Recent Submissions

  • xCELLanalyzer: A Framework for the Analysis of Cellular Impedance Measurements for Mode of Action Discovery

    Franke, Raimo; Hinkelmann, Bettina; Fetz, Verena; Stradal, Theresia; Sasse, Florenz; Klawonn, Frank; Brönstrup, Mark; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Sage, 2019-01-25)
    Mode of action (MoA) identification of bioactive compounds is very often a challenging and time-consuming task. We used a label-free kinetic profiling method based on an impedance readout to monitor the time-dependent cellular response profiles for the interaction of bioactive natural products and other small molecules with mammalian cells. Such approaches have been rarely used so far due to the lack of data mining tools to properly capture the characteristics of the impedance curves. We developed a data analysis pipeline for the xCELLigence Real-Time Cell Analysis detection platform to process the data, assess and score their reproducibility, and provide rank-based MoA predictions for a reference set of 60 bioactive compounds. The method can reveal additional, previously unknown targets, as exemplified by the identification of tubulin-destabilizing activities of the RNA synthesis inhibitor actinomycin D and the effects on DNA replication of vioprolide A. The data analysis pipeline is based on the statistical programming language R and is available to the scientific community through a GitHub repository.
  • Künstliche Intelligenz zur diagnostischen Unterstützung ausgewählter seltener lysosomaler Speichererkrankungen: Ergebnisse einer Pilotstudie.

    Sieg, Anna-Lena; Anibh, Martin; Muschol, Nicole Maria; Köhn, Anja; Lampe, Christina; Kortum, Xiauwei; Mehmecke, Sandra; Blöß, Susanne; Lechner, Werner; Klawonn, Frank; Grigull, Lorenz; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Thieme, 2019-02-10)
    Hintergrund: Die Diagnosestellung einer seltenen Stoffwechselerkrankung stellt eine Herausforderung für Familien und betreuende Ärzte dar. Um den Weg zur Diagnose zu unterstützen, wurde ein diagnostisches Werkzeug entwickelt, welches die Erfahrungen Betroffener nutzt.
  • A functional interplay between intein and extein sequences in protein splicing compensates for the essential block B histidine

    Friedel, Kristina; Popp, Monika A.; Matern, Julian C. J.; Gazdag, Emerich M.; Thiel, Ilka V.; Volkmann, Gerrit; Blankenfeldt, Wulf; Mootz, Henning D.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Royal Society of Chemistry, 2018-10-03)
    Inteins remove themselves from a precursor protein by protein splicing. Due to the concomitant structural changes of the host protein, this self-processing reaction has enabled many applications in protein biotechnology and chemical biology. We show that the evolved M86 mutant of the Ssp DnaB intein displays a significantly improved tolerance towards non-native amino acids at the N-terminally flanking (−1) extein position compared to the parent intein, in the form of both an artificially trans-splicing split intein and the cis-splicing mini-intein. Surprisingly, side chains with increased steric bulk compared to the native Gly(−1) residue, including D-amino acids, were found to compensate for the essential block B histidine in His73Ala mutants in the initial N–S acyl shift of the protein splicing pathway. In the case of the M86 intein, large (−1) side chains can even rescue protein splicing activity as a whole. With the comparison of three crystal structures, namely of the M86 intein as well as of its Gly(−1)Phe and Gly(−1)Phe/His73Ala mutants, our data supports a model in which the intein's active site can exert a strain by varying mechanisms on the different angles of the scissile bond at the extein–intein junction to effect a ground-state destabilization. The compensatory mechanism of the block B histidine is the first example for the direct functional role of an extein residue in protein splicing. It sheds new light on the extein–intein interplay and on possible consequences of their co-evolution as well as on the laboratory engineering of improved inteins.
  • Improving the Decision Support in Diagnostic Systems using Classifier Probability Calibration

    Kortum, Xiaowei; Grigull, Lorenz; Lechner, Werner; Klawonn, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2018-11-09)
    In modern medical diagnoses, classifying a patient’s disease is often realized with the help of a system-aided symptoms interpreter. Most of these systems rely on supervised learning algorithms, which can statistically extend the doctor’s logic capabilities for interpreting and examining symptoms, thus supporting the doctor to find the correct diagnosis. Besides, these algorithms compute classifier scores and class labels that are used to statistically characterize the system’s confidence level on a patient’s type of disease. Unfortunately, most classifier scores are based on an arbitrary scale but not uniformed, thus the interpretations often lack of clinical significance and evaluation criterion. Especially combining multiple classifier scores within a diagnostic system, it is essential to apply a calibration process to make the different scores comparable. As a frequently used calibration technique, we adapted isotonic regression for our medical diagnostic support system, to provide a flexible and effective scaling process that consequently calibrates the arbitrary scales of classifiers’ scores. In a comparative evaluation, we show that our disease diagnostic system with isotonic regression can actively improve the diagnostic result based on an ensemble of classifiers, also effectively remove outliers from data, thus optimize the decision support system to obtain better diagnostic results.
  • Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.

