic comparative studies to date, the number n of independent observations (independent contrasts) has been greater than the number p of variables (characters). The behavior of comparative methods for these classic problems is now well understood under a wide variety of conditions. In studies of gene expression, and in studies based on other high-throughput tools, the number n of samples is dwarfed by the number p of variables. The estimated covariance matrices will be singular, complicating their analysis and interpretation, and prone to spurious results. Third, new approaches are needed to investigate the expression of the many genes whose phylogenies are not congruent with species phylogenies due to gene loss, gene duplication, and incomplete lineage sorting. Here we outline general considerations of project design for phylogenetic analyses of gene expression and suggest solutions to these three categories of challenges. These topics are relevant to high-throughput phenotypic data well beyond gene expression.

  15. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    and Bmp-2a loci. The corresponding mRNA (3.0 kilobases) is expressed in most murine tissues and most abundantly expressed in brain and kidney. Antibodies against a synthetic peptide of R-PTP-alpha identified a 130-kDa protein in cells transfected with the R-PTP-alpha cDNA.......We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  16. Studying Emotional Expression in Music Performance.

    Science.gov (United States)

    Gabrielsson, Alf

    1999-01-01

    Explores the importance of emotional expression in music performance. Performers played music to express different emotions and then listening tests were conducted in order to determine whether the intended expressions were perceived. Presents and discusses the results. (CMK)

  17. iFace: Facial Expression Training System

    OpenAIRE

    Ito, Kyoko; Kurose, Hiroyuki; Takami, Ai; Nishida, Shogo

    2008-01-01

    In this study, a target facial expression selection interface for a facial expression training system and a facial expression training system were both proposed and developed. Twelve female dentists used the facial expression training system, and evaluations and opinions about the facial expression training system were obtained from these participants. In the future, we will attempt to improve both the target facial expression selection interface and the comparison of a current and a target f...

  18. An Expressive Extension of TLC

    DEFF Research Database (Denmark)

    Henriksen, Jesper Gulmann

    2002-01-01

    A temporal logic of causality (TLC) was introduced by Alur, Penczek and Peled in [1]. It is basically a linear time temporal logic interpreted over Mazurkiewicz traces which allows quantification over causal chains. Through this device one can directly formulate causality properties of distributed...... systems. In this paper we consider an extension of TLC by strengthening the chain quantification operators. We show that our logic TLC* adds to the expressive power of TLC. We do so by defining an Ehrenfeucht-Fraïssé game to capture the expressive power of TLC. We then exhibit a property and by means...... of this game prove that the chosen property is not definable in TLC. We then show that the same property is definable in TLC*. We prove in fact the stronger result that TLC* is expressively stronger than TLC exactly when the dependency relation associated with the underlying trace alphabet is not transitive....

  19. Sleep deprivation and gene expression.

    Science.gov (United States)

    da Costa Souza, Annie; Ribeiro, Sidarta

    2015-01-01

    Sleep occurs in a wide range of animal species as a vital process for the maintenance of homeostasis, metabolic restoration, physiological regulation, and adaptive cognitive functions in the central nervous system. Long-term perturbations induced by the lack of sleep are mostly mediated by changes at the level of transcription and translation. This chapter reviews studies in humans, rodents, and flies to address the various ways by which sleep deprivation affects gene expression in the nervous system, with a focus on genes related to neuronal plasticity, brain function, and cognition. However, the effects of sleep deprivation on gene expression and the functional consequences of sleep loss are clearly not restricted to the cognitive domain but may include increased inflammation, expression of stress-related genes, general impairment of protein translation, metabolic imbalance, and thermal deregulation.

  20. Construction of PVX virus-expression vector to express enterotoxin ...

    African Journals Online (AJOL)

    Potato X potyvirus (PVX)-based vector has been comprehensively applied in transient expression system. In order to produce the heterologous proteins more quickly and stably, the ClaI and NotI enzyme sites were introduced into the Enterotoxin fusion gene LTB-ST by polymerase chain reaction (PCR) and the LTB-ST ...

