TY - JOUR A1 - Wegert, Jenny A1 - Vokuhl, Christian A1 - Collord, Grace A1 - Del Castillo Velasco-Herrera, Martin A1 - Farndon, Sarah J. A1 - Guzzo, Charlotte A1 - Jorgensen, Mette A1 - Anderson, John A1 - Slater, Olga A1 - Duncan, Catriona A1 - Bausenwein, Sabrina A1 - Streitenberger, Heike A1 - Ziegler, Barbara A1 - Furtwängler, Rhoikos A1 - Graf, Norbert A1 - Stratton, Michael R. A1 - Campbell, Peter J. A1 - Jones, David TW A1 - Koelsche, Christian A1 - Pfister, Stefan M. A1 - Mifsud, William A1 - Sebire, Neil A1 - Sparber-Sauer, Monika A1 - Koscielniak, Ewa A1 - Rosenwald, Andreas A1 - Gessler, Manfred A1 - Behjati, Sam T1 - Recurrent intragenic rearrangements of EGFR and BRAF in soft tissue tumors of infants JF - Nature Communications N2 - Soft tissue tumors of infancy encompass an overlapping spectrum of diseases that pose unique diagnostic and clinical challenges. We studied genomes and transcriptomes of cryptogenic congenital mesoblastic nephroma (CMN), and extended our findings to five anatomically or histologically related soft tissue tumors: infantile fibrosarcoma (IFS), nephroblastomatosis, Wilms tumor, malignant rhabdoid tumor, and clear cell sarcoma of the kidney. A key finding is recurrent mutation of EGFR in CMN by internal tandem duplication of the kinase domain, thus delineating CMN from other childhood renal tumors. Furthermore, we identify BRAF intragenic rearrangements in CMN and IFS. Collectively these findings reveal novel diagnostic markers and therapeutic strategies and highlight a prominent role of isolated intragenic rearrangements as drivers of infant tumors. KW - cancer KW - genetics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233446 VL - 9 ER - TY - JOUR A1 - Markert, Sebastian Matthias A1 - Britz, Sebastian A1 - Proppert, Sven A1 - Lang, Marietta A1 - Witvliet, Daniel A1 - Mulcahy, Ben A1 - Sauer, Markus A1 - Zhen, Mei A1 - Bessereau, Jean-Louis A1 - Stigloher, Christian T1 - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome JF - Neurophotonics N2 - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses. KW - caenorhabditis elegans KW - localization micoscopy KW - fluorescent-probes KW - junction proteins KW - resolution limit KW - direct stochasticoptical reconstruction microscopy KW - structured illumination microscopy KW - correlative light and electron microscopy KW - gap junction KW - neural circuits KW - nervous-system KW - image data KW - reconstruction KW - innexins KW - super-resolution microscopy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292 VL - 3 IS - 4 ER - TY - JOUR A1 - Schneider, Johannes A1 - Klein, Teresa A1 - Mielich-Süss, Benjamin A1 - Koch, Gudrun A1 - Franke, Christian A1 - Kuipers, Oskar P. A1 - Kovács, Ákos T. A1 - Sauer, Markus A1 - Lopez, Daniel T1 - Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium JF - PLoS Genetics N2 - Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium. KW - membrane proteins KW - gene expression KW - bacillus subtilis KW - fluorescence microscopy KW - cell fusion KW - signal transduction KW - gene regulation KW - lipids Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125577 VL - 11 IS - 4 ER - TY - JOUR A1 - Schwitter, Juerg A1 - Wacker, Christian M. A1 - Wilke, Norbert A1 - Al-Saadi, Nidal A1 - Sauer, Ekkehart A1 - Huettle, Kalman A1 - Schönberg, Stefan O. A1 - Debl, Kurt A1 - Strohm, Oliver A1 - Ahlstrom, Hakan A1 - Dill, Thorsten A1 - Hoebel, Nadja A1 - Simor, Tamas T1 - Superior diagnostic performance of perfusion-cardiovascular magnetic resonance versus SPECT to detect coronary artery disease: The secondary endpoints of the multicenter multivendor MR-IMPACT II (Magnetic Resonance Imaging for Myocardial Perfusion Assessment in Coronary Artery Disease Trial) JF - Journal of Cardiovascular Magnetic Resonance N2 - Background: Perfusion-cardiovascular magnetic resonance (CMR) is generally accepted as an alternative to SPECT to assess myocardial ischemia non-invasively. However its performance vs gated-SPECT and in sub-populations is not fully established. The goal was to compare in a multicenter setting the diagnostic performance of perfusion-CMR and gated-SPECT for the detection of CAD in various populations using conventional x-ray coronary angiography (CXA) as the standard of reference. Methods: In 33 centers (in US and Europe) 533 patients, eligible for CXA or SPECT, were enrolled in this multivendor trial. SPECT and CXA were performed within 4 weeks before or after CMR in all patients. Prevalence of CAD in the sample was 49% and 515 patients received MR contrast medium. Drop-out rates for CMR and SPECT were 5.6% and 3.7%, respectively (ns). The study was powered for the