@article{WagnerEikenHaubitzetal.2019, author = {Wagner, Johanna and Eiken, Barbara and Haubitz, Imme and Lichthardt, Sven and Matthes, Niels and L{\"o}b, Stefan and Klein, Ingo and Germer, Christoph-Thomas and Wiegering, Armin}, title = {Suprapubic bladder drainage and epidural catheters following abdominal surgery—a risk for urinary tract infections?}, series = {PLoS ONE}, volume = {14}, journal = {PLoS ONE}, number = {1}, doi = {10.1371/journal.pone.0209825}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-177731}, pages = {e0209825}, year = {2019}, abstract = {Background Epidural catheters are state of the art for postoperative analgesic in abdominal surgery. Due to neurolysis it can lead to postoperative urinary tract retention (POUR), which leads to prolonged bladder catheterization, which has an increased risk for urinary tract infections (UTI). Our aim was to identify the current perioperative management of urinary catheters and, second, to identify the optimal time of suprapubic bladder catheter removal in regard to the removal of the epidural catheter. Methods We sent a questionnaire to 102 German hospitals and analyzed the 83 received answers to evaluate the current handling of bladder drainage and epidural catheters. Then, we conducted a retrospective study including 501 patients, who received an epidural and suprapubic catheter after abdominal surgery at the University Hospital W{\"u}rzburg. We divided the patients into three groups according to the point in time of suprapubic bladder drainage removal in regard to the removal of the epidural catheter and analyzed the onset of a UTI. Results Our survey showed that in almost all hospitals (98.8\%), patients received an epidural catheter and a bladder drainage after abdominal surgery. The point in time of urinary catheter removal was equally distributed between before, simultaneously and after the removal of the epidural catheter (respectively: ~28-29\%). The retrospective study showed a catheter-associated UTI in 6.7\%. Women were affected significantly more often than men (10,7\% versus 2,5\%, p<0.001). There was a non-significant trend to more UTIs when the suprapubic catheter was removed after the epidural catheter (before: 5.7\%, after: 8.4\%). Conclusion The point in time of suprapubic bladder drainage removal in relation to the removal of the epidural catheter does not seem to correlate with the rate of UTIs. The current handling in Germany is inhomogeneous, so further studies to standardize treatment are recommended.}, language = {en} } @article{HovestadtThomasMitesseretal.2019, author = {Hovestadt, Thomas and Thomas, Jeremy A. and Mitesser, Oliver and Sch{\"o}nrogge, Karsten}, title = {Multiple host use and the dynamics of host-switching in host-parasite systems}, series = {Insect Conservation and Diversity}, volume = {12}, journal = {Insect Conservation and Diversity}, number = {6}, doi = {10.1111/icad.12374}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204747}, pages = {511-522}, year = {2019}, abstract = {The link between multi-host use and host switching in host-parasite interactions is a continuing area of debate. Lycaenid butterflies in the genus Maculinea, for example, exploit societies of different Myrmica ant species across their ranges, but there is only rare evidence that they simultaneously utilise multiple hosts at a local site, even where alternative hosts are present. We present a simple population-genetic model accounting for the proportion of two alternative hosts and the fitness of parasite genotypes on each host. In agreement with standard models, we conclude that simultaneous host use is possible whenever fitness of heterozygotes on alternative hosts is not too low. We specifically focus on host-shifting dynamics when the frequency of hosts changes. We find that (i) host shifting may proceed so rapidly that multiple host use is unlikely to be observed, (ii) back and forth transition in host use can exhibit a hysteresis loop, (iii) the parasites' host use may not be proportional to local host frequencies and be restricted to the rarer host under some conditions, and (iv) that a substantial decline in parasite abundance may typically precede a shift in host use. We conclude that focusing not just on possible equilibrium conditions but also considering the dynamics of host shifting in non-equilibrium situations may provide added insights into host-parasite systems.