  1. Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

    Directory of Open Access Journals (Sweden)

    Hiroshi Hanagata

    2014-05-01

    Full Text Available Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production.

  2. Osteocalcin expression in pulp inflammation.

    Science.gov (United States)

    Abd-Elmeguid, Ashraf; Abdeldayem, Marwa; Kline, Loren W; Moqbel, Redwan; Vliagoftis, Harrisios; Yu, Donald C

    2013-07-01

    Dental pulp inflammation and repair are closely related. Osteocalcin (OCN), a glycoprotein present in dentin matrix, is expressed by odontoblasts. Although OCN is considered a reparative molecule inside the dental pulp, it is not clear if it is involved in pulpal inflammation. The objective of this study was to localize OCN in reversible and irreversible pulpitis and to describe its possible function in inflammation. Pulp tissues in the form of reversible and irreversible pulpitis were collected from the endodontic clinic. Those from impacted teeth were used as controls. Immunohistochemistry was used to localize OCN. Samples were analyzed for OCN and inflammatory mediator expression using multiplex assay. OCN in inflamed tissues was localized in cells and matrix around calcification areas and in cells around blood vessels but not in normal tissues. The plex assay (Bio-Plex 200, Bio-Rad Laboratories Ltd, Mississauga, ON, Canada) showed OCN expression in reversible pulpitis significantly higher than in irreversible pulpitis, and both were significantly higher than in the controls. A panel of inflammatory mediators showed an increase in reversible and irreversible pulpitis. Another panel was decreased in both stages compared with the controls. OCN expression in reversible pulpitis was positively correlated to the expression of vascular endothelial growth factor, fibroblast growth factor, macrophage inflammatory protein-1β, monocyte-derived chemokine, monocyte chemoattractant protein-1, interleukin (IL)-17, and soluble IL-2 receptor α and negatively correlated to that of IL-1α, IL-1β, IL-8, granulocyte macrophage colony-stimulating factor, and macrophage inflammatory protein-1α. Profound understanding of the pulp inflammatory process would lead to new molecular treatment strategies. Our data indicate that OCN expression in reversible pulpitis is associated with angiogenic markers, suggesting its potential use in regenerative treatment. Copyright © 2013 American

  3. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

  4. Microsoft Expression Web for dummies

    CERN Document Server

    Hefferman, Linda

    2013-01-01

    Expression Web is Microsoft's newest tool for creating and maintaining dynamic Web sites. This FrontPage replacement offers all the simple ""what-you-see-is-what-you-get"" tools for creating a Web site along with some pumped up new features for working with Cascading Style Sheets and other design options. Microsoft Expression Web For Dummies arrives in time for early adopters to get a feel for how to build an attractive Web site. Author Linda Hefferman teams up with longtime FrontPage For Dummies author Asha Dornfest to show the easy way for first-time Web designers, FrontPage ve

  5. Advanced express web application development

    CERN Document Server

    Keig, Andrew

    2013-01-01

    A practical book, guiding the reader through the development of a single page application using a feature-driven approach.If you are an experienced JavaScript developer who wants to build highly scalable, real-world applications using Express, this book is ideal for you. This book is an advanced title and assumes that the reader has some experience with node, Javascript MVC web development frameworks, and has heard of Express before, or is familiar with it. You should also have a basic understanding of Redis and MongoDB. This book is not a tutorial on Node, but aims to explore some of the more

  6. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each......%). Fifteen nuclear encoded mitochondrial proteins were all down-regulated in CRC. We identified several chromosomal locations with clusters of either potential oncogenes or potential tumor suppressors. Some of these, such as aminopeptidase N/CD13 and sigma B3 protein on chromosome 15q25, coincided...