primary endpoint of non-inferiority of CMR vs SPECT for both, sensitivity and specificity for the detection of CAD (using a single-threshold reading), the results for the primary endpoint were reported elsewhere. In this article secondary endpoints are presented, i.e. the diagnostic performance of CMR versus SPECT in subpopulations such as multi-vessel disease (MVD), in men, in women, and in patients without prior myocardial infarction (MI). For diagnostic performance assessment the area under the receiver-operator-characteristics-curve (AUC) was calculated. Readers were blinded versus clinical data, CXA, and imaging results. Results: The diagnostic performance (= area under ROC = AUC) of CMR was superior to SPECT (p = 0.0004, n = 425) and to gated-SPECT (p = 0.018, n = 253). CMR performed better than SPECT in MVD (p = 0.003 vs all SPECT, p = 0.04 vs gated-SPECT), in men (p = 0.004, n = 313) and in women (p = 0.03, n = 112) as well as in the non-infarct patients (p = 0.005, n = 186 in 1-3 vessel disease and p = 0.015, n = 140 in MVD). Conclusion: In this large multicenter, multivendor study the diagnostic performance of perfusion-CMR to detect CAD was superior to perfusion SPECT in the entire population and in sub-groups. Perfusion-CMR can be recommended as an alternative for SPECT imaging. KW - coronary disease KW - perfusion KW - contrast KW - ischemia KW - women KW - emission-computed-tomography KW - noninvasive detection KW - intervention KW - randomized-trial KW - cardiovascular magnetic resonance KW - stress perfusion KW - prognostic value KW - angiography KW - scintigraphy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134256 VL - 14 IS - 61 ER - TY - JOUR A1 - Wolf, Annette A1 - Akrap, Nina A1 - Marg, Berenice A1 - Galliardt, Helena A1 - Heiligentag, Martyna A1 - Humpert, Fabian A1 - Sauer, Markus A1 - Kaltschmidt, Barbara A1 - Kaltschmidt, Christian A1 - Seidel, Thorsten T1 - Elements of Transcriptional Machinery Are Compatible among Plants and Mammals JF - PLoS ONE N2 - In the present work, the objective has been to analyse the compatibility of plant and human transcriptional machinery. The experiments revealed that nuclear import and export are conserved among plants and mammals. Further it has been shown that transactivation of a human promoter occurs by human transcription factor NF-\(\kappa\) B in plant cells, demonstrating that the transcriptional machinery is highly conserved in both kingdoms. Functionality was also seen for regulatory elements of NF-\(\kappa\) B such as its inhibitor I\(\kappa\)B isoform \(\alpha\) that negatively regulated the transactivation activity of the p50/RelA heterodimer by interaction with NF-\(\kappa\)B in plant cells. Nuclear export of RelA could be demonstrated by FRAP-measurements so that RelA shows nucleo-cytoplasmic shuttling as reported for RelA in mammalian cells. The data reveals the high level of compatibility of human transcriptional elements with the plant transcriptional machinery. Thus, Arabidopsis thaliana mesophyll protoplasts might provide a new heterologous expression system for the investigation of the human NF-\(\kappa\)B signaling pathways. The system successfully enabled the controlled manipulation of NF-\(\kappa\)B activity. We suggest the plant protoplast system as a tool for reconstitution and analyses of mammalian pathways and for direct observation of responses to e. g. pharmaceuticals. The major advantage of the system is the absence of interference with endogenous factors that affect and crosstalk with the pathway. KW - complexes KW - in vivo KW - DNA-binding KW - nuclear proe KW - gene expression KW - NF-KAPPA-B KW - RNA-binding protein KW - alpha KW - inflammation KW - homodimers Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131203 VL - 8 IS - 1 ER - TY - THES A1 - Sauer, Christian T1 - Untersuchungen zu den genetischen Ursachen hereditärer Netzhautdegenerationen des Menschen T1 - Analyses of the genetic causes of hereditary retinal degenerations in human N2 - Die Positionsklonierung hat sich als erfolgreiche Strategie zur Identifizierung und Isolierung von Genen erwiesen. Da ihre Anwendung im Allgemeinen keine Informationen über den zugrundeliegenden Pathomechanismus einer Erkrankung voraussetzt, eignen sich die Methoden der Positionsklonierung in besonderem Maße für die Erforschung hereditärer Netzhauterkrankungen. Im Rahmen der hier vorliegenden Arbeit wurden sie zur Untersuchung ausgewählter retinaler Degenerationen eingesetzt. Dabei konnten wichtige Beiträge für die Aufklärung der genetische Ursachen dieser Erkrankungen geleistet werden. Die autosomal dominante North Carolina Makuladystrophie (NCMD) oder die