28,114 research outputs found

    Construction and characterization of H5N1-recombinant fowlpox viruses co-expressing host cytokines

    Get PDF
    Possessing a large double stranded DNA genome up to 300 kb, fowlpox virus (FWPV) has been developed to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to coexpress host cytokines from such recombinants. This thesis describes the strategy to construct H5N1-recombinant FWPV (rFWPV) coexpressing chicken Interleukin 12 (IL-12) or Interleukin 15 (IL-15), and discusses the immunogenicity of the recombinants following inoculation into specific-pathogen-free (SPF) chickens. Previously cloned and sequenced cDNAs encoding full-length H5 and N1 of influenza strain A/Chicken/Malaysia/5858/2004 genes were amplified by PCR and inserted into plasmid pEFL29, under the control of a copy of the vaccinia virus p7.5 early/late promoter. The expression cassettes were recombined into the genome of the FP9 strain of FWPV at the fpv002 locus. Recombinant viruses were produced by transfection of the plasmid into chicken embryo fibroblasts (CEFs) after infection with FP9, and isolated by six fold plaque purification on CEFs using X-Gal selection. Chicken IL-12 or IL-15 genes, under control of a synthetic/hybrid poxvirus promoter, were inserted into a ‘transient dominant selection’ recombination plasmid, pPC1.X. The cytokine expression cassettes were then recombined, at the fpPC1 (fpv030) locus, into rFWPV already carrying AIV genes. Following three rounds of passage in CEFs in the presence of mycophenolic acid (MPA), recombinant viruses carrying the gpt gene were isolated. These unstable recombinants were plaque-purified in the absence of MPA until they lost the gpt gene spontaneously, verified by their failure to replicate in the presence of MPA. Recombinant proteins were successfully detected using western blotting and indirect immunofluorescence assay (IFAT). Parental and rFWPV (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. Sera from chickens immunized with rFWPV/H5 and rFWPV/H5/IL-15 demonstrated viral neutralizing activities, based on the haemagglutation inhibition (HI) test, in which reached a peak at Week 3. A competitive enzyme-linked immunosorbent (ELISA) assay detected N1-specific antibodies induced by rFWPV/N1 and rFWPV/N1/IL-12 at Weeks 4 and 5. Non-specific cellular immune responses were assessed by flow cytometric analysis to enumerate CD4+ and CD8+ T-lymphocytes in peripheral blood. Results of Experiment 2 showed chickens vaccinated with rFWPV/H5, rFWPV/H5/IL-15, rFWPV/N1 and rFWPV/N1/IL-12 demonstrated a higher increase in CD8+ than CD4+ T cell population, relative to control and chickens vaccinated with parental FWPV. Weekly weighing showed that chickens vaccinated with rFWPV/H5/IL-15 had the highest body weight compared to other groups, while the rFWPV/N1/IL-12 group showed the significantly lowest body weight. In summary, this study showed diverse immunogenicity of H5N1-rFWPV coexpressing IL-12 or IL-15. It also demonstrated a weight sparing effect of co-expressing IL-15 in rFWPV vaccines. The results provide the basis for future homologous challenge studies, using live H5N1 virus to evaluate the protective efficacy of the rFWPV vaccines

    Development of a Next-Generation NIL Library in Arabidopsis Thaliana for Dissecting Complex Traits