  7. Beginning Oracle Application Express 4

    CERN Document Server

    Gault, Doug; Cimolini, Patrick; D'Souza, Martin; Hilaire, Timothy St

    2011-01-01

    Beginning Oracle Application Express 4 introduces one of the most talked-about development platforms to come out of Oracle Corporation in years. Oracle Application Express, called APEX for short, enables rapid and easy development of web-based applications that make full use of Oracle Database. The release of APEX 4 brings a huge leap forward in terms of functionality and usability for both the developer and the end user. Power users and programmers alike can quickly put together robust and scalable applications for use by one person, by a department, by an entire company. Whether you're new t

  8. Mars Express wins unanimous support

    Science.gov (United States)

    1998-11-01

    "The green light for Mars Express shows that Europe is perfectly capable of seizing special chances in exploring space," said Roger Bonnet, ESA's director of science. "At a cost to ESA of 150 million ECU, Mars Express is the cheapest Mars mission ever, yet its importance and originality are far greater than the price tag suggests." Bonnet continued: "Mars Express has been advertised by the Science Programme Committee as a test case for new approaches in procuring and managing future science projects, with a view to achieving major savings. In the international arena, Mars Express will confirm Europe's interest in a major target for space research in the new century, when we make our forceful debut at the Red Planet. In fact, Mars Express is designed to be a pivotal element of an international multi-mission, global effort for the exploration of Mars." Development of the spacecraft will now proceed swiftly, to meet the deadline of an exceptionally favourable launch window early in June 2003. Mars Express will go into orbit around Mars at Christmas 2003. Seven scientific instruments on board will include a high-resolution camera, a range of spectrometers, and a radar to penetrate below the surface. For the first time in the history of the exploration of the Red Planet, scientists can hope to detect sub-surface water, whether it exists in the form of undergound rivers, pools, glaciers or permafrost. Signs of life on Mars, whether extinct or continuing today, may reveal themselves to a lander carried by Mars Express. This is Beagle 2, a project led by the Open University in the United Kingdom, with contributions from many other European countries. The lander also promises invaluable information about the chemistry of the Martian surface and atmosphere. Beagle 2 is to be independently funded. Some of the necessary funds have already been raised and ESA has agreed with the principal investigator to keep a place for Beagle 2 aboard Mars Express. The financial situation

  9. Spirit Boxes: Expressions of Culture.

    Science.gov (United States)

    DeMuro, Ted

    1984-01-01

    After studying the culture and art of the ancient civilizations of South America, Mesopotamia, Greece, and Egypt, secondary level art students made spirit boxes as expressions of the various cultures. How to make the boxes and how to prepare the face molds are described. (RM)

  10. Visualizing structures of speech expressiveness

    DEFF Research Database (Denmark)

    Herbelin, Bruno; Jensen, Karl Kristoffer; Graugaard, Lars

    2008-01-01

    vowels and consonants, and which converts the speech energy into visual particles that form complex visual structures, provides us with a mean to present the expressiveness of speech into a visual mode. This system is presented in an artwork whose scenario is inspired from the reasons of language...

  11. Lysozyme expression in Lactococcus lactis

    NARCIS (Netherlands)

    Guchte, Maarten van de; Wal, Fimme Jan van der; Kok, Jan; Venema, Gerhardus

    Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence

  12. ASPM gene expression in medulloblastoma.

    Science.gov (United States)

    Vulcani-Freitas, Tânia M; Saba-Silva, Najsla; Cappellano, Andréa; Cavalheiro, Sérgio; Marie, Sueli K N; Oba-Shinjo, Sueli M; Malheiros, Suzana M F; de Toledo, Sílvia Regina Caminada