zentral areoläre Pigmentepitheldystrophie (CAPED) sind allelische Erkrankungen mit allenfalls gering progredientem Verlauf. Ihr Genlokus liegt in einem etwa 7,2 cM großen Bereich auf 6q14-q16.2 zwischen den DNA-Markern D6S424 und D6S1671. Mit Hilfe von 21 polymorphen DNA-Markern welche den NCMD-Lokus (MCDR1) flankieren, wurden Kopplungsanalysen in drei deutschen NCMD-Familien durchgeführt. Die Analyse der krankheitsassoziierten Haplotypen erbrachte Hinweise auf einen gemeinsamen Vorfahren aller drei Familien. Darüber hinaus konnte der MCDR1-Lokus auf 3,2 cM eingeengt werden und wird von den Markern D6S249 und D6S475 flankiert. Dies bedeutet einen wichtigen Schritt auf dem Weg zur Klonierung des zugrundeliegenden Krankheitsgens. Eine häufige Ursache für den frühzeitigen Verlust der zentralen Sehschärfe bei Jungen ist die X-gebundene juvenile Retinoschisis (RS). Ihr Genlokus wurde in einen etwa 900 kb großen Bereich auf dem kurzen Arm des X-Chromosoms (Xp22.2) kartiert, wo er von den DNA-Markern DXS418 und DXS999/DXS7161 flankiert wird. Die Analyse von EST-Sequenzen aus dieser Region ermöglichte die Isolierung eines neuen retinaspezifischen Transkriptes, welches als RS1 bezeichnet wurde. Das RS1-Gen besteht aus sechs Exonen und codiert ein Protein, welches eine in der Evolution hoch konservierte Discoidin-Domäne enthält. Diese Domäne ist in anderen Proteinen u.a. an der Ausbildung von Zell-Zell-Interaktionen beteiligt. Mutationsanalysen in betroffenen Personen aus neun nicht-verwandten RS-Familien ergaben neun verschiedene Sequenzveränderungen die mit dem Krankheitsbild der jeweiligen Familie segregierten. Einen ersten Einblick in die zeitliche und räumliche Expression ergab die Untersuchung des murinen Orthologs Rs1h mit Hilfe von Northern Blot, RT-PCR und RNA in situ-Hybridisierungen. Rs1h wird in der Maus hauptsächlich in den Photorezeptoren exprimiert. Die Expression beginnt erst postnatal und ist mit der Entwicklung der Photorezeptoren korreliert. Das Auftreten zahlreicher weißlich-gelber Flecken, sogenannter Drusen, in radiärer Anordung am hinteren Augenpol ist das charakteristische Merkmal einer Gruppe von Netzhauterkrankungen mit gemeinsamer Ätiologie, die unter dem Begriffen Doynsche Honigwaben Dystrophie (DHRD), Malattia Leventinese (MLVT) oder radiäre Drusen zusammengefasst werden. Der Genlokus dieser Erkrankung wurde auf den kurzen Arm von Chromosom 2 in den Bereich 2p16 kartiert. Die Durchsuchung von EST-Datenbanken führte zur Identifizierung des neuronal exprimerten Gens pNEU60. Dieses besteht aus zwei Exonen, wobei der vollständige codierende Bereich im zweiten Exon liegt. Die Analyse des pNEU60-Proteins ergab eine Struktur aus sieben Transmembrandomänen, dem gemeinsamen Merkmal G-Protein gekoppelter Rezeptoren, wie z.B. Rhodopsin. Patienten mit radiären Drusen zeigten keinerlei Sequenzveränderungen in pNEU60. Die Untersuchung von fast 200 Patienten mit der phänotypisch sehr ähnlichen altersbedingten Makuladegeneration (AMD), führte zur Identifizierung von drei potentiellen Mutationen, darunter eine nonsense-Mutation, sowie zwei polymorphen Veränderungen. Die Assoziation einer einzigen missense-Mutation (R345W) im ubiquitär exprimierten Gen EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1) mit der DHRD und MLVT wurde von einer amerikanischen Arbeitsgruppe nachgewiesen. Die R345W Mutation in diesem proximal zu pNEU60 liegenden Gen wurde in den zur Verfügung stehenden zwei MLVT-Familien sowie einer DHRD-Familie nachgewiesen. Bei der Analyse von 14 Patienten mit sporadischen radiären Drusen konnte weder die R345W Mutation, noch irgendeine andere krankheitsassoziierte Mutation nachgewiesen werden. Es wurden jedoch drei polymorphe Sequenzvarianten, sowie zwei polymorphe Di- bzw. Trinukleotidsequenzen identifiziert. Die Klonierung des orthologen EFEMP1-Gens des Rinds diente als Voraussetzung zur Untersuchung der Interaktionsfähigkeit von EFEMP1 mit anderen Proteinen. Mit der Anwendung des Hefe Zwei-Hybrid Systems konnte gezeigt werden, dass die EGF-Domänen von EFEMP1 eine Interaktion mit sich selbst ermöglichen. Die Einführung der R345W Mutation hatte dabei keinen Einfluss auf diese Wechselwirkungen. Die beschriebene Interaktion mit dem zur Familie der Ubiquiline gehörenden Protein DA41 konnte nicht reproduziert werden. Das Gen welches mit der inkompletten Form der X-gebundenen kongenitalen stationären Nachtblindheit (CSNB2) assoziiert ist, codiert die a1-Untereinheit des retinaspezifischen spannungsabhängigen L-Typ Kalziumkanals (CACNA1F). Mit Hilfe von RT-PCR Analysen und RNA in situ-Hybridisierungen wurde die räumliche Expression dieses Gens in der Netzhaut untersucht. Dabei wurde