    Get PDF
    The identification of the loci and specific alleles underlying variation in quantitative traits is an important goal for evolutionary biologists and breeders. Despite major advancements in genomics technology, moving from QTL to causal alleles remains a major challenge in genetics research. Near-isogenic lines are the ideal raw material for QTL validation, refinement of QTL location and, ultimately, gene discovery. Results: In this study, a population of 75 Arabidopsis thaliana near-isogenic lines was developed from an existing recombinant inbred line (RIL) population derived from a cross between physiologically divergent accessions Kas-1 and Tsu-1. First, a novel algorithm was developed to utilize genome-wide marker data in selecting RILs fully isogenic to Kas-1 for a single chromosome. Seven such RILs were used in 2 generations of crossing to Tsu-1 to create BC1 seed. BC1 plants were genotyped with SSR markers so that lines could be selected that carried Kas-1 introgressions, resulting in a population carrying chromosomal introgressions spanning the genome. BC1 lines were genotyped with 48 genome-wide SSRs to identify lines with a targeted Kas-1 introgression and the fewest genomic introgressions elsewhere. 75 such lines were selected and genotyped at an additional 41 SNP loci and another 930 tags using 2b-RAD genotyping by sequencing. The final population carried an average of 1.35 homozygous and 2.49 heterozygous introgressions per line with average introgression sizes of 5.32 and 5.16 Mb, respectively. In a simple case study, we demonstrate the advantage of maintaining heterozygotes in our library whereby fine-mapping efforts are conducted simply by self-pollination. Crossovers in the heterozygous interval during this single selfing generation break the introgression into smaller, homozygous fragments (sub-NILs). Additionally, we utilize a homozygous NIL for validation of a QTL underlying stomatal conductance, a low heritability trait. Conclusions: The present results introduce a new and valuable resource to the Brassicaceae research community that enables rapid fine-mapping of candidate loci in parallel with QTL validation. These attributes along with dense marker coverage and genome-wide chromosomal introgressions make this population an ideal starting point for discovery of genes underlying important complex traits of agricultural and ecological significance.NSF DEB-1022196, DEB-0618302, DEB-0618347, IOS-09221457Integrative Biolog

    Inactivation of pathogens on food and contact surfaces using ozone as a biocidal agent

    Get PDF
    This study focuses on the inactivation of a range of food borne pathogens using ozone as a biocidal agent. Experiments were carried out using Campylobacter jejuni, E. coli and Salmonella enteritidis in which population size effects and different treatment temperatures were investigate

    Purification and Reconstitution of the Glutamate Carrier GltT of the Thermophilic Bacterium Bacillus stearothermophilus

    Get PDF
    An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of expression were observed in E. coli strain DH5α grown on rich medium. The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100. Purified GltT was reconstituted in an active state in liposomes prepared from E. coli phospholipids. The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads. Active reconstitution was realized with a wide range of Triton X100 concentrations. Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state. In B. stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions. In membrane vesicles derived from E. coli cells expressing GltT, the Na+ ion dependency seems to be lost, suggesting a role for the lipid environment in the cation specificity. In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E. coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+.

    Novel expression of Haemonchus contortus vaccine candidate aminopeptidase H11 using the free-living nematode Caenorhabditis elegans

    Get PDF
    With the problem of parasitic nematode drug resistance increasing, vaccine development offers an alternative sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly protective. However, recombinant forms of these proteins have failed to replicate this protection. This is thought to be due to differences in glycosylation and/or conformation between native and recombinant proteins. We have exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus aminopeptidase H11 glycoprotein, which is enriched in a gut membrane fraction capable of inducing significant protection against this important ovine gastrointestinal nematode. We show that H. contortus H11 expressed in C. elegans is enzymatically active and MALDI mass spectrometry identifies similar di- and tri-fucosylated structures to those on native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on rH11 do not contribute to the protective effect of H11 and that additional components present in native H11-enriched extract are likely required for enhancing the antibody response necessary for protection

    Immunogenicity evaluation of a DNA vaccine expressing the hepatitis C virus non-structural protein 2 gene in C57BL/6 Mice