    2011-01-01

    Medulloblastomas are the most common malignant tumors of the central nervous system in childhood. The incidence is about 19-20% between children younger than 16 years old with peak incidence between 4 and 7 years. Despite its sensibility to no specific therapeutic means like chemotherapy and radiotherapy, the treatment is very aggressive and frequently results in regression, growth deficit, and endocrine dysfunction. From this point of view, new treatment approaches are needed such as molecular targeted therapies. Studies in glioblastoma demonstrated that ASPM gene was overexpressed when compared to normal brain and ASPM inhibition by siRNA-mediated inhibits tumor cell proliferation and neural stem cell proliferation, supporting ASPM gene as a potential molecular target in glioblastoma. The aim of this work was to evaluate ASPM expression in medulloblastoma fragment samples, and to compare the results with the patient clinical features. Analysis of gene expression was performed by quantitative PCR real time using SYBR Green system in tumor samples from 37 children. The t test was used to analyze the gene expression, and Mann-Whitney test was performed to analyze the relationship between gene expressions and clinical characteristics. Kaplan-Meier test evaluated curve survival. All samples overexpressed ASPM gene more than 40-fold. However, we did not find any association between the overexpressed samples and the clinical parameters. ASPM overexpression may modify the ability of stem cells to differentiate during the development of the central nervous system, contributing to the development of medulloblastoma, a tumor of embryonic origin from cerebellar progenitor cells.

  13. Construction of the expression plasmid

    African Journals Online (AJOL)

    nausch

    2012-10-09

    Oct 9, 2012 ... Sepsis is one of the top 10 causes of death worldwide. (Dombrovskiy et al., 2007; Parrish et al., 2008). .... may protect recombinant C5a from proteolytic degradation as observed for unmodified C5a expressed ..... crosslinking of the 3 disulfide bridges that stabilize the four core α-helices of C5a are crucial for ...

  14. 'Endurance' Courtesy of Mars Express

    Science.gov (United States)

    2004-01-01

    NASA's Mars Exploration Rover Opportunity used its panoramic camera to capture this false-color image of the interior of 'Endurance Crater' on the rover's 188th martian day (Aug. 4, 2004). The image data were relayed to Earth by the European Space Agency's Mars Express orbiter. The image was generated from separate frames using the cameras 750-, 530- and 480-nanometer filters.

  15. The Change of Expression Configuration Affects Identity-Dependent Expression Aftereffect but Not Identity-Independent Expression Aftereffect

    OpenAIRE

    Song, Miao; Shinomori, Keizo; Qian, Qian; Yin, Jun; Zeng, Weiming

    2015-01-01

    The present study examined the influence of expression configuration on cross-identity expression aftereffect. The expression configuration refers to the spatial arrangement of facial features in a face for conveying an emotion, e.g., an open-mouth smile vs. a closed-mouth smile. In the first of two experiments, the expression aftereffect is measured using a cross-identity/cross-expression configuration factorial design. The facial identities of test faces were the same or different from the ...

  16. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  17. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  18. EXPRESS: EXPressing REstful Semantic Services Using Domain Ontologies

    Science.gov (United States)

    Alowisheq, Areeb; Millard, David E.; Tiropanis, Thanassis

    Existing approaches to Semantic Web Services (SWS) require a domain ontology and a semantic description of the service. In the case of lightweight SWS approaches, such as SAWSDL, service description is achieved by semantically annotating existing web service interfaces. Other approaches such as OWL-S and WSMO describe services in a separate ontology. So, existing approaches separate service description from domain description, therefore increasing design efforts. We propose EXPRESS a lightweight approach to SWS that requires the domain ontology definition only. Its simplicity stems from the similarities between REST and the Semantic Web such as resource realization, self describing representations, and uniform interfaces. The semantics of a service is elicited from a resource's semantic description in the domain ontology and the semantics of the uniform interface, hence eliminating the need for ontologically describing services. We provide an example that illustrates EXPRESS and then discuss how it compares to SA-REST and WSMO.

  19. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  20. Decomposing Path : The Nanosyntax of Directional Expressions

    OpenAIRE

    Pantcheva, Marina Blagoeva

    2011-01-01

    In my thesis, I investigate directional expressions cross-linguistically. I examine the morpho-syntactic structure of expressions of Goal (to the house), Source (from the house), Route (through the house), non-transitional paths (towards the house) and, finally, delimited paths (up to the house). I conclude that all these types of directional expressions are of different syntactic complexity. Precisely, Source expressions (from) are formed on the basis of Goal expressions (to) and Route expr...