    Muthukumar, Yazh; Münkemer, Johanna; Mathieu, Daniel; Richter, Christian; Schwalbe, Harald; Steinmetz, Heinrich; Kessler, Wolfgang; Reichelt, Joachim; Beutling, Ulrike; Frank, Ronald; Büssow, Konrad; van den Heuvel, Joop; Brönstrup, Mark; Taylor, Richard E; Laschat, Sabine; Sasse, Florenz (PLOS, 2018-01-01)
    The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
  • Induced B Cell Development in Adult Mice.

    Brennecke, Anne-Margarete; Düber, Sandra; Roy, Bishnudeo; Thomsen, Irene; Garbe, Annette I; Klawonn, Frank; Pabst, Oliver; Kretschmer, Karsten; Weiss, Siegfried; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-01-01)
    We employed the B-Indu-Rag1 model in which the coding exon of recombination-activating gene 1 (Rag1) is inactivated by inversion. It is flanked by inverted loxP sites. Accordingly, B cell development is stopped at the pro/pre B-I cell precursor stage. A B cell-specific Cre recombinase fused to a mutated estrogen receptor allows the induction of RAG1 function and B cell development by application of Tamoxifen. Since Rag1 function is recovered in a non-self-renewing precursor cell, only single waves of development can be induced. Using this system, we could determine that B cells minimally require 5 days to undergo development from pro/preB-I cells to the large and 6 days to the small preB-II cell stage. First immature transitional (T) 1 and T2 B cells could be detected in the bone marrow at day 6 and day 7, respectively, while their appearance in the spleen took one additional day. We also tested a contribution of adult bone marrow to the pool of B-1 cells. Sublethally irradiated syngeneic WT mice were adoptively transferred with bone marrow of B-Indu-Rag1 mice and B cell development was induced after 6 weeks. A significant portion of donor derived B-1 cells could be detected in such adult mice. Finally, early VH gene usage was tested after induction of B cell development. During the earliest time points the VH genes proximal to D/J were found to be predominantly rearranged. At later time points, the large family of the most distal VH prevailed.
  • Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non-redundant Functions in Vivo.

    Schaks, Matthias; Singh, Shashi P; Kage, Frieda; Thomason, Peter; Klünemann, Thomas; Steffen, Anika; Blankenfeldt, Wulf; Stradal, Theresia E; Insall, Robert H; Rottner, Klemens; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-10-25)
    Cell migration often involves the formation of sheet-like lamellipodia generated by branched actin filaments. The branches are initiated when Arp2/3 complex [1] is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family [2]. Recent structural studies defined two independent Rac binding sites on WRC within the Sra-1/PIR121 subunit of the pentameric WRC [3, 4], but the functions of these sites in vivo have remained unknown. Here we dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra-1, using CRISPR/Cas9-mediated gene disruption of Sra-1 and its paralog PIR121 in murine B16-F1 cells combined with Sra-1 mutant rescue. We show that the A site, positioned adjacent to the binding region of WAVE-WCA mediating actin and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site toward the C terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium cells. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants; we conclude these residues are important for Rac-D site interaction. Finally, constitutively activated WRC was able to induce lamellipodia even after both Rac interaction sites were lost, showing that Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but is not mandatory for WRC accumulation in the lamellipodium.
  • Structural insights into antigen recognition of an anti-β-(1,6)-β-(1,3)-D-glucan antibody.