das CACNA1F-Transkript in der äußeren und inneren Körnerschicht, sowie in der Ganglienzellschicht nachgewiesen. N2 - The positional cloning strategy is a powerful tool for the identification and isolation of genes. The application of positional cloning methods to the investigation of hereditary retinal disorders proved to be suitable as they require no informations about the pathology underlying the disease. For the investigations described in this thesis several retinal degenerations were selected for the examination with the positional cloning strategy. The findings of those researches contribute to the further enlightenment of the genetic causes of those disorders. Autosomal dominant North Carolina macular dystrophy (NCMD) or central areolar pigment epithelial dystrophy (CAPED) is an allelic disorder with slow progression. It maps to an approximately 7.2 cM interval between DNA markers at D6S424 and D6S1671 on 6q14-q16.2. A total of 21 polymorphic DNA markers flanking the NCMD locus (MCDR1) were used for genetic linkage analysis in three multigeneration families of German descent expressing the NCMD phenotype. The analysis of the disease associated haplotypes provide evidence for an ancestral founder for all three families. In addition the haplotype analysis refined the MCDR1 locus to a 3.2 cM interval flanked by markers D6S249 and D6S475. This facilitates further approaches in cloning the gene underlying NCMD. X-linked juvenile retinoschisis (RS) is an important cause of early vision lost in males. The RS gene has been localized to Xp22.2 to an approximately 900 kb interval between DXS418 and DXS999/DXS7161. The analysis of expressed sequence tags (ESTs) have identified a novel transcript, designated RS1, within the RS locus that is exclusively expressed in retina. RS1 consists of six exons and encodes for a protein containing the highly conserved discoidin-domain which is implicated in cell-cell interaction. Mutational analyses of RS1 in affected individuals from nine unrelated RS families revealed nine different sequence variations segregating with the disease phenotype in the respective families. The temporal and spatial expression of the murine ortholog Rs1h was studied by northern blot and RT-PCR analyses as well as RNA in situ hybridizations. Predominant expression of Rs1h was found in photoreceptor cells starting postnatal with correlation to photoreceptor development. The appearance of multiple yellowish-white drusen in the posterior pole of the retina is characteristic of a group of retinal disorders with a common etiology, often referred to a Doyne honeycomb retinal dystrophy (DHRD), Malattia Leventinese (MLVT) or radial drusen. The gene underlying this disorder has been mapped to the short arm of chromosome 2 at 2p16. EST database searches led to the identification of the neuronal tissue specific gene pNEU60. The complete coding sequence of this gene is located in the second of two exons. Motif searches in protein databases revealed homology to a seven transmembrane domain which is a hallmark for G-protein coupled receptors like rhodopsin. While mutational analyses in patients with radial drusen identified no sequence variation in pNEU60, three potential pathogenic variants and two frequent polymorphic changes were found in a cohort of almost 200 patients with phenotypically similar age-related macular degeneration (AMD). The association of DHRD and MLVT with a single missense mutation (R345W) in the ubiquitously expressed gene encoding the EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) has been recently demonstrated by a research group from the United States. The presence of the R345W mutation has been demonstrated for our two MLVT-families and the one DRHD-family. The mutational analyses in 14 unrelated individuals with sporadic early onset drusen did not detect the R345W mutation or any other disease-associated mutation. Three different polymorphic sequence variations and two intragenic polymorphic repeats were present in similar frequencies in the patients and control individuals. As a prerequisite to the analysis of the interaction capability of EFEMP1, he bovine ortholog of this gene has been cloned. The use of a yeast two hybrid system demonstrated that the EGF-motifs of EFEMP1 could interact with each other. The introduction of the R345W mutation had no effect on that interaction. The described interaction of EFEMP1 with DA41, a family member of the ubiquilin protein family could not be reproduced. The gene associated with the incomplete form of the X-linked congenital stationary nightblindness encodes the a1-subunit of a retina specific L-type calcium-channel (CACNA1F). The spatial expression of this gene within the retina has been investigated by RT-PCR analyses and RNA in situ hybridizations. Transcriptional activity could be detected in the outer an inner nuclear layer as well as in the ganglion cell layer. KW - Netzhautdegeneration KW - Erbkrankheit KW - Molekulargenetik KW - Netzhaut KW - Degeneration KW - Makula KW - Positionsklonierung KW - North Carolina Makuladystrophie KW - Retinoschisis KW - Radiäre Drusen KW - Nachtblindheit KW - retina KW - degeneration KW - makula KW - positional cloning KW - North Carolina Makuladystrophy KW - Retinoschisis KW - radial drusen KW - night blindness Y1 - 2001 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-1936 ER - TY - BOOK A1 - Bock, Stefanie A1 - Gauch, Fabian A1 - Giernat, Yannik A1 - Hillebrand, Frank A1 - Kozlova, Darja A1 - Linck, Lisa A1 - Moschall, Rebecca A1 - Sauer, Markus A1 - Schenk, Christian A1 - Ulrich, Kristina A1 - Bodem, Jochen T1 - HIV-1 : Lehrbuch von Studenten für Studenten T1 - HIV-1 : a textbook for students written by students N2 - Dies ist ein Lehrbuch über die HIV-1 Replikation, Pathogenese und Therapie. Es richtet sich an Studenten der Biologie und der Medizin, die etwas mehr über HIV erfahren wollen und stellt neben virologischen Themen auch die zellulären Grundlagen dar. Es umfasst den Viruseintritt, die reverse Transkription, Genom-Integration, Transkriptionsregualtion, die Kotrolle des Spleißens, der Polyadenylierung und des RNA-Exportes. Die Darstellung wird abgerundet mit Kapiteln zum intrazellulärem Transport, zu Nef und zum Virusassembly. In zwei weiteren Kapitel wird die HIV-1 Pathogenese und die Therapie besprochen. Zur Lernkontrolle sind den Kapiteln Fragen und auch Klausurfragen angefügt. KW - HIV KW - Retroviren KW - Lehrbuch KW - Viren KW - Virologie KW - Transkription KW - RNS KW - Therapie KW - Pathogenese KW - Epidemiologie KW - RNA-Export KW - Polyadenylierung KW - Reverse Transkription KW - Transkription Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78980 SN - 978-3-923959-90-7 ER - TY - JOUR A1 - Andreska, Thomas A1 - Lüningschrör, Patrick A1 - Wolf, Daniel A1 - McFleder, Rhonda L. A1 - Ayon-Olivas, Maurilyn A1 - Rattka, Marta A1 - Drechsler, Christine A1 - Perschin, Veronika A1 - Blum, Robert A1 - Aufmkolk, Sarah A1 - Granado, Noelia A1 - Moratalla, Rosario A1 - Sauer, Markus A1 - Monoranu, Camelia A1 - Volkmann, Jens A1 - Ip, Chi Wang A1 - Stigloher, Christian A1 - Sendtner, Michael T1 - DRD1 signaling modulates TrkB turnover and BDNF sensitivity in direct pathway striatal medium spiny neurons JF - Cell Reports N2 - Highlights • Dopamine receptor-1 activation induces TrkB cell-surface expression in striatal neurons • Dopaminergic deficits cause TrkB accumulation and clustering in the ER • TrkB clusters colocalize with cargo receptor SORCS-2 in direct pathway striatal neurons • Intracellular TrkB clusters fail to fuse with lysosomes after dopamine depletion Summary Disturbed motor control is a hallmark of Parkinson’s disease (PD). Cortico-striatal synapses play a central role in motor learning and adaption, and brain-derived neurotrophic factor (BDNF) from cortico-striatal afferents modulates their plasticity via TrkB in striatal medium spiny projection neurons (SPNs). We studied the role of dopamine in modulating the sensitivity of direct pathway SPNs (dSPNs) to BDNF in cultures of fluorescence-activated cell sorting (FACS)-enriched D1-expressing SPNs and 6-hydroxydopamine (6-OHDA)-treated rats. DRD1 activation causes enhanced TrkB translocation to the cell surface and increased sensitivity for BDNF. In contrast, dopamine depletion in cultured dSPN neurons, 6-OHDA-treated rats, and postmortem brain of patients with PD reduces BDNF responsiveness and causes formation of intracellular TrkB clusters. These clusters associate with sortilin related VPS10 domain containing receptor 2 (SORCS-2) in multivesicular-like structures, which apparently protects them from lysosomal degradation. Thus, impaired TrkB processing might contribute to disturbed motor function in PD. KW - motor learning KW - cortico-striatal synapse KW - basal ganglia KW - direct pathway KW - DRD1 KW - dSPN KW - BDNF KW - TrkB KW - synaptic plasticity KW - GPCR Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-349932 VL - 42 IS - 6 ER - TY - JOUR A1 - Endres, Leo M. A1 - Jungblut, Marvin A1 - Divyapicigil, Mustafa A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Christodoulides, Myron A1 - Kim, Brandon J. A1 - Schubert-Unkmeir, Alexandra T1 - Development of a multicellular in vitro model of the meningeal blood-CSF barrier to study Neisseria meningitidis infection JF - Fluids