  1. Short communication

    African Journals Online (AJOL)

    abp

    2013-09-17

    Sep 17, 2013 ... fractions (total bacterial cell lysate before purification, flow-through, first wash, second Wash and protein elutions fractions) was evaluated by SDS-PAGE. The SDS-PAGE was stained with Coomassie. Blue dye. Results. HIV-1 tat and nef genes were successfully cloned into a prokaryotic expression plasmid ...

  2. Silencing of HIV-1 with RNA interference: a multiple shRNA approach

    NARCIS (Netherlands)

    ter Brake, Olivier; Konstantinova, Pavlina; Ceylan, Mustafa; Berkhout, Ben

    2006-01-01

    Double-stranded RNA can induce gene silencing via a process known as RNA interference (RNAi). Previously, we have shown that stable expression of a single shRNA targeting the HIV-1 Nef gene strongly inhibits HIV-1 replication. However, this was not sufficient to maintain inhibition. One of the

  3. Summer Oral Expression English Course

    CERN Multimedia

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol here. Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  4. Gene expression based cancer classification

    OpenAIRE

    Sara Tarek; Reda Abd Elwahab; Mahmoud Shoman

    2017-01-01

    Cancer classification based on molecular level investigation has gained the interest of researches as it provides a systematic, accurate and objective diagnosis for different cancer types. Several recent researches have been studying the problem of cancer classification using data mining methods, machine learning algorithms and statistical methods to reach an efficient analysis for gene expression profiles. Studying the characteristics of thousands of genes simultaneously offered a deep in...

  5. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  6. Gene expression profile of pulpitis

    Science.gov (United States)

    Galicia, Johnah C.; Henson, Brett R.; Parker, Joel S.; Khan, Asma A.

    2016-01-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the Significance Analysis of Microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (≥30mm on VAS) compared to those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology. PMID:27052691

  7. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  8. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology.

  9. Children's expression through play therapy

    Directory of Open Access Journals (Sweden)

    Ljubomirović Nataša

    2015-01-01

    Full Text Available Play as a child's expression, is a skill through which children speaks to adults. Play therapy is a broad field of therapeutic intervention based on the play in order to help the child to cope with problems. Through play, children learn to communicate with others, to express their feelings. Through play they learn and can improve their cognitive, emotional and social capabilities. Play therapy is a nondirective technique focused on the child. It is not focused on the problem, at present even the past, but focused on the expression of the child feelings, accepting the child, rather than correction. The focus has been on the wisdom of a child, not on expertise therapists, guiding the child through play rather than instructing. The aim of play therapy is to encourage healthy growth and development, developing skills in problem solving, reduction of undesirable behavior, confidence building and the development of self-control. This method is effective for a wide range of children's problems, such as the state of stress, anxiety, problem behavior, hyperkinetic syndrome, depression, loss, trauma, the problem of bonding situations parents divorced, somatic disorders, autism spectrum disorders, social problems.

  10. Children's Representations of Facial Expression and Identity: Identity-Contingent Expression Aftereffects

    Science.gov (United States)

    Vida, Mark D.; Mondloch, Catherine J.

    2009-01-01

    This investigation used adaptation aftereffects to examine developmental changes in the perception of facial expressions. Previous studies have shown that adults' perceptions of ambiguous facial expressions are biased following adaptation to intense expressions. These expression aftereffects are strong when the adapting and probe expressions share…

  11. Misrecognition of facial expressions in delinquents

    Directory of Open Access Journals (Sweden)

    Matsuura Naomi

    2009-09-01

    Full Text Available Abstract Background Previous reports have suggested impairment in facial expression recognition in delinquents, but controversy remains with respect to how such recognition is impaired. To address this issue, we investigated facial expression recognition in delinquents in detail. Methods We tested 24 male adolescent/young adult delinquents incarcerated in correctional facilities. We compared their performances with those of 24 age- and gender-matched control participants. Using standard photographs of facial expressions illustrating six basic emotions, participants matched each emotional facial expression with an appropriate verbal label. Results Delinquents were less accurate in the recognition of facial expressions that conveyed disgust than were control participants. The delinquents misrecognized the facial expressions of disgust as anger more frequently than did controls. Conclusion These results suggest that one of the underpinnings of delinquency might be impaired recognition of emotional facial expressions, with a specific bias toward interpreting disgusted expressions as hostile angry expressions.