    Sung, Kwang Hoon; Josewski, Jörn; Dübel, Stefan; Blankenfeldt, Wulf; Rau, Udo; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-12)
    Schizophyllan (SCH) is a high molecular weight homopolysaccharide composed of a β-(1,3)-D-glucan main chain with branching β-(1,6)-bound D-glucose residues. It forms triple helices that are highly stable towards heat and extreme pH, which provides SCH with interesting properties for industrial and medical applications. The recombinant anti-SCH antibody JoJ48C11 recognizes SCH and related β-(1,6)-branched β-(1,3)-D-glucans, but details governing its specificity are not known. Here, we fill this gap by determining crystal structures of the antigen binding fragment (Fab) of JoJ48C11 in the apo form and in complex with the unbranched β-(1,3)-D-glucose hexamer laminarihexaose 3.0 and 2.4 Å resolution, respectively. Together with docking studies, this allowed construction of a JoJ48C11/triple-helical SCH complex, leading to the identification of eight amino acid residues of JoJ48C11 (Tyr27
  • External quality assessment schemes for glucose measurements in Germany: factors for successful participation, analytical performance and medical impact.

    Bietenbeck, Andreas; Geilenkeuser, Wolf J; Klawonn, Frank; Spannagl, Michael; Nauck, Matthias; Petersmann, Astrid; Thaler, Markus A; Winter, Christof; Luppa, Peter B; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-07-26)
    Determination of blood glucose concentration is one of the most important measurements in clinical chemistry worldwide. Analyzers in central laboratories (CL) and point-of-care tests (POCT) are both frequently used. In Germany, regular participation in external quality assessment (EQA) schemes is mandatory for laboratories performing glucose testing. Glucose testing data from the two German EQAs "Reference Institute for Bioanalytics" (RfB) and "INSTAND - Gesellschaft zur Förderung der Qualitätssicherung in medizinischen Laboratorien" (Instand) were analyzed from 2012 to 2016. Multivariable odds ratios (OR) for the probability to reach a "good" result were calculated. Imprecision and bias were determined and clinical risk of measurement errors estimated. The device employed was the most important variable required for a "good" performance in all EQAs. Additional participation in an EQA for CL automated analyzers improved performance in POCT EQAs. The reciprocal effect was less pronounced. New participants performed worse than experienced participants especially in CL EQAs. Imprecision was generally smaller for CL, but some POCT devices reached a comparable performance. Large lot-to-lot differences occurred in over 10% of analyzed cases. We propose the "bias budget" as a new metric to express the maximum allowable bias that still carries acceptable medical risk. Bias budgets were smallest and clinical risks of errors greatest in the low range of measurement 60-115 mg/dL (3.3-6.4 mmol/L) for most devices. EQAs help to maintain high analytical performances. They generate important data that serve as the foundation for learning and improvement in the laboratory healthcare system.
  • Pseudomonas aeruginosa pyoverdine maturation enzyme PvdP has a noncanonical domain architecture and affords insight into a new subclass of tyrosinases

    Poppe, Juliane; Reichelt, Joachim; Blankenfeldt, Wulf; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (2018-09-21)
    Pyoverdines (PVDs) are important chromophore-containing siderophores of fluorescent pseudomonad bacteria such as the opportunistic human pathogen
  • Rapid access to RNA resonances by proton-detected solid-state NMR at >100 kHz MAS.

    Marchanka, Alexander; Stanek, Jan; Pintacuda, Guido; Carlomagno, Teresa; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-21)
    Fast (4100 kHz) magic angle spinning solid-state NMR allows combining high-sensitive proton detection with the absence of an intrinsic molecular weight limit. Using this technique we observe for the first time narrow 1H RNA resonances and assign nucleotide spin systems with only 200 lg of uniformly 13C,15N-labelled RNA.
  • Exchange of amino acids in the H1-haemagglutinin to H3 residues is required for efficient influenza A virus replication and pathology in Tmprss2 knock-out mice.

    Lambertz, Ruth L O; Pippel, Jan; Gerhauser, Ingo; Kollmus, Heike; Anhlan, Darisuren; Hrincius, Eike R; Krausze, Joern; Kühn, Nora; Schughart, Klaus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-01)
    The haemagglutinin (HA) of H1N1 and H3N2 influenza A virus (IAV) subtypes has to be activated by host proteases. Previous studies showed that H1N1 virus cannot replicate efficiently in Tmprss2/ knock-out mice whereas H3N2 viruses are able to replicate to the same levels in Tmprss2/ as in wild type (WT) mice. Here, we investigated the sequence requirements for the HA molecule that allow IAV to replicate efficiently in the absence of TMPRSS2. We showed that replacement of the H3 for the H1-loop sequence (amino acids 320 to 329, at the C-terminus of HA1) was not sufficient for equal levels of virus replication or severe pathology in Tmprss2/ knock-out mice compared to WT mice. However, exchange of a distant amino acid from H1 to H3 sequence (E31D) in addition to the HA-loop substitution resulted in virus replication in Tmprss2/ knockout mice that was comparable to WT mice. The higher virus replication and lung damage was associated with increased epithelial damage and higher mortality. Our results provide further evidence and insights into host proteases as a promising target for therapeutic intervention of IAV infections.
  • Chronic Toxoplasma infection is associated with distinct alterations in the synaptic protein composition.

