Now showing items 21-40 of 78

    • Efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics: an animal study in rabbits.

      Lensing, Rebecca; Bleich, André; Smoczek, Anna; Glage, Silke; Ehlert, Nina; Luessenhop, Tammo; Behrens, Peter; Müller, Peter Paul; Kietzmann, Manfred; Stieve, Martin; et al. (2013-01)
      Nanoporous silica layers are able to host molecules and release them over a certain period of time. These local drug delivery systems for antibiotics could be a new approach in the treatment of chronic otitis media. The aim of this study was to examine the efficacy of nanoporous silica coatings on middle ear prostheses as a delivery system for antibiotics in vivo. Pseudomonas aeruginosa was inoculated into the middle ear of rabbits to induce an otitis media. The control group received coated Bioverit®II implants without antibiotics. Coated prostheses with loaded ciprofloxacin were implanted into the middle ears of the study group. After 1 week, the rabbits were sacrificed. The clinical examination as well as the microbiological and histological examinations of organs and middle ear irrigation revealed clear differences between the two groups. P. aeruginosa was detected in every middle ear of the control group and was almost completely eliminated in the study group. Organ examinations revealed the presence of P. aeruginosa in the control group and a prevention of a bacterial spread in the study group. The nanoporous silica layer as antibiotic delivery system showed convincing efficacy in induced pseudomonal otitis media in the rabbit.
    • Comparison of in vitro and in vivo protein release from hydrogel systems.

      Wöhl-Bruhn, Stefanie; Badar, Muhammad; Bertz, Andreas; Tiersch, Brigitte; Koetz, Joachim; Menzel, Henning; Mueller, Peter P; Bunjes, Heike; Technische Universität Braunschweig, Institute of Pharmaceutical Technology, Mendelssohnstraße 1, 38106 Braunschweig, Germany. (2012-08-20)
      Hydrogel systems based on hydroxyethyl starch-polyethylene glycol methacrylate (HES-P(EG)(6)MA) or hydroxyethyl starch methacrylate (HES-MA) were used to assess the protein release behavior. Here, we analyzed the in vitro release of FITC-anti-human antibodies incorporated in either HES-P(EG)(6)MA or HES-MA hydrogel delivery systems in PBS or human serum. In addition, hydrogel disks and microparticles prepared from the two polymers were subcutaneously implanted in BALB/c mice. The in vivo release of FITC-IgG was non-invasively monitored by an in vivo imaging system (IVIS 200) over a time period of up to 3 months. The imaging system allowed to asses individual animals over time, therefore only a small number of animals was required to obtain high quality data. The reduction in fluorescence intensity at the site of administration was compared to in vitro release profiles. These investigations demonstrated a sustained release from HES-MA hydrogel disks compared to rapidly degrading HES-P(EG)(6)MA disks and microparticles. The sustained release from HES-MA disks could be further optimized by using increased polymer concentrations. Human serum as in vitro release medium reflected better the in vivo release from HES-P(EG)(6)MA systems than PBS, suggesting that the presence of organic substances like proteins or lipids may play a significant role for the release kinetics.
    • Histological and molecular evaluation of iron as degradable medical implant material in a murine animal model.

      Mueller, Peter P; Arnold, Sylvia; Badar, Muhammad; Bormann, Dirk; Bach, Friedrich-Wilhelm; Drynda, Andreas; Meyer-Lindenberg, Andrea; Hauser, Hansjörg; Peuster, Matthias; Helmholtz Centre for Infection Research, Braunschweig, Germany. pmu@gbf.de (2012-11)
      A small animal model was established to evaluate the potential of iron as a degradable implant material. After insertion into the tail of mice, the implants gradually degraded over a clinically relevant time period of several months. Histological analysis and gene expression data from whole-genome microarray analyses indicated a limited inflammatory reaction. No evidence of cellular responses to excess iron ions was detected, suggesting that the iron degradation products were metabolically inactive. Iron-rich compounds could be detected in the vicinity of the implant and in individual cells distant from the implantation site. These results demonstrate that the mouse model could be useful for the primary in vivo evaluation of novel implant materials and that iron degradation products can accumulate in diverse organs of the body.
    • The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis.

      Cudmore, Melissa J; Hewett, Peter W; Ahmad, Shakil; Wang, Ke-Qing; Cai, Meng; Al-Ani, Bahjat; Fujisawa, Takeshi; Ma, Bin; Sissaoui, Samir; Ramma, Wenda; et al. (2012)
      VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.
    • Characterisation of bovine leukocyte Ig-like receptors.