}, language = {en} } @phdthesis{Wilde2019, author = {Wilde, Sabrina}, title = {Einsatz von mechanistischen Biomarkern zur Charakterisierung und Bewertung von \(in\) \(vitro\) Genotoxinen}, doi = {10.25972/OPUS-18278}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182782}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Die verf{\"u}gbaren in vitro Genotoxizit{\"a}tstests weisen hinsichtlich ihrer Spezifit{\"a}t und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschr{\"a}nkungen auf. Um diese M{\"a}ngel zu {\"u}berwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Pr{\"u}fung auf Genotoxizit{\"a}t in der Arzneimittelentwicklung beitragen. 1. Etablierung und Bewertung einer neuen in vitro Genotoxizit{\"a}tsmethode (MultiFlow Methode) Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbr{\"u}che), phosphorylierten H3 (S10) (mitotische Zellen), nukle{\"a}ren Protein p53 (Genotoxizit{\"a}t) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und erg{\"a}nzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre F{\"a}higkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen" gegen{\"u}ber der „alten" Methode f{\"u}hrte zu einer verbesserten Sensitivit{\"a}t von 95\% gegen{\"u}ber 90\%, Spezifit{\"a}t von 90\% gegen{\"u}ber 72\% und einer MoA-Klassifizierungsrate von 85\% gegen{\"u}ber 45\% (Aneugen vs. Klastogen). 2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen Die Analyse 67 ausgew{\"a}hlter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen k{\"o}nnen. Die Kombination der h{\"o}chstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 erm{\"o}glichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivit{\"a}t, Spezifit{\"a}t und Pr{\"a}diktivit{\"a}t von 86\%, 83\% und 85\%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizit{\"a}t mit TP53I3, Klastogenit{\"a}t mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann. Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen f{\"u}r BubR1 (S670) und pH3 (S28) mit Aneugenit{\"a}t, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenit{\"a}t, p53 (K373) mit Genotoxizit{\"a}t und Nrf2 (S40) mit oxidativem Stress identifiziert. Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinver{\"a}nderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivit{\"a}ten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden k{\"o}nnen und daher eine bessere Risikobewertung von Wirkstoffkandidaten erm{\"o}glichen.}, subject = {Genotoxizit{\"a}t}, language = {de} } @article{ThiemHesbacherKneitzetal.2019, author = {Thiem, Alexander and Hesbacher, Sonja and Kneitz, Hermann and di Primio, Teresa and Heppt, Markus V. and Hermanns, Heike M. and Goebeler, Matthias and Meierjohann, Svenja and Houben, Roland and Schrama, David}, title = {IFN-gamma-induced PD-L1 expression in melanoma depends on p53 expression}, series = {Journal of Experimental \& Clinical Cancer Research}, volume = {38}, journal = {Journal of Experimental \& Clinical Cancer Research}, doi = {10.1186/s13046-019-1403-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201016}, pages = {397}, year = {2019}, abstract = {Background Immune checkpoint inhibition and in particular anti-PD-1 immunotherapy have revolutionized the treatment of advanced melanoma. In this regard, higher tumoral PD-L1 protein (gene name: CD274) expression is associated with better clinical response and increased survival to anti-PD-1 therapy. Moreover, there is increasing evidence that tumor suppressor proteins are involved in immune regulation and are capable of modulating the expression of immune checkpoint proteins. Here, we determined the role of p53 protein (gene name: TP53) in the regulation of PD-L1 expression in melanoma. Methods We analyzed publicly available mRNA and protein expression data from the cancer genome/proteome atlas and performed immunohistochemistry on tumors with known TP53 status. Constitutive and IFN-ɣ-induced PD-L1 expression upon p53 knockdown in wildtype, TP53-mutated or JAK2-overexpressing melanoma cells or in cells, in which p53 was rendered transcriptionally inactive by CRISPR/Cas9, was determined by immunoblot or flow cytometry. Similarly, PD-L1 expression was investigated after overexpression of a transcriptionally-impaired p53 (L22Q, W23S) in TP53-wt or a TP53-knockout melanoma cell line. Immunoblot was applied to analyze the IFN-ɣ signaling pathway. Results For TP53-mutated tumors, an increased CD274 mRNA expression and a higher frequency of PD-L1 positivity was observed. Interestingly, positive correlations of IFNG mRNA and PD-L1 protein in both TP53-wt and -mutated samples and of p53 and PD-L1 protein suggest a non-transcriptional mode of action of p53. Indeed, cell line experiments revealed a diminished IFN-ɣ-induced PD-L1 expression upon p53 knockdown in both wildtype and TP53-mutated melanoma cells, which was not the case when p53 wildtype protein was rendered transcriptionally inactive or by ectopic