    Lang, Daniel; Schott, Björn H; van Ham, Marco; Morton, Lorena; Kulikovskaja, Leonora; Herrera-Molina, Rodrigo; Pielot, Rainer; Klawonn, Frank; Montag, Dirk; Jänsch, Lothar; Gundelfinger, Eckart D; Smalla, Karl Heinz; Dunay, Ildiko Rita; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-01)
    Chronic infection with the neurotropic parasite Toxoplasma gondii has been implicated in the risk for several neuropsychiatric disorders. The mechanisms, by which the parasite may alter neural function and behavior of the host, are not yet understood completely. Here, a novel proteomic approach using mass spectrometry was employed to investigate the alterations in synaptic protein composition in a murine model of chronic toxoplasmosis. In a candidate-based strategy, immunoblot analysis and immunohistochemistry were applied to investigate the expression levels of key synaptic proteins in glutamatergic signaling. A comparison of the synaptosomal protein composition revealed distinct changes upon infection, with multiple proteins such as EAAT2, Shank3, AMPA receptor, and NMDA receptor subunits being downregulated, whereas inflammation-related proteins showed an upregulation. Treatment with the antiparasitic agent sulfadiazine strongly reduced tachyzoite levels and diminished neuroinflammatory mediators. However, in both conditions, a significant number of latent cysts persisted in the brain. Conversely, infection-related alterations of key synaptic protein levels could be partly reversed by the treatment. These results provide evidence for profound changes especially in synaptic protein composition in T. gondii-infected mice with a downregulation of pivotal components of glutamatergic neurotransmission. Our results suggest that the detected synaptic alterations are a consequence of the distinct neuroinflammatory milieu caused by the neurotropic parasite.
  • TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1.

    Zmora, Pawel; Hoffmann, Markus; Kollmus, Heike; Moldenhauer, Anna-Sophie; Danov, Olga; Braun, Armin; Winkler, Michael; Schughart, Klaus; Pöhlmann, Stefan; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-09-07)
    The influenza virus hemagglutinin (HA) facilitates viral entry into target cells. Cleavage of HA by host cell proteases is essential for viral infectivity, and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease (TTSP) TMPRSS2 has been identified as an HA activator in cell culture and in the infected host. However, it is less clear whether TMPRSS2-related enzymes can also activate HA for spread in target cells. Moreover, the activity of cellular serine protease inhibitors against HA-activating TTSPs is poorly understood. Here, we show that TMPRSS11A, another member of the TTSP family, cleaves and activates the influenza A virus (FLUAV) HA and the Middle East respiratory syndrome coronavirus spike protein (MERS-S). Moreover, we demonstrate that TMPRSS11A is expressed in murine tracheal epithelium, which is a target of FLUAV infection, and in human trachea, suggesting that the protease could support FLUAV spread in patients. Finally, we show that HA activation by the TMPRSS11A-related enzymes human airway tryptase and DESC1, but not TMPRSS11A itself, is blocked by the cellular serine protease inhibitor hepatocyte growth factor activator inhibitor type-1 (HAI-1). Our results suggest that TMPRSS11A could promote FLUAV spread in target cells and that HA-activating TTSPs exhibit differential sensitivity to blockade by cellular serine protease inhibitors.
  • Fate of the UPR marker protein Kar2/Bip and autophagic processes in fed-batch cultures of secretory insulin precursor producing Pichia pastoris.