  12. Summer Oral Expression English course

    CERN Multimedia

    2013-01-01

    An English Oral Expression course will take place this summer at some time between August 19 and October 4.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch.

  13. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer from 20 August to 29 September.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  14. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer at some time between 25 June and 28 September. The exact dates will be decided according to the preferences of the students.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  15. Summer Oral Expression English Course

    CERN Multimedia

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol through the following link https://cta.cern.ch/cta2/f?p=110:9:1576796470009589::::X_STATUS,XS_COURSE_NAME,XS_PROGRAMME,XS_SUBCATEGORY,X_COURSE_ID,XS_LANGUAGE,XS_SESSION:D,,1,,4368,B, Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  16. [Perception, expression and psychosomatic functions].

    Science.gov (United States)

    Bühler, K E

    1988-01-01

    The investigation starts with the mind/body-problem and with epistemological peculiarities of psychophysical processes. Every monism has to be conceived at least as a dualism of properties. As a special kind of dualism the functionalism is an example against the type/type-identity thesis but not against the token-/token-identity thesis. But the last one is no serious alternative for genuine psychological methods. But also the functionalism does not offer a complete psychological theory: there is an ambivalence concerning the secondary qualities and also concerning intentionality. Bodily expressions were analyzed according the semiotic theory of Charles Sanders Peirce.

  17. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  18. Mediated Interactions and Musical Expression - A Survey

    NARCIS (Netherlands)

    Reidsma, Dennis; Radha, Mustafa; Nijholt, Antinus; Lee, Newton

    2014-01-01

    This chapter surveys the field of technologically mediated musical interaction and technologically enhanced musical expression. We look at several new technologies that enable new ways of musical expression and interaction, explore the micro-coordination that occurs in collaborative musical

  19. Expressed haplotypes and polymorphisms in prunus species

    Science.gov (United States)

    Expressed sequence tags (ESTs) represent collective and redundant gene transcripts that contain alleles and paralogs of genes expressed in various genotypes, tissues, developmental stages, and environmental conditions. The former are primarily responsible for distinct individual phenotypes in a segr...

  20. Gene Expression Analysis of Breast Cancer Progression

    National Research Council Canada - National Science Library

    Gerald, Wiliam L

    2004-01-01

    ... to identify genes, gene expression profiles and molecular pathways associated with metastatic BC we have performed genome-wide gene expression analysis of a large number of breast cancer samples...

  1. Kivy and Langer on expressiveness in music

    Directory of Open Access Journals (Sweden)

    van der Schoot Albert

    2013-01-01

    Full Text Available From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  2. Kivy and Langer on expressiveness in music

    OpenAIRE

    van der Schoot Albert

    2013-01-01

    From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  3. Interaction between facial expression and color

    OpenAIRE

    Kae Nakajima; Tetsuto Minami; Shigeki Nakauchi

    2017-01-01

    Facial color varies depending on emotional state, and emotions are often described in relation to facial color. In this study, we investigated whether the recognition of facial expressions was affected by facial color and vice versa. In the facial expression task, expression morph continua were employed: fear-anger and sadness-happiness. The morphed faces were presented in three different facial colors (bluish, neutral, and reddish color). Participants identified a facial expression between t...

  4. The gene expression signatures of melanoma progression

    OpenAIRE

    Haqq, Christopher; Nosrati, Mehdi; Sudilovsky, Daniel; Crothers, Julia; Khodabakhsh, Daniel; Pulliam, Brian L.; Federman, Scot; Miller, James R.; Allen, Robert E.; Singer, Mark I.; Leong, Stanley P L; Ljung, Britt-Marie; Sagebiel, Richard W.; Kashani-Sabet, Mohammed

    2005-01-01

    Because of the paucity of available tissue, little information has previously been available regarding the gene expression profiles of primary melanomas. To understand the molecular basis of melanoma progression, we compared the gene expression profiles of a series of nevi, primary melanomas, and melanoma metastases. We found that metastatic melanomas exhibit two dichotomous patterns of gene expression, which unexpectedly reflect gene expression differences already apparent in comparing laser...