    Get PDF
    Backgrounds: Most of the hepatitis C virus (HCV) infections elicit poor immune responses and 75% to 85% of cases become chronic; therefore, the development of an effective vaccine against HCV is of paramount importance. In this study, we aimed to evaluate co-administration of HCV non-Structural Protein 2 and IL-12 DNA vaccines in C57BL/6 mice. Methods: A plasmid encoding full-length HCV NS2 protein (non-structural protein 2) was generated and used to vaccinate mice. Negative control (an empty expression vector) was also employed to evaluate the background response. To investigate immune responses against vaccine, C57BL/6 mice received three doses of the vaccine with a two-week interval. Cellular immunity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay for lymphocyte proliferation, lactate dehydrogenase release for cytotoxic T lymphocyte (CTL) activity and cytokine assay. Results: The findings demonstrated that immunization of mice with plasmid expressing HCV NS2 induced CTL response, interferon gamma production, and lymphocyte proliferation compared to negative control. The results also demonstrated that co-administration of IL-12 with the HCV NS2 plasmid induced significantly better immune response in C57BL/6 mice. Conclusion: DNA vaccine encoding HCV NS2 is an effective candidate that can trigger CTL-based immune response against HCV. In addition, the results suggested that combining the DNA vaccine approach with immune stimulatory cytokines may significantly enhance antigen-specific immune responses

    Mutations in the RB1 Gene in Argentine Retinoblastoma Patients and Uncommon Clinical Presentations

    Get PDF
    Background: Retinoblastoma, the most common ocular cancer of childhood, is caused by inactivation of the RB1 tumor suppressor gene in the developing retina. It may occur as unilateral, bilateral or rarely as multicentric retinoblastoma, including pineal or suprasellar tumors. Being the retinoblastoma a hereditary cancer, identification of the causative mutation is important for risk prediction in the family members. An early detection of tumor is critical for survival and eye preservation. Screening for RB1 mutations is important for early tumor detection, critical for survival and eye preservation. Purpose: To identify causative RB1 mutations in retinoblastoma patients with different clinical presentations, some of them with a rare multicentric retinoblastoma or with a second non ocular malignancy, as well as the rare association with down syndrome. A comprehensive approach was used to identify the mutations and to detect children with a hereditary condition. Methods: A cohort of 20 patients with unilateral, bilateral and multicentric retinoblastoma was studied. Blood and tumor DNA was analyzed by sequencing, segregation of polymorphisms and MLPA analyses. Some of the rare mutations were validated by cloning or by Real-Time PCR. Results: Six germline and seven somatic mutations were identified; they include nonsense, frameshift, splice mutations and gross rearrangements, four of them novel. Three out of four nonsense/ frameshift germline mutations were associated with severe phenotype: bilateral and multicentric retinoblastomas. The at-riskhaplotype was identified in a familial case including one patient with osteosarcoma; it was useful for detection of mutation carriers. Conclusions: This study allowed us to identify causative RB1 mutations, including several novels. Some patients showed uncommon clinical presentations of retinoblastoma. These data are significant for genetic counseling. Our results support the relevance of carrying out complete genetic screening for RB1 mutations in both constitutional and tumor tissues.Fil: Ottaviani, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cåtedra de Genética y Biología Molecular; ArgentinaFil: Parma, Diana Lidia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cåtedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Ferrer, Marcela Maria. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Florencia Giliberto. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cåtedra de Genética y Biología Molecular; ArgentinaFil: Luce, Leonela Natalia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cåtedra de Genética y Biología Molecular; ArgentinaFil: Alonso, Cristina. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan". Servicio de Hemato-Oncología; ArgentinaFil: Szijan, Irena. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cåtedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

    Characterization of the Hemagglutinin Cleaving Transmembrane Serine Proteases Matriptase and TMPRSS2