      Hogan, Louise; Bhuju, Sabin; Jones, Des C; Laing, Ken; Trowsdale, John; Butcher, Philip; Singh, Mahavir; Vordermeier, Martin; Allen, Rachel L; Centre for Infection, Division of Clinical Sciences, St George's, University of London, Cranmer Terrace, London, United Kingdom. p0904768@sgul.ac.uk (2012)
      Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors (PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping, indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in other species.
    • Industrial biotechnology of Pseudomonas putida and related species.

      Poblete-Castro, Ignacio; Becker, Judith; Dohnt, Katrin; dos Santos, Vitor Martins; Wittmann, Christoph; HZI-Helmholtz Centre for Infection Research, Systems and Synthetic Biology, Braunschweig, Germany. (2012-03)
      Since their discovery many decades ago, Pseudomonas putida and related subspecies have been intensively studied with regard to their potential application in industrial biotechnology. Today, these Gram-negative soil bacteria, traditionally known as well-performing xenobiotic degraders, are becoming efficient cell factories for various products of industrial relevance including a full range of unnatural chemicals. This development is strongly driven by systems biotechnology, integrating systems metabolic engineering approaches with novel concepts from bioprocess engineering, including novel reactor designs and renewable feedstocks.
    • VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.

      Thiele, Wilko; Krishnan, Jaya; Rothley, Melanie; Weih, Debra; Plaumann, Diana; Kuch, Vanessa; Quagliata, Luca; Weich, Herbert A; Sleeman, Jonathan P; Universität Heidelberg, Medizinische Fakultät Mannheim, Mannheim, Germany; (2012-08-30)
      VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41(+) cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41(+) cells in the BM and a significant increase in diploid CD41(+) cells, whereas the number of polyploid CD41(+) cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.
    • Programmable bacterial catalysis - designing cells for biosynthesis of value-added compounds.

      Lam, Carolyn M C; Suárez Diez, María; Godinho, Miguel; Martins Dos Santos, Vítor A P; Systems and Synthetic Biology Group, Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124 Braunschweig, Germany; Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, Building number 316, 6703 HB Wageningen, The Netherlands. (2012-07-16)
      Bacteria have long been used for the synthesis of a wide range of useful proteins and compounds. The developments of new bioprocesses and improvements of existing strategies for syntheses of valuable products in various bacterial cell hosts have their own challenges and limitations. The field of synthetic biology has combined knowledge from different science and engineering disciplines and facilitated the advancement of novel biological components which has inspired the design of targeted biosynthesis. Here we discuss recent advances in synthetic biology with relevance to biosynthesis in bacteria and the applications of computational algorithms and tools for manipulation of cellular components. Continuous improvements are necessary to keep up with increasing demands in terms of complexity, scale, and predictability of biosynthesis products.
    • The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach.

      Poblete-Castro, Ignacio; Escapa, Isabel F; Jäger, Christian; Puchalka, Jacek; Lam, Carolyn Ming Chi; Schomburg, Dietmar; Prieto, María Auxiliadora; Martins dos Santos, Vítor A P; Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany. ignacio.pobletecastro@helmholtz-hzi.de (2012)
      Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.
    • Random and cyclical deletion of large DNA segments in the genome of Pseudomonas putida.

      Leprince, Audrey; de Lorenzo, Víctor; Völler, Petra; van Passel, Mark W J; Martins dos Santos, Vitor A P; Systems and Synthetic Biology Group, Helmholtz-Centre for Infection Research, Braunschweig, Germany. (2012-06)
      Cumulative site-directed mutagenesis is of limited suitability for the global analysis of the gene functions in the microbe's cellular network. In order to simplify and stabilize the genome of the soil bacterium Pseudomonas putida, we developed a recyclable three-step excision method based on the combination of customized mini-transposons and the FLP-FRT site-specific recombination system. To demonstrate the powerful potential of these tools, we first established insertion mutant libraries that allow users to study gene functions with respect either to phenotypic characteristics (single insertions) or to their involvement in predicted networks (double insertions). Based on these libraries, we generated as a proof-of-principle, single-deletion mutants lacking ~4.1% of the genome (~3.7% of the gene repertoire). A cyclical application of the method generated four double-deletion mutants of which a maximum of ~7.4% of the chromosome (~6.9% of the gene count) was excised. This procedure demonstrates a new strategy for rapid genome streamlining and gain of new insights into the molecular interactions and regulations.
    • Implantatoberflächen

      Windhagen, Henning; Dempwolf, Wiebke; Gross, Gerhard; Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany. (Druckerei der Medizinischen Hochschule Hannover, 2010)
    • Funktionalisierte Mittelohrprothesen

      Behrens, Peter; Müller, Peter P.; Stieve, Martin; Besdo, Silke; Ehlert, Nina; Lenarz, Thomas; Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Braunschweig, Germany. (Druckerei der Medizinischen Hochschule, 2010)
    • Directing neuronal cell growth on implant material surfaces by microstructuring.