and Barriers of the CNS N2 - Background Bacterial meningitis is a life-threatening disease that occurs when pathogens such as Neisseria meningitidis cross the meningeal blood cerebrospinal fluid barrier (mBCSFB) and infect the meninges. Due to the human-specific nature of N. meningitidis, previous research investigating this complex host–pathogen interaction has mostly been done in vitro using immortalized brain endothelial cells (BECs) alone, which often do not retain relevant barrier properties in culture. Here, we developed physiologically relevant mBCSFB models using BECs in co-culture with leptomeningeal cells (LMCs) to examine N. meningitidis interaction. Methods We used BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in co-culture with LMCs derived from tumor biopsies. We employed TEM and structured illumination microscopy to characterize the models as well as bacterial interaction. We measured TEER and sodium fluorescein (NaF) permeability to determine barrier tightness and integrity. We then analyzed bacterial adherence and penetration of the cell barrier and examined changes in host gene expression of tight junctions as well as chemokines and cytokines in response to infection. Results Both cell types remained distinct in co-culture and iBECs showed characteristic expression of BEC markers including tight junction proteins and endothelial markers. iBEC barrier function as determined by TEER and NaF permeability was improved by LMC co-culture and remained stable for seven days. BEC response to N. meningitidis infection was not affected by LMC co-culture. We detected considerable amounts of BEC-adherent meningococci and a relatively small number of intracellular bacteria. Interestingly, we discovered bacteria traversing the BEC-LMC barrier within the first 24 h post-infection, when barrier integrity was still high, suggesting a transcellular route for N. meningitidis into the CNS. Finally, we observed deterioration of barrier properties including loss of TEER and reduced expression of cell-junction components at late time points of infection. Conclusions Here, we report, for the first time, on co-culture of human iPSC derived BECs or hCMEC/D3 with meningioma derived LMCs and find that LMC co-culture improves barrier properties of iBECs. These novel models allow for a better understanding of N. meningitidis interaction at the mBCSFB in a physiologically relevant setting. KW - brain endothelial cells KW - bacterial meningitis KW - meningeal blood-csf barrier KW - induced pluripotent stem cells KW - neisseria meningitidis KW - leptomeningeal cells Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300208 VL - 19 IS - 1 ER - TY - JOUR A1 - Kuhlemann, Alexander A1 - Beliu, Gerti A1 - Janzen, Dieter A1 - Petrini, Enrica Maria A1 - Taban, Danush A1 - Helmerich, Dominic A. A1 - Doose, Sören A1 - Bruno, Martina A1 - Barberis, Andrea A1 - Villmann, Carmen A1 - Sauer, Markus A1 - Werner, Christian T1 - Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy JF - Frontiers in Synaptic Neuroscience N2 - Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft. KW - super-resolution microscopy (SRM) KW - click-chemistry KW - dSTORM KW - GABA-A receptor KW - genetic code expansion Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-251035 SN - 1663-3563 VL - 13 ER - TY - JOUR A1 - Peters, Simon A1 - Kaiser, Lena A1 - Fink, Julian A1 - Schumacher, Fabian A1 - Perschin, Veronika A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Stigloher, Christian A1 - Kleuser, Burkhard A1 - Seibel, Juergen A1 - Schubert-Unkmeir, Alexandra T1 - Click-correlative light and electron microscopy (click-AT-CLEM) for imaging and tracking azido-functionalized sphingolipids in bacteria JF - Scientific Reports N2 - Sphingolipids, including ceramides, are a diverse group of structurally related lipids composed of a sphingoid base backbone coupled to a fatty acid side chain and modified terminal hydroxyl group. Recently, it has been shown that sphingolipids show antimicrobial activity against a broad range of pathogenic microorganisms. The antimicrobial mechanism, however, remains so far elusive. Here, we introduce 'click-AT-CLEM', a labeling technique for correlated light and electron microscopy (CLEM) based on the super-resolution array tomography (srAT) approach and bio-orthogonal click chemistry for imaging of azido-tagged sphingolipids to directly visualize their interaction with the model Gram-negative bacterium Neisseria meningitidis at subcellular level. We observed ultrastructural damage of bacteria and disruption of the bacterial outer membrane induced by two azido-modified sphingolipids by scanning electron