    Get PDF
    Influenza is one of the commonest infectious diseases affecting millions of people every year including 290,000 – 650,000 heavy casualties. Influenza viruses undergo constant genetic changes and every 10 – 50 years new influenza virus strains emerge that potentially cause a severe pandemic. In this modern interconnected world, experts believe the next influenza pandemic will be a “devastating global health event with far-reaching consequences” [1]. Novel effective anti-influenza drugs are in need. One strategy of influenza research is to focus on host-specific proteases that are essential for virus activation and spread. Trypsin-like serine proteases are crucial for influenza activation by mediating the cleavage of the viral surface glycoprotein HA and hence promoting the fusion potential of the virus. Therefore, their inhibition provides a promising therapeutic approach. The present work focused on the characterization of two relevant HA cleaving type-II transmembrane serine proteases matriptase and TMPRSS2. Chapter 3 and chapter 4 of this thesis engaged with the recombinant production of matriptase (chapter 3) in order to obtain a pure functional enzyme of high quality for a SAR study with novel monobasic (hence potentially bioavailable) matriptase inhibitors of the 3-amidinophenylalanine type (chapter 4). Adequate amounts of high-quality matriptase enzymes were isolated using a new expression system and in total 5 matriptase crystals were available at the end of this thesis for structural analysis. The matriptase inhibitor design in this thesis focused on matriptase-affine compounds with a fair selectivity profile against the blood coagulation enzymes thrombin and fXa. In total, 18 new monobasic and potentially bioavailable, as well as four new dibasic compounds of the 3-amidinophenylalanine types were tested. Based on the last published crystal structure of this inhibitor type in complex with matriptase from 2006 (PDB code 2GV6) docking was used as a structure-based virtual screening method for lead optimization of the compounds N-terminus. Selected compounds were suggested to interact with the carbonyl side chain of Gln175 of matriptase to achieve a higher affinity of matriptase compared to fXa. The 4-tert-butylureido-piperidine could be identified as suitable C-terminus in combination with 3-fluoro-4-hydroxymethyl biphenylsulphonyl N-terminally in order to obtain excellent selectivity over thrombin. The binding mode of this compound (compound 55) was crystallographically determined in complex with matriptase as well as trypsin. Trypsin proved as a suitable alternative to matriptase for detailed binding mode analysis of the compounds N-terminus. However, different preferences were detected for the C-terminus. Dibasic compounds showed higher matriptase affinity and selectivity in comparison with the monobasic analogues. However, the tested monobasic compounds were still decent matriptase inhibitors that are additionally suitable for cell culture and animal studies in their benzamidine prodrug forms, which are well established from related inhibitors of thrombin. In addition, selected monobasic as well as dibasic compounds demonstrated strong suppression of the replication of certain H9N2 influenza viruses in a matriptase-expressing MDCK II cell model. These matriptase inhibitors could be potential lead structures for the development of new drugs against H9 strains for influenza. TMPRSS2 is widely discussed for its role in influenza activation. With a TMPRSS2 dependancy of HA-activation of certain subtypes, the characterization of this protease is an important prerequisite for being available as a target for influenza drug design. However, only little is known about the physiological function of TMPRSS2 and no experimental structure data are available at the moment to enable a structure-based drug development. Therefore, chapter 5 of this thesis focused on the characterization of TMPRSS2 in order to develop a strategy for the isolation of proteolytically active TMPRSS2 from cell culture. Even though, no functional TMPRSS2 could be recovered at the end of this work some new structural characteristics of TMPRSS2 were identified as crucial for functionality insight the cell. In general, TMPRSS2 without the cytosolic part, the transmembrane domain and the LDLRA domain is able to undergo autocatalytically activation if an artificial signal peptide was added N-terminal to enable entry into the endoplasmic reticulum. The presence of the cysteine-rich SRCR domain and the presence of the disulfide chain that connects the SPD and the stem region after activation cleavage have been identified as crucial for activity. N-terminal truncation of TMPRSS2 did not result in obvious dislocation within the cell: as the full-length positive control truncated TMPRSS2 was exclusively found in cell compartments surrounding the nucleus in immunofluorescence experiments. However, a reduced proteolytic cleavage activity towards H3-HA in co-expression experiments has been observed and might be a result of dislocation, since truncated TMPRSS2 is not bound to the biomembrane anymore. In addition, TMPRSS2 has been identified as a potential substrate of matriptase in vitro, which suggests possible participation in several zymogen cascades
    • 

    corecore