      Reich, Uta; Fadeeva, Elena; Warnecke, Athanasia; Paasche, Gerrit; Müller, Peter; Chichkov, Boris; Stöver, Timo; Lenarz, Thomas; Reuter, Günter; Department of Otorhinolaryngology, Head and Neck Surgery, Hannover Medical School, Hannover, Germany. (2012-05)
      For best hearing sensation, electrodes of auditory prosthesis must have an optimal electrical contact to the respective neuronal cells. To improve the electrode-nerve interface, microstructuring of implant surfaces could guide neuronal cells toward the electrode contact. To this end, femtosecond laser ablation was used to generate linear microgrooves on the two currently relevant cochlear implant materials, silicone elastomer and platinum. Silicone surfaces were structured by two different methods, either directly, by laser ablation or indirectly, by imprinting using laser-microstructured molds. The influence of surface structuring on neurite outgrowth was investigated utilizing a neuronal-like cell line and primary auditory neurons. The pheochromocytoma cell line PC-12 and primary spiral ganglion cells were cultured on microstructured auditory implant materials. The orientation of neurite outgrowth relative to the microgrooves was determined. Both cell types showed a preferred orientation in parallel to the microstructures on both, platinum and on molded silicone elastomer. Interestingly, microstructures generated by direct laser ablation of silicone did not influence the orientation of either cell type. This shows that differences in the manufacturing procedures can affect the ability of microstructured implant surfaces to guide the growth of neurites. This is of particular importance for clinical applications, since the molding technique represents a reproducible, economic, and commercially feasible manufacturing procedure for the microstructured silicone surfaces of medical implants.
    • Multi-layered stochasticity and paracrine signal propagation shape the type-I interferon response.

      Rand, Ulfert; Rinas, Melanie; Schwerk, Johannes; Nöhren, Gesa; Linnes, Melanie; Kröger, Andrea; Flossdorf, Michael; Kály-Kullai, Kristóf; Hauser, Hansjörg; Höfer, Thomas; et al. (2012)
      The cellular recognition of viruses evokes the secretion of type-I interferons (IFNs) that induce an antiviral protective state. By live-cell imaging, we show that key steps of virus-induced signal transduction, IFN-β expression, and induction of IFN-stimulated genes (ISGs) are stochastic events in individual cells. The heterogeneity in IFN production is of cellular-and not viral-origin, and temporal unpredictability of IFN-β expression is largely due to cell-intrinsic noise generated both upstream and downstream of the activation of nuclear factor-κB and IFN regulatory factor transcription factors. Subsequent ISG induction occurs as a stochastic all-or-nothing switch, where the responding cells are protected against virus replication. Mathematical modelling and experimental validation show that reliable antiviral protection in the face of multi-layered cellular stochasticity is achieved by paracrine response amplification. Achieving coherent responses through intercellular communication is likely to be a more widely used strategy by mammalian cells to cope with pervasive stochasticity in signalling and gene expression.
    • Towards rational engineering of cells: Recombinant gene expression in defined chromosomal loci.

      Nehlsen, Kristina; da Gama-Norton, Leonor; Schucht, Roland; Hauser, Hansjörg; Wirth, Dagmar; Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany. dagmar.wirth@helmholtz-hzi.de. (2011-11-22)
    • High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

      Hemmen, Katherina; Reinl, Tobias; Buttler, Kerstin; Behler, Friederike; Dieken, Hauke; Jänsch, Lothar; Wilting, Jörg; Weich, Herbert A; Department of Gene Regulation, HZI, Build. D, Inhoffenstr. 7, 38124, Braunschweig, Germany. katharina.hemmen@ewetel.net (2011-05)
      Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis.
    • Linking genes to microbial growth kinetics: an integrated biochemical systems engineering approach.