  5. Nongenomic regulation of gene expression.

    Science.gov (United States)

    Iglesias-Platas, Isabel; Monk, David

    2016-08-01

    The purpose of this review is to highlight the recent advances in epigenetic regulation and chromatin biology for a better understanding of gene regulation related to human disease. Alterations to chromatin influence genomic function, including gene transcription. At its most simple level, this involves DNA methylation and posttranscriptional histone modifications. However, recent developments in biochemical and molecular techniques have revealed that transcriptional regulation is far more complex, involving combinations of histone modifications and discriminating transcription factor binding, and long-range chromatin loops with enhancers, to generate a multifaceted code. Here, we describe the most recent advances, culminating in the example of genomic imprinting, the parent-of-origin monoallelic expression that utilizes the majority of these mechanisms to attain one active and one repressed allele. It is becoming increasingly evident that epigenetic mechanisms work in unison to maintain tight control of gene expression and genome function. With the wealth of knowledge gained from recent molecular studies, future goals should focus on the application of this information in deciphering their role in developmental diseases.

  6. Systems Biophysics of Gene Expression

    Science.gov (United States)

    Vilar, Jose M.G.; Saiz, Leonor

    2013-01-01

    Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses. PMID:23790365

  7. Moxibustion upregulates hippocampal progranulin expression

    Directory of Open Access Journals (Sweden)

    Tao Yi

    2016-01-01

    Full Text Available In China, moxibustion is reported to be useful and has few side effects for chronic fatigue syndrome, but its mechanisms are largely unknown. More recently, the focus has been on the wealth of information supporting stress as a factor in chronic fatigue syndrome, and largely concerns dysregulation in the stress-related hypothalamic-pituitary-adrenal axis. In the present study, we aimed to determine the effect of moxibustion on behavioral symptoms in chronic fatigue syndrome rats and examine possible mechanisms. Rats were subjected to a combination of chronic restraint stress and forced swimming to induce chronic fatigue syndrome. The acupoints Guanyuan (CV4 and Zusanli (ST36, bilateral were simultaneously administered moxibustion. Untreated chronic fatigue syndrome rats and normal rats were used as controls. Results from the forced swimming test, open field test, tail suspension test, real-time PCR, enzyme-linked immunosorbent assay, and western blot assay showed that moxibustion treatment decreased mRNA expression of corticotropin-releasing hormone in the hypothalamus, and adrenocorticotropic hormone and corticosterone levels in plasma, and markedly increased progranulin mRNA and protein expression in the hippocampus. These findings suggest that moxibustion may relieve the behavioral symptoms of chronic fatigue syndrome, at least in part, by modulating the hypothalamic-pituitary-adrenal axis and upregulating hippocampal progranulin.

  8. Control of Renin Gene Expression

    Science.gov (United States)

    Glenn, Sean T.; Jones, Craig A.; Gross, Kenneth W.; Pan, Li

    2015-01-01

    Renin, as part of the renin-angiotensin system, plays a critical role in the regulation of blood pressure, electrolyte homeostasis, mammalian renal development and progression of fibrotic/hypertrophic diseases. Renin gene transcription is subject to complex developmental and tissue-specific regulation. Initial studies using the mouse As4.1 cell line, which has many characteristics of the renin-expressing juxtaglomerular cells of the kidney, have identified a proximal promoter region (−197 to −50 bp) and an enhancer (−2866 to −2625 bp) upstream of the Ren-1c gene, which are critical for renin gene expression. The proximal promoter region contains several transcription factor-binding sites including a binding site for the products of the developmental control genes Hox. The enhancer consists of at least 11 transcription factor-binding sites and is responsive to various signal transduction pathways including cAMP, retinoic acid, endothelin-1, and cytokines, all of which are known to alter renin mRNA levels. Furthermore, in vivo models have validated several of these key components found within the proximal promoter region and the enhancer as well as other key sites necessary for renin gene transcription. PMID:22576577

  9. Facial displays, emotional expressions and conversational acts

    NARCIS (Netherlands)

    Heylen, Dirk K.J.; Trappl, R.

    2006-01-01

    “Emotional expression is multifaceted – expression is determined both by a person’s reaction to an event and by the attempt to manipulate this expression for strategic reasons in social interaction.��? (Scherer, 2001). In this paper we present some thoughts on the relation between emotion, facial

  10. Vocal Emotion Expressions Effects on Cooperation Behavior

    Science.gov (United States)

    Caballero Meneses, Jonathan Azael; Menez Díaz, Judith Marina

    2017-01-01

    Emotional expressions have been proposed to be important for regulating social interaction as they can serve as cues for behavioral intentions. The issue has been mainly addressed analyzing the effects of facial emotional expressions in cooperation behavior, but there are contradictory results regarding the impact of emotional expressions on that…

  11. Learning to Be Creatively Expressive Performers

    Science.gov (United States)

    Strand, Katherine; Brenner, Brenda

    2017-01-01

    Research conducted on the development of expressive performance capabilities suggests that children can learn to demonstrate expressiveness in their music-making. Expressivity includes musical interpretation, performance technique, and musical and personal creativity. This article examines creativity as an important component of musical…

  12. Generating Expressive Speech for Storytelling Applications

    NARCIS (Netherlands)

    Bailly, G.; Theune, Mariet; Meijs, Koen; Campbell, N.; Hamza, W.; Heylen, Dirk K.J.; Ordelman, Roeland J.F.; Hoge, H.; Jianhua, T.

    Work on expressive speech synthesis has long focused on the expression of basic emotions. In recent years, however, interest in other expressive styles has been increasing. The research presented in this paper aims at the generation of a storytelling speaking style, which is suitable for

  13. Ectopic expression of Crambe abyssinica lysophosphatidic acid ...

    African Journals Online (AJOL)

    To study its function in vivo, CaLPAAT was introduced into Brassica napus by Agrobacterium. The expression profile of several genes in the glycerolipids synthesis pathway was determined by quantitative RT-PCR. Interestingly, higher expression of CaLPAAT led to elevated expression of these genes. Further analysis of ...

  14. Endogenous retrovirus sequences expressed in male mammalian ...

    African Journals Online (AJOL)

    In humans, one ERV family, human endogenous retrovirus- K (HERV-K) is abundantly expressed, and is associated with germ cell tumours, while ERV3 env is expressed in normal human testis. Conclusion: The expression of ERVs in male reproductive tissues suggests a possible role in normal and disease conditions ...

  15. 7 CFR 27.99 - Values; expression.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Values; expression. 27.99 Section 27.99 Agriculture... CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Price Quotations and Differences § 27.99 Values; expression. For the purpose of this subpart values shall be expressed in terms of cents and hundredths of a...

  16. Expression and sequence characterization of growth hormone ...

    African Journals Online (AJOL)

    An expression plasmid was constructed for the production of BbGHBP in Escherichia coli BL21 (RIPL) CodonPlus under the control of T7lac promoter. On induction with isopropyl β-D thiogalactopyranoside, the BbGHBP was expressed at levels >30% of the total E. coli proteins. The target protein expressed as inclusion ...

  17. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene ...

  18. Expression, purification and characterization of the interferon ...

    Indian Academy of Sciences (India)

    Earlier, we had reported that expression of recombinant human RNase L caused RNA-degradation and cell-growth inhibition in E. coli without the need for exogenous 2-5A. Expression of human RNase L in E. coli usually leads to problems of leaky expression, low yield and degradation of the recombinant protein, which ...

  19. Inhibition of bromodomain and extra-terminal (BET proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay

    Directory of Open Access Journals (Sweden)

    Maria Pia Abruzzese

    2016-12-01

    Full Text Available Abstract Background Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the impo