    Roth, Gustavo; Vanz, Ana Letícia; Lünsdorf, Heinrich; Nimtz, Manfred; Rinas, Ursula; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-08-09)
    Secretory recombinant protein production with Pichia (syn. Komagataella) pastoris is commonly associated with the induction of an unfolded protein response (UPR) usually apparent through increased intracellular levels of endoplasmic reticulum (ER) resident chaperones such as Kar2/Bip. During methanol-induced secretory production of an insulin precursor (IP) under industrially relevant fed-batch conditions the initially high level of intracellular Kar2/Bip after batch growth on glycerol unexpectedly declined in the following methanol fed-batch phase misleadingly suggesting that IP production had a low impact on UPR activation. Analysis of the protein production independent level of Kar2/Bip revealed that high Kar2/Bip levels were reached in the exponential growth phase of glycerol batch cultures followed by a strong decline of Kar2/Bip during entry into stationary phase. Ultra-structural cell morphology studies revealed autophagic processes (e.g. ER phagy) at the end of the glycerol batch phase most likely responsible for the degradation of ER resident chaperones such as Kar2/Bip. The pre-induction level of Kar2/Bip did not affect the IP secretion efficiency in the subsequent methanol-induced IP production phase. During growth on methanol intracellular Kar2/Bip levels declined in IP producing and non-producing host cells. However, extracellular accumulation of Kar2/Bip was observed in IP-producing cultures but not in non-producing controls. Most importantly, the majority of the extracellular Kar2/Bip accumulated in the culture supernatant of IP producing cells as truncated protein (approx. 35 kDa). Rapid growth leads to higher basal levels of the major UPR marker protein Kar2/Bip independent of recombinant protein production. Entry into stationary phase or slower growth on poorer substrate, e.g. methanol, leads to a lower basal Kar2/Bip level. Methanol-induced secretory IP production elicits a strong UPR activation which counteracts the reduced UPR during slow growth on methanol. The major ER chaperone Kar2/Bip is found together with recombinant IP in the culture medium where full-length Kar2/Bip accumulates in addition to large amounts of truncated Kar2/Bip. Thus, for judging UPR activating properties of the produced protein it is important to additionally analyze the medium not only for intact Kar2/Bip but also for truncated versions of this UPR reporter protein.
  • Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From in the Pre-assembled State.

    Schwemmlein, Nina; Pippel, Jan; Gazdag, Emerich-Mihai; Blankenfeldt, Wulf (2018-01-01)
    iffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.
  • Chronic lung inflammation primes humoral immunity and augments antipneumococcal resistance.

    Boehme, Julia D; Stegemann-Koniszewski, Sabine; Autengruber, Andrea; Peters, Nicole; Wissing, Josef; Jänsch, Lothar; Jeron, Andreas; Bruder, Dunja; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-07-10)
    Airway epithelial cells (AECs) display remarkable plasticity in response to infectious stimuli and their functional adaptations are critical for antimicrobial immunity. However, the roles of AECs and humoral mediators to host defense in non-communicable lung inflammation remain elusive. We dissected pulmonary defense against Streptococcus pneumoniae in hosts with pre-existing inflammatory conditions (SPC-HAxTCR-HA mice). Lung tissue transcriptomics and bronchoalveolar lavage fluid (BALF) proteomics revealed an induction of humoral defense mechanisms in inflamed lungs. Accordingly, besides antibacterial proteins and complement components being overrepresented in inflamed lungs, elevated polymeric immunoglobulin receptor (pIgR)-expression in AECs correlated with increased secretory immunoglobulin (SIg) transport. Consequently, opsonization assays revealed augmented pneumococcal coverage by SIgs present in the BALF of SPC-HAxTCR-HA mice, which was associated with enhanced antipneumococcal resistance. These findings emphasize the immunologic potential of AECs as well as their central role in providing antibacterial protection and put forward pIgR as potential target for therapeutic manipulation in infection-prone individuals.
  • Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products.

    Guttenberger, Nikolaus; Blankenfeldt, Wulf; Breinbauer, Rolf; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-11-15)
    Phenazines are natural products which are produced by bacteria or by archaeal Methanosarcina species. The tricyclic ring system enables redox processes, which producing organisms use for oxidation of NADH or for the generation of reactive oxygen species (ROS), giving them advantages over other microorganisms. In this review we summarize the progress in the field since 2005 regarding the isolation of new phenazine natural products, new insights in their biological function, and particularly the now almost completely understood biosynthesis. The review is complemented by a description of new synthetic methods and total syntheses of phenazines.
  • Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    Böldicke, Thomas; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-05-01)
    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.
  • Properties of dimeric, disulfide-linked rhBMP-2 recovered from E. coli derived inclusion bodies by mild extraction or chaotropic solubilization and subsequent refolding

    Quaas, Bastian; Burmeister, Laura; Li, Zhaopeng; Nimtz, Manfred; Hoffmann, Andrea; Rinas, Ursula; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.

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