microscopy and transmission electron microscopy. Click-AT-CLEM imaging and mass spectrometry clearly revealed efficient incorporation of azido-tagged sphingolipids into the outer membrane of Gram-negative bacteria as underlying cause of their antimicrobial activity. KW - antimicrobials KW - biological techniques KW - imaging KW - microbiology KW - microbiology techniques KW - microscopy Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259147 VL - 11 IS - 1 ER - TY - JOUR A1 - Scheer, Monika A1 - Vokuhl, Christian A1 - Blank, Bernd A1 - Hallmen, Erika A1 - von Kalle, Thekla A1 - Münter, Marc A1 - Wessalowski, Rüdiger A1 - Hartwig, Maite A1 - Sparber-Sauer, Monika A1 - Schlegel, Paul-Gerhardt A1 - Kramm, Christof M. A1 - Kontny, Udo A1 - Spriewald, Bernd A1 - Kegel, Thomas A1 - Bauer, Sebastian A1 - Kazanowska, Bernarda A1 - Niggli, Felix A1 - Ladenstein, Ruth A1 - Ljungman, Gustaf A1 - Jahnukainen, Kirsi A1 - Fuchs, Jörg A1 - Bielack, Stefan S. A1 - Klingebiel, Thomas A1 - Koscielniak, Ewa T1 - Desmoplastic small round cell tumors: Multimodality treatment and new risk factors JF - Cancer Medicine N2 - Background To evaluate optimal therapy and potential risk factors. Methods Data of DSRCT patients <40 years treated in prospective CWS trials 1997-2015 were analyzed. Results Median age of 60 patients was 14.5 years. Male:female ratio was 4:1. Tumors were abdominal/retroperitoneal in 56/60 (93%). 6/60 (10%) presented with a localized mass, 16/60 (27%) regionally disseminated nodes, and 38/60 (63%) with extraperitoneal metastases. At diagnosis, 23/60 (38%) patients had effusions, 4/60 (7%) a thrombosis, and 37/54 (69%) elevated CRP. 40/60 (67%) patients underwent tumor resection, 21/60 (35%) macroscopically complete. 37/60 (62%) received chemotherapy according to CEVAIE (ifosfamide, vincristine, actinomycin D, carboplatin, epirubicin, etoposide), 15/60 (25%) VAIA (ifosfamide, vincristine, adriamycin, actinomycin D) and, 5/60 (8%) P6 (cyclophosphamide, doxorubicin, vincristine, ifosfamide, etoposide). Nine received high-dose chemotherapy, 6 received regional hyperthermia, and 20 received radiotherapy. Among 25 patients achieving complete remission, 18 (72%) received metronomic therapies. Three-year event-free (EFS) and overall survival (OS) were 11% (±8 confidence interval [CI] 95%) and 30% (±12 CI 95%), respectively, for all patients and 26.7% (±18.0 CI 95%) and 56.9% (±20.4 CI 95%) for 25 patients achieving remission. Extra-abdominal site, localized disease, no effusion or ascites only, absence of thrombosis, normal CRP, complete tumor resection, and chemotherapy with VAIA correlated with EFS in univariate analysis. In multivariate analysis, significant factors were no thrombosis and chemotherapy with VAIA. In patients achieving complete remission, metronomic therapy with cyclophosphamide/vinblastine correlated with prolonged time to relapse. Conclusion Pleural effusions, venous thrombosis, and CRP elevation were identified as potential risk factors. The VAIA scheme showed best outcome. Maintenance therapy should be investigated further. KW - C-reactive protein KW - desmoplastic small round cell tumor KW - maintenance therapy KW - soft tissue sarcoma KW - Trousseau's syndrome Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228444 VL - 8 IS - 2 ER - TY - THES A1 - Sauer, Christian T1 - Development, Simulation and Evaluation of Mobile Wireless Networks in Industrial Applications T1 - Entwicklung, Simulation und Bewertung von Mobilen Kabellosen Netzwerken in Industriellen Anwendungen N2 - Manyindustrialautomationsolutionsusewirelesscommunicationandrelyontheavail- ability and quality of the wireless channel. At the same time the wireless medium is highly congested and guaranteeing the availability of wireless channels is becoming increasingly difficult. In this work we show, that ad-hoc networking solutions can be used to provide new communication channels and improve the performance of mobile automation systems. These ad-hoc networking solutions describe different communi- cation strategies, but avoid relying on network infrastructure by utilizing the Peer-to- Peer (P2P) channel between communicating entities. This work is a step towards the effective implementation of low-range communication technologies(e.g. VisibleLightCommunication(VLC), radarcommunication, mmWave communication) to the industrial application. Implementing infrastructure networks with these technologies is unrealistic, since the low communication range would neces- sitate a high number of Access Points (APs) to yield full coverage. However, ad-hoc networks do not require any network infrastructure. In this work different ad-hoc