• SUMOylation of the polycomb group protein L3MBTL2 facilitates repression of its target genes.

      Stielow, Christina; Stielow, Bastian; Finkernagel, Florian; Scharfe, Maren; Jarek, Michael; Suske, Guntram (2013-12-24)
      Lethal(3) malignant brain tumour like 2 (L3MBTL2) is an integral component of the polycomb repressive complex 1.6 (PRC1.6) and has been implicated in transcriptional repression and chromatin compaction. Here, we show that L3MBTL2 is modified by SUMO2/3 at lysine residues 675 and 700 close to the C-terminus. SUMOylation of L3MBTL2 neither affected its repressive activity in reporter gene assays nor it's binding to histone tails in vitro. In order to analyse whether SUMOylation affects binding of L3MBTL2 to chromatin, we performed ChIP-Seq analysis with chromatin of wild-type HEK293 cells and with chromatin of HEK293 cells stably expressing either FLAG-tagged SUMOylation-competent or SUMOylation-defective L3MBTL2. Wild-type FLAG-L3MBTL2 and the SUMOylation-defective FLAG-L3MBTL2 K675/700R mutant essentially occupied the same sites as endogenous L3MBTL2 suggesting that SUMOylation of L3MBTL2 does not affect chromatin binding. However, a subset of L3MBTL2-target genes, particularly those with low L3MBTL2 occupancy including pro-inflammatory genes, was de-repressed in cells expressing the FLAG-L3MBTL2 K675/700R mutant. Finally, we provide evidence that SUMOylation of L3MBTL2 facilitates repression of these PRC1.6-target genes by balancing the local H2Aub1 levels established by the ubiquitinating enzyme RING2 and the de-ubiquitinating PR-DUB complex.
    • Susceptibility to experimental biliary atresia linked to different hepatic gene expression profiles in two mouse strains.

      Leonhardt, Johannes; Kuebler, Joachim F; Turowski, Carmen; Tschernig, Thomas; Geffers, Robert; Petersen, Claus; Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany. (2010-02)
      Aim: To compare hepatic gene expression during the development of experimental biliary atresia (BA) in two different mouse strains. Methods: Balb/c mice and C57Black/6 (Black/6) mice were infected with rhesus rotavirus (RRV) postpartum, clinical signs of BA and survival were noted. Liver sections were assessed for cluster of differentiation antigen (CD) 3, CD4 and CD8 expression, and the hepatic virus load was determined. Second, mice of both strains were sacrificed three days after infection. Isolated hepatic RNA was subjected to gene expression analysis using Affymetrix Gene Chip MOE 430 2.0. Results: The incidence of BA was significantly lower in Black/6 mice compared to Balb/c mice (13.5% vs. 67%, P < 0.05). The mean virus titers were higher in mice with BA compared to mice without BA. Different gene profiles three days after virus infection were noted, with differential expression of 201 genes, including those regulating apoptosis, nucleic acid binding, transport function and particularly the immune response (chemokine C-C motif ligand 2, toll-like receptor 3, CD antigen 14, chemokine (C-X-C motif) ligands 10 and 11). This correlated with a significant increase of CD4 positive cells only in Balb/c mice with BA compared to healthy mice (13.5 vs. 5.0; P < 0.05). Black/6 mice did not exhibit any significant increase of CD3 or CD4 leukocytes despite cholestasis. Conclusion: The different susceptibility to experimental BA was associated with an increase of CD4 T-cells in the liver of Balb/c mice, which is linked to different gene profiles at the onset of bile duct obstruction.
    • Synergistic activity of IDH1 inhibitor BAY1436032 with azacitidine in IDH1 mutant acute myeloid leukemia.

      Chaturvedi, Anuhar; Gupta, Charu; Gabdoulline, Razif; Borchert, Nora M; Goparaju, Ramya; Kaulfuss, Stefan; Görlich, Kerstin; Schottmann, Renate; Othman, Basem; Welzenbach, Julia; et al. (Ferrraata Storti Foundation, 2020-04-02)
      Mutant IDH1 (mIDH1) inhibitors have shown single-agent activity in relapsed/refractory AML, though most patients eventually relapse. We evaluated the efficacy and molecular mechanism of the combination treatment with azacitidine, which is currently the standard of care in older AML patients, and mIDH1 inhibitor BAY1436032. Both compounds were evaluated in vivo as single agents and in combination with sequential (azacitidine, followed by BAY1436032) or simultaneous application in two human IDH1 mutated AML xenograft models. Combination treatment significantly prolonged survival compared to single agent or control treatment (P<.005). The sequential combination treatment depleted leukemia stem cells (LSC) by 470-fold. Interestingly, the simultaneous combination treatment depleted LSCs by 33,150-fold compared to control mice. This strong synergy is mediated through inhibition of MAPK/ERK and RB/E2F signaling. Our data strongly argues for the concurrent application of mIDH1 inhibitors and azacitidine and predicts improved outcome of this regimen in IDH1 mutated AML patients.
    • Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.