      Koutinas, Michalis; Kiparissides, Alexandros; Silva-Rocha, Rafael; Lam, Ming-Chi; Martins Dos Santos, Vitor A P; de Lorenzo, Victor; Pistikopoulos, Efstratios N; Mantalaris, Athanasios; Centre for Process Systems Engineering, Department of Chemical Engineering, South Kensington Campus, Imperial College London, UK. (2011-07)
      The majority of models describing the kinetic properties of a microorganism for a given substrate are unstructured and empirical. They are formulated in this manner so that the complex mechanism of cell growth is simplified. Herein, a novel approach for modelling microbial growth kinetics is proposed, linking biomass growth and substrate consumption rates to the gene regulatory programmes that control these processes. A dynamic model of the TOL (pWW0) plasmid of Pseudomonas putida mt-2 has been developed, describing the molecular interactions that lead to the transcription of the upper and meta operons, known to produce the enzymes for the oxidative catabolism of m-xylene. The genetic circuit model was combined with a growth kinetic model decoupling biomass growth and substrate consumption rates, which are expressed as independent functions of the rate-limiting enzymes produced by the operons. Estimation of model parameters and validation of the model's predictive capability were successfully performed in batch cultures of mt-2 fed with different concentrations of m-xylene, as confirmed by relative mRNA concentration measurements of the promoters encoded in TOL. The growth formation and substrate utilisation patterns could not be accurately described by traditional Monod-type models for a wide range of conditions, demonstrating the critical importance of gene regulation for the development of advanced models closely predicting complex bioprocesses. In contrast, the proposed strategy, which utilises quantitative information pertaining to upstream molecular events that control the production of rate-limiting enzymes, predicts the catabolism of a substrate and biomass formation and could be of central importance for the design of optimal bioprocesses.
    • Rapid establishment of G-protein-coupled receptor-expressing cell lines by site-specific integration.

      Schucht, Roland; Lydford, Simon; Andzinski, Lisa; Zauers, Jeannette; Cooper, James; Hauser, Hansjörg; Wirth, Dagmar; May, Tobias; Department of Gene Regulation and Differentiation, HZI-Helmholtz Centre for Infection Research, Braunschweig, Germany. roland.schucht@inscreenex.com (2011-03)
      The establishment of mammalian cell lines reliably expressing G-protein-coupled receptors (GPCRs) can be a tedious and often time-consuming process. A strategy has been developed to allow the rapid production of such cell lines. The first step of this approach was the generation of a specialized master cell line, characterized by optimized stable expression of a membrane-bound reporter protein. In the second step, this reporter gene was exchanged for that of the GPCR of interest by a DNA recombinase "cut-and-paste" engineering step. It has been demonstrated that the resulting GPCR cell lines inherit the advantages of the master cell line, expressing the GPCR in a homogeneous and stable manner. The case studies presented demonstrate the functionality of the established GPCR cell lines, and most important, because of the highly efficient integration event, these recombinant GPCR-expressing cell lines were generated within a timeframe of 2 to 4 weeks. The advantages of this cut-and-paste approach versus other strategies such as Flp-In or Jump-In are compared.
    • Novel nonviral bioassays for mouse type I and type III interferon.

      Kugel, Daniela; Pulverer, Julia Elisabeth; Köster, Mario; Hauser, Hansjörg; Staeheli, Peter; Department of Virology, University of Freiburg, Freiburg, Germany. (2011-04)
      We used embryo fibroblasts from Mx2-Luc transgenic mice that express Firefly luciferase under control of the interferon (IFN)-regulated mouse Mx2 promoter to develop simple nonviral bioassays for type I and type III IFN. Since type III IFN is acid-labile, Mx2-Luc fibroblasts detected the presence of type I IFN in acid-treated biological samples with high sensitivity and selectivity. For selective detection of type III IFN, we employed embryo fibroblasts from Mx2-Luc mutant mice that lack functional receptors for type I IFN. The sensitivity of this latter assay remained comparatively low, presumably because type III IFN receptors are not abundantly present on fibroblasts. The main advantages of our novel IFN assays are that they are easy to perform, yield fast results, and can be used in laboratories that are not licensed for work with infectious agents. Further, the type I IFN assay has superior sensitivity than commercially available enzyme-linked immunosorbent assay systems.
    • Reconciliation of genome-scale metabolic reconstructions for comparative systems analysis.

      Oberhardt, Matthew A; Puchałka, Jacek; Martins dos Santos, Vítor A P; Papin, Jason A; Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, United States of America. (2011-03)
      In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.