net- working solutions for the industrial use case are presented and tools and models for their examination are proposed. The main use case investigated in this work are Automated Guided Vehicles (AGVs) for industrial applications. These mobile devices drive throughout the factory trans- porting crates, goods or tools or assisting workers. In most implementations they must exchange data with a Central Control Unit (CCU) and between one another. Predicting if a certain communication technology is suitable for an application is very challenging since the applications and the resulting requirements are very heterogeneous. The proposed models and simulation tools enable the simulation of the complex inter- action of mobile robotic clients and a wireless communication network. The goal is to predict the characteristics of a networked AGV fleet. Theproposedtoolswereusedtoimplement, testandexaminedifferentad-hocnetwork- ing solutions for industrial applications using AGVs. These communication solutions handle time-critical and delay-tolerant communication. Additionally a control method for the AGVs is proposed, which optimizes the communication and in turn increases the transport performance of the AGV fleet. Therefore, this work provides not only tools for the further research of industrial ad-hoc system, but also first implementations of ad-hoc systems which address many of the most pressing issues in industrial applica- tions. N2 - Viele industrielle Automatisierungslösungen verwenden drahtlose Kommunikations- systeme und sind daher auf die Verfügbarkeit und Qualität des drahtlosen Kanals an- gewiesen. Gleichzeitig ist das drahtlose Medium stark belastet und die Gewährleis- tung der Verfügbarkeit der drahtlosen Kanäle wird zunehmends herrausfordernder. In dieser Arbeit wird gezeigt, dass Ad-hoc-Netzwerklösungen genutzt werden können, um neue Kommunikationskanäle bereitzustellen und die Leistung von mobilen Au- tomatisierungssystemen zu verbessern. Diese Ad-hoc-Netzwerklösungen können un- terschiedliche Kommunikationsstrategien bezeichnen. In all diesen Strategien wird der Peer-to-Peer (P2P)-Kanal zwischen zwei kommunizierenden Systemen verwendet statt Netzwerk-Infrastruktur. Diese Arbeit ist ein Schritt hin zur effektiven Implementierung von Kommunikations- technologien mit geringer Reichweite (z.B. Visible Light Communication (VLC), Radar- kommunikation, mmWave-Kommunikation) in der industriellen Anwendung. Die Im- plementierung von Infrastrukturnetzen mit diesen Technologien ist unrealistisch, da die geringe Kommunikationsreichweite eine hohe Anzahl von Access Points (APs) er- fordern würde um eine flächendeckende Bereitstellung von Kommunikationskanälen zu gewährleisten. Ad-hoc-Netzwerke hingegen benötigen keine Netzwerkinfrastruk- tur. In dieser Arbeit werden verschiedene Ad-hoc-Netzwerklösungen für den industri- ellenAnwendungsfallvorgestelltundWerkzeugeundModellefürderenUntersuchung vorgeschlagen. Der Hauptanwendungsfall, der in dieser Arbeit untersucht wird, sind Fahrerlose Trans- portSysteme (FTS) (fortführend als Automated Guided Vehicles (AGVs)) für industri- elle Anwendungen. Diese FTS fahren durch die Produktionsanlage um Kisten, Waren oder Werkzeuge zu transportieren oder um Mitarbeitern zu assistieren. In den meisten Implementierungen müssen sie Daten mit einer Central Control Unit (CCU) und unter- einander austauschen. Die Vorhersage, ob eine bestimmte Kommunikationstechnologie für eine Anwendung geeignet ist, ist sehr anspruchsvoll, da sowohl Anwendungen als auch Anforderungen sehr heterogen sind. Die präsentierten Modelle und Simulationswerkzeuge ermöglichen die Simulation der komplexen Interaktion von mobilen Robotern und drahtlosen Kommunikationsnetz- werken. Das Ziel ist die Vorhersage der Eigenschaften einer vernetzten FTS-Flotte. Mit den vorgestellten Werkzeugen wurden verschiedene Ad-hoc-Netzwerklösungen für industrielle Anwendungen mit FTS implementiert, getestet und untersucht. Die- se Kommunikationssysteme übertragen zeitkritische und verzögerungstolerante Nach- richten. Zusätzlich wird eine Steuerungsmethode für die FTS vorgeschlagen, die die KommunikationoptimiertunddamiteinhergehenddieTransportleistungderFTS-Flotte erhöht. Dieses Werk führt also nicht nur neue Werkzeuge ein um die Entwicklung in- dustrieller Ad-hoc Systeme zu ermöglichen, sondern schlägt auch einige Systeme für die kritischsten Kommunikationsprobleme industrieller Anwendungen vor. KW - Industrie KW - Routing KW - Funknetz KW - Autonomer Roboter KW - Drahtloses vermaschtes Netzwerk KW - Industrie-Roboter KW - Kabellose Netzwerke KW - Simulation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-299238 ER -