      Osterloh, Anke; Kalinke, Ulrich; Weiss, Siegfried; Fleischer, Bernhard; Breloer, Minka; Department of Immunology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany. osterloh@bni.uni-hamburg.de (2007-02-16)
      Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation of an efficient immune response. In this study we show that Hsp60 mediates immune stimulation by different mechanisms, dependent and independent of lipopolysaccharide (LPS). We have demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific interferon (IFN) gamma release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha production in professional APC. Neutralization of IFNalpha as well as the absence of functional IFNalphabeta receptor on APC and T cells interfered with Hsp60-mediated IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS and both molecules synergistically enhanced IL-12p40 production in APC and IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively binds to macrophages and DC but not to T or B lymphocytes and that both, T cell stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence of professional APC and is not mediated by B cells. Taken together, our data support an extension of the concept of Hsp60 as an endogenous danger signal: besides its function as a classical danger signal indicating unplanned tissue destruction to the innate immune system, in the incident of bacterial infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by lowering the threshold of pathogen-associated molecular pattern (PAMP) detection and enhancing Toll-like receptor (TLR) signaling.
    • SynTReN: a generator of synthetic gene expression data for design and analysis of structure learning algorithms.

      Van den Bulcke, Tim; Van Leemput, Koenraad; Naudts, Bart; van Remortel, Piet; Ma, Hongwu; Verschoren, Alain; De Moor, Bart; Marchal, Kathleen (2006)
      BACKGROUND: The development of algorithms to infer the structure of gene regulatory networks based on expression data is an important subject in bioinformatics research. Validation of these algorithms requires benchmark data sets for which the underlying network is known. Since experimental data sets of the appropriate size and design are usually not available, there is a clear need to generate well-characterized synthetic data sets that allow thorough testing of learning algorithms in a fast and reproducible manner. RESULTS: In this paper we describe a network generator that creates synthetic transcriptional regulatory networks and produces simulated gene expression data that approximates experimental data. Network topologies are generated by selecting subnetworks from previously described regulatory networks. Interaction kinetics are modeled by equations based on Michaelis-Menten and Hill kinetics. Our results show that the statistical properties of these topologies more closely approximate those of genuine biological networks than do those of different types of random graph models. Several user-definable parameters adjust the complexity of the resulting data set with respect to the structure learning algorithms. CONCLUSION: This network generation technique offers a valid alternative to existing methods. The topological characteristics of the generated networks more closely resemble the characteristics of real transcriptional networks. Simulation of the network scales well to large networks. The generator models different types of biological interactions and produces biologically plausible synthetic gene expression data.
    • Systemic and Mucosal Immune Reactivity upon Mycobacterium avium ssp. paratuberculosis Infection in Mice.

      Koc, Arzu; Bargen, Imke; Suwandi, Abdulhadi; Roderfeld, Martin; Tschuschner, Annette; Rath, Timo; Gerlach, Gerald F; Hornef, Mathias; Goethe, Ralph; Weiss, Siegfried; et al. (2014)
      Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn's disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2-/- mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4+ and CD8+ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4+ T cells. Whereby inflammation in infected and CD4+CD45RBhi T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8+ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4+CD45RBlo/int T cells. We propose, the usual non-colitogenic CD4+CD45RBlo/int T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne's diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.
    • t(8;9)(p22;p24)/PCM1-JAK2 Activates SOCS2 and SOCS3 via STAT5.

      Ehrentraut, Stefan; Nagel, Stefan; Scherr, Michaela E; Schneider, Björn; Quentmeier, Hilmar; Geffers, Robert; Kaufmann, Maren; Meyer, Corinna; Prochorec-Sobieszek, Monika; Ketterling, Rhett P; et al. (2013)
      Fusions of the tyrosine kinase domain of JAK2 with multiple partners occur in leukemia/lymphoma where they reportedly promote JAK2-oligomerization and autonomous signalling, Affected entities are promising candidates for therapy with JAK2 signalling inhibitors. While JAK2-translocations occur in myeloid, B-cell and T-cell lymphoid neoplasms, our findings suggest their incidence among the last group is low. Here we describe the genomic, transcriptional and signalling characteristics of PCM1-JAK2 formed by t(8;9)(p22;p24) in a trio of cell lines established at indolent (MAC-1) and aggressive (MAC-2A/2B) phases of a cutaneous T-cell lymphoma (CTCL). To investigate signalling, PCM1-JAK2 was subjected to lentiviral knockdown which inhibited 7 top upregulated genes in t(8;9) cells, notably SOCS2/3. SOCS3, but not SOCS2, was also upregulated in a chronic eosinophilic leukemia bearing PCM1-JAK2, highlighting its role as a central signalling target of JAK2 translocation neoplasia. Conversely, expression of GATA3, a key T-cell developmental gene silenced in aggressive lymphoma cells, was partially restored by PCM1-JAK2 knockdown. Treatment with a selective JAK2 inhibitor (TG101348) to which MAC-1/2A/2B cells were conspicuously sensitive confirmed knockdown results and highlighted JAK2 as the active moiety. PCM1-JAK2 signalling required pSTAT5, supporting a general paradigm of STAT5 activation by JAK2 alterations in lymphoid malignancies. MAC-1/2A/2B - the first JAK2-translocation leukemia/lymphoma cell lines described - display conspicuous JAK/STAT signalling accompanied by T-cell developmental and autoimmune disease gene expression signatures, confirming their fitness as CTCL disease models. Our data support further investigation of SOCS2/3 as signalling effectors, prognostic indicators and potential therapeutic targets in cancers with JAK2 rearrangements.
    • Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria.

      Donnert, Monique; Elsheikh, Sarah; Arce-Rodriguez, Alejandro; Pawar, Vinay; Braubach, Peter; Jonigk, Danny; Haverich, Axel; Weiss, Siegfried; Müsken, Mathias; Häussler, Susanne; et al. (PLOS, 2020-12-22)
      Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell's bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.
    • Th17 cytokine differentiation and loss of plasticity after SOCS1 inactivation in a cutaneous T-cell lymphoma.

      Ehrentraut, Stefan; Schneider, Björn; Nagel, Stefan; Pommerenke, Claudia; Quentmeier, Hilmar; Geffers, Robert; Feist, Maren; Kaufmann, Maren; Meyer, Corinna; Kadin, Marshall E; et al. (2016-04-28)
      We propose that deregulated T-helper-cell (Th) signaling underlies evolving Th17 cytokine expression seen during progression of cutaneous T-cell lymphoma (CTCL). Accordingly, we developed a lymphoma progression model comprising cell lines established at indolent (MAC-1) and aggressive (MAC-2A) CTCL stages. We discovered activating JAK3 (V722I) mutations present at indolent disease, reinforced in aggressive disease by novel compound heterozygous SOCS1 (G78R/D105N) JAK-binding domain inactivating mutations. Though isogenic, indolent and aggressive-stage cell lines had diverged phenotypically, the latter expressing multiple Th17 related cytokines, the former a narrower profile. Importantly, indolent stage cells remained poised for Th17 cytokine expression, readily inducible by treatment with IL-2 - a cytokine which mitigates Th17 differentiation in mice. In indolent stage cells JAK3 expression was boosted by IL-2 treatment. Th17 conversion of MAC-1 cells by IL-2 was blocked by pharmacological inhibition of JAK3 or STAT5, implicating IL2RG - JAK3 - STAT5 signaling in plasticity responses. Like IL-2 treatment, SOCS1 knockdown drove indolent stage cells to mimic key aggressive stage properties, notably IL17F upregulation. Co-immunoprecipitation experiments showed that SOCS1 mutations abolished JAK3 binding, revealing a key role for SOCS1 in regulating JAK3/STAT5 signaling. Collectively, our results show how JAK/STAT pathway mutations contribute to disease progression in CTCL cells by potentiating inflammatory cytokine signaling, widening the potential therapeutic target range for this intractable entity. MAC-1/2A cells also provide a candidate human Th17 laboratory model for identifying potentally actionable CTCL markers or targets and testing their druggability in vitro.
    • Therapeutic benefit of Salmonella attributed to LPS and TNF-α is exhaustible and dictated by tumor susceptibility.