expression of p53\(^{L22Q,W23S}\), a transcriptionally-impaired variant, in TP53-wt cells. Accordingly, expression of p53\(^{L22Q,W23S}\) in a TP53-knockout melanoma cell line boosted IFN-ɣ-induced PD-L1 expression. The impaired PD-L1-inducibility after p53 knockdown was associated with a reduced JAK2 expression in the cells and was almost abrogated by JAK2 overexpression. Conclusions While having only a small impact on basal PD-L1 expression, both wildtype and mutated p53 play an important positive role for IFN-ɣ-induced PD-L1 expression in melanoma cells by supporting JAK2 expression. Future studies should address, whether p53 expression levels might influence response to anti-PD-1 immunotherapy.}, language = {en} } @article{VeyKapsnerFuchsetal.2019, author = {Vey, Johannes and Kapsner, Lorenz A. and Fuchs, Maximilian and Unberath, Philipp and Veronesi, Giulia and Kunz, Meik}, title = {A toolbox for functional analysis and the systematic identification of diagnostic and prognostic gene expression signatures combining meta-analysis and machine learning}, series = {Cancers}, volume = {11}, journal = {Cancers}, number = {10}, issn = {2072-6694}, doi = {10.3390/cancers11101606}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193240}, year = {2019}, abstract = {The identification of biomarker signatures is important for cancer diagnosis and prognosis. However, the detection of clinical reliable signatures is influenced by limited data availability, which may restrict statistical power. Moreover, methods for integration of large sample cohorts and signature identification are limited. We present a step-by-step computational protocol for functional gene expression analysis and the identification of diagnostic and prognostic signatures by combining meta-analysis with machine learning and survival analysis. The novelty of the toolbox lies in its all-in-one functionality, generic design, and modularity. It is exemplified for lung cancer, including a comprehensive evaluation using different validation strategies. However, the protocol is not restricted to specific disease types and can therefore be used by a broad community. The accompanying R package vignette runs in ~1 h and describes the workflow in detail for use by researchers with limited bioinformatics training.}, language = {en} } @article{LyutovaSelchoPfeufferetal.2019, author = {Lyutova, Radostina and Selcho, Mareike and Pfeuffer, Maximilian and Segebarth, Dennis and Habenstein, Jens and Rohwedder, Astrid and Frantzmann, Felix and Wegener, Christian and Thum, Andreas S. and Pauls, Dennis}, title = {Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-11092-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202161}, pages = {3097}, year = {2019}, abstract = {Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells we provide evidence that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.}, language = {en} } @phdthesis{Breitenbach2019, author = {Breitenbach, Tim}, title = {A mathematical optimal control based approach to pharmacological modulation with regulatory networks and external stimuli}, doi = {10.25972/OPUS-17436}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174368}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {In this work models for molecular networks consisting of ordinary differential equations are extended by terms that include the interaction of the corresponding molecular network with the environment that the molecular network is embedded in. These terms model the effects of the external stimuli on the molecular network. The usability of this extension is demonstrated with a model of a circadian clock that is extended with certain terms and reproduces data from several experiments at the same time. Once the model including external stimuli is set up, a framework is developed in order to calculate external stimuli that have a predefined desired effect on the molecular network. For this purpose the task of finding appropriate external stimuli is formulated as a mathematical optimal control problem for which in order to solve it a lot of mathematical methods are available. Several methods are discussed and worked out in order to calculate a solution for the corresponding optimal control problem. The application of the framework to find pharmacological intervention points or effective drug combinations is pointed out and discussed. Furthermore the framework is related to existing network analysis tools and their combination for network analysis in order to find dedicated external stimuli is discussed. The total framework is verified with biological examples by comparing the calculated results with data from literