Sample records for ubiquitous gp63-like metalloprotease

  1. Impact of Leishmania metalloprotease GP63 on macrophage signaling

    PubMed Central

    Isnard, Amandine; Shio, Marina T.; Olivier, Martin

    2012-01-01

    The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions. PMID:22919663

  2. Impact of Leishmania metalloprotease GP63 on macrophage signaling.

    PubMed

    Isnard, Amandine; Shio, Marina T; Olivier, Martin

    2012-01-01

    The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions.

  3. Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

    PubMed

    Elias, Camila G R; Chagas, Michel G; Souza-Gonçalves, Ana Luiza; Pascarelli, Bernardo M O; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

    2012-01-01

    Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Gp63-like molecules in Phytomonas serpens: possible role in the insect interaction.

    PubMed

    d'Avila-Levy, Claudia M; Santos, Lívia O; Marinho, Fernanda A; Dias, Felipe A; Lopes, Angela H; Santos, André L S; Branquinha, Marta H

    2006-06-01

    In this study, we demonstrated that metallopeptidase inhibitors (EDTA, EGTA, and 1,10-phenanthroline) were able to arrest Phytomonas serpens growth in distinct patterns. This parasite released exclusively metallopeptidases to the extracellular environment, whereas in cellular extracts only cysteine peptidases were detected. In addition, an extracellular polypeptide of 60 kDa reacted in Western blotting probed with polyclonal antibody raised against gp63 of Leishmania amazonensis. In the cellular parasite extract, this antibody recognized bands migrating at 63 and 52 kDa, which partitioned on both aqueous and membrane-rich fractions. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. Moreover, phospholipase C (PLC)-treated parasites reduced the number of gp63-positive cells. The anti-cross-reacting determinant (CRD) and anti-gp63 antibodies recognized the 60-kDa band in the supernatant from PLC-treated cells, suggesting that this protein is glycosylphosphatidylinositol-anchored to the plasma membrane. This is the first report on the presence of gp63-like molecules in members of the Phytomonas genus. The pretreatment of the parasites with anti-gp63 antibody significantly diminished their adhesion index to explanted salivary glands of the phytophagous insect Oncopeltus fasciatus, suggesting a potential involvement of the gp63-like molecules in the adhesive process of this plant trypanosomatid.

  5. PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

    PubMed Central

    Jung, Jee Yong; Chang, Kwang-Poo; Olivier, Martin

    2015-01-01

    Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface

  6. GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in Leishmania.

    PubMed

    Parashar, Smriti; Mukhopadhyay, Amitabha

    2017-07-21

    Metalloprotease gp63 ( Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His 6 -LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63 L594A/L595A or GFP-Ldgp63 V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis.

    PubMed

    Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C

    1997-06-01

    The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

  8. Expression and immunological cross-reactivity of LALP3, a novel astacin-like metalloprotease from brown spider (Loxosceles intermedia) venom.

    PubMed

    Morgon, Adriano M; Belisario-Ferrari, Matheus R; Trevisan-Silva, Dilza; Meissner, Gabriel O; Vuitika, Larissa; Marin, Brenda; Tashima, Alexandre K; Gremski, Luiza H; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Veiga, Silvio S; Chaim, Olga M

    2016-01-01

    Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  9. In Vitro Inhibition of Leishmania Attachment to Sandfly Midguts and LL-5 Cells by Divalent Metal Chelators, Anti-gp63 and Phosphoglycans.

    PubMed

    Soares, Rodrigo Pedro; Altoé, Ellen Cristina Félix; Ennes-Vidal, Vítor; da Costa, Simone M; Rangel, Elizabeth Ferreira; de Souza, Nataly Araújo; da Silva, Vanderlei Campos; Volf, Petr; d'Avila-Levy, Claudia Masini

    2017-07-01

    Leishmania braziliensis and Leishmania infantum are the causative agents of cutaneous and visceral leishmaniasis, respectively. Several aspects of the vector-parasite interaction involving gp63 and phosphoglycans have been individually assayed in different studies. However, their role under the same experimental conditions was not studied yet. Here, the roles of divalent metal chelators, anti-gp63 antibodies and purified type I phosphoglycans (PGs) were evaluated during in vitro parasite attachment to the midgut of the vector. Parasites were treated with divalent metal chelators or anti-gp63 antibodies prior to the interaction with Lutzomyia longipalpis/Lutzomyia intermedia midguts or sand fly LL-5 cells. In vitro binding system was used to examine the role of PG and gp63 in parallel. Treatment with divalent metal chelators reduced Le. infantum adhesion to the Lu. longipalpis midguts. The most effective compound (Phen) inhibited the binding in both vectors. Similar results were observed in the interaction between both Leishmania species and the cell line LL-5. Finally, parallel experiments using anti-gp63-treated parasites and PG-incubated midguts demonstrated that both approaches substantially inhibited attachment in the natural parasite-vector pairs Le. infantum/Lu. longipalpis and Le. braziliensis/Lu. intermedia. Our results suggest that gp63 and/or PG are involved in parasite attachment to the midgut of these important vectors. Copyright © 2017 Elsevier GmbH. All rights reserved.

  10. Potentiating Effects of MPL on DSPC Bearing Cationic Liposomes Promote Recombinant GP63 Vaccine Efficacy: High Immunogenicity and Protection

    PubMed Central

    Mazumder, Saumyabrata; Maji, Mithun; Ali, Nahid

    2011-01-01

    Background Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice. Methodology/Principal Findings Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo. Conclusion Our results define the

  11. Downregulation of DNA-PKcs suppresses P-gp expression via inhibition of the Akt/NF-κB pathway in CD133-positive osteosarcoma MG-63 cells.

    PubMed

    Li, Ka; Li, Xin; Tian, Jiguang; Wang, Hongliang; Pan, Jingbo; Li, Jianmin

    2016-10-01

    The development of chemoresistance is closely linked to the plateau of the survival rate in osteosarcoma (OS) patients. CD133-positive (CD133+) OS cells are known as cancer stem cells (CSCs) in OS and exhibit the characteristic of chemoresistance. In this study, CD133+ and CD133‑negative (CD133‑) MG‑63 cells were isolated by magnetic activated cell sorting (MACS). We verified that CD133+ MG‑63 cells were more resistant to cisplatin (CDDP) than CD133‑ MG‑63 cells. DNA‑dependent protein kinase catalytic subunit (DNA‑PKcs) and P‑glycoprotein (P‑gp) were expressed at higher levels in the CD133+ MG‑63 cells compared with those levels in the CD133‑ MG‑63 cells, whereas downregulation of DNA‑PKcs by small interfering RNA (siRNA) decreased chemoresistance to CDDP and P‑gp expression at the mRNA and protein levels in these cells. This indicated that DNA‑PKcs was correlated with P‑gp expression in the CD133+ MG‑63 cells. The Akt/NF‑κB pathway was hyperactivated in the CD133+ MG‑63 cells, whereas inhibition of the Akt/NF‑κB pathway downregulated P‑gp expression. In addition, downregulation of DNA‑PKcs suppressed the activity of the Akt/NF‑κB pathway. These results revealed that downregulation of DNA‑PKcs could decrease P‑gp expression via suppression of the Akt/NF‑κB pathway in CD133+ MG‑63 cells. Therefore, inhibition of DNA‑PKcs decreases P‑gp expression and sensitizes OS CSCs to chemotherapeutic agents in vitro, which needs to be further validated in vivo.

  12. copia-like retrotransposons are ubiquitous among plants.

    PubMed Central

    Voytas, D F; Cummings, M P; Koniczny, A; Ausubel, F M; Rodermel, S R

    1992-01-01

    Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes. Images PMID:1379734

  13. Ursolic acid reduces the metalloprotease/anti-metalloprotease imbalance in cerebral ischemia and reperfusion injury

    PubMed Central

    Wang, Yanzhe; He, Zhiyi; Deng, Shumin

    2016-01-01

    Background Activators of PPARs, particularly PPARγ, may be effective neuroprotective drugs against inflammatory responses in cerebral ischemia and reperfusion injury. Ursolic acid (UA) may act as a PPARγ agonist and serve as an anti-inflammatory agent. In this study, we used a rat middle cerebral artery occlusion and reperfusion model to examine how UA acts as a neuroprotective agent to modulate the metalloprotease/anti-metalloprotease balance. Methods The middle cerebral artery occlusion and reperfusion model (occlusion for 2 hours followed by reperfusion for 48 hours) was induced in male Sprague Dawley rats. UA was administered intragastrically 0.5, 24, and 47 hours after reperfusion. Bisphenol A diglycidyl ether (a PPARγ antagonist) was intraperitoneally administered 1, 24.5, and 47.5 hours after reperfusion. Forty-eight hours after reperfusion, neurological deficits and infarct volume were estimated. The PPARγ level and the metalloprotease/anti-metalloprotease balance were examined by Western blotting and immunohistochemistry. The activation of MAPK signaling pathways was also assessed. Results UA-treated (5, 10, or 20 mg/kg) rats showed significant improvement in neurological deficit score, infarct volume, and the number of intact neurons compared with control rats (P<0.01). Both the PPARγ protein level and the percentage of PPARγ-positive cells were increased in the UA-treated groups (P<0.01). Compared with the control group, the UA-treated groups exhibited reduced protein levels of MMP2, MMP9, and activated MAPKs (P<0.01) but an increased level of TIMP1 (P<0.01). UA exerted its protective effects in a dose-dependent manner. Co-treatment with UA and bisphenol A diglycidyl ether completely abolished the UA-induced changes in PPARγ expression; however UA continued to exert a significant but partial neuroprotective effect. Conclusion UA can act as a PPARγ agonist to improve the metalloprotease/anti-metalloprotease balance, possibly by inhibiting the

  14. Ursolic acid reduces the metalloprotease/anti-metalloprotease imbalance in cerebral ischemia and reperfusion injury.

    PubMed

    Wang, Yanzhe; He, Zhiyi; Deng, Shumin

    2016-01-01

    Activators of PPARs, particularly PPARγ, may be effective neuroprotective drugs against inflammatory responses in cerebral ischemia and reperfusion injury. Ursolic acid (UA) may act as a PPARγ agonist and serve as an anti-inflammatory agent. In this study, we used a rat middle cerebral artery occlusion and reperfusion model to examine how UA acts as a neuroprotective agent to modulate the metalloprotease/anti-metalloprotease balance. The middle cerebral artery occlusion and reperfusion model (occlusion for 2 hours followed by reperfusion for 48 hours) was induced in male Sprague Dawley rats. UA was administered intragastrically 0.5, 24, and 47 hours after reperfusion. Bisphenol A diglycidyl ether (a PPARγ antagonist) was intraperitoneally administered 1, 24.5, and 47.5 hours after reperfusion. Forty-eight hours after reperfusion, neurological deficits and infarct volume were estimated. The PPARγ level and the metalloprotease/anti-metalloprotease balance were examined by Western blotting and immunohistochemistry. The activation of MAPK signaling pathways was also assessed. UA-treated (5, 10, or 20 mg/kg) rats showed significant improvement in neurological deficit score, infarct volume, and the number of intact neurons compared with control rats (P<0.01). Both the PPARγ protein level and the percentage of PPARγ-positive cells were increased in the UA-treated groups (P<0.01). Compared with the control group, the UA-treated groups exhibited reduced protein levels of MMP2, MMP9, and activated MAPKs (P<0.01) but an increased level of TIMP1 (P<0.01). UA exerted its protective effects in a dose-dependent manner. Co-treatment with UA and bisphenol A diglycidyl ether completely abolished the UA-induced changes in PPARγ expression; however UA continued to exert a significant but partial neuroprotective effect. UA can act as a PPARγ agonist to improve the metalloprotease/anti-metalloprotease balance, possibly by inhibiting the activation of the MAPK signaling pathway

  15. Leishmania mexicana Gp63 cDNA Using Gene Gun Induced Higher Immunity to L. mexicana Infection Compared to Soluble Leishmania Antigen in BALB/C

    PubMed Central

    Rezvan, H; Rees, R; Ali, SA

    2011-01-01

    Background Leishmaniasis is a worldwide disease prevalent in tropical and sub tropical countries. Many attempts have been made and different strategies have been approached to develop a potent vaccine against Leishmania. DNA immunisation is a method, which is shown to be effective in Leishmania vaccination. Leishmania Soluble Antigen (SLA) has also recently been used Leishmania vaccination. Methods The immunity generated by SLA and L. mexicana gp63 cDNA was compared in groups of 6 mice, which were statistically analysed by student t- test with the P-value of 0.05. SLA was administered by two different methods; intramuscular injection and injection of dendritic cells (DCs) loaded with SLA. L. mexicana gp63 cDNA was administered by the gene gun. Results Immunisation of BALB/c mice with L. mexicana gp63 resulted in high levels of Th1-type immune response and cytotoxic T lymphocytes (CTL) activity, which were accompanied with protection induced by the immunisation against L. mexicana infection. In contrast, administration of SLA, produced a mixed Th1/Th2-type immune responses as well as a high level of CTL activity but did not protect mice from the infection. Conclusion The results indicate higher protection by DNA immunisation using L. mexicana gp63 cDNA compared to SLA, which is accompanied by a high level of Th1 immune response. However, the CTL activity does not necessarily correlate with the protection induced by the vaccine. Also, gene gun immunisation is a potential approach in Leishmania vaccination. These findings would be helpful in opening new windows in Leishmania vaccine research. PMID:22347315

  16. Deep sequencing of the Trypanosoma cruzi GP63 surface proteases reveals diversity and diversifying selection among chronic and congenital Chagas disease patients.

    PubMed

    Llewellyn, Martin S; Messenger, Louisa A; Luquetti, Alejandro O; Garcia, Lineth; Torrico, Faustino; Tavares, Suelene B N; Cheaib, Bachar; Derome, Nicolas; Delepine, Marc; Baulard, Céline; Deleuze, Jean-Francois; Sauer, Sascha; Miles, Michael A

    2015-04-01

    Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target--ND5--was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I gene family suggests a link between genetic diversity within this gene

  17. Deep Sequencing of the Trypanosoma cruzi GP63 Surface Proteases Reveals Diversity and Diversifying Selection among Chronic and Congenital Chagas Disease Patients

    PubMed Central

    Llewellyn, Martin S.; Messenger, Louisa A.; Luquetti, Alejandro O.; Garcia, Lineth; Torrico, Faustino; Tavares, Suelene B. N.; Cheaib, Bachar; Derome, Nicolas; Delepine, Marc; Baulard, Céline; Deleuze, Jean-Francois; Sauer, Sascha; Miles, Michael A.

    2015-01-01

    Background Chagas disease results from infection with the diploid protozoan parasite Trypanosoma cruzi. T. cruzi is highly genetically diverse, and multiclonal infections in individual hosts are common, but little studied. In this study, we explore T. cruzi infection multiclonality in the context of age, sex and clinical profile among a cohort of chronic patients, as well as paired congenital cases from Cochabamba, Bolivia and Goias, Brazil using amplicon deep sequencing technology. Methodology/ Principal Findings A 450bp fragment of the trypomastigote TcGP63I surface protease gene was amplified and sequenced across 70 chronic and 22 congenital cases on the Illumina MiSeq platform. In addition, a second, mitochondrial target—ND5—was sequenced across the same cohort of cases. Several million reads were generated, and sequencing read depths were normalized within patient cohorts (Goias chronic, n = 43, Goias congenital n = 2, Bolivia chronic, n = 27; Bolivia congenital, n = 20), Among chronic cases, analyses of variance indicated no clear correlation between intra-host sequence diversity and age, sex or symptoms, while principal coordinate analyses showed no clustering by symptoms between patients. Between congenital pairs, we found evidence for the transmission of multiple sequence types from mother to infant, as well as widespread instances of novel genotypes in infants. Finally, non-synonymous to synonymous (dn:ds) nucleotide substitution ratios among sequences of TcGP63Ia and TcGP63Ib subfamilies within each cohort provided powerful evidence of strong diversifying selection at this locus. Conclusions/Significance Our results shed light on the diversity of parasite DTUs within each patient, as well as the extent to which parasite strains pass between mother and foetus in congenital cases. Although we were unable to find any evidence that parasite diversity accumulates with age in our study cohorts, putative diversifying selection within members of the TcGP63I

  18. Molecular detection and analysis of a novel metalloprotease gene of entomopathogenic Serratia marcescens strains in infected Galleria mellonella.

    PubMed

    Tambong, J T; Xu, R; Sadiku, A; Chen, Q; Badiss, A; Yu, Q

    2014-04-01

    Serratia marcescens strains isolated from entomopathogenic nematodes (Rhabditis sp.) were examined for their pathogenicity and establishment in wax moth (Galleria mellonella) larvae. All the Serratia strains were potently pathogenic to G. mellonella larvae, leading to death within 48 h. The strains were shown to possess a metalloprotease gene encoding for a novel serralysin-like protein. Rapid establishment of the bacteria in infected larvae was confirmed by specific polymerase chain reaction (PCR) detection of a DNA fragment encoding for this protein. Detection of the viable Serratia strains in infected larvae was validated using the SYBR Green reverse transcriptase real-time PCR assay targeting the metalloprotease gene. Nucleotide sequences of the metalloprotease gene obtained in our study showed 72 single nucleotide polymorphisms (SNP) and 3 insertions compared with the metalloprotease gene of S. marcescens E-15. The metalloprotease gene had 60 synonymous and 8 nonsynonymous substitutions relative to the closest GenBank entry, S. marcescens E-15. A comparison of the amino acid composition of the new serralysin-like protein with that of the serralysin protein of S. marcescens E-15 revealed differences at 11 positions and a new aspartic acid residue. Analysis of the effect of protein variation suggests that a new aspartic acid residue resulting from nonsynonymous nucleotide mutations in the protein structure could have the most significant effect on its biological function. The new metalloprotease gene and (or) its product could have applications in plant agricultural biotechnology.

  19. Mini-P-gp and P-gp Co-Expression in Brown Trout Erythrocytes: A Prospective Blood Biomarker of Aquatic Pollution

    PubMed Central

    Valton, Emeline; Amblard, Christian; Desmolles, François; Combourieu, Bruno; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2015-01-01

    In aquatic organisms, such as fish, blood is continually exposed to aquatic contaminants. Multidrug Resistance (MDR) proteins are ubiquitous detoxification membrane pumps, which recognize various xenobiotics. Moreover, their expression is induced by a large class of drugs and pollutants. We have highlighted the co-expression of a mini P-gp of 75 kDa and a P-gp of 140 kDa in the primary culture of brown trout erythrocytes and in the erythrocytes of wild brown trout collected from three rivers in the Auvergne region of France. In vitro experiments showed that benzo[a]pyrene, a highly toxic pollutant model, induced the co-expression of mini-P-gp and P-gp in trout erythrocytes in a dose-dependent manner and relay type response. Similarly, in the erythrocytes of wild brown trout collected from rivers contaminated by a mixture of PAH and other multi-residues of pesticides, mini-P-gp and P-gp were able to modulate their expression, according to the nature of the pollutants. The differential and complementary responses of mini-P-gp and P-gp in trout erythrocytes suggest the existence in blood cells of a real protective network against xenobiotics/drugs. This property could be exploited to develop a blood biomarker of river pollution. PMID:26854141

  20. SlpE is a calcium-dependent cytotoxic metalloprotease associated with clinical isolates of Serratia marcescens.

    PubMed

    Stella, Nicholas A; Callaghan, Jake D; Zhang, Liang; Brothers, Kimberly M; Kowalski, Regis P; Huang, Jean J; Thibodeau, Patrick H; Shanks, Robert M Q

    Serralysin-like proteases are found in a wide variety of bacteria. These metalloproteases are frequently implicated in virulence and are members of the widely conserved RTX-toxin family. We identified a serralysin-like protease in the genome of a clinical isolate of Serratia marcescens that is highly similar to the canonical serralysin protein, PrtS. This gene was named serralysin-like protease E, SlpE, and was found in the majority (67%) of tested clinical isolates, but was absent from most tested non-clinical isolates including the insect pathogen and reference S. marcescens strain Db11. Purified recombinant SlpE exhibited calcium-dependent protease activity similar to metalloproteases PrtS and SlpB. Induction of slpE in the low-protease-producing S. marcescens strain PIC3611 highly elevated extracellular protease activity, and extracellular secretion required the lipD type 1 secretion system gene. Transcription of slpE was highly reduced in an eepR transcription factor mutant. Mutation of the slpE gene in a highly proteolytic clinical isolate reduced its protease activity, and evidence suggests that SlpE confers cytotoxicity of S. marcescens to the A549 airway carcinoma cell line. Together, these data reveal SlpE to be an EepR-regulated cytotoxic metalloprotease associated with clinical isolates of an important opportunistic pathogen. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  1. AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Yong, E-mail: dr_yongchen@hotmail.com; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612; Zeng, Gucheng

    2011-04-08

    Highlights: {yields} The unbinding force of sCD4-gp120 interaction was 25.45 {+-} 20.46 pN. {yields} The unbinding force of CD4 antigen-antibody interaction was 51.22 {+-} 34.64 pN. {yields} The unbinding force of gp120 antigen-antibody interaction was 89.87 {+-} 44.63 pN. {yields} The interaction forces between various HIV inhibitors and the target molecules are significantly different. {yields} Functionalizing on AFM tip or substrate of an interaction pair caused different results. -- Abstract: Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors andmore » corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4-gp120 interaction, CD4 antigen-antibody interaction, and gp120 antigen-antibody interaction were 25.45 {+-} 20.46, 51.22 {+-} 34.64, and 89.87 {+-} 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.« less

  2. Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.

    PubMed

    Vollenweider, F; Benjannet, S; Decroly, E; Savaria, D; Lazure, C; Thomas, G; Chrétien, M; Seidah, N G

    1996-03-01

    We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases.

  3. Zn-metalloprotease sequences in extremophiles

    NASA Astrophysics Data System (ADS)

    Holden, T.; Dehipawala, S.; Golebiewska, U.; Cheung, E.; Tremberger, G., Jr.; Williams, E.; Schneider, P.; Gadura, N.; Lieberman, D.; Cheung, T.

    2010-09-01

    The Zn-metalloprotease family contains conserved amino acid structures such that the nucleotide fluctuation at the DNA level would exhibit correlated randomness as described by fractal dimension. A nucleotide sequence fractal dimension can be calculated from a numerical series consisting of the atomic numbers of each nucleotide. The structure's vibration modes can also be studied using a Gaussian Network Model. The vibration measure and fractal dimension values form a two-dimensional plot with a standard vector metric that can be used for comparison of structures. The preference for amino acid usage in extremophiles may suppress nucleotide fluctuations that could be analyzed in terms of fractal dimension and Shannon entropy. A protein level cold adaptation study of the thermolysin Zn-metalloprotease family using molecular dynamics simulation was reported recently and our results show that the associated nucleotide fluctuation suppression is consistent with a regression pattern generated from the sequences's fractal dimension and entropy values (R-square { 0.98, N =5). It was observed that cold adaptation selected for high entropy and low fractal dimension values. Extension to the Archaemetzincin M54 family in extremophiles reveals a similar regression pattern (R-square = 0.98, N = 6). It was observed that the metalloprotease sequences of extremely halophilic organisms possess high fractal dimension and low entropy values as compared with non-halophiles. The zinc atom is usually bonded to the histidine residue, which shows limited levels of vibration in the Gaussian Network Model. The variability of the fractal dimension and entropy for a given protein structure suggests that extremophiles would have evolved after mesophiles, consistent with the bias usage of non-prebiotic amino acids by extremophiles. It may be argued that extremophiles have the capacity to offer extinction protection during drastic changes in astrobiological environments.

  4. Expression and Purification of a Matrix Metalloprotease Transmembrane Domain in Escherichia coli.

    PubMed

    Galea, Charles A

    2017-01-01

    Membrane tethered matrix metalloproteases are bound to the plasma membrane by a glycosylphosphatidylinositol-anchor or a transmembrane domain. To date, most studies of membrane-bound matrix metalloprotease have focused on the globular catalytic and protein-protein interaction domains of these enzymes. However, the transmembrane domains have been poorly studied even though they are known to mediate intracellular signaling via interaction with various cellular proteins. The expression and purification of the transmembrane domain of these proteins can be challenging due to their hydrophobic nature. In this chapter we describe the purification of a transmembrane domain for a membrane-bound matrix metalloprotease expressed in E. coli and its initial characterization by NMR spectroscopy.

  5. P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes.

    PubMed

    Valton, Emeline; Amblard, Christian; Wawrzyniak, Ivan; Penault-Llorca, Frederique; Bamdad, Mahchid

    2013-12-05

    Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous "membrane detoxification proteins" implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality.

  6. Matrix metalloproteases and PAR1 activation

    PubMed Central

    Austin, Karyn M.; Covic, Lidija

    2013-01-01

    Cardiovascular diseases, including atherothrombosis, are the leading cause of morbidity and mortality in the United States, Europe, and the developed world. Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet and endothelial function, and atherothrombotic disease. Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that is classically activated through cleavage of the N-terminal exodomain by the serine protease thrombin. Most recently, 2 MMPs have been discovered to have agonist activity for PAR1. Unexpectedly, MMP-1 and MMP-13 cleave the N-terminal exodomain of PAR1 at noncanonical sites, which result in distinct tethered ligands that activate G-protein signaling pathways. PAR1 exhibits metalloprotease-specific signaling patterns, known as biased agonism, that produce distinct functional outputs by the cell. Here we contrast the mechanisms of canonical (thrombin) and noncanonical (MMP) PAR1 activation, the contribution of MMP-PAR1 signaling to diseases of the vasculature, and the therapeutic potential of inhibiting MMP-PAR1 signaling with MMP inhibitors, including atherothrombotic disease, in-stent restenosis, heart failure, and sepsis. PMID:23086754

  7. Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers

    PubMed Central

    Morris, Charles D.; Azadnia, Parisa; de Val, Natalia; Vora, Nemil; Honda, Andrew; Giang, Erick; Saye-Francisco, Karen; Cheng, Yushao; Lin, Xiaohe; Mann, Colin J.; Tang, Jeffrey; Sok, Devin; Burton, Dennis R.; Law, Mansun; Ward, Andrew B.

    2017-01-01

    ABSTRACT Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. PMID:28246356

  8. Optional part-time and longer GP training modules in GP practices associated with more trainees becoming GPs - a cohort study in Switzerland.

    PubMed

    Studerus, Lara; Ahrens, Regina; Häuptle, Christian; Goeldlin, Adrian; Streit, Sven

    2018-01-05

    Switzerland, like many other countries, has a shortage of General Practitioners (GPs). Optional GP training modules in GP practices were offered during the at least 5-year GP training program to increase student and trainee interest in becoming a GP. The training modules had not yet been evaluated. We determined how many Swiss GP trainees became practicing GPs after they completed optional training modules, and if longer modules were associated with higher rates of GP specialization. In this population-based cohort study, we included GP trainees who chose an optional GP training module in GP practice, provided by the Foundation to Promote Training in General Practice (WHM) between 2006 and 2015. GP trainees were invited to complete an online survey to assess the primary outcome (becoming a practicing GP by 2016). Data on non-responders was collected via an internet search. We calculated univariate time-to-event curves to become a practicing GP, stratified by trainee's gender, length, part-time training, and number of years after graduation until training modules were completed. We used a multivariate model to adjust for characteristics of participants, training, and satisfaction with training modules. We assessed primary outcome for 351 (92.1%) of 381 former GP trainees who participated in a WHM program between 2006 and 2015. Of these 218 (57%) were practicing GPs by 2016. When focusing on the trainees who had completed training between 2006 and 2010, the rate of practicing GPs was even 73%. Longer (p = 0.018) and part-time training modules (p = 0.003) were associated with higher rates of being a practicing GP. Most (81%) practicing GPs thought their optional GP training module was (very) important in their choice of specialty. GP trainees who spent more time training in a GP practice, or who trained part-time were more likely to become practicing GPs. Most (80%) rated their training module as (very) important in their choice of career, highlighting that

  9. Matrix metalloprotease-3 expression in the medial plica and pannus-like tissue in knees from patients with medial compartment osteoarthritis.

    PubMed

    Wang, Hwai-Shi; Kuo, Pei-Yin; Yang, Chih-Chang; Lyu, Shaw-Ruey

    2011-03-01

    The severity of cartilage degeneration is positively correlated with the severity of the pathologic change of medial plica. However, knowledge of the pathogenic mechanisms and the impact of plica on cartilage destruction is limited. The aim of the present study was therefore to investigate matrix metalloprotease-3 (MMP-3) expression in the plica isolated from patients with medial compartment osteoarthritis of the knee. Immunohistochemistry showed that MMP-3 was highly expressed in pannus-like tissue and the plica. Western blotting of culture supernatants showed that interleukin-1β (IL-1β) treatment induced MMP-3 release by cells isolated from pannus tissue or the plica. Furthermore, reverse transcriptase polymerase chain reaction and real-time polymerase chain reaction analysis showed that MMP-3 mRNA levels were increased after IL-1β treatment of the cultured cells. MMP-3 and IL-1β mRNAs were expressed in the plica and pannus-like tissue, with MMP-3 mRNA being expressed at significantly higher levels in the plica than in normal synovial membrane and highly expressed in the plica at different stages in osteoarthritis (OA) patients. Pannus-like tissue and the plica express IL-1β and MMP-3. Moreover, MMP-3 mRNA and protein expression in the plica may contribute to the pathogenesis of OA. © 2011 Blackwell Publishing Limited.

  10. Period change in GP And

    NASA Astrophysics Data System (ADS)

    Rodriguez, E.; Rolland, A.; Lopez de Coca, P.

    1993-05-01

    GP And is a high amplitude d Scuti type star with V~10.m75, DV~0.m55, P=0.d0787 and spectral type A3 (Lopez de Coca et al. 1990, A & A 83, 51). To study the stability of its fundamental pulsation we have carried out simultaneous uvby photometry of this star in the years 1987 and 1992 at Sierra Nevada and Caltar Alto observatories, both in Spain. Ten new times of light maxima were obtained. In total, forty-one time s of light maxima (from 1973 to 1992, collected from Splittgerber 1976, Mitt. Veraend. Sterne 7, 137; Eggen 1978, IBVS 1517; Gieseking et al. 1979, A & AS 36, 457; Burchi et al. 1992, Mem. Soc. Astron. Ital. 63, 87 and us) were used to determinate the ephemeris of the light curve of GP And by means of the classical O-C method.

  11. Occurrence and Evolution of the Paralogous Zinc Metalloproteases IgA1 Protease, ZmpB, ZmpC, and ZmpD in Streptococcus pneumoniae and Related Commensal Species

    PubMed Central

    Bek-Thomsen, Malene; Poulsen, Knud; Kilian, Mogens

    2012-01-01

    ABSTRACT The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host. PMID:23033471

  12. Differential Evolution and Neofunctionalization of Snake Venom Metalloprotease Domains*

    PubMed Central

    Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A.B.; Vetter, Irina; Yang, Daryl C.; Casewell, Nicholas R.; Jackson, Timothy N. W.; Koludarov, Ivan; Alewood, Paul F.; Hodgson, Wayne C.; Lewis, Richard J.; King, Glenn F.; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G.

    2013-01-01

    Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands. PMID:23242553

  13. Differential evolution and neofunctionalization of snake venom metalloprotease domains.

    PubMed

    Brust, Andreas; Sunagar, Kartik; Undheim, Eivind A B; Vetter, Irina; Yang, Daryl C; Yang, Dary C; Casewell, Nicholas R; Jackson, Timothy N W; Koludarov, Ivan; Alewood, Paul F; Hodgson, Wayne C; Lewis, Richard J; King, Glenn F; Antunes, Agostinho; Hendrikx, Iwan; Fry, Bryan G

    2013-03-01

    Snake venom metalloproteases (SVMP) are composed of five domains: signal peptide, propeptide, metalloprotease, disintegrin, and cysteine-rich. Secreted toxins are typically combinatorial variations of the latter three domains. The SVMP-encoding genes of Psammophis mossambicus venom are unique in containing only the signal and propeptide domains. We show that the Psammophis SVMP propeptide evolves rapidly and is subject to a high degree of positive selection. Unlike Psammophis, some species of Echis express both the typical multidomain and the unusual monodomain (propeptide only) SVMP, with the result that a lower level of variation is exerted upon the latter. We showed that most mutations in the multidomain Echis SVMP occurred in the protease domain responsible for proteolytic and hemorrhagic activities. The cysteine-rich and disintegrin-like domains, which are putatively responsible for making the P-III SVMPs more potent than the P-I and P-II forms, accumulate the remaining variation. Thus, the binding sites on the molecule's surface are evolving rapidly whereas the core remains relatively conserved. Bioassays conducted on two post-translationally cleaved novel proline-rich peptides from the P. mossambicus propeptide domain showed them to have been neofunctionalized for specific inhibition of mammalian a7 neuronal nicotinic acetylcholine receptors. We show that the proline rich postsynaptic specific neurotoxic peptides from Azemiops feae are the result of convergent evolution within the precursor region of the C-type natriuretic peptide instead of the SVMP. The results of this study reinforce the value of studying obscure venoms for biodiscovery of novel investigational ligands.

  14. Structure and evolution of N-domains in AAA metalloproteases.

    PubMed

    Scharfenberg, Franka; Serek-Heuberger, Justyna; Coles, Murray; Hartmann, Marcus D; Habeck, Michael; Martin, Jörg; Lupas, Andrei N; Alva, Vikram

    2015-02-27

    Metalloproteases of the AAA (ATPases associated with various cellular activities) family play a crucial role in protein quality control within the cytoplasmic membrane of bacteria and the inner membrane of eukaryotic organelles. These membrane-anchored hexameric enzymes are composed of an N-terminal domain with one or two transmembrane helices, a central AAA ATPase module, and a C-terminal Zn(2+)-dependent protease. While the latter two domains have been well studied, so far, little is known about the N-terminal regions. Here, in an extensive bioinformatic and structural analysis, we identified three major, non-homologous groups of N-domains in AAA metalloproteases. By far, the largest one is the FtsH-like group of bacteria and eukaryotic organelles. The other two groups are specific to Yme1: one found in plants, fungi, and basal metazoans and the other one found exclusively in animals. Using NMR and crystallography, we determined the subunit structure and hexameric assembly of Escherichia coli FtsH-N, exhibiting an unusual α+β fold, and the conserved part of fungal Yme1-N from Saccharomyces cerevisiae, revealing a tetratricopeptide repeat fold. Our bioinformatic analysis showed that, uniquely among these proteins, the N-domain of Yme1 from the cnidarian Hydra vulgaris contains both the tetratricopeptide repeat region seen in basal metazoans and a region of homology to the N-domains of animals. Thus, it is a modern-day representative of an intermediate in the evolution of animal Yme1 from basal eukaryotic precursors. Copyright © 2015. Published by Elsevier Ltd.

  15. TssA forms a gp6-like ring attached to the type VI secretion sheath.

    PubMed

    Planamente, Sara; Salih, Osman; Manoli, Eleni; Albesa-Jové, David; Freemont, Paul S; Filloux, Alain

    2016-08-01

    The type VI secretion system (T6SS) is a supra-molecular bacterial complex that resembles phage tails. It is a killing machine which fires toxins into target cells upon contraction of its TssBC sheath. Here, we show that TssA1 is a T6SS component forming dodecameric ring structures whose dimensions match those of the TssBC sheath and which can accommodate the inner Hcp tube. The TssA1 ring complex binds the T6SS sheath and impacts its behaviour in vivo In the phage, the first disc of the gp18 sheath sits on a baseplate wherein gp6 is a dodecameric ring. We found remarkable sequence and structural similarities between TssA1 and gp6 C-termini, and propose that TssA1 could be a baseplate component of the T6SS Furthermore, we identified similarities between TssK1 and gp8, the former interacting with TssA1 while the latter is found in the outer radius of the gp6 ring. These observations, combined with similarities between TssF and gp6N-terminus or TssG and gp53, lead us to propose a comparative model between the phage baseplate and the T6SS. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  16. Faecal matrix metalloprotease-9 is a more sensitive marker for diagnosing pouchitis than faecal calprotectin: results from a pilot study.

    PubMed

    Farkas, Klaudia; Bálint, Anita; Bor, Renáta; Földesi, Imre; Szűcs, Mónika; Nagy, Ferenc; Szepes, Zoltán; Annaházi, Anita; Róka, Richárd; Molnár, Tamás

    2015-03-01

    Potential non-invasive markers of pouchitis would have a great deal of significance within clinical practice. This study is aimed at assessing the diagnostic accuracy of fecal calprotectin and matrix metalloprotease-9 as potential markers in patients both with and without pouchitis. Stool and blood samples were collected from 33 ileal pouch-anal anastomosis patients before a follow-up pouchoscopy. Biopsy samples were taken for histological purposes. The presence of cuffitis and stenosis was evaluated with an endoscopy. Calprotectin and matrix metalloprotease-9 were quantified with an enzyme-linked immunosorbent assay. Pouchitis was detected in 30.3% of the patients. The levels of fecal calprotectin and matrix metalloprotease-9 increased significantly in patients with pouchitis. The sensitivity and specificity of matrix metalloprotease-9 was higher than that of fecal calprotectin. Only matrix metalloprotease-9 correlated significantly with the severity of pouchitis. Fecal matrix metalloprotease-9 has a high specificity in the diagnosis of pouchitis.

  17. [The role of metalloprotease in pathogenesis of nervous system diseases].

    PubMed

    Mirowska, D; Członkowska, A

    2001-01-01

    Matrix Metalloproteases (MMPs) comprise a big family of proteolytic enzymes secreted into extracellular matrix and involved in remodelling of many tissues. The MMPs' activity is regulated on many levels. It is also determined by specific inhibitors known as tissue inhibitors of metalloproteases (TIMPs). Several studies revealed that MMPs have a role not only in physiological processes but also in pathophysiology of nervous system diseases, such as multiplex sclerosis, Guillan-Barré syndrome and strokes. Concerning demyelination MMPs are responsible for degradation of myelin components and facilitation of immune cells migration into inflammatory sites by degrading vascular basement membrane. We still investigate substances with positive clinical effect on the nervous system diseases due to MMPs inactivation.

  18. Technologies for Achieving Field Ubiquitous Computing

    NASA Astrophysics Data System (ADS)

    Nagashima, Akira

    Although the term “ubiquitous” may sound like jargon used in information appliances, ubiquitous computing is an emerging concept in industrial automation. This paper presents the author's visions of field ubiquitous computing, which is based on the novel Internet Protocol IPv6. IPv6-based instrumentation will realize the next generation manufacturing excellence. This paper focuses on the following five key issues: 1. IPv6 standardization; 2. IPv6 interfaces embedded in field devices; 3. Compatibility with FOUNDATION fieldbus; 4. Network securities for field applications; and 5. Wireless technologies to complement IP instrumentation. Furthermore, the principles of digital plant operations and ubiquitous production to support the above key technologies to achieve field ubiquitous systems are discussed.

  19. Impact of Ebola mucin-like domain on antiglycoprotein antibody responses induced by Ebola virus-like particles.

    PubMed

    Martinez, Osvaldo; Tantral, Lee; Mulherkar, Nirupama; Chandran, Kartik; Basler, Christopher F

    2011-11-01

    Ebola virus (EBOV) glycoprotein (GP), responsible for mediating host-cell attachment and membrane fusion, contains a heavily glycosylated mucin-like domain hypothesized to shield GP from neutralizing antibodies. To test whether the mucin-like domain inhibits the production and function of anti-GP antibodies, we vaccinated mice with Ebola virus-like particles (VLPs) that express vesicular stomatitis virus G, wild-type EBOV GP (EBGP), EBOV GP without its mucin-like domain (ΔMucGP), or EBOV GP with a Crimean-Congo hemorrhagic fever virus mucin-like domain substituted for the EBOV mucin-like domain (CMsubGP). EBGP-VLP immunized mice elicited significantly higher serum antibody titers toward EBGP or its mutants, as detected by western blot analysis, than did VLP-ΔMucGP. However, EBGP-, ΔMucGP- and CMsubGP-VLP immunized mouse sera contained antibodies that bound to cell surface-expressed GP at similar levels. Furthermore, low but similar neutralizing antibody titers, measured against a vesicular stomatitis virus (VSV) expressing EBGP or ΔMucGP, were present in EBGP, ΔMucGP, and CMsubGP sera, although a slightly higher neutralizing titer (2- to 2.5-fold) was detected in ΔMucGP sera. We conclude that the EBOV GP mucin-like domain can increase relative anti-GP titers, however these titers appear to be directed, at least partly, to denatured GP. Furthermore, removing the mucin-like domain from immunizing VLPs has modest impact on neutralizing antibody titers in serum.

  20. Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.

    PubMed

    Pallesen, Jesper; Murin, Charles D; de Val, Natalia; Cottrell, Christopher A; Hastie, Kathryn M; Turner, Hannah L; Fusco, Marnie L; Flyak, Andrew I; Zeitlin, Larry; Crowe, James E; Andersen, Kristian G; Saphire, Erica Ollmann; Ward, Andrew B

    2016-08-08

    The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.

  1. The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression*

    PubMed Central

    Haining, Elizabeth J.; Yang, Jing; Bailey, Rebecca L.; Khan, Kabir; Collier, Richard; Tsai, Schickwann; Watson, Steve P.; Frampton, Jon; Garcia, Paloma; Tomlinson, Michael G.

    2012-01-01

    A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane “partner” proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting. PMID:23035126

  2. Immunohistochemical evidence for ubiquitous distribution of metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines

    PubMed Central

    Weirich, Gregor; Mengele, Karin; Yfanti, Christina; Gkazepis, Apostolos; Hellmann, Daniela; Welk, Anita; Giersig, Cecylia; Kuo, Wen-Liang; Rosner, Marsha Rich; Tang, Wei-Jen; Schmitt, Manfred

    2013-01-01

    Immunohistochemical evidence for ubiquitous distribution of metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, spleen) and on a cell microarray encompassing 31 tumor cell lines of different origin plus trophoblast cells, and normal blood lymphocytes and granulocytes. IDE protein is expressed by all of the tissues assessed and in all of the tumor cell lines except Raji and HL-60; trophoblast cells and granulocytes but not normal lymphocytes are also IDE-positive. PMID:18783335

  3. The Extracellular Metalloprotease of Vibrio tubiashii Is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae▿

    PubMed Central

    Hasegawa, Hiroaki; Lind, Erin J.; Boin, Markus A.; Häse, Claudia C.

    2008-01-01

    Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae. PMID:18456850

  4. Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

    PubMed

    Morris, Charles D; Azadnia, Parisa; de Val, Natalia; Vora, Nemil; Honda, Andrew; Giang, Erick; Saye-Francisco, Karen; Cheng, Yushao; Lin, Xiaohe; Mann, Colin J; Tang, Jeffrey; Sok, Devin; Burton, Dennis R; Law, Mansun; Ward, Andrew B; He, Linling; Zhu, Jiang

    2017-02-28

    Broadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches. IMPORTANCE Both epitope-focused and trimer-based strategies are currently being explored in HIV-1 vaccine development, which aims to elicit broadly neutralizing

  5. Immunohistochemical evidence of ubiquitous distribution of the metalloendoprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cell lines.

    PubMed

    Weirich, Gregor; Mengele, Karin; Yfanti, Christina; Gkazepis, Apostolos; Hellmann, Daniela; Welk, Anita; Giersig, Cecylia; Kuo, Wen-Liang; Rosner, Marsha Rich; Tang, Wei-Jen; Schmitt, Manfred

    2008-11-01

    Immunohistochemical evidence of ubiquitous distribution of the metalloprotease insulin-degrading enzyme (IDE; insulysin) in human non-malignant tissues and tumor cells is presented. Immunohistochemical staining was performed on a multi-organ tissue microarray (pancreas, lung, kidney, central/peripheral nervous system, liver, breast, placenta, myocardium, striated muscle, bone marrow, thymus, and spleen) and on a cell microarray of 31 tumor cell lines of different origin, as well as trophoblast cells and normal blood lymphocytes and granulocytes. IDE protein was expressed in all the tissues assessed and all the tumor cell lines except for Raji and HL-60. Trophoblast cells and granulocytes, but not normal lymphocytes, were also IDE-positive.

  6. GP workforce participation in Tasmania.

    PubMed

    Gartlan, Jan; Male, Sarah; Donaldson, Lawrence; Nelson, Mark; Winzenberg, Tania

    2007-05-01

    Predicting future general practitioner workforce requires information about how demographic factors affect GP workforce participation. Regional differences might not be accounted for in national studies. The authors aimed to determine GP characteristics associated with workforce participation in Tasmania. A self administered census of Tasmanian GPs measured GP demographics and the number of 3.5 hour sessions worked in 1 week in 2005. Four hundred and three GPs responded (76% response rate). Six percent of GPs were on leave at the time of the census. Age, gender and graduation outside of Australia, the United Kingdom or Ireland were associated with workforce participation, but rurality had no effect. The effect of age was modified by gender with women aged over 55 years being more likely to work full time (p=0.03). Factors affecting workforce participation may vary across regions. Predictions based on national models may need to be interpreted in the context of local circumstances.

  7. Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

    PubMed Central

    Zhang, Mei-Yun; Yuan, Tingting; Li, Jingjing; Rosa Borges, Andrew; Watkins, Jennifer D.; Guenaga, Javier; Yang, Zheng; Wang, Yanping; Wilson, Richard; Li, Yuxing; Polonis, Victoria R.; Pincus, Seth H.; Ruprecht, Ruth M.; Dimitrov, Dimiter S.

    2012-01-01

    Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics. PMID:22970187

  8. Characterization and role of a metalloprotease induced by chitin in Serratia sp. KCK.

    PubMed

    Kim, Hyun-Soo; Golyshin, Peter N; Timmis, Kenneth N

    2007-11-01

    A metalloprotease induced by chitin in a new chitinolytic bacterium Serratia sp. Strain KCK was purified and characterized. Compared with other Serratia enzymes, it exhibited a rather broad pH activity range (pH 5.0-8.0), and thermostability. The cognate ORF, mpr, was cloned and expressed. Its deduced amino acid sequence showed high similarity to those of bacterial zinc-binding metalloproteases and a well-conserved serralysin family motif. Pretreatment of chitin with the Mpr protein promoted chitin degradation by chitinase A, which suggests that Mpr participates in, and facilitates, chitin degradation by this microorganism.

  9. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an inmore » vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.« less

  10. The first non Clostridial botulinum-like toxin cleaves VAMP within the juxtamembrane domain.

    PubMed

    Zornetta, Irene; Azarnia Tehran, Domenico; Arrigoni, Giorgio; Anniballi, Fabrizio; Bano, Luca; Leka, Oneda; Zanotti, Giuseppe; Binz, Thomas; Montecucco, Cesare

    2016-07-22

    The genome of Weissella oryzae SG25T was recently sequenced and a botulinum neurotoxin (BoNT) like gene was identified by bioinformatics methods. The typical three-domains organization of BoNTs with a N-terminal metalloprotease domain, a translocation and a cell binding domains could be identified. The BoNT family of neurotoxins is rapidly growing, but this was the first indication of the possible expression of a BoNT toxin outside the Clostridium genus. We performed molecular modeling and dynamics simulations showing that the 50 kDa N-terminal domain folds very similarly to the metalloprotease domain of BoNT/B, whilst the binding part is different. However, neither the recombinant metalloprotease nor the binding domains showed cross-reactivity with the standard antisera that define the seven serotypes of BoNTs. We found that the purified Weissella metalloprotease cleaves VAMP at a single site untouched by the other VAMP-specific BoNTs. This site is a unique Trp-Trp peptide bond located within the juxtamembrane segment of VAMP which is essential for neurotransmitter release. Therefore, the present study identifies the first non-Clostridial BoNT-like metalloprotease that cleaves VAMP at a novel and relevant site and we propose to label it BoNT/Wo.

  11. HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.

    PubMed

    Krell, Tino; Greco, Frédéric; Engel, Olivier; Dubayle, Jean; Dubayle, Joseline; Kennel, Audrey; Charloteaux, Benoit; Brasseur, Robert; Chevalier, Michel; Sodoyer, Regis; El Habib, Raphaëlle

    2004-04-01

    HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.

  12. Evidence for metalloprotease involvement in the in vivo effects of big endothelin 1.

    PubMed

    Pollock, D M; Opgenorth, T J

    1991-07-01

    The potent vasoconstrictor endothelin 1 (ET-1) is thought to arise from the proteolytic processing of big endothelin 1 (Big ET) by a unique endothelin-converting enzyme, possibly a metalloprotease. Experiments were conducted to determine the effects of Big ET on cardiovascular and renal functions during inhibition of metalloprotease activity in vivo. Intravenous infusion of Big ET (0.1 nmol.kg-1.min-1) in anesthetized euvolemic rats produced a significant increase in mean arterial pressure (MAP; 39 +/- 8%) and a decrease in effective renal plasma flow (ERPF; -39 +/- 2%), whereas glomerular filtration rate (GFR) remained unchanged (-8 +/- 8%). Simultaneous intravenous infusion of phosphoramidon (0.25 mg.kg-1.min-1), an inhibitor of metalloprotease activity including neutral endopeptidase EC 3.4.24.11 (NEP), completely prevented these effects of Big ET. Thiorphan (0.1 mg.kg-1.min-1), also an inhibitor of NEP, had absolutely no effect on either the renal or cardiovascular response to Big ET. Similarly, the response to Big ET was unaffected by infusion of enalaprilat (0.1 mg.kg-1.min-1), an inhibitor of the angiotensin-converting enzyme, which is also a metalloprotease. To determine whether the effect of phosphoramidon was due to antagonism of ET-1, an identical series of experiments was performed using ET-1 infusion (0.02 nmol.kg-1.min-1). Although the increase in MAP (24 +/- 5%) produced by ET-1 was less than that observed for the given dose of Big ET, the renal vasoconstriction was much more severe; the smaller peptide changed ERPF and GFR by -66 +/- 7 and -54 +/- 9%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. P-gp expression levels in the erythrocytes of brown trout: a new tool for aquatic sentinel biomarker development.

    PubMed

    Valton, Emeline; Wawrzyniak, Ivan; Amblard, Christian; Combourieu, Bruno; Bayle, Marie-Laure; Desmolles, François; Kwiatkowski, Fabrice; Penault-Llorca, Frédérique; Bamdad, Mahchid

    2017-09-01

    P-glycoprotein (P-gp) is a ubiquitous membrane detoxification pump involved in cellular defence against xenobiotics. Blood is a hub for the trade and transport of physiological molecules and xenobiotics. Our recent studies have highlighted the expression of a 140-kDa P-gp in brown trout erythrocytes in primary cell culture and its dose-dependent response to Benzo[a]pyrene pollutant. The purpose of this study was focused on using P-gp expression in brown trout erythrocytes as a biomarker for detecting the degree of river pollution. abcb1 gene and P-gp expression level were analysed by reverse transcriptase-PCR and Western blot, in the erythrocytes of brown trouts. The latter were collected in upstream and downstream of four rivers in which 17 polycyclic aromatic hydrocarbons and 348 varieties of pesticides micro-residues were analysed by liquid chromatography and mass spectrometry. The abcb1 gene and the 140-kDa P-gp were not expressed in trout erythrocytes from uncontaminated river. In contrast, they are clearly expressed in contaminated rivers, in correlation with the river pollution degree and the nature of the pollutants. This biological tool may offer considerable advantages since it provides an effective response to the increasing need for an early biomarker.

  14. Psychosocial factors in GP work: the effects of taking a GP position or leaving GP work.

    PubMed

    Heponiemi, Tarja; Kouvonen, Anne; Aalto, Anna-Mari; Elovainio, Marko

    2013-06-01

    We examined the effects of leaving public sector general practitioner (GP) work and of taking a GP position on changes in work-related psychosocial factors, such as time pressure, patient-related stress, distress and work interference with family. In addition, we examined whether changes in time pressure and patient-related stress mediated the association of employment change with changes of distress and work interference with family. Participants were 1705 Finnish physicians (60% women) who responded to surveys in 2006 and 2010. Analyses of covariance were conducted to examine the effect of employment change to outcome changes adjusted for gender, age and response format. Mediational effects were tested following the procedures outlined by Baron and Kenny. Employment change was significantly associated with all the outcomes. Leaving public sector GP work was associated with substantially decreased time pressure, patient-related stress, distress and work interference with family. In contrast, taking a position as a public sector GP was associated with an increase in these factors. Mediation tests suggested that the associations of employment change with distress change and work interference with family change were partially explained by the changes in time pressure and patient-related stress. Our results showed that leaving public sector GP work is associated with favourable outcomes, whereas taking a GP position in the public sector is associated with adverse effects. Primary health-care organizations should pay more attention to the working conditions of their GPs, in particular, to time pressure and patient-related stress.

  15. Integrated musculoskeletal service design by GP consortia

    PubMed Central

    2011-01-01

    Background Musculoskeletal conditions are common in primary care and are associated with significant co-morbidity and impairment of quality of life. Traditional care pathways combined community-based physiotherapy with GP referral to hospital for a consultant opinion. Locally, this model led to only 30% of hospital consultant orthopaedic referrals being listed for surgery, with the majority being referred for physiotherapy. The NHS musculoskeletal framework proposed the use of interface services to provide expertise in diagnosis, triage and management of musculoskeletal problems not requiring surgery. The White Paper Equity and Excellence: Liberating the NHS has replaced PCT commissioning with GP consortia, who will lead future service development. Setting Primary and community care, integrated with secondary care, in the NHS in England. Question How can GP consortia lead the development of integrated musculoskeletal services? Review: The Ealing experience We explore here how Ealing implemented a ‘See and Treat’ interface clinic model to improve surgical conversion rates, reduce unnecessary hospital referrals and provide community treatment more efficiently than a triage model. A high-profile GP education programme enabled GPs to triage in their practices and manage patients without referral. Conclusion In Ealing, we demonstrated that most patients with musculoskeletal conditions can be managed in primary care and community settings. The integrated musculoskeletal service provides clear and fast routes to secondary care. This is both clinically effective and cost-effective, reserving hospital referral for patients most likely to need surgery. GP consortia, in conjunction with strong clinical leadership, inbuilt organisational and professional learning, and a GP champion, are well placed to deliver service redesign by co-ordinating primary care development, local commissioning of community services and the acute commissioning vehicles responsible for secondary

  16. Murine serum glycoprotein gp70 behaves as an acute phase reactant

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hara, I.; Izui, S.; Dixon, F.J.

    1982-02-01

    A single intraperitoneal injection of bacterial lipopolysaccharide (LPS) or its lipid A component induced high levels of glycoprotein, gp70, in sera of several strains of mice within 24 h. This serum gp70 response induced by LPS was independent of the activation of B cells and the presence of T cells. However, serological and immunohistochemical studies demonstrated the production of gp70 by hepatic parenchymal cells and its subsequent release into the circulating blood. The expression of gp70 in the serum was enhanced not only by LPS but also other inducers of acute phase reactants (APR) such as turpentine oil or polyriboinosinic-polyribocytidylicmore » acid. Further, the serum gp70 response was kinetically identical to those of APR. These results strongly suggest that (a) the liver may be the major source for serum gp70, (b) serum gp70 behaves like an APR, (c) its expression may be controlled by a mechanism similar to that for other APR, and (d) this glycoprotein apparently behaves as a normal host constituent and not a product of a viral genome.« less

  17. Purification and characterization of a novel fibrinolytic α chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji.

    PubMed

    Moon, Sung-Min; Kim, Jae-Sung; Kim, Heung-Joong; Choi, Mi Suk; Park, Bo Ram; Kim, Su-Gwan; Ahn, Hoon; Chun, Hong Sung; Shin, Yong Kook; Kim, Jong-Jin; Kim, Do Kyung; Lee, Sook-Young; Seo, Young-Woo; Kim, Yong Hwan; Kim, Chun Sung

    2014-05-01

    A novel fibrinolytic enzyme was purified from Lyophyllum shimeji, a popular edible mushroom in Asia. The enzyme was purified using combination of anion exchange chromatography on a Mono Q 5/5 column and size exclusion gel filtration chromatography on Superdex 200 100/300 column. This purification protocol resulted 80.9-fold purification of the enzyme and a final yield of 5.7%. The molecular weight of the purified enzyme was estimated to be 21 kDa by SDS-PAGE and size exclusion gel filtration. The N-terminal amino acid sequence was found to be ITFQSASP, which is dissimilar from that of known fibrinolytic enzymes. The purified enzyme was a neutral protease with an optimal reaction pH and temperature of 8.0 and 37°C, respectively. Enzymatic activity was inhibited by Cu(2+) and Co(2+). It was also significantly inhibited by PMSF and TPCK. Furthermore, it was found to exhibit a higher specificity for S-7388, a well-known chymotrypsin chromogenic substrate, indicating chymotrypsin like serine metalloprotease. The relative fibrinolytic activity of 5 μg purified enzyme have two fold more activity than 1 unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the Aα-chain followed by the Bβ- and γ-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L. shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. Recognition of similar epitopes on varicella-zoster virus gpI and gpIV by monoclonal antibodies.

    PubMed Central

    Vafai, A; Wroblewska, Z; Mahalingam, R; Cabirac, G; Wellish, M; Cisco, M; Gilden, D

    1988-01-01

    Two monoclonal antibodies, MAb43.2 and MAb79.0, prepared against varicella-zoster virus (VZV) proteins were selected to analyze VZV gpIV and gpI, respectively. MAb43.2 reacted only with cytoplasmic antigens, whereas MAb79.0 recognized both cytoplasmic and membrane antigens in VZV-infected cells. Immunoprecipitation of in vitro translation products with MAb43.2 revealed only proteins encoded by the gpIV gene, whereas MAb79.0 precipitated proteins encoded by the gpIV and gpI genes. Pulse-chase analysis followed by immunoprecipitation of VZV-infected cells indicated reactivity of MAb43.2 with three phosphorylated precursor species of gpIV and reactivity of MAb79.0 with the precursor and mature forms of gpI and gpIV. These results indicated that (i) MAb43.2 and MAb79.0 recognize different epitopes on VZV gpIV, (ii) glycosylation of gpIV ablates recognition by MAb43.2, and (iii) gpIV is phosphorylated. To map the binding site of MAb79.0 on gpI, the pGEM transcription vector, containing the coding region of the gpI gene, was linearized, and three truncated gpI DNA fragments were generated. RNA was transcribed from each truncated fragment by using SP6 RNA polymerase, translated in vitro in a rabbit reticulocyte lysate, and immunoprecipitated with MAb79.0 and human sera. The results revealed the existence of an antibody-binding site within 14 amino acid residues located between residues 109 to 123 on the predicted amino acid sequences of gpI. From the predicted amino acid sequences, 14 residues on gpI (residues 107 to 121) displayed a degree of similarity (36%) to two regions (residues 55 to 69 and 245 to 259) of gp IV. Such similarities may account for the binding of MAb79.0 to both VZV gpI and gpIV. Images PMID:2455814

  19. Vibrio Zinc-Metalloprotease Causes Photoinactivation of Coral Endosymbionts and Coral Tissue Lesions

    PubMed Central

    Sussman, Meir; Mieog, Jos C.; Doyle, Jason; Victor, Steven; Willis, Bette L.; Bourne, David G.

    2009-01-01

    Background Coral diseases are emerging as a serious threat to coral reefs worldwide. Of nine coral infectious diseases, whose pathogens have been characterized, six are caused by agents from the family Vibrionacae, raising questions as to their origin and role in coral disease aetiology. Methodology/Principal Findings Here we report on a Vibrio zinc-metalloprotease causing rapid photoinactivation of susceptible Symbiodinium endosymbionts followed by lesions in coral tissue. Symbiodinium photosystem II inactivation was diagnosed by an imaging pulse amplitude modulation fluorometer in two bioassays, performed by exposing Symbiodinium cells and coral juveniles to non-inhibited and EDTA-inhibited supernatants derived from coral white syndrome pathogens. Conclusion/Significance These findings demonstrate a common virulence factor from four phylogenetically related coral pathogens, suggesting that zinc-metalloproteases may play an important role in Vibrio pathogenicity in scleractinian corals. PMID:19225559

  20. Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases.

    PubMed

    Wang, Ying; Ha, Seung-Wook; Zhang, Tianpeng; Kho, Dhong-Hyo; Raz, Avraham; Xie, Youming

    2014-04-30

    gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.

  1. Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases

    PubMed Central

    Kho, Dhong-Hyo; Raz, Avraham; Xie, Youming

    2014-01-01

    gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation. PMID:24810856

  2. Influence of elastin-derived peptides on metalloprotease production in endothelial cells.

    PubMed

    Siemianowicz, Krzysztof; Gminski, Jan; Goss, Malgorzata; Francuz, Tomasz; Likus, Wirginia; Jurczak, Teresa; Garczorz, Wojciech

    2010-11-01

    Matrix metalloproteases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. MMP-1 and MMP-2 are produced by endothelial cells and are involved in specific vascular pathologies, including atherosclerosis and aortal aneurysm. One of the most important differences between these two metalloproteases is the possibility of hydrolysis of elastin and collagen type IV by MMP-2, but not by MMP-1. Elastin-derived peptides are generated as a result of the degradation of elastin fibers. The aim of our study was to compare the production of MMP-1 and MMP-2 in cultured human arterial endothelial cells derived from vascular pathologies localized at three different sites, the coronary artery, iliac artery and aorta, measured as their concentration in cell culture medium. The second aim was to evaluate the influence of κ-elastin (at concentrations 0.1, 0.4, 1.0, 2.5 or 5.0 μg/ml) on the production of the evaluated metalloproteases in three endothelial cell lines. The production of MMP-1 was statistically significantly greater in endothelial cells derived from the aorta compared to that in the endothelium obtained from the coronary and iliac arteries. There were no statistically significant differences in the production of MMP-2 among the endothelial cell lines tested. The addition of κ-elastin at all evaluated concentrations did not statistically significantly influence the concentration of MMP-1 in the cultured coronary artery endothelium. Furthermore, no statistically significant differences were observed in the cultured iliac artery endothelium. In the cultured endothelium derived from the aorta, κ-elastin at concentrations of 0.1 and 0.4 μg/ml significantly increased the amount of MMP-1.

  3. A phage display selected 7-mer peptide inhibitor of the Tannerella forsythia metalloprotease-like enzyme Karilysin can be truncated to Ser-Trp-Phe-Pro.

    PubMed

    Skottrup, Peter Durand; Sørensen, Grete; Ksiazek, Miroslaw; Potempa, Jan; Riise, Erik

    2012-01-01

    Tannerella forsythia is a gram-negative bacteria, which is strongly associated with the development of periodontal disease. Karilysin is a newly identified metalloprotease-like enzyme, that is secreted from T. forsythia. Karilysin modulates the host immune response and is therefore considered a likely drug target. In this study peptides were selected towards the catalytic domain from Karilysin (Kly18) by phage display. The peptides were linear with low micromolar binding affinities. The two best binders (peptide14 and peptide15), shared the consensus sequence XWFPXXXGGG. A peptide15 fusion with Maltose Binding protein (MBP) was produced with peptide15 fused to the N-terminus of MBP. The peptide15-MBP was expressed in E. coli and the purified fusion-protein was used to verify Kly18 specific binding. Chemically synthesised peptide15 (SWFPLRSGGG) could inhibit the enzymatic activity of both Kly18 and intact Karilysin (Kly48). Furthermore, peptide15 could slow down the autoprocessing of intact Kly48 to Kly18. The WFP motif was important for inhibition and a truncation study further demonstrated that the N-terminal serine was also essential for Kly18 inhibition. The SWFP peptide had a Ki value in the low micromolar range, which was similar to the intact peptide15. In conclusion SWFP is the first reported inhibitor of Karilysin and can be used as a valuable tool in structure-function studies of Karilysin.

  4. Ubiquitous information for ubiquitous computing: expressing clinical data sets with openEHR archetypes.

    PubMed

    Garde, Sebastian; Hovenga, Evelyn; Buck, Jasmin; Knaup, Petra

    2006-01-01

    Ubiquitous computing requires ubiquitous access to information and knowledge. With the release of openEHR Version 1.0 there is a common model available to solve some of the problems related to accessing information and knowledge by improving semantic interoperability between clinical systems. Considerable work has been undertaken by various bodies to standardise Clinical Data Sets. Notwithstanding their value, several problems remain unsolved with Clinical Data Sets without the use of a common model underpinning them. This paper outlines these problems like incompatible basic data types and overlapping and incompatible definitions of clinical content. A solution to this based on openEHR archetypes is motivated and an approach to transform existing Clinical Data Sets into archetypes is presented. To avoid significant overlaps and unnecessary effort during archetype development, archetype development needs to be coordinated nationwide and beyond and also across the various health professions in a formalized process.

  5. Human Immunodeficiency Virus Envelope Protein Gp120 Induces Proliferation but Not Apoptosis in Osteoblasts at Physiologic Concentrations

    PubMed Central

    Cummins, Nathan W.; Klicpera, Anna; Sainski, Amy M.; Bren, Gary D.; Khosla, Sundeep; Westendorf, Jennifer J.; Badley, Andrew D.

    2011-01-01

    Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism. PMID:21931863

  6. Family effects on the rurality of GP's work location: a longitudinal panel study.

    PubMed

    McGrail, Matthew R; Russell, Deborah J; O'Sullivan, Belinda G

    2017-10-19

    Reduced opportunities for children's schooling and spouse's/partner's employment are identified internationally as key barriers to general practitioners (GPs) working rurally. This paper aims to measure longitudinal associations between the rurality of GP work location and having (i) school-aged children and (ii) a spouse/partner in the workforce. Participants included 4377 GPs responding to at least two consecutive annual surveys of the Medicine in Australia: Balancing Employment and Life (MABEL) national longitudinal study between 2008 and 2014. The main outcome, GP work location, was categorised by remoteness and population size. Five sequential binary school-age groupings were defined according to whether a GP had no children, only preschool children (aged 0-4 years), at least one primary-school child (aged 5-11 years), at least one child in secondary school (aged 12-18 years), and all children older than secondary school (aged ≥ 19). Partner in the workforce was defined by whether a GP had a partner who was either currently working or looking for work, or not. Separate generalised estimating equation models, which aggregated consecutive annual observations per GP, tested associations between work location and (i) educational stages and (ii) partner employment, after adjusting for key covariates. Male GPs with children in secondary school were significantly less likely to work rurally (inclusive of > 50 000 regional centres through to the smallest rural towns of < 5000) compared to male GPs with children in primary school. In contrast, female GPs' locations were not significantly associated with the educational stage of their children. Having a partner in the workforce was not associated with work location for male GPs, whereas female GPs with a partner in the workforce were significantly less likely to work in smaller rural/remote communities (< 15 000 population). This is the first systematic, national-level longitudinal study showing that GP

  7. Influence of elastin-derived peptides on metalloprotease production in endothelial cells

    PubMed Central

    SIEMIANOWICZ, KRZYSZTOF; GMINSKI, JAN; GOSS, MALGORZATA; FRANCUZ, TOMASZ; LIKUS, WIRGINIA; JURCZAK, TERESA; GARCZORZ, WOJCIECH

    2010-01-01

    Matrix metalloproteases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins. MMP-1 and MMP-2 are produced by endothelial cells and are involved in specific vascular pathologies, including atherosclerosis and aortal aneurysm. One of the most important differences between these two metalloproteases is the possibility of hydrolysis of elastin and collagen type IV by MMP-2, but not by MMP-1. Elastin-derived peptides are generated as a result of the degradation of elastin fibers. The aim of our study was to compare the production of MMP-1 and MMP-2 in cultured human arterial endothelial cells derived from vascular pathologies localized at three different sites, the coronary artery, iliac artery and aorta, measured as their concentration in cell culture medium. The second aim was to evaluate the influence of κ-elastin (at concentrations 0.1, 0.4, 1.0, 2.5 or 5.0 μg/ml) on the production of the evaluated metalloproteases in three endothelial cell lines. The production of MMP-1 was statistically significantly greater in endothelial cells derived from the aorta compared to that in the endothelium obtained from the coronary and iliac arteries. There were no statistically significant differences in the production of MMP-2 among the endothelial cell lines tested. The addition of κ-elastin at all evaluated concentrations did not statistically significantly influence the concentration of MMP-1 in the cultured coronary artery endothelium. Furthermore, no statistically significant differences were observed in the cultured iliac artery endothelium. In the cultured endothelium derived from the aorta, κ-elastin at concentrations of 0.1 and 0.4 μg/ml significantly increased the amount of MMP-1. PMID:22993640

  8. ΔNp63α is an oncogene that induces Lsh expression and promotes stem-like proliferation

    PubMed Central

    Keyes, William M.; Pecoraro, Matteo; Aranda, Victoria; Vernersson-Lindahl, Emma; Li, Wangzhi; Vogel, Hannes; Guo, Xuecui; Garcia, Elvin L.; Michurina, Tatyana V.; Enikolopov, Grigori; Muthuswamy, Senthil K.; Mills, Alea A.

    2014-01-01

    SUMMARY The p53 homolog p63 is essential for development, yet its role in cancer is not clear. We discovered that p63 deficiency evokes the tumor suppressive mechanism of cellular senescence, causing a striking absence of stratified epithelia such as the skin. Here we identify the predominant p63 isoform, ΔNp63α, as a protein that bypasses oncogene induced senescence to drive tumorigenesis in vivo. Interestingly, bypass of senescence promotes stem-like proliferation and maintains survival of the keratin 15-positive stem cell population. Furthermore, we identify the chromatin remodeling protein Lsh as a new target of ΔNp63α that is an essential mediator of senescence bypass. These findings indicate that ΔNp63α is an oncogene that cooperates with Ras to promote tumor-initiating stem-like proliferation, and suggest that Lsh-mediated chromatin remodeling events are critical to this process. PMID:21295273

  9. Experimental anti-inflammatory drug Semapimod inhibits Toll-like receptor signaling by targeting the TLR chaperone gp961

    PubMed Central

    Wang, Jin; Grishin, Anatoly V.; Ford, Henri R.

    2016-01-01

    Semapimod, a tetravalent guanylhydrazone, suppresses inflammatory cytokine production and has potential in a variety of inflammatory and autoimmune disorders. The mechanism of action of Semapimod is not well understood. Here we demonstrate that in rat IEC-6 intestinal epithelioid cells, Semapimod inhibits activation of p38 MAPK, NF-kB and induction of COX-2 by TLR ligands, but not by IL-1β or stresses. Semapimod inhibits TLR4 signaling (IC50≈0.3 μM) and acts by desensitizing cells to LPS; it fails to block responses to LPS concentrations of 5 μg/ml or higher. Inhibition of TLR signaling by Semapimod is almost instantaneous: the drug is effective when applied simultaneously with LPS. Semapimod blocks cell surface recruitment of the MyD88 adapter, one of the earliest events in TLR signaling. gp96, the ER-localized chaperone of the HSP90 family critically involved in the biogenesis of TLRs, was identified as a target of Semapimod using ATP-desthiobiotin pull-down and mass spectroscopy. Semapimod inhibits ATP-binding and ATPase activities of gp96 in vitro (IC50≈0.2-0.4 μM). On prolonged exposure, Semapimod causes accumulation of TLR4 and TLR9 in perinuclear space, consistent with ER retention, an anticipated consequence of impaired gp96 chaperone function. Our data indicate that Semapimod desensitizes TLR signaling via its effect on the TLR chaperone gp96. Fast inhibition by Semapimod is consistent with gp96 participating in high affinity sensing of TLR ligands in addition to its role as a TLR chaperone. PMID:27194788

  10. Chlorination of lignin by ubiquitous fungi has a likely role in global organochlorine production

    PubMed Central

    Ortiz-Bermúdez, Patricia; Hirth, Kolby C.; Srebotnik, Ewald; Hammel, Kenneth E.

    2007-01-01

    Soils and decayed plant litter contain significant quantities of chlorinated aromatic polymers that have a natural but largely unknown origin. We used cupric oxide ligninolysis coupled with gas chromatography/mass spectrometry to show that Curvularia inaequalis, a widely distributed litter ascomycete, chlorinated the aromatic rings of lignin in wood that it was degrading. In aspen wood decayed for 24 weeks, two chlorolignin fragments, 5-chlorovanillin and 2-chlorosyringaldehyde, were each found at ≈10 μg/g of wood (dry weight). These levels resemble those of similar structures generally found in unpolluted environmental samples. Fractionation of the extractable proteins followed by tandem mass spectrometric analysis showed that the colonized wood contained a previously described C. inaequalis chloroperoxidase that very likely catalyzed lignin chlorination. Chlorolignin produced by this route and humus derived from it are probably significant components of the global chlorine cycle because chloroperoxidase-producing fungi are ubiquitous in decaying lignocellulose and lignin is the earth's most abundant aromatic substance. PMID:17360449

  11. HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine.

    PubMed

    Shen, Xiaoying; Basu, Rahul; Sawant, Sheetal; Beaumont, David; Kwa, Sue Fen; LaBranche, Celia; Seaton, Kelly E; Yates, Nicole L; Montefiori, David C; Ferrari, Guido; Wyatt, Linda S; Moss, Bernard; Alam, S Munir; Haynes, Barton F; Tomaras, Georgia D; Robinson, Harriet L

    2017-12-15

    An important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4 + T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4 + T cell responses. IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with

  12. Production, purification, and characterization of metalloprotease from Candida kefyr 41 PSB.

    PubMed

    Yavuz, Sevgi; Kocabay, Samet; Çetinkaya, Serap; Akkaya, Birnur; Akkaya, Recep; Yenidunya, Ali Fazil; Bakıcı, Mustafa Zahir

    2017-01-01

    A thermostable metalloprotease, produced from an environmental strain of Candida kefyr 41 PSB, was purified 16 fold with a 60% yield by cold ethanol precipitation and affinity chromatography (bentonite-acrylamide-cysteine microcomposite). The purified enzyme appeared as a single protein band at 43kDa. Its optimum pH and temperature points were found to be 7.0 and 105°C, respectively. K m and V max values of the enzyme were determined to be 3.5mg/mL and 4.4μmolmL -1 min -1 , 1.65mg/mL and 6.1μmolmL -1 min -1 , using casein and gelatine as the substrates, respectively. The activity was inhibited by using ethylenediamine tetraacetic acid (EDTA), indicating that the enzyme was a metalloprotease. Stability of the enzyme was investigated by using thermodynamic and kinetic parameters. The thermal inactivation profile of the enzyme conformed to the first order kinetics. The half life of the enzyme at 95, 105, 115, 125 and 135°C was 1310, 610, 220, 150, and 86min, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Identification and activity of inhibitors of the essential nematode-specific metalloprotease DPY-31.

    PubMed

    France, David J; Stepek, Gillian; Houston, Douglas R; Williams, Lewis; McCormack, Gillian; Walkinshaw, Malcolm D; Page, Antony P

    2015-12-15

    Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several μM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect 'Dumpy' (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  14. GP trainers and trainees - trait and gender differences in personality: implications for GP training.

    PubMed

    Joffe, Megan; MacLeod, Sheona; Kedziora, Marta; Main, Paul

    2016-05-01

    This study looked at differences between established GP trainers and current GP trainees in relation to personality traits. Personality differences are particularly important for training in the UK context where the attributes of successful GPs may be evolving as the context changes, and where there is a unique one-to-one relationship between trainer and trainee. GP trainers and trainees attending educational events were invited to participate in this study by completing the NEO-PI-R, a personality measure. Correlation and multiple regression analysis demonstrated differences between these groups; some in line with expected differences relating to age and gender. Others, such as lower reported levels of emotional resilience, may be particular to this trainee population. Overall the gender differences are significant given the trend towards the feminisation of the medical profession. Generational differences may also explain some behaviour and attitudinal differences which can aid trainers' understanding of training issues. The findings have important implications for training, particularly in relation to the development of emotional resilience for GP trainees, and for recruitment. Further research correlating educational outcomes and perceived satisfaction with a GP career and GP training would indicate if trainer/trainee personality differences have a direct bearing on educational outcomes and future practice.

  15. Rapid autocatalytic activation of the M4 metalloprotease aureolysin is controlled by a conserved N-terminal fungalysin-thermolysin-propeptide domain.

    PubMed

    Nickerson, Nicholas N; Joag, Vineet; McGavin, Martin J

    2008-09-01

    The Staphylococcus aureus proteolytic cascade consists of a metalloprotease aureolysin (Aur), which activates a serine protease zymogen proSspA, which in turn activates the SspB cysteine protease. As with other M4 metalloproteases, including elastase of Pseudomonas aeruginosa, the propeptide of proAur contains an N-terminal fungalysin-thermolysin-propeptide (FTP) domain. Autocatalytic activation of proAur was initiated by processing at T85 downward arrowL(86) in the FTP domain. This differed from the mechanism described for proElastase, where the FTP domain has an RY motif in place of TL(86), and processing occurred at the junction of the propeptide and metalloprotease domains, which remained as an inactive complex during passage across the outer membrane. When TL(86) in the FTP domain was replaced with RY, an intact N-terminal propeptide was secreted, but the M4 metalloprotease domain was degraded. Consequently, this segment of the FTP domain promotes intramolecular processing of proAur while bestowing a chaperone function, but discourages processing within the FTP domain of proElastase, where activation must be co-ordinated with passage across a second membrane. We conclude that the FTP domain of proAur is adapted to facilitate a rapid autocatalytic activation mechanism, consistent with the role or proAur as initiator of the staphylococcal proteolytic cascade.

  16. RNA Editing of the GP Gene of Ebola Virus is an Important Pathogenicity Factor.

    PubMed

    Volchkova, Valentina A; Dolnik, Olga; Martinez, Mikel J; Reynard, Olivier; Volchkov, Viktor E

    2015-10-01

    Synthesis of the surface glycoprotein GP of Ebola virus (EBOV) is dependent on transcriptional RNA editing, whereas direct expression of the GP gene results in synthesis of nonstructural secreted glycoprotein sGP. In this study, we investigate the role of RNA editing in the pathogenicity of EBOV using a guinea pig model and recombinant guinea pig-adapted EBOV containing mutations at the editing site, allowing expression of surface GP without the need for RNA editing, and also preventing synthesis of sGP. We demonstrate that the elimination of the editing site leads to EBOV attenuation in vivo, explained by lower virus spread caused by the higher virus cytotoxicity and, most likely, by an increased ability of the host defense systems to recognize and eliminate virus-infected cells. We also demonstrate that expression of sGP does not affect pathogenicity of EBOV in guinea pigs. In conclusion, data obtained indicate that downregulation of the level of surface GP expression through a mechanism of GP gene RNA editing plays an important role in the high pathogenicity of EBOV. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer.

    PubMed

    He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L; Mann, Colin J; Augst, Ryan; Morris, Charles D; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B; Burton, Dennis R; Zhu, Jiang

    2017-01-01

    Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.

  18. Hidden Lineage Complexity of Glycan-Dependent HIV-1 Broadly Neutralizing Antibodies Uncovered by Digital Panning and Native-Like gp140 Trimer

    PubMed Central

    He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L.; Mann, Colin J.; Augst, Ryan; Morris, Charles D.; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B.; Burton, Dennis R.; Zhu, Jiang

    2017-01-01

    Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development. PMID:28883821

  19. Involvement of caspases and transmembrane metalloproteases in sulphur mustard-induced microvesication in adult human skin in organ culture: directions for therapy.

    PubMed

    Mol, Marijke A E; van den Berg, Roland M; Benschop, Henk P

    2009-04-05

    While skin is a major target for sulphur mustard (HD), a therapy to limit HD-induced vesication is currently not available. Since it is supposed that apoptotic cell death and proteolytic digestion of extracellular matrix proteins by metalloproteases are initiating factors for blister formation, we have explored whether inhibition of these processes could prevent HD-induced epidermal-dermal separation using adult human skin in organ culture. Involvement of the caspase and the metalloprotease families was confirmed by the observation that their respective broad spectrum inhibitors, Z-VAD-fmk and GM6001, each suppressed HD-induced microvesication. The lowest effective concentrations were 10 and 100microM, respectively. Using specific inhibitors for caspase-8 (> or =10microM) and caspase-9 (> or =10microM) we learned that HD-induced apoptosis is initiated by the death receptor pathway as well as by the mitochondrial pathway. Remarkably, blocking caspase-8 activity resulted in morphologically better conserved cells than blocking caspase-9 activity. We zoomed in on the role of metalloproteases in HD-induced microvesication by testing the effects of two inhibitors: dec-RVKR-cmk and TAPI-2. Dec-RVKR-cmk is an inhibitor of furin, which activates transmembrane enzymes of the 'a disintegrin and metalloproteinase' (ADAM)-family as well as the membrane-type metalloproteases (MTx-MMP). TAPI-2 specifically inhibits TNFalpha-converting enzyme (TACE/ADAM17), which is involved in pericellular proteolysis. Both inhibitors prevented microvesication at concentrations of > or =500 and > or =20microM, respectively. This confirms that ADAMs and MT-MMPs play a role in HD-induced epidermal-dermal separation, with a particular role for TACE/ADAM17. Since TACE is involved not only in degradation of cell-matrix adhesion structures, but also in ectodomain shedding of ligands for epidermal growth factor receptor (EGFR) and in release of TNFalpha, these results imply TACE-mediated pathways as a

  20. Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

    PubMed Central

    Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

  1. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility

    PubMed Central

    Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E.; Schief, William R.; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D.

    2009-01-01

    The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded β-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate—and structurally plastic—layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated β-sandwich and providing for conformational diversity used in immune evasion. A “layered” gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a β-sandwich clamp maintains gp120–gp41 interaction and regulates gp41 transitions. PMID:20080564

  2. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility.

    PubMed

    Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Kwon, Young Do; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E; Schief, William R; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D

    2010-01-19

    The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded beta-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated beta-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a beta-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.

  3. Cultural competency training of GP Registrars-exploring the views of GP Supervisors.

    PubMed

    Watt, Kelly; Abbott, Penny; Reath, Jenny

    2015-10-06

    An equitable multicultural society requires General Practitioners (GPs) to be proficient in providing health care to patients from diverse backgrounds. This requires a certain set of attitudes, knowledge and skills known as cultural competence. While training in cultural competence is an important part of the Australian GP Registrar training curriculum, it is unclear who provides this training apart from in Aboriginal and Torres Strait Islander training posts. The majority of Australian GP Registrar training takes place in a workplace setting facilitated by the GP Supervisor. In view of the central role of GP Supervisors, their views on culturally competent practice, and their role in its development in Registrars, are important to ascertain. We conducted 14 semi-structured interviews with GP Supervisors. These were audiotaped, transcribed verbatim and thematically analyzed using an iterative approach. The Supervisors interviewed frequently viewed cultural competence as adequately covered by using patient-centered approaches. The Supervisor role in promoting cultural competence of Registrars was affirmed, though training was noted to occur opportunistically and focused largely on patient-centered care rather than health disparities. Formal training for both Registrars and Supervisors may be beneficial not only to develop a deeper understanding of cultural competence and its relevance to practice but also to promote more consistency in training from Supervisors in the area, particularly with respect to self-reflection, non-conscious bias and utilizing appropriate cultural knowledge without stereotyping and assumption-making.

  4. Ubiquitous human computing.

    PubMed

    Zittrain, Jonathan

    2008-10-28

    Ubiquitous computing means network connectivity everywhere, linking devices and systems as small as a drawing pin and as large as a worldwide product distribution chain. What could happen when people are so readily networked? This paper explores issues arising from two possible emerging models of ubiquitous human computing: fungible networked brainpower and collective personal vital sign monitoring.

  5. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

    DTIC Science & Technology

    2008-12-23

    glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain... peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the...terminal signal peptide (SP), which directs the precursor to the endoplasmic retic- ulum (ER) for further processing [11]. The SP, which has been

  6. GP management of dementia--a consumer perspective.

    PubMed

    Millard, Fiona

    2008-01-01

    As the population ages, the general practitioner is likely to have an increasingly important role in diagnosing and managing dementia. Many people in the community dealing with dementia have little help until a diagnosis is made, and their experiences produce a profile of their met and unmet needs. A nondirected interview technique elicits a wide range of themes from individual life phenomena, with each person attributing their own meaning to events and experiences. Open ended discussion can produce unexpected information that is outside the experience of the researcher but relevant to the people being studied, reflecting the unique nature of each person's experience with dementia. Patients expect their GP to know about dementia and look to their GP for help with the disease, but these expectations may not always be matched by the doctor's knowledge about dementia or their perceived role in dementia care.

  7. Determinants of the choice of GP practice registration in England: evidence from a discrete choice experiment.

    PubMed

    Lagarde, Mylene; Erens, Bob; Mays, Nicholas

    2015-04-01

    There have been growing concerns that general practitioner (GP) services in England, which are based on registration with a single practice located near the patient's home, are not sufficiently convenient for patients. To inform the decision as to whether to change registration rules allowing patients to register 'out-of-area' and to estimate the demand for this wider choice, we undertook a discrete choice experiment with 1706 respondents. Latent class models were used to analyse preferences for GP practice registration comparing preferences for neighbourhood and non-neighbourhood practices. We find that there is some appetite for registering outside the neighbourhood, but this preference is not uniformly shared across the population. Specifically individuals who are less mobile (e.g. older people and those with caring responsibilities), or satisfied with their local practice are less likely to be interested in registering at a practice outside their neighbourhood. Overall, people feel most strongly about obtaining an appointment with a GP as quickly as possible. Respondents regarded weekend opening as less important than other factors, and particularly less important than extended practice opening hours from Monday to Friday. Assuming a constant demand for GP services, a policy encouraging GP practices to extend their opening hours during the week is likely to decrease the average patient waiting time for an appointment and is likely to be preferred by patients. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Comprehensive functional analysis of N-linked glycans on Ebola virus GP1.

    PubMed

    Lennemann, Nicholas J; Rhein, Bethany A; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

    2014-01-28

    Ebola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca(2+)-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. Filovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus

  9. IL-1β-induced matrix metalloproteinase-13 is activated by a disintegrin and metalloprotease-28-regulated proliferation of human osteoblast-like cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ozeki, Nobuaki; Kawai, Rie; Yamaguchi, Hideyuki

    2014-04-15

    We reported previously that matrix metalloproteinase (MMP)-13 accelerates bone remodeling in oral periradicular lesions, and indicated a potentially unique role for MMP-13 in wound healing and regeneration of alveolar bone. The ADAM (a disintegrin and metalloprotease) family is a set of multifunctional cell surface and secreted glycoproteins, of which ADAM-28 has been localized in bone and bone-like tissues. In this study, we show that interleukin (IL)-1β induces the expression of MMP-13 and ADAM-28 in homogeneous α7 integrin-positive human skeletal muscle stem cell (α7{sup +}hSMSC)-derived osteoblast-like (α7{sup +}hSMSC-OB) cells, and promotes proliferation while inhibiting apoptosis in these cells. At higher concentrations,more » however, IL-1β failed to induce the expression of these genes and caused an increase in apoptosis. We further employed ADAM-28 small interfering RNA (siRNA) to investigate whether IL-1β-induced MMP-13 expression is linked to this IL-1β-mediated changes in cell proliferation and apoptosis. Silencing ADAM-28 expression potently suppressed IL-1β-induced MMP-13 expression and activity, decreased cell proliferation and increased apoptosis in α7{sup +}hSMSC-OB cells. In contrast, MMP-13 siRNA had no effect on ADAM-28 expression, suggesting ADAM-28 regulates MMP-13. Exogenous MMP-13 induced α7{sup +}hSMSC-OB cell proliferation and could rescue ADAM-28 siRNA-induced apoptosis, and we found that proMMP-13 is partially cleaved into its active form by ADAM-28 in vitro. Overall, our results suggest that IL-1β-induced MMP-13 expression and changes in cell proliferation and apoptosis in α7{sup +}hSMSC-OB cells are regulated by ADAM-28. - Highlights: • IL-1β induces the MMP-13 and ADAM-28 expression in human osteoblast-like cells. • IL-1β-induced MMP-13 expression increases proliferation and decreased apoptosis. • MMP-13 expression induced by IL-1β is regulated by ADAM-28. • proMMP-13 appears to be cleaved into its active form

  10. Metalloprotease Peptide Inhibitors: A Semester-Long Organic Synthetic Research Project for the Introductory Laboratory Course

    ERIC Educational Resources Information Center

    Pontrello, Jason K.

    2015-01-01

    A semester-long research project to synthesize unique compounds designed after published metalloprotease peptide inhibitors is presented. The research project encompasses a set of nine organic chemistry reactions traditionally taught in the second semester lab course, and the procedures are derived from scientific literature. The two principle…

  11. Ubiquitous Versus One-to-One

    ERIC Educational Resources Information Center

    McAnear, Anita

    2006-01-01

    When we planned the editorial calendar with the topic ubiquitous computing, we were thinking of ubiquitous computing as the one-to-one ratio of computers to students and teachers and 24/7 access to electronic resources. At the time, we were aware that ubiquitous computing in the computer science field had more to do with wearable computers. Our…

  12. Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cingolani, Gino, E-mail: cingolag@upstate.edu; Andrews, Dewan; Casjens, Sherwood

    2006-05-01

    The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26more » forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.« less

  13. ADAM metalloproteases promote a developmental switch in responsiveness to the axonal repellant Sema3A.

    PubMed

    Romi, Erez; Gokhman, Irena; Wong, Eitan; Antonovsky, Niv; Ludwig, Andreas; Sagi, Irit; Saftig, Paul; Tessier-Lavigne, Marc; Yaron, Avraham

    2014-06-05

    During embryonic development, axons can gain and lose sensitivity to guidance cues, and this flexibility is essential for the correct wiring of the nervous system. Yet, the underlying molecular mechanisms are largely unknown. Here we show that receptor cleavage by ADAM (A Disintegrin And Metalloprotease) metalloproteases promotes murine sensory axons loss of responsiveness to the chemorepellant Sema3A. Genetic ablation of ADAM10 and ADAM17 disrupts the developmental downregulation of Neuropilin-1 (Nrp1), the receptor for Sema3A, in sensory axons. Moreover, this is correlated with gain of repulsive response to Sema3A. Overexpression of Nrp1 in neurons reverses axonal desensitization to Sema3A, but this is hampered in a mutant Nrp1 with high susceptibility to cleavage. Lastly, we detect guidance errors of proprioceptive axons in ADAM knockouts that are consistent with enhanced response to Sema3A. Our results provide the first evidence for involvement of ADAMs in regulating developmental switch in responsiveness to axonal guidance cues.

  14. A fibrinolytic, alkaline and thermostable metalloprotease from the newly isolated Serratia sp RSPB11.

    PubMed

    Lakshmi Bhargavi, P; Prakasham, R S

    2013-10-01

    This study shows the purification and characterization of metalloprotease (serralysin) with fibrin and fibrinogenolytic property, from the newly isolated Serratia marcescens RSPB11. This protein macro molecule was more stable over a wide range of pH (6-10) and the temperatures up to 60 °C. It showed optimum enzyme activity at pH 9.0 and at a temperature of 37 °C. Inhibitory analysis revealed that this enzyme is metalloprotease and its enzyme activity could be regained by the addition of Co(2+), Cu(2+), Fe(2+), Mg(2+)and Zn(2+) ions after chelation of ions with EDTA. This enzyme showed the Michaelis-Menten's constant Km (1.261 mg/ml) for its substrate, casein and the observed maximum attainable velocity was Vmax (24,842 U/min). The purified enzyme showed an apparent molecular mass of approximately 50 kDa in SDS-PAGE. The results also suggested that this serralysin is having potential application thrombolytic therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells.

    PubMed

    Fan, J Z; Yang, X; Bi, Z G

    2015-07-01

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation.

  16. The effects of 6-gingerol on proliferation, differentiation, and maturation of osteoblast-like MG-63 cells

    PubMed Central

    Fan, J.Z.; Yang, X.; Bi, Z.G.

    2015-01-01

    We investigated whether 6-gingerol affects the maturation and proliferation of osteoblast-like MG63 cells in vitro. Osteoblast-like MG63 cells were treated with 6-gingerol under control conditions, and experimental inflammation was induced by tumor necrosis factor-α (TNF-α). Expression of different osteogenic markers and cytokines was analyzed by real-time PCR, Western blotting, and enzyme-linked immunosorbent assay. In addition, alkaline phosphatase (ALP) enzyme activity and biomineralization as markers for differentiation were measured. Treatment with 6-gingerol resulted in insignificant effects on the proliferation rate. 6-Gingerol induced the differentiation of osteoblast-like cells with increased transcription levels of osteogenic markers, upregulated ALP enzyme activity, and enhanced mineralized nodule formation. Stimulation with TNF-α led to enhanced interleukin-6 and nuclear factor-κB expression and downregulated markers of osteoblastic differentiation. 6-Gingerol reduced the degree of inflammation in TNF-α-treated MG-63 cells. In conclusion, 6-gingerol stimulated osteoblast differentiation in normal physiological and inflammatory settings, and therefore, 6-gingerol represents a promising agent for treating osteoporosis or bone inflammation. PMID:25923459

  17. Template-directed synthesis on the pentanucleotide CpCpGpCpC

    NASA Technical Reports Server (NTRS)

    Inoue, T.; Joyce, G. F.; Grzeskowiak, K.; Orgel, L. E.; Brown, J. M.; Reese, C. B.

    1984-01-01

    Experiments in which CpCpGpCpC is used as a template to facilitate the co-oligomerization of 2-MeImpG and 2-MeImpC are described. It is shown that 3' to 5' prime-linked pGpGpCpGpG, whose sequence is complementary to that of the template, is substantially the most adundant pentameric product of the template-directed reaction. The yield of pGpGpCpGpG is never large (less than 20 percent), presumably becauase off-template reactions consume template-directed products. Thus pGpGpCpGpG is converted to the various isomers of G5C and G4C2 by off-template terminal addition of G or C. The 3' to 5' isomer of GpG is elongated on the template to give GpGpC, GpGpCpG, and GpGpCpGpG, while the 2' to 5' isomer does not initiate the synthesis of detectable amounts of longer oligomers.

  18. A profile of PMS salaried GP contracts and their impact on recruitment.

    PubMed

    Williams, J; Petchey, R; Gosden, T; Leese, B; Sibbald, B

    2001-06-01

    Personal medical services (PMS) pilot sites aim to use salaried GP schemes to improve GP recruitment and retention and enhance the quality of service provision, particularly in underserved areas. Our objectives were to (i) compare the work incentives of salaried compared with standard GP contracts; (ii) assess recruitment success to salaried posts; and (iii) describe the types of GPs attracted to these new posts. All first wave PMS pilot sites with salaried GP posts known to be 'live' in October 1998 were included in the analysis of employment contracts and job descriptions. Information on recruitment was obtained by a questionnaire survey of PMS sites that were intending to recruit a salaried GP. The mean full-time equivalent salary was 43,674 pounds sterling with additional benefits in terms of sick leave, maternity leave and paid expenses. Eighty-nine percent of posts were eligible for the NHS pension scheme. Posts were mainly full time (40.8 hours per week). GPs were responsible for providing services equivalent in scope to general medical services. One-fifth of contracts freed GPs from out-of-hours responsibility and most freed them from practice management. Forty-three of the pilot sites actively recruited to fill 63 salaried posts, which involved a total of 51 recruitment 'rounds', with some pilots advertising more than once. There were 291 applications. The median number of applicants per post was three and the median time to recruitment was 6 weeks. Eighty-five percent of sites were satisfied with the quality of their applicants and 64% with the quantity. Eighty-five percent of applicants previously had been working in general practice, most in locum or salaried posts. Applicants tended to be young and male. Sixty posts were filled. Salaried contracts offer positive incentives to recruitment in terms of reduced hours of work and freedom from administrative responsibility. Recruitment success was similar to that achieved by inner city practices generally

  19. Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

    PubMed Central

    Fatehi Hassanabad, Mostafa; Chang, Tom; Pirani, Nawaz; Bona, Diane; Edwards, Aled M.

    2013-01-01

    A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis. PMID:24097944

  20. A rapid and sensitive fluorometric method for the quantitative analysis of snake venom metalloproteases and their inhibitors.

    PubMed

    Biardi, J E; Nguyen, K T; Lander, S; Whitley, M; Nambiar, K P

    2011-02-01

    Metalloproteases are responsible for the hemorrhagic effects of many snake venoms and contribute to other pathways that lead to local tissue damage. Methods that quantify snake venom metalloproteases (SVMP) are therefore valuable tools in research on the clinical, physiological, and biochemical effects of envenomation. Comparative analysis of individual, population, and species differences requires screening of large numbers of samples and treatments, and therefore require a method of quantifying SVMP activity that is simple, rapid, and sensitive. This paper demonstrates the properties of a new fluorometric assay of SVMP activity that can provide a measure of metalloprotease activity in 1 h. The assay is reliable, with variation among replicates sufficiently small to reliably detect differences in between species (F(19,60) = 2924, p < 0.001), even for those venoms with low overall activity. It is also sensitive enough to detect differences among venoms using <2 ng of whole venom protein. We provide an example use of this assay to detect the presence of natural SVMP inhibitors in minute samples of blood plasma from rock squirrels (S. variegatus), a natural prey species for North American rattlesnakes. We propose this assay is a useful addition to the set of tools used to characterize venoms, as well as high-throughput screening of natural or synthetic inhibitors, or other novel therapeutic agents against SVMP effects. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs.

    PubMed

    Jiang, Xiaohong; Dalebout, Tim J; Bredenbeek, Peter J; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S; Lukashevich, Igor S

    2011-02-01

    Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. Copyright © 2010 Elsevier Ltd. All rights reserved.

  2. Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

    PubMed Central

    Jiang, Xiaohong; Dalebout, Tim J.; Bredenbeek, Peter J.; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S.; Lukashevich, Igor S.

    2010-01-01

    Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV GP1 and GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and –GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF and LASV GP proteins in infected cells. YF17D/LASV-GP1&GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1&GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

  3. Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

    PubMed Central

    Sloma, A; Rudolph, C F; Rufo, G A; Sullivan, B J; Theriault, K A; Ally, D; Pero, J

    1990-01-01

    The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation. Images FIG. 2 FIG. 7 PMID:2105291

  4. Learning with Ubiquitous Computing

    ERIC Educational Resources Information Center

    Rosenheck, Louisa

    2008-01-01

    If ubiquitous computing becomes a reality and is widely adopted, it will inevitably have an impact on education. This article reviews the background of ubiquitous computing and current research projects done involving educational "ubicomp." Finally it explores how ubicomp may and may not change education in both formal and informal settings and…

  5. gp-91 mediates histone deacetylase inhibition-induced cardioprotection

    PubMed Central

    Zhao, Ting C; Zhang, Ling X; Cheng, Guangmao; Liu, Jun T

    2010-01-01

    We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91−/− mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30 min of ischemia and 30 min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/L) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91+/+cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91phox-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91+/+cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect. PMID:20433879

  6. Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes.

    PubMed

    Lee, Sang Jin; Choi, Jin San; Park, Ki Suk; Khang, Gilson; Lee, Young Moo; Lee, Hai Bang

    2004-08-01

    Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (P<0.05) between different micropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.

  7. Enhanced Immune Responses to HIV-1 Envelope Elicited by a Vaccine Regimen Consisting of Priming with Newcastle Disease Virus Expressing HIV gp160 and Boosting with gp120 and SOSIP gp140 Proteins.

    PubMed

    Khattar, Sunil K; DeVico, Anthony L; LaBranche, Celia C; Panda, Aruna; Montefiori, David C; Samal, Siba K

    2016-02-01

    Newcastle disease virus (NDV) expressing HIV-1 BaL gp160 was evaluated either alone or with monomeric BaL gp120 and BaL SOSIP gp140 protein in a prime-boost combination in guinea pigs to enhance envelope (Env)-specific humoral and mucosal immune responses. We showed that a regimen consisting of an NDV prime followed by a protein boost elicited stronger serum and mucosal Th-1-biased IgG responses and neutralizing antibody responses than NDV-only immunizations. Additionally, these responses were higher after the gp120 than after the SOSIP gp140 protein boost. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Comparative Glycoprofiling of HIV gp120 Immunogens by Capillary Electrophoresis and MALDI Mass Spectrometry

    PubMed Central

    Guttman, Miklós; Váradi, Csaba; Lee, Kelly K.; Guttman, András

    2015-01-01

    The Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host's humoral immune response. To assess how the global glycosylation patterns vary between gp120 constructs, the glycan profiles of several gp120s were examined by capillary electrophoresis with laser induced fluorescence detection and MALDI-MS. The glycosylation profiles were found to be similar for chronic vs. transmitter/founder isolates and only varied moderately between gp120s from different clades. This study revealed that the addition of specific tags, such as the gD tag used in the RV144 trial, had significant effects on the overall glycosylation patterns. Such effects are likely to influence the immunogenicity of various Env immunogens and should be considered for future vaccine strategies, emphasizing the importance of the glycosylation analysis approach described in this paper. PMID:25809283

  9. A Collaborative Model for Ubiquitous Learning Environments

    ERIC Educational Resources Information Center

    Barbosa, Jorge; Barbosa, Debora; Rabello, Solon

    2016-01-01

    Use of mobile devices and widespread adoption of wireless networks have enabled the emergence of Ubiquitous Computing. Application of this technology to improving education strategies gave rise to Ubiquitous e-Learning, also known as Ubiquitous Learning. There are several approaches to organizing ubiquitous learning environments, but most of them…

  10. Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs

    PubMed Central

    Lopes, Anne; Amarir-Bouhram, Jihane; Faure, Guilhem; Petit, Marie-Agnès; Guerois, Raphaël

    2010-01-01

    Homologous recombination is a key in contributing to bacteriophages genome repair, circularization and replication. No less than six kinds of recombinase genes have been reported so far in bacteriophage genomes, two (UvsX and Gp2.5) from virulent, and four (Sak, Redβ, Erf and Sak4) from temperate phages. Using profile–profile comparisons, structure-based modelling and gene-context analyses, we provide new views on the global landscape of recombinases in 465 bacteriophages. We show that Sak, Redβ and Erf belong to a common large superfamily adopting a shortcut Rad52-like fold. Remote homologs of Sak4 are predicted to adopt a shortcut Rad51/RecA fold and are discovered widespread among phage genomes. Unexpectedly, within temperate phages, gene-context analyses also pinpointed the presence of distant Gp2.5 homologs, believed to be restricted to virulent phages. All in all, three major superfamilies of phage recombinases emerged either related to Rad52-like, Rad51-like or Gp2.5-like proteins. For two newly detected recombinases belonging to the Sak4 and Gp2.5 families, we provide experimental evidence of their recombination activity in vivo. Temperate versus virulent lifestyle together with the importance of genome mosaicism is discussed in the light of these novel recombinases. Screening for these recombinases in genomes can be performed at http://biodev.extra.cea.fr/virfam. PMID:20194117

  11. A Disintegrin and Metalloprotease 17 in the Cardiovascular and Central Nervous Systems.

    PubMed

    Xu, Jiaxi; Mukerjee, Snigdha; Silva-Alves, Cristiane R A; Carvalho-Galvão, Alynne; Cruz, Josiane C; Balarini, Camille M; Braga, Valdir A; Lazartigues, Eric; França-Silva, Maria S

    2016-01-01

    ADAM17 is a metalloprotease and disintegrin that lodges in the plasmatic membrane of several cell types and is able to cleave a wide variety of cell surface proteins. It is somatically expressed in mammalian organisms and its proteolytic action influences several physiological and pathological processes. This review focuses on the structure of ADAM17, its signaling in the cardiovascular system and its participation in certain disorders involving the heart, blood vessels, and neural regulation of autonomic and cardiovascular modulation.

  12. Chemical synthesis of 5'-pyrophosphate and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. CD study of the effect of 5'-phosphate groups on the conformation of 3'-5' GpG.

    PubMed Central

    Tomasz, J; Simoncsits, A; Kajtár, M; Krug, R M; Shatkin, A J

    1978-01-01

    A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups. PMID:211490

  13. Abnormalities in the zinc-metalloprotease-BDNF axis may contribute to megalencephaly and cortical hyperconnectivity in young autism spectrum disorder patients.

    PubMed

    Koh, Jae-Young; Lim, Joon Seo; Byun, Hyae-Ran; Yoo, Min-Heui

    2014-09-03

    Whereas aberrant brain connectivity is likely the core pathology of autism-spectrum disorder (ASD), studies do not agree as to whether hypo- or hyper-connectivity is the main underlying problem. Recent functional imaging studies have shown that, in most young ASD patients, cerebral cortical regions appear hyperconnected, and cortical thickness/brain size is increased. Collectively, these findings indicate that developing ASD brains may exist in an altered neurotrophic milieu. Consistently, some ASD patients, as well as some animal models of ASD, show increased levels of brain-derived neurotrophic factor (BDNF). However, how BDNF is upregulated in ASD is unknown. To address this question, we propose the novel hypothesis that a putative zinc-metalloprotease-BDNF (ZMB) axis in the forebrain plays a pivotal role in the development of hyperconnectivity and megalencephaly in ASD. We have previously demonstrated that extracellular zinc at micromolar concentrations can rapidly increase BDNF levels and phosphorylate the receptor tyrosine kinase TrkB via the activation of metalloproteases. The role of metalloproteases in ASD is still uncertain, but in fragile X syndrome, a monogenic disease with an autistic phenotype, the levels of MMP are increased. Early exposure to lipopolysaccharides (LPS) and other MMP activators such as organic mercurials also have been implicated in ASD pathogenesis. The resultant increases in BDNF levels at synapses, especially those involved in the zinc-containing, associative glutamatergic system may produce abnormal brain circuit development. Various genetic mutations that lead to ASD are also known to affect BDNF signaling: some down-regulate, and others up-regulate it. We hypothesize that, although both up- and down-regulation of BDNF may induce autism symptoms, only BDNF up-regulation is associated with the hyperconnectivity and large brain size observed in most young idiopathic ASD patients. To test this hypothesis, we propose to examine the

  14. Abnormalities in the zinc-metalloprotease-BDNF axis may contribute to megalencephaly and cortical hyperconnectivity in young autism spectrum disorder patients

    PubMed Central

    2014-01-01

    Whereas aberrant brain connectivity is likely the core pathology of autism-spectrum disorder (ASD), studies do not agree as to whether hypo- or hyper-connectivity is the main underlying problem. Recent functional imaging studies have shown that, in most young ASD patients, cerebral cortical regions appear hyperconnected, and cortical thickness/brain size is increased. Collectively, these findings indicate that developing ASD brains may exist in an altered neurotrophic milieu. Consistently, some ASD patients, as well as some animal models of ASD, show increased levels of brain-derived neurotrophic factor (BDNF). However, how BDNF is upregulated in ASD is unknown. To address this question, we propose the novel hypothesis that a putative zinc-metalloprotease-BDNF (ZMB) axis in the forebrain plays a pivotal role in the development of hyperconnectivity and megalencephaly in ASD. We have previously demonstrated that extracellular zinc at micromolar concentrations can rapidly increase BDNF levels and phosphorylate the receptor tyrosine kinase TrkB via the activation of metalloproteases. The role of metalloproteases in ASD is still uncertain, but in fragile X syndrome, a monogenic disease with an autistic phenotype, the levels of MMP are increased. Early exposure to lipopolysaccharides (LPS) and other MMP activators such as organic mercurials also have been implicated in ASD pathogenesis. The resultant increases in BDNF levels at synapses, especially those involved in the zinc-containing, associative glutamatergic system may produce abnormal brain circuit development. Various genetic mutations that lead to ASD are also known to affect BDNF signaling: some down-regulate, and others up-regulate it. We hypothesize that, although both up- and down-regulation of BDNF may induce autism symptoms, only BDNF up-regulation is associated with the hyperconnectivity and large brain size observed in most young idiopathic ASD patients. To test this hypothesis, we propose to examine the

  15. Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

    PubMed Central

    Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

    2011-01-01

    Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

  16. A Disintegrin and Metalloproteases (ADAMs) in Cardiovascular, Metabolic and Inflammatory Diseases: Aspects for Theranostic Approaches.

    PubMed

    van der Vorst, Emiel P C; Weber, Christian; Donners, Marjo M P C

    2018-06-06

    A disintegrin and metalloproteases (ADAMs) are membrane-bound enzymes responsible for the shedding or cleavage of various cell surface molecules, such as adhesion molecules, cytokines/chemokines and growth factors. This shedding can result in the release of soluble proteins that can exert agonistic or antagonistic functions. Additionally, ADAM-mediated cleavage can render these membrane proteins inactive. This review will describe the role and association of ADAMs in various pathologies with a main focus on ADAM10 and ADAM17 in atherosclerosis, including a brief overview of atherosclerosis-related ADAM substrates. Furthermore, ADAMs involvement in other metabolic and inflammatory diseases like diabetes, sepsis, Alzheimer's disease and rheumatoid arthritis will be highlighted. Subsequently, we will briefly discuss an interesting emerging field of research, i.e. the potential implications of ADAM expression in extracellular vesicles. Finally, several ADAM-based therapeutic and diagnostic (theranostic) opportunities will be discussed, while focusing on key questions about the required specificity and selectivity. Schattauer GmbH Stuttgart.

  17. Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

    DTIC Science & Technology

    2008-12-23

    glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain...consistent with viral TM fusion proteins [9,10]. GPC con- tains a 58 residue hydrophobic N-terminal signal peptide (SP), which directs the precursor to the...including GPC, GP1, and GP2. Various signal peptides , purification tags, and modifications to internal domains were employed for the generation and

  18. A Dynamic Ubiquitous Learning Resource Model with Context and Its Effects on Ubiquitous Learning

    ERIC Educational Resources Information Center

    Chen, Min; Yu, Sheng Quan; Chiang, Feng Kuang

    2017-01-01

    Most ubiquitous learning researchers use resource recommendation and retrieving based on context to provide contextualized learning resources, but it is the kind of one-way context matching. Learners always obtain fixed digital learning resources, which present all learning contents in any context. This study proposed a dynamic ubiquitous learning…

  19. CGP lil-gp 2.1;1.02 User's Manual

    NASA Technical Reports Server (NTRS)

    Janikow, Cezary Z.; DeWeese, Scott W.

    1997-01-01

    This document describes extensions provided to lil-gp facilitating dealing with constraints. This document deals specifically with lil-gp 1.02, and the resulting extension is referred to as CGP lil-gp 2.1; 1.02 (the first version is for the extension, the second for the utilized lil-gp version). Unless explicitly needed to avoid confusion, version numbers are omitted.

  20. TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

    NASA Astrophysics Data System (ADS)

    Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

    2014-04-01

    Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53-/- mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53-/- mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

  1. TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

    PubMed Central

    Munne, Pauliina M.; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G.

    2014-01-01

    Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53−/− mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53−/− mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers. PMID:24722541

  2. TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli.

    PubMed

    Munne, Pauliina M; Gu, Yuexi; Tumiati, Manuela; Gao, Ping; Koopal, Sonja; Uusivirta, Sanna; Sawicki, Janet; Wei, Gong-Hong; Kuznetsov, Sergey G

    2014-04-11

    Multiple observations suggest a cell type-specific role for TP53 in mammary epithelia. We developed an in vitro assay, in which primary mouse mammary epithelial cells (mMECs) progressed from lumenal to basal-like phenotypes based on expression of Krt18 or ΔNp63, respectively. Such transition was markedly delayed in Trp53(-/-) mMECs suggesting that Trp53 is required for specification of the basal, but not lumenal cells. Evidence from human basal-like cell lines suggests that TP53 may support the activity of ΔNp63 by preventing its translocation from nucleoplasm into nucleoli. In human lumenal cells, activation of TP53 by inhibiting MDM2 or BRCA1 restored the nucleoplasmic expression of ΔNp63. Trp53(-/-) mMECs eventually lost epithelial features resulting in upregulation of MDM2 and translocation of ΔNp63 into nucleoli. We propose that TP63 may contribute to TP53-mediated oncogenic transformation of epithelial cells and shed light on tissue- and cell type-specific biases observed for TP53-related cancers.

  3. Meprin metalloproteases inactivate interleukin 6.

    PubMed

    Keiffer, Timothy R; Bond, Judith S

    2014-03-14

    Meprins have been implicated in the pathogenesis of several inflammatory diseases, including inflammatory bowel disease, in which the cytokine IL-6 is a prominent effector molecule. Because IL-6 levels are elevated markedly in meprin α and α/β knockout mice in an experimental model of inflammatory bowel disease, the interaction between meprins and IL-6 was studied. The results demonstrate that rodent and human meprin A and B cleave IL-6 to a smaller product and, subsequently, are capable of extensive degradation of the cytokine. Analysis of the limited degradation product formed by meprin A indicated that three to five amino acids are removed from the C terminus of the cytokine. Meprin A and meprin B cleaved IL-6 with micromolar affinities (Km of 4.7 and 12.0 μM, respectively) and with high efficiencies (kcat/Km of 0.2 and 2.5 (M(-1)/s(-1)) × 10(6), respectively). These efficiency constants are among the highest for known meprin substrates. Madin-Darby canine kidney cells transiently transfected with meprin α or meprin β constructs also cleave exogenous IL-6. Both human and murine IL-6 cleaved by meprin A or B are inactivated, as demonstrated by their decreased capability to stimulate proliferation of B9 cells. These results are consistent with the proposition that one function of meprin metalloproteases is to modulate inflammation by inactivating IL-6.

  4. Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

    PubMed Central

    Ren, Shoufeng; Wei, Qimei; Cai, Liya; Yang, Xuejing; Xing, Cuicui; Tan, Feng; Leavenworth, Jianmei W.; Liang, Shaohui; Liu, Wenquan

    2018-01-01

    Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention. PMID:29375526

  5. Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice

    PubMed Central

    Yao, Longbiao; Yago, Tadayuki; Shao, Bojing; Liu, Zhenghui; Silasi-Mansat, Robert; Setiadi, Hendra; Lupu, Florea

    2013-01-01

    Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin–dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti–P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation. PMID:24081661

  6. Secretome analysis to elucidate metalloprotease-dependent ectodomain shedding of glycoproteins during neuronal differentiation.

    PubMed

    Tsumagari, Kazuya; Shirakabe, Kyoko; Ogura, Mayu; Sato, Fuminori; Ishihama, Yasushi; Sehara-Fujisawa, Atsuko

    2017-02-01

    Many membrane proteins are subjected to limited proteolyses at their juxtamembrane regions, processes referred to as ectodomain shedding. Shedding ectodomains of membrane-bound ligands results in activation of downstream signaling pathways, whereas shedding those of cell adhesion molecules causes loss of cell-cell contacts. Secreted proteomics (secretomics) using high-resolution mass spectrometry would be strong tools for both comprehensive identification and quantitative measurement of membrane proteins that undergo ectodomain shedding. In this study, to elucidate the ectodomain shedding events that occur during neuronal differentiation, we establish a strategy for quantitative secretomics of glycoproteins released from differentiating neuroblastoma cells into culture medium with or without GM6001, a broad-spectrum metalloprotease inhibitor. Considering that most of transmembrane and secreted proteins are N-glycosylated, we include a process of N-glycosylated peptides enrichment as well as isotope tagging in our secretomics workflow. Our results show that differentiating N1E-115 neurons secrete numerous glycosylated polypeptides in metalloprotease-dependent manners. They are derived from cell adhesion molecules such as NCAM1, CADM1, L1CAM, various transporters and receptor proteins. These results show the landscape of ectodomain shedding and other secretory events in differentiating neurons and/or during axon elongation, which should help elucidate the mechanism of neurogenesis and the pathogenesis of neurological disorders. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  7. Kunjin Virus Replicon-Based Vaccines Expressing Ebola Virus Glycoprotein GP Protect the Guinea Pig Against Lethal Ebola Virus Infection

    PubMed Central

    Reynard, O.; Mokhonov, V.; Mokhonova, E.; Leung, J.; Page, A.; Mateo, M.; Pyankova, O.; Georges-Courbot, M. C.; Raoul, H.; Khromykh, A. A.

    2011-01-01

    Pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. We evaluated, in a guinea pig model, the immunogenic potential of Kunjin virus (KUN)–derived replicons as a vaccine candidate against Ebola virus (EBOV). Virus like particles (VLPs) containing KUN replicons expressing EBOV wild-type glycoprotein GP, membrane anchor-truncated GP (GP/Ctr), and mutated GP (D637L) with enhanced shedding capacity were generated and assayed for their protective efficacy. Immunization with KUN VLPs expressing full-length wild-type and D637L-mutated GPs but not membrane anchor–truncated GP induced dose-dependent protection against a challenge of a lethal dose of recombinant guinea pig-adapted EBOV. The surviving animals showed complete clearance of the virus. Our results demonstrate the potential for KUN replicon vectors as vaccine candidates against EBOV infection. PMID:21987742

  8. Characteristics and career intentions of Scottish rural and urban GP registrars: cause for concern?

    PubMed

    Ross, S; Gillies, J C

    1999-01-01

    To investigate the differences between the characteristics and career intentions of GP registrars in urban and rural areas, and to make recommendations to reduce a potential work force crisis in rural practice. Postal survey. All general practices in Scotland. In February 1996, 40/196 (20%) of urban and 45/150 (30%) of rural GP registrar places available in Scotland, were vacant (chi 2 = 4.22, df = 1, p = 0.02). Postal questionnaires were sent to all 261 GP registrars in post. Of 235 respondents (90%), the majority wished to remain in general practice (63% of urban and 53% of rural registrars), but only 22% of urban and 18% of rural registrars intended to apply for principal posts immediately after training. Fewer urban (8%) than rural registrars (21%) stated an intention to go abroad to work after training. Rural registrars tended to want to work in rural areas, and vice versa. Part-time and job-sharing were attractive employment options for both groups, and more flexible career structures were favoured by over 80%. Though much more attention has been paid to recruitment in inner cities, the findings from this study suggest that in Scotland difficulties in finding principals may occur first in rural areas. As general practitioners have an extended role in rural areas, including that of emergency care, shortages could have a serious impact on patient care.

  9. Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus

    PubMed Central

    2014-01-01

    Background The European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) has recently emerged in China. The coexistence of Genotype-I and -II PRRSV strains could cause seriously affect PRRSV diagnosis and management. Current vaccines are not able to protect against PRRSV infection completely and have inherent drawbacks. Thus, genetically engineered vaccines, including DNA vaccine and live vector engineered vaccines, have been developed. This study aimed to determine the enhanced immune responses of mice inoculated with a DNA vaccine coexpressing GP3 and GP5 of a Genotype-I PRRSV. Results To evaluate the immunogenicity of GP3 and GP5 proteins from European-type PRRSV, three DNA vaccines, pVAX1-EU-ORF3-ORF5, pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed, which were based on a Genotype-I LV strain (GenBank ID: M96262). BALB/c mice were immunized with the DNA vaccines; delivered in the form of chitosan-DNA nanoparticles. To increase the efficiency of the vaccine, Quil A (Quillaja) was used as an adjuvant. GP3 and GP5-specific antibodies, neutralizing antibodies and cytokines (IL-2, IL-4, IL-10 and IFN gamma) from the immunized mice sera, and other immune parameters, were examined, including T-cell proliferation responses and subgroups of spleen T-lymphocytes. The results showed that ORF3 and ORF5 proteins of Genotype-I PRRSV induced GP3 and GP5-specific antibodies that could neutralize the virus. The levels of Cytokines IL-2, IL-4, IL-10, and IFN–γ of the experimental groups were significantly higher than those of control groups after booster vaccination (P < 0.05). The production of CD3+CD4+ and CD3+CD8+ T lymphocyte was also induced. T lymphocyte proliferation assays showed that the PRRSV LV strain virus could stimulate the proliferation of T lymphocytes in mice in the experimental group. Conclusions Using Quil A as adjuvant, Genotype-I PRRSV GP3 and GP5 proteins produced good immunogenicity and reactivity. More

  10. GP Workbench Manual: Technical Manual, User's Guide, and Software Guide

    USGS Publications Warehouse

    Oden, Charles P.; Moulton, Craig W.

    2006-01-01

    GP Workbench is an open-source general-purpose geophysical data processing software package written primarily for ground penetrating radar (GPR) data. It also includes support for several USGS prototype electromagnetic instruments such as the VETEM and ALLTEM. The two main programs in the package are GP Workbench and GP Wave Utilities. GP Workbench has routines for filtering, gridding, and migrating GPR data; as well as an inversion routine for characterizing UXO (unexploded ordinance) using ALLTEM data. GP Workbench provides two-dimensional (section view) and three-dimensional (plan view or time slice view) processing for GPR data. GP Workbench can produce high-quality graphics for reports when Surfer 8 or higher (Golden Software) is installed. GP Wave Utilities provides a wide range of processing algorithms for single waveforms, such as filtering, correlation, deconvolution, and calculating GPR waveforms. GP Wave Utilities is used primarily for calibrating radar systems and processing individual traces. Both programs also contain research features related to the calibration of GPR systems and calculating subsurface waveforms. The software is written to run on the Windows operating systems. GP Workbench can import GPR data file formats used by major commercial instrument manufacturers including Sensors and Software, GSSI, and Mala. The GP Workbench native file format is SU (Seismic Unix), and subsequently, files generated by GP Workbench can be read by Seismic Unix as well as many other data processing packages.

  11. The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo.

    PubMed

    Li, Ming; de Graaf, Inge A M; van de Steeg, Evita; de Jager, Marina H; Groothuis, Geny M M

    2017-04-01

    Intestinal P-gp and CYP3A4 work coordinately to reduce the intracellular concentration of drugs, and drug-drug interactions (DDIs) based on this interplay are of clinical importance and require pre-clinical investigation. Using precision-cut intestinal slices (PCIS) of human jejunum, ileum and colon, we investigated the P-gp/CYP3A4 interplay and related DDIs with P-gp inhibitors at the different regions of the human intestine with quinidine (Qi), dual substrate of P-gp and CYP3A4, as probe. All the P-gp inhibitors increased the intracellular concentrations of Qi by 2.1-2.6 fold in jejunum, 2.6-3.8 fold in ileum but only 1.2-1.3 fold in colon, in line with the different P-gp expression in these intestinal regions. The selective P-gp inhibitors (CP100356 and PSC833) enhanced 3-hydroxy-quinidine (3OH-Qi) in jejunum and ileum, while dual inhibitors of P-gp and CYP3A4 (verapamil and ketoconazole) decreased the 3OH-Qi production, despite of the increased intracellular Qi concentration, due to inhibition of CYP3A4. The outcome of DDIs based on P-gp/CYP3A4 interplay, shown as remarkable changes in the intracellular concentration of both the parent drug and the metabolite, varied among the intestinal regions, probably due to the different expression of P-gp and CYP3A4, and were different from those found in rat PCIS, which may have important implications for the disposition and toxicity of drugs and their metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Cuscuta chinensis extract promotes osteoblast differentiation and mineralization in human osteoblast-like MG-63 cells.

    PubMed

    Yang, Hyun Mo; Shin, Hyun-Kyung; Kang, Young-Hee; Kim, Jin-Kyung

    2009-02-01

    The aim of the present study was to investigate whether the aqueous extract of To-Sa-Za (TSZ-AE), the seed of Cuscuta chinensis Lam., which is a traditional medicinal herb commonly used in Korea and other oriental countries, could induce osteogenic activity in human osteoblast-like MG-63 cells. TSZ-AE treatment mildly promoted the proliferation of MG-63 cells at doses of 500 and 1,000 microg/mL in the 24-hour culture period. Dose-dependent increases in alkaline phosphatase (ALP) activity and collagen synthesis were shown at 48 and 72 hours of incubation. The release of bone morphogenetic protein (BMP)-2 but not osteocalcin in the MG-63 cells was induced by TSZ-AE at 72 hours (100-1,000 microg/mL). In addition, TSZ-AE markedly increased mRNA expression of ALP, collagen, and BMP-2 in the MG-63 cells in a dose-dependent manner. Mineralization in the culture of MG-63 cells was significantly induced at 500 and 1,000 microg/mL TSZ-AE treatment. In conclusion, this study shows that TSZ-AE enhanced ALP activity, collagen synthesis, BMP-2 expression, and mineralization in MG-63 cells. These results strongly suggest that C. chinensis can play an important role in osteoblastic bone formation and may possibly lead to the development of bone-forming drugs.

  13. T198. A SCHIZOPHRENIA-LIKE BIRTH SEASONALITY AMONG MATHEMATICIANS AND AN OPPOSITE SEASONALITY AMONG BIOLOGISTS: MORE EVIDENCE IMPLICATING BIMODAL RHYTHMS OF GENERAL BIRTHS

    PubMed Central

    Marzullo, Giovanni

    2018-01-01

    Abstract Background Based on early-20th century births, a pre-electric illumination time of comparatively normal human exposure to sunlight, studies of schizophrenia (SCZ) found a birth seasonality with two opposite effects: a SCZ-liability peak among subjects born around late-February and an equally significant SCZ-resistance peak among those born six months later, around late-August. We previously investigated this rhythm in connection with a sunlight-dependent bimodal rhythm of general births that, prior to the full advent of electric lighting (but not later), occurred ubiquitously in non-equatorial parts of the world. We found that the SCZ-liability peak coincided with a first, Feb-Mar peak of general-population births (the GP1) while the SCZ-resistance peak coincided with a second, Aug-Sep peak of those births (the GP2). Moreover, in a study of hand and visual-field preferences among professional baseball players, we found the SCZ-liability, GP1-coincident seasonality among players with preferences denoting cerebral asymmetry “deficits” (CADs) and the SCZ-resistance, GP2-coincident seasonality among those with preferences denoting cerebral asymmetry “excesses.” Also, in a study suggested by associations of CADs with artistic abilities, we found the SCZ-liability, GP1-coincident seasonality among groups representing visual, performing and literary art “creators” (VPL-Artists) and the SCZ-resistance, GP2-coincident seasonality among groups representing art critics, historians, curators and other art “observers” (Para-Artists). Together, these findings suggested, as one possibility (but see later), that the SCZ-liability, CAD effects and artistic abilities could all three represent traits genetically or otherwise selected into the GP1 excess population of newborns and out of the GP2 population. The present study of “scientists” was initially aimed at the purported arts/science antithesis. Methods Birth seasonalities were examined among early

  14. Novel Apigenin Based Small Molecule that Targets Snake Venom Metalloproteases

    PubMed Central

    Anusha, Sebastian; Hemshekhar, Mahadevappa; Chandra Nayaka, Siddaiah; Kemparaju, Kempaiah; Basappa; Girish, Kesturu S.; Rangappa, Kanchugarakoppal S.

    2014-01-01

    The classical antivenom therapy has appreciably reduced snakebite mortality rate and thus is the only savior drug available. Unfortunately, it considerably fails to shield the viper bite complications like hemorrhage, local tissue degradation and necrosis responsible for severe morbidity. Moreover, the therapy is also tagged with limitations including anaphylaxis, serum sickness and poor availability. Over the last decade, snake venom metalloproteases (SVMPs) are reported to be the primary component responsible for hemorrhage and tissue degradation at bitten site. Thus, antivenom inability to offset viper venom-induced local toxicity has been a basis for an insistent search for SVMP inhibitors. Here we report the inhibitory effect of compound 5d, an apigenin based molecule against SVMPs both in silico and in vivo. Several apigenin analogues are synthesized using multicomponent Ugi reactions. Among them, compound 5d effectively abrogated Echis carinatus (EC) venom-induced local hemorrhage, tissue necrosis and myotoxicity in a dose dependant fashion. The histopathological study further conferred effective inhibition of basement membrane degradation, and accumulation of inflammatory leucocytes at the site of EC venom inoculation. The compound also protected EC venom-induced fibrin and fibrinogen degradation. The molecular docking of compound 5d and bothropasin demonstrated the direct interaction of hydroxyl group of compound with Glu146 present in hydrophobic pocket of active site and does not chelate Zn2+. Hence, it is concluded that compound 5d could be a potent agent in viper bite management. PMID:25184206

  15. Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates.

    PubMed

    Ozgür, Burak; Saaby, Lasse; Langthaler, Kristine; Brodin, Birger

    2018-01-15

    Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm 2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14 C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [ 3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. K m and V max of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124μM and 111±16pmol·cm -2 ·min -1 (n=3, total N=6), respectively. V max and K m of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC 50 ) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01μM (n=3, total N=6) and 0.04±0.01μM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC 50 values of inhibition, K i values were only possible to obtain using

  16. Ubiquitous computing in the military environment

    NASA Astrophysics Data System (ADS)

    Scholtz, Jean

    2001-08-01

    Increasingly people work and live on the move. To support this mobile lifestyle, especially as our work becomes more intensely information-based, companies are producing various portable and embedded information devices. The late Mark Weiser coined the term, 'ubiquitous computing' to describe an environment where computers have disappeared and are integrated into physical objects. Much industry research today is concerned with ubiquitous computing in the work and home environments. A ubiquitous computing environment would facilitate mobility by allowing information users to easily access and use information anytime, anywhere. As war fighters are inherently mobile, the question is what effect a ubiquitous computing environment would have on current military operations and doctrine. And, if ubiquitous computing is viewed as beneficial for the military, what research would be necessary to achieve a military ubiquitous computing environment? What is a vision for the use of mobile information access in a battle space? Are there different requirements for civilian and military users of this technology? What are those differences? Are there opportunities for research that will support both worlds? What type of research has been supported by the military and what areas need to be investigated? Although we don't yet have all the answers to these questions, this paper discusses the issues and presents the work we are doing to address these issues.

  17. gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing.

    PubMed

    Merkle, R K; Helland, D E; Welles, J L; Shilatifard, A; Haseltine, W A; Cummings, R D

    1991-10-01

    The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.

  18. Models for the Binary Complex of Bacteriophage T4 Gp59 Helicase Loading Protein. GP32 Single-Stranded DNA-Binding Protein and Ternary Complex with Pseudo-Y Junction DNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hinerman, Jennifer M.; Dignam, J. David; Mueser, Timothy C.

    2012-04-05

    The bacteriophage T4 gp59 helicase assembly protein (gp59) is required for loading of gp41 replicative helicase onto DNA protected by gp32 single-stranded DNA-binding protein. The gp59 protein recognizes branched DNA structures found at replication and recombination sites. Binding of gp32 protein (full-length and deletion constructs) to gp59 protein measured by isothermal titration calorimetry demonstrates that the gp32 protein C-terminal A-domain is essential for protein-protein interaction in the absence of DNA. Sedimentation velocity experiments with gp59 protein and gp32ΔB protein (an N-terminal B-domain deletion) show that these proteins are monomers but form a 1:1 complex with a dissociation constant comparable withmore » that determined by isothermal titration calorimetry. Small angle x-ray scattering (SAXS) studies indicate that the gp59 protein is a prolate monomer, consistent with the crystal structure and hydrodynamic properties determined from sedimentation velocity experiments. SAXS experiments also demonstrate that gp32ΔB protein is a prolate monomer with an elongated A-domain protruding from the core. Moreover, fitting structures of gp59 protein and the gp32 core into the SAXS-derived molecular envelope supports a model for the gp59 protein-gp32ΔB protein complex. Our earlier work demonstrated that gp59 protein attracts full-length gp32 protein to pseudo-Y junctions. A model of the gp59 protein-DNA complex, modified to accommodate new SAXS data for the binary complex together with mutational analysis of gp59 protein, is presented in the accompanying article (Dolezal, D., Jones, C. E., Lai, X., Brister, J. R., Mueser, T. C., Nossal, N. G., and Hinton, D. M. (2012) J. Biol. Chem. 287, 18596–18607).« less

  19. Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel

    A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stainmore » electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly

  20. Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

    PubMed Central

    Elliott, Debra H.; Rouelle, Julie; Smira, Ashley; Ndabambi, Nonkululeko; Druz, Aliaksandr; Williamson, Carolyn

    2017-01-01

    A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing

  1. The Future of Ubiquitous Elearning

    ERIC Educational Resources Information Center

    Arndt, Timothy

    2014-01-01

    Post-secondary students are increasingly receiving instruction by eLearning. Many or these are part-time students or are working while taking classes. In such circumstances, students may find themselves short of time to study. One mechanism that can be exploited to make the best use of available time is ubiquitous eLearning. Ubiquitous eLearning…

  2. The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1+ lymph node macrophages

    PubMed Central

    Park, Chung; Arthos, James; Cicala, Claudia; Kehrl, John H

    2015-01-01

    The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1+ sinus macrophages located in interfollicular pockets and underlying SIGN-R1+ macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells. DOI: http://dx.doi.org/10.7554/eLife.06467.001 PMID:26258881

  3. Ubiquitous Accessibility for People with Visual Impairments: Are We There Yet?

    PubMed Central

    Billah, Syed Masum; Ashok, Vikas; Porter, Donald E.; Ramakrishnan, IV

    2017-01-01

    Ubiquitous access is an increasingly common vision of computing, wherein users can interact with any computing device or service from anywhere, at any time. In the era of personal computing, users with visual impairments required special-purpose, assistive technologies, such as screen readers, to interact with computers. This paper investigates whether technologies like screen readers have kept pace with, or have created a barrier to, the trend toward ubiquitous access, with a specific focus on desktop computing as this is still the primary way computers are used in education and employment. Towards that, the paper presents a user study with 21 visually-impaired participants, specifically involving the switching of screen readers within and across different computing platforms, and the use of screen readers in remote access scenarios. Among the findings, the study shows that, even for remote desktop access—an early forerunner of true ubiquitous access—screen readers are too limited, if not unusable. The study also identifies several accessibility needs, such as uniformity of navigational experience across devices, and recommends potential solutions. In summary, assistive technologies have not made the jump into the era of ubiquitous access, and multiple, inconsistent screen readers create new practical problems for users with visual impairments. PMID:28782061

  4. Ubiquitous Accessibility for People with Visual Impairments: Are We There Yet?

    PubMed

    Billah, Syed Masum; Ashok, Vikas; Porter, Donald E; Ramakrishnan, I V

    2017-05-01

    Ubiquitous access is an increasingly common vision of computing, wherein users can interact with any computing device or service from anywhere, at any time. In the era of personal computing, users with visual impairments required special-purpose, assistive technologies, such as screen readers, to interact with computers. This paper investigates whether technologies like screen readers have kept pace with, or have created a barrier to, the trend toward ubiquitous access, with a specific focus on desktop computing as this is still the primary way computers are used in education and employment. Towards that, the paper presents a user study with 21 visually-impaired participants, specifically involving the switching of screen readers within and across different computing platforms, and the use of screen readers in remote access scenarios. Among the findings, the study shows that, even for remote desktop access-an early forerunner of true ubiquitous access-screen readers are too limited, if not unusable. The study also identifies several accessibility needs, such as uniformity of navigational experience across devices, and recommends potential solutions. In summary, assistive technologies have not made the jump into the era of ubiquitous access, and multiple, inconsistent screen readers create new practical problems for users with visual impairments.

  5. Internalization and Axonal Transport of the HIV Glycoprotein gp120

    PubMed Central

    Berth, Sarah; Caicedo, Hector Hugo; Sarma, Tulika; Morfini, Gerardo

    2015-01-01

    The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity. PMID:25636314

  6. Immunological activation following transcutaneous delivery of HR-gp100 protein

    PubMed Central

    Frankenburg, Shoshana; Grinberg, Igor; Bazak, Ziva; Fingerut, Lena; Pitcovski, Jacob; Gorodetsky, Raphael; Peretz, Tamar; Spira, Ram M.; Skornik, Yehuda; Goldstein, Ronald S.

    2009-01-01

    Transcutaneous immunization aims at taking advantage of the skin’s immune system for the purpose of immunoprotection. In the present study we evaluated the potential of topical delivery of a recombinant melanoma protein, HR-gp100, derived from a shorter sequence of the native gp100 gene. The protein was applied on the skin, with and without the addition of two forms of heat labile enterotoxin (nLT and LTB). HR-gp100 fused to Haptide, a cell penetrating 20mer peptide (HR-gp100H) was also tested. Topical HR-gp100 and HR-gp100H application on the ears of mice elicited the production of specific antibodies, and transcutaneous delivery to intact human skin induced dose-dependent LC activation. nLT and LTB also activated LC, but did not further increase the activation induced by HR-gp100. These results show that HR-gp100, an antigenic tumor-derived protein, activates the immune system following transcutaneous delivery, as shown by both Langerhans cell activation and induction of antibody production. PMID:17493711

  7. Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Ming; Huang, Cui; Qian, Duo-Duo

    2014-09-15

    To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST{sub 50} to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with themore » nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm. - Highlights: • gp16 knockout and repair mutants of AcMNPV were constructed and characterized. • AcMNPV gp16 is not essential to virus replication. • Deletion of gp16 resulted in time lengthening to kill S. exigua larvae. • GP16 was localized close around the nuclear membrane of infected cells. • GP16 was fractionated in the light membrane fraction in subcellular fractionation.« less

  8. User Fees in General Practice: Willingness to Pay and Potential Substitution Patterns-Results from a Danish GP Patient Survey.

    PubMed

    Kronborg, Christian; Pedersen, Line Bjørnskov; Fournaise, Anders; Kronborg, Christel Nøhr

    2017-10-01

    Increases in public expenditures to general practitioner (GP) services and specialist care have spurred debate over whether to implement user fees for healthcare services such as GP consultations in Denmark. The objective of this study was to examine Danish patients' attitudes towards user fees and their willingness to pay (WTP) for a consultation, and to investigate how user charges may impact patients' behaviour. A questionnaire survey was conducted in a GP clinic. A total of 343 individual persons answered the questionnaire. One hundred and seventy (50%) persons were not willing to pay for a consultation. Among patients reporting positive WTP values, the mean WTP was 137 (standard deviation 140) Danish kroner (DKK). Patients who were 65 years old or older were more likely to be willing to pay for a GP consultation than patients under the age of 65 years. Furthermore, patients with a personal annual income of more than 200,000 DKK were more likely to be willing to pay for a consultation than other income groups. With respect to patients with a positive WTP value, their own assessment of the seriousness of the consultation and their self-assessed health influenced the amount they would be willing to pay. Finally, we observed a stated willingness to substitute GP consultations with alternatives that are free of charge. About half of the patients with an appointment for a GP consultation are willing to pay for the consultation. User charges may potentially influence the patients' behaviour. ClinicalTrials.gov NCT01784731.

  9. Characterization of the Serralysin-like gene of 'Ca. Liberibacter solanacearum' associated with Potato Zebra Chip disease

    USDA-ARS?s Scientific Manuscript database

    The non-culturable bacterium ‘Candidatus Liberibacter solanacearum’ (Lso) is the causative agent of zebra chip disease in potato. Computational analysis of the Lso genome revealed a serralysin-like gene based on conserved domains characteristic of genes encoding metalloprotease enzymes similar to se...

  10. The multiple roles of sGP in Ebola pathogenesis.

    PubMed

    de La Vega, Marc-Antoine; Wong, Gary; Kobinger, Gary P; Qiu, Xiangguo

    2015-02-01

    Ebola causes severe hemorrhagic fever in humans and nonhuman primates, and there are currently no approved therapeutic countermeasures. The virulence of Ebola virus (EBOV) may be partially attributed to the secreted glycoprotein (sGP), which is the main product transcribed from its GP gene. sGP is secreted from infected cells and can be readily detected in the serum of EBOV-infected hosts. This review summarizes the multiple roles that sGP may play during infection and highlights the implications for the future design of vaccines and treatments.

  11. The Multiple Roles of sGP in Ebola Pathogenesis

    PubMed Central

    de La Vega, Marc-Antoine; Wong, Gary; Kobinger, Gary P.

    2015-01-01

    Abstract Ebola causes severe hemorrhagic fever in humans and nonhuman primates, and there are currently no approved therapeutic countermeasures. The virulence of Ebola virus (EBOV) may be partially attributed to the secreted glycoprotein (sGP), which is the main product transcribed from its GP gene. sGP is secreted from infected cells and can be readily detected in the serum of EBOV-infected hosts. This review summarizes the multiple roles that sGP may play during infection and highlights the implications for the future design of vaccines and treatments. PMID:25354393

  12. Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis

    PubMed Central

    2012-01-01

    Background Haemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. Results Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome

  13. Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis.

    PubMed

    Zehr, Emilie S; Tabatabai, Louisa B; Bayles, Darrell O

    2012-07-23

    Haemophilus parasuis, the causative agent of Glässer's disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer's disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate. Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome's G + C content was

  14. HIV-1 gp120 Induces Expression of IL-6 through a Nuclear Factor-Kappa B-Dependent Mechanism: Suppression by gp120 Specific Small Interfering RNA

    PubMed Central

    Shah, Ankit; Verma, Ashish S.; Patel, Kalpeshkumar H.; Noel, Richard; Rivera-Amill, Vanessa; Silverstein, Peter S.; Chaudhary, Suman; Bhat, Hari K.; Stamatatos, Leonidas; Singh, Dhirendra P.; Buch, Shilpa; Kumar, Anil

    2011-01-01

    In addition to its role in virus entry, HIV-1 gp120 has also been implicated in HIV-associated neurocognitive disorders. However, the mechanism(s) responsible for gp120-mediated neuroinflammation remain undefined. In view of increased levels of IL-6 in HIV-positive individuals with neurological manifestations, we sought to address whether gp120 is involved in IL-6 over-expression in astrocytes. Transfection of a human astrocyte cell line with a plasmid encoding gp120 resulted in increased expression of IL-6 at the levels of mRNA and protein by 51.3±2.1 and 11.6±2.2 fold respectively; this effect of gp120 on IL-6 expression was also demonstrated using primary human fetal astrocytes. A similar effect on IL-6 expression was observed when primary astrocytes were treated with gp120 protein derived from different strains of X4 and R5 tropic HIV-1. The induction of IL-6 could be abrogated by use of gp120-specific siRNA. Furthermore, this study showed that the NF-κB pathway is involved in gp120-mediated IL-6 over-expression, as IKK-2 and IKKβ inhibitors inhibited IL-6 expression by 56.5% and 60.8%, respectively. These results were also confirmed through the use of NF-κB specific siRNA. We also showed that gp120 could increase the phosphorylation of IκBα. Furthermore, gp120 transfection in the SVGA cells increased translocation of NF-κB from cytoplasm to nucleus. These results demonstrate that HIV-1 gp120-mediated over-expression of IL-6 in astrocytes is one mechanism responsible for neuroinflammation in HIV-infected individuals and this is mediated by the NF-κB pathway. PMID:21712995

  15. Designing a Soluble Near Full-Length HIV-1 GP41 Trimer

    DTIC Science & Technology

    2012-11-26

    envelope; gp41 trimer; bacteriophage T4 display; prehairpin fusion intermediate. Background: The envelope glycoprotein gp41 is a key component of...protein into trimers and defined oligomers. These gp41 trimers were displayed on bacteriophage T4 capsid nanoparticles by attaching to the small...Construction of the Expression Vectors —All the gp41 constructs were generated by splicing-by- overlap extension PCR using wild-type HXB2 gp41 DNA

  16. A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

    PubMed

    Forster, Brian M; Bitar, Alan Pavinski; Marquis, Hélène

    2014-01-01

    Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

  17. Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

    PubMed

    Blattner, Claudia; Lee, Jeong Hyun; Sliepen, Kwinten; Derking, Ronald; Falkowska, Emilia; de la Peña, Alba Torrents; Cupo, Albert; Julien, Jean-Philippe; van Gils, Marit; Lee, Peter S; Peng, Wenjie; Paulson, James C; Poignard, Pascal; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Wilson, Ian A; Ward, Andrew B

    2014-05-15

    All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. The chaotrope-soluble glycoprotein GP1 is a constituent of the insoluble glycoprotein framework of the Chlamydomonas cell wall.

    PubMed

    Voigt, Jürgen; Frank, Ronald; Wöstemeyer, Johannes

    2009-02-01

    Chlamydomonas reinhardtii wild-type cells are surrounded by the insoluble cell wall component, a sac-like framework of cross-linked glycoproteins containing 22% hydroxyproline. The chaotrope-soluble cell wall glycoprotein GP1 is the only polypeptide with an even higher proportion of hydroxyproline (35%) occurring in vegetative C. reinhardtii cells. Mass spectrometric analyses of peptides released from the purified insoluble cell wall fraction by trypsin treatment and epitope analyses of polyclonal antibodies raised against different deglycosylation products of this particular wall fraction using 181 chemically synthesized GP1-derived pentadecapeptides revealed evidence that GP1 is indeed a constituent of the insoluble wall component.

  19. VanT, a Homologue of Vibrio harveyi LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in Vibrio anguillarum

    PubMed Central

    Croxatto, Antony; Chalker, Victoria J.; Lauritz, Johan; Jass, Jana; Hardman, Andrea; Williams, Paul; Cámara, Miguel; Milton, Debra L.

    2002-01-01

    Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production. PMID:11872713

  20. VanT, a homologue of Vibrio harveyi LuxR, regulates serine, metalloprotease, pigment, and biofilm production in Vibrio anguillarum.

    PubMed

    Croxatto, Antony; Chalker, Victoria J; Lauritz, Johan; Jass, Jana; Hardman, Andrea; Williams, Paul; Cámara, Miguel; Milton, Debra L

    2002-03-01

    Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum Delta vanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the Delta vanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an L-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum Delta vanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.

  1. Ubiquitin-Like Proteasome System Represents a Eukaryotic-Like Pathway for Targeted Proteolysis in Archaea

    DOE PAGES

    Fu, Xian; Liu, Rui; Sanchez, Iona; ...

    2016-05-17

    The molecular mechanisms of targeted proteolysis in archaea are poorly understood, yet they may have deep evolutionary roots shared with the ubiquitin-proteasome system of eukaryotic cells. Here, we demonstrate in archaea that TBP2, a TATA-binding protein (TBP) modified by ubiquitin-like isopeptide bonds, is phosphorylated and targeted for degradation by proteasomes. Rapid turnover of TBP2 required the functions of UbaA (the E1/MoeB/ThiF homolog of archaea), AAA ATPases (Cdc48/p97 and Rpt types), a type 2 JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) homolog (JAMM2), and 20S proteasomes. The ubiquitin-like protein modifier small archaeal modifier protein 2 (SAMP2) stimulated the degradation of TBP2, but SAMP2 itself wasmore » not degraded. Analysis of the TBP2 fractions that were not modified by ubiquitin-like linkages revealed that TBP2 had multiple N termini, including Met1-Ser2, Ser2, and Met1-Ser2(p) [where (p) represents phosphorylation]. The evidence suggested that the Met1-Ser2(p) form accumulated in cells that were unable to degrade TBP2. We propose a model in archaea in which the attachment of ubiquitin-like tags can target proteins for degradation by proteasomes and be controlled by N-terminal degrons. In support of a proteolytic mechanism that is energy dependent and recycles the ubiquitin-like protein tags, we find that a network of AAA ATPases and a JAMM/MPN+ metalloprotease are required, in addition to 20S proteasomes, for controlled intracellular proteolysis. IMPORTANCEThis study advances the fundamental knowledge of signal-guided proteolysis in archaea and sheds light on components that are related to the ubiquitin-proteasome system of eukaryotes. In archaea, the ubiquitin-like proteasome system is found to require function of an E1/MoeB/ThiF homolog, a type 2 JAMM/MPN+ metalloprotease, and a network of AAA ATPases for the targeted destruction of proteins. We provide evidence that the attachment of the ubiquitin-like protein is controlled by an N

  2. Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model: Inclusion of GP83 reduces gB antibody response but both vaccine approaches provide equivalent protection against pup mortality.

    PubMed

    Swanson, Elizabeth C; Gillis, Pete; Hernandez-Alvarado, Nelmary; Fernández-Alarcón, Claudia; Schmit, Megan; Zabeli, Jason C; Wussow, Felix; Diamond, Don J; Schleiss, Mark R

    2015-07-31

    Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p<0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p<0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Dashboard for Analyzing Ubiquitous Learning Log

    ERIC Educational Resources Information Center

    Lkhagvasuren, Erdenesaikhan; Matsuura, Kenji; Mouri, Kousuke; Ogata, Hiroaki

    2016-01-01

    Mobile and ubiquitous technologies have been applied to a wide range of learning fields such as science, social science, history and language learning. Many researchers have been investigating the development of ubiquitous learning environments; nevertheless, to date, there have not been enough research works related to the reflection, analysis…

  4. Purification of a toxic metalloprotease produced by the pathogenic Photobacterium damselae subsp. piscicida isolated from cobia (Rachycentron canadum).

    PubMed

    Liu, Ping-Chung; Chuang, Wen-Hsiao; Lee, Kuo-Kau

    2011-01-01

    The aim of the present study was to purify and characterize a toxic protease secreted by the pathogenic Photobacterium damselae subsp. piscicida strain CP1 originally isolated from diseased cobia (Rachycentron canadum). The toxin isolated by anion exchange chromatography, was a metalloprotease, inhibited by L-cysteine, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 1,10-phenanthroline, N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK), and showed maximal activity at pH 6.0-8.0 and an apparent molecular mass of about 34.3 kDa. The toxin was also completely inhibited by HgCl2, and partially by sodium dodecyl sulfate (SDS) and CuCl2. The extracellular products and the partially purified protease were lethal to cobia with LD50 values of 1.26 and 6.8 microg protein/g body weight, respectively. The addition of EDTA completely inhibited the lethal toxicity of the purified protease, indicating that this metalloprotease was a lethal toxin produced by the bacterium.

  5. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function

    PubMed Central

    Kuhn, Peer-Hendrik; Colombo, Alessio Vittorio; Schusser, Benjamin; Dreymueller, Daniela; Wetzel, Sebastian; Schepers, Ute; Herber, Julia; Ludwig, Andreas; Kremmer, Elisabeth; Montag, Dirk; Müller, Ulrike; Schweizer, Michaela; Saftig, Paul; Bräse, Stefan; Lichtenthaler, Stefan F

    2016-01-01

    Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain. DOI: http://dx.doi.org/10.7554/eLife.12748.001 PMID:26802628

  6. Design requirements for ubiquitous computing environments for healthcare professionals.

    PubMed

    Bång, Magnus; Larsson, Anders; Eriksson, Henrik

    2004-01-01

    Ubiquitous computing environments can support clinical administrative routines in new ways. The aim of such computing approaches is to enhance routine physical work, thus it is important to identify specific design requirements. We studied healthcare professionals in an emergency room and developed the computer-augmented environment NOSTOS to support teamwork in that setting. NOSTOS uses digital pens and paper-based media as the primary input interface for data capture and as a means of controlling the system. NOSTOS also includes a digital desk, walk-up displays, and sensor technology that allow the system to track documents and activities in the workplace. We propose a set of requirements and discuss the value of tangible user interfaces for healthcare personnel. Our results suggest that the key requirements are flexibility in terms of system usage and seamless integration between digital and physical components. We also discuss how ubiquitous computing approaches like NOSTOS can be beneficial in the medical workplace.

  7. Vascular Induction of a Disintegrin and Metalloprotease 17 by Angiotensin II Through Hypoxia Inducible Factor 1α

    PubMed Central

    Obama, Takashi; Takayanagi, Takehiko; Kobayashi, Tomonori; Bourne, Allison M.; Elliott, Katherine J.; Charbonneau, Martine; Dubois, Claire M.

    2015-01-01

    BACKGROUND A disintegrin and metalloprotease 17 (ADAM17) is a membrane-spanning metalloprotease overexpressed in various cardiovascular diseases such as hypertension and atherosclerosis. However, little is known regarding the regulation of ADAM17 expression in the cardiovascular system. Here, we test our hypothesis that angiotensin II induces ADAM17 expression in the vasculature. METHODS Cultured vascular smooth muscle cells were stimulated with 100nM angiotensin II. Mice were infused with 1 μg/kg/minute angiotensin II for 2 weeks. ADAM17 expression was evaluated by a promoter–reporter construct, quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS In vascular smooth muscle cells, angiotensin II increased ADAM17 protein expression, mRNA, and promoter activity. We determined that the angiotensin II response involves hypoxia inducible factor 1α and a hypoxia responsive element. In angiotensin II–infused mice, marked induction of ADAM17 and hypoxia inducible factor 1α was seen in vasculatures in heart and kidney, as well as in aortae, by immunohistochemistry. CONCLUSIONS Angiotensin II induces ADAM17 expression in the vasculatures through a hypoxia inducible factor 1α–dependent transcriptional upregulation, potentially contributing to end-organ damage in the cardiovascular system. PMID:24871629

  8. Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α)*

    PubMed Central

    Sun, Ruili; Zhang, Yu; Lv, Qingshan; Liu, Bei; Jin, Miao; Zhang, Weijia; He, Qing; Deng, Minjie; Liu, Xueting; Li, Guancheng; Li, Yuehui; Zhou, Guohua; Xie, Pingli; Xie, Xiumei; Hu, Jinyue; Duan, Zhaojun

    2011-01-01

    Toll-like receptor 3 (TLR3), a member of the pathogen recognition receptors, is widely expressed in various cells and has been shown to activate immune signaling pathways by recognizing viral double-stranded RNA. Recently, it was reported that the activation of TLR3 induced apoptosis in some cells, but the detailed molecular mechanism is not fully understood. In this study, we found that in endothelial cells polyinosinic-polycytidylic acid (poly(I-C)) induced dose- and time-dependent cell apoptosis, which was elicited by TLR3 activation, as TLR3 neutralization and down-regulation repressed the apoptosis. Poly(I-C) induced the activation of both caspases 8 and 9, indicating that TLR3 triggered the signaling of both the extrinsic and intrinsic apoptotic pathways. Poly(I-C) up-regulated tumor necrosis factor-related apoptosis-inducing ligand and its receptors, death receptors 4/5, resulting in initiating the extrinsic pathway. Furthermore, poly(I-C) down-regulated anti-apoptotic protein, B cell lymphoma 2 (Bcl-2), and up-regulated Noxa, a key Bcl-2 homology 3-only antagonist of Bcl-2, leading to the priming of the intrinsic pathway. A p53-related protein, the transactivating p63 isoform α (TAp63α), was induced by TLR3 activation and contributed to the activation of both the intrinsic and extrinsic apoptotic pathways. Both the cells deficient in p63 gene expression by RNA interference and cells that overexpressed the N-terminally truncated p63 isoform α (ΔNp63α), a dominant-negative variant of TAp63α, by gene transfection, survived TLR3 activation. Taken together, TAp63α is a crucial regulator downstream of TLR3 to induce cell death via death receptors and mitochondria. PMID:21367858

  9. Male reproductive fitness and queen polyandry are linked to variation in the supergene Gp-9 in the fire ant Solenopsis invicta.

    PubMed

    Lawson, Lucinda P; Vander Meer, Robert K; Shoemaker, Dewayne

    2012-08-22

    Supergenes are clusters of tightly linked loci maintained in specific allelic combinations to facilitate co-segregation of genes governing adaptive phenotypes. In species where strong selection potentially operates at different levels (e.g. eusocial Hymenoptera), positive selection acting within a population to maintain specific allelic combinations in supergenes may have unexpected consequences for some individuals, including the preservation of disadvantageous traits. The nuclear gene Gp-9 in the invasive fire ant Solenopsis invicta is part of a non-recombining, polymorphic supergene region associated with polymorphism in social organization as well as traits affecting physiology, fecundity and behaviour. We show that both male reproductive success and facultative polyandry in queens have a simple genetic basis and are dependent on male Gp-9 genotype. Gp-9(b) males are unable to maintain exclusive reproductive control over their mates such that queens mated to Gp-9(b) males remain highly receptive to remating. Queens mated to multiple Gp-9(B) males are rare. This difference appears to be independent of mating plug production in fertile males of each Gp-9 genotype. However, Gp-9(b) males have significantly lower sperm counts than Gp-9(B) males, which could be a cue to females to seek additional mates. Despite the reduced fitness of Gp-9(b) males, polygyne worker-induced selective mortality of sexuals lacking b-like alleles coupled with the overall success of the polygyne social form act to maintain the Gp-9(b) allele within nature. Our findings highlight how strong worker-induced selection acting to maintain the Gp-9(b) allele in the polygyne social form may simultaneously result in reduced reproductive fitness for individual sexual offspring.

  10. Male reproductive fitness and queen polyandry are linked to variation in the supergene Gp-9 in the fire ant Solenopsis invicta

    PubMed Central

    Lawson, Lucinda P.; Vander Meer, Robert K.; Shoemaker, DeWayne

    2012-01-01

    Supergenes are clusters of tightly linked loci maintained in specific allelic combinations to facilitate co-segregation of genes governing adaptive phenotypes. In species where strong selection potentially operates at different levels (e.g. eusocial Hymenoptera), positive selection acting within a population to maintain specific allelic combinations in supergenes may have unexpected consequences for some individuals, including the preservation of disadvantageous traits. The nuclear gene Gp-9 in the invasive fire ant Solenopsis invicta is part of a non-recombining, polymorphic supergene region associated with polymorphism in social organization as well as traits affecting physiology, fecundity and behaviour. We show that both male reproductive success and facultative polyandry in queens have a simple genetic basis and are dependent on male Gp-9 genotype. Gp-9b males are unable to maintain exclusive reproductive control over their mates such that queens mated to Gp-9b males remain highly receptive to remating. Queens mated to multiple Gp-9B males are rare. This difference appears to be independent of mating plug production in fertile males of each Gp-9 genotype. However, Gp-9b males have significantly lower sperm counts than Gp-9B males, which could be a cue to females to seek additional mates. Despite the reduced fitness of Gp-9b males, polygyne worker-induced selective mortality of sexuals lacking b-like alleles coupled with the overall success of the polygyne social form act to maintain the Gp-9b allele within nature. Our findings highlight how strong worker-induced selection acting to maintain the Gp-9b allele in the polygyne social form may simultaneously result in reduced reproductive fitness for individual sexual offspring. PMID:22535783

  11. 77 FR 73635 - Northwest Storage GP, LLC; Notice of Application

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-11

    ... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. CP13-18-000; PF12-2-000] Northwest Storage GP, LLC; Notice of Application Take notice that on November 21, 2012, Northwest Storage GP..., Northwest Storage GP, LLC., 295 Chipeta Way, Salt Lake City, Utah 84108, by phone at 801-584-6857 or by...

  12. Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

    PubMed

    Coleman, James L J; Ngo, Tony; Schmidt, Johannes; Mrad, Nadine; Liew, Chu Kong; Jones, Nicole M; Graham, Robert M; Smith, Nicola J

    2016-04-12

    Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue. Copyright © 2016, American Association for the Advancement of Science.

  13. Differences in signal peptide processing between GP3 glycoproteins of Arteriviridae.

    PubMed

    Zhang, Minze; Veit, Michael

    2018-04-01

    We reported previously that carbohydrate attachment to an overlapping glycosylation site adjacent to the signal peptide of GP3 from equine arteritis virus (EAV) prevents cleavage. Here we investigated whether this unusual processing scheme is a feature of GP3s of other Arteriviridae, which all contain a glycosylation site at a similar position. Expression of GP3 from type-1 and type-2 porcine reproductive and respiratory syndrome virus (PRRSV) and from lactate dehydrogenase-elevating virus (LDV) revealed that the first glycosylation site is used, but has no effect on signal peptide cleavage. Comparison of the SDS-PAGE mobility of deglycosylated GP3 from PRRSV and LDV with mutants having or not having a signal peptide showed that GP3´s signal peptide is cleaved. Swapping the signal peptides between GP3 of EAV and PRRSV revealed that the information for co-translational processing is not encoded in the signal peptide, but in the remaining part of GP3. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Sickness certification for common mental disorders and GP return-to-work advice.

    PubMed

    Gabbay, Mark; Shiels, Chris; Hillage, Jim

    2016-09-01

    Aim To report the types and duration of sickness certification for different common mental disorders (CMDs) and the prevalence of GP advice aimed at returning the patient to work. In the United Kingdom, common mental health problems, such and depression and stress, have become the main reasons for patients requesting a sickness certificate to abstain from usual employment. Increasing attention is being paid to mental health and its impact on employability and work capacity in all parts of the welfare system. However, relatively little is known about the extent to which different mental health diagnoses impact upon sickness certification outcomes, and how the GP has used the new fit note (introduced in 2010) to support a return to work for patients with mental health diagnoses. Sickness certification data was collected from 68 UK-based general practices for a period of 12 months. Findings The study found a large part of all sickness absence certified by GPs was due to CMDs (29% of all sickness absence episodes). Females, younger patients and those living in deprived areas were more likely to receive a fit note for a CMD (compared with one for a physical health problem). The highest proportion of CMD fit notes were issued for 'stress'. However, sickness certification for depression contributed nearly half of all weeks certified for mental health problems. Only 7% of CMD fit notes included any 'may be fit' advice from the GP, with type of advice varying by mental health diagnostic category. Patients living in the most socially deprived neighbourhoods were less likely to receive 'may be fit' advice on their CMD fit notes.

  15. Baculovirus GP64-mediated entry into mammalian cells.

    PubMed

    Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

    2012-03-01

    The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

  16. Chaotic Stochasticity: A Ubiquitous Source of Unpredictability in Epidemics

    NASA Astrophysics Data System (ADS)

    Rand, D. A.; Wilson, H. B.

    1991-11-01

    We address the question of whether or not childhood epidemics such as measles and chickenpox are chaotic, and argue that the best explanation of the observed unpredictability is that it is a manifestation of what we call chaotic stochasticity. Such chaos is driven and made permanent by the fluctuations from the mean field encountered in epidemics, or by extrinsic stochastic noise, and is dependent upon the existence of chaotic repellors in the mean field dynamics. Its existence is also a consequence of the near extinctions in the epidemic. For such systems, chaotic stochasticity is likely to be far more ubiquitous than the presence of deterministic chaotic attractors. It is likely to be a common phenomenon in biological dynamics.

  17. Exploring resilience in rural GP registrars--implications for training.

    PubMed

    Walters, Lucie; Laurence, Caroline O; Dollard, Joanne; Elliott, Taryn; Eley, Diann S

    2015-07-02

    Resilience can be defined as the ability to rebound from adversity and overcome difficult circumstances. General Practice (GP) registrars face many challenges in transitioning into general practice, and additional stressors and pressures apply for those choosing a career in rural practice. At this time of international rural generalist medical workforce shortages, it is important to focus on the needs of rural GP registrars and how to support them to become resilient health care providers. This study sought to explore GP registrars' perceptions of their resilience and strategies they used to maintain resilience in rural general practice. In this qualitative interpretive research, semi-structured interviews were recorded, transcribed and analysed using an inductive approach. Initial coding resulted in a coding framework which was refined using constant comparison and negative case analysis. Authors developed consensus around the final conceptual model. Eighteen GP registrars from: Australian College of Rural and Remote Medicine Independent Pathway, and three GP regional training programs with rural training posts. Six main themes emerged from the data. Firstly, rural GP registrars described four dichotomous tensions they faced: clinical caution versus clinical courage; flexibility versus persistence; reflective practice versus task-focused practice; and personal connections versus professional commitment. Further themes included: personal skills for balance which facilitated resilience including optimistic attitude, self-reflection and metacognition; and finally GP registrars recognised the role of their supervisors in supporting and stretching them to enhance their clinical resilience. Resilience is maintained as on a wobble board by balancing professional tensions within acceptable limits. These limits are unique to each individual, and may be expanded through personal growth and professional development as part of rural general practice training.

  18. Privacy-related context information for ubiquitous health.

    PubMed

    Seppälä, Antto; Nykänen, Pirkko; Ruotsalainen, Pekka

    2014-03-11

    Ubiquitous health has been defined as a dynamic network of interconnected systems. A system is composed of one or more information systems, their stakeholders, and the environment. These systems offer health services to individuals and thus implement ubiquitous computing. Privacy is the key challenge for ubiquitous health because of autonomous processing, rich contextual metadata, lack of predefined trust among participants, and the business objectives. Additionally, regulations and policies of stakeholders may be unknown to the individual. Context-sensitive privacy policies are needed to regulate information processing. Our goal was to analyze privacy-related context information and to define the corresponding components and their properties that support privacy management in ubiquitous health. These properties should describe the privacy issues of information processing. With components and their properties, individuals can define context-aware privacy policies and set their privacy preferences that can change in different information-processing situations. Scenarios and user stories are used to analyze typical activities in ubiquitous health to identify main actors, goals, tasks, and stakeholders. Context arises from an activity and, therefore, we can determine different situations, services, and systems to identify properties for privacy-related context information in information-processing situations. Privacy-related context information components are situation, environment, individual, information technology system, service, and stakeholder. Combining our analyses and previously identified characteristics of ubiquitous health, more detailed properties for the components are defined. Properties define explicitly what context information for different components is needed to create context-aware privacy policies that can control, limit, and constrain information processing. With properties, we can define, for example, how data can be processed or how components

  19. Imbalanced gp130 signalling in ApoE-deficient mice protects against atherosclerosis.

    PubMed

    Jones, Gareth W; McLeod, Louise; Kennedy, Catherine L; Bozinovski, Steven; Najdovska, Meri; Jenkins, Brendan J

    2015-02-01

    Interleukin (IL)-6 is a key modulator of the acute phase response (APR), and while both are implicated in atherosclerosis, the pathological role of specific IL-6 signalling cascades is ill-defined. Since IL-6 employs the cytokine receptor gp130 to primarily activate the STAT3 pathway, here we evaluate whether gp130-dependent STAT3 activation modulates atherosclerosis. High-fat diet-induced atherosclerosis was established in ApoE(-/-) mice crossed with gp130(F/F) knock-in mice displaying elevated gp130-dependent STAT3 activation and production of the APR protein, serum amyloid A (SAA). Also generated were gp130(F/F):Stat3(-/+):ApoE(-/-) mice displaying genetically-normalised STAT3 activation and SAA levels, and bone marrow chimeras involving ApoE(-/-) and gp130(F/F):ApoE(-/-) mice. At 10 weeks post high-fat diet, aortic atherosclerotic lesions, including the presence of CD68(+) macrophages, and plasma lipid and SAA profiles, were assessed. Aortic plaque development and plasma triglyceride levels in gp130(F/F):ApoE(-/-) mice were significantly reduced (3-fold, P < 0.001) compared to ApoE(-/-) littermates. By contrast, in gp130(F/F):ApoE(-/-) mice, atherosclerotic plaques contained augmented CD68(+) macrophage infiltrates, and plasma SAA levels were elevated, compared to ApoE(-/-) mice. Atherosclerotic lesion development and plasma triglyceride levels in gp130(F/F):ApoE(-/-) and gp130(F/F):Stat3(-/+):ApoE(-/-) mice were comparable, despite a significant (P < 0.05) reduction in macrophage numbers in lesions, and also plasma SAA levels, in gp130(F/F):Stat3(-/+):ApoE(-/-) mice. Aortic plaque development and plasma triglyceride levels were comparable in ApoE(-/-) mice reconstituted with gp130(F/F):ApoE(-/-) (ApoE(F/F:ApoE)) or ApoE(-/-) (ApoE(ApoE)) bone marrow cells. Deregulation of gp130/STAT3 signalling augments the APR and macrophage infiltration during atherosclerosis without impacting on the development of aortic plaques. Copyright © 2014 Elsevier Ireland Ltd

  20. International variation in GP treatment strategies for subclinical hypothyroidism in older adults: a case-based survey.

    PubMed

    den Elzen, Wendy P J; Lefèbre-van de Fliert, Anne A; Virgini, Vanessa; Mooijaart, Simon P; Frey, Peter; Kearney, Patricia M; Kerse, Ngaire; Mallen, Christian D; McCarthy, Vera J C; Muth, Christiane; Rosemann, Thomas; Russell, Audrey; Schers, Henk; Stott, David J; de Waal, Margot W M; Warner, Alex; Westendorp, Rudi G J; Rodondi, Nicolas; Gussekloo, Jacobijn

    2015-02-01

    There is limited evidence about the impact of treatment for subclinical hypothyroidism, especially among older people. To investigate the variation in GP treatment strategies for older patients with subclinical hypothyroidism depending on country and patient characteristics. Case-based survey of GPs in the Netherlands, Germany, England, Ireland, Switzerland, and New Zealand. The treatment strategy of GPs (treatment yes/no, starting-dose thyroxine) was assessed for eight cases presenting a woman with subclinical hypothyroidism. The cases differed in the patient characteristics of age (70 versus 85 years), vitality status (vital versus vulnerable), and thyroid-stimulating hormone (TSH) concentration (6 versus 15 mU/L). A total of 526 GPs participated (the Netherlands n = 129, Germany n = 61, England n = 22, Ireland n = 21, Switzerland n = 262, New Zealand n = 31; overall response 19%). Across countries, differences in treatment strategy were observed. GPs from the Netherlands (mean treatment percentage 34%), England (40%), and New Zealand (39%) were less inclined to start treatment than GPs in Germany (73%), Ireland (62%), and Switzerland (52%) (P = 0.05). Overall, GPs were less inclined to start treatment in 85-year-old than in 70-year-old females (pooled odds ratio [OR] 0.74 [95% confidence interval [CI] = 0.63 to 0.87]). Females with a TSH of 15 mU/L were more likely to get treated than those with a TSH of 6 mU/L (pooled OR 9.49 [95% CI = 5.81 to 15.5]). GP treatment strategies of older people with subclinical hypothyroidism vary largely by country and patient characteristics. This variation underlines the need for a new generation of international guidelines based on the outcomes of randomised clinical trials set within primary care. © British Journal of General Practice 2015.

  1. International variation in GP treatment strategies for subclinical hypothyroidism in older adults: a case-based survey

    PubMed Central

    den Elzen, Wendy PJ; Lefèbre-van de Fliert, Anne A; Virgini, Vanessa; Mooijaart, Simon P; Frey, Peter; Kearney, Patricia M; Kerse, Ngaire; Mallen, Christian D; McCarthy, Vera JC; Muth, Christiane; Rosemann, Thomas; Russell, Audrey; Schers, Henk; Stott, David J; de Waal, Margot WM; Warner, Alex; Westendorp, Rudi GJ; Rodondi, Nicolas; Gussekloo, Jacobijn

    2015-01-01

    Background There is limited evidence about the impact of treatment for subclinical hypothyroidism, especially among older people. Aim To investigate the variation in GP treatment strategies for older patients with subclinical hypothyroidism depending on country and patient characteristics. Design and setting Case-based survey of GPs in the Netherlands, Germany, England, Ireland, Switzerland, and New Zealand. Method The treatment strategy of GPs (treatment yes/no, starting-dose thyroxine) was assessed for eight cases presenting a woman with subclinical hypothyroidism. The cases differed in the patient characteristics of age (70 versus 85 years), vitality status (vital versus vulnerable), and thyroid-stimulating hormone (TSH) concentration (6 versus 15 mU/L). Results A total of 526 GPs participated (the Netherlands n = 129, Germany n = 61, England n = 22, Ireland n = 21, Switzerland n = 262, New Zealand n = 31; overall response 19%). Across countries, differences in treatment strategy were observed. GPs from the Netherlands (mean treatment percentage 34%), England (40%), and New Zealand (39%) were less inclined to start treatment than GPs in Germany (73%), Ireland (62%), and Switzerland (52%) (P = 0.05). Overall, GPs were less inclined to start treatment in 85-year-old than in 70-year-old females (pooled odds ratio [OR] 0.74 [95% confidence interval [CI] = 0.63 to 0.87]). Females with a TSH of 15 mU/L were more likely to get treated than those with a TSH of 6 mU/L (pooled OR 9.49 [95% CI = 5.81 to 15.5]). Conclusion GP treatment strategies of older people with subclinical hypothyroidism vary largely by country and patient characteristics. This variation underlines the need for a new generation of international guidelines based on the outcomes of randomised clinical trials set within primary care. PMID:25624308

  2. The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

    PubMed

    Jefferson, Tamara; Auf dem Keller, Ulrich; Bellac, Caroline; Metz, Verena V; Broder, Claudia; Hedrich, Jana; Ohler, Anke; Maier, Wladislaw; Magdolen, Viktor; Sterchi, Erwin; Bond, Judith S; Jayakumar, Arumugam; Traupe, Heiko; Chalaris, Athena; Rose-John, Stefan; Pietrzik, Claus U; Postina, Rolf; Overall, Christopher M; Becker-Pauly, Christoph

    2013-01-01

    The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

  3. A wearable context aware system for ubiquitous healthcare.

    PubMed

    Kang, Dong-Oh; Lee, Hyung-Jik; Ko, Eun-Jung; Kang, Kyuchang; Lee, Jeunwoo

    2006-01-01

    Recent developments of information technologies are leading the advent of the era of ubiquitous healthcare, which means healthcare services at any time and at any places. The ubiquitous healthcare service needs a wearable system for more continual measurement of biological signals of a user, which gives information of the user from wearable sensors. In this paper, we propose a wearable context aware system for ubiquitous healthcare, and its systematic design process of a ubiquitous healthcare service. Some wearable sensor systems are introduced with Zigbee communication. We develop a context aware framework to send information from wearable sensors to healthcare service entities as a middleware to solve the interoperability problem between sensor makers and healthcare service providers. And, we propose a systematic process of design of ubiquitous healthcare services with the context aware framework. In order to show the feasibility of the proposed system, some application examples are given, which are applied to remote monitoring, and a self check service.

  4. Complete genome sequence of 285P, a novel T7-like polyvalent E. coli bacteriophage.

    PubMed

    Xu, Bin; Ma, Xiangyu; Xiong, Hongyan; Li, Yafei

    2014-06-01

    Bacteriophages are considered potential biological agents for the control of infectious diseases and environmental disinfection. Here, we describe a novel T7-like polyvalent Escherichia coli bacteriophage, designated "285P," which can lyse several strains of E. coli. The genome, which consists of 39,270 base pairs with a G+C content of 48.73 %, was sequenced and annotated. Forty-three potential open reading frames were identified using bioinformatics tools. Based on whole-genome sequence comparison, phage 285P was identified as a novel strain of subgroup T7. It showed strongest sequence similarity to Kluyvera phage Kvp1. The phylogenetic analyses of both non-structural proteins (endonuclease gp3, amidase gp3.5, DNA primase/helicase gp4, DNA polymerase gp5, and exonuclease gp6) and structural protein (tail fiber protein gp17) led to the identification of 285P as T7-like phage. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analyses verified the annotation of the structural proteins (major capsid protein gp10a, tail protein gp12, and tail fiber protein gp17).

  5. Inhibition of biosynthesis of metalloprotease of Aeromonas sobria by sodium chloride in the medium.

    PubMed

    Takahashi, Eizo; Kobayashi, Hidetomo; Yamanaka, Hiroyasu; Nair, Gopinath Balakrish; Takeda, Yoshifumi; Arimoto, Sakae; Negishi, Tomoe; Okamoto, Keinosuke

    2011-01-01

    The present authors have previously shown that the serine protease activity of Aeromonas sobria is markedly decreased when A. sobria is cultured in medium containing 3.0% sodium chloride (NaCl, concentration almost equivalent to sea water salinity), and that this occurs because, although the synthesis of ASP is not disturbed by the salt in the medium, the maturation pathway of serine protease of A. sobria (ASP) does not proceed successfully in such a medium. In this study, the effect of salt in the medium on the production of metalloprotease by A. sobria (AMP) was examined. A. sobria produced AMP in the milieu when the bacteria were cultured in medium containing (NaCl) at a concentration of 0.5%. However, AMP was not produced when the bacteria were cultured in salty medium containing 1.5% or more NaCl. To examine how NaCl reduces the production of metalloprotease by A. sobria, the amount of amp mRNA in the cell was measured and it was found that this decreased in proportion to the concentration of NaCl in the medium. The mRNA of amp was not detected in cells cultured in medium containing 1.5% or more NaCl. This means that the transcription of amp is inhibited in salty condition. As described, NaCl in the medium disturbs the maturation pathway of ASP. The mode of action whereby NaCl suppresses AMP activity in A. sobria differs from the mechanism for suppressing ASP activity. © 2010 The Societies and Blackwell Publishing Asia Pty Ltd.

  6. The Australian Defence Force Post‑discharge GP Health Assessment.

    PubMed

    Reed, Richard L; Masters, Stacey; Roeger, Leigh S

    2016-03-01

    All former serving members of the Australian Defence Force (ADF) can receive a comprehensive health assessment from their general practitioners (GPs). The aim of this article is to describe the ADF Post-discharge GP Health Assessment and introduce a tool that assists GPs in performing the assessment. The ADF Post-discharge GP Health Assessment is intended to promote the early detection and intervention of potential mental or physical health concerns in the veteran population and facilitate the establishment of ongoing care with a GP.

  7. Integrating Collaborative and Decentralized Models to Support Ubiquitous Learning

    ERIC Educational Resources Information Center

    Barbosa, Jorge Luis Victória; Barbosa, Débora Nice Ferrari; Rigo, Sandro José; de Oliveira, Jezer Machado; Rabello, Solon Andrade, Jr.

    2014-01-01

    The application of ubiquitous technologies in the improvement of education strategies is called Ubiquitous Learning. This article proposes the integration between two models dedicated to support ubiquitous learning environments, called Global and CoolEdu. CoolEdu is a generic collaboration model for decentralized environments. Global is an…

  8. The Construction of an Ontology-Based Ubiquitous Learning Grid

    ERIC Educational Resources Information Center

    Liao, Ching-Jung; Chou, Chien-Chih; Yang, Jin-Tan David

    2009-01-01

    The purpose of this study is to incorporate adaptive ontology into ubiquitous learning grid to achieve seamless learning environment. Ubiquitous learning grid uses ubiquitous computing environment to infer and determine the most adaptive learning contents and procedures in anytime, any place and with any device. To achieve the goal, an…

  9. Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model.

    PubMed

    Hatting, M; Spannbauer, M; Peng, J; Al Masaoudi, M; Sellge, G; Nevzorova, Y A; Gassler, N; Liedtke, C; Cubero, F J; Trautwein, C

    2015-03-05

    Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130(Δhepa)) and control animals (gp130(f/f)) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0-144 h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130(Δhepa) animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130(Δhepa) mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130(f/f) and gp130(Δhepa) animals. However, gp130(Δhepa) livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-β (TGFβ), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.

  10. Molecular mechanism of transcription inhibition by phage T7 gp2 protein.

    PubMed

    Mekler, Vladimir; Minakhin, Leonid; Sheppard, Carol; Wigneshweraraj, Sivaramesh; Severinov, Konstantin

    2011-11-11

    Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β' jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the -10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β' jaw. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Hairpin Folding of HIV gp41 Abrogates Lipid Mixing Function at Physiologic pH and Inhibits Lipid Mixing by Exposed gp41 Constructs†

    PubMed Central

    Sackett, Kelly; Nethercott, Matthew J.; Shai, Yechiel; Weliky, David P.

    2009-01-01

    Conformational changes in the HIV gp41 protein are directly correlated with fusion between the HIV and target cell plasma membranes which is the initial step of infection. Key gp41 fusion conformations include an early extended conformation termed pre-hairpin which contains exposed regions and a final low energy conformation termed hairpin which has compact six-helix bundle structure. Current fusion models debate the roles of hairpin and pre-hairpin conformations in the process of membrane merger. In the present work, gp41 constructs have been engineered which correspond to fusion relevant parts of both pre-hairpin and hairpin conformations, and have been analyzed for their ability to induce lipid mixing between membrane vesicles. The data correlate membrane fusion function with the pre-hairpin conformation and suggest that one of the roles of the final hairpin conformation is sequestration of membrane perturbing gp41 regions with consequent loss of the membrane disruption induced earlier by the pre-hairpin structure. To our knowledge, this is the first biophysical study to delineate the membrane fusion potential of gp41 constructs modeling key fusion conformations. PMID:19222185

  12. Privacy-Related Context Information for Ubiquitous Health

    PubMed Central

    Nykänen, Pirkko; Ruotsalainen, Pekka

    2014-01-01

    Background Ubiquitous health has been defined as a dynamic network of interconnected systems. A system is composed of one or more information systems, their stakeholders, and the environment. These systems offer health services to individuals and thus implement ubiquitous computing. Privacy is the key challenge for ubiquitous health because of autonomous processing, rich contextual metadata, lack of predefined trust among participants, and the business objectives. Additionally, regulations and policies of stakeholders may be unknown to the individual. Context-sensitive privacy policies are needed to regulate information processing. Objective Our goal was to analyze privacy-related context information and to define the corresponding components and their properties that support privacy management in ubiquitous health. These properties should describe the privacy issues of information processing. With components and their properties, individuals can define context-aware privacy policies and set their privacy preferences that can change in different information-processing situations. Methods Scenarios and user stories are used to analyze typical activities in ubiquitous health to identify main actors, goals, tasks, and stakeholders. Context arises from an activity and, therefore, we can determine different situations, services, and systems to identify properties for privacy-related context information in information-processing situations. Results Privacy-related context information components are situation, environment, individual, information technology system, service, and stakeholder. Combining our analyses and previously identified characteristics of ubiquitous health, more detailed properties for the components are defined. Properties define explicitly what context information for different components is needed to create context-aware privacy policies that can control, limit, and constrain information processing. With properties, we can define, for example, how

  13. Targeting HIV-1 gp41-induced fusion and pathogenesis for anti-viral therapy.

    PubMed

    Garg, Himanshu; Viard, Mathias; Jacobs, Amy; Blumenthal, Robert

    2011-12-01

    HIV gp41 is a metastable protein whose native conformation is maintained in the form of a heterodimer with gp120. The non-covalently associated gp41/gp120 complex forms a trimer on the virus surface. As gp120 engages with HIV's receptor, CD4, and coreceptor, CXCR4 or CCR5, gp41 undergoes several conformational changes resulting in fusion between the viral and cellular membranes. Several lipophilic and amphiphilic domains have been shown to be critical in that process. While the obvious function of gp41 in viral entry is well-established its role in cellular membrane fusion and the link with pathogenesis are only now beginning to appear. Recent targeting of gp41 via fusion inhibitors has revealed an important role of this protein not only in viral entry but also in bystander apoptosis and HIV pathogenesis. Studies by our group and others have shown that the phenomenon of gp41-mediated hemifusion initiates apoptosis in bystander cells and correlates with virus pathogenesis. More interestingly, recent clinical evidence suggests that gp41 mutants arising after Enfuvirtide therapy are associated with CD4 cell increase and immunological benefits. This has in turn been correlated to a decrease in bystander apoptosis in our in vitro as well as in vivo assays. Although a great deal of work has been done to unravel HIV-1 gp41-mediated fusion mechanisms, the factors that regulate gp41-mediated fusion versus hemifusion and the mechanism by which hemifusion initiates bystander apoptosis are not fully understood. Further insight into these issues will open new avenues for drug development making gp41 a critical anti-HIV target both for neutralization and virus attenuation.

  14. Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis.

    PubMed

    Zhu, Fucheng; Liu, Feng; Wu, Bin; He, Bingfang

    2016-12-28

    Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.

  15. The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells

    PubMed Central

    Gram, Anna M.; Oosenbrug, Timo; Lindenbergh, Marthe F. S.; Büll, Christian; Comvalius, Anouskha; Dickson, Kathryn J. I.; Wiegant, Joop; Vrolijk, Hans; Lebbink, Robert Jan; Wolterbeek, Ron; Adema, Gosse J.; Griffioen, Marieke; Heemskerk, Mirjam H. M.; Tscharke, David C.; Hutt-Fletcher, Lindsey M.; Ressing, Maaike E.

    2016-01-01

    Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150’s cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle. PMID:27077376

  16. GP3 is a structural component of the PRRSV type II (US) virion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lima, M. de; Departamento de Microbiologia e Parasitologia, Universidade Federal Fluminense, Niteroi, RJ; Ansari, I.H.

    2009-07-20

    Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen tomore » generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, {sup 35}S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20-60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.« less

  17. Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries

    PubMed Central

    Jonasson, Anette; Eriksson, Christer; Jenkinson, Howard F; Källestål, Carina; Johansson, Ingegerd; Strömberg, Nicklas

    2007-01-01

    Background Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of Streptococcus mutans (implicated in caries), harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of S. mutans. Methods A case-referent study was performed in 12-year-old Swedish children with high (n = 19) or low (n = 19) caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of S. mutans using the partial least squares (PLS) multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms. Results All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively). The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37) and saliva adhesion of S. mutans Ingbritt (VIP = 1.47). The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of S. mutans strains Ingbritt and NG8 and Lactococcus lactis expressing AgI/II adhesins (SpaP or PAc) compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins. Conclusion Gp-340 I behaves as a caries susceptibility protein. PMID

  18. Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells.

    PubMed

    Yu, Kai-Wen; Yao, Chung-Chen; Jeng, Jiiang-Huei; Shieh, Hao-Ying; Chen, Yi-Jane

    2018-04-01

    Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1 Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. The expression of periostin in MG-63 cells is involved in the TGF-β signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells. Copyright © 2017. Published by Elsevier B.V.

  19. CILT2000: Ubiquitous Computing--Spanning the Digital Divide.

    ERIC Educational Resources Information Center

    Tinker, Robert; Vahey, Philip

    2002-01-01

    Discusses the role of ubiquitous and handheld computers in education. Summarizes the contributions of the Center for Innovative Learning Technologies (CILT) and describes the ubiquitous computing sessions at the CILT2000 Conference. (Author/YDS)

  20. p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

    PubMed

    Patel, Vivekkumar; Singh, Vivek P; Pinnamaneni, Jaya Pratap; Sanagasetti, Deepthi; Olive, Jacqueline; Mathison, Megumi; Cooney, Austin; Flores, Elsa R; Crystal, Ronald G; Yang, Jianchang; Rosengart, Todd K

    2018-04-13

    Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming. p63 Knockout ( -/- ) and knockdown murine embryonic fibroblasts (MEFs), p63 -/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2-Myocardin (HM). Flow cytometry revealed that a significantly greater number of p63 -/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63 -/- MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63 -/- adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure. Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  1. The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro.

    PubMed

    Lu, Lin; Dong, Haixia; Liu, Guixiang; Yuan, Bin; Li, Yizhao; Liu, Huaxiang

    2014-11-01

    Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies.

  2. The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro

    PubMed Central

    Lu, Lin; Dong, Haixia; Liu, Guixiang; Yuan, Bin; Li, Yizhao; Liu, Huaxiang

    2014-01-01

    Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies. PMID:25489421

  3. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

    PubMed Central

    Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

  4. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

    PubMed

    Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

  5. Altered plasma levels of cytokines, soluble adhesion molecules and matrix metalloproteases in venous thrombosis.

    PubMed

    Mosevoll, Knut Anders; Lindås, Roald; Tvedt, Tor Henrik Anderson; Bruserud, Øystein; Reikvam, Håkon

    2015-07-01

    Recent studies have emphasized the importance of the inflammatory response mediated by monocyte and neutrophil activation in deep venous thrombosis (DVT); we therefore investigated whether this response was reflected in the plasma profile of inflammatory mediators in patients with suspected DVT. We included a group of 169 consecutive patients admitted to hospital from the primary health care service with suspected lower limb DVT. Plasma levels of 43 mediators were examined for a cohort of 89 consecutive patients and 20 healthy controls by Luminex multiplex analyses, i.e. 13 interleukins, 3 immunomodulatory cytokines, 8 chemokines, 8 growth factors, 3 adhesion molecules and 8 matrix metalloproteases. Selected mediators were analyzed for a second cohort of 80 consecutive patients. Thirty-five of 169 (21%) of referred patients were diagnosed with DVT. Only P-selectin (p<0.0001), vascular cell adhesion protein 1 (VCAM-1, p=0.0009), matrix metalloprotease 8 (MMP-8, p=0.0151) and hepatocyte growth factor (HGF, p=0.0415) differed significantly when comparing patients with and without DVT. When comparing DVT patients with healthy controls we observed significant differences for several mediators, where P-selectin (p=0.0009), VCAM-1 (p<0.0001), all the MMPs (all p<0.0014) and HGF (p<0.0001) showed the strongest significant differences. Unsupervised hierarchical clustering analyses based on biomarkers showing differences between patients with and without DVT could be used to identify patient subsets that differed significantly in DVT frequency. Plasma biomarker profiling of selected soluble mediators can be used to identify subsets among patients with suspected lower limb thrombosis that differ significantly in their frequencies of DVT. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. HIV-1 infection: functional competition between gp41 and interleukin-2.

    PubMed

    Sanhadji, Kamel; Tardy, Jean-Claude; Touraine, Jean-Louis

    2010-08-01

    To determine whether the gp41 of HIV-1 could adhere to the interleukin (IL)-2 receptor at the surface of target cells in vitro, we analysed in vitro the possible functional competition between various forms of the HIV-1 gp41 molecule (i.e. peptides, trimeric or primary structures) and IL-2. This competition has been analysed in a test involving the proliferation of an IL-2-dependent cell line (CTLL2). The putative interaction between the IL-2 molecule and HIV-1 has also been assayed on MT4 cells (CD4(+) T lymphocytes) in culture. The gp41 trimeric molecule and an HIV-1 gp41 peptide (578-590 aminoacid sequence) dramatically inhibited CTLL2 cell proliferation, despite the presence of IL-2. The addition of serum, containing anti-gp41 antibodies, from HIV-1 patients resulted in a significant abolition of this inhibition. The concomitant incubation of IL-2 and HIV-1 with MT4 cells resulted in a strong decrease (70%) in HIV-1 p24 release. These data suggest that the gp41 of HIV-1 can use the IL-2 receptor during the process of HIV-1 infection and that there is some functional mimesis between gp41 and IL-2. Copyright 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  7. Inhibition of the Human ABC Efflux Transporters P-gp and ...

    EPA Pesticide Factsheets

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

  8. The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies.

    PubMed

    Brinkmann, Constantin; Nehlmeier, Inga; Walendy-Gnirß, Kerstin; Nehls, Julia; González Hernández, Mariana; Hoffmann, Markus; Qiu, Xiangguo; Takada, Ayato; Schindler, Michael; Pöhlmann, Stefan

    2016-12-15

    present study shows that LLOV, like EBOV, counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Biosynthesis and processing of the Autographa californica nuclear polyhedrosis virus gp64 protein.

    PubMed

    Jarvis, D L; Garcia, A

    1994-11-15

    gp64 is a major virion envelope glycoprotein of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). gp64 plays an important role in AcMNPV infection, probably mediating penetration of one form of the virus into host cells through the endocytic pathway. gp64 also represents an excellent probe for studying the membrane glycoprotein processing capabilities of baculovirus-infected insect cells, which are used widely as a eucaryotic expression system. The goals of this study were to characterize gp64 biosynthesis and processing and determine how N-glycosylation and N-linked oligosaccharide processing influence the fate and function of gp64 in AcMNPV-infected insect cells. We found that gp64 was synthesized in a biphasic fashion, with peaks at 8 and 24 hr postinfection in both the intracellular and extracellular fractions. Interestingly, the first peak preceded detectable budded virus (BV) production, suggesting that gp64 is shed from infected cells early in infection. Transcriptional regulation accounted for the biphasic mode of gp64 protein synthesis, as transcription initiated at a consensus early motif during early times of infection, at a late motif during late times of infection, and there was a lag between the peak of early and the onset of late transcription. In vitro transcription-translation assays showed that the second ATG in the AcMNPV gp64 long open reading frame is used as the translational initiation codon and that downstream sequences encode a functional signal peptide. Pulse-chase analyses, endoglycosidases, and various inhibitors were used to show that some N-linked oligosaccharides on gp64 are processed by glucosidases and alpha-mannosidases in AcMNPV-infected insect cells. These experiments also revealed that at least two differentially processed gp64 glycoforms are produced in these cells and that both can reach the cell surface and assemble into progeny BV. However, N-linked oligosaccharide processing was not

  10. Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

    PubMed

    Lumen, Annie Albin; Li, Libin; Li, Jiben; Ahmed, Zeba; Meng, Zhou; Owen, Albert; Ellens, Harma; Hidalgo, Ismael J; Bentz, Joe

    2013-01-01

    We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable

  11. Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

    PubMed Central

    Lumen, Annie Albin; Li, Libin; Li, Jiben; Ahmed, Zeba; Meng, Zhou; Owen, Albert; Ellens, Harma; Hidalgo, Ismael J.; Bentz, Joe

    2013-01-01

    We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable

  12. InhA1-Mediated Cleavage of the Metalloprotease NprA Allows Bacillus cereus to Escape From Macrophages.

    PubMed

    Haydar, Abbass; Tran, Seav-Ly; Guillemet, Elisabeth; Darrigo, Claire; Perchat, Stéphane; Lereclus, Didier; Coquet, Laurent; Jouenne, Thierry; Ramarao, Nalini

    2018-01-01

    Bacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation in the host despite the induction of inflammation. To circumvent inflammation, bacteria must resist the bactericidal activity of professional phagocytes, which constitute a first line of host defense against pathogens. Interactions between phagocytic cells and B. cereus are still poorly characterized and the mechanism of resistance to the host immune system is not known yet. We have previously shown that the spores are phagocytosed by macrophages but survive and escape from these cells. The metalloprotease InhA1 is a key effector involved in these processes. inhA1 -deficient spores are retained intracellularly, in contrast to the wild type strain spores. NprA is also a B. cereus metalloprotease able to cleave tissue components such as fibronectin, laminin, and collagen. Here, we show that NprA, concomitantly secreted with InhA1 in the B. cereus secretome, is essential to promote bacterial escape from macrophages. We show that InhA1 cleaves NprA at specific sites. This cleavage allows liberation of the mature form of the NprA protein in the supernatant of the wild type strain. This mature form of NprA is actually the principal effector allowing bacterial escape from host macrophages.

  13. Deletion of Interleukin-6 Signal Transducer gp130 in Small Sensory Neurons Attenuates Mechanonociception and Down-Regulates TRPA1 Expression

    PubMed Central

    Malsch, Philipp; Andratsch, Manfred; Vogl, Christian; Link, Andrea S.; Alzheimer, Christian; Brierley, Stuart M.; Hughes, Patrick A.

    2014-01-01

    Glycoprotein 130 (gp130) is the signal transducing receptor subunit for cytokines of the interleukin-6 (IL-6) family, and it is expressed in a multitude of cell types of the immune and nervous system. IL-6-like cytokines are not only key regulators of innate immunity and inflammation but are also essential factors for the differentiation and development of the somatosensory system. Mice with a null mutation of gp130 in primary nociceptive afferents (SNS-gp130−/−) are largely protected from hypersensitivity to mechanical stimuli in mouse models of pathological pain. Therefore, we set out to investigate how neuronal gp130 regulates mechanonociception. SNS-gp130−/− mice revealed reduced mechanosensitivity to high mechanical forces in the von Frey assay in vivo, and this was associated with a reduced sensitivity of nociceptive primary afferents in vitro. Together with these findings, transient receptor potential ankyrin 1 (TRPA1) mRNA expression was significantly reduced in DRG from SNS-gp130−/− mice. This was also reflected by a reduced number of neurons responding with calcium transients to TRPA1 agonists in primary DRG cultures. Downregulation of Trpa1 expression was predominantly discovered in nonpeptidergic neurons, with the deficit becoming evident during stages of early postnatal development. Regulation of Trpa1 mRNA expression levels downstream of gp130 involved the classical Janus kinase family-signal transducer and activator of transcription pathway. Our results closely link proinflammatory cytokines to the expression of TRPA1, both of which have been shown to contribute to hypersensitive pain states. We suggest that gp130 has an essential role in mechanonociception and in the regulation of TRPA1 expression. PMID:25057188

  14. Plasma deposited composite coatings to control biological response of osteoblast-like MG-63 cells

    NASA Astrophysics Data System (ADS)

    Keremidarska, M.; Radeva, E.; Eleršič, K.; Iglič, A.; Pramatarova, L.; Krasteva, N.

    2014-12-01

    The successful osseointegration of a bone implant is greatly dependent on its ability to support cellular adhesion and functions. Deposition of thin composite coatings onto the implant surface is a promising approach to improve interactions with cells without compromising implant bulk properties. In this work, we have developed composite coatings, based on hexamethyldisiloxane (HMDS) and detonation nanodiamond (DND) particles and have studied adhesion, growth and function of osteoblast-like MG-63 cells. PPHMDS/DND composites are of interest for orthopedics because they combine superior mechanical properties and good biocompatibility of DND with high adherence of HMDS to different substrata including glass, metals and plastics. We have used two approaches of the implementation of DND particles into a polymer matrix: pre-mixture of both components followed by plasma polymerization and layer-by-layer deposition of HMDS and DND particles and found that the deposition approach affects significantly the surface properties of the resulting layers and cell behaviour. The composite, prepared by subsequent deposition of monomer and DND particles was hydrophilic, with a rougher surface and MG-63 cells demonstrated better spreading, growth and function compared to the other composite which was hydrophobic with a smooth surface similarly to unmodified polymer. Thus, by varying the deposition approach, different PPHMDS/DND composite coatings, enhancing or inhibiting osteoblast adhesion and functions, can be obtained. In addition, the effect of fibronectin pre-adsorption was studied and was found to increase greatly MG-63 cell spreading.

  15. Identification of a de novo variant in CHUK in a patient with an EEC/AEC syndrome-like phenotype and hypogammaglobulinemia.

    PubMed

    Khandelwal, Kriti D; Ockeloen, Charlotte W; Venselaar, Hanka; Boulanger, Cécile; Brichard, Bénédicte; Sokal, Etienne; Pfundt, Rolph; Rinne, Tuula; van Beusekom, Ellen; Bloemen, Marjon; Vriend, Gerrit; Revencu, Nicole; Carels, Carine E L; van Bokhoven, Hans; Zhou, Huiqing

    2017-05-17

    The cardinal features of Ectrodactyly, Ectodermal dysplasia, Cleft lip/palate (EEC), and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndromes are ectodermal dysplasia (ED), orofacial clefting, and limb anomalies. EEC and AEC are caused by heterozygous mutations in the transcription factor p63 encoded by TP63. Here, we report a patient with an EEC/AEC syndrome-like phenotype, including ankyloblepharon, ED, cleft palate, ectrodactyly, syndactyly, additional hypogammaglobulinemia, and growth delay. Neither pathogenic mutations in TP63 nor CNVs at the TP63 locus were identified. Exome sequencing revealed de novo heterozygous variants in CHUK (conserved helix-loop-helix ubiquitous kinase), PTGER4, and IFIT2. While the variant in PTGER4 might contribute to the immunodeficiency and growth delay, the variant in CHUK appeared to be most relevant for the EEC/AEC-like phenotype. CHUK is a direct target gene of p63 and encodes a component of the IKK complex that plays a key role in NF-κB pathway activation. The identified CHUK variant (g.101980394T>C; c.425A>G; p.His142Arg) is located in the kinase domain which is responsible for the phosphorylation activity of the protein. The variant may affect CHUK function and thus contribute to the disease phenotype in three ways: (1) the variant exhibits a dominant negative effect and results in an inactive IKK complex that affects the canonical NF-κB pathway; (2) it affects the feedback loop of the canonical and non-canonical NF-κB pathways that are CHUK kinase activity-dependent; and (3) it disrupts NF-κB independent epidermal development that is often p63-dependent. Therefore, we propose that the heterozygous CHUK variant is highly likely to be causative to the EEC/AEC-like and additional hypogammaglobulinemia phenotypes in the patient presented here. © 2017 Wiley Periodicals, Inc.

  16. Ubiquitous Learning Environments in Higher Education: A Scoping Literature Review

    ERIC Educational Resources Information Center

    Virtanen, Mari Aulikki; Haavisto, Elina; Liikanen, Eeva; Kääriäinen, Maria

    2018-01-01

    Ubiquitous learning and the use of ubiquitous learning environments heralds a new era in higher education. Ubiquitous learning environments enhance context-aware and seamless learning experiences available from any location at any time. They support smooth interaction between authentic and digital learning resources and provide personalized…

  17. Ubiquitous geospatial concept in evolution of the macro and micro spatial planning

    NASA Astrophysics Data System (ADS)

    Sabri, S.; Ludin, A. N. M.; Majid, M. R.

    2014-02-01

    There are many examples of GIS application in planning such as urban land-use planning, cultural heritage conservation, coastal zone management, and the design of structure plans for sustainable economic development. All these applications are dealing with systems in which natural and human factors are interconnected. But an issue that should be addressed is to what extent the current information technology is able to connect all these parts together? Contemporary improvement in information technology made the computer so imbedded in our everyday practices that we use it without having to think about it. Thus, computing is becoming truly ubiquitous and is available anywhere anytime. Advances in the internet facilities and devices, such as high speed wireless networks, mobile middleware, and smart technologies, has pushed the concept of ubiquitous computing to the forefront of GIS research and development. There are developments in this regards, these are such as GeoWeb 2.0, voluntarily geographic Information (VGI), and Mashups, whereby the application of cloud computing was possible in visualizing urban air pollution and emergency responses to ensure the safety and security. These advancements therefore, have changed the conventional facet of macro and micro spatial planning. Every possible information system such as residential, medical, business, environmental, governmental, and the like can be linked through ubiquitous computing technologies and acts as a virtually one system which works for society. However, the journey to achieve a true ubiquitous GIS is not without challenges. Despite the current potentials there are many issues and obstacles need to be addressed before GIS can to be truly ubiquitous in planning context. Perhaps four criteria as explained by Goodchild et al (1997) can be applied to ubiquitous GIS in planning very well: the system must be distributed (data storage, processing and user interaction can occur at locations that are potentially

  18. Pancreatic Autoantibodies Against CUZD1 and GP2 Are Associated with Distinct Clinical Phenotypes of Crohn's Disease.

    PubMed

    Michaels, Maike Anna; Jendrek, Sebastian Torben; Korf, Tobias; Nitzsche, Thomas; Teegen, Bianca; Komorowski, Lars; Derer, Stefanie; Schröder, Torsten; Baer, Florian; Lehnert, Henrik; Büning, Jürgen; Fellerman, Klaus; Sina, Christian

    2015-12-01

    Inflammatory bowel disease (IBD) is characterized by a broad spectrum of clinical phenotypes with different outcomes. In the last decades, several IBD-associated autoantibodies have been identified and investigated for their diagnostic relevance. Autoantibodies against the pancreatic glycoproteins (PAB) CUB and zona pellucida-like domains-containing protein 1 (CUZD1), and glycoprotein 2 (GP2) have been demonstrated to possess high specificity for the diagnosis of IBD. Although several studies have shown significant interrelations of anti-GP2 positivity with disease phenotype, associations of clinical phenotypes with anti-CUZD1 are still unknown. The aim was to identify the association of clinical phenotypes with anti-CUZD1 and anti-GP2 in a well-defined German IBD cohort. Patients with IBD (224 patients with Crohn's disease and 136 patients with ulcerative colitis), who were tested for anti-GP2 and anti-CUZD1 immunoglobulin G and immunoglobulin A by indirect immunofluorescence on transfected cells between 2005 and 2013, were included. Serotype and specified phenotypic data were collected in retrospect and statistically analyzed. Both anti-GP2 (P < 0.001) and anti-CUZD1 (P < 0.001) were significantly more prevalent in patients with Crohn's disease than in ulcerative colitis. PAB positivity was associated with ileocolonic disease (P = 0.002), perianal disease (P = 0.011), immunosuppressive treatment (P = 0.036), and ASCA positivity (P = 0.036). Anti-CUZD1 positivity was associated with ileocolonic (P = 0.016) and perianal disease (P = 0.002), whereas anti-GP2 positivity was positively associated with stricturing behavior (P = 0.016). We found distinct clinical phenotypes to be associated with PAB positivity. Therefore, determination of PABs and their subgroup analysis might identify patients with complicated disease behavior. However, the clinical relevance of our findings should be further evaluated in prospective cohorts.

  19. New functionalities in abundant element oxides: ubiquitous element strategy

    PubMed Central

    Hosono, Hideo; Hayashi, Katsuro; Kamiya, Toshio; Atou, Toshiyuki; Susaki, Tomofumi

    2011-01-01

    While most ceramics are composed of ubiquitous elements (the ten most abundant elements within the Earth's crust), many advanced materials are based on rare elements. A ‘rare-element crisis’ is approaching owing to the imbalance between the limited supply of rare elements and the increasing demand. Therefore, we propose a ‘ubiquitous element strategy’ for materials research, which aims to apply abundant elements in a variety of innovative applications. Creation of innovative oxide materials and devices based on conventional ceramics is one specific challenge. This review describes the concept of ubiquitous element strategy and gives some highlights of our recent research on the synthesis of electronic, thermionic and structural materials using ubiquitous elements. PMID:27877391

  20. Fluctuation Dynamics Analysis of gp120 Envelope Protein Reveals a Topologically Based Communication Network

    PubMed Central

    Shrivastava, Indira; LaLonde, Judith M.

    2012-01-01

    HIV infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. Upon CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of Gaussian Network Model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. . These results provide a new context for interpreting gp120 core envelope structure-function relationships. PMID:20718047

  1. GP Section selects Best Student Paper

    NASA Astrophysics Data System (ADS)

    The AGU Geomagnetism and Paleomagnetism (GP) Section has announced its selection of a paper entitled “Multicomponent Magnetization of the Upper Silurian-Lower Devonian Ringerike Sandstone, Adjacent Dikes, and Permian Lavas, Oslo, Norway” as the best GP student paper presented at the 1986 AGU Spring Meeting. The primary author, Dartmouth College Ph.D. candidate David Douglass, was assisted on the paper by a colleague from Lamont-Doherty Geological Observatory. Douglass received his B.S. in geology from the University of California, Los Angeles, in 1980, and in 1984, he received his M.S. in earth sciences at Dartmouth. His current studies examine the paleomagnetism, structure, and sedimentation of several North Atlantic old red sandstone basins.

  2. GP registrar well-being: a cross-sectional survey

    PubMed Central

    2010-01-01

    Objectives To investigate the major stressors affecting GP registrars, how those at risk can be best identified and the most useful methods of managing or reducing their stress. Design, setting and participants Cross-sectional postal questionnaire of all GP registrars in one large regional training provider's catchment area. Main outcome measures The Depression, Anxiety and Stress Scale (DASS), a specifically developed Registrar Stressor Scale consisting of five subscales of potential stressors, plus closed questions on how to identify and manage stress in GP registrars. Results Survey response rate of 51% (102/199). Rural difficulties followed by achieving a work/life balance were the principal stressors. Ten percent of registrars were mildly or moderately depressed or anxious (DASS) and 7% mild to moderately anxious (DASS). Registrars preferred informal means of identifying those under stress (a buddy system and talks with their supervisors); similarly, they preferred to manage stress by discussions with family and friends, debriefing with peers and colleagues, or undertaking sport and leisure activities. Conclusions This study supports research which confirms that poor psychological well-being is an important issue for a significant minority of GP trainees. Regional training providers should ensure that they facilitate formal and informal strategies to identify those at risk and assist them to cope with their stress. PMID:20181138

  3. GP registrar well-being: a cross-sectional survey.

    PubMed

    Schattner, Peter; Mazalin, Dennis; Pier, Ciaran; Wainer, Jo; Ling, Mee Yoke

    2010-02-09

    To investigate the major stressors affecting GP registrars, how those at risk can be best identified and the most useful methods of managing or reducing their stress. Cross-sectional postal questionnaire of all GP registrars in one large regional training provider's catchment area. The Depression, Anxiety and Stress Scale (DASS), a specifically developed Registrar Stressor Scale consisting of five subscales of potential stressors, plus closed questions on how to identify and manage stress in GP registrars. Survey response rate of 51% (102/199). Rural difficulties followed by achieving a work/life balance were the principal stressors. Ten percent of registrars were mildly or moderately depressed or anxious (DASS) and 7% mild to moderately anxious (DASS). Registrars preferred informal means of identifying those under stress (a buddy system and talks with their supervisors); similarly, they preferred to manage stress by discussions with family and friends, debriefing with peers and colleagues, or undertaking sport and leisure activities. This study supports research which confirms that poor psychological well-being is an important issue for a significant minority of GP trainees. Regional training providers should ensure that they facilitate formal and informal strategies to identify those at risk and assist them to cope with their stress.

  4. Privacy Policy Enforcement for Ambient Ubiquitous Services

    NASA Astrophysics Data System (ADS)

    Oyomno, Were; Jäppinen, Pekka; Kerttula, Esa

    Ubiquitous service providers leverage miniaturised computing terminals equipped with wireless capabilities to avail new service models. These models are pivoted on personal and inexpensive terminals to customise services to individual preferences. Portability, small sizes and compact keyboards are few features popularising mobile terminals. Features enable storing and carrying of ever increasing proportions of personal data and ability to use them in service adaptations. Ubiquitous services automate deeper soliciting of personal data transparently without the need for user interactions. Transparent solicitations, acquisitions and handling of personal data legitimises privacy concerns regarding disclosures, retention and re-use of the data. This study presents a policy enforcement for ubiquitous services that safeguards handling of users personal data and monitors adherence to stipulated privacy policies. Enforcement structures towards usability and scalability are presented.

  5. Positive evolution of the glycoprotein (GP) gene is related to transmission of the Ebola virus.

    PubMed

    Jing, Y X; Wang, L N; Wu, X M; Song, C X

    2016-03-28

    Ebola hemorrhagic fever is a fatal disease caused by the negative-strand RNA of the Ebola virus. A high-intensity outbreak of this fever was reported in West Africa last year; however, there is currently no definitive treatment strategy available for this disease. In this study, we analyzed the molecular evolutionary history and attempted to determine the positive selection sites in the Ebola genes using multiple-genomic sequences of the various Ebola virus subtypes, in order to gain greater clarity into the evolution of the virus and its various subtypes. Only the glycoprotein (GP) gene was positively selected among the 8 Ebola genes, with the other genes remaining in the purification stage. The positive selection sites in the GP gene were identified by a random-site model; these sites were found to be located in the mucin-like region, which is associated with transmembrane protein binding. Additionally, different branches of the phylogenetic tree displayed different positive sites, which in turn was responsible for differences in the cell adhesion ability of the virus. In conclusion, the pattern of positive sites in the GP gene is associated with the epidemiology and prevalence of Ebola in different areas.

  6. Structural Plasticity and Conformational Transitions of HIV Envelope Glycoprotein gp120

    PubMed Central

    Korkut, Anil; Hendrickson, Wayne A.

    2012-01-01

    HIV envelope glycoproteins undergo large-scale conformational changes as they interact with cellular receptors to cause the fusion of viral and cellular membranes that permits viral entry to infect targeted cells. Conformational dynamics in HIV gp120 are also important in masking conserved receptor epitopes from being detected for effective neutralization by the human immune system. Crystal structures of HIV gp120 and its complexes with receptors and antibody fragments provide high-resolution pictures of selected conformational states accessible to gp120. Here we describe systematic computational analyses of HIV gp120 plasticity in such complexes with CD4 binding fragments, CD4 mimetic proteins, and various antibody fragments. We used three computational approaches: an isotropic elastic network analysis of conformational plasticity, a full atomic normal mode analysis, and simulation of conformational transitions with our coarse-grained virtual atom molecular mechanics (VAMM) potential function. We observe collective sub-domain motions about hinge points that coordinate those motions, correlated local fluctuations at the interfacial cavity formed when gp120 binds to CD4, and concerted changes in structural elements that form at the CD4 interface during large-scale conformational transitions to the CD4-bound state from the deformed states of gp120 in certain antibody complexes. PMID:23300605

  7. Aminopeptidase N1 (EtAPN1), an M1 Metalloprotease of the Apicomplexan Parasite Eimeria tenella, Participates in Parasite Development

    PubMed Central

    Gras, Simon; Byzia, Anna; Gilbert, Florence B.; McGowan, Sheena; Drag, Marcin; Niepceron, Alisson; Lecaille, Fabien; Lalmanach, Gilles; Brossier, Fabien

    2014-01-01

    Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs. PMID:24839124

  8. Feasibility, acceptability and effectiveness of an online alternative to face-to-face consultation in general practice: a mixed-methods study of webGP in six Devon practices

    PubMed Central

    Carter, Mary; Fletcher, Emily; Sansom, Anna; Warren, Fiona C; Campbell, John L

    2018-01-01

    Objectives To evaluate the feasibility, acceptability and effectiveness of webGP as piloted by six general practices. Methods Mixed-methods evaluation, including data extraction from practice databases, general practitioner (GP) completion of case reports, patient questionnaires and staff interviews. Setting General practices in NHS Northern, Eastern and Western Devon Clinical Commissioning Group’s area approximately 6 months after implementing webGP (February–July 2016). Participants Six practices provided consultations data; 20 GPs completed case reports (regarding 61 e-consults); 81 patients completed questionnaires; 5 GPs and 5 administrators were interviewed. Outcome measures Attitudes and experiences of practice staff and patients regarding webGP. Results WebGP uptake during the evaluation was small, showing no discernible impact on practice workload. The completeness of cross-sectional data on consultation workload varied between practices. GPs judged 41/61 (72%) of webGP requests to require a face-to-face or telephone consultation. Introducing webGP appeared to be associated with shifts in responsibility and workload between practice staff and between practices and patients. 81/231 patients completed a postal survey (35.1% response rate). E-Consulters were somewhat younger and more likely to be employed than face-to-face respondents. WebGP appeared broadly acceptable to patients regarding timeliness and quality/experience of care provided. Similar problems were presented by all respondents. Both groups appeared equally familiar with other practice online services; e-consulters were somewhat more likely to have used them. From semistructured staff interviews, it appeared that, while largely acceptable within practice, introducing e-consults had potential for adverse interactions with pre-existing practice systems. Conclusions There is potential to assess the impact of new systems on consultation patterns by extracting routine data from practice

  9. The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles

    PubMed Central

    Alvarez, Diego E.

    2016-01-01

    Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207–238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207–238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. PMID:27068088

  10. The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

    PubMed

    Alvarez, Diego E; Agaisse, Hervé

    2016-06-01

    Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  11. Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

    PubMed

    Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

    2012-03-02

    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

  12. Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

    PubMed Central

    2012-01-01

    The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

  13. Nuclease digestion and mass spectrometric characterization of oligodeoxyribonucleotides containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG cisplatin intrastrand cross-links.

    PubMed

    Williams, Renee T; Nalbandian, Jenifer N; Tu, Audrey; Wang, Yinsheng

    2013-05-01

    The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, toward the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with (15)N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5' or 3' terminus revealed that the dinucleotide carries a terminal 5' phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Nuclease Digestion and Mass Spectrometric Characterization of Oligodeoxyribonucleotides Containing 1,2-GpG, 1,2-ApG, and 1,3-GpXpG Cisplatin Intrastrand Cross-links

    PubMed Central

    Williams, Renee T.; Nalbandian, Jenifer; Tu, Audrey; Wang, Yinsheng

    2013-01-01

    Background The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, towards the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. Methods We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with 15N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). Results 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5′ or 3′ terminus revealed that the dinucleotide carries a terminal 5′ phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. Results The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA. PMID:23266768

  15. Polyacrylic resins regulate transcriptional control of interleukin-6, gp80, and gp130 genes in human gingival fibroblasts.

    PubMed

    Borelli, Bruna; Zarone, Fernando; Rivieccio, Virginia; Riccitiello, Francesco; Simeone, Michele; Sorrentino, Roberto; Rengo, Sandro; Spagnuolo, Gianrico; Procino, Alfredo

    2017-03-31

    Studies have failed to identify the molecular mechanisms that regulate the genotoxic and cytotoxic effects of methacrylate resins, which are important in the biocompatibility of dental materials. Interleukin (IL)-6 has a crucial role in the control of acute-phase protein response during inflammation. In humans, the synthesis and release of two major acute-phase proteins, C-reactive protein and serum amyloid A, are regulated by IL-6. This study focused on IL-6 and activation of its receptors gp80 and gp130 in human gingival fibroblasts in order to assess the effects of the commercial acid resins Jet Kit, Unifast, and Duralay on control of inflammation.

  16. A Bipartite Signal Regulates the Faithful Delivery of Apical Domain Marker Podocalyxin/Gp135

    PubMed Central

    Yu, Chun-Ying; Chen, Jen-Yau; Lin, Yu-Yu; Shen, Kuo-Fang; Lin, Wei-Ling; Chien, Chung-Liang; ter Beest, Martin B.A.

    2007-01-01

    Podocalyxin/Gp135 was recently demonstrated to participate in the formation of a preapical complex to set up initial polarity in MDCK cells, a function presumably depending on the apical targeting of Gp135. We show that correct apical sorting of Gp135 depends on a bipartite signal composed of an extracellular O-glycosylation–rich region and the intracellular PDZ domain–binding motif. The function of this PDZ-binding motif could be substituted with a fusion construct of Gp135 with Ezrin-binding phosphoprotein 50 (EBP50). In accordance with this observation, EBP50 binds to newly synthesized Gp135 at the Golgi apparatus and facilitates oligomerization and sorting of Gp135 into a clustering complex. A defective connection between Gp135 and EBP50 or EBP50 knockdown results in a delayed exit from the detergent-resistant microdomain, failure of oligomerization, and basolateral missorting of Gp135. Furthermore, the basolaterally missorted EBP50-binding defective mutant of Gp135 was rapidly retrieved via a PKC-dependent mechanism. According to these findings, we propose a model by which a highly negative charged transmembrane protein could be packed into an apical sorting platform with the aid of its cytoplasmic partner EBP50. PMID:17332505

  17. Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

    PubMed Central

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

    2014-01-01

    Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

  18. HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces gp41 Antibody Immunodominance in Rhesus Macaques

    PubMed Central

    Williams, Wilton B.; Saunders, Kevin O.; Seaton, Kelly E.; Wiehe, Kevin J.; Vandergrift, Nathan; Von Holle, Tarra A.; Trama, Ashley M.; Parks, Robert J.; Luo, Kan; Gurley, Thaddeus C.; Kepler, Thomas B.; Marshall, Dawn J.; Montefiori, David C.; Sutherland, Laura L.; Alam, Munir S.; Whitesides, John F.; Bowman, Cindy M.; Permar, Sallie R.; Graham, Barney S.; Mascola, John R.; Seed, Patrick C.; Van Rompay, Koen K. A.; Tomaras, Georgia D.; Moody, M. Anthony

    2017-01-01

    ABSTRACT Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response. IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41

  19. The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity.

    PubMed

    Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

    2015-03-15

    Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. © 2015. Published by The Company of Biologists Ltd.

  20. The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity

    PubMed Central

    Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T.; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

    2015-01-01

    ABSTRACT Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895

  1. GP and pharmacist inter-professional learning - a grounded theory study.

    PubMed

    Cunningham, David E; Ferguson, Julie; Wakeling, Judy; Zlotos, Leon; Power, Ailsa

    2016-05-01

    Practice Based Small Group Learning (PBSGL) is an established learning resource for primary care clinicians in Scotland and is used by one-third of general practitioners (GPs). Scottish Government and UK professional bodies have called for GPs and pharmacists to work more closely together to improve care. To gain GPs' and pharmacists' perceptions and experiences of learning together in an inter-professional PBSGL pilot. Qualitative research methods involving established GP PBSGL groups in NHS Scotland recruiting one or two pharmacists to join them. A grounded theory method was used. GPs were interviewed in focus groups by a fellow GP, and pharmacists were interviewed individually by two researchers, neither being a GP or a pharmacist. Interviews were audio-recorded, transcribed and analysed using grounded theory methods. Data saturation was achieved and confirmed. Three themes were identified: GPs' and pharmacists' perceptions and experiences of inter-professional learning; Inter-professional relationships and team-working; Group identity and purpose of existing GP groups. Pharmacists were welcomed into GP groups and both professions valued inter-professional PBSGL learning. Participants learned from each other and both professions gained a wider perspective of the NHS and of each others' roles in the organisation. Inter-professional relationships, communication and team-working were strengthened and professionals regarded each other as peers and friends.

  2. GP96 is a GARP chaperone and controls regulatory T cell functions.

    PubMed

    Zhang, Yongliang; Wu, Bill X; Metelli, Alessandra; Thaxton, Jessica E; Hong, Feng; Rachidi, Saleh; Ansa-Addo, Ephraim; Sun, Shaoli; Vasu, Chenthamarakshan; Yang, Yi; Liu, Bei; Li, Zihai

    2015-02-01

    Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.

  3. Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene.

    PubMed

    Wang, Wenbing; Zhu, Shanying; Wang, Liqun; Yu, Feng; Shen, Weide

    2005-12-01

    Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

  4. Trust information-based privacy architecture for ubiquitous health.

    PubMed

    Ruotsalainen, Pekka Sakari; Blobel, Bernd; Seppälä, Antto; Nykänen, Pirkko

    2013-10-08

    Ubiquitous health is defined as a dynamic network of interconnected systems that offers health services independent of time and location to a data subject (DS). The network takes place in open and unsecure information space. It is created and managed by the DS who sets rules that regulate the way personal health information is collected and used. Compared to health care, it is impossible in ubiquitous health to assume the existence of a priori trust between the DS and service providers and to produce privacy using static security services. In ubiquitous health features, business goals and regulations systems followed often remain unknown. Furthermore, health care-specific regulations do not rule the ways health data is processed and shared. To be successful, ubiquitous health requires novel privacy architecture. The goal of this study was to develop a privacy management architecture that helps the DS to create and dynamically manage the network and to maintain information privacy. The architecture should enable the DS to dynamically define service and system-specific rules that regulate the way subject data is processed. The architecture should provide to the DS reliable trust information about systems and assist in the formulation of privacy policies. Furthermore, the architecture should give feedback upon how systems follow the policies of DS and offer protection against privacy and trust threats existing in ubiquitous environments. A sequential method that combines methodologies used in system theory, systems engineering, requirement analysis, and system design was used in the study. In the first phase, principles, trust and privacy models, and viewpoints were selected. Thereafter, functional requirements and services were developed on the basis of a careful analysis of existing research published in journals and conference proceedings. Based on principles, models, and requirements, architectural components and their interconnections were developed using system

  5. Trust Information-Based Privacy Architecture for Ubiquitous Health

    PubMed Central

    2013-01-01

    Background Ubiquitous health is defined as a dynamic network of interconnected systems that offers health services independent of time and location to a data subject (DS). The network takes place in open and unsecure information space. It is created and managed by the DS who sets rules that regulate the way personal health information is collected and used. Compared to health care, it is impossible in ubiquitous health to assume the existence of a priori trust between the DS and service providers and to produce privacy using static security services. In ubiquitous health features, business goals and regulations systems followed often remain unknown. Furthermore, health care-specific regulations do not rule the ways health data is processed and shared. To be successful, ubiquitous health requires novel privacy architecture. Objective The goal of this study was to develop a privacy management architecture that helps the DS to create and dynamically manage the network and to maintain information privacy. The architecture should enable the DS to dynamically define service and system-specific rules that regulate the way subject data is processed. The architecture should provide to the DS reliable trust information about systems and assist in the formulation of privacy policies. Furthermore, the architecture should give feedback upon how systems follow the policies of DS and offer protection against privacy and trust threats existing in ubiquitous environments. Methods A sequential method that combines methodologies used in system theory, systems engineering, requirement analysis, and system design was used in the study. In the first phase, principles, trust and privacy models, and viewpoints were selected. Thereafter, functional requirements and services were developed on the basis of a careful analysis of existing research published in journals and conference proceedings. Based on principles, models, and requirements, architectural components and their interconnections

  6. Enhancing field GP engagement in hospital-based studies. Rationale, design, main results and participation in the Diagest 3-GP motivation study.

    PubMed

    Berkhout, Christophe; Vandaele-Bétancourt, Marie; Robert, Stéphane; Lespinasse, Solène; Mitha, Gamil; Bradier, Quentin; Vambergue, Anne; Fontaine, Pierre

    2012-06-21

    Diagest 3 was a study aimed at lowering the risk of developing type 2 diabetes within 3 years after childbirth. Women with gestational diabetes were enrolled in the study. After childbirth, the subjects showed little interest in the structured education programme and did not attend workshops. Their general practitioners (GPs) were approached to help motivate the subjects to participate in Diagest 3, but the GPs were reluctant. The present study aimed to understand field GPs' attitudes towards hospital-based studies, and to develop strategies to enhance their involvement and reduce subject drop-out rates. We used a three-step process: step one used a phenomenological approach exploring the beliefs, attitudes, motivations and environmental factors contributing to the GPs' level of interest in the study. Data were collected in face-to-face interviews and coded by hand and with hermeneutic software to develop distinct GP profiles. Step two was a cross-sectional survey by questionnaire to determine the distribution of the profiles in the GP study population and whether completion of an attached case report form (CRF) was associated with a particular GP profile. In step three, we assessed the impact of the motivation study on participation rates in the main study. Fifteen interviews were conducted to achieve data saturation. Theorisation led to the definition of 4 distinct GP profiles. The response rate to the questionnaire was 73%, but dropped to 52% when a CRF was attached. The link between GP profiles and the rate of CRF completion remains to be verified. The GPs provided data on the CRF that was of comparable quality to those collected in the main trial. Our analysis showed that the motivation study increased overall participation in the main study by 23%, accounting for 16% (24/152) of all final visits for 536 patients who were initially enrolled in the Diagest 3 study. When a hospital-led study explores issues in primary care, its design must anticipate GP

  7. Mining the preferences of patients for ubiquitous clinic recommendation.

    PubMed

    Chen, Tin-Chih Toly; Chiu, Min-Chi

    2018-03-06

    A challenge facing all ubiquitous clinic recommendation systems is that patients often have difficulty articulating their requirements. To overcome this problem, a ubiquitous clinic recommendation mechanism was designed in this study by mining the clinic preferences of patients. Their preferences were defined using the weights in the ubiquitous clinic recommendation mechanism. An integer nonlinear programming problem was solved to tune the values of the weights on a rolling basis. In addition, since it may take a long time to adjust the values of weights to their asymptotic values, the back propagation network (BPN)-response surface method (RSM) method is applied to estimate the asymptotic values of weights. The proposed methodology was tested in a regional study. Experimental results indicated that the ubiquitous clinic recommendation system outperformed several existing methods in improving the successful recommendation rate.

  8. Are there practical opportunities for developing leadership skills during GP training and beyond? A survey of GP trainees and trainers in South East Scotland.

    PubMed

    Curry, Nicola; Denney, MeiLing

    2016-01-01

    There is currently a lack of formal training in leadership skills, particularly during GP training. This study aimed to explore the current training and practical opportunities which exist, specifically exploring the views of GP trainees and trainers. An electronic questionnaire was sent to 266 GP trainees and trainers in south-east Scotland. Questions focused on respondents' experience of leadership-specific training and opportunities to engage with leadership roles. There were a total of 76 respondents (28.6% response rate). Response rate was 19.0% in trainees and 34.6% in trainers. A majority of respondents (80.0%) were established GPs. Of those who had received training in leadership, most (72.1%) underwent this after qualifying as a GP. Respondents identified a range of leadership roles within and outside the practice covering clinical and non-clinical areas. Most were interested in future leadership roles (46.7% moderately interested; 28% very interested). More time, training opportunities and the presence of GP role models were motivating factors in terms of participants' readiness to take on future leadership roles. Signposting trainees, trainers and general practitioners to leadership opportunities and training would be relatively easy but addressing a lack of motivating factors at a local level is essential. The effectiveness of such training and opportunities for experiential learning in leadership roles requires further research.

  9. Peptides designed to spatially depict the Epstein-Barr virus major virion glycoprotein gp350 neutralization epitope elicit antibodies that block virus-neutralizing antibody 72A1 interaction with the native gp350 molecule.

    PubMed

    Tanner, Jerome E; Coinçon, Mathieu; Leblond, Valérie; Hu, Jing; Fang, Janey M; Sygusch, Jurgen; Alfieri, Caroline

    2015-05-01

    Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas, and other cancers. The EBV major virion surface glycoprotein gp350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 revealed only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of the 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic, or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV-neutralizing antibody 72A1 and the major virion surface protein gp350. gp350 mimetic peptides that spatially depict the EBV-neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope. The production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the first in silico map of the gp350 interaction with a virus-blocking monoclonal antibody

  10. Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

    PubMed

    Lázaro-Frías, Adrián; Gómez-Medina, Sergio; Sánchez-Sampedro, Lucas; Ljungberg, Karl; Ustav, Mart; Liljeström, Peter; Muñoz-Fontela, César; Esteban, Mariano; García-Arriaza, Juan

    2018-06-01

    Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV. IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths

  11. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    PubMed

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. Published by Elsevier Inc.

  12. Construction of Course Ubiquitous Learning Based on Network

    ERIC Educational Resources Information Center

    Wang, Xue; Zhang, Wei; Yang, Xinhui

    2017-01-01

    Ubiquitous learning has been more and more recognized, which describes a new generation of learning from a new point of view. Ubiquitous learning will bring the new teaching practice and teaching reform, which will become an essential way of learning in 21st century. Taking translation course as a case study, this research constructed a system of…

  13. Differential role of gp130-dependent STAT and Ras signalling for haematopoiesis following bone-marrow transplantation.

    PubMed

    Kroy, Daniela C; Hebing, Lisa; Sander, Leif E; Gassler, Nikolaus; Erschfeld, Stephanie; Sackett, Sara; Galm, Oliver; Trautwein, Christian; Streetz, Konrad L

    2012-01-01

    Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(ΔMx)), or to selectively disrupt gp130-dependent Ras (gp130(ΔMxRas)) or STAT signalling (gp130(ΔMxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. BM derived from gp130 deficient donor mice (gp130(ΔMx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(ΔMxRas) and gp130(ΔMxSTAT) donor BM. BMT of gp130(ΔMxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(ΔMxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on

  14. Sex of the GP--20 years on.

    PubMed

    Harrison, Christopher M; Britt, Helena C; Charles, Janice

    2011-08-15

    Previous research with the Australian Morbidity and Treatment Survey (1990-1991) showed significant differences in general practitioner characteristics and patient mix of male and female GPs. Even after adjusting for these, it was seen that male and female GPs managed different types of medical conditions. The proportion of female GPs increased from 19.6% in 1990-1991 to 37.1% in 2009-2010. This study investigates whether differences remain two decades later. Analysis of 2009-2010 Bettering the Evaluation and Care of Health (BEACH) data examining GP characteristics, patient encounter characteristics, patient reasons for encounter (RFE), problem types managed and management methods used, by GP sex. Whether GP sex was an independent predictor of problem types being managed, or management methods used, was tested using multiple logistic regressions and Poisson regression. 988 GPs recorded 98 800 GP-patient encounters. Adjusted differences in clinical activity of male and female GPs. After adjustment, compared with male GPs, females recorded more RFEs about general and unspecified issues and endocrine, female genital, pregnancy and family planning problems; and fewer concerning the musculoskeletal, respiratory, skin and male genital systems. Female GPs managed more general and unspecified, digestive, circulatory, psychological, endocrine, female genital and social problems; recorded nearly 20% more clinical treatments and referrals; recorded nearly 10% more imaging and pathology tests; and 4.3% fewer medications. After two decades, even with increased numbers of female GPs, the differences in problems managed by male and female GPs remain, and will probably continue. Female GPs use more resources per encounter, but may not use more resources in terms of annual patient care.

  15. Molecular cloning and sequence analysis of the Anticarsia gemmatalis multicapsid nuclear polyhedrosis virus GP64 glycoprotein.

    PubMed

    Pilloff, Marcela Gabriela; Bilen, Marcos Fabián; Belaich, Mariano Nicolás; Lozano, Mario Enrique; Ghiringhelli, Pablo Daniel

    2003-01-01

    The gp64 locus of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus isolate Santa Fe (AgMNPV-SF) was characterised molecularly in our laboratory. To this end, we have located and cloned a AgMNPV-SF genomic DNA fragment containing the gp64 gene and sequenced the complete gp64 locus. Nucleotide sequence analysis indicated that the AgMNPV gp64 gene consists of a 1500 nucleotide open reading frame (ORF), encoding a protein of 499 amino acids. Of the seven gp64 homologues identified to date, the AgMNPV gp64 ORF shared most sequence similarity with the gp64 gene of Orgyia pseudotsugata MNPV. The GP64 from AgMNPV is the smallest baculoviral envelope glycoprotein found to date, differing in 10 or more residues from the other group I nucleopolyhedroviruses. The biological activity of AgMNPV GP64 protein was assessed by cell fusion assays in UFL-AG-286 cells using the obtained recombinant plasmids. In the upstream and downstream regions, relative to the gp64 ORF, we found different conserved transcriptional and post-transcriptional regulatory elements, respectively.

  16. Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

    PubMed

    Liu, Nina; Tao, Yisong; Brenowitz, Michael D; Girvin, Mark E; Lai, Jonathan R

    2015-10-01

    Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Ubiquitous CM and DM

    NASA Technical Reports Server (NTRS)

    Crowley, Sandra L.

    2000-01-01

    Ubiquitous is a real word. I thank a former Total Quality Coach for my first exposure some years ago to its existence. My version of Webster's dictionary defines ubiquitous as "present, or seeming to be present, everywhere at the same time; omnipresent." While I believe that God is omnipresent, I have come to discover that CM and DM are present everywhere. Oh, yes; I define CM as Configuration Management and DM as either Data or Document Management. Ten years ago, I had my first introduction to the CM world. I had an opportunity to do CM for the Space Station effort at the NASA Lewis Research Center. I learned that CM was a discipline that had four areas of focus: identification, control, status accounting, and verification. I was certified as a CMIl graduate and was indoctrinated about clear, concise, and valid. Off I went into a world of entirely new experiences. I was exposed to change requests and change boards first hand. I also learned about implementation of changes, and then of technical and CM requirements.

  18. Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

    PubMed Central

    Cai, Lifeng; Gochin, Miriam; Liu, Keliang

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

  19. Internet messenger based smart virtual class learning using ubiquitous computing

    NASA Astrophysics Data System (ADS)

    Umam, K.; Mardi, S. N. S.; Hariadi, M.

    2017-06-01

    Internet messenger (IM) has become an important educational technology component in college education, IM makes it possible for students to engage in learning and collaborating at smart virtual class learning (SVCL) using ubiquitous computing. However, the model of IM-based smart virtual class learning using ubiquitous computing and empirical evidence that would favor a broad application to improve engagement and behavior are still limited. In addition, the expectation that IM based SVCL using ubiquitous computing could improve engagement and behavior on smart class cannot be confirmed because the majority of the reviewed studies followed instructions paradigms. This article aims to present the model of IM-based SVCL using ubiquitous computing and showing learners’ experiences in improved engagement and behavior for learner-learner and learner-lecturer interactions. The method applied in this paper includes design process and quantitative analysis techniques, with the purpose of identifying scenarios of ubiquitous computing and realize the impressions of learners and lecturers about engagement and behavior aspect and its contribution to learning

  20. Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain.

    PubMed

    Ou, Wu; Delisle, Josie; Jacques, Jerome; Shih, Joanna; Price, Graeme; Kuhn, Jens H; Wang, Vivian; Verthelyi, Daniela; Kaplan, Gerardo; Wilson, Carolyn A

    2012-01-25

    The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system.

  1. Introducing a GP copayment in Australia: Who would carry the cost burden?

    PubMed

    Elkins, Rosemary Kate; Schurer, Stefanie

    2017-05-01

    Recent policy changes designed to contain unsustainable health expenditure growth imply that many more Australians may soon be charged a copayment to consult a GP. We explore the distributional consequences associated with a range of hypothetical GP copayment scenarios using nationally-representative Australian survey data. For each scenario, we estimate the cost burden that individuals and households across the income distribution would need to absorb to maintain their current GP service utilisation. Even when concessional patients are charged a third or a quarter of the non-concessional copayment rate, the average estimated cost burden in the lowest income quartile is typically between three and six times that of the highest, and the average cost burden for women is significantly higher than for men within every income quartile. These disparities are intensified for those with a chronic illness. We conclude that the widespread implementation of GP copayments would disproportionately burden lower-income families, who experience higher rates of chronic illness, higher demand for GP services, and lower capacity to absorb price increases. The regressive nature of GP copayments is reduced when concessional and child patients are exempted entirely, highlighting the importance of supporting GPs-particularly in disadvantaged areas-to maintain bulk-billing arrangements for vulnerable patient groups. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. The Env-like open reading frame of the baculovirus-integrated retrotransposon TED encodes a retrovirus-like envelope protein.

    PubMed

    Ozers, M S; Friesen, P D

    1996-12-15

    TED is a 7.5-kbp member of the gypsy family of retrotransposons that was first identified by its integration within the baculovirus DNA genome. This lepidopteran (moth) transposon contains three retrovirus-like genes, including functional gag and pol that yield reverse transcriptase-containing virus-like particles. To identify and characterize the product(s) of the third env-like open reading frame, TED ORF3 was expressed in homologous lepidopteran cells by using a baculovirus vector, vENV. Immunoblots and immunoprecipitations with antiserum raised against a bacterial ORF3-fusion protein detected two ORF3-encoded proteins, p68env and gp75env. On the basis of selective incorporation of [3H]mannose and inhibition of modification by tunicamycin which blocks N-linked glycosylation, gp75env is a glycoprotein derived from core precursor p68env. As predicted by the presence of a transmembrane domain near the carboxyl terminus, both p68env and gp75env were associated with heavy membranes of vENV-infected cells. Thus, TED ORF3 encodes a membrane glycoprotein with properties characteristic of retroviral env proteins. These data are consistent with the hypothesis that TED is an invertebrate retrovirus. Moreover, TED integration within the baculovirus genome provides an example of retroelement-mediated acquisition of host genes that may contribute to virus evolution.

  3. Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

    PubMed Central

    Kesavardhana, Sannula

    2014-01-01

    ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers

  4. The Effects of IGF-1 on Trk Expressing DRG Neurons with HIV-gp120- Induced Neurotoxicity.

    PubMed

    Li, Hao; Liu, Zhen; Chi, Heng; Bi, Yanwen; Song, Lijun; Liu, Huaxiang

    2016-01-01

    HIV envelope glycoprotein gp120 is the main protein that causes HIVassociated sensory neuropathy. However, the underlying mechanisms of gp120-induced neurotoxicity are still unclear. There are lack effective treatments for relieving HIV-related neuropathic symptoms caused by gp120-induced neurotoxicity. In the present study, tyrosine kinase receptor (Trk)A, TrkB, and TrkC expression in primary cultured dorsal root ganglion (DRG) neurons with gp120-induced neurotoxicity was investigated. The effects of IGF-1 on distinct Trk-positive DRG neurons with gp120-induced neurotoxicity were also determined. The results showed that gp120 not only dose-dependently induced DRG neuronal apoptosis and inhibited neuronal survival and neurite outgrowth, but also decreased distinct Trk expression levels. IGF-1 rescued DRG neurons from apoptosis and improved neuronal survival of gp120 neurotoxic DRG neurons in vitro. IGF-1 also improved TrkA and TrkB, but not TrkC, expression in gp120 neurotoxic conditions. The effects of IGF-1 could be blocked by preincubation with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results suggested that gp120 may have a wide range of neurotoxicity on different subpopulations of DRG neurons, while IGF-1 might only relieve some subpopulations of DRG neurons with gp120-induced neurotoxicity. These data provide novel information of mechanisms of gp120 neurotoxicity on primary sensory neurons and the potential therapeutic effects of IGF-1 on gp120-induced neurotoxicity.

  5. Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease.

    PubMed

    Gardner, Andrew F; Prangishvili, David; Jack, William E

    2011-09-01

    The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.

  6. Deletion of fusion peptide or destabilization of fusion core of HIV gp41 enhances antigenicity and immunogenicity of 4E10 epitope

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li Jing; Beijing Key Laboratory for Protein Therapeutics, Beijing 100084; Chen Xi

    2008-11-07

    The human monoclonal antibody 4E10 against the membrane-proximal external region (MPER) of HIV-1 gp41 demonstrates broad neutralizing activity across various strains, and makes its epitope an attractive target for HIV-1 vaccine development. Although the contiguous epitope of 4E10 has been identified, attempts to re-elicit 4E10-like antibodies have failed, possibly due to the lack of proper conformation of the 4E10 epitope. Here we used pIg-tail expression system to construct a panel of eukaryotic cell-surface expression plasmids encoding the extracellular domain of gp41 with deletion of fusion peptide and/or introduction of L568P mutation that may disrupt the gp41 six-helix bundle core conformationmore » as DNA vaccines for immunization of mice. We found that these changes resulted in significant increase of the antigenicity and immunogenicity of 4E10 epitope. This information is thus useful for rational design of vaccines targeting the HIV-1 gp41 MPER.« less

  7. Feasibility, acceptability and effectiveness of an online alternative to face-to-face consultation in general practice: a mixed-methods study of webGP in six Devon practices.

    PubMed

    Carter, Mary; Fletcher, Emily; Sansom, Anna; Warren, Fiona C; Campbell, John L

    2018-02-15

    To evaluate the feasibility, acceptability and effectiveness of webGP as piloted by six general practices. Mixed-methods evaluation, including data extraction from practice databases, general practitioner (GP) completion of case reports, patient questionnaires and staff interviews. General practices in NHS Northern, Eastern and Western Devon Clinical Commissioning Group's area approximately 6 months after implementing webGP (February-July 2016). Six practices provided consultations data; 20 GPs completed case reports (regarding 61 e-consults); 81 patients completed questionnaires; 5 GPs and 5 administrators were interviewed. Attitudes and experiences of practice staff and patients regarding webGP. WebGP uptake during the evaluation was small, showing no discernible impact on practice workload. The completeness of cross-sectional data on consultation workload varied between practices.GPs judged 41/61 (72%) of webGP requests to require a face-to-face or telephone consultation. Introducing webGP appeared to be associated with shifts in responsibility and workload between practice staff and between practices and patients.81/231 patients completed a postal survey (35.1% response rate). E-Consulters were somewhat younger and more likely to be employed than face-to-face respondents. WebGP appeared broadly acceptable to patients regarding timeliness and quality/experience of care provided. Similar problems were presented by all respondents. Both groups appeared equally familiar with other practice online services; e-consulters were somewhat more likely to have used them.From semistructured staff interviews, it appeared that, while largely acceptable within practice, introducing e-consults had potential for adverse interactions with pre-existing practice systems. There is potential to assess the impact of new systems on consultation patterns by extracting routine data from practice databases. Staff and patients noticed subtle changes to responsibilities associated with

  8. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions.

    PubMed

    Dong, Jing-fei; Moake, Joel L; Nolasco, Leticia; Bernardo, Aubrey; Arceneaux, Wendy; Shrimpton, Corie N; Schade, Alicia J; McIntire, Larry V; Fujikawa, Kazuo; López, José A

    2002-12-01

    Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibalpha, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm(2)) and arterial (20 and 50 dyne/cm(2)) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.

  9. [NF-kappaB-induced gp96 up-regulation promotes hepatocyte growth, cell cycle progression and transition].

    PubMed

    Feng, Cong; Wu, Bo; Fan, Hongxia; Li, Changfei; Meng, Songdong

    2014-10-04

    To investigate the mechanism of gp96 raised during hepatitis B virus (HBV) infection and the pathological mechanism. The mechanism of NF-KB activating gp96 expression was determined by bioinformatics analysis, luciferase reporter assay, real-time PCR and Western blot. The effect of over-expression and knockdown gp96 expression by transfection or RNA interference on hepatocyte proliferation, apoptosis and cell cycle was examined by CCK-8 and flow cytometry. The role of gp96 for HCC development was determined by epithelial-mesenchymal transition (EMT) and colony formation assay. NF-kB significantly increased the gp96 expression by binding to the NF-kappaB binding site. Over-expression and knockdown studies both show that gp96 promoted hepatocyte proliferation, inhibited apoptosis, and induced G0/G1 to S phase cell cycle progression. Moreover, gp96 induced epithelial-mesenchymal transition and increased colony formation ability of hepatocytes. Our results therefore provide insights in chronic HBV infection-induced gp96 expression, and indicate that elevated gp96 may contribute to HCC development during chronic inflammation.

  10. RpA, an extracellular protease similar to the metalloprotease of serralysin family, is required for pathogenicity of Ralstonia pickettii.

    PubMed

    Chen, C-M; Liu, J-J; Chou, C-W; Lai, C-H; Wu, L-T

    2015-10-01

    To investigate the biochemical and functional properties of an extracellular protease, RpA, in Ralstonia pickettii WP1 isolated from water supply systems. An extracellular protease was identified and characterized from R. pickettii WP1. A mutant strain WP1M2 was created from strain WP1 by mini-Tn5 transposition. The culture filtrates from WP1M2 had a lower cytotoxic effect than the parental WP1 on several mammalian cell lines. Cloning and sequence analysis revealed the Tn5 transposon inserted at a protease gene (rpA) which is 81% homologous to prtA and aprX genes of Pseudomonas fluorescens. The rpA gene encodes a 482-residue protein showing sequence similarity to metalloproteases of the serralysin family. The RpA protein was expressed in Escherichia coli using a pET expression vector and purified as a 55 kDa molecular weight protein. Furthermore, the protease activity of RpA was inhibited by protease inhibitor and heat treatment. The in vitro cytotoxic activity of R. pickettii culture filtrates was attributed to RpA protease. An extracellular protease, RpA, was identified from R. pickettii WP1 isolated from water supply system. The RpA metalloproteases is required for the pathogenicity of R. pickettii to mammalian cell lines. © 2015 The Society for Applied Microbiology.

  11. The Genital Tract Virulence Factor pGP3 Is Essential for Chlamydia muridarum Colonization in the Gastrointestinal Tract.

    PubMed

    Shao, Lili; Zhang, Tianyuan; Melero, Jose; Huang, Yumeng; Liu, Yuanjun; Liu, Quanzhong; He, Cheng; Nelson, David E; Zhong, Guangming

    2018-01-01

    The cryptic plasmid is essential for Chlamydia muridarum dissemination from the genital tract to the gastrointestinal (GI) tract. Following intravaginal inoculation, a C. muridarum strain deficient in plasmid-encoded pGP3 or pGP4 but not pGP5, pGP7, or pGP8 failed to spread to the mouse gastrointestinal tract, although mice infected with these strains developed productive genital tract infections. pGP3- or pGP4-deficient strains also failed to colonize the gastrointestinal tract when delivered intragastrically. pGP4 regulates pGP3, while pGP3 does not affect pGP4 expression, indicating that pGP3 is critical for C. muridarum colonization of the gastrointestinal tract. Mutants deficient in GlgA, a chromosome-encoded protein regulated by pGP4, also consistently colonized the mouse gastrointestinal tract. Interestingly, C. muridarum colonization of the gastrointestinal tract positively correlated with pathogenicity in the upper genital tract. pGP3-deficient C. muridarum strains did not induce hydrosalpinx or spread to the GI tract even when delivered to the oviduct by intrabursal inoculation. Thus, the current study not only has revealed that pGP3 is a novel chlamydial colonization factor in the gastrointestinal tract but also has laid a foundation for investigating the significance of gastrointestinal Chlamydia . Copyright © 2017 American Society for Microbiology.

  12. Qualitative study of depression management in primary care: GP and patient goals, and the value of listening.

    PubMed

    Johnston, Olwyn; Kumar, Satinder; Kendall, Kathleen; Peveler, Robert; Gabbay, John; Kendrick, Tony

    2007-11-01

    Guidelines for depression management have been developed but little is known about GP and patient goals, which are likely to influence treatment offers, uptake, and adherence. To identify issues of importance to GPs, patients, and patients' supporters regarding depression management. GP and patient goals for depression management became a focus of the study. Grounded theory-based qualitative study. GPs were drawn from 28 practices. The majority of patients and supporters were recruited from 10 of these practices. Sixty-one patients (28 depressed, 18 previously depressed, 15 never depressed), 18 supporters, and 32 GPs were interviewed. GPs described encouraging patients to view depression as separate from the self and 'normal' sadness. Patients and supporters often questioned such boundaries, rejecting the notion of a medical cure and emphasising self-management. The majority of participants who were considering depression-management strategies wanted to 'get out' of their depression. However, a quarter did not see this as immediately relevant or achievable. They focused on getting by from day to day, which had the potential to clash with GP priorities. GP frustration and uncertainty could occur when depression was resistant to cure. Participants identified the importance of GPs listening to patients, but often felt that this did not happen. Physicians need greater awareness of the extent to which their goals for the management of depression are perceived as relevant or achievable by patients. Future research should explore methods of negotiating agreed strategies for management.

  13. Implications of Ubiquitous Computing for the Social Studies Curriculum

    ERIC Educational Resources Information Center

    van Hover, Stephanie D.; Berson, Michael J.; Bolick, Cheryl Mason; Swan, Kathleen Owings

    2004-01-01

    In March 2002, members of the National Technology Leadership Initiative (NTLI) met in Charlottesville, Virginia to discuss the potential effects of ubiquitous computing on the field of education. Ubiquitous computing, or "on-demand availability of task-necessary computing power," involves providing every student with a handheld computer--a…

  14. Metabolism of AGEs--bacterial AGEs are degraded by metallo-proteases.

    PubMed

    Cohen-Or, Ifat; Katz, Chen; Ron, Eliora Z

    2013-01-01

    Advanced Glycation End Products (AGEs) are the final products of non-enzymatic protein glycation that results in loss of protein structure and function. We have previously shown that in E. coli AGEs are continually formed as high-molecular weight protein complexes. Moreover, we showed that AGEs are removed from the cells by an active, ATP-dependent secretion and that these secreted molecules have low molecular weight. Taken together, these results indicate that E. coli contains a fraction of low molecular weight AGEs, in addition to the high-molecular weight AGEs. Here we show that the low-molecular weight AGEs originate from high-molecular weight AGEs by proteolytic degradation. Results of in-vitro and in vivo experiments indicated that this degradation is carried out not by the major ATP-dependent proteases that are responsible for the main part of bacterial protein quality control but by an alternative metal-dependent proteolysis. This proteolytic reaction is essential for the further secretion of AGEs from the cells. As the biochemical reactions involving AGEs are not yet understood, the implication of a metalloprotease in breakdown of high molecular weight AGEs and their secretion constitutes an important step in the understanding of AGEs metabolism.

  15. Diversion of HIV-1 Vaccine-induced Immunity by gp41-Microbiota Cross-reactive Antibodies

    PubMed Central

    Williams, Wilton B; Liao, Hua-Xin; Moody, M. Anthony; Kepler, Thomas B.; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M.; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E.; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C.; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, Julie; Mascola, John R.; Koup, Richard A; Corey, Lawrence; Nabel, Gary J.; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S.; Baden, Lindsey R.; Tomaras, Georgia D.; Haynes, Barton F.

    2015-01-01

    A HIV-1 DNA prime-recombinant Adenovirus Type 5 (rAd5) boost vaccine failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells was to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies (mAbs) were non-neutralizing, and frequently polyreactive with host and environmental antigens including intestinal microbiota (IM). Next generation sequencing of an IGHV repertoire prior to vaccination revealed an Env-IM cross-reactive Ab that was clonally-related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. PMID:26229114

  16. Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage.

    PubMed

    Ploss, Martin; Kuhn, Andreas

    2011-09-26

    Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

  17. Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

    PubMed Central

    2011-01-01

    Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies. PMID:21943062

  18. Socio-technical Issues for Ubiquitous Information Society in 2010

    NASA Astrophysics Data System (ADS)

    Funabashi, Motohisa; Homma, Koichi; Sasaki, Toshiro; Sato, Yoshinori; Kido, Kunihiko; Fukumoto, Takashi; Yano, Koujin

    Impact of the ubiquitous information technology on our society is so significant that directing technological development and preparing institutional apparatus are quite important and urgent. The present paper elaborates, with the efforts by both humanity and engineering disciplines, to find out the socio-technical issues of ubiquitous information society in 2010 by inspecting social implications of emerging technology as well as social expectations. In order to deliberate the issues, scenarios are developed that describes possible life in ubiquitous information society. The derived issues cover integrating information technology and human body, producing smart sharable environment, protecting individual rights, fostering new service business, and forming community.

  19. What weekday? How acute? An analysis of reported planned and unplanned GP visits by older multi-morbid patients in the Patient Journey Record System database.

    PubMed

    Surate Solaligue, David Emanuel; Hederman, Lucy; Martin, Carmel Mary

    2014-08-01

    . The PaJR project Care Guides advised patients to make unplanned visits in 6.3% of calls and advised planned GP visits in 2.5% of calls. Unplanned GP visits consistently indicated a significant change to worse health with planned visits presenting less acuity in this study of older multi-morbid patients in general practice, when monitored by regular calls at about every 3 days. The PaJR study actively prompted GP visits according to its algorithms. Assessing and predicting acuity in older multi-morbid patients appears to be a promising strategy to improve access to primary care, and thus to reducing avoidable hospital utilization. Further research is needed to investigate the topic on a wider scale. © 2014 John Wiley & Sons, Ltd.

  20. Expression of gp120 in mice evokes anxiety behavior: Co-occurrence with increased dendritic spines and brain-derived neurotrophic factor in the amygdala.

    PubMed

    Bachis, Alessia; Forcelli, Patrick; Masliah, Eliezer; Campbell, Lee; Mocchetti, Italo

    2016-05-01

    Human immunodeficiency virus type 1 (HIV) infection of the brain produces cognitive and motor disorders. In addition, HIV positive individuals exhibit behavioral alterations, such as apathy, and a decrease in spontaneity or emotional responses, typically seen in anxiety disorders. Anxiety can lead to psychological stress, which has been shown to influence HIV disease progression. These considerations underscore the importance of determining if anxiety in HIV is purely psychosocial, or if by contrast, there are the molecular cascades associated directly with HIV infection that may mediate anxiety. The present study had two goals: (1) to determine if chronic exposure to viral proteins would induce anxiety-like behavior in an animal model and (2) to determine if this exposure results in anatomical abnormalities that could explain increased anxiety. We have used gp120 transgenic mice, which display behavior and molecular deficiencies similar to HIV positive subjects with cognitive and motor impairments. In comparison to wild type mice, 6 months old gp120 transgenic mice demonstrated an anxiety like behavior measured by open field, light/dark transition task, and prepulse inhibition tests. Moreover, gp120 transgenic mice have an increased number of spines in the amygdala, as well as higher levels of brain-derived neurotrophic factor and tissue plasminogen activator when compared to age-matched wild type. Our data support the hypothesis that HIV, through gp120, may cause structural changes in the amygdala that lead to maladaptive responses to anxiety. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. A safety and feasibility report of combined direct thrombin and GP IIb/IIIa inhibition with bivalirudin and tirofiban in peripheral vascular disease intervention: treating critical limb ischemia like acute coronary syndrome.

    PubMed

    Allie, David E; Hebert, Chris J; Lirtzman, Mitchell D; Wyatt, Charles H; Keller, V Antoine; Khan, Mohamed H; Khan, Muhammad A; Fail, Peter S; Vivekananthan, Krishnamoorthy; Allie, Sonja E; Mitran, Elena V; Chaisson, Gary; Stagg, Samuel J; Allie, Adam A; McElderry, Michael W; Barker, Esmond A; Walker, Craig M

    2005-08-01

    The combination of glycoprotein (GP) IIb-IIIa inhibition and direct thrombin inhibition (DTI) with bivalirudin (Angiomax, The Medicines Company, Cambridge, Massachusetts) have shown ischemic and hemorrhagic outcomes benefit in coronary interventions and may have similar benefits in percutaneous peripheral interventions (PPI). The high incidence of diabetes, chronic renal disease, platelet dysfunction, hypercoagulability, inflammation and a thrombus-rich environment make a GP IIb-IIIa and DTI combination with tirofiban (Aggrastat Merck and Company, Inc., Whitehouse Station, New Jersey) an attractive anticoagulation strategy in the PPI treatment of critical limb ischemia (CLI). Between May 1, 2001 and January 31, 2003, a CLI treatment group of 149 patients received PPI with bivalirudin (0.75 mg per kg bolus with 1.75 mg per kg per hour periprocedural infusion) and tirofiban (10 mcg per kg per minute bolus with 12-hour 0.1 mcg per kg per minute infusion) as an anticoagulation and antiplatelet strategy, and were compared to a matched unfractionated heparin (UFH) control group without GP IIb-IIIa inhibitors. Clinical and hemostasis outcomes were analyzed, including distal embolization (DE). Procedural success was 95.9% and 97.3% in the UFH control group and DTI-GP IIb-IIIa group, respectively. Significant differences were observed in the sheath removal time < 2 hours (60.5% UFH group versus 19.4% DTI-GP IIb-IIIa group; p = < 0.0001). Vascular closure devices were used equally in both groups. No statistical significance was observed in major and minor complications, femoral access complications, acute (< 48 hours) or subacute (30 days) vessel thrombosis, and 6-month duplex ultrasound restenosis rate between the DTI-GP IIb-IIIa versus the UFH group. A trend towards statistical significance was observed in the 6-month secondary re-intervention and limb salvage rates (10.7% versus 18.8%; p = 0.0501 and 93.9% versus 88.5%; p = 0.053) in the DTI-GP IIb-IIIa versus the UFH

  2. Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

    PubMed

    Shilling, F M; Krätzschmar, J; Cai, H; Weskamp, G; Gayko, U; Leibow, J; Myles, D G; Nuccitelli, R; Blobel, C P

    1997-06-15

    Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

  3. Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

    The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

  4. Deciding who gets treatment for depression and anxiety: a study of consecutive GP attenders

    PubMed Central

    Hyde, Julia; Evans, Jonathan; Sharp, Debbie; Croudace, Tim; Harrison, Glynn; Lewis, Glyn; Araya, Ricardo

    2005-01-01

    Background Most research has focused on recognition by GPs of the common mental disorders: depression and anxiety. However, less is known about the factors that determine whether patients with those disorders that are recognised receive any active treatment. Aim To investigate factors associated with receiving active treatment among consecutive attenders identified by GPs as having a common mental disorder. Setting Data were collected as part of a cluster randomised controlled trial in 30 general practices in the south of Bristol, UK, on the impact of mental health guidelines in primary care. Method We studied 439 consecutive general practice attenders aged 16–64 years who were given a diagnosis of depression, anxiety, or chronic mixed anxiety and depression by their GP. The main outcome measure was the provision of any active treatment, whether pharmacological or psychological, for these disorders. Patient, GP, and practice level data, including sociodemographic, clinical, and administrative data were explored as predictors in a logistic regression model. Huber White variance estimates were used to account for hierarchical clustering. Results Of those patients identified as having a common mental disorder by the GP, 54% were offered active treatment. Higher symptom score, as measured by the General Health Questionnaire (GHQ) (odds ratio [OR] = 1.09; 95% confidence interval [CI] = 1.06 to 1.13; P<0.001) and being male (OR = 1.54; 95% CI = 1.13 to 2.09; P = 0.006), were both associated with an increased likelihood of being offered active treatment. Patients with anxiety (OR = 0.24; 95% = CI 0.14 to 0.41; P<0.001), or chronic mixed anxiety/depression (OR = 0.41; 95% CI = 0.23 to 0.73; P = 0.003) were less likely to be offered active treatment than those considered to have depression. Conclusion When deciding to offer active treatment for common mental disorders, GPs appear to be influenced by the severity of symptoms rather than their ‘understandability’ in

  5. Trust models in ubiquitous computing.

    PubMed

    Krukow, Karl; Nielsen, Mogens; Sassone, Vladimiro

    2008-10-28

    We recapture some of the arguments for trust-based technologies in ubiquitous computing, followed by a brief survey of some of the models of trust that have been introduced in this respect. Based on this, we argue for the need of more formal and foundational trust models.

  6. The Status of Ubiquitous Computing.

    ERIC Educational Resources Information Center

    Brown, David G.; Petitto, Karen R.

    2003-01-01

    Explains the prevalence and rationale of ubiquitous computing on college campuses--teaching with the assumption or expectation that all faculty and students have access to the Internet--and offers lessons learned by pioneering institutions. Lessons learned involve planning, technology, implementation and management, adoption of computer-enhanced…

  7. Evaluation of a disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) activity enzyme-linked immunosorbent assay for measuring plasma ADAMTS13 activity in dogs.

    PubMed

    Maruyama, Haruhiko; Kaneko, Michiko; Otake, Taiga; Kano, Rui; Yamaya, Yoshiki; Watari, Toshihiro; Hasegawa, Atsuhiko; Kamata, Hiroshi

    2014-03-01

    A disintegrin-like and metalloprotease with thrombospondin type 1 repeat motifs 13 (ADAMTS13) is a von Willebrand factor (vWF)-cleaving protease. Deficiencies in ADAMTS13 activity are known to cause thrombotic diseases in human beings. The present study evaluated whether the human ADAMTS13 activity enzyme-linked immunosorbent assay (ELISA) kit containing human vWF73 (a minimal substrate) and anti-N10 antibody (which specifically recognizes the decapeptide of the C-terminal edge of cleaved vWF by human ADAMTS13) is applicable to the measurement of canine plasma ADAMTS13 activity. Human vWF73 fused with a GST-tag and a His-tag (GST-hvWF73-His) was reacted with recombinant canine (rc)ADAMTS13, canine plasma, and human plasma, and then used in Western blotting using anti-N10 antibody. Linearity and intra- and interassay reproducibility of the human ADAMTS13 activity ELISA kit in canine plasma were further evaluated. Finally, plasma ADAMTS13 activity was measured in 13 healthy dogs and 6 dogs with bacteremia using the human ADAMTS13 activity ELISA kit. Cleaved products with a 28-kDa GST-hvWF73-His were detected specifically in rcADAMTS13 as well as in human ADAMTS13, and also in canine plasma by anti-N10 antibody, showing excellent linearity. Intra-assay coefficient of variation (CV) was 3.0-12.4%, and interassay CV was 11.5-12.5%. The ADAMTS13 activity was significantly lower in dogs with bacteremia than in healthy dogs (P = 0.0025). The current study revealed that the human ADAMTS13 activity ELISA kit is applicable for measurement of canine plasma ADAMTS13 activity to elucidate the pathology of thrombotic diseases in dogs.

  8. Ubiquitous Total Station Development using Smartphone, RSSI and Laser Sensor providing service to Ubi-GIS

    NASA Astrophysics Data System (ADS)

    Shoushtari, M. A.; Sadeghi-Niaraki, H.

    2014-10-01

    The growing trend in technological advances and Micro Electro Mechanical Systems (MEMS) has targeted for intelligent human lives. Accordingly, Ubiquitous Computing Approach was proposed by Mark Weiser. This paper proposes an ubiquitous surveying solution in Geometrics and surveying field. Ubiquitous Surveying provides cost-effective, smart and available surveying techniques while traditional surveying equipment are so expensive and have small availability specially in indoor and daily surveying jobs. In order to have a smart surveying instrument, different information technology methods and tools like Triangle method, Received Signal Strength Indicator (RSSI) method and laser sensor are used. These new ways in combine with surveying equations introduces a modern surveying equipment called Ubi-Total Station that also employed different sensors embedded in smartphone and mobile stand. RSSI-based localization and Triangle method technique are easy and well known methods to predict the position of an unknown node in indoor environments whereas additional measures are required for a sufficient accuracy. In this paper the main goal is to introduce the Ubiquitous Total Station as a development in smart and ubiquitous GIS. In order to public use of the surveying equipment, design and implementation of this instrument has been done. Conceptual model of Smartphone-based system is designed for this study and based on this model, an Android application as a first sample is developed. Finally the evaluations shows that absolute errors in X and Y calculation are 0.028 and 0.057 meter respectively. Also RMSE of 0.26 was calculated in RSSI method for distance measurement. The high price of traditional equipment and their requirement for professional surveyors has given way to intelligent surveying. In the suggested system, smartphones can be used as tools for positioning and coordinating geometric information of objects.

  9. Transcriptional activation of the human inducible nitric-oxide synthase promoter by Kruppel-like factor 6.

    PubMed

    Warke, Vishal G; Nambiar, Madhusoodana P; Krishnan, Sandeep; Tenbrock, Klaus; Geller, David A; Koritschoner, Nicolas P; Atkins, James L; Farber, Donna L; Tsokos, George C

    2003-04-25

    Nitric oxide is a ubiquitous free radical that plays a key role in a broad spectrum of signaling pathways in physiological and pathophysiological processes. We have explored the transcriptional regulation of inducible nitric-oxide synthase (iNOS) by Krüppel-like factor 6 (KLF6), an Sp1-like zinc finger transcription factor. Study of serial deletion constructs of the iNOS promoter revealed that the proximal 0.63-kb region can support a 3-6-fold reporter activity similar to that of the full-length 16-kb promoter. Within the 0.63-kb region, we identified two CACCC sites (-164 to -168 and -261 to -265) that bound KLF6 in both electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutation of both these sites abrogated the KLF6-induced enhancement of the 0.63-kb iNOS promoter activity. The binding of KLF6 to the iNOS promoter was significantly increased in Jurkat cells, primary T lymphocytes, and COS-7 cells subjected to NaCN-induced hypoxia, heat shock, serum starvation, and phorbol 12-myristate 13-acetate/ ionophore stimulation. Furthermore, in KLF6-transfected and NaCN-treated COS-7 cells, there was a 3-4-fold increase in the expression of the endogenous iNOS mRNA and protein that correlated with increased production of nitric oxide. These findings indicate that KLF6 is a potential transactivator of the human iNOS promoter in diverse pathophysiological conditions.

  10. IgG Antibody Responses to Recombinant gp120 Proteins, gp70V1/V2 Scaffolds, and a CyclicV2 Peptide in Thai Phase I/II Vaccine Trials Using Different Vaccine Regimens.

    PubMed

    Karasavvas, Nicos; Karnasuta, Chitraporn; Savadsuk, Hathairat; Madnote, Sirinan; Inthawong, Dutsadee; Chantakulkij, Somsak; Rittiroongrad, Surawach; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Thongcharoen, Prasert; Siriyanon, Vinai; Andrews, Charla A; Barnett, Susan W; Tartaglia, James; Sinangil, Faruk; Francis, Donald P; Robb, Merlin L; Michael, Nelson L; Ngauy, Viseth; de Souza, Mark S; Paris, Robert M; Excler, Jean-Louis; Kim, Jerome H; O'Connell, Robert J

    2015-11-01

    RV144 correlates of risk analysis showed that IgG antibodies to gp70V1V2 scaffolds inversely correlated with risk of HIV acquisition. We investigated IgG antibody responses in RV135 and RV132, two ALVAC-HIV prime-boost vaccine trials conducted in Thailand prior to RV144. Both trials used ALVAC-HIV (vCP1521) at 0, 1, 3, and 6 months and HIV-1 gp120MNgD and gp120A244gD in alum (RV135) or gp120SF2 and gp120CM235 in MF59 (RV132) at 3 and 6 months. We assessed ELISA binding antibodies to the envelope proteins (Env) 92TH023, A244gD and MNgD, cyclicV2, and gp70V1V2 CaseA2 (subtype B) and 92TH023 (subtype CRF01_AE), and Env-specific IgG1 and IgG3. Antibody responses to gp120 A244gD, MNgD, and gp70V1V2 92TH023 scaffold were significantly higher in RV135 than in RV132. Antibodies to gp70V1V2 CaseA2 were detected only in RV135 vaccine recipients and IgG1 and IgG3 antibody responses to A244gD were significantly higher in RV135. IgG binding to gp70V1V2 CaseA2 and CRF01_AE scaffolds was higher with the AIDSVAX(®)B/E boost but both trials showed similar rates of antibody decline post-vaccination. MF59 did not result in higher IgG antibody responses compared to alum with the antigens tested. However, notable differences in the structure of the recombinant proteins and dosage used for immunizations may have contributed to the magnitude and specificity of IgG induced by the two trials.

  11. Live attenuated measles vaccine expressing HIV-1 Gag virus like particles covered with gp160DELTAV1V2 is strongly immunogenic

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guerbois, Mathilde; Moris, Arnaud; Combredet, Chantal

    Although a live attenuated HIV vaccine is not currently considered for safety reasons, a strategy inducing both T cells and neutralizing antibodies to native assembled HIV-1 particles expressed by a replicating virus might mimic the advantageous characteristics of live attenuated vaccine. To this aim, we generated a live attenuated recombinant measles vaccine expressing HIV-1 Gag virus-like particles (VLPs) covered with gp160DELTAV1V2 Env protein. The measles-HIV virus replicated efficiently in cell culture and induced the intense budding of HIV particles covered with Env. In mice sensitive to MV infection, this recombinant vaccine stimulated high levels of cellular and humoral immunity tomore » both MV and HIV with neutralizing activity. The measles-HIV virus infected human professional antigen-presenting cells, such as dendritic cells and B cells, and induced efficient presentation of HIV-1 epitopes and subsequent activation of human HIV-1 Gag-specific T cell clones. This candidate vaccine will be next tested in non-human primates. As a pediatric vaccine, it might protect children and adolescents simultaneously from measles and HIV.« less

  12. Induction of ebolavirus cross-species immunity using retrovirus-like particles bearing the Ebola virus glycoprotein lacking the mucin-like domain

    PubMed Central

    2012-01-01

    Background The genus Ebolavirus includes five distinct viruses. Four of these viruses cause hemorrhagic fever in humans. Currently there are no licensed vaccines for any of them; however, several vaccines are under development. Ebola virus envelope glycoprotein (GP1,2) is highly immunogenic, but antibodies frequently arise against its least conserved mucin-like domain (MLD). We hypothesized that immunization with MLD-deleted GP1,2 (GPΔMLD) would induce cross-species immunity by making more conserved regions accessible to the immune system. Methods To test this hypothesis, mice were immunized with retrovirus-like particles (retroVLPs) bearing Ebola virus GPΔMLD, DNA plasmids (plasmo-retroVLP) that can produce such retroVLPs in vivo, or plasmo-retroVLP followed by retroVLPs. Results Cross-species neutralizing antibody and GP1,2-specific cellular immune responses were successfully induced. Conclusion Our findings suggest that GPΔMLD presented through retroVLPs may provide a strategy for development of a vaccine against multiple ebolaviruses. Similar vaccination strategies may be adopted for other viruses whose envelope proteins contain highly variable regions that may mask more conserved domains from the immune system. PMID:22273269

  13. Role of Passive Capturing in a Ubiquitous Learning Environment

    ERIC Educational Resources Information Center

    Ogata, Hiroaki; Hou, Bin; Li, MengMeng; Uosaki, Noriko; Mouri, Kousuke

    2013-01-01

    Ubiquitous Learning Log (ULL) is defined as a digital record of what you have learned in the daily life using ubiquitous technologies. This paper focuses on how to capture learning experiences in our daily life for vocabulary learning. In our previous works, we developed a system named SCROLL (System for Capturing and Reminding Of Learning Log) in…

  14. Ubiquitous Learning Project Using Life-Logging Technology in Japan

    ERIC Educational Resources Information Center

    Ogata, Hiroaki; Hou, Bin; Li, Mengmeng; Uosaki, Noriko; Mouri, Kosuke; Liu, Songran

    2014-01-01

    A Ubiquitous Learning Log (ULL) is defined as a digital record of what a learner has learned in daily life using ubiquitous computing technologies. In this paper, a project which developed a system called SCROLL (System for Capturing and Reusing Of Learning Log) is presented. The aim of developing SCROLL is to help learners record, organize,…

  15. An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the -Helical Trimeric Coiled Coil

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bhardwaj, A.; Walker-Kopp, N; Casjens, S

    2009-01-01

    Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution.more » All gp26-like factors contain an elongated {alpha}-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 {angstrom}. gp26 tail needles display a high level of structural stability in solution, with Tm (temperature of melting) between 85 and 95 C. To determine how the structural stability of these phage fibers correlates with the length of the {alpha}-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the {alpha}-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the {alpha}-helical core.« less

  16. Describing team development within a novel GP-led urgent care centre model: a qualitative study.

    PubMed

    Morton, Sarah; Ignatowicz, Agnieszka; Gnani, Shamini; Majeed, Azeem; Greenfield, Geva

    2016-06-23

    Urgent care centres (UCCs) co-located within an emergency department were developed to reduce the numbers of inappropriate emergency department admissions. Since then various UCC models have developed, including a novel general practitioner (GP)-led UCC that incorporates both GPs and emergency nurse practitioners (ENPs). Traditionally these two groups do not work alongside each other within an emergency setting. Although good teamwork is crucial to better patient outcomes, there is little within the literature about the development of a team consisting of different healthcare professionals in a novel healthcare setting. Our aim was therefore to describe staff members' perspectives of team development within the GP-led UCC model. Open-ended semistructured interviews, analysed using thematic content analysis. GP-led urgent care centres in two academic teaching hospitals in London. 15 UCC staff members including six GPs, four ENPs, two receptionists and three managers. Overall participants were positive about the interprofessional team that had developed and recognised that this process had taken time. Hierarchy within the UCC setting has diminished with time, although some residual hierarchical beliefs do appear to remain. Staff appreciated interdisciplinary collaboration was likely to improve patient care. Eight key facilitating factors for the team were identified: appointment of leaders, perception of fair workload, education on roles/skill sets and development of these, shared professional understanding, interdisciplinary working, ED collaboration, clinical guidelines and social interactions. A strong interprofessional team has evolved within the GP-led UCCs over time, breaking down traditional professional divides. Future implementation of UCC models should pro-actively incorporate the eight facilitating factors identified from the outset, to enable effective teams to develop more quickly. Published by the BMJ Publishing Group Limited. For permission to use

  17. 65 nm LP/GP mix low cost platform for multi-media wireless and consumer applications

    NASA Astrophysics Data System (ADS)

    Tavel, B.; Duriez, B.; Gwoziecki, R.; Basso, M. T.; Julien, C.; Ortolland, C.; Laplanche, Y.; Fox, R.; Sabouret, E.; Detcheverry, C.; Boeuf, F.; Morin, P.; Barge, D.; Bidaud, M.; Biénacel, J.; Garnier, P.; Cooper, K.; Chapon, J. D.; Trouiller, Y.; Belledent, J.; Broekaart, M.; Gouraud, P.; Denais, M.; Huard, V.; Rochereau, K.; Difrenza, R.; Planes, N.; Marin, M.; Boret, S.; Gloria, D.; Vanbergue, S.; Abramowitz, P.; Vishnubhotla, L.; Reber, D.; Stolk, P.; Woo, M.; Arnaud, F.

    2006-04-01

    A complete 65 nm CMOS platform, called LP/GP Mix, has been developed employing thick oxide transistor (IO), Low Power (LP) and General Purpose (GP) devices on the same chip. Dedicated to wireless multi-media and consumer applications, this new triple gate oxide platform is low cost (+1mask only) and saves over 35% of dynamic power with the use of the low operating voltage GP. The LP/GP mix shows competitive digital performance with a ring oscillator (FO = 1) speed equal to 7 ps per stage (GP) and 6T-SRAM static power lower than 10 pA/cell (LP). Compatible with mixed-signal design requirements, transistors show high voltage gain, low mismatch factor and low flicker noise. Moreover, to address mobile phone demands, excellent RF performance has been achieved with FT = 160 GHz for LP and 280 GHz for GP nMOS transistors.

  18. Structural analysis of a highly glycosylated and unliganded gp120-based antigen using mass spectrometry†

    PubMed Central

    Wang, Liwen; Qin, Yali; Ilchenko, Serguei; Bohon, Jen; Shi, Wuxian; Cho, Michael W.; Takamoto, Keiji; Chance, Mark R.

    2010-01-01

    Structural characterization of the HIV envelope protein gp120 is very important to provide an understanding of the protein's immunogenicity and it's binding to cell receptors. So far, crystallographic structure determination of gp120 with an intact V3 loop (in the absence of CD4 co-receptor or antibody) has not been achieved. The third variable region (V3) of the gp120 is immunodominant and contains glycosylation signatures that are essential for co-receptor binding and viral entry to T-cells. In this study, we characterized the structure of the outer domain of gp120 with an intact V3 loop (gp120-OD8) purified from Drosophila S2 cells utilizing mass spectrometry-based approaches. We mapped the glycosylation sites and calculated glycosylation occupancy of gp120-OD8; eleven sites from fifteen glycosylation motifs were determined as having high mannose or hybrid glycosylation structures. The specific glycan moieties of nine glycosylation sites from eight unique glycopeptides were determined by a combination of ECD and CID MS approaches. Hydroxyl radical-mediated protein footprinting coupled with mass spectrometry analysis was employed to provide detailed information on protein structure of gp120-OD8 by directly identifying accessible and hydroxyl radical-reactive side chain residues. Comparison of gp120-OD8 experimental footprinting data with a homology model derived from the ligated CD4/ gp120-OD8 crystal structure revealed a flexible V3 loop structure where the V3 tip may provide contacts with the rest of the protein while residues in the V3 base remain solvent accessible. In addition, the data illustrate interactions between specific sugar moieties and amino acid side chains potentially important to the gp120-OD8 structure. PMID:20825246

  19. Qualitative study of depression management in primary care: GP and patient goals, and the value of listening

    PubMed Central

    Johnston, Olwyn; Kumar, Satinder; Kendall, Kathleen; Peveler, Robert; Gabbay, John; Kendrick, Tony

    2007-01-01

    Background Guidelines for depression management have been developed but little is known about GP and patient goals, which are likely to influence treatment offers, uptake, and adherence. Aim To identify issues of importance to GPs, patients, and patients' supporters regarding depression management. GP and patient goals for depression management became a focus of the study. Design of study Grounded theory-based qualitative study. Setting GPs were drawn from 28 practices. The majority of patients and supporters were recruited from 10 of these practices. Method Sixty-one patients (28 depressed, 18 previously depressed, 15 never depressed), 18 supporters, and 32 GPs were interviewed. Results GPs described encouraging patients to view depression as separate from the self and ‘normal’ sadness. Patients and supporters often questioned such boundaries, rejecting the notion of a medical cure and emphasising self-management. The majority of participants who were considering depression-management strategies wanted to ‘get out’ of their depression. However, a quarter did not see this as immediately relevant or achievable. They focused on getting by from day to day, which had the potential to clash with GP priorities. GP frustration and uncertainty could occur when depression was resistant to cure. Participants identified the importance of GPs listening to patients, but often felt that this did not happen. Conclusion Physicians need greater awareness of the extent to which their goals for the management of depression are perceived as relevant or achievable by patients. Future research should explore methods of negotiating agreed strategies for management. PMID:17976282

  20. "...they should be offering it": a qualitative study to investigate young peoples' attitudes towards chlamydia screening in GP surgeries

    PubMed Central

    2010-01-01

    Background Despite the known health and healthcare costs of untreated chlamydia infection and the efforts of the National Chlamydia Screening Programme (NCSP) to control chlamydia through early detection and treatment of asymptomatic infection, the rates of screening are well below the 2010-2011 target rate of 35%. General Practitioner (GP) surgeries are a key venue within the NCSP however; previous studies indicate that GP surgery staff are concerned that they may offend their patients by offering a screen. This study aimed to identify the attitudes to, and preferences for, chlamydia screening in 15-24 year old men and women attending GP surgeries (the target group). Methods We undertook 36 interviews in six surgeries of differing screening rates. Our participants were 15-24 year olds attending a consultation with a staff member. Data were analysed thematically. Results GP surgeries are acceptable to young people as a venue for opportunistic chlamydia screening and furthermore they think it is the duty of GP surgery staff to offer it. They felt strongly that it is important for surgery staff to have a non-judgemental attitude and they did not want to be singled out as 'needing' a chlamydia screen. Furthermore, our sample reported a strong preference for being offered a screen by staff and providing the sample immediately at the surgery rather than taking home a testing kit. The positive attitude and subjective norms demonstrated by interviewees suggest that young peoples' behaviour would be to accept a screen if it was offered to them. Conclusion Young people attending GP surgeries have a positive attitude towards chlamydia screening and given the right environment are likely to take up the offer in this setting. The right environment involves normalising screening by offering a chlamydia screen to all 15-24 year olds at every interaction with staff, offering screening with a non-judgemental attitude and minimising barriers to screening such as embarrassment. The

  1. Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.

    2008-04-02

    Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The foldmore » of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.« less

  2. Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

    PubMed

    Schweneker, Marc; Laimbacher, Andrea S; Zimmer, Gert; Wagner, Susanne; Schraner, Elisabeth M; Wolferstätter, Michael; Klingenberg, Marieken; Dirmeier, Ulrike; Steigerwald, Robin; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

    2017-06-01

    There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant. IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified

  3. gp49B-mediated negative regulation of antibody production by memory and marginal zone B cells.

    PubMed

    Fukao, Saori; Haniuda, Kei; Nojima, Takuya; Takai, Toshiyuki; Kitamura, Daisuke

    2014-07-15

    The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases. Copyright © 2014 by The American Association of Immunologists, Inc.

  4. Multimodal and ubiquitous computing systems: supporting independent-living older users.

    PubMed

    Perry, Mark; Dowdall, Alan; Lines, Lorna; Hone, Kate

    2004-09-01

    We document the rationale and design of a multimodal interface to a pervasive/ubiquitous computing system that supports independent living by older people in their own homes. The Millennium Home system involves fitting a resident's home with sensors--these sensors can be used to trigger sequences of interaction with the resident to warn them about dangerous events, or to check if they need external help. We draw lessons from the design process and conclude the paper with implications for the design of multimodal interfaces to ubiquitous systems developed for the elderly and in healthcare, as well as for more general ubiquitous computing applications.

  5. A model for ubiquitous care of noncommunicable diseases.

    PubMed

    Vianna, Henrique Damasceno; Barbosa, Jorge Luis Victória

    2014-09-01

    The ubiquitous computing, or ubicomp, is a promising technology to help chronic diseases patients managing activities, offering support to them anytime, anywhere. Hence, ubicomp can aid community and health organizations to continuously communicate with patients and to offer useful resources for their self-management activities. Communication is prioritized in works of ubiquitous health for noncommunicable diseases care, but the management of resources is not commonly employed. We propose the UDuctor, a model for ubiquitous care of noncommunicable diseases. UDuctor focuses the resources offering, without losing self-management and communication supports. We implemented a system and applied it in two practical experiments. First, ten chronic patients tried the system and filled out a questionnaire based on the technology acceptance model. After this initial evaluation, an alpha test was done. The system was used daily for one month and a half by a chronic patient. The results were encouraging and show potential for implementing UDuctor in real-life situations.

  6. Navigation studies based on the ubiquitous positioning technologies

    NASA Astrophysics Data System (ADS)

    Ye, Lei; Mi, Weijie; Wang, Defeng

    2007-11-01

    This paper summarized the nowadays positioning technologies, such as absolute positioning methods and relative positioning methods, indoor positioning and outdoor positioning, active positioning and passive positioning. Global Navigation Satellite System (GNSS) technologies were introduced as the omnipresent out-door positioning technologies, including GPS, GLONASS, Galileo and BD-1/2. After analysis of the shortcomings of GNSS, indoor positioning technologies were discussed and compared, including A-GPS, Cellular network, Infrared, Electromagnetism, Computer Vision Cognition, Embedded Pressure Sensor, Ultrasonic, RFID (Radio Frequency IDentification), Bluetooth, WLAN etc.. Then the concept and characteristics of Ubiquitous Positioning was proposed. After the ubiquitous positioning technologies contrast and selection followed by system engineering methodology, a navigation system model based on Incorporate Indoor-Outdoor Positioning Solution was proposed. And this model was simulated in the Galileo Demonstration for World Expo Shanghai project. In the conclusion, the prospects of ubiquitous positioning based navigation were shown, especially to satisfy the public location information acquiring requirement.

  7. The glycan-mediated mechanism on the interactions of gp120 with CD4 and antibody: Insights from molecular dynamics simulation.

    PubMed

    Zhang, Yan; Niu, Yuzhen; Tian, Jiaqi; Liu, Xuewei; Yao, Xiaojun; Liu, Huanxiang

    2017-12-01

    N-linked glycans such as 234 and 276 gp 120 glycans are vital components of HIV evasion from humoral immunity and important for HIV-1 neutralization of many broadly neutralizing antibodies (bNAbs). However, it is unknown the action mechanism of two glycans. To investigate the roles of the glycans on the interactions of gp120 with CD4 and antibody, molecular dynamic simulations based on gp120-CD4-8ANC195 complex with 234 and 276 gp 120 glycans, 234 gp 120 glycan, 276 gp 120 glycan, and without glycan were performed. Our results reveal that 276 gp 120 glycan can enhance gp120-CD4 and gp120-antibody interactions through the formation of hydrogen bonds of the glycan with CD4 and antibody and make the binding interface of gp120, CD4 and antibody stable; 234 gp 120 glycan primarily reinforces gp120-antibody interactions and weakly affects gp120-CD4 interactions as it mainly lies between gp120 and antibody. The co-operating of two glycans can enhance gp120-CD4 and gp120-antibody associations. Through the structural analysis, it can be seen that 234 gp 120 glycan leads to moving upward of two glycans and the variable region of heavy chain, which is favorable for the interactions of gp120 with CD4 and antibody. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors. © 2017 John Wiley & Sons A/S.

  8. Distinct Stabilities of the Structurally Homologous Heptameric Co-Chaperonins GroES and gp31

    NASA Astrophysics Data System (ADS)

    Dyachenko, Andrey; Tamara, Sem; Heck, Albert J. R.

    2018-05-01

    The GroES heptamer is the molecular co-chaperonin that partners with the tetradecamer chaperonin GroEL, which assists in the folding of various nonnative polypeptide chains in Escherichia coli. Gp31 is a structural and functional analogue of GroES encoded by the bacteriophage T4, becoming highly expressed in T4-infected E. coli, taking over the role of GroES, favoring the folding of bacteriophage proteins. Despite being slightly larger, gp31 is quite homologous to GroES in terms of its tertiary and quaternary structure, as well as in its function and mode of interaction with the chaperonin GroEL. Here, we performed a side-by-side comparison of GroES and gp31 heptamer complexes by (ion mobility) tandem mass spectrometry. Surprisingly, we observed quite distinct fragmentation mechanisms for the GroES and gp31 heptamers, whereby GroES displays a unique and unusual bimodal charge distribution in its released monomers. Not only the gas-phase dissociation but also the gas-phase unfolding of GroES and gp31 were found to be very distinct. We rationalize these observations with the similar discrepancies we observed in the thermal unfolding characteristics and surface contacts within GroES and gp31 in the solution. From our data, we propose a model that explains the observed simultaneous dissociation pathways of GroES and the differences between GroES and gp31 gas-phase dissociation and unfolding. We conclude that, although GroES and gp31 exhibit high homology in tertiary and quaternary structure, they are quite distinct in their solution and gas-phase (un)folding characteristics and stability. [Figure not available: see fulltext.

  9. Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1.

    PubMed

    Gohain, Neelakshi; Tolbert, William D; Acharya, Priyamvada; Yu, Lei; Liu, Tongyun; Zhao, Pingsen; Orlandi, Chiara; Visciano, Maria L; Kamin-Lewis, Roberta; Sajadi, Mohammad M; Martin, Loïc; Robinson, James E; Kwong, Peter D; DeVico, Anthony L; Ray, Krishanu; Lewis, George K; Pazgier, Marzena

    2015-09-01

    Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes). Our previous studies defined the A32-like epitope within the cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions of gp120. Here, we elucidate additional cluster A epitope structures, including an A32-like epitope, recognized by human monoclonal antibody (MAb) N60-i3, and a hybrid A32-C11-like epitope, recognized by rhesus macaque MAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like subregion and the seven-layered β-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function in the cluster A region depends on precise epitope targeting--a combination of epitope footprint and mode of antibody attachment. All together these findings help further an understanding of how cluster A epitopes are targeted by humoral responses. HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication, no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV

  10. Systemic blockade of dopamine D2-like receptors increases high-voltage spindles in the globus pallidus and motor cortex of freely moving rats.

    PubMed

    Yang, Chen; Ge, Shun-Nan; Zhang, Jia-Rui; Chen, Lei; Yan, Zhi-Qiang; Heng, Li-Jun; Zhao, Tian-Zhi; Li, Wei-Xin; Jia, Dong; Zhu, Jun-Ling; Gao, Guo-Dong

    2013-01-01

    High-voltage spindles (HVSs) have been reported to appear spontaneously and widely in the cortical-basal ganglia networks of rats. Our previous study showed that dopamine depletion can significantly increase the power and coherence of HVSs in the globus pallidus (GP) and motor cortex of freely moving rats. However, it is unclear whether dopamine regulates HVS activity by acting on dopamine D₁-like receptors or D₂-like receptors. We employed local-field potential and electrocorticogram methods to simultaneously record the oscillatory activities in the GP and primary motor cortex (M1) in freely moving rats following systemic administration of dopamine receptor antagonists or saline. The results showed that the dopamine D₂-like receptor antagonists, raclopride and haloperidol, significantly increased the number and duration of HVSs, and the relative power associated with HVS activity in the GP and M1 cortex. Coherence values for HVS activity between the GP and M1 cortex area were also significantly increased by dopamine D₂-like receptor antagonists. On the contrary, the selective dopamine D₁-like receptor antagonist, SCH23390, had no significant effect on the number, duration, or relative power of HVSs, or HVS-related coherence between M1 and GP. In conclusion, dopamine D₂-like receptors, but not D₁-like receptors, were involved in HVS regulation. This supports the important role of dopamine D₂-like receptors in the regulation of HVSs. An siRNA knock-down experiment on the striatum confirmed our conclusion.

  11. Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

    PubMed Central

    Xue, Gondga; von Schubert, Conrad; Hermann, Pascal; Peyer, Martina; Maushagen, Regina; Schmuckli-Maurer, Jacqueline; Bütikofer, Peter; Langsley, Gordon; Dobbelaere, Dirk A.E.

    2010-01-01

    Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite–host interactions during host cell division. PMID:20381541

  12. Lessons from the GP-B Experience for Future Fundamental Physics Missions in Space

    NASA Technical Reports Server (NTRS)

    Kolodziejczak, Jeffery

    2006-01-01

    Gravity Probe B launched in April 2004 and completed its science data collection in September 2005, with the objective of sub-milliarcsec measurement of two General Relativistic effects on the spin axis orientation of orbiting gyroscopes. Much of the technology required by GP-B has potential application in future missions intended to make precision measurements. The philosophical approach and experiment design principles developed for GP-B are equally adaptable to these mission concepts. This talk will discuss GP-B's experimental approach and the technological and philosophical lessons learned that apply to future experiments in fundamental physics. Measurement of fundamental constants to high precision, probes of short-range forces, searches for equivalence principle violations, and detection of gravitational waves are examples of concepts and missions that will benefit kern GP-B's experience.

  13. Which positive factors determine the GP satisfaction in clinical practice? A systematic literature review.

    PubMed

    Le Floch, B; Bastiaens, H; Le Reste, J Y; Lingner, H; Hoffman, R D; Czachowski, S; Assenova, R; Koskela, T H; Klemenc-Ketis, Z; Nabbe, P; Sowinska, A; Montier, T; Peremans, L

    2016-09-13

    Looking at what makes General Practitioners (GPs) happy in their profession, may be important in increasing the GP workforce in the future. The European General Practice Research Network (EGPRN) created a research team (eight national groups) in order to clarify the factors involved in GP job satisfaction throughout Europe. The first step of this study was a literature review to explore how the satisfaction of GPs had been studied before. The research question was "Which factors are related to GP satisfaction in Clinical Practice?" Systematic literature review according to the PRISMA statement. The databases searched were Pubmed, Embase and Cochrane. All articles were identified, screened and included by two separate research teams, according to inclusion or exclusion criteria. Then, a qualitative appraisal was undertaken. Next, a thematic analysis process was undertaken to capture any issue relevant to the research question. The number of records screened was 458. One hundred four were eligible. Finally, 17 articles were included. The data revealed 13 subthemes, which were grouped into three major themes for GP satisfaction. First there were general profession-related themes, applicable to many professions. A second group of issues related specifically to a GP setting. Finally, a third group was related to professional life and personal issues. A number of factors leading to GP job satisfaction, exist in literature They should be used by policy makers within Europe to increase the GP workforce. The research team needs to undertake qualitative studies to confirm or enhance those results.

  14. Deformation behavior of additively manufactured GP1 stainless steel

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clausen, B.; Brown, D. W.; Carpenter, J. S.

    In-situ neutron diffraction measurements were performed in this paper during heat-treating and uniaxial loading of additively manufactured (AM) GP1 material. Although the measured chemical composition of the GP1 powder falls within the composition specifications of 17-4 PH steel, a fully martensitic alloy in the wrought condition, the crystal structure of the as-built GP1 material is fully austenitic. Chemical analysis of the as-built material shows high oxygen and nitrogen content, which then significantly decreased after heat-treating in a vacuum furnace at 650 °C for one hour. Significant austenite-to-martensite phase transformation is observed during compressive and tensile loading of the as-built andmore » heat-treated material with accompanied strengthening as martensite volume fraction increases. During loading, the initial average phase stress state in the martensite is hydrostatic compression independent of the loading direction. Finally, preferred orientation transformation in austenite and applied load accommodation by variant selection in martensite are observed via measurements of the texture development.« less

  15. Deformation behavior of additively manufactured GP1 stainless steel

    DOE PAGES

    Clausen, B.; Brown, D. W.; Carpenter, J. S.; ...

    2017-04-22

    In-situ neutron diffraction measurements were performed in this paper during heat-treating and uniaxial loading of additively manufactured (AM) GP1 material. Although the measured chemical composition of the GP1 powder falls within the composition specifications of 17-4 PH steel, a fully martensitic alloy in the wrought condition, the crystal structure of the as-built GP1 material is fully austenitic. Chemical analysis of the as-built material shows high oxygen and nitrogen content, which then significantly decreased after heat-treating in a vacuum furnace at 650 °C for one hour. Significant austenite-to-martensite phase transformation is observed during compressive and tensile loading of the as-built andmore » heat-treated material with accompanied strengthening as martensite volume fraction increases. During loading, the initial average phase stress state in the martensite is hydrostatic compression independent of the loading direction. Finally, preferred orientation transformation in austenite and applied load accommodation by variant selection in martensite are observed via measurements of the texture development.« less

  16. 'Just a GP': a mixed method study of undermining of general practice as a career choice in the UK.

    PubMed

    Alberti, Hugh; Banner, Kimberley; Collingwood, Helen; Merritt, Kymberlee

    2017-11-03

    Failure to recruit sufficient applicants to general practice (GP) training has been a problem both nationally and internationally for many years and undermining of GP is one possible contributing factor. The aim of our study was to ascertain what comments, both negative and positive, are being made in UK clinical settings to GP trainees about GP and to further explore these comments and their influence on career choice. We conducted a mixed methods study. We surveyed all foundation doctors and GP trainees within one region of Health Education England regarding any comments they experienced relating to a career in GP. We also conducted six focus groups with early GP trainees to discuss any comments that they experienced and whether these comments had any influence on their or others career choice. Positive comments reported by trainees centred around the concept that choosing GP is a positive, family-focused choice which facilities a good work-life balance. Workload was the most common negative comment, alongside the notion of being 'just a GP'; the belief that GP is boring, a waste of training and a second-class career choice. The reasons for and origin of the comments are multifactorial in nature. Thematic analysis of the focus groups identified key factors such as previous exposure to and experience of GP, family members who were GPs, GP role models, demographics of the clinician and referral behaviour. Trainees perceived that negative comments may be discouraging others from choosing GP as a career. Our study demonstrates that negative comments towards GP as a career do exist within clinical settings and are having a potential impact on poor recruitment rates to GP training. We have identified areas in which further negative comments could be prevented by changing perceptions of GP as a career. Additional time spent in GP as undergraduates and postgraduates, and positive GP role models, could particularly benefit recruitment. We recommend that undermining of GP

  17. Initial antibodies binding to HIV-1 gp41 in acutely infected subjects are polyreactive and highly mutated

    PubMed Central

    Chen, Xi; Munshaw, Supriya; Zhang, Ruijun; Marshall, Dawn J.; Vandergrift, Nathan; Whitesides, John F.; Lu, Xiaozhi; Yu, Jae-Sung; Hwang, Kwan-Ki; Gao, Feng; Markowitz, Martin; Heath, Sonya L.; Bar, Katharine J.; Goepfert, Paul A.; Montefiori, David C.; Shaw, George C.; Alam, S. Munir; Margolis, David M.; Denny, Thomas N.; Boyd, Scott D.; Marshal, Eleanor; Egholm, Michael; Simen, Birgitte B.; Hanczaruk, Bozena; Fire, Andrew Z.; Voss, Gerald; Kelsoe, Garnett; Tomaras, Georgia D.; Moody, M. Anthony; Kepler, Thomas B.

    2011-01-01

    The initial antibody response to HIV-1 is targeted to envelope (Env) gp41, and is nonneutralizing and ineffective in controlling viremia. To understand the origins and characteristics of gp41-binding antibodies produced shortly after HIV-1 transmission, we isolated and studied gp41-reactive plasma cells from subjects acutely infected with HIV-1. The frequencies of somatic mutations were relatively high in these gp41-reactive antibodies. Reverted unmutated ancestors of gp41-reactive antibodies derived from subjects acutely infected with HIV-1 frequently did not react with autologous HIV-1 Env; however, these antibodies were polyreactive and frequently bound to host or bacterial antigens. In one large clonal lineage of gp41-reactive antibodies, reactivity to HIV-1 Env was acquired only after somatic mutations. Polyreactive gp41-binding antibodies were also isolated from uninfected individuals. These data suggest that the majority of gp41-binding antibodies produced after acute HIV-1 infection are cross-reactive responses generated by stimulating memory B cells that have previously been activated by non–HIV-1 antigens. PMID:21987658

  18. In vitro investigation of anodization and CaP deposited titanium surface using MG63 osteoblast-like cells

    NASA Astrophysics Data System (ADS)

    Lee, J. M.; Lee, J. I.; Lim, Y. J.

    2010-03-01

    The aim of the present study was to investigate surface characteristics in four different titanium surfaces (AN: anodized at 270 V; AN-CaP: anodic oxidation and CaP deposited; SLA: sandblasted and acid etched; MA: machined) and to evaluate biological behaviors such as cell adhesion, cell proliferation, cytoskeletal organization, and osteogenic protein expression of MG63 osteoblast-like cells at the early stage. Surface analysis was performed using scanning electron microscopy, thin-film X-ray diffractometry, and a confocal laser scanning microscope. In order to evaluate cellular responses, MG63 osteoblast-like cells were used. The cell viability was evaluated by MTT assay. Immunofluorescent analyses of actin, type I collagen, osteonectin and osteocalcin were performed. The anodized and CaP deposited specimen showed homogeneously distributed CaP particles around micropores and exhibited anatase type oxides, titanium, and HA crystalline structures. This experiment suggests that CaP particles on the anodic oxidation surface affect cellular attachment and spreading. When designing an in vitro biological study for CaP coated titanium, it must be taken into account that preincubation in medium prior to cell seeding and the cell culture medium may affect the CaP coatings. All these observations illustrate the importance of the experimental conditions and the physicochemical parameters of the CaP coating. It is considered that further evaluations such as long-term in vitro cellular assays and in vivo experiments should be necessary to figure out the effect of CaP deposition to biological responses.

  19. GpIIb/IIIa+ subpopulation of rat megakaryocyte progenitor cells exhibits high responsiveness to human thrombopoietin.

    PubMed

    Kato, T; Horie, K; Hagiwara, T; Maeda, E; Tsumura, H; Ohashi, H; Miyazaki, H

    1996-08-01

    The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIIb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa- population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/IIIa- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIIa+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

  20. P-glycoprotein Inhibition Increases the Brain Distribution and Antidepressant-Like Activity of Escitalopram in Rodents

    PubMed Central

    O'Brien, Fionn E; O'Connor, Richard M; Clarke, Gerard; Dinan, Timothy G; Griffin, Brendan T; Cryan, John F

    2013-01-01

    Despite the clinical prevalence of the antidepressant escitalopram, over 30% of escitalopram-treated patients fail to respond to treatment. Recent gene association studies have highlighted a potential link between the drug efflux transporter P-glycoprotein (P-gp) and response to escitalopram. The present studies investigated pharmacokinetic and pharmacodynamic interactions between P-gp and escitalopram. In vitro bidirectional transport studies revealed that escitalopram is a transported substrate of human P-gp. Microdialysis-based pharmacokinetic studies demonstrated that administration of the P-gp inhibitor cyclosporin A resulted in increased brain levels of escitalopram without altering plasma escitalopram levels in the rat, thereby showing that P-gp restricts escitalopram transport across the blood–brain barrier (BBB) in vivo. The tail suspension test (TST) was carried out to elucidate the pharmacodynamic impact of P-gp inhibition on escitalopram effect in a mouse model of antidepressant activity. Pre-treatment with the P-gp inhibitor verapamil enhanced the response to escitalopram in the TST. Taken together, these data indicate that P-gp may restrict the BBB transport of escitalopram in humans, potentially resulting in subtherapeutic brain concentrations in certain patients. Moreover, by verifying that increasing escitalopram delivery to the brain by P-gp inhibition results in enhanced antidepressant-like activity, we suggest that adjunctive treatment with a P-gp inhibitor may represent a beneficial approach to augment escitalopram therapy in depression. PMID:23670590

  1. Use of Ubiquitous Technologies in Military Logistic System in Iran

    NASA Astrophysics Data System (ADS)

    Jafari, P.; Sadeghi-Niaraki, A.

    2013-09-01

    This study is about integration and evaluation of RFID and ubiquitous technologies in military logistic system management. Firstly, supply chain management and the necessity of a revolution in logistic systems especially in military area, are explained. Secondly RFID and ubiquitous technologies and the advantages of their use in supply chain management are introduced. Lastly a system based on these technologies for controlling and increasing the speed and accuracy in military logistic system in Iran with its unique properties, is presented. The system is based on full control of military logistics (supplies) from the time of deployment to replenishment using sensor network, ubiquitous and RFID technologies.

  2. Ubiquitous Computing: The Universal Use of Computers on College Campuses.

    ERIC Educational Resources Information Center

    Brown, David G., Ed.

    This book is a collection of vignettes from 13 universities where everyone on campus has his or her own computer. These 13 institutions have instituted "ubiquitous computing" in very different ways at very different costs. The chapters are: (1) "Introduction: The Ubiquitous Computing Movement" (David G. Brown); (2) "Dartmouth College" (Malcolm…

  3. Ubiquitous Learning: Determinants Impacting Learners' Satisfaction and Performance with Smartphones

    ERIC Educational Resources Information Center

    Jung, Hee-Jung

    2014-01-01

    Although the concept of ubiquitous technologies has been introduced to many parts of society, there have been limited applications, and little is known about learners' behavior toward ubiquitous technologies, particularly in the context of English learning. This study considers a sample of Korean students to identify the key factors that influence…

  4. Influences on GP coping and resilience: a qualitative study in primary care.

    PubMed

    Cheshire, Anna; Ridge, Damien; Hughes, John; Peters, David; Panagioti, Maria; Simon, Chantal; Lewith, George

    2017-06-01

    'Neoliberal' work policies, austerity, NHS restructuring, and increased GP consultation rates provide the backdrop against increasing reports of GP burnout and an impending shortage of GPs. To explore GPs' experiences of workplace challenges and stresses, and their coping strategies, particularly focusing on understanding the impact of recent NHS workplace change. Study design was qualitative, with data collected from two focus groups and seven one-to-one telephone interviews. Focus groups and one-to-one telephone interviews explored the experiences of GPs currently practising in England, recruited through convenience sampling. Data were collected using a semi-structured interview approach and analysed using thematic analysis. There were 22 GP participants recruited: focus groups ( n = 15) and interviews ( n = 7). Interviewees understood GPs to be under intense and historically unprecedented pressures, which were tied to the contexts in which they work, with important moral implications for 'good' doctoring. Many reported that being a full-time GP was too stressful: work-related stress led to mood changes, sleep disruption, increases in anxiety, and tensions with loved ones. Some had subsequently sought ways to downsize their clinical workload. Workplace change resulted in little time for the things that helped GP resilience: a good work-life balance and better contact with colleagues. Although some GPs were coping better than others, GPs acknowledged that there was only so much an individual GP could do to manage their stress, given the external work issues they faced. GPs experience their emotional lives and stresses as being meaningfully shaped by NHS factors. To support GPs to provide effective care, resilience building should move beyond the individual to include systemic work issues. © British Journal of General Practice 2017.

  5. Blended learning in CME: the perception of GP trainers.

    PubMed

    Te Pas, E; Meinema, J G; Visser, M R M; van Dijk, N

    2016-05-01

    Blended learning (the combination of electronic methods with traditional teaching methods) has the potential to combine the best of traditional education with the best of computer-mediated training. We chose to develop such an intervention for GP trainers who were undertaking a Continuing Medical Education (CME) course in evidence-based medicine (EBM). This study reports on our experience and investigated the factors influencing the perception on usefulness and logistics of blended learning for learners in CME. In total, 170 GP trainers participated in the intervention. We used questionnaires, observations during the four face-to-face meetings and evaluations in the e-course over one year. Additionally we organised focus groups to gain insight in some of the outcomes of the questionnaires and interpretations of the observations. The GP trainers found the design and the educational method (e-course in combination with meetings) attractive, instructive and complementary. Factors influencing their learning were (1) educational design, (2) educational method, (3) topic of the intervention, (4) time (planning), (5) time (intervention), (6) learning style, (7) technical issues, (8) preconditions and (9) level of difficulty. A close link between daily practice and the educational intervention was considered an important precondition for the success of the intervention in this group of learners. GP trainers were positive about blended learning: they found e-learning a useful way to gain knowledge and the meetings a pleasant way of transferring the knowledge into practice. Although some preconditions should be taken into consideration during its development and implementation, they would participate in similarly designed learning in the future.

  6. Co-distribution of cysteine cathepsins and matrix metalloproteases in human dentin.

    PubMed

    Scaffa, Polliana Mendes Candia; Breschi, Lorenzo; Mazzoni, Annalisa; Vidal, Cristina de Mattos Pimenta; Curci, Rosa; Apolonio, Fabianni; Gobbi, Pietro; Pashley, David; Tjäderhane, Leo; Tersariol, Ivarne Luis Dos Santos; Nascimento, Fábio Dupart; Carrilho, Marcela Rocha

    2017-02-01

    It has been hypothesized that cysteine cathepsins (CTs) along with matrix metalloproteases (MMPs) may work in conjunction in the proteolysis of mature dentin matrix. The aim of this study was to verify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralized dentin; and further to evaluate the activity of CTs and MMPs in the same tissue. The distribution of CT-B and CT-K in sound human dentin was assessed by immunohistochemistry. A double-immunolabeling technique was used to identify, at once, the occurrence of those enzymes in dentin. Activities of CTs and MMPs in dentin extracts were evaluated spectrofluorometrically. In addition, in situ gelatinolytic activity of dentin was assayed by zymography. The results revealed the distribution of CT-B and CT-K along the dentin organic matrix and also indicated co-occurrence of MMPs and CTs in that tissue. The enzyme kinetics studies showed proteolytic activity in dentin extracts for both classes of proteases. Furthermore, it was observed that, at least for sound human dentin matrices, the activity of MMPs seems to be predominant over the CTs one. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. What is a good general practitioner (GP)? The development and evaluation of a multi-source feedback instrument for GP appraisal.

    PubMed

    Shepherd, Annabel; Lough, Murray

    2010-05-01

    Although multi-source feedback (MSF) has been used in primary healthcare, the development of an MSF instrument specific to this setting in the UK has not been previously described. The aims of this study were to develop and evaluate an MSF instrument for GPs in Scotland taking part in appraisal. The members of ten primary healthcare teams in the west of Scotland were asked to provide comments in answer to the question, 'What is a good GP?'. The data were reduced and coded by two researchers and questions were devised. Following content validity testing the MSF process was evaluated with volunteers using face-to-face interviews and a postal survey. Thirty-seven statements covering the six domains of communication skills, professional values, clinical care, working with colleagues, personality issues and duties and responsibilities were accepted as relevant by ten primary healthcare teams using a standard of 80 percent agreement. The evaluation found the MSF process to be feasible and acceptable and participants provided some evidence of educational impact. An MSF instrument for GPs has been developed based on the concept of 'the good GP' as described by the primary healthcare team. The evaluation of the resultant MSF process illustrates the potential of MSF, when delivered in the supportive environment of GP appraisal, to provide feedback which has the possibility of improving working relationships between GPs and their colleagues.

  8. HIV-1 gp120 envelope glycoprotein determinants for cytokine burst in human monocytes

    PubMed Central

    Coutu, Mathieu; Prévost, Jérémie; Brassard, Nathalie; Peres, Adam; Stegen, Camille; Madrenas, Joaquín; Kaufmann, Daniel E.; Finzi, Andrés

    2017-01-01

    The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy. PMID:28346521

  9. How can educators support general practice (GP) trainees to develop resilience to prevent burnout?

    PubMed

    Sales, Bryony; Macdonald, Alexandra; Scallan, Samantha; Crane, Sue

    2016-11-01

    Burnout impacts adversely on professional and personal life, and holds implications for patient care. Current research on burnout mainly focuses on established general practitioners but it is unclear how early the signs of burnout really start. This work seeks to identify whether specific GP trainee groups are particularly at risk of burnout and the aspects of training they find stressful. A longitudinal cohort study, collecting qualitative and quantitative data through a single mode of data collection (questionnaire) took place with trainees from all GP training years (ST1-3), across a vocational training scheme (n = 48). Data gathered included the Oldenburg Burnout Inventory (OLBI). Higher than anticipated levels of burnout were displayed by all trainees. A sub-group self reporting higher levels of burnout comprised all-female, UK-trained-at-undergraduate GP trainees, with a partner but no children. Top reported stressors included knowledge/uncertainty, workload/time pressures and ePortfolio. Less than 50% of trainees perceived their burnout levels to be as high as their OLBI showing potential lack of insight. This research demonstrates that high levels of burnout are experienced in GP trainees as early as the first year of training. Early identification of burnout amongst trainees is essential by GP educators to help protect the future GP workforce.

  10. Expression and characterization of human group C rotavirus virus-like particles in insect cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clark, Kristina B.; Lin, S.-C.; Humphrey, Charles

    2009-05-10

    Group C rotavirus (GpC RV) is a causative agent of acute gastroenteritis in children and adults. We expressed the three major capsid proteins VP2, VP6 and VP7 of human GpC RV in baculovirus and demonstrated the self-assembly of VP2/6/7 or VP6/7 virus-like particles (VLPs) in insect cells. We examined a number of parameters, including the kinetics of protein synthesis in different cell lines and media, to optimize the most favorable conditions for the synthesis of recombinant viral proteins and the production of VLPs in Sf9 cells. Hyperimmune serum to VP2/6/7 and VP6/7 VLPs recognized individual recombinant proteins of human GpCmore » RV by Western blot analysis. This serum also showed specific reactivities with the corresponding GpC VLPs but not GpA RV by using immune electron microscopy (IEM) and enzyme immunoassay (EIA). The ability to produce an unlimited amount of GpC RV antigen and the availability of high quality antibody will allow us to develop sensitive and specific diagnostic assays to better determine the epidemiology and disease burden of GpC RV in humans.« less

  11. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

    PubMed Central

    Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

    2012-01-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  12. Resveratrol-decreased hyperalgesia mediated by the P2X7 receptor in gp120-treated rats.

    PubMed

    Wu, Bing; Ma, Yucheng; Yi, Zhihua; Liu, Shuangmei; Rao, Shenqiang; Zou, Lifang; Wang, Shouyu; Xue, Yun; Jia, Tianyu; Zhao, Shanhong; Shi, Liran; Li, Lin; Yuan, Huilong; Liang, Shangdong

    2017-01-01

    Background Chronic pain is a common symptom in human immunodeficiency virus (HIV)-1 infection/acquired immunodeficiency syndrome patients. The literature shows that the HIV envelope glycoprotein 120 (gp120) can directly cause hyperalgesia by stimulating primary sensory afferent nerves. The P2X 7 receptor in the dorsal root ganglia (DRG) is closely related to neuropathic and inflammatory pain. In this study, we aimed to explore the effect of resveratrol (RES) on gp120-induced neuropathic pain that is mediated by the P2X 7 receptor in the rat DRG. Results Mechanical hyperalgesia in rats treated with gp120 was increased compared with that in the sham group. The P2X 7 expression levels in rats treated with gp120 were higher than those in the sham group. Co-localization of the P2X 7 receptor and glial fibrillary acidic protein (GFAP, a marker of satellite glial cells [SGCs]) in the DRG SGCs of the gp120 group exhibited more intense staining than that of the sham group. RES decreased the mechanical hyperalgesia and P2X 7 expression levels in gp120 treatment rats. Co-localization of the P2X 7 receptor and GFAP in the gp120+ RES group was significantly decreased compared to the gp120 group. RES decreased the IL-1β and TNF-α receptor (R) expression levels and ERK1/2 phosphorylation levels as well as increased IL-10 expression in the DRG of gp120-treated rats. Whole cell clamping demonstrated that RES significantly inhibited adenosine triphosphate-activated currents in HEK293 cells that were transfected with the P2X 7 plasmid. Conclusions RES relieved mechanical hyperalgesia in gp120-treated rats by inhibiting the P2X 7 receptor.

  13. Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor.

    PubMed

    Perrone, Maria Grazia; Inglese, Carmela; Berardi, Francesco; Leopoldo, Marcello; Perrone, Roberto; Colabufo, Nicola Antonio

    2013-01-01

    P-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood-brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by in vivo experiments. Here we validate the new dye-probe beta-amyloid (1-40) HiLyte Fluor™ TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using MC18 or MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine. Male Wistar rats' everted intestines were divided into sacs, each sac was filled with 10μM Ab-HiLyte with or without 50μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594nm at each appropriate time. The Ab-HiLyte P-gp mediated efflux had a K=1.00×10(-2)min(-1) and t(1/2)=68.74min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K=1.65×10(-3)min(-1)) and the half life is extremely increased (t(1/2)=419min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K=1.81×10(-2)min(-1) and t(1/2)=38.28min). The results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Ancylostoma ceylanicum Excretory-Secretory Protein 2 Adopts a Netrin-Like Fold and Defines a Novel Family of Nematode Proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    K Kucera; L Harrison; M Cappello

    2011-12-31

    Hookworms are human parasites that have devastating effects on global health, particularly in underdeveloped countries. Ancylostoma ceylanicum infects humans and animals, making it a useful model organism to study disease pathogenesis. A. ceylanicum excretory-secretory protein 2 (AceES-2), a highly immunoreactive molecule secreted by adult worms at the site of intestinal attachment, is partially protective when administered as a mucosal vaccine against hookworm anemia. The crystal structure of AceES-2 determined at 1.75 {angstrom} resolution shows that it adopts a netrin-like fold similar to that found in tissue inhibitors of matrix metalloproteases (TIMPs) and in complement factors C3 and C5. However, recombinantmore » AceES-2 does not significantly inhibit the 10 most abundant human matrix metalloproteases or complement-mediated cell lysis. The presence of a highly acidic surface on AceES-2 suggests that it may function as a cytokine decoy receptor. Several small nematode proteins that have been annotated as TIMPs or netrin-domain-containing proteins display sequence homology in structurally important regions of AceES-2's netrin-likefold. Together, our results suggest that AceES-2 defines a novel family of nematode netrin-like proteins, which may function to modulate the host immune response to hookworm and other parasites.« less

  15. Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Duan, Liangwei; Du, Jiansen; Wang, Xuefeng

    Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesismore » of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine. - Highlights: • The crystal structure of EIAV gp45 was determined. • The fusion peptide proximal region adopts a novel conformation different to HIV-1. • The asparagine-rich layer includes an extensive hydrogen-bond network. • These regions of EIAV are highly tolerant to mutations. • The results provide insight into the mechanism of gp41/gp45-mediated membrane fusion.« less

  16. HIV Glycoprotein Gp120 Impairs Fast Axonal Transport by Activating Tak1 Signaling Pathways

    PubMed Central

    Berth, Sarah H.; Mesnard-Hoaglin, Nichole; Wang, Bin; Kim, Hajwa; Song, Yuyu; Sapar, Maria; Morfini, Gerardo

    2016-01-01

    Sensory neuropathies are the most common neurological complication of HIV. Of these, distal sensory polyneuropathy (DSP) is directly caused by HIV infection and characterized by length-dependent axonal degeneration of dorsal root ganglion (DRG) neurons. Mechanisms for axonal degeneration in DSP remain unclear, but recent experiments revealed that the HIV glycoprotein gp120 is internalized and localized within axons of DRG neurons. Based on these findings, we investigated whether intra-axonal gp120 might impair fast axonal transport (FAT), a cellular process critical for appropriate maintenance of the axonal compartment. Significantly, we found that gp120 severely impaired both anterograde and retrograde FAT. Providing a mechanistic basis for these effects, pharmacological experiments revealed an involvement of various phosphotransferases in this toxic effect, including members of mitogen-activated protein kinase pathways (Tak-1, p38, and c-Jun N-terminal Kinase (JNK)), inhibitor of kappa-B-kinase 2 (IKK2), and PP1. Biochemical experiments and axonal outgrowth assays in cell lines and primary cultures extended these findings. Impairments in neurite outgrowth in DRG neurons by gp120 were rescued using a Tak-1 inhibitor, implicating a Tak-1 mitogen-activated protein kinase pathway in gp120 neurotoxicity. Taken together, these observations indicate that kinase-based impairments in FAT represent a novel mechanism underlying gp120 neurotoxicity consistent with the dying-back degeneration seen in DSP. Targeting gp120-based impairments in FAT with specific kinase inhibitors might provide a novel therapeutic strategy to prevent axonal degeneration in DSP. PMID:27872270

  17. The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41.

    PubMed

    Squeglia, Flavia; Bachert, Beth; De Simone, Alfonso; Lukomski, Slawomir; Berisio, Rita

    2014-02-21

    The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

  18. The role of the T7 Gp2 inhibitor of host RNA polymerase in phage development.

    PubMed

    Savalia, Dhruti; Robins, William; Nechaev, Sergei; Molineux, Ian; Severinov, Konstantin

    2010-09-10

    Bacteriophage T7 relies on its own RNA polymerase (RNAp) to transcribe its middle and late genes. Early genes, which include the viral RNAp gene, are transcribed by the host RNAp from three closely spaced strong promoters-A1, A2, and A3. One middle T7 gene product, gp2, is a strong inhibitor of the host RNAp. Gp2 is essential and is required late in infection, during phage DNA packaging. Here, we explore the role of gp2 in controlling host RNAp transcription during T7 infection. We demonstrate that in the absence of gp2, early viral transcripts continue to accumulate throughout the infection. Decreasing transcription from early promoter A3 is sufficient to make gp2 dispensable for phage infection. Gp2 also becomes dispensable when an antiterminating element boxA, located downstream of early promoters, is deleted. The results thus suggest that antiterminated transcription by host RNAp from the A3 promoter is interfering with phage development and that the only essential role for gp2 is to prevent this transcription. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. Patients' perceptions of GP non-verbal communication: a qualitative study.

    PubMed

    Marcinowicz, Ludmila; Konstantynowicz, Jerzy; Godlewski, Cezary

    2010-02-01

    During doctor-patient interactions, many messages are transmitted without words, through non-verbal communication. To elucidate the types of non-verbal behaviours perceived by patients interacting with family GPs and to determine which cues are perceived most frequently. In-depth interviews with patients of family GPs. Nine family practices in different regions of Poland. At each practice site, interviews were performed with four patients who were scheduled consecutively to see their family doctor. Twenty-four of 36 studied patients spontaneously perceived non-verbal behaviours of the family GP during patient-doctor encounters. They reported a total of 48 non-verbal cues. The most frequent features were tone of voice, eye contact, and facial expressions. Less frequent were examination room characteristics, touch, interpersonal distance, GP clothing, gestures, and posture. Non-verbal communication is an important factor by which patients spontaneously describe and evaluate their interactions with a GP. Family GPs should be trained to better understand and monitor their own non-verbal behaviours towards patients.

  20. Ubiquitous technologies in health: new challenges of opportunity, expectation, and responsibility.

    PubMed

    Rigby, Michael

    2006-01-01

    In spite of their name, 'ubiquitous' technologies are not yet ubiquitous in the true sense of the word, but rather are 'novel', being at the research, pilot, and selective use stages. In future, the proliferation in types of application, the major increase in cases and data volumes, and above all the dependence on ubiquitous technologies will raise practical, ethical, and liability issues. Equally significantly, it will require health service redesign, including new response services. Health informaticians need to be active in stimulating consideration of all these issues, as part of both social and professional responsibility.

  1. VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

    PubMed

    Argemi, X; Prévost, G; Riegel, P; Keller, D; Meyer, N; Baldeyrou, M; Douiri, N; Lefebvre, N; Meghit, K; Ronde Oustau, C; Christmann, D; Cianférani, S; Strub, J M; Hansmann, Y

    2017-05-01

    Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  2. ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling

    PubMed Central

    Chakrabarti, Rumela; Wei, Yong; Hwang, Julie; Hang, Xiang; Blanco, Mario Andres; Choudhury, Abrar; Tiede, Benjamin; Romano, Rose-Anne; DeCoste, Christina; Mercatali, Laura; Ibrahim, Toni; Amadori, Dino; Kannan, Nagarajan; Eaves, Connie J; Sinha, Satrajit; Kang, Yibin

    2014-01-01

    Emerging evidence suggests that cancer is populated and maintained by tumor initiating cells (TICs) with stem-like properties similar to that of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the ΔNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7, thereby enhancing Wnt signaling. Importantly, Fzd7-dependent enhancement of Wnt signaling by ΔNp63 also governs tumor initiating activity of the basal subtype of breast cancer. These findings establish ΔNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms. PMID:25241036

  3. The learner's perspective in GP teaching practices with multi-level learners: a qualitative study.

    PubMed

    Thomson, Jennifer S; Anderson, Katrina; Haesler, Emily; Barnard, Amanda; Glasgow, Nicholas

    2014-03-19

    Medical students, junior hospital doctors on rotation and general practice (GP) registrars are undertaking their training in clinical general practices in increasing numbers in Australia. Some practices have four levels of learner. This study aimed to explore how multi-level teaching (also called vertical integration of GP education and training) is occurring in clinical general practice and the impact of such teaching on the learner. A qualitative research methodology was used with face-to-face, semi-structured interviews of medical students, junior hospital doctors, GP registrars and GP teachers in eight training practices in the region that taught all levels of learners. Interviews were audio-recorded and transcribed. Qualitative analysis was conducted using thematic analysis techniques aided by the use of the software package N-Vivo 9. Primary themes were identified and categorised by the co-investigators. 52 interviews were completed and analysed. Themes were identified relating to both the practice learning environment and teaching methods used.A practice environment where there is a strong teaching culture, enjoyment of learning, and flexible learning methods, as well as learning spaces and organised teaching arrangements, all contribute to positive learning from a learners' perspective.Learners identified a number of innovative teaching methods and viewed them as positive. These included multi-level learner group tutorials in the practice, being taught by a team of teachers, including GP registrars and other health professionals, and access to a supernumerary GP supervisor (also termed "GP consultant teacher"). Other teaching methods that were viewed positively were parallel consulting, informal learning and rural hospital context integrated learning. Vertical integration of GP education and training generally impacted positively on all levels of learner. This research has provided further evidence about the learning culture, structures and teaching

  4. Influences on GP coping and resilience: a qualitative study in primary care

    PubMed Central

    Cheshire, Anna; Ridge, Damien; Hughes, John; Peters, David; Panagioti, Maria; Simon, Chantal; Lewith, George

    2017-01-01

    Background ‘Neoliberal’ work policies, austerity, NHS restructuring, and increased GP consultation rates provide the backdrop against increasing reports of GP burnout and an impending shortage of GPs. Aim To explore GPs’ experiences of workplace challenges and stresses, and their coping strategies, particularly focusing on understanding the impact of recent NHS workplace change. Design and setting Study design was qualitative, with data collected from two focus groups and seven one-to-one telephone interviews. Method Focus groups and one-to-one telephone interviews explored the experiences of GPs currently practising in England, recruited through convenience sampling. Data were collected using a semi-structured interview approach and analysed using thematic analysis. Results There were 22 GP participants recruited: focus groups (n = 15) and interviews (n = 7). Interviewees understood GPs to be under intense and historically unprecedented pressures, which were tied to the contexts in which they work, with important moral implications for ‘good’ doctoring. Many reported that being a full-time GP was too stressful: work-related stress led to mood changes, sleep disruption, increases in anxiety, and tensions with loved ones. Some had subsequently sought ways to downsize their clinical workload. Workplace change resulted in little time for the things that helped GP resilience: a good work–life balance and better contact with colleagues. Although some GPs were coping better than others, GPs acknowledged that there was only so much an individual GP could do to manage their stress, given the external work issues they faced. Conclusion GPs experience their emotional lives and stresses as being meaningfully shaped by NHS factors. To support GPs to provide effective care, resilience building should move beyond the individual to include systemic work issues. PMID:28483822

  5. [Active participation in research and teaching during post-graduate GP training: perspectives of future general practitioners].

    PubMed

    Haumann, Hannah; Flum, Elisabeth; Joos, Stefanie

    2016-12-01

    Academic institutions of general practice at German medical faculties have grown during the past years. This leads to an increase in the need of qualified young researchers and teachers in general practice (GP). Little is known about the interest in research and teaching skills and their training among general practice trainees and young GPs. This cross-sectional survey among GP trainees and young GPs examined 1. if there is an interest in the training in research and teaching skills during post-graduate GP training, 2. which fostering and hindering factors have an effect on this interest and 3. which roles are attributed to academic institutions of general practice. A web-based cross-sectional study was performed among members of "Verbundweiterbildung plus" , a network of GP trainees, as well as "Junge Allgemeinmedizin Deutschland", the German network of young GPs. Descriptive analysis was conducted. 148 GP trainees and young GPs participated in the study, 76% (n=109) of them were GP trainees. There was interest in a position in research and teaching during post-graduate GP training among 55% (n=78). Factors associated with the interest in a position in research and teaching during post-graduate GP training were (MV 5-point Likert scale ± SD): compatibility of clinical work and research/teaching and of family and career (4.4±0.8; 4.7±0.6 respectively). The roles of academic institutions of general practice were attributed to training of medical students (4.6±0.6), post-graduate GP training (4.5±0.7) and research (4.5±0.7). GP trainees assessed the importance of training in research and teaching skills during post-graduate GP training and of the compatibility of family and career differently from young GPs (3.7±1.0 vs. 4.1±0.8 p=0.027; 4.8±0.5 vs. 4.3±0.9, p=0.016). Those interested in a position in research and teaching during post-graduate GP training showed a stronger interest in specific training in research skills (3.7±1.1 vs. 2.8±1.1, p<0.001), a

  6. Verification of 'learning credits' by GP appraisers.

    PubMed

    Murie, Jill; Wakeling, Judy

    2011-11-01

    The RCGP CPD Learning Credits system aims to enable GPs to demonstrate knowledge and skills relevant to their daily practice. Credits are self-assessed and will form part of the 'evidence' necessary for successful revalidation. At an appraisal, GP appraisers verify the credits in terms of the time spent on the CPD activity and its impact on the GP's practice. The purpose of this study was to examine the extent to which GPs (as appraisees) are able to self-assess their own learning and, as appraisers, verify credits in a standardised way. All 17 GP appraisers in NHS Lanarkshire were invited to participate in a study, which triangulated three sources of evidence on credits: self-rating, peer-assessment and workshop discussion. The resultant data were analysed on an Excel spreadsheet. Outcomes included self-assessed credit value, peer-assessed mean score (range) and free text. Of the 17 appraisers, 15 completed the paperwork and 13 attended the workshop. GPs' self-assessed learning credits were equivalent to peer-assessed score in 5/15 cases, but considered overestimates in 4/15 and underestimates in 6/15 cases. The most extreme variance was for an oncology module, where the variance ranged from 28% to 200% of the self-assessed score. GPs have a variable understanding of how to award themselves learning credits and of how to judge the credits of potential appraisees. Without adequate resources for appraisal training, validated instruments, calibration and reliability, verification of the learning credit system will be flawed by its subjective and arbitrary nature.

  7. An improved anonymous authentication scheme for roaming in ubiquitous networks.

    PubMed

    Lee, Hakjun; Lee, Donghoon; Moon, Jongho; Jung, Jaewook; Kang, Dongwoo; Kim, Hyoungshick; Won, Dongho

    2018-01-01

    With the evolution of communication technology and the exponential increase of mobile devices, the ubiquitous networking allows people to use our data and computing resources anytime and everywhere. However, numerous security concerns and complicated requirements arise as these ubiquitous networks are deployed throughout people's lives. To meet the challenge, the user authentication schemes in ubiquitous networks should ensure the essential security properties for the preservation of the privacy with low computational cost. In 2017, Chaudhry et al. proposed a password-based authentication scheme for the roaming in ubiquitous networks to enhance the security. Unfortunately, we found that their scheme remains insecure in its protection of the user privacy. In this paper, we prove that Chaudhry et al.'s scheme is vulnerable to the stolen-mobile device and user impersonation attacks, and its drawbacks comprise the absence of the incorrect login-input detection, the incorrectness of the password change phase, and the absence of the revocation provision. Moreover, we suggest a possible way to fix the security flaw in Chaudhry et al's scheme by using the biometric-based authentication for which the bio-hash is applied in the implementation of a three-factor authentication. We prove the security of the proposed scheme with the random oracle model and formally verify its security properties using a tool named ProVerif, and analyze it in terms of the computational and communication cost. The analysis result shows that the proposed scheme is suitable for resource-constrained ubiquitous environments.

  8. Cognitive deficits associated with combined HIV gp120 expression and chronic methamphetamine exposure in mice

    PubMed Central

    Kesby, James P.; Markou, Athina; Semenova, Svetlana

    2014-01-01

    Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e., similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult. PMID:25476577

  9. A review of a GP registrar-run mobile health clinic for homeless people.

    PubMed

    O'Carroll, A; Irving, N; O'Neill, J; Flanagan, E

    2017-08-01

    Homeless people have excessively high morbidity and mortality rates, yet they face barriers accessing primary care. A mobile health clinic, staffed by GP registrars, was developed to provide services to homeless people, particularly rough sleepers and sex workers. The aims were to improve access to primary care and to challenge the stereotypes and prejudices of GP registrars through direct contact with homeless people. This was a qualitative study; questionnaires were completed on the mobile health clinic and two focus groups were conducted. All service users were asked to complete a questionnaire over a 3 month period. Two focus groups were conducted with 6 and 14 GP registrars who had worked on the bus. There was an 80% response rate (116 of 145). Fifty-two percent had no Medical Card meaning that they had no way to access the free primary care to which they are entitled. Had the clinic not been available, over half would not have sought further treatment and 16% would have gone to an Emergency Department. Ninety-one percent of users rated the service 10/10. The focus groups found that GP registrars who worked on the mobile health clinic had decreased negative stereotypes, increased empathy, and more knowledge of homeless issues. Furthermore, they intended to ensure that homeless people will not face discrimination in their future practice. A GP Registrar-run Mobile Health Clinic achieved its aims of improving access to primary care for rough sleepers and sex workers, and challenging stereotypes of GP Registrars.

  10. Context Aware Ubiquitous Learning Environments for Peer-to-Peer Collaborative Learning

    ERIC Educational Resources Information Center

    Yang, Stephen J. H.

    2006-01-01

    A ubiquitous learning environment provides an interoperable, pervasive, and seamless learning architecture to connect, integrate, and share three major dimensions of learning resources: learning collaborators, learning contents, and learning services. Ubiquitous learning is characterized by providing intuitive ways for identifying right learning…

  11. Social relationships and GP use of middle-aged and older adults in Europe: a moderator analysis.

    PubMed

    Bremer, Daniel; Lüdecke, Daniel; Vonneilich, Nico; von dem Knesebeck, Olaf

    2018-04-07

    This paper investigates (1) how social relationships (SRs) relate to the frequency of general practitioner (GP) visits among middle-aged and older adults in Europe, (2) if SRs moderate the association between self-rated health and GP visits, and (3) how the associations vary regarding employment status. Data stem from the Survey of Health, Ageing and Retirement in Europe project (wave 4, 56 989 respondents, 50 years or older). GP use was assessed by frequency of contacts with GPs in the last 12 months. Predictors were self-rated health and structural (Social Integration Index (SII), social contact frequency) and functional (emotional closeness) aspects of SR. Regressions were used to measure the associations between GP use and those predictors. Sociodemographic and socioeconomic factors were used as covariates. Additional models were computed with interactions. Analyses did not reveal significant associations of functional and structural aspects of SR with frequency of GP visits (SII: incidence rate ratio (IRR)=0.99, 95% CI 0.97 to 1.01, social contact frequency: IRR=1.04, 95% CI 1.00 to 1.07, emotional closeness: IRR=1.02, 95% CI 1.00 to 1.04). Moderator analyses showed that 'high social contact frequency people' with better health had more statistically significant GP visits than 'low social contact frequency people' with better health. Furthermore, people with poor health and an emotionally close network showed a significantly higher number of GP visits compared with people with same health, but less close networks. Three-way interaction analyses indicated employment status specific behavioural patterns with regard to SR and GP use, but coefficients were mostly not significant. All in all, the not employed groups showed a higher number of GP visits. Different indicators of SR showed statistically insignificantly associations with GP visits. Consequently, the relevance of SR may be rated rather low in quantitative terms for investigating GP use behaviour

  12. Patient satisfaction with out-of-hours GP cooperatives: a longitudinal study.

    PubMed

    Smits, Marleen; Huibers, Linda; Oude Bos, Anita; Giesen, Paul

    2012-12-01

    For over a decade, out-of-hours primary care in the Netherlands has been provided by general practitioner (GP) cooperatives. In the past years, quality improvements have been made and patients have become acquainted with the service. This may have increased patient satisfaction. The objective of this study was to examine changes in patient satisfaction with GP cooperatives over time. Longitudinal observational study. A validated patient satisfaction questionnaire was distributed in 2003-2004 (T1) and 2007-2008 (T2). Items were rated on a scale from 0 to 10 (1 = very bad; 10 = excellent). Eight GP cooperatives in the Netherlands. Stratified sample of 9600 patients. Response was 55% at T1 (n = 2634) and 51% at T2 (n = 2462). Expectations met; satisfaction with triage nurses, GPs, and organization. For most patients the care received at the GP cooperative met their expectations (T1: 86.1% and T2: 88.4%). Patients were satisfied with the triage nurses (overall grade T1: 7.73 and T2: 7.99), GPs (T1: 8.04 and T2: 8.25), and organization (overall grade T1: 7.60 and T2: 7.78). Satisfaction with triage nurses showed the largest increase over time. The quality and effectiveness of advice or treatment were given relatively low grades. Of all organizational aspects, the lowest grades were given for waiting times and information about the cooperative. In general, patients were initially satisfied with GP cooperatives and satisfaction had even increased four years later. However, there is room for improvement in the content of the advice, waiting times, and information supply. More research is needed into satisfaction of specific patient groups.

  13. Using photography to enhance GP trainees' reflective practice and professional development.

    PubMed

    Rutherford; Forde, Emer; Priego-Hernandez, Jacqueline; Butcher, Aurelia; Wedderburn, Clare

    2018-02-08

    The capacity and the commitment to reflect are integral to the practice of medicine and are core components of most general practitioners (GP) training programmes. Teaching through the humanities is a growing area within medical education, but one which is often considered a voluntary 'add-on' for the interested doctor. This article describes an evaluation of a highly innovative pedagogical project which used photography as a means to enhance GP trainees' reflective capacity, self-awareness and professional development. Photography was used as a tool to develop GP trainees' skills in recognising and articulating the attitudes, feelings and values that might impact on their clinical work and to enhance their confidence in their ability to deal with these concerns/issues. We submit that photography is uniquely well suited for facilitating insight and self-reflection because it provides the ability to record 'at the touch of a button' those scenes and images to which our attention is intuitively drawn without the need for-or the interference of-conscious decisions. This allows us the opportunity to reflect later on the reasons for our intuitive attraction to these scenes. These photography workshops were a compulsory part of the GP training programme and, despite the participants' traditional scientific backgrounds, the results clearly demonstrate the willingness of participants to accept-even embrace-the use of art as a tool for learning. The GP trainees who took part in this project acknowledged it to be beneficial for both their personal and professional development. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  14. Patient influences on satisfaction and loyalty for GP services.

    PubMed

    Rundle-Thiele, Sharyn; Russell-Bennett, Rebekah

    2010-04-01

    Little is known about the influence that patients themselves have on their loyalty to a general practitioner (GP). Consequently, a theoretical framework that draws on diverse literature is proposed to suggest that along with satisfaction, patient loyalty is an important outcome for GPs. Comprising 174 Australian patients, this study identified that knowledgeable patients reported lower levels of loyalty while older patients and patients visiting a GP more frequently reported higher levels of loyalty. The results suggest that extending patient-centered care practices to encompass all patients may be warranted in order to improve patient satisfaction and loyalty. Further, future research opportunities abound, with intervention and dyadic research methodologies recommended.

  15. optGpSampler: an improved tool for uniformly sampling the solution-space of genome-scale metabolic networks.

    PubMed

    Megchelenbrink, Wout; Huynen, Martijn; Marchiori, Elena

    2014-01-01

    Constraint-based models of metabolic networks are typically underdetermined, because they contain more reactions than metabolites. Therefore the solutions to this system do not consist of unique flux rates for each reaction, but rather a space of possible flux rates. By uniformly sampling this space, an estimated probability distribution for each reaction's flux in the network can be obtained. However, sampling a high dimensional network is time-consuming. Furthermore, the constraints imposed on the network give rise to an irregularly shaped solution space. Therefore more tailored, efficient sampling methods are needed. We propose an efficient sampling algorithm (called optGpSampler), which implements the Artificial Centering Hit-and-Run algorithm in a different manner than the sampling algorithm implemented in the COBRA Toolbox for metabolic network analysis, here called gpSampler. Results of extensive experiments on different genome-scale metabolic networks show that optGpSampler is up to 40 times faster than gpSampler. Application of existing convergence diagnostics on small network reconstructions indicate that optGpSampler converges roughly ten times faster than gpSampler towards similar sampling distributions. For networks of higher dimension (i.e. containing more than 500 reactions), we observed significantly better convergence of optGpSampler and a large deviation between the samples generated by the two algorithms. optGpSampler for Matlab and Python is available for non-commercial use at: http://cs.ru.nl/~wmegchel/optGpSampler/.

  16. Immunodiagnosis of Paracoccidioidomycosis due to Paracoccidioides brasiliensis Using a Latex Test: Detection of Specific Antibody Anti-gp43 and Specific Antigen gp43

    PubMed Central

    dos Santos, Priscila Oliveira; Rodrigues, Anderson Messias; Fernandes, Geisa Ferreira; da Silva, Silvia Helena Marques; Burger, Eva; de Camargo, Zoilo Pires

    2015-01-01

    Background Paracoccidioidomycosis (PCM) is a life-threatening systemic disease and is a neglected public health problem in many endemic regions of Latin America. Though several diagnostic methods are available, almost all of them present with some limitations. Method/Principle Findings A latex immunoassay using sensitized latex particles (SLPs) with gp43 antigen, the immunodominant antigen of Paracoccidioides brasiliensis, or the monoclonal antibody mAb17c (anti-gp43) was evaluated for antibody or antigen detection in sera, cerebrospinal fluid (CSF), and bronchoalveolar lavage (BAL) from patients with PCM due to P. brasiliensis. The gp43-SLPs performed optimally to detect specific antibodies with high levels of sensitivity (98.46%, 95% CI 91.7–100.0), specificity (93.94%, 95% CI 87.3–97.7), and positive (91.4%) and negative (98.9%) predictive values. In addition, we propose the use of mAb17c-SLPs to detect circulating gp43, which would be particularly important in patients with immune deficiencies who fail to produce normal levels of immunoglobulins, achieving good levels of sensitivity (96.92%, 95% CI 89.3–99.6), specificity (88.89%, 95% CI 81.0–94.3), and positive (85.1%) and negative (97.8%) predictive values. Very good agreement between latex tests and double immune diffusion was observed for gp43-SLPs (k = 0.924) and mAb17c-SLPs (k = 0.850), which reinforces the usefulness of our tests for the rapid diagnosis of PCM in less than 10 minutes. Minor cross-reactivity occurred with sera from patients with other fungal infections. We successfully detected antigens and antibodies from CSF and BAL samples. In addition, the latex test was useful for monitoring PCM patients receiving therapy. Conclusions/Significance The high diagnostic accuracy, low cost, reduced assay time, and simplicity of this new latex test offer the potential to be commercialized and makes it an attractive diagnostic assay for use not only in clinics and medical mycology laboratories, but

  17. Distinct effects of Broncho-Vaxom (OM-85 BV) on gp130 binding cytokines

    PubMed Central

    Roth, M; Block, L

    2000-01-01

    BACKGROUND—Broncho-Vaxom (OM-85 BV) is known to support respiratory tract resistance to bacterial infections. In vivo and in vitro studies in animals and humans have shown that the action of the drug is based on the modulation of the host immune response, and it has been found to upregulate interferon γ (IFN-γ) and interleukin (IL)-2, IL-6, and IL-8. These immunomodulatory effects of the compound may explain its stimulation on T helper cells and natural killer cells. Following earlier findings that OM-85 BV induces the synthesis of IL-6, a study was undertaken to investigate its possible effect on other gp130 binding cytokines including IL-11, IL-12, leukaemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neutrophil factor (CNTF). Its modulation of the corresponding receptors of the above mentioned cytokines and of the signal transducer gp130 in human pulmonary fibroblasts and peripheral blood lymphocytes was also studied.
METHODS—Transcription of cytokines was assessed by Northern blot analysis. Secretion of cytokines was analysed using commercially available enzyme linked immunosorbent assay kits. Cytokine receptors and gp130 proteins were determined by Western blot analysis.
RESULTS—OM-85 BV increased the expression of IL-11 in human lung fibroblasts, but not in lymphocytes, in a dose and time dependent manner by maximal fivefold within 20 hours. The compound inhibited serum induced IL-12 expression in peripheral blood lymphocytes but did not induce OSM, LIF, or CNTF at any concentration. In lung fibroblasts the expression of the IL-6 receptor was enhanced fourfold at a concentration of 10 µg/ml OM-85 BV while that of the IL-11 receptor was not altered. In peripheral blood lymphocytes LIF receptor α expression was downregulated in the presence of 10 µg/ml OM-85 BV. At a concentration of 10 µg/ml OM-85 BV enhanced gp130 gene transcription fivefold and increased gp130 protein accumulation in cell membranes by 2.5times

  18. Antibody responses to prime-boost vaccination with an HIV-1 gp145 envelope protein and chimpanzee adenovirus vectors expressing HIV-1 gp140.

    PubMed

    Emmer, Kristel L; Wieczorek, Lindsay; Tuyishime, Steven; Molnar, Sebastian; Polonis, Victoria R; Ertl, Hildegund C J

    2016-10-23

    Over 2 million individuals are infected with HIV type 1 (HIV-1) each year, yet an effective vaccine remains elusive. The most successful HIV-1 vaccine to date demonstrated 31% efficacy. Immune correlate analyses associated HIV-1 envelope (Env)-specific antibodies with protection, thus providing a path toward a more effective vaccine. We sought to test the antibody response from novel prime-boost vaccination with a chimpanzee-derived adenovirus (AdC) vector expressing a subtype C Env glycoprotein (gp)140 combined with either a serologically distinct AdC vector expressing gp140 of a different subtype C isolate or an alum-adjuvanted, partially trimeric gp145 from yet another subtype C isolate. Three different prime-boost regimens were tested in mice: AdC prime-protein boost, protein prime-AdC boost, and AdC prime-AdC boost. Each regimen was tested at two different doses of AdC vector in a total of six experimental groups. Sera were collected at various time points and evaluated by ELISA for Env-specific antibody binding, isotype, and avidity. Antibody functionality was assessed by pseudovirus neutralization assay. Priming with AdC followed by a protein boost or sequential immunizations with two AdC vectors induced HIV-1 Env-specific binding antibodies, including those to the variable region 2, whereas priming with protein followed by an AdC boost was relatively ineffective. Antibodies that cross-neutralized tier 1 HIV-1 from different subtypes were elicited with vaccine regimens that included immunizations with protein. Our study warrants further investigation of AdC vector and gp145 protein prime-boost vaccines and their ability to protect against acquisition in animal challenge studies.

  19. Investigation of the function of the putative self-association site of Epstein-Barr virus (EBV) glycoprotein 42 (gp42)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rowe, Cynthia L., E-mail: c-rowe@northwestern.edu; Matsuura, Hisae, E-mail: hisaem@stanford.edu; Interdepartmental Biological Sciences Program, Northwestern University, Evanston, IL 60208

    The Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a type II membrane protein essential for entry into B cells but inhibits entry into epithelial cells. X-ray crystallography suggests that gp42 may form dimers when bound to human leukocyte antigen (HLA) class II receptor (Mullen et al., 2002) or multimerize when not bound to HLA class II (Kirschner et al., 2009). We investigated this self-association of gp42 using several different approaches. We generated soluble mutants of gp42 containing mutations within the self-association site and found that these mutants have a defect in fusion. The gp42 mutants bound to gH/gL and HLAmore » class II, but were unable to bind wild-type gp42 or a cleavage mutant of gp42. Using purified gp42, gH/gL, and HLA, we found these proteins associate 1:1:1 by gel filtration suggesting that gp42 dimerization or multimerization does not occur or is a transient event undetectable by our methods.« less

  20. The impact of standalone call centres and GP cooperatives on access to after hours GP care: a before and after study adjusted for secular trend.

    PubMed

    Dunt, David; Day, Susan E; Kelaher, Margaret; Montalto, Michael

    2006-08-01

    The After Hours Primary Medical Trials were initiated by the Australian government to redress difficulties in after hours (AH) GP care in areas of high need. The study's objective is to study the impact of two standalone call centres and one GP cooperative offering comprehensive services, in improving consumer access to services for residents of a defined geographic area. A pre-post design was used to evaluate their impact after adjusting for secular trend at a national level. Access was considered in terms of availability, accessibility, affordability, acceptability and responsiveness of care. Unmet need and ease of obtaining AH telephone professional medical advice were also considered. Pre-trial and post-trial telephone surveys of two separate random samples of approximately 350 households using AH services in each trial area as well as in a national sample outside the trial areas. Consumer acceptability and affordability increased in residents in the area served by the GP cooperative. Access, however measured, did not improve in either of the standalone call centre areas. Reduction in unmet need approached but did not achieve statistical significance in most but not all trial areas. Improvements in access in the GP cooperative conformed to expectations based on current and pre-existing AH care arrangements put in place. Absence of improvements in access in the standalone call centres did not conform to expectations but may be partly explained by the reductions in consumer acceptability, following introduction of telephone triage systems reported elsewhere.

  1. Tryptophan 375 stabilizes the outer-domain core of gp120 for HIV vaccine immunogen design.

    PubMed

    Hu, Duoyi; Bowder, Dane; Wei, Wenzhong; Thompson, Jesse; Wilson, Mark A; Xiang, Shi-Hua

    2017-05-25

    The outer-domain core of gp120 may serve as a better HIV vaccine immunogen than the full-length gp120 because of its greater stability and immunogenicity. In our previous report, we introduced two disulfide bonds to the outer-domain core of gp120 to fix its conformation into a CD4-bound state, which resulted in a significant increase in its immunogenicity when compared to the wild-type outer-domain core. In this report, to further improve the immunogenicity of the outer-domain core based immunogen, we have introduced a Tryptophan residue at gp120 amino acid sequence position 375 (375S/W). Our data from immunized guinea pigs indeed shows a striking increase in the immune response due to this stabilized core outer-domain. Therefore, we conclude that the addition of 375W to the outer-domain core of gp120 further stabilizes the structure of immunogen and increases the immunogenicity. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  2. The Fast-Paced iPad Revolution: Can Educators Stay up to Date and Relevant about These Ubiquitous Devices?

    ERIC Educational Resources Information Center

    Peluso, Deanna C. C.

    2012-01-01

    Stepping into a modern day classroom, one will find that it is filled with a ubiquitous array of multimodal and digital resources, yet a majority of these revolutionary resources are likely not school issued, rather they were brought by the young people themselves. Digital mediums for communication, expression and multimodally engaging in one's…

  3. Cognitive deficits associated with combined HIV gp120 expression and chronic methamphetamine exposure in mice.

    PubMed

    Kesby, James P; Markou, Athina; Semenova, Svetlana

    2015-01-01

    Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e. similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

  4. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    PubMed Central

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

  5. GP50 as a promising early diagnostic antigen for Taenia multiceps infection in goats by indirect ELISA.

    PubMed

    Huang, Xing; Xu, Jing; Wang, Yu; Guo, Cheng; Chen, Lin; Gu, Xiaobin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

    2016-12-01

    Coenurosis is caused by coenurus, the metacestode of Taenia multiceps, which mainly parasitizes the brain and spinal cord of cattle, sheep and goats. To date, no widely-approved methods are available to identify early coenurus infection. In this study, we identified a full-length cDNA that encodes GP50 (TmGP50) from the transcriptome of T. multiceps, and then cloned and expressed in E. coli. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of recombinant TmGP50 protein (rTmGP50) for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, we orally infected 20 goats with mature T. multiceps eggs. Praziquantel (10%) was given to 10 of the goats 45 days post-infection (p.i.). Blood samples were collected for 17 weeks p.i. from the 20 goats and anti-rTmGP50 antibodies were evaluated using the indirect ELISA established here. The TmGP50 contains an 897 bp open reading frame, in which signal sequence resides in 1 ~ 48 sites and mature polypeptide consists of 282 amino acid residues. Immunofluorescence staining showed that native TmGP50 was localized to the microthrix and parenchymatous zone of the adult parasite and coenurus, and the coenurus cystic wall. The indirect ELISA based on rTmGP50 exhibited a sensitivity of 95.0% and a specificity of 92.6% when detecting GP50 antibodies in sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmGP50 antibody was detectable from 2 to 17 weeks p.i. in the control group, while the antibody fell below the cut-off value about 3 weeks after praziquantel treatment. Our results indicate that recombinant TmGP50 is a suitable early diagnostic antigen for coenurus infection in goats.

  6. The Teamwork Study: enhancing the role of non-GP staff in chronic disease management in general practice.

    PubMed

    Black, D A; Taggart, J; Jayasinghe, U W; Proudfoot, J; Crookes, P; Beilby, J; Powell-Davis, G; Wilson, L A; Harris, M F

    2013-01-01

    There is evidence for a team-based approach in the management of chronic disease in primary health care. However, the standard of care is variable, probably reflecting the limited organisational capacity of health services to provide the necessary structured and organised care for this group of patients. This study aimed to evaluate the impact of a structured intervention involving non-GP staff in GP practices on the quality of care for patients with diabetes or cardiovascular disease. A cluster randomised trial was undertaken across 60 GP practices. The intervention was implemented in 30 practices with staff and patients interviewed at baseline and at 12-15 months follow up. The change in team roles was evaluated using a questionnaire completed by practice staff. The quality of care was evaluated using the Patient Assessment of Chronic Illness Care questionnaire. We found that although the team roles of staff improved in the intervention practices and there were significant differences between practices, there was no significant difference between those in the intervention and control groups in patient-assessed quality of care after adjusting for baseline-level score and covariates at the 12-month follow up. Practice team roles were not significantly associated with change in Patient Assessment of Chronic Illness Care scores. Patients with multiple conditions were more likely to assess their quality of care to be better. Thus, although previous research has shown a cross-sectional association between team work and quality of care, we were unable to replicate these findings in the present study. These results may be indicative of insufficient time for organisational change to result in improved patient-assessed quality of care, or because non-GP staff roles were not sufficiently focussed on the aspects of care assessed. The findings provide important information for researchers when designing similar studies.

  7. An improved anonymous authentication scheme for roaming in ubiquitous networks

    PubMed Central

    Lee, Hakjun; Lee, Donghoon; Moon, Jongho; Jung, Jaewook; Kang, Dongwoo; Kim, Hyoungshick

    2018-01-01

    With the evolution of communication technology and the exponential increase of mobile devices, the ubiquitous networking allows people to use our data and computing resources anytime and everywhere. However, numerous security concerns and complicated requirements arise as these ubiquitous networks are deployed throughout people’s lives. To meet the challenge, the user authentication schemes in ubiquitous networks should ensure the essential security properties for the preservation of the privacy with low computational cost. In 2017, Chaudhry et al. proposed a password-based authentication scheme for the roaming in ubiquitous networks to enhance the security. Unfortunately, we found that their scheme remains insecure in its protection of the user privacy. In this paper, we prove that Chaudhry et al.’s scheme is vulnerable to the stolen-mobile device and user impersonation attacks, and its drawbacks comprise the absence of the incorrect login-input detection, the incorrectness of the password change phase, and the absence of the revocation provision. Moreover, we suggest a possible way to fix the security flaw in Chaudhry et al’s scheme by using the biometric-based authentication for which the bio-hash is applied in the implementation of a three-factor authentication. We prove the security of the proposed scheme with the random oracle model and formally verify its security properties using a tool named ProVerif, and analyze it in terms of the computational and communication cost. The analysis result shows that the proposed scheme is suitable for resource-constrained ubiquitous environments. PMID:29505575

  8. Different oligosaccharide processing of the membrane-integrated and the secretory form of gp 80 in rat liver.

    PubMed

    Tauber, R; Schenck, I; Josić, D; Gross, V; Heinrich, P C; Gerok, W; Reutter, W

    1986-09-01

    Rat liver synthesizes a glycoprotein with Mr of 80.000 (gp 80) which is partly inserted into the plasma membrane and partly secreted into the serum. The membrane-integrated and the secretory form of this glycoprotein have an identical peptide pattern, but different N-linked glycans. Whereas gp 80 from the serum is glycosylated with complex-type oligosaccharides, gp 80 from the plasma membrane has high mannose glycans. Phase separation with Triton X-114 showed that membrane-integrated gp 80 contains hydrophobic portions, whereas secretory gp 80 has hydrophilic properties. Intracellular transport and oligosaccharide processing of gp 80 were studied in vivo in the endoplasmic reticulum, the Golgi apparatus and plasma membranes of rat liver and in serum using pulse-chase labeling with L-[35S]methionine and immunoprecipitation. Peak labeling of gp 80 was reached in the endoplasmic reticulum 10 min after the pulse, in the Golgi apparatus 20 min later, and in the plasma membrane after 2 h; in the serum the specific radioactivity was steadily increasing during the experiment. Gp 80 of the endoplasmic reticulum was completely sensitive to endo-beta-N-glucosaminidase H (endo H), but simultaneously occurred in the Golgi apparatus in an endo H-sensitive and endo H-resistant form. The endo H-sensitive form was transported to the plasma membrane, the endo H-resistant species secreted into the serum. Conversion from the endo H-sensitive to the endo H-resistant form was completed within 10 min after transfer of gp 80 to the Golgi apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

    PubMed

    d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

    2015-04-16

    ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Protective effects of naringin against gp120-induced injury mediated by P2X7 receptors in BV2 microglial cells.

    PubMed

    Chen, Q; Hu, J; Qin, S S; Liu, C L; Wu, H; Wang, J R; Lu, X M; Wang, J; Chen, G Q; Liu, Y; Liu, B Y; Xu, C S; Liang, S D

    2016-05-13

    This study was aimed at exploring the effects of P2X7 receptors on gp120-induced injury and naringin's protective effects against gp120-induced injury in BV2 microglia. BV2 microglia injury model was established by gp120 treatment and MTS assay was used to verify whether naringin has a cell-protective effect against gp120-induced injury. Changes in P2X7 receptor expression were assayed using RT-PCR, qPCR, and western blot. Results showed that the ODs of the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.91 ± 0.10, 0.71 ± 0.09, 0.83 ± 0.10, and 0.83 ± 0.10, respectively. Compared to the control group, the gp120 group showed a significantly decreased cell survival rate. Cell survival rates of the gp120+naringin group increased significantly compared to those of the gp120 group, while no difference was observed when compared to the gp120+BBG group. The relative P2X7 mRNA expression levels in the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.73 ± 0.06, 1.05 ± 0.06, 0.78 ± 0.05, and 0.81 ± 0.04, respectively. The corresponding P2X7 protein expression levels were 0.46 ± 0.04, 0.79 ± 0.04, 0.38 ± 0.07, and 0.42 ± 0.06. P2X7 mRNA and protein expression in the gp120 group increased significantly compared to those in the control group, and declined in the gp120+naringin group compared to those in the gp120 group. Therefore, P2X7 receptors might be involved in gp120-induced injury in BV2 microglia, and naringin might play a protective role by inhibiting the up-regulated expression of P2X7 receptors.

  11. P-gp Protein Expression and Transport Activity in Rodent Seizure Models and Human Epilepsy.

    PubMed

    Hartz, Anika M S; Pekcec, Anton; Soldner, Emma L B; Zhong, Yu; Schlichtiger, Juli; Bauer, Bjoern

    2017-04-03

    A cure for epilepsy is currently not available, and seizure genesis, seizure recurrence, and resistance to antiseizure drugs remain serious clinical problems. Studies show that the blood-brain barrier is altered in animal models of epilepsy and in epileptic patients. In this regard, seizures increase expression of blood-brain barrier efflux transporters such as P-glycoprotein (P-gp), which is thought to reduce brain uptake of antiseizure drugs, and thus, contribute to antiseizure drug resistance. The goal of the current study was to assess the viability of combining in vivo and ex vivo preparations of isolated brain capillaries from animal models of seizures and epilepsy as well as from patients with epilepsy to study P-gp at the blood-brain barrier. Exposing isolated rat brain capillaries to glutamate ex vivo upregulated P-gp expression to levels that were similar to those in capillaries isolated from rats that had status epilepticus or chronic epilepsy. Moreover, the fold-increase in P-gp protein expression seen in animal models is consistent with the fold-increase in P-gp observed in human brain capillaries isolated from patients with epilepsy compared to age-matched control individuals. Overall, the in vivo/ex vivo approach presented here allows detailed analysis of the mechanisms underlying seizure-induced changes of P-gp expression and transport activity at the blood-brain barrier. This approach can be extended to other blood-brain barrier proteins that might contribute to drug-resistant epilepsy or other CNS disorders as well.

  12. Patient satisfaction with out-of-hours GP cooperatives: A longitudinal study

    PubMed Central

    Smits, Marleen; Huibers, Linda; Oude Bos, Anita; Giesen, Paul

    2012-01-01

    Objective For over a decade, out-of-hours primary care in the Netherlands has been provided by general practitioner (GP) cooperatives. In the past years, quality improvements have been made and patients have become acquainted with the service. This may have increased patient satisfaction. The objective of this study was to examine changes in patient satisfaction with GP cooperatives over time. Design Longitudinal observational study. A validated patient satisfaction questionnaire was distributed in 2003–2004 (T1) and 2007–2008 (T2). Items were rated on a scale from 0 to 10 (1 = very bad; 10 = excellent). Setting Eight GP cooperatives in the Netherlands. Subjects Stratified sample of 9600 patients. Response was 55% at T1 (n = 2634) and 51% at T2 (n = 2462). Main outcome measures Expectations met; satisfaction with triage nurses, GPs, and organization. Results For most patients the care received at the GP cooperative met their expectations (T1: 86.1% and T2: 88.4%). Patients were satisfied with the triage nurses (overall grade T1: 7.73 and T2: 7.99), GPs (T1: 8.04 and T2: 8.25), and organization (overall grade T1: 7.60 and T2: 7.78). Satisfaction with triage nurses showed the largest increase over time. The quality and effectiveness of advice or treatment were given relatively low grades. Of all organizational aspects, the lowest grades were given for waiting times and information about the cooperative. Conclusion In general, patients were initially satisfied with GP cooperatives and satisfaction had even increased four years later. However, there is room for improvement in the content of the advice, waiting times, and information supply. More research is needed into satisfaction of specific patient groups. PMID:23113756

  13. GP training in out-of-hours care: implications for the future workforce.

    PubMed

    Hayward, Gail; Drinkwater, Jessica; El-Gohary, Magdy; Burgess, Hana; Heneghan, Carl

    2015-03-01

    To understand GP trainees' experience of out-of-hours (OOH) training in England; whether it is achieving educational aims, and to highlight potential improvements. Additionally to explore factors that influence the decision to work in OOH care. An online survey was sent to 1091 GP trainees in England. Odds ratios were calculated for factors correlating with intention to work in OOH, or confidence and effectiveness in OOH work. Free text responses were coded and organised thematically. Trainees' experience of OOH care influences the decision to work there once qualified. Although this experience has positively influenced over three-quarters of trainees, it can be improved. Training is not achieving competencies in managing psychiatric emergencies and personal safety. Half of trainees received formal teaching in OOH skills; 3% receiving assessments in telephone triage. Only a quarter of trainees had worked with their usual GP trainer. Influential features of training included trainer enthusiasm and continuity, familiarity with the workplace, and confidence in OOH skills. Financial and lifestyle considerations were also important. OOH training in England has an impact on the future workforce and could be improved. The planned transition to a 4-year GP training structure offers an opportunity to address this.

  14. Biosynthesis and intracellular movement of the melanosomal membrane glycoprotein gp75, the human b (brown) locus product

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vijayasaradhi, S.; Doskoch, P.M.; Houghton, A.N.

    1991-10-01

    A 75-kDa melanosomal glycoprotein (gp75) is the product of a gene that maps to the b (brown) locus, a genetic locus that determines coat color in the mouse. The b locus is conserved (88% identity) between mouse and human. The mouse monoclonal antibody TA99 was used to study the biosynthesis and processing of gp75. gp75 was synthesized as a 55-kDa polypeptide, glycosylated by addition and processing of five or more Asn-linked carbohydrate chains through the cis and trans Golgi, and transported to melanosomes as a mature 75-kDa form. Synthesis and processing of gp75 was rapid (T{sub 1/2} < 30 min),more » and early steps in processing were required for efficient export of gp75 was quite stable in the melanosome. Studies with inhibitors of steps in oligosaccharide processing showed that alternative forms of gp75 were generated during trimming reactions by mannosidase IA/IB and that further maturation resulted in the two mature forms of gp75. The authors purpose that the kinetics of biosynthesis and processing reflect events in the biogenesis and maturation of melanosomes.« less

  15. Metabolism of AGEs – Bacterial AGEs Are Degraded by Metallo-Proteases

    PubMed Central

    Cohen-Or, Ifat; Katz, Chen; Ron, Eliora Z.

    2013-01-01

    Advanced Glycation End Products (AGEs) are the final products of non-enzymatic protein glycation that results in loss of protein structure and function. We have previously shown that in E. coli AGEs are continually formed as high-molecular weight protein complexes. Moreover, we showed that AGEs are removed from the cells by an active, ATP-dependent secretion and that these secreted molecules have low molecular weight. Taken together, these results indicate that E. coli contains a fraction of low molecular weight AGEs, in addition to the high-molecular weight AGEs. Here we show that the low-molecular weight AGEs originate from high-molecular weight AGEs by proteolytic degradation. Results of in-vitro and in vivo experiments indicated that this degradation is carried out not by the major ATP-dependent proteases that are responsible for the main part of bacterial protein quality control but by an alternative metal-dependent proteolysis. This proteolytic reaction is essential for the further secretion of AGEs from the cells. As the biochemical reactions involving AGEs are not yet understood, the implication of a metalloprotease in breakdown of high molecular weight AGEs and their secretion constitutes an important step in the understanding of AGEs metabolism. PMID:24130678

  16. Relevance of matrix metalloproteases in non-small cell lung cancer diagnosis.

    PubMed

    Blanco-Prieto, Sonia; Barcia-Castro, Leticia; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco Javier; Vázquez-Iglesias, Lorena; Botana-Rial, María Isabel; Fernández-Villar, Alberto; De Chiara, Loretta

    2017-12-05

    The need for novel biomarkers that could aid in non-small cell lung cancer (NSCLC) detection, together with the relevance of Matrix Metalloproteases (MMPs) -1, -2, -7, -9 and -10 in lung tumorigenesis, prompted us to assess the diagnostic usefulness of these MMPs and the Tissue Inhibitor of Metalloproteinase (TIMP) -1 in NSCLC patients. Markers were evaluated in an initial study cohort (19 NSCLC cases and 19 healthy controls). Those that better performed were analyzed in a larger sample including patients with benign lung diseases. Serum MMPs and TIMP-1 were determined by multiplexed immunoassays. Logistic regression was employed for multivariate analysis of biomarker combinations. MMPs and TIMP-1 were elevated in the serum of NSCLC patients compared to healthy controls. MMP-1, -7 and -9 performed at best and were further evaluated in the sample including benign pathologies, corroborating the superiority of MMP-9 in NSCLC discrimination, also at early-stage NSCLC. The optimal diagnostic value was obtained with the model including MMP-9, gender, age and smoking history, that demonstrated an AUC of 0.787, 85.54% sensitivity and 64.89% specificity. Our results suggest that MMP-9 is a potential biomarker for NSCLC diagnosis and its combined measurement with other biomarkers could improve NSCLC detection.

  17. Investigation of Learning Behaviors and Achievement of Vocational High School Students Using an Ubiquitous Physics Tablet PC App

    ERIC Educational Resources Information Center

    Purba, Siska Wati Dewi; Hwang, Wu-Yuin

    2017-01-01

    In this study, we designed and developed an app called Ubiquitous-Physics (U-Physics) for mobile devices like tablet PC or smart phones to help students learn the principles behind a simple pendulum in Physics. The unique characteristic of U-Physics is the use of sensors on mobile devices to collect acceleration and velocity data during pendulum…

  18. Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stingele, Julian; Bellelli, Roberto; Alte, Ferdinand

    Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled bymore » several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.« less

  19. Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

    DOE PAGES

    Stingele, Julian; Bellelli, Roberto; Alte, Ferdinand; ...

    2016-10-27

    Covalent DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with essential chromatin transactions, such as replication and transcription. Little was known about DPC-specific repair mechanisms until the recent identification of a DPC-processing protease in yeast. The existence of a DPC protease in higher eukaryotes is inferred from data in Xenopus laevis egg extracts, but its identity remains elusive. Here we identify the metalloprotease SPRTN as the DPC protease acting in metazoans. Loss of SPRTN results in failure to repair DPCs and hypersensitivity to DPC-inducing agents. SPRTN accomplishes DPC processing through a unique DNA-induced protease activity, which is controlled bymore » several sophisticated regulatory mechanisms. Cellular, biochemical, and structural studies define a DNA switch triggering its protease activity, a ubiquitin switch controlling SPRTN chromatin accessibility, and regulatory autocatalytic cleavage. Our data also provide a molecular explanation on how SPRTN deficiency causes the premature aging and cancer predisposition disorder Ruijs-Aalfs syndrome.« less

  20. Structure of a Complex Phosphoglycan Epitope from gp72 of Trypanosoma cruzi *

    PubMed Central

    Allen, Simon; Richardson, Julia M.; Mehlert, Angela; Ferguson, Michael A. J.

    2013-01-01

    The parasitic protozoan organism Trypanosoma cruzi is the causative agent of Chagas disease. The insect vector-dwelling epimastigote form of the organism expresses a low abundance glycoprotein associated with the flagellum adhesion zone, called gp72. The gp72 glycoprotein was first identified with an anti-carbohydrate IgG3 monoclonal antibody called WIC29.26 and has been shown to have an unusual sugar composition. Here, we describe a new way to isolate the WIC29.26 carbohydrate epitope of gp72. Using 1H NMR and mass spectrometry before and after derivatization, we provide an almost complete primary chemical structure for the epitope, which is that of a complex phosphosaccharide: Galfβ1–4Rhapα1–2Fucpα1-4(Galpβ1–3)(Galpα1–2)Xylpβ1–4Xylpβ1–3(Xylpβ1–2Galpα1-4(Galpβ1–3)(Rhapα1–2)Fucpα1–4)GlcNAcp, with phosphate attached to one or other of the two Galp terminal residues and in which all residues are of the d-absolute configuration, except for fucose and rhamnose which are l. Combined with previous data (Haynes, P. A., Ferguson, M. A., and Cross, G. A. (1996) Glycobiology 6, 869–878), we postulate that this complex structure and its variants lacking one or more residues are linked to Thr and Ser residues in gp72 via a phosphodiester linkage (GlcNAcpα1-P-Thr/Ser) and that these units may form phosphosaccharide repeats through GlcNAcpα1-P-Galp linkages. The gp72 glycoprotein is associated with the flagellum adhesion zone on the parasite surface, and its ligation has been implicated in inhibiting parasite differentiation from the epimastigote to the metacyclic trypomastigote stage. The detailed structure of the unique phosphosaccharide component of gp72 reported here provides a template for future biosynthetic and functional studies. PMID:23436655

  1. The learner’s perspective in GP teaching practices with multi-level learners: a qualitative study

    PubMed Central

    2014-01-01

    Background Medical students, junior hospital doctors on rotation and general practice (GP) registrars are undertaking their training in clinical general practices in increasing numbers in Australia. Some practices have four levels of learner. This study aimed to explore how multi-level teaching (also called vertical integration of GP education and training) is occurring in clinical general practice and the impact of such teaching on the learner. Methods A qualitative research methodology was used with face-to-face, semi-structured interviews of medical students, junior hospital doctors, GP registrars and GP teachers in eight training practices in the region that taught all levels of learners. Interviews were audio-recorded and transcribed. Qualitative analysis was conducted using thematic analysis techniques aided by the use of the software package N-Vivo 9. Primary themes were identified and categorised by the co-investigators. Results 52 interviews were completed and analysed. Themes were identified relating to both the practice learning environment and teaching methods used. A practice environment where there is a strong teaching culture, enjoyment of learning, and flexible learning methods, as well as learning spaces and organised teaching arrangements, all contribute to positive learning from a learners’ perspective. Learners identified a number of innovative teaching methods and viewed them as positive. These included multi-level learner group tutorials in the practice, being taught by a team of teachers, including GP registrars and other health professionals, and access to a supernumerary GP supervisor (also termed “GP consultant teacher”). Other teaching methods that were viewed positively were parallel consulting, informal learning and rural hospital context integrated learning. Conclusions Vertical integration of GP education and training generally impacted positively on all levels of learner. This research has provided further evidence about the

  2. Feasibility trial of GP and case-managed support for workplace sickness absence.

    PubMed

    Rannard, Anne; Gabbay, Mark; Sen, Dil; Riley, Richard; Britt, David

    2014-07-01

    Aim Our aim was to compare the return-to-work rates between individuals supported by their GP plus workplace health advisers (intervention group) and those supported by their GP alone. Workplace sickness absence places a significant cost burden on individuals and the wider economy. Previous research shows better outcomes for individuals if they are supported while still in employment, or have been on sick leave for four weeks or less. Those helped back to work at an early stage are more likely to remain at work. A non-medicalised case-managed approach appears to have the best outcomes and can prevent or reduce the slide onto out-of-work benefits, but UK literature on its effectiveness is sparse. The design was a feasibility-controlled trial in which participants were sickness absentees, or presentees in employment with work-related health problems. Individuals completed health status measures (SF-36; EQ-5D) and a Job Content Questionnaire at baseline and again at four-month follow-up. Findings In the intervention group, 29/60 participants completed both phases of the trial. GP practices referred two control patients, and, despite various attempts by the research team, GPs failed to engage with the trial. This finding is of concern, although not unique in primary care research. In earlier studies, GPs reported a lack of knowledge and confidence in dealing with workplace health issues. Despite this, we report interesting findings from the case-managed group, the majority of whom returned to work within a month. Age and length of sickness absence at recruitment were better predictors of return-to-work rates than the number of case-managed contacts. The traditional randomised controlled trial approach was unsuitable for this study. GPs showed low interest in workplace sickness absence, despite their pivotal role in the process. This study informed a larger Department for Work and Pensions study of case-managed support.

  3. Single-Chain Soluble BG505.SOSIP gp140 Trimers as Structural and Antigenic Mimics of Mature Closed HIV-1 Env.

    PubMed

    Georgiev, Ivelin S; Joyce, M Gordon; Yang, Yongping; Sastry, Mallika; Zhang, Baoshan; Baxa, Ulrich; Chen, Rita E; Druz, Aliaksandr; Lees, Christopher R; Narpala, Sandeep; Schön, Arne; Van Galen, Joseph; Chuang, Gwo-Yu; Gorman, Jason; Harned, Adam; Pancera, Marie; Stewart-Jones, Guillaume B E; Cheng, Cheng; Freire, Ernesto; McDermott, Adrian B; Mascola, John R; Kwong, Peter D

    2015-05-01

    Similar to other type I fusion machines, the HIV-1 envelope glycoprotein (Env) requires proteolytic activation; specifically, cleavage of a gp160 precursor into gp120 and gp41 subunits creates an N-terminal gp41 fusion peptide and permits folding from an immature uncleaved state to a mature closed state. While the atomic-level consequences of cleavage for HIV-1 Env are still being determined, the uncleaved state is antigenically distinct from the mature closed state, and cleavage has been reported to be essential for mimicry of the mature viral spike by soluble versions of Env. Here we report the redesign of a current state-of-the-art soluble Env mimic, BG505.SOSIP, to make it cleavage independent. Specifically, we replaced the furin cleavage site between gp120 and gp41 with Gly-Ser linkers of various lengths. The resultant linked gp120-gp41 constructs, termed single-chain gp140 (sc-gp140), exhibited different levels of structural and antigenic mimicry of the parent cleaved BG505.SOSIP. When constructs were subjected to negative selection to remove subspecies recognized by poorly neutralizing antibodies, trimers of high antigenic mimicry of BG505.SOSIP could be obtained; negative-stain electron microscopy indicated these to resemble the mature closed state. Higher proportions of BG505.SOSIP-trimer mimicry were observed in sc-gp140s with linkers of 6 or more residues, with a linker length of 15 residues exhibiting especially promising traits. Overall, flexible linkages between gp120 and gp41 in BG505.SOSIP can thus substitute for cleavage, and sc-gp140s that closely mimicked the vaccine-preferred mature closed state of Env could be obtained. The trimeric HIV-1 envelope glycoprotein (Env) is the sole target of virus-directed neutralizing antibody responses and a primary focus of vaccine design. Soluble mimics of Env have proven challenging to obtain and have been thought to require proteolytic cleavage into two-component subunits, gp120 and gp41, to achieve

  4. The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species*

    PubMed Central

    Bien, Jessica; Jefferson, Tamara; Čaušević, Mirsada; Jumpertz, Thorsten; Munter, Lisa; Multhaup, Gerd; Weggen, Sascha; Becker-Pauly, Christoph; Pietrzik, Claus U.

    2012-01-01

    The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase. PMID:22879596

  5. Lessons learned from an Internet GP information system.

    PubMed

    Briggs, J S; Bradley, M P

    1998-01-01

    We describe the prototype of an application that in actual use would allow GPs to find out more information about consultants at hospitals. This would aid the GP in making the decision about which consultant a patient should be referred to. The requirements of the application from the GP's perspective are described, together with some of the issues that have to be resolved before hospitals can provide the necessary information in a standard format. The application is implemented as a client--server system using standard Internet technologies such as Java and HTML. This architecture has a number of advantages but also revealed some issues concerning security and the format of data, among other things. The project showed that there is a desire for such a system and that that desire can be fulfilled at a relatively low cost.

  6. Ubiquitous Robotic Technology for Smart Manufacturing System.

    PubMed

    Wang, Wenshan; Zhu, Xiaoxiao; Wang, Liyu; Qiu, Qiang; Cao, Qixin

    2016-01-01

    As the manufacturing tasks become more individualized and more flexible, the machines in smart factory are required to do variable tasks collaboratively without reprogramming. This paper for the first time discusses the similarity between smart manufacturing systems and the ubiquitous robotic systems and makes an effort on deploying ubiquitous robotic technology to the smart factory. Specifically, a component based framework is proposed in order to enable the communication and cooperation of the heterogeneous robotic devices. Further, compared to the service robotic domain, the smart manufacturing systems are often in larger size. So a hierarchical planning method was implemented to improve the planning efficiency. A test bed of smart factory is developed. It demonstrates that the proposed framework is suitable for industrial domain, and the hierarchical planning method is able to solve large problems intractable with flat methods.

  7. Ubiquitous Robotic Technology for Smart Manufacturing System

    PubMed Central

    Zhu, Xiaoxiao; Wang, Liyu; Qiu, Qiang; Cao, Qixin

    2016-01-01

    As the manufacturing tasks become more individualized and more flexible, the machines in smart factory are required to do variable tasks collaboratively without reprogramming. This paper for the first time discusses the similarity between smart manufacturing systems and the ubiquitous robotic systems and makes an effort on deploying ubiquitous robotic technology to the smart factory. Specifically, a component based framework is proposed in order to enable the communication and cooperation of the heterogeneous robotic devices. Further, compared to the service robotic domain, the smart manufacturing systems are often in larger size. So a hierarchical planning method was implemented to improve the planning efficiency. A test bed of smart factory is developed. It demonstrates that the proposed framework is suitable for industrial domain, and the hierarchical planning method is able to solve large problems intractable with flat methods. PMID:27446206

  8. Bovine papillomavirus-like particles presenting conserved epitopes from membrane-proximal external region of HIV-1 gp41 induced mucosal and systemic antibodies

    PubMed Central

    Zhai, Yougang; Zhong, Zhenyu; Zariffard, Mohammadreza; Spear, Gregory T.; Qiao, Liang

    2013-01-01

    Two conserved epitopes, located in the membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41, are recognized by two HIV-1 broadly neutralizing antibodies 2F5 and 4E10, and are promising targets for vaccine design in efforts to elicit anti-HIV-1 broadly neutralizing antibodies. Since most HIV-1 infections initiate at mucosal surfaces, induction of mucosal neutralizing antibodies is necessary and of utmost importance to counteract HIV-1 infection. Here, we utilized a mucosal vaccine vector, bovine papillomavirus (BPV) virus-like particles (VLPs), as a platform to present HIV-1 neutralizing epitopes by inserting the extended 2F5 or 4E10 epitope or the MPER domain into D-E loop of BPV L1 respectively. The chimeric VLPs presenting MPER domain resembled the HIV-1 natural epitopes better than the chimeric VLPs presenting single epitopes. Oral immunization of mice with the chimeric VLPs displaying the 2F5 epitope or MPER domain elicited epitope-specific serum IgGs and mucosal secretory IgAs. The induced antibodies specifically recognized the native conformation of MPER in the context of HIV-1 envelope protein. The antibodies induced by chimeric VLPs presenting MPER domain are able to partially neutralize HIV-1 viruses from clade B and clade C. PMID:24055348

  9. U-ALS: A Ubiquitous Learning Environment

    ERIC Educational Resources Information Center

    Piovesan, Sandra Dutra; Passerino, Liliana Maria; Medina, Roseclea Duarte

    2012-01-01

    The diffusion of the use of the learning virtual environments presents a great potential for the development of an application which meet the necessities in the education area. In view of the importance of a more dynamic application and that can adapt itself continuously to the students' necessities, the "U-ALS" (Ubiquitous Adapted Learning…

  10. Evaluation of effect on skills of GP trainees taking time out of programme (OOP) in developing countries.

    PubMed

    Kiernan, Patrick; O'Dempsey, Tim; Kwalombota, Kwalombota; Elliott, Lynne; Cowan, Lesley

    2014-03-01

    The London School of General Practice Time Out of Programme (OOP) provides general practice (GP) trainees with an opportunity to enhance clinical experience and develop a range of skills and competencies, which are often not achievable in a three-year training programme, that are relevant and transferable to their practice in the UK. The programme offers one-year posts in the developing world to trainees between years ST2/3. This study builds on the work of the International Health Links Centre and London Deanery report (2011) and is designed to assess the skills and competencies of GP trainees on an OOP scheme. The study evaluated the impact of the OOP scheme on: • GP trainees? clinical skills • GP trainees' decision-making, management and leadership skills • Any other competencies. London GP trainees and trainers. Data were gathered using structured interview schedules developed for GP trainees and GP trainers and mapped against the RCGP Trainee e-portfolio Competence Areas. Our findings show that trainees and trainers reported an increase in skill levels in the more generic competencies. The study shows that the OOP scheme provides GP trainees with an excellent opportunity to develop clinical skills and more generic skills such as leadership, management and decision-making, as well as effective use of resources. However, not all clinical skill improvements were directly transferable to trainees' clinical work on return to the UK.

  11. Using Context-Aware Ubiquitous Learning to Support Students' Understanding of Geometry

    ERIC Educational Resources Information Center

    Crompton, Helen

    2015-01-01

    In this study, context-aware ubiquitous learning was used to support 4th grade students as they learn angle concepts. Context-aware ubiquitous learning was provided to students primarily through the use of iPads to access real-world connections and a Dynamic Geometry Environment. Gravemeijer and van Eerde's (2009), design-based research (DBR)…

  12. 'I need her to be a doctor': patients' experiences of presenting health information from the internet in GP consultations.

    PubMed

    Bowes, Parvathy; Stevenson, Fiona; Ahluwalia, Sanjiv; Murray, Elizabeth

    2012-11-01

    Patients are increasingly using the internet for health-related information and may bring this to a GP consultation. There is scant information about why patients do this and what they expect from their GP. The aim was to explore patients' motivation in presenting information, their perception of the GP's response and what they wanted from their doctor. Qualitative study based in North London involving patients with experience of bringing health information from the internet to their GP. Semi-structured face-to-face and telephone interviews using a critical incident technique, recorded, transcribed verbatim, and subjected to thematic analysis by a multidisciplinary team of researchers. Twenty-six interviews were completed. Participants reported using the internet to become better informed about their health and hence make best use of the limited time available with the GP and to enable the GP to take their problem more seriously. Patients expected their GP to acknowledge the information; discuss, explain, or contextualise it; and offer a professional opinion. Patients tended to prioritise the GP opinion over the internet information. However, if the GP appeared disinterested, dismissive or patronising patients reported damage to the doctor-patient relationship, occasionally to the extent of seeking a second opinion or changing their doctor. This is the first in-depth qualitative study to explore why patients present internet information to their GP within the consultation and what they want when they do this. This information should help GPs respond appropriately in such circumstances.

  13. GPs' experiences with out-of-hours GP cooperatives: a survey study from the Netherlands.

    PubMed

    Smits, Marleen; Keizer, Ellen; Huibers, Linda; Giesen, Paul

    2014-09-01

    Out-of-hours primary care has been provided by general practitioner (GP) cooperatives since the year 2000 in the Netherlands. Early studies in countries with similar organizational structures showed positive GP experiences. However, nowadays it is said that GPs experience a high workload at the cooperative and that they outsource a considerable part of their shifts. To examine positive and negative experiences of GPs providing out-of-hours primary care, and the frequency and reasons for outsourcing shifts. A cross-sectional observational survey among 688 GPs connected to six GP cooperatives in the Netherlands, using a web-based questionnaire. The response was 55% (n = 378). The main reasons for working in GP cooperatives were to retain registration as GP (79%) and remain experienced in acute care (74%). GPs considered the peak hours (81%) and the high number of patients (73%) as the most negative aspects. Most GPs chose to provide the out-of-hours shifts themselves: 85% outsourced maximally 25% of their shifts. The percentage of outsourced shifts increased with age. Main reasons for outsourcing were the desire to have more private time (76%); the high workload in daytime practice (71%); and less the workload during out-of-hours (46%). GPs are motivated to work in out-of-hours GP cooperatives, and they outsource few shifts. GPs consider the peak load and the large number of (non-urgent) help requests as the most negative aspects. To motivate and involve GPs for 7 × 24-h primary care, it is important to set limits on their workload.

  14. Mutations in gp41 are correlated with coreceptor tropism but do not improve prediction methods substantially.

    PubMed

    Thielen, Alexander; Lengauer, Thomas; Swenson, Luke C; Dong, Winnie W Y; McGovern, Rachel A; Lewis, Marilyn; James, Ian; Heera, Jayvant; Valdez, Hernan; Harrigan, P Richard

    2011-01-01

    The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations. Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort (n=444) and from patients screened for the MOTIVATE studies (n=1,916; 859 with maraviroc outcome data). Correlations of mutations with tropism were assessed using Fisher's exact test and prediction models trained using support vector machines. Models were validated by cross-validation, by testing models from one dataset on the other, and by analysing virological outcome. Several mutations within gp41 were highly significant for CXCR4 usage; most strikingly an insertion occurring in 7.7% of HOMER-R5 and 46.3% of HOMER-X4 samples (MOTIVATE 5.7% and 25.2%, respectively). Models trained on gp41 sequence alone achieved relatively high areas under the receiver-operating characteristic curve (AUCs; HOMER 0.713 and MOTIVATE 0.736) that were almost as good as V3 models (0.773 and 0.884, respectively). However, combining the two regions improved predictions only marginally (0.813 and 0.902, respectively). Similar results were found when models were trained on HOMER and validated on MOTIVATE or vice versa. The difference in median log viral load decrease at week 24 between patients with R5 and X4 virus was 1.65 (HOMER 2.45 and MOTIVATE 0.79) for V3 models, 1.59 for gp41-models (2.42 and 0.83, respectively) and 1.58 for the combined predictor (2.44 and 0.86, respectively). Several mutations within gp41 showed strong correlation with tropism in two independent datasets. However, incorporating gp41 mutations into prediction models is not mandatory because they do not improve substantially on models trained on V3 sequences alone.

  15. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

    PubMed

    Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

    2016-02-23

    The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

  16. Anti-GP2 IgA autoantibodies are associated with poor survival and cholangiocarcinoma in primary sclerosing cholangitis.

    PubMed

    Jendrek, Sebastian Torben; Gotthardt, Daniel; Nitzsche, Thomas; Widmann, Laila; Korf, Tobias; Michaels, Maike Anna; Weiss, Karl-Heinz; Liaskou, Evaggelia; Vesterhus, Mette; Karlsen, Tom Hemming; Mindorf, Swantje; Schemmer, Peter; Bär, Florian; Teegen, Bianca; Schröder, Torsten; Ehlers, Marc; Hammers, Christoph Matthias; Komorowski, Lars; Lehnert, Hendrik; Fellermann, Klaus; Derer, Stefanie; Hov, Johannes Roksund; Sina, Christian

    2017-01-01

    Pancreatic autoantibodies (PABs), comprising antibodies against glycoprotein 2 (anti-GP2), are typically associated with complicated phenotypes in Crohn's disease, but have also been observed with variable frequencies in patients with UC. In a previous study, we observed a high frequency of primary sclerosing cholangitis (PSC) in patients with anti-GP2-positive UC. We therefore aimed to characterise the role of anti-GP2 in PSC. In an evaluation phase, sera from 138 well-characterised Norwegian patients with PSC were compared with healthy controls (n=52), and patients with UC without PSC (n=62) for the presence of PABs by indirect immunofluorescence. Further, 180 German patients with PSC served as a validation cohort together with 56 cases of cholangiocarcinoma without PSC, 20 of secondary sclerosing cholangitis (SSC) and 18 of autoimmune hepatitis. Anti-GP2 IgA specifically occurred at considerable rates in large bile duct diseases (cholangiocarcinoma=36%, PSC and SSC about 50%). In PSC, anti-GP2 IgA consistently identified patients with poor survival during follow-up (Norwegian/German cohort: p Log Rank=0.016/0.018). Anti-GP2 IgA was associated with the development of cholangiocarcinoma in both PSC cohorts, yielding an overall OR of cholangiocarcinoma in patients with anti-GP2 IgA-positive PSC of 5.0 (p=0.001). Importantly, this association remained independent of disease duration, bilirubin level and age. Anti-GP2 IgA can be hypothesised as a novel marker in large bile duct diseases. In particular, in PSC, anti-GP2 IgA identified a subgroup of patients with severe phenotype and poor survival due to cholangiocarcinoma. Anti-GP2 IgA may therefore be a clinically valuable tool for risk stratification in PSC. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  17. [Expression and significance of P-gp/mdr1 mRNA, MRP and LRP in non-Hodgkin's lymphoma].

    PubMed

    Li, Le; Su, Li-ping; Ma, Li; Zhao, Jin; Zhu, Lei; Zhou, Yong-an

    2009-03-01

    To explore the expression and clinical significance of P-glycoprotein (P-gp)/mdr1mRNA, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in newly diagnosed non-Hodgkin's lymphoma. mdr1 mRNA of in 41 patients with non-Hodgkin's lymphoma was assayed by semi-quantitative RT-PCR. The expressions of P-gp, MRP and LRP proteins in lymph node viable blasts were identified by flow cytometry. The results were compared with those obtained from control cases, and the correlation of the changes with clinical outcomes was analyzed. (1) Among the 41 cases, the positive expression of P-gp protein was detected in 8 cases, MRP in 7 cases, LRP in 15 cases, and mdr 1 mRNA in 11 cases. (2) The P-gp and LRP levels in NHL were significantly higher than those in control group, but MRP wasn't. The P-gp over-expression was significantly associated with mdr1mRNA (r = 0.396, P = 0.01). No correlation was showed among the expressions of P-gp, MRP and LRP. (3) Patients with P-gp expression had a poorer outcome of chemotherapy than those with P-gp-negative (P = 0.005). P-gp expression was significantly associated with higher clinical stage (P = 0.046) and elevated serum lactate dehydrogenase level (P = 0.032), but not associated with malignant degree (P = 0.298). MRP had no impact on the outcome of chemotherapy (P = 0.212), and wasn't significantly associated with higher clinical stage (P = 0.369), elevated LDH (P = 0.762) and higher malignant degree (P = 0.451). Patients with LRP expression had a poorer outcome of chemotherapy than those LRP-negative (P = 0.012). LRP expression was significantly associated with higher clinical stage (P = 0.0019), elevated LDH (P = 0.02) and higher malignant degree (P = 0.01). The data of this study indicate that P-gp and LRP expressions but not MRP expression are important in the mechanism of drug resistance associated with a poor clinical outcome in previously untreated NHL.

  18. Reproducing SIVΔnef vaccine correlates of protection: trimeric gp41 antibody concentrated at mucosal front lines

    PubMed Central

    Voss, James E.; Macauley, Matthew S.; Rogers, Kenneth A.; Villinger, Francois; Duan, Lijie; Shang, Liang; Fink, Elizabeth A.; Andrabi, Raiees; Colantonio, Arnaud D.; Robinson, James E.; Johnson, R. Paul; Burton, Dennis R.; Haase, Ashley T.

    2016-01-01

    Vaccination with SIVmac239Δnef provides robust protection against subsequent challenge with wild type SIV, but safety issues have precluded designing an HIV-1 vaccine based on a live attenuated virus concept. Safe immunogens and adjuvants that could reproduce identified immune correlates of SIVmac239Δnef protection therefore offer an alternative path for development of an HIV vaccine. Here we describe SIV envelope trimeric gp41 (gp41t) immunogens based on a protective correlate of antibodies to gp41t concentrated on the path of virus entry by the neonatal Fc receptor (FcRn) in cervical vaginal epithelium. We developed a gp41t immunogen-MPLA adjuvant liposomal nanoparticle for intra-muscular immunization and a gp41t-Fc immunogen for intranasal immunization for pilot studies in mice, rabbits, and rhesus macaques. Repeated immunizations to mimic persistent antigen exposure in infection elicited gp41t antibodies in rhesus macaques that were detectable in FcRn+ cervical vaginal epithelium, thus recapitulating one key feature of SIVmac239Δnef vaccinated and protected animals. While this strategy did not reproduce the system of local production of antibody in SIVmac239Δnef-vaccinated animals, passive immunization experiments supported the concept that sufficiently high levels of antibody can be concentrated by the FcRn at mucosal frontlines, thus setting the stage for assessing protection against vaginal challenge by gp41t immunization. PMID:27428745

  19. Marburg Virus Glycoprotein GP2: pH-Dependent Stability of the Ectodomain α-Helical Bundle†

    PubMed Central

    Harrison, Joseph S.; Koellhoffer, Jayne F.; Chandran, Kartik; Lai, Jonathan R.

    2012-01-01

    Marburg virus (MARV) and Ebola virus (EBOV) constitute the family Filoviridae of enveloped viruses (filoviruses) that cause severe hemorrhagic fever. Infection by MARV is required for fusion between the host cell and viral membranes, a process that is mediated by the two subunits of the envelope glycoprotein GP1 (surface subunit) and GP2 (transmembrane subunit). Upon viral attachment and uptake, it is believed that the MARV viral fusion machinery is triggered by host factors and environmental conditions found in the endosome. Next, conformational rearrangements in the GP2 ectodomain result in the formation of a highly stable six-helix bundle; this refolding event provides the energetic driving force for membrane fusion. Both GP1 and GP2 from EBOV have been extensively studied, but there is little information available for the MARV glycoproteins. Here we have expressed two variants of the MARV GP2 ectodomain in Escherichia coli and analyzed their biophysical properties. Circular dichroism indicates that the MARV GP2 ectodomain adopts an α-helical conformation, and one variant sediments as a trimer by equilibrium analytical ultracentrifugation. Denaturation studies indicate the α-helical structure is highly stable at pH 5.3 (unfolding energy, ΔGunf H2O, of 33.4 ± 2.5 kcal/mol and melting temperature, Tm, of 75.3 ± 2.1 °C for one variant). Furthermore, we found the α-helical stability to be strongly dependent on pH with higher stability under lower pH conditions (Tm values ranging from ~92 °C at pH 4.0 to ~38 °C at pH 8.0). Mutational analysis suggests two glutamic acid residues (E579 and E580) are partially responsible for this pH-dependent behavior. Based on these results, we hypothesize that pH-dependent folding stability of the MARV GP2 ectodomain provides a mechanism to control conformational preferences such that the six-helix bundle ‘post-fusion’ state is preferred under conditions of appropriately matured endosomes. PMID:22369502

  20. Multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) inhibition by tariquidar impacts on neuroendocrine and behavioral processing of stress.

    PubMed

    Thoeringer, Christoph K; Wultsch, Thomas; Shahbazian, Anaid; Painsipp, Evelin; Holzer, Peter

    2007-01-01

    The multidrug-resistance gene 1-type p-glycoprotein (MDR1 p-gp) is a major gate-keeper at the blood-brain barrier (BBB), protecting the central nervous system from accumulation of toxic xenobiotics and drugs. In addition, MDR1 p-gp has been found to control the intracerebral access of glucocorticoid hormones and thus to modulate the activity of the hypothalamic-pituitary-adrenocortical (HPA) system. In view of the implication of glucocorticoids in the control of behavior, we examined how acute pharmacological inhibition of MDR1 p-gp at the BBB by tariquidar (XR9576; 12 mg/kg, PO) impacts the neuroendocrine and behavioral processing of stress in C57BL/6JIcoHim inbred mice. Inhibition of MDR1 p-gp at the BBB did not alter emotional behavior at baseline. However, mice that were sensitized by water-avoidance stress, a mild psychological stressor, displayed significantly reduced anxiety-related behavior in the elevated plus-maze test when treated with tariquidar. Tariquidar, however, had no effect on stress-coping performance assessed in the forced swim test. Investigating the impact of acute MDR1 p-gp inhibition on the glucocorticoid system, we observed a significant attenuation of the mild stress-induced increase of plasma corticosterone after tariquidar administration. In order to examine whether the anti-anxiety effect of tariquidar in sensitized animals is mediated by glucocorticoids, the animals were treated with corticosterone (1mg/kg, SC) immediately after exposure to water-avoidance stress. Corticosterone caused a significant anxiolytic-like effect in this stress-related anxiety protocol, whereas tariquidar could not further enhance corticosterone's anti-anxiety effects. The current data show for the first time that pharmacological inhibition of MDR1 p-gp at the murine BBB by tariquidar alters emotional behavior and HPA axis activity. By facilitating the entry of corticosterone into the brain, tariquidar enhances feedback inhibition of the HPA system and in

  1. Electrochemical corrosion behavior and MG-63 osteoblast-like cell response of surface-treated titanium

    NASA Astrophysics Data System (ADS)

    Kim, Hak-Kwan; Jang, Ju-Woong

    2004-10-01

    Commercially pure titanium is used as a clinical implant material for many orthopedic and dental implant devices owing to its excellent corrosion resistance and good biocompatibility. However, there remains concern over the release of metal ions from prostheses and unresolved questions about its behavior in a biological environment. Our research investigated the influence of surface oxide thickness and phase on the corrosion resistance in 0.9% NaCl solution by potentiostat and XRD. Also, the MG-63 osteoblast like cell morphology and proliferation were studied to evaluate the biocompatibility in terms of surface treatment. It is demonstrated that a substantial decrease in the current density may be attained due to surface oxide thickening and phase transformation by thermal oxidation. The osteoblast adhesion morphology and proliferation data indicated that the osteoblast cell response is not conspicuously influenced by the thermal oxidation and nitric acid passivation treatments but by surface roughness and porosity of 3rd networking.

  2. Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Arthur, L.O.; Pyle, S.W.; Nara, P.L.

    1987-12-01

    The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/more » cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.« less

  3. Hyperion 5113/GP Infrasound Sensor Evaluation.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Merchant, Bion J.

    2015-08-01

    Sandia National Laboratories has tested and evaluated an infrasound sensor, the 5113/GP manufactured by Hyperion. These infrasound sensors measure pressure output by a methodology developed by the University of Mississippi. The purpose of the infrasound sensor evaluation was to determine a measured sensitivity, transfer function, power, self-noise, dynamic range, and seismic sensitivity. These sensors are being evaluated prior to deployment by the U.S. Air Force.

  4. After Installation: Ubiquitous Computing and High School Science in Three Experienced, High-Technology Schools

    ERIC Educational Resources Information Center

    Drayton, Brian; Falk, Joni K.; Stroud, Rena; Hobbs, Kathryn; Hammerman, James

    2010-01-01

    There are few studies of the impact of ubiquitous computing on high school science, and the majority of studies of ubiquitous computing report only on the early stages of implementation. The present study presents data on 3 high schools with carefully elaborated ubiquitous computing systems that have gone through at least one "obsolescence cycle"…

  5. The Elementary Mass Action Rate Constants of P-gp Transport for a Confluent Monolayer of MDCKII-hMDR1 Cells

    PubMed Central

    Tran, Thuy Thanh; Mittal, Aditya; Aldinger, Tanya; Polli, Joseph W.; Ayrton, Andrew; Ellens, Harma; Bentz, Joe

    2005-01-01

    The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been

  6. Activation of the GP130-STAT3 axis and its potential implications in nonalcoholic fatty liver disease

    PubMed Central

    Min, Hae-Ki; Mirshahi, Faridoddin; Verdianelli, Aurora; Pacana, Tommy; Patel, Vaishali; Park, Chun-Geon; Choi, Aejin; Lee, Jeong-Hoon; Park, Chung-Berm; Ren, Shunlin

    2015-01-01

    The status of the GP130-STAT3 signaling pathway in humans with nonalcoholic fatty liver disease (NAFLD) and its relevance to disease pathogenesis are unknown. The expression of the gp130-STAT3 axis and gp130 cytokine receptors were studied in subjects with varying phenotypes of NAFLD including nonalcoholic steatohepatitis (NASH) and compared with lean and weight-matched controls without NAFLD. Gp130 and its downstream signaling element (Tyk2 and STAT3) expression were inhibited in obese controls whereas they were increased in NAFLD. IL-6 levels were increased in NASH and correlated with gp130 expression (P < 0.01). Palmitate inhibited gp130-STAT3 expression and signaling. IL-6 and palmitate inhibited hepatic insulin signaling via STAT3-dependent and independent mechanisms, respectively. STAT3 overexpression reversed palmitate-induced lipotoxicity by increasing autophagy (ATG7) and decreasing endoplasmic reticulum stress. These data demonstrate that the STAT3 pathway is activated in NAFLD and can worsen insulin resistance while protecting against other lipotoxic mechanisms of disease pathogenesis. PMID:25747354

  7. HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.

    PubMed

    Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly E; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, M Juliana; Mascola, John R; Koup, Richard A; Corey, Lawrence; Nabel, Gary J; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S; Baden, Lindsey R; Tomaras, Georgia D; Haynes, Barton F

    2015-08-14

    An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. Copyright © 2015, American Association for the Advancement of Science.

  8. Resveratrol strongly enhances the retinoic acid-induced superoxide generating activity via up-regulation of gp91-phox gene expression in U937 cells.

    PubMed

    Kikuchi, Hidehiko; Mimuro, Hitomi; Kuribayashi, Futoshi

    2018-01-01

    The membrane bound cytochrome b 558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O 2 - )-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O 2 - -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and resveratrol, the O 2 - -generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O 2 - -generating activity via up-regulation of gp91-phox gene expression in U937 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120

    PubMed Central

    Schoeniger-Skinner, Diana K.; Ledeboer, Annemarie; Frank, Matthew G.; Milligan, Erin D.; Poole, Stephen; Martin, David; Maier, Steven F.; Watkins, Linda R.

    2007-01-01

    Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-α (TNF), and interleukin-1β (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other proinflammatory cytokines. PMID:17204394

  10. Income-related inequity in the use of GP services by children: a comparison of Ireland and Scotland.

    PubMed

    Layte, Richard; Nolan, Anne

    2015-06-01

    Equity of access to health care is a key component of national and international health policy, with most countries subscribing to the principle that health care should be allocated on the basis of need, rather than ability to pay or other criteria. The issue of health care entitlements for children is particularly pertinent given the strong causal links that have been demonstrated between eligibility for free care, utilisation and health outcomes. The Irish health care system is unusual in requiring the majority of the population to pay the full out-of-pocket cost of GP care. In contrast, all Scottish residents are entitled to free GP care at the point of use. This difference in public health care entitlements between Ireland and Scotland allows us to examine the impact of differences in financing structures on equity in GP care. In this paper, we use data from two nationally representative surveys of children in Ireland and Scotland to examine the degree of income-related inequity in the utilisation of GP services in both countries. We find that while the distribution of GP care is significantly pro-poor in Ireland, even after adjustment for health need, there is little or no significant inequity in GP utilisation among Scottish children. However, focusing just on children who pay the full price of GP care in Ireland, we find some evidence for a significant pro-rich distribution of GP visits. These results reflect the particular structure of health care entitlements that exist in two systems.

  11. How patients perceive the therapeutic communications skills of their general practitioners, and how that perception affects adherence: use of the TCom-skill GP scale in a specific geographical area.

    PubMed

    Baumann, Michèle; Baumann, Cédric; Le Bihan, Etienne; Chau, Nearkasen

    2008-12-01

    To study: (1) the structure and test-retest reliability of a measure of how patients perceive the therapeutic communications skills of their general practitioners (TCom-skill GP), and (2) the associations of that scale with socio-demographic and health-related characteristics, and adherence. A total of 393 people who lived in the same geographic area and invited to attend a preventive medical centre for a check up were asked to complete a self-administered questionnaire concerning TCom-skill GP (15 items), socio-demographic and health-related characteristics, and to answer two questions on perceived adherence. The average age of respondents was 46.8 years (SD 14), and 50.4% were men. The TCom-skill GP score was one-dimensional, had high internal coherence (Cronbach alpha 0.92), and good test-retest reliability (intra-class correlation coefficient 0.74). The overall score was positively related to increasing age. Respondents aged 60+ were more likely to be adherent. The higher the score, the higher the probability of adherence. Multivariate analysis showed that the TCom-skill score was associated with advancing age and the number of consultations with the GP during the previous 3 months, but not with gender, living alone, being employed, job category or educational level. Multivariate analysis also showed that adherence was associated with TCom-skill GP score which concealed the association between adherence and advancing age observed in univariate analysis. The TCom-skill GP scale probably has value in assessing the quality of doctor-patient relationships and therapeutic communications. The psychometric properties of the TCom-skill GP scale were appropriate for its use in this context. Adherence related to the TCom-skill GP and the latter related to the age of patients and the number of their previous consultations. The TCom-skill GP scale may be a useful way to assess, in a specific geographical location, the impact of medical professional training on therapeutic

  12. Matrix metalloproteases inhibition and biocompatibility of gold and platinum nanoparticles.

    PubMed

    Hashimoto, Masanori; Kawai, Koji; Kawakami, Hayato; Imazato, Satoshi

    2016-01-01

    Matrix metalloprotease (MMP) inhibitors improve the longevity of dental adhesives/tooth bonds; however, biocompatibility is required for their clinical use. This study evaluated the inhibition of MMPs and toxicity of two gold (AuNPs) and platinum nanoparticles (PtNPs) as possible compounds for use in dental adhesives. The MMP assay for studying the interaction of MMPs and nanoparticles (NPs) was evaluated by an MMP assay kit and gelatin zymography. Cultured L929 fibroblast cells or RAW264 macrophages were exposed to NPs. The cellular responses to NPs were examined using cytotoxic (cell viability) and genotoxic assays (comet assay), and transmission electron microscopic (TEM) analysis. The mechanical properties (elastic modulus) of the experimental resin loaded with NPs were examined using thermomechanical analysis. All NPs inhibited MMP activity at relatively low concentrations. The NPs inhibit MMPs by chelating with the Zn(2+) bound in the active sites of MMPs. No cytotoxic and genotoxic effects were found in AuNPs, whereas the PtNPs possessed both adverse effects. In TEM analysis, the NPs were localized mainly in lysosomes without penetration into nuclei. The mechanical properties of the resins increased when AuNPs were added in resins, but not by PtNPs. AuNPs are attractive candidates to inhibit MMPs and improve the mechanical properties of resins without cytotoxic/genotoxic effects to cells, and therefore should be suitable for applications in adhesive resin systems. © 2015 Wiley Periodicals, Inc.

  13. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko

    2010-09-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinantmore » AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.« less

  14. Is hyperactivity ubiquitous in ADHD or dependent on environmental demands? Evidence from meta-analysis

    PubMed Central

    Kofler, Michael J.; Raiker, Joseph S.; Sarver, Dustin E.; Wells, Erica L.; Soto, Elia F.

    2016-01-01

    Hyperactivity, or excess gross motor activity, is considered a core and ubiquitous characteristic of ADHD. Alternate models question this premise, and propose that hyperactive behavior reflects, to a large extent, purposeful behavior to cope with environmental demands that interact with underlying neurobiological vulnerabilities. The present review critically evaluates the ubiquity and environmental modifiability of hyperactivity in ADHD through meta-analysis of 63 studies of mechanically measured activity level in children, adolescents, and adults with ADHD relative to typically developing (TD) groups. Random effects models corrected for publication bias confirmed elevated gross motor activity in ADHD (d = 0.86); surprisingly, neither participant age (child vs. adult) nor the proportion of each ADHD sample diagnosed with the Inattentive subtype/presentation moderated this effect. In contrast, activity level assessed during high cognitive load conditions in general (d = 1.14) and high executive functioning demands in particular (d = 1.39) revealed significantly higher effect sizes than activity level during low cognitive load (d = 0.36) and in-class schoolwork (d = 0.50) settings. Low stimulation environments, more rigorous diagnostic practices, actigraph measurement of movement frequency and intensity, and ADHD samples that included fewer females were also associated with larger effects. Overall, the results are inconsistent with DSM-5 and ADHD models that a) describe hyperactivity as ubiquitous behavior, b) predict a developmental decline in hyperactivity, or c) differentiate subtypes/presentations according to perceived differences in hyperactive behavior. Instead, results suggest that the presence and magnitude of hyperactive behavior in ADHD may be influenced to a considerable extent by environmental factors in general, and cognitive/executive functioning demands in particular. PMID:27131918

  15. Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

    PubMed Central

    Liao, Hua-Xin; Tomaras, Georgia D.; Bonsignori, Mattia; Tsao, Chun-Yen; Hwang, Kwan-Ki; Chen, Haiyan; Lloyd, Krissey E.; Bowman, Cindy; Sutherland, Laura; Jeffries, Thomas L.; Kozink, Daniel M.; Stewart, Shelley; Anasti, Kara; Jaeger, Frederick H.; Parks, Robert; Yates, Nicole L.; Overman, R. Glenn; Sinangil, Faruk; Berman, Phillip W.; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Karasavva, Nicos; Rerks-Ngarm, Supachai; Kim, Jerome H.; Michael, Nelson L.; Zolla-Pazner, Susan; Santra, Sampa; Letvin, Norman L.; Harrison, Stephen C.

    2013-01-01

    An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity. PMID:23175357

  16. Depression management within GP-centered health care - A case-control study based on claims data.

    PubMed

    Freytag, Antje; Krause, Markus; Lehmann, Thomas; Schulz, Sven; Wolf, Florian; Biermann, Janine; Wasem, Jürgen; Gensichen, Jochen

    For most patients with depression, GPs are the first and long-term medical providers. GP-centered health care (GPc-HC) programs target patients with chronic diseases. What are the effects of GPc-HC on primary care depression management? An observational retrospective case-control study was conducted using health insurance claims data of patients with depressive disorder from July 2011 to December 2012. From 40,298 patients insured with the largest health plan in Central Germany participating in the GPc-HC program (intervention group, IG), we observed 4645 patients with depression over 18months: 72.2% women; 66.6years (mean); multiple conditions (morbidity-weight 2.50 (mean), 86%>1.0). We compared them with 4013 patients who did not participate (control group). In participants we found lower number of incomplete/non-specified depression diagnoses (4.46vs.4.82;MD-0.36; p<0.01); lower rate of patients consulting more than one GP-practice (49.1%vs.58.0%;PP-8.9;p<0.01); more GP-contacts (18.19vs.15.59;MD+2.60;p<0.01); more GP-initiated referrals to specialists (82.9%vs.79.3%;PP+3.6;p<0.05), more antidepressant pharmacotherapy prescribed by a GP (37.9%vs.35.4%;PP+2.5;p<0.05), more frequent guideline-concordant therapy duration (19.2%vs.13.1%;PP+6.1;p<0.01) and more patients receiving "GP-psychosomatic basic care" (38.2%vs.30.2%;PP+8.0;p<0.01). Depressive patients participating in a GPc-HC program may be more often diagnosed by a GP, receive symptom-monitoring and appropriate depression treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ.

    PubMed

    Sborgi, Lorenzo; Verma, Abhinav; Muñoz, Victor; de Alba, Eva

    2011-01-01

    GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein structures.

  18. CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

    PubMed Central

    Hu, Shuxian; Sheng, Wen S.; Rock, R. Bryan

    2013-01-01

    Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB1/CB2 agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB2 receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety

  19. Potable Water Treatment Facility General Permit (PWTF GP) ...

    EPA Pesticide Factsheets

    2017-08-28

    The Final PWTF GP establishes permit eligibility conditions, Notice of Intent (NOI) requirements, effluent limitations, standards, prohibitions, and best management practices for facilities that discharge to waters in the Commonwealth of Massachusetts (including both Commonwealth and Indian country lands) and the State of New Hampshire.

  20. A new method measuring the interaction of radiotracers with the human P-glycoprotein (P-gp) transporter.

    PubMed

    Vraka, Chrysoula; Dumanic, Monika; Racz, Teresa; Pichler, Florian; Philippe, Cecile; Balber, Theresa; Klebermass, Eva-Maria; Wagner, Karl-Heinz; Hacker, Marcus; Wadsak, Wolfgang; Mitterhauser, Markus

    2018-05-01

    In drug development, biomarkers for cerebral applications have a lower success rate compared to cardiovascular drugs or tumor therapeutics. One reason is the missing blood brain barrier penetration, caused by the tracer's interaction with efflux transporters such as the P-gp (MDR1 or ABCB1). Aim of this study was the development of a reliable model to measure the interaction of radiotracers with the human efflux transporter P-gp in parallel to the radiolabeling process. LigandTracer® Technology was used with the wildtype cell line MDCKII and the equivalent cell line overexpressing human P-gp (MDCKII-hMDR1). The method was evaluated based on established PET tracers with known interaction with the human P-gp transporter and in nanomolar concentration (15 nM). [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP were used as P-gp substrates by comparing the real-time model with an uptake assay and μPET images. [ 11 C]DASB [ 11 C]Harmine, [ 18 F]FMeNER,[ 18 F]FE@SUPPY and [ 11 C]Me@HAPTHI were used as tracers without interactions with P-gp in vitro. However, [ 11 C]Me@HAPTHI shows a significant increase in SUV levels after blocking with Tariquidar. The developed real-time kinetic model uses directly PET tracers in a compound concentration, which is reflecting the in vivo situation. This method may be used at an early stage of radiopharmaceutical development to measure interactions to P-gp before conducting animal experiments. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. People with multiple unhealthy lifestyles are less likely to consult primary healthcare.

    PubMed

    Feng, Xiaoqi; Girosi, Federico; McRae, Ian S

    2014-06-26

    Behavioural interventions are often implemented within primary healthcare settings to prevent type 2 diabetes and other lifestyle-related diseases. Although smoking, alcohol consumption, physical inactivity and poor diet are associated with poorer health that may lead a person to consult a general practitioner (GP), previous work has shown that unhealthy lifestyles cluster among low socioeconomic groups who are less likely to seek primary healthcare. Therefore, it is uncertain whether behavioural interventions in primary healthcare are reaching those in most need. This study investigated patterns of GP consultations in relation to the clustering of unhealthy lifestyles among a large sample of adults aged 45 years and older in New South Wales, Australia. A total of 267,153 adults participated in the 45 and Up Study between 2006 and 2009, comprising 10% of the equivalent demographic in the state of New South Wales, Australia (response rate: 18%). All consultations with GPs within 6 months prior and post survey completion were identified (with many respondents attending multiple GPs) via linkage to Medicare Australia data. An index of unhealthy lifestyles was constructed from self-report data on adherence to published guidelines on smoking, alcohol consumption, diet and physical activity. Logistic and zero-truncated negative binomial regression models were used to analyse: (i) whether or not a person had at least one GP consultation within the study period; (ii) the count of GP consultations attended by each participant who visited a GP at least once. Analyses were adjusted for measures of health status, socioeconomic circumstances and other confounders. After adjustment, participants scoring 7 unhealthy lifestyles were 24% more likely than persons scoring 0 unhealthy lifestyles not to have attended any GP consultation in the 12-month time period. Among those who attended at least one consultation, those with 7 unhealthy lifestyles reported 7% fewer consultations than

  2. Connecting Effective Immune Response, Fluorescent Granzyme B-like Peptide, Specific Peptide Binding Patterns, Patients with Cancer and Viral Infection, in Remission, Clinical Significance, and Liquid Biopsy.

    PubMed

    Lo, Wai Chun Jennifer; Luther, Donald Gene

    2016-11-01

    Functional cytotoxic-T-lymphocytes (CTL) with granzyme B play an important role in an effective immune response to tumor growth and infection progression. Tumor cells and platelets in peripheral whole blood smears of cancer patients have shown the presence of innate binding targets for GP1R, a fluorescent synthetic Granzyme B-like peptide. It is not known if similar GP1R-binding targets and specific binding patterns are detectable in peripheral blood of patients with viral infection. It is also not known if a specific binding pattern may be associated with an effective immune response to indicate a favorable prognosis. We reviewed the GP1R-binding patterns in the peripheral blood smears of 5 patients in remission at the time of sampling (3 with cancer and 2 with flu-like symptoms) and a negative control. We show with fluoroscopic images that there are: 1) fluorescent GP1R-binding targets mostly in the cytoplasmic areas of nucleated cells in patients with breast and lung cancer who have longer survival, 2) intense fluorescent deposits mostly in the nuclear areas of segmented neutrophils in patients recovered from severe to mild flu-like symptoms, 3) discernible fluorescent deposits in the cytoplasmic areas of small lymphocyte-like elements and overall intense fluorescent stain in large cells in the patient with advanced pancreatic cancer who had shorter survival, 4) GP1R-binding targets in numerous platelet-like elements in all 5 patients. The control sample did not show similar binding patterns. The potential association between specific GP1R-binding patterns in peripheral blood samples and prognostic significance, and its use as liquid biopsy are discussed.

  3. The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

    2010-08-17

    The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 {angstrom} crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophagemore » SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.« less

  4. The Crystal Structure of Bacteriophage HK97 gp6: Defining a Large Family of Head-Tail Connector Proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cardarelli, Lia; Lam, Robert; Tuite, Ashleigh

    2011-11-23

    The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 Å crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophagemore » SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.« less

  5. From Virtual Environments to Physical Environments: Exploring Interactivity in Ubiquitous-Learning Systems

    ERIC Educational Resources Information Center

    Peng, Hsinyi; Chou, Chien; Chang, Chun-Yu

    2008-01-01

    Computing devices and applications are now used beyond the desktop, in diverse environments, and this trend toward ubiquitous computing is evolving. In this study, we re-visit the interactivity concept and its applications for interactive function design in a ubiquitous-learning system (ULS). Further, we compare interactivity dimensions and…

  6. Effects of naringin on learning and memory dysfunction induced by gp120 in rats.

    PubMed

    Qin, Shanshan; Chen, Qiang; Wu, Hui; Liu, Chenglong; Hu, Jing; Zhang, Dalei; Xu, Changshui

    2016-06-01

    The aim of the present study was to investigate the effects of naringin on learning and memory dysfunction induced by HIV-1-enveloped protein gp120 in rats, and to identify its potential mechanisms of action. Learning and memory ability was evaluated via Morris water maze test, P2X7 receptor and P65 protein expressions in the rat hippocampus were detected by western blot analysis, and P2X7 mRNA expression in the hippocampus was measured by RT-PCR. We also recorded P2X7 agonist BzATP-activated current in the hippocampus via patch clamp technique. The results showed that naringin treatment (30mg/kg/day) markedly decreased the escape latency and target platform errors of rats treated with gp120 (50ng/day), and further, that naringin treatment significantly decreased the expression of P2X7 and P65 protein and P2X7 mRNA in the hippocampus of gp120-treated rats. In addition, naringin treatment reduced BzATP-activated current in the hippocampus of gp120-treated rats. These results altogether demonstrated that naringin can improve gp120-induced learning and memory dysfunction via mechanisms involving the inhibition of P2X7 expression in the hippocampus. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia

    PubMed Central

    Kamarlis, Reno K; Lubis, Muhammad ND; Hernowo, Bethy S; Kar, Azmi S

    2018-01-01

    Background: Using immunohistochemical stains to target specific breast cancer markers has become indispensable for evaluation of small diagnostic tissue specimens, and therefore novel marker cocktails for specific breast cancers are required. This study was conducted to assess the immunoexpression of P63 and SOX2 in triple negative breast cancer (TNBC), and to evaluate the predictive diagnostic value of these markers for specific types of TNBC. Methods: Histological slides and paraffin blocks of TNBC cases were collected from Dr. Hasan Sadikin Hospital, Bandung, Indonesia from 5-years period (2011-2015). Each histological slide was subjected to immunohistochemical staining for P63 (nucleus and cytoplasm) and SOX2 (nucleus), with specific primer antibodies. Immunoexpression of P63 and SOX2 was evaluated using immunoreactivity scoring. Associations between P63 and SOX2 immunoexpression and TNBC types were assessed using Mann Whitney tests. In addition, the predictive diagnostic values of these markers were assessed. Results: Forty TNBC histological slides were included, and 23 (57.5%) were Basal-like type TNBC and 17 (42.5%) were Non basal-like type TNBC. Immunoexpression of P63 nucleus and SOX2 was not different between types of TNBC. However, immunoexpression of P63 in the cytoplasm in Basal-like type TNBC was significantly higher than in Non basal-like type TNBC ( p=0.021). Predictor diagnostic value analysis suggested that immunoexpression of P63 in cytoplasm had 56.5% sensitivity and 70.6% specificity for diagnosing Basal-like type TNBC, with area under curve of 0.64.    Conclusions: Immunoexpression of P63 in the cytoplasm has a relatively weak diagnostic value to discriminate Basal-like and Non basal-like types of TNBC. PMID:29527291

  8. The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

    PubMed

    Bellantuono, I; Lashford, L S; Rafferty, J A; Fairbairn, L J

    2000-05-01

    As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order

  9. Ubiquitous Mobile Knowledge Construction in Collaborative Learning Environments

    PubMed Central

    Baloian, Nelson; Zurita, Gustavo

    2012-01-01

    Knowledge management is a critical activity for any organization. It has been said to be a differentiating factor and an important source of competitiveness if this knowledge is constructed and shared among its members, thus creating a learning organization. Knowledge construction is critical for any collaborative organizational learning environment. Nowadays workers must perform knowledge creation tasks while in motion, not just in static physical locations; therefore it is also required that knowledge construction activities be performed in ubiquitous scenarios, and supported by mobile and pervasive computational systems. These knowledge creation systems should help people in or outside organizations convert their tacit knowledge into explicit knowledge, thus supporting the knowledge construction process. Therefore in our understanding, we consider highly relevant that undergraduate university students learn about the knowledge construction process supported by mobile and ubiquitous computing. This has been a little explored issue in this field. This paper presents the design, implementation, and an evaluation of a system called MCKC for Mobile Collaborative Knowledge Construction, supporting collaborative face-to-face tacit knowledge construction and sharing in ubiquitous scenarios. The MCKC system can be used by undergraduate students to learn how to construct knowledge, allowing them anytime and anywhere to create, make explicit and share their knowledge with their co-learners, using visual metaphors, gestures and sketches to implement the human-computer interface of mobile devices (PDAs). PMID:22969333

  10. Ubiquitous mobile knowledge construction in collaborative learning environments.

    PubMed

    Baloian, Nelson; Zurita, Gustavo

    2012-01-01

    Knowledge management is a critical activity for any organization. It has been said to be a differentiating factor and an important source of competitiveness if this knowledge is constructed and shared among its members, thus creating a learning organization. Knowledge construction is critical for any collaborative organizational learning environment. Nowadays workers must perform knowledge creation tasks while in motion, not just in static physical locations; therefore it is also required that knowledge construction activities be performed in ubiquitous scenarios, and supported by mobile and pervasive computational systems. These knowledge creation systems should help people in or outside organizations convert their tacit knowledge into explicit knowledge, thus supporting the knowledge construction process. Therefore in our understanding, we consider highly relevant that undergraduate university students learn about the knowledge construction process supported by mobile and ubiquitous computing. This has been a little explored issue in this field. This paper presents the design, implementation, and an evaluation of a system called MCKC for Mobile Collaborative Knowledge Construction, supporting collaborative face-to-face tacit knowledge construction and sharing in ubiquitous scenarios. The MCKC system can be used by undergraduate students to learn how to construct knowledge, allowing them anytime and anywhere to create, make explicit and share their knowledge with their co-learners, using visual metaphors, gestures and sketches to implement the human-computer interface of mobile devices (PDAs).

  11. In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Han Yi; Chin Tan, Theresa May; Lim, Lee-Yong

    2008-08-01

    Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 {mu}M, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [{sup 3}H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [{sup 3}H]-digoxin polarized transport attained at 50 {mu}M of piperine. In contrast, prolongedmore » (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 {mu}M) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [{sup 3}H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 {mu}g/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.« less

  12. Energetics of dendrimer binding to HIV-1 gp120-CD4 complex and mechanismic aspects of its role as an entry-inhibitor

    NASA Astrophysics Data System (ADS)

    Saurabh, Suman; Sahoo, Anil Kumar; Maiti, Prabal K.

    2016-10-01

    Experiments and computational studies have established that de-protonated dendrimers (SPL7013 and PAMAM) act as entry-inhibitors of HIV. SPL7013 based Vivagel is currently under clinical development. The dendrimer binds to gp120 in the gp120-CD4 complex, destabilizes it by breaking key contacts between gp120 and CD4 and prevents viral entry into target cells. In this work, we provide molecular details and energetics of the formation of the SPL7013-gp120-CD4 ternary complex and decipher modes of action of the dendrimer in preventing viral entry. It is also known from experiments that the dendrimer binds weakly to gp120 that is not bound to CD4. It binds even more weakly to the CD4-binding region of gp120 and thus cannot directly block gp120-CD4 complexation. In this work, we examine the feasibility of dendrimer binding to the gp120-binding region of CD4 and directly blocking gp120-CD4 complex formation. We find that the process of the dendrimer binding to CD4 can compete with gp120-CD4 binding due to comparable free energy change for the two processes, thus creating a possibility for the dendrimer to directly block gp120-CD4 complexation by binding to the gp120-binding region of CD4.

  13. Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate

    PubMed Central

    2013-01-01

    Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity. PMID:23835244

  14. Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate.

    PubMed

    Visciano, Maria Luisa; Tagliamonte, Maria; Stewart-Jones, Guillaume; Heyndrickx, Leo; Vanham, Guido; Jansson, Marianne; Fomsgaard, Anders; Grevstad, Berit; Ramaswamy, Meghna; Buonaguro, Franco M; Tornesello, Maria Lina; Biswas, Priscilla; Scarlatti, Gabriella; Buonaguro, Luigi

    2013-07-08

    Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

  15. Ubiquitous computing in shared-care environments.

    PubMed

    Koch, S

    2006-07-01

    In light of future challenges, such as growing numbers of elderly, increase in chronic diseases, insufficient health care budgets and problems with staff recruitment for the health-care sector, information and communication technology (ICT) becomes a possible means to meet these challenges. Organizational changes such as the decentralization of the health-care system lead to a shift from in-hospital to both advanced and basic home health care. Advanced medical technologies provide solutions for distant home care in form of specialist consultations and home monitoring. Furthermore, the shift towards home health care will increase mobile work and the establishment of shared care teams which require ICT-based solutions that support ubiquitous information access and cooperative work. Clinical documentation and decision support systems are the main ICT-based solutions of interest in the context of ubiquitous computing for shared care environments. This paper therefore describes the prerequisites for clinical documentation and decision support at the point of care, the impact of mobility on the documentation process, and how the introduction of ICT-based solutions will influence organizations and people. Furthermore, the role of dentistry in shared-care environments is discussed and illustrated in the form of a future scenario.

  16. Immune modulation with high-dose heat-shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis.

    PubMed

    Chandawarkar, Rajiv Y; Wagh, Mihir S; Kovalchin, Joseph T; Srivastava, Pramod

    2004-04-01

    Immunization with heat-shock protein (HSP) gp96 elicits protective immunity to the cancer or virus-infected cells from which it is derived. Low doses of gp96 generate immunity, while doses 10 times the immunizing dose do not. We show here that injection of high doses of gp96 generates CD4(+) T cells that down-regulate a variety of ongoing immune responses. Immunization with high doses of gp96 prevents myelin basic protein- or proteolipid protein-induced autoimmune encephalomyelitis in SJL mice and the onset of diabetes in non-obese diabetic mice. The suppression of immune response can be adoptively transferred with CD4(+) cells and does not partition with the CD25 phenotype. The immunomodulatory properties of gp96 (and possibly other HSP) may be used for antigen-specific activation or suppression of cellular immune responses. The latter may form the basis for novel immunotherapies for autoimmune diseases.

  17. [Zinc-dependent metalloprotease 1 promotes apoptosis of RAW264.7 macrophages].

    PubMed

    Li, Peng; He, Yonglin; Zhang, Jiming; Fang, Chencheng

    2015-12-01

    To construct the eukaryotic expression vector of zinc-dependent metalloprotease 1 (zmp1) gene from Bacillus Calmette-Guerin (BCG) and investigate its impact on the apoptosis of RAW264.7 macrophages. Zmp1 gene was amplified from the genome of BCG by PCR. The zmp1 gene fragment was inserted into multiple cloning sites of pEGFP-N1 to construct the eukaryotic expression vector pEGFP-N1-zmp1. The constructed pEGFP-N1-zmp1 was transfected into RAW264.7 cells by Lipofectamine(TM) 2000. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The zmp1 mRNA was detected by quantitative real-time PCR (qR-PCR). The effect of Zmp1 protein on the apoptosis of RAW264.7 macrophages was detected by flow cytometry (FCM). With zmp1 gene amplified by PCR, we successfully constructed the recombinant vector pEGFP-N1-zmp1 as demonstrated by restriction enzyme analysis and sequencing. GFP was seen in RAW264.7 cells 24 hours after transfected with the recombinant plasmid. As qRT-PCR showed, the expression level of zmp1 mRNA was up-regulated. The early apoptotic rate increased 48 hours after transfection. The increased expression of Zmp1 in RAW264.7 cells promotes the apoptosis of RAW264.7 cells.

  18. Knowledge-transfer learning for prediction of matrix metalloprotease substrate-cleavage sites.

    PubMed

    Wang, Yanan; Song, Jiangning; Marquez-Lago, Tatiana T; Leier, André; Li, Chen; Lithgow, Trevor; Webb, Geoffrey I; Shen, Hong-Bin

    2017-07-18

    Matrix Metalloproteases (MMPs) are an important family of proteases that play crucial roles in key cellular and disease processes. Therefore, MMPs constitute important targets for drug design, development and delivery. Advanced proteomic technologies have identified type-specific target substrates; however, the complete repertoire of MMP substrates remains uncharacterized. Indeed, computational prediction of substrate-cleavage sites associated with MMPs is a challenging problem. This holds especially true when considering MMPs with few experimentally verified cleavage sites, such as for MMP-2, -3, -7, and -8. To fill this gap, we propose a new knowledge-transfer computational framework which effectively utilizes the hidden shared knowledge from some MMP types to enhance predictions of other, distinct target substrate-cleavage sites. Our computational framework uses support vector machines combined with transfer machine learning and feature selection. To demonstrate the value of the model, we extracted a variety of substrate sequence-derived features and compared the performance of our method using both 5-fold cross-validation and independent tests. The results show that our transfer-learning-based method provides a robust performance, which is at least comparable to traditional feature-selection methods for prediction of MMP-2, -3, -7, -8, -9 and -12 substrate-cleavage sites on independent tests. The results also demonstrate that our proposed computational framework provides a useful alternative for the characterization of sequence-level determinants of MMP-substrate specificity.

  19. Caterpillar, Inc. — Converter GP-Catalytic Technology (CGP) system

    EPA Pesticide Factsheets

    This EPA memo approves Caterpillar Inc. request for Caterpillar Converter GP-Catalytic Technology (CGP) system for certain wheeled hydraulic excavators in reducing emissions and will be posted on the National Clean Diesel Verified Technologies List.

  20. HIV-gp120 and physical dependence to buprenorphine.

    PubMed

    Palma, J; Abood, M E; Benamar, K

    2015-05-01

    Opioids are among the most effective and commonly used analgesics in clinical practice for severe pain. However, the use of opioid medications is clinically limited by several adverse properties including dependence. While opioid dependence is a complex health condition, the treatment of HIV-infected individuals with opioid dependence presents additional challenges. The goal of this study was to examine the physical dependence to buprenorphine in the context of HIV. Young adult male rats (Sprague-Dawley) were pretreated with HIV-1 envelope glycoprotein 120 (gp120) injected into the periaqueductal gray area (PAG) and we examined the impact on physical dependence to opioid. It was found that the physical dependence to methadone occurred earlier than that to buprenorphine, and that gp120 did not enhance or precipitate the buprenorphine withdrawal. The results suggest that buprenorphine could be the better therapeutic option to manage opioid dependence in HIV. Copyright © 2015. Published by Elsevier Ireland Ltd.

  1. Mdm2 mediates FMRP- and Gp1 mGluR-dependent protein translation and neural network activity.

    PubMed

    Liu, Dai-Chi; Seimetz, Joseph; Lee, Kwan Young; Kalsotra, Auinash; Chung, Hee Jung; Lu, Hua; Tsai, Nien-Pei

    2017-10-15

    Activating Group 1 (Gp1) metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, elicits translation-dependent neural plasticity mechanisms that are crucial to animal behavior and circuit development. Dysregulated Gp1 mGluR signaling has been observed in numerous neurological and psychiatric disorders. However, the molecular pathways underlying Gp1 mGluR-dependent plasticity mechanisms are complex and have been elusive. In this study, we identified a novel mechanism through which Gp1 mGluR mediates protein translation and neural plasticity. Using a multi-electrode array (MEA) recording system, we showed that activating Gp1 mGluR elevates neural network activity, as demonstrated by increased spontaneous spike frequency and burst activity. Importantly, we validated that elevating neural network activity requires protein translation and is dependent on fragile X mental retardation protein (FMRP), the protein that is deficient in the most common inherited form of mental retardation and autism, fragile X syndrome (FXS). In an effort to determine the mechanism by which FMRP mediates protein translation and neural network activity, we demonstrated that a ubiquitin E3 ligase, murine double minute-2 (Mdm2), is required for Gp1 mGluR-induced translation and neural network activity. Our data showed that Mdm2 acts as a translation suppressor, and FMRP is required for its ubiquitination and down-regulation upon Gp1 mGluR activation. These data revealed a novel mechanism by which Gp1 mGluR and FMRP mediate protein translation and neural network activity, potentially through de-repressing Mdm2. Our results also introduce an alternative way for understanding altered protein translation and brain circuit excitability associated with Gp1 mGluR in neurological diseases such as FXS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Impact of Leishmania Infection on Host Macrophage Nuclear Physiology and Nucleopore Complex Integrity

    PubMed Central

    Isnard, Amandine; Christian, Jan G.; Kodiha, Mohamed; Stochaj, Ursula; McMaster, W. Robert; Olivier, Martin

    2015-01-01

    The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites. PMID:25826301

  3. The characteristics of general practice and the attractiveness of working as a GP: medical students' views.

    PubMed

    Landstrom, Bjorn; Mattsson, Bengt; Nordin, Per; Rudebeck, Carl E

    2014-03-15

    The aim of the study was to investigate medical students' views on general practice based on their experiences in training, and to find out whether there were certain views associated with the intention to become a GP. A questionnaire, based on our earlier studies about GP working behaviour, was handed out to medical students in terms 1, 3, 5, 7, 10 and 11 of undergraduate studies in Gothenburg, Sweden. The analysis comprised statistical descriptions and comparisons. The students regarded general practice positively. They found the work environment good, the GP's awareness of patients' living conditions necessary, and that GP work requires medical breadth. The status of the GP in the medical profession was not considered high. One-fourth of the students strongly agreed with the possibility of a future as a GP. This attitude was statistically associated with support to the statements that general practice offers a good work environment and should be a major component in undergraduate training. Students with a negative attitude to working as GPs were also negative to having a major component of general practice in undergraduate training. Medical students with a positive stated attitude towards becoming GPs support changes in undergraduate training to include more general practice. The risk of increasing a negative attitude should be considered when changes are discussed.

  4. 25 CFR 63.5-63.9 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false [Reserved] 63.5-63.9 Section 63.5-63.9 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR TRIBAL GOVERNMENT INDIAN CHILD PROTECTION AND FAMILY VIOLENCE PREVENTION Purpose, Policy, and Definitions §§ 63.5-63.9 [Reserved] ...

  5. Commentary: Ubiquitous Computing Revisited--A New Perspective

    ERIC Educational Resources Information Center

    Bull, Glen; Garofalo, Joe

    2006-01-01

    In 2002, representatives from the teacher educator associations representing the core content areas (science, mathematics, language arts, and social studies) and educational technology met at the National Technology Leadership Retreat (NTLR) to discuss potential implications of ubiquitous computing for K-12 schools. This paper re-examines some of…

  6. Towards ubiquitous access of computer-assisted surgery systems.

    PubMed

    Liu, Hui; Lufei, Hanping; Shi, Weishong; Chaudhary, Vipin

    2006-01-01

    Traditional stand-alone computer-assisted surgery (CAS) systems impede the ubiquitous and simultaneous access by multiple users. With advances in computing and networking technologies, ubiquitous access to CAS systems becomes possible and promising. Based on our preliminary work, CASMIL, a stand-alone CAS server developed at Wayne State University, we propose a novel mobile CAS system, UbiCAS, which allows surgeons to retrieve, review and interpret multimodal medical images, and to perform some critical neurosurgical procedures on heterogeneous devices from anywhere at anytime. Furthermore, various optimization techniques, including caching, prefetching, pseudo-streaming-model, and compression, are used to guarantee the QoS of the UbiCAS system. UbiCAS enables doctors at remote locations to actively participate remote surgeries, share patient information in real time before, during, and after the surgery.

  7. A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1.

    PubMed

    Wilson, D J; Kim, D S; Clarke, G A; Marshall-Clarke, S; Moss, D J

    1996-12-01

    We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a

  8. CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael

    2011-02-20

    CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained bymore » the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.« less

  9. From Many-to-One to One-to-Many: The Evolution of Ubiquitous Computing in Education

    ERIC Educational Resources Information Center

    Chen, Wenli; Lim, Carolyn; Tan, Ashley

    2011-01-01

    Personal, Internet-connected technologies are becoming ubiquitous in the lives of students, and ubiquitous computing initiatives are already expanding in educational contexts. Historically in the field of education, the terms one-to-one (1:1) computing and ubiquitous computing have been interpreted in a number of ways and have at times been used…

  10. 25 CFR 63.25-63.29 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false [Reserved] 63.25-63.29 Section 63.25-63.29 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR TRIBAL GOVERNMENT INDIAN CHILD PROTECTION AND FAMILY VIOLENCE PREVENTION Minimum Standards of Character and Suitability for Employment §§ 63.25-63.29 [Reserved] ...

  11. 25 CFR 63.37-63.50 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false [Reserved] 63.37-63.50 Section 63.37-63.50 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR TRIBAL GOVERNMENT INDIAN CHILD PROTECTION AND FAMILY VIOLENCE PREVENTION Indian Child Protection and Family Violence Prevention Program §§ 63.37-63.50 [Reserved] ...

  12. The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α1→2 Mannose Residues on the Outer Face of gp120

    PubMed Central

    Scanlan, Christopher N.; Pantophlet, Ralph; Wormald, Mark R.; Ollmann Saphire, Erica; Stanfield, Robyn; Wilson, Ian A.; Katinger, Hermann; Dwek, Raymond A.; Rudd, Pauline M.; Burton, Dennis R.

    2002-01-01

    2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N→A substitution produced significant decreases in 2G12 binding affinity to gp120JR-CSF. Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120JR-CSF. Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Manα1→2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Manα1→2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Manα1→2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation

  13. A highly conserved metalloprotease effector enhances virulence in the maize anthracnose fungus Colletotrichum graminicola.

    PubMed

    Sanz-Martín, José M; Pacheco-Arjona, José Ramón; Bello-Rico, Víctor; Vargas, Walter A; Monod, Michel; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

    2016-09-01

    Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine whether Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants is normal, but they exhibit delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitro chitinase activity assays of leaves infected with wild-type and null mutant strains show that, in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain-containing homologues of Ecp6, suggesting that this fungus employs both Cgfl-mediated and LysM protein-mediated strategies to control chitin signalling. © 2015 BSPP and John Wiley & Sons Ltd.

  14. P-gp is involved in the intestinal absorption and biliary excretion of afatinib in vitro and in rats.

    PubMed

    Zhang, Yan; Wang, Changyuan; Liu, Zhihao; Meng, Qiang; Huo, Xiaokui; Liu, Qi; Sun, Pengyuan; Yang, Xiaobo; Sun, Huijun; Ma, Xiaodong; Liu, Kexin

    2018-04-01

    Afatinib is an irreversible multi-targeted TKI, used in the treatment with EGFR mutated non-small cell lung cancer (NSCLC). The purpose of this study is to explore the molecular pharmacokinetic mechanism underlying the effect of P-gp inhibitors on the intestinal absorption and biliary excretion and to understand how P-gp inhibitors affect afatinib pharmacokinetics. Pharmacokinetics in vivo, in situ intestinal perfusion, perfused rat liver in situ, Caco-2 cells, P-gp ATPase activity, sandwich-cultured rat hepatocytes (SCRH) and transfected-cell transport were used in the evaluation. P-gp inhibitor verapamil (Ver) markedly increased the plasma concentrations and significantly decreased the biliary excretion of afatinib in vivo. Ver increased the intestinal absorption and decreased biliary excretion of afatinib in situ single-pass intestinal perfusion studies and in situ perfused rat liver, respectively. The accumulation of afatinib in Caco-2 cells was enhanced by Ver and Cyclosporin A (CsA). The biliary excretion index (BEI) of afatinib in SCRH was decreased by Ver and CsA, respectively. The net efflux ratio of afatinib was 2.3 across vector-/MDR1-MDCKII cell monolayers and was decreased by P-gp inhibitor. The activity of P-gp ATPase was induced by afatinib and the K m and V max were 1.05μM and 59.88nmol ATP/mg hP-gp/min, respectively. At least partly P-gp is involved in increasing the intestinal absorption and decreasing the biliary excretion of afatinib in rats. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

  15. Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction.

    PubMed

    Barrera, Daniel; Llanos, Ricardo J; Miceli, Dora C

    2012-05-01

    The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.

  16. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

    PubMed

    Lim, HooiCheng; Yu, Chun-Ying; Jou, Tzuu-Shuh

    2017-11-01

    Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O -glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O -glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. © FASEB.

  17. Towards Ubiquitous Blood Pressure Monitoring via Pulse Transit Time: Theory and Practice

    PubMed Central

    Hahn, Jin-Oh; Inan, Omer T.; Mestha, Lalit K.; Kim, Chang-Sei; Töreyin, Hakan; Kyal, Survi

    2015-01-01

    Ubiquitous blood pressure (BP) monitoring is needed to improve hypertension detection and control and is becoming feasible due to recent technological advances such as in wearable sensing. Pulse transit time (PTT) represents a well-known, potential approach for ubiquitous BP monitoring. The goal of this review is to facilitate the achievement of reliable, ubiquitous BP monitoring via PTT. We explain the conventional BP measurement methods and their limitations; present models to summarize the theory of the PTT-BP relationship; outline the approach while pinpointing the key challenges; overview the previous work towards putting the theory to practice; make suggestions for best practice and future research; and discuss realistic expectations for the approach. PMID:26057530

  18. Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120

    PubMed Central

    Kong, Leopold; Lee, Jeong Hyun; Doores, Katie J.; Murin, Charles D.; Julien, Jean-Philippe; McBride, Ryan; Liu, Yan; Marozsan, Andre; Cupo, Albert; Klasse, Per-Johan; Hoffenberg, Simon; Caulfield, Michael; King, C. Richter; Hua, Yuanzi; Le, Khoa M.; Khayat, Reza; Deller, Marc C.; Clayton, Thomas; Tien, Henry; Feizi, Ten; Sanders, Rogier W.; Paulson, James C.; Moore, John P.; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.

    2013-01-01

    A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization. PMID:23708606

  19. Differential binding of neutralizing and non-neutralizing antibodies to native-like soluble HIV-1 Env trimers, uncleaved Env proteins, and monomeric subunits

    PubMed Central

    2014-01-01

    Background The trimeric envelope glycoproteins (Env) on the surface of HIV-1 virions are the targets for neutralizing antibodies (NAbs). No candidate HIV-1 immunogen has yet induced potent, broadly active NAbs (bNAbs). Part of the explanation may be that previously tested Env proteins inadequately mimic the functional, native Env complex. Trimerization and the proteolytic processing of Env precursors into gp120 and gp41 profoundly alter antigenicity, but soluble cleaved trimers are too unstable to serve as immunogens. By introducing stabilizing mutations (SOSIP), we constructed soluble, cleaved Env trimers derived from the HIV-1 subtype A isolate BG505 that resemble native Env spikes on virions both structurally and antigenically. Results We used surface plasmon resonance (SPR) to quantify antibody binding to different forms of BG505 Env: the proteolytically cleaved SOSIP.664 trimers, cleaved gp120-gp41ECTO protomers, and gp120 monomers. Non-NAbs to the CD4-binding site bound only marginally to the trimers but equally well to gp120-gp41ECTO protomers and gp120 monomers, whereas the bNAb VRC01, directed to the CD4bs, bound to all three forms. In contrast, bNAbs to V1V2 glycan-dependent epitopes bound preferentially (PG9 and PG16) or exclusively (PGT145) to trimers. We also explored the antigenic consequences of three different features of SOSIP.664 gp140 trimers: the engineered inter-subunit disulfide bond, the trimer-stabilizing I559P change in gp41ECTO, and proteolytic cleavage at the gp120-gp41ECTO junction. Each of these three features incrementally promoted native-like trimer antigenicity. We compared Fab and IgG versions of bNAbs and validated a bivalent model of IgG binding. The NAbs showed widely divergent binding kinetics and degrees of binding to native-like BG505 SOSIP.664. High off-rate constants and low stoichiometric estimates of NAb binding were associated with large amounts of residual infectivity after NAb neutralization of the corresponding BG505.T

  20. Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

    PubMed Central

    Hinz, Andreas; Lutje Hulsik, David; Forsman, Anna; Koh, Willie Wee-Lee; Belrhali, Hassan; Gorlani, Andrea; de Haard, Hans; Weiss, Robin A.; Verrips, Theo; Weissenhorn, Winfried

    2010-01-01

    HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site. PMID:20463957

  1. Membrane-type matrix metalloproteases as diverse effectors of cancer progression.

    PubMed

    Turunen, S Pauliina; Tatti-Bugaeva, Olga; Lehti, Kaisa

    2017-11-01

    Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity.

    PubMed

    Scirè, Andrea; Tanfani, Fabio; Bertoli, Enrico; Furlani, Emiliano; Nadozie, Hope-Onyekwere N; Cerutti, Helena; Cortelazzo, Alessio; Bini, Luca; Guerranti, Roberto

    2011-07-15

    In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds. Copyright © 2011 Elsevier GmbH. All rights reserved.

  3. The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

    PubMed Central

    Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

    2008-01-01

    Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

  4. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC 50) <50 μg ml -1. The median IC 50 of neutralized viruses was 0.033 μg ml -1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and amore » reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.« less

  5. Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

    PubMed

    Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

    2017-05-15

    The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

  6. Development of classification models for identifying "true" P-glycoprotein (P-gp) inhibitors through inhibition, ATPase activation and monolayer efflux assays.

    PubMed

    Rapposelli, Simona; Coi, Alessio; Imbriani, Marcello; Bianucci, Anna Maria

    2012-01-01

    P-glycoprotein (P-gp) is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external) validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as "true" P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

  7. Heterogeneous transport of digitalis-like compounds by P-glycoprotein in vesicular and cellular assays.

    PubMed

    Gozalpour, Elnaz; Wilmer, Martijn J; Bilos, Albert; Masereeuw, Rosalinde; Russel, Frans G M; Koenderink, Jan B

    2016-04-01

    Digitalis-like compounds (DLCs), the ancient medication of heart failure and Na,K-ATPase inhibitors, are characterized by their toxicity. Drug-drug interactions (DDIs) at absorption and excretion levels play a key role in their toxicity, hence, knowledge about the transporters involved might prevent these unwanted interactions. In the present study, the transport of fourteen DLCs with human P-glycoprotein (P-gp; ABCB1) was studied using a liquid chromatography-mass spectrometry (LC-MS) quantification method. DLC transport by P-gp overexpressing Madin-Darby canine kidney (MDCK) and immortalized human renal cells (ciPTEC) was compared to vesicular DLC transport. Previously, we identified convallatoxin as a substrate using membrane vesicles overexpressing P-gp; however, we could not measure transport of other DLCs in this assay (Gozalpour et al., 2014a). Here, we showed that lipophilic digitoxin, digoxigenin, strophanthidin and proscillaridin A are P-gp substrates in cellular accumulation assays, whereas the less lipophilic convallatoxin was not. P-gp function in the cellular accumulation assays depends on the entrance of lipophilic compounds by passive diffusion, whereas the vesicular transport assay is more appropriate for hydrophilic substrates. In conclusion, we identified digitoxin, digoxigenin, strophanthidin and proscillaridin A as P-gp substrates using cellular accumulation assays and recognized lipophilicity as an important factor in selecting a suitable transport assay. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    PubMed Central

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  9. The personal computer and GP-B management. [Gravity Probe experiment

    NASA Technical Reports Server (NTRS)

    Neighbors, A. K.

    1986-01-01

    The Gravity Probe-B (GP-B) experiment is one of the most sophisticated and challenging developments to be undertaken by NASA. Its objective is to measure the relativistic drift of gyroscopes in orbit about the earth. In this paper, the experiment is described, and the strategy of phased procurements for accomplishing the engineering development of the hardware is discussed. The microcomputer is a very convenient and powerful tool in the management of GP-B. It is used in creating and monitoring such project data as schedules, budgets, hardware procurements and technical and interface requirements. Commercially available software in word processing, database management, communications, spreadsheet, graphics and program management are used. Examples are described of the efficacy of the application of the computer by the management team.

  10. Degradation of 1,2-Dibromoethane by Mycobacterium sp. Strain GP1

    PubMed Central

    Poelarends, Gerrit J.; van Hylckama Vlieg, Johan E. T.; Marchesi, Julian R.; Freitas Dos Santos, Luisa M.; Janssen, Dick B.

    1999-01-01

    The newly isolated bacterial strain GP1 can utilize 1,2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a dhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the hheB gene found in Corynebacterium sp. strain N-1074. PMID:10094681

  11. Degradation of 1,2-dibromoethane by Mycobacterium sp. strain GP1.

    PubMed

    Poelarends, G J; van Hylckama Vlieg, J E; Marchesi, J R; Freitas Dos Santos, L M; Janssen, D B

    1999-04-01

    The newly isolated bacterial strain GP1 can utilize 1, 2-dibromoethane as the sole carbon and energy source. On the basis of 16S rRNA gene sequence analysis, the organism was identified as a member of the subgroup which contains the fast-growing mycobacteria. The first step in 1,2-dibromoethane metabolism is catalyzed by a hydrolytic haloalkane dehalogenase. The resulting 2-bromoethanol is rapidly converted to ethylene oxide by a haloalcohol dehalogenase, in this way preventing the accumulation of 2-bromoethanol and 2-bromoacetaldehyde as toxic intermediates. Ethylene oxide can serve as a growth substrate for strain GP1, but the pathway(s) by which it is further metabolized is still unclear. Strain GP1 can also utilize 1-chloropropane, 1-bromopropane, 2-bromoethanol, and 2-chloroethanol as growth substrates. 2-Chloroethanol and 2-bromoethanol are metabolized via ethylene oxide, which for both haloalcohols is a novel way to remove the halide without going through the corresponding acetaldehyde intermediate. The haloalkane dehalogenase gene was cloned and sequenced. The dehalogenase (DhaAf) encoded by this gene is identical to the haloalkane dehalogenase (DhaA) of Rhodococcus rhodochrous NCIMB 13064, except for three amino acid substitutions and a 14-amino-acid extension at the C terminus. Alignments of the complete dehalogenase gene region of strain GP1 with DNA sequences in different databases showed that a large part of a dhaA gene region, which is also present in R. rhodochrous NCIMB 13064, was fused to a fragment of a haloalcohol dehalogenase gene that was identical to the last 42 nucleotides of the hheB gene found in Corynebacterium sp. strain N-1074.

  12. Ubiquitous Computing--Are We Crazy? Point/Counterpoint

    ERIC Educational Resources Information Center

    DeWitt, Scott W.; Horn, Patricia S.

    2005-01-01

    The push for ubiquitous computing (UC) relies on an understandable and well-intentioned belief that teaching and schooling need to be transformed. This view appears credible based on large-scale criteria, such as test scores relative to other countries, drop-out rates, and economic changes. And the use of technology to achieve this goal is…

  13. A Ubiquitous English Vocabulary Learning System: Evidence of Active/Passive Attitudes vs. Usefulness/Ease-of-Use

    ERIC Educational Resources Information Center

    Huang, Yueh-Min; Huang, Yong-Ming; Huang, Shu-Hsien; Lin, Yen-Ting

    2012-01-01

    English vocabulary learning and ubiquitous learning have separately received considerable attention in recent years. However, research on English vocabulary learning in ubiquitous learning contexts has been less studied. In this study, we develop a ubiquitous English vocabulary learning (UEVL) system to assist students in experiencing a systematic…

  14. The Influences of Glycosylation on the Antigenicity, Immunogenicity, and Protective Efficacy of Ebola Virus GP DNA Vaccines

    DTIC Science & Technology

    2006-11-22

    multiple muta- tions were not studied, (iii) a vaccinia virus (VACV)- T7 system was used for transient expression, (iv) pseudotyped retrovi- ruses were used...those studies produced little to no detectable GP1 or GP2 in the transient VACV- T7 expression assays, whereas in our studies with the DNA con- structs...type GP2 was detected in pseudotyped retroviruses, a result seemingly in conflict with these authors’ findings with the VACV- T7 expression. Although

  15. Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

    PubMed Central

    Zhao, Bingchun; Zhang, Xiao; Krummenacher, Claude; Song, Shuo; Gao, Ling; Zhang, Haojiong; Xu, Miao; Feng, Lin; Feng, Qisheng; Zeng, Musheng; Xu, Yuting; Zeng, Yixin

    2018-01-01

    Epstein–Barr virus (EBV) was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb) and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications. PMID:29765376

  16. Uterine Deletion of Gp130 or Stat3 Shows Implantation Failure with Increased Estrogenic Responses

    PubMed Central

    Sun, Xiaofei; Bartos, Amanda; Whitsett, Jeffrey A.

    2013-01-01

    Leukemia inhibitory factor (LIF), a downstream target of estrogen, is essential for implantation in mice. LIF function is thought to be mediated by its binding to LIF receptor (LIFR) and recruitment of coreceptor GP130 (glycoprotein 130), and this receptor complex then activates signal transducer and activator of transcription (STAT)1/3. However, the importance of LIFR and GP130 acting via STAT3 in implantation remains uncertain, because constitutive inactivation of Lifr, Gp130, or Stat3 shows embryonic lethality in mice. To address this issue, we generated mice with conditional deletion of uterine Gp130 or Stat3 and show that both GP130 and STAT3 are critical for uterine receptivity and implantation. Implantation failure in these deleted mice is associated with higher uterine estrogenic responses prior to the time of implantation. These heightened estrogenic responses are not due to changes in ovarian hormone levels or expression of their nuclear receptors. In the deleted mice, estrogen-responsive gene, Lactoferrin (Ltf), and Mucin 1 protein, were up-regulated in the uterus. In addition, progesterone-responsive genes, Hoxa10 and Indian hedgehog (Ihh), were markedly down-regulated in STAT3-inactivated uteri. These changes in uteri of deleted mice were reflected by the failure of differentiation of the luminal epithelium, which is essential for blastocyst attachment. PMID:23885093

  17. Ebola virus-like particles produced in insect cells exhibit dendritic cell stimulating activity and induce neutralizing antibodies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ye Ling; Lin Jianguo; Sun Yuliang

    2006-08-01

    Recombinant baculoviruses (rBV) expressing Ebola virus VP40 (rBV-VP40) or GP (rBV-GP) proteins were generated. Infection of Sf9 insect cells by rBV-VP40 led to assembly and budding of filamentous particles from the cell surface as shown by electron microscopy. Ebola virus-like particles (VLPs) were produced by coinfection of Sf9 cells with rBV-VP40 and rBV-GP, and incorporation of Ebola GP into VLPs was demonstrated by SDS-PAGE and Western blot analysis. Recombinant baculovirus infection of insect cells yielded high levels of VLPs, which were shown to stimulate cytokine secretion from human dendritic cells similar to VLPs produced in mammalian cells. The immunogenicity ofmore » Ebola VLPs produced in insect cells was evaluated by immunization of mice. Analysis of antibody responses showed that most of the GP-specific antibodies were of the IgG2a subtype, while no significant level of IgG1 subtype antibodies specific for GP was induced, indicating the induction of a Th1-biased immune response. Furthermore, sera from Ebola VLP immunized mice were able to block infection by Ebola GP pseudotyped HIV virus in a single round infection assay, indicating that a neutralizing antibody against the Ebola GP protein was induced. These results show that production of Ebola VLPs in insect cells using recombinant baculoviruses represents a promising approach for vaccine development against Ebola virus infection.« less

  18. Technology in practice – GP computer use by age.

    PubMed

    Henderson, Joan; Pollack, Allan; Gordon, Julie; Miller, Graeme

    2014-12-01

    Since 2005, more than 95% of general practitioners (GPs) have had access to computers in their clinical work. We have analysed the most recent 2 years of BEACH data (April 2012-March 2014) to determine whether GP age affects clinical computer use.

  19. The GP problem: quantifying gene-to-phenotype relationships.

    PubMed

    Cooper, Mark; Chapman, Scott C; Podlich, Dean W; Hammer, Graeme L

    2002-01-01

    In this paper we refer to the gene-to-phenotype modeling challenge as the GP problem. Integrating information across levels of organization within a genotype-environment system is a major challenge in computational biology. However, resolving the GP problem is a fundamental requirement if we are to understand and predict phenotypes given knowledge of the genome and model dynamic properties of biological systems. Organisms are consequences of this integration, and it is a major property of biological systems that underlies the responses we observe. We discuss the E(NK) model as a framework for investigation of the GP problem and the prediction of system properties at different levels of organization. We apply this quantitative framework to an investigation of the processes involved in genetic improvement of plants for agriculture. In our analysis, N genes determine the genetic variation for a set of traits that are responsible for plant adaptation to E environment-types within a target population of environments. The N genes can interact in epistatic NK gene-networks through the way that they influence plant growth and development processes within a dynamic crop growth model. We use a sorghum crop growth model, available within the APSIM agricultural production systems simulation model, to integrate the gene-environment interactions that occur during growth and development and to predict genotype-to-phenotype relationships for a given E(NK) model. Directional selection is then applied to the population of genotypes, based on their predicted phenotypes, to simulate the dynamic aspects of genetic improvement by a plant-breeding program. The outcomes of the simulated breeding are evaluated across cycles of selection in terms of the changes in allele frequencies for the N genes and the genotypic and phenotypic values of the populations of genotypes.

  20. Integrated Environment for Ubiquitous Healthcare and Mobile IPv6 Networks

    NASA Astrophysics Data System (ADS)

    Cagalaban, Giovanni; Kim, Seoksoo

    The development of Internet technologies based on the IPv6 protocol will allow real-time monitoring of people with health deficiencies and improve the independence of elderly people. This paper proposed a ubiquitous healthcare system for the personalized healthcare services with the support of mobile IPv6 networks. Specifically, this paper discusses the integration of ubiquitous healthcare and wireless networks and its functional requirements. This allow an integrated environment where heterogeneous devices such a mobile devices and body sensors can continuously monitor patient status and communicate remotely with healthcare servers, physicians, and family members to effectively deliver healthcare services.

  1. Ageing of the baby boomer generation: how demographic change will impact on city and rural GP and nursing workforce.

    PubMed

    Schofield, D J; Page, S L; Lyle, D M; Walker, T J

    2006-01-01

    To compare the impact of ageing on the GP and nursing rural and city workforce. Cohort analysis of Australian Bureau of Statistics census data. The data was used to examine the age distribution of the city and rural GP and nursing workforce; patterns of attrition for those 50 years and over; and the impact of changes in working hours. The rural GP and nursing workforce is significantly older than their city counterparts (p<0.001) with the 'baby boomer' generation making up 52% of city GPs but 59% of rural GPs in 2001. While a large proportion of city and rural GPs continued to work past the age of 65 years, rural GPs left the workforce at a significantly younger age than city doctors (p<0.001). Rural nurses are older than their city peers (p<0.001) but retire at an older age than city nurses (p<0.001). In 1986, a significantly higher proportion of rural GPs in all age cohorts worked more than 41 hours per week compared with their city counterparts (p<0.001). By 2001, rural 'generation X' GPs were no more likely to work long hours than those in the city (p<0.001). However, significantly more rural than city 'baby boomers' continued to work long hours. Rural GPs are retiring faster than city GPs and strategies to attract rural GPs and nurses will be critical to ensure adequate rural health care and that current rural workforce shortage do not worsen.

  2. An efficient assisted history matching and uncertainty quantification workflow using Gaussian processes proxy models and variogram based sensitivity analysis: GP-VARS

    NASA Astrophysics Data System (ADS)

    Rana, Sachin; Ertekin, Turgay; King, Gregory R.

    2018-05-01

    Reservoir history matching is frequently viewed as an optimization problem which involves minimizing misfit between simulated and observed data. Many gradient and evolutionary strategy based optimization algorithms have been proposed to solve this problem which typically require a large number of numerical simulations to find feasible solutions. Therefore, a new methodology referred to as GP-VARS is proposed in this study which uses forward and inverse Gaussian processes (GP) based proxy models combined with a novel application of variogram analysis of response surface (VARS) based sensitivity analysis to efficiently solve high dimensional history matching problems. Empirical Bayes approach is proposed to optimally train GP proxy models for any given data. The history matching solutions are found via Bayesian optimization (BO) on forward GP models and via predictions of inverse GP model in an iterative manner. An uncertainty quantification method using MCMC sampling in conjunction with GP model is also presented to obtain a probabilistic estimate of reservoir properties and estimated ultimate recovery (EUR). An application of the proposed GP-VARS methodology on PUNQ-S3 reservoir is presented in which it is shown that GP-VARS provides history match solutions in approximately four times less numerical simulations as compared to the differential evolution (DE) algorithm. Furthermore, a comparison of uncertainty quantification results obtained by GP-VARS, EnKF and other previously published methods shows that the P50 estimate of oil EUR obtained by GP-VARS is in close agreement to the true values for the PUNQ-S3 reservoir.

  3. Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

    PubMed

    Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

    2018-06-05

    Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

  4. From Learning Object to Learning Cell: A Resource Organization Model for Ubiquitous Learning

    ERIC Educational Resources Information Center

    Yu, Shengquan; Yang, Xianmin; Cheng, Gang

    2013-01-01

    The key to implementing ubiquitous learning is the construction and organization of learning resources. While current research on ubiquitous learning has primarily focused on concept models, supportive environments and small-scale empirical research, exploring ways to organize learning resources to make them available anywhere on-demand is also…

  5. Involvement of viral envelope GP2 in Ebola virus entry into cells expressing the macrophage galactose-type C-type lectin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Usami, Katsuaki; Matsuno, Keita; Igarashi, Manabu

    2011-04-01

    Highlights: {yields} Ebola virus infection is mediated by binding to and fusion with the target cells. {yields} Structural feature of the viral glycoprotein determines the infectivity. {yields} Surface C-type lectin, MGL, of macrophages and dendritic cells mediate the infection. {yields} GP2, one of glycoprotein subunits, plays an essential role in MGL-mediated infection. {yields} There is a critical amino acid residue involved in high infectivity. -- Abstract: Ebola virus (EBOV) infection is initiated by the interaction of the viral surface envelope glycoprotein (GP) with the binding sites on target cells. Differences in the mortality among different species of the Ebola viruses,more » i.e., Zaire ebolavirus (ZEBOV) and Reston ebolavirus (REBOV), correspond to the in vitro infectivity of the pseudo-typed virus constructed with the GPs in cells expressing macrophage galactose-type calcium-type lectin (MGL/CD301). Through mutagenesis of GP2, the transmembrane-anchored subunit of GP, we found that residues 502-527 of the GP2 sequence determined the different infectivity between VSV-ZEBOV GP and -REBOV GP in MGL/CD301-expressing cells and a histidine residue at position 516 of ZEBOV GP2 appeared essential in the differential infectivity. These findings may provide a clue to clarify a molecular basis of different pathogenicity among EBOV species.« less

  6. Using Ubiquitous Games in an English Listening and Speaking Course: Impact on Learning Outcomes and Motivation

    ERIC Educational Resources Information Center

    Liu, Tsung-Yu; Chu, Yu-Ling

    2010-01-01

    This paper reports the results of a study which aimed to investigate how ubiquitous games influence English learning achievement and motivation through a context-aware ubiquitous learning environment. An English curriculum was conducted on a school campus by using a context-aware ubiquitous learning environment called the Handheld English Language…

  7. Network architecture test-beds as platforms for ubiquitous computing.

    PubMed

    Roscoe, Timothy

    2008-10-28

    Distributed systems research, and in particular ubiquitous computing, has traditionally assumed the Internet as a basic underlying communications substrate. Recently, however, the networking research community has come to question the fundamental design or 'architecture' of the Internet. This has been led by two observations: first, that the Internet as it stands is now almost impossible to evolve to support new functionality; and second, that modern applications of all kinds now use the Internet rather differently, and frequently implement their own 'overlay' networks above it to work around its perceived deficiencies. In this paper, I discuss recent academic projects to allow disruptive change to the Internet architecture, and also outline a radically different view of networking for ubiquitous computing that such proposals might facilitate.

  8. Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia

    PubMed Central

    Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun

    2017-01-01

    Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308–331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner. PMID:28832643

  9. Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia.

    PubMed

    Chen, Qiang; Wu, Hui; Tao, Jia; Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun; Xu, Changshui

    2017-01-01

    Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308-331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner.

  10. A system for ubiquitous health monitoring in the bedroom via a Bluetooth network and wireless LAN.

    PubMed

    Choi, J M; Choi, B H; Seo, J W; Sohn, R H; Ryu, M S; Yi, W; Park, K S

    2004-01-01

    Advances in information technology have enabled ubiquitous health monitoring at home, which is particularly useful for patients, who have to live alone. We have focused on the automatic and unobtrusive measurement of biomedical signals and activities of patients. We have constructed wireless communication networks in order to transfer data. The networks consist of Bluetooth and Wireless Local Area Network (WLAN). In this paper, we present the concept of a ubiquitous-Bedroom (u-Bedroom) which is a part of a ubiquitous-House (u-House) and we present our systems for ubiquitous health monitoring.

  11. Systemic Functional Linguistic Genre Pedagogy ((SFL GP) in a Tertiary EFL Writing Context in Indonesia

    ERIC Educational Resources Information Center

    Emilia, Emi; Hamied, Fuad Abdul

    2015-01-01

    This article reports on the results of a study aiming to investigate whether systemic functional linguistic genre pedagogy (SFL GP) can help students develop their writing ability in English and the students' opinions about the teaching program using SFL GP. The study was conducted in one semester with 19 student teachers taking a writing course…

  12. Measurement of Circulating Progranulin (PGRN/GP88/GEP) by Enzyme-Linked Immunosorbent Assay and Application in Human Diseases.

    PubMed

    Serrero, Ginette; Hicks, David

    2018-01-01

    The enzyme-linked immunosorbent assay (ELISA) is a well-established methodology for detection of analytes in various biological fluids. The assay described herein has been validated for the detection of PGRN/GP88/GEP in blood (serum/ plasma), urine and cerebrospinal fluid (CSF), and synovial fluid and may also be used for breast milk, ductal lavage, nipple aspirates, and saliva. The ability to measure circulating levels of PGRN/GP88/GEP has proven to have clinical utility for several human diseases such as cancer where changes of PGRN/GP88/GEP can be determined as a mean to monitor disease status or response to therapy. In the case of frontotemporal dementia (FTD), the ability to measure PGRN/GP88/GEP levels in plasma and cerebrospinal fluid may be useful in distinguishing PGRN mutation carriers among FTD populations at large. The assay used is a sandwich ELISA where a highly specific antihuman PGRN/GP88/GEP monoclonal antibody is employed as a capture antibody coated on 96-well microplates. After contact with serum (or other bodily fluid), unbound material is washed away before application of another PGRN/GP88/GEP detecting antibody which in turn is detected by a horseradish peroxidase (HRP) conjugated antibody. After further washing to remove all unbound HRP, a substrate (TMB) is added, and after approximately 6 min, a color is developed and can be read as optical density at 620 nm (or 450 nm if using HCL as a stop solution) in a microplate reader. The test described herein is capable of measuring very low levels of PGRN/GP88/GEP such as 0.2 ng/mL as found in CSF of certain FTD patients. Additionally, we have demonstrated the potential clinical utility of measuring the changes of PGRN/GP88/GEP blood levels in cancer patients undergoing therapy.

  13. People with multiple unhealthy lifestyles are less likely to consult primary healthcare

    PubMed Central

    2014-01-01

    Background Behavioural interventions are often implemented within primary healthcare settings to prevent type 2 diabetes and other lifestyle-related diseases. Although smoking, alcohol consumption, physical inactivity and poor diet are associated with poorer health that may lead a person to consult a general practitioner (GP), previous work has shown that unhealthy lifestyles cluster among low socioeconomic groups who are less likely to seek primary healthcare. Therefore, it is uncertain whether behavioural interventions in primary healthcare are reaching those in most need. This study investigated patterns of GP consultations in relation to the clustering of unhealthy lifestyles among a large sample of adults aged 45 years and older in New South Wales, Australia. Methods A total of 267,153 adults participated in the 45 and Up Study between 2006 and 2009, comprising 10% of the equivalent demographic in the state of New South Wales, Australia (response rate: 18%). All consultations with GPs within 6 months prior and post survey completion were identified (with many respondents attending multiple GPs) via linkage to Medicare Australia data. An index of unhealthy lifestyles was constructed from self-report data on adherence to published guidelines on smoking, alcohol consumption, diet and physical activity. Logistic and zero-truncated negative binomial regression models were used to analyse: (i) whether or not a person had at least one GP consultation within the study period; (ii) the count of GP consultations attended by each participant who visited a GP at least once. Analyses were adjusted for measures of health status, socioeconomic circumstances and other confounders. Results After adjustment, participants scoring 7 unhealthy lifestyles were 24% more likely than persons scoring 0 unhealthy lifestyles not to have attended any GP consultation in the 12-month time period. Among those who attended at least one consultation, those with 7 unhealthy lifestyles

  14. HIV gp41 fusion peptide increases membrane ordering in a cholesterol-dependent fashion.

    PubMed

    Lai, Alex L; Freed, Jack H

    2014-01-07

    Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. A vision for ubiquitous sequencing

    PubMed Central

    Erlich, Yaniv

    2015-01-01

    Genomics has recently celebrated reaching the $1000 genome milestone, making affordable DNA sequencing a reality. With this goal successfully completed, the next goal of the sequencing revolution can be sequencing sensors—miniaturized sequencing devices that are manufactured for real-time applications and deployed in large quantities at low costs. The first part of this manuscript envisions applications that will benefit from moving the sequencers to the samples in a range of domains. In the second part, the manuscript outlines the critical barriers that need to be addressed in order to reach the goal of ubiquitous sequencing sensors. PMID:26430149

  16. Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II recepter HLA-DR1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mullen, M.; Haan, K.M.; Longnecker, R.

    Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms amore » large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.« less

  17. Glycyrrhetinic Acid Accelerates the Clearance of Triptolide through P-gp In Vitro.

    PubMed

    Li, Zhihua; Yan, Miao; Cao, Lingjuan; Fang, Pingfei; Guo, Zhaohui; Hou, Zhenyan; Zhang, Bikui

    2017-07-01

    Triptolide (TP) is an active ingredient isolated from Tripterygium wilfordii Hook. f. (TWHF), which is a traditional herbal medicine widely used for the treatment of rheumatoid arthritis and autoimmune disease in the clinic. However, its adverse reactions of hepatotoxicity and nephrotoxicity have been frequently reported which limited its clinical application. The aim of this study was to investigate the mechanism of glycyrrhetinic acid (GA) effecting on the elimination of TP in HK-2 cells and the role of the efflux transporters of P-gp and multidrug resistance-associated proteins (MRPs) in this process. An ultra performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) analytical method was established to determine the intracellular concentration of TP. In order to study the role of efflux transporters of P-gp and MRPs in GA impacting on the accumulation of TP, the inhibitors of efflux transporters (P-gp: verapamil; MRPs: MK571) were used in this study. The results showed that GA could enhance the elimination of TP and reduce the TP accumulation in HK-2 cells. Verapamil and MK571 could increase the intracellular concentration of TP; in addition, GA co-incubation with verapamil significantly increased the TP cellular concentration compared with the control group. In conclusion, GA could reduce the accumulation of TP in HK-2 cells, which was related to P-gp. This is probably one of the mechanisms that TP combined with GA to detoxify its toxicity. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  18. Effects of local application of methylprednisolone delivered by the C/GP-hydrogel on the recovery of facial nerves.

    PubMed

    Chao, Xiuhua; Fan, Zhaomin; Han, Yuechen; Wang, Yan; Li, Jianfeng; Chai, Renjie; Xu, Lei; Wang, Haibo

    2015-01-01

    Local administration of MP delivered by the C/GP-MP-hydrogel can improve the recovery of facial nerve following crush injury. The findings suggested that locally injected MP delivered by C/GP-hydrogel might be a promising treatment for facial nerve damage. In this study, the aim is to assess the effectiveness of locally administrating methylprednisolone(MP) loaded by chitosan-β-glycerophosphate hydrogel (C/GP-hydrogel) on the regeneration of facial nerve crush injury. After the crush of left facial nerves, Wistar rats were randomly divided into four different groups. Then, four different therapies were used to treat the damaged facial nerves. At the 1(st), 2(nd), 3(rd), and 4(th) week after injury, the functional recovery of facial nerves and the morphological changes of facial nerves were assessed. The expression of growth associated protein-43 (GAP-43) protein in the facial nucleus were also evaluated. Locally injected MP delivered by C/GP-hydrogel effectively accelerated the facial functional recovery. In addition, the regenerated facial nerves in the C/GP-MP group were more mature than those in the other groups. The expression of GAP-43 protein was also improved by the MP, especially in the C/GP-MP group.

  19. Screening of primary gp120 immunogens to formulate the next generation polyvalent DNA prime-protein boost HIV-1 vaccines

    PubMed Central

    Wang, Shixia; Chou, Te-hui; Hackett, Anthony; Efros, Veronica; Wang, Yan; Han, Dong; Wallace, Aaron; Chen, Yuxin; Hu, Guangnan; Liu, Shuying; Clapham, Paul; Arthos, James; Montefiori, David; Lu, Shan

    2017-01-01

    ABSTRACT Our previous preclinical studies and a Phase I clinical trial DP6-001 have indicated that a polyvalent Env formulation was able to elicit broadly reactive antibody responses including low titer neutralizing antibody responses against viral isolates of subtypes A, B, C and AE. In the current report, a panel of 62 gp120 immunogens were screened in a rabbit model to identify gp120 immunogens that can elicit improved binding and neutralizing antibody responses and some of them can be included in the next polyvalent formulation. Only about 19% of gp120 immunogens in this panel were able to elicit neutralizing antibodies against greater than 50% of the viruses included in a high throughput PhenoSense neutralization assay when these immuongens were tested as a DNA prime followed by a fixed 5-valent gp120 protein vaccine boost. The new polyvalent formulation, using five gp120 immunogens selected from this subgroup, elicited improved quality of antibody responses in rabbits than the previous DP6-001 formulation. More significantly, this new polyvalent formulation elicited higher antibody responses against a panel of gp70V1/V2 antigens expressing V1/V2 sequences from diverse subtypes. Bioinformatics analysis supports the design of a 4-valent or 5-valent formulation using gp120 immunogens from this screening study to achieve a broad coverage against 16 HIV-1 subtypes. PMID:28933684

  20. MHC class II presentation of gp100 epitopes in melanoma cells requires the function of conventional endosomes, and is influenced by melanosomes1

    PubMed Central

    Robila, Valentina; Ostankovitch, Marina; Altrich-VanLith, Michelle L.; Theos, Alexander C.; Drover, Sheila; Marks, Michael S.; Restifo, Nicholas; Engelhard, Victor H.

    2009-01-01

    Many human solid tumors express MHC II molecules, and proteins normally localized to melanosomes give rise to MHC II restricted epitopes in melanoma. However, the pathways by which this occurs have not been defined. We analyzed the processing of one such epitope, gp10044-59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1hi/MHC II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044-59 presentation. By depletion of the AP2 adaptor protein using siRNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044-59 epitope production. Gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC II molecules. Gp10044-59 presentation is dramatically reduced, and processing occurs entirely in early endosomes / stage I melanosomes. This suggests that melanosomes are inefficient antigen processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition. PMID:19017974