      Kocijancic, Dino; Leschner, Sara; Felgner, Sebastian; Komoll, Ronja-Melinda; Frahm, Michael; Pawar, Vinay; Weiss, Siegfried; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-30)
      The potential of bacteria-mediated tumor therapy (BMTT) is highlighted by more than a century of investigation. Attenuated Salmonella has prevailed as promising therapeutic agents. For BMTT - categorized as an immune therapy - the exact contribution of particular immune reactions to the therapeutic effect remains ambiguous. In addition, one could argue for or against the requirement of bacterial viability and tumor targeting. Herein we evaluate the isolated therapeutic efficacy of purified LPS and TNF-α, which together account for a dominant immunogenic pathway of gram negative bacteria like Salmonella. We show that therapeutic efficacy against CT26 tumors does not require bacterial viability. Analogous to viable Salmonella SL7207, tumor regression by a specific CD8+ T cell response can be induced by purified LPS or recombinant TNF-α (rTNF-α). Conversely, therapeutic effects against RenCa tumors were abrogated upon bacterial avitalization and limited using isolated adjuvants. This argues for an alternative mechanistic explanation for SL7207 against RenCa that depends on viability and persistence. Unable to boost bacterial therapies by co-injection of rTNF-α suggested therapeutic effects along this axis are exhausted by the intrinsic adjuvanticity of bacteria alone. However, the importance of TNF-α for BMTT was highlighted by its support of tumor invasion and colonization in concert with lower infective doses of Salmonella. In consideration, bacterial therapeutic effectiveness along the axis of LPS and TNF-α appears limited, and does not offer the necessary plasticity for different tumors. This emphasizes a need for recombinant strengthening and vehicular exploitation to accommodate potency, plasticity and distinctiveness in BMTT.
    • Therapeutic modulation of RNA-binding protein Rbm38 facilitates re-endothelialization after arterial injury.

      Sonnenschein, Kristina; Fiedler, Jan; Pfanne, Angelika; Just, Annette; Mitzka, Saskia; Geffers, Robert Robert; Pich, Andreas; Bauersachs, Johann; Thum, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Oxford Academic, 2019-03-07)
      Aims Delayed re-endothelialization after balloon angioplasty in patients with coronary or peripheral artery disease impairs vascular healing and leads to neointimal proliferation. In the present study, we examined the effect of RNA-binding motif protein 38 (Rbm38) during re-endothelialization in a murine model of experimental vascular injury. Methods and results Left common carotid arteries of C57BL/6 mice were electrically denudated and endothelial regeneration was evaluated. Profiling of RNA-binding proteins revealed dysregulated expression of Rbm38 in the denudated and regenerated areas. We next tested the importance of Rbm38 in human umbilical vein endothelial cells (HUVECS) and analysed its effects on cellular proliferation, migration and apoptosis. Rbm38 silencing in vitro demonstrated important beneficial functional effects on migratory capacity and proliferation of endothelial cells. In vivo, local silencing of Rbm38 also improved re-endothelialization of denuded carotid arteries. Luciferase reporter assay identified miR-98 and let-7f to regulate Rbm38 and the positive proliferative properties of Rbm38 silencing in vitro and in vivo were mimicked by therapeutic overexpression of these miRNAs. Conclusion The present data identified Rbm38 as an important factor of the regulation of various endothelial cell functions. Local inhibition of Rbm38 as well as overexpression of the upstream regulators miR-98 and let-7f improved endothelial regeneration in vivo and thus may be a novel therapeutic entry point to avoid endothelial damage after balloon angioplasty.
    • Therapeutic Potential of Bacteria against Solid Tumors.

      Hatzikirou, Haralampos; López Alfonso, Juan Carlos; Leschner, Sara; Weiss, Siegfried; Meyer-Hermann, Michael; BRICS, Braunschweiger Zentrum für Systembiologie, Rebenring 56, 38106 Braunschweig, Germany. (2017-04-01)
      Intentional bacterial infections can produce efficacious antitumor responses in mice, rats, dogs, and humans. However, low overall success rates and intense side effects prevent such approaches from being employed clinically. In this work, we titered bacteria and/or the proinflammatory cytokine TNFα in a set of established murine models of cancer. To interpret the experiments conducted, we considered and calibrated a tumor-effector cell recruitment model under the influence of functional tumor-associated vasculature. In this model, bacterial infections and TNFα enhanced immune activity and altered vascularization in the tumor bed. Information to predict bacterial therapy outcomes was provided by pretreatment tumor size and the underlying immune recruitment dynamics. Notably, increasing bacterial loads did not necessarily produce better long-term tumor control, suggesting that tumor sizes affected optimal bacterial loads. Short-term treatment responses were favored by high concentrations of effector cells postinjection, such as induced by higher bacterial loads, but in the longer term did not correlate with an effective restoration of immune surveillance. Overall, our findings suggested that a combination of intermediate bacterial loads with low levels TNFα administration could enable more favorable outcomes elicited by bacterial infections in tumor-bearing subjects. Cancer Res; 77(7); 1553-63. ©2017 AACR.
    • Therapy of solid tumors using probiotic Symbioflor-2: restraints and potential.

      Kocijancic, Dino; Felgner, Sebastian; Frahm, Michael; Komoll, Ronja-Melinda; Iljazovic, Aida; Pawar, Vinay; Rohde, M; Heise, Ulrike; Zimmermann, Kurt; Gunzer, Florian; et al. (2016-04-19)
      To date, virulent bacteria remain the basis of most bacteria mediated cancer therapies. For clinical application attenuation is required. However, this might result in a drastically lowered therapeutic capacity. Herein we argue that the E. coli probiotic Symbioflor-2, with a history of safe application may constitute a viable tumor therapeutic candidate. We demonstrate that Symbioflor-2 displays a highly specific tumor targeting ability as determined in murine CT26 and RenCa tumor models. The excellent specificity was ascribed to reduced levels of adverse colonization. A high safety standard was demonstrated in WT and Rag1-/- mice. Thus, Symbioflor-2 may represent an ideal tumor targeting delivery system for therapeutic molecules. Moreover, Symbioflor-2 was capable of inducing CT26 tumor clearance as result of an adjuvant effect on tumor specific CD8+ T cells analogous to the Salmonella variant SL7207. However, lower therapeutic efficacy against RenCa tumors suggested a generally reduced therapeutic potency for probiotics. Interestingly, concurrent depletion of Gr-1+ or Ly6G+ cells installed therapeutic efficacy equal to SL7207, thus highlighting the role of innate effector cells in restraining the anti-tumor effects of Symbioflor-2. Collectively, our findings argue for a strategy of safe strain application and a more sustainable use of bacteria as a delivery system for therapeutic molecules.
    • Thermoplasmatales and sulfur-oxidizing bacteria dominate the microbial community at the surface water of a CO2-rich hydrothermal spring located in Tenorio Volcano National Park, Costa Rica

      Arce-Rodríguez, Alejandro; Puente-Sánchez, Fernando; Avendaño, Roberto; Martínez-Cruz, María; de Moor, J Maarten; Pieper, Dietmar H; Chavarría, Max; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2019-01-01)
      Here we report the chemical and microbial characterization of the surface water of a CO2-rich hydrothermal vent known in Costa Rica as Borbollones, located at Tenorio Volcano National Park. The Borbollones showed a temperature surrounding 60 °C, a pH of 2.4 and the gas released has a composition of ~ 97% CO2, ~ 0.07% H2S, ~ 2.3% N2 and ~ 0.12% CH4. Other chemical species such as sulfate and iron were found at high levels with respect to typical fresh water bodies. Analysis by 16S rRNA gene metabarcoding revealed that in Borbollones predominates an archaeon from the order Thermoplasmatales and one bacterium from the genus Sulfurimonas. Other sulfur- (genera Thiomonas, Acidithiobacillus, Sulfuriferula, and Sulfuricurvum) and iron-oxidizing bacteria (genera Sideroxydans, Gallionella, and Ferrovum) were identified. Our results show that CO2-influenced surface water of Borbollones contains microorganisms that are usually found in acid rock drainage environments or sulfur-rich hydrothermal vents. To our knowledge, this is the first microbiological characterization of a CO2-dominated hydrothermal spring from Central America and expands our understanding of those extreme ecosystems.
    • Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding.

      Agren, Daniel; Stehr, Matthias; Berthold, Catrine L; Kapoor, Shobhna; Oehlmann, Wulf; Singh, Mahavir; Schneider, Gunter; Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden. (2008-04-04)
      L-alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to L-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.3 and 2.0 A resolution, respectively. The enzyme forms a hexamer of identical subunits, with the NAD-binding domains building up the core of the molecule and the substrate-binding domains located at the apical positions of the hexamer. Coenzyme binding stabilizes a closed conformation where the substrate-binding domains are rotated by about 16 degrees toward the dinucleotide-binding domains, compared to the open structure of the apo-enzyme. In the structure of the abortive ternary complex with NAD+ and pyruvate, the substrates are suitably positioned for hydride transfer between the nicotinamide ring and the C2 carbon atom of the substrate. The approach of the nucleophiles water and ammonia to pyruvate or the reaction intermediate iminopyruvate, respectively, is, however, only possible through conformational changes that make the substrate binding site more accessible. The crystal structures identified the conserved active-site residues His96 and Asp270 as potential acid/base catalysts in the reaction. Amino acid replacements of these residues by site-directed mutagenesis led to inactive mutants, further emphasizing their essential roles in the enzymatic reaction mechanism.
    • Topical imiquimod yields systemic effects due to unintended oral uptake.

      Grine, Lynda; Steeland, Sophie; Van Ryckeghem, Sara; Ballegeer, Marlies; Lienenklaus, Stefan; Weiss, Siegfried; Sanders, Niek N; Vandenbroucke, Roosmarijn E; Libert, Claude; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.
    • Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

      Wiemann, Stefan; Weil, Bernd; Wellenreuther, Ruth; Gassenhuber, Johannes; Glassl, Sabine; Ansorge, Wilhelm; Böcher, Michael; Blöcker, Helmut; Bauersachs, Stefan; Blum, Helmut; et al. (Cold Spring Harbor Laboratory Press, 2001-03)
    • Towards individualized diagnostics of biofilm-associated infections: a case study.

      Müsken, Mathias; Klimmek, Kathi; Sauer-Heilborn, Annette; Donnert, Monique; Sedlacek, Ludwig; Suerbaum, Sebastian; Häussler, Susanne; H-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      Organized within biofilm communities, bacteria exhibit resistance towards a broad spectrum of antibiotics. Thus, one might argue that bacteria isolated from biofilm-associated chronic infections should be subjected to resistance profiling under biofilm growth conditions. Various test systems have been developed to determine the biofilm-associated resistance; however, it is not clear to what extent the in vitro results reflect the situation in vivo, and whether the biofilm-resistance profile should guide clinicians in their treatment choice. To address this issue, we used confocal microscopy in combination with live/dead staining, and profiled biofilm-associated resistance of a large number (>130) of clinical Pseudomonas aeruginosa isolates from overall 15 cystic fibrosis patients. Our results demonstrate that in addition to a general non-responsiveness of bacteria when grown under biofilm conditions, there is an isolate-specific and antibiotic-specific biofilm-resistance profile. This individual resistance profile is independent on the structural properties of the biofilms. Furthermore, biofilm resistance is not linked to the resistance profile under planktonic growth conditions, or a mucoid, or small colony morphology of the tested isolates. Instead, it seems that individual biofilm structures evolve during biofilm-associated growth and are shaped by environment-specific cues. In conclusion, our results demonstrate that biofilm resistance profiles are isolate specific and cannot be deduced from commonly studied phenotypes. Further clinical studies will have to show the added value of biofilm-resistance profiling. Individualized diagnosis of biofilm resistance might lead to more rational recommendations for antimicrobial therapy and, thus, increased effectiveness of the treatment of chronically infected patients.
    • Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy.

      Khera, Tanvi; Todt, Daniel; Vercauteren, Koen; McClure, C Patrick; Verhoye, Lieven; Farhoudi, Ali; Bhuju, Sabin; Geffers, Robert; Baumert, Thomas F; Steinmann, Eike; et al. (2017-03)
      Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations (<1%) was detectable in the donor inoculum and recipient mice, with single nucleotide variants (SNVs) > 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.
    • TRANSCompel®: a database on composite regulatory elements in eukaryotic genes

      Kel-Margoulis, Olga V.; Kel, Alexander E.; Reuter, Ingmar; Deineko, Igor V.; Wingender, Edgar (Oxford University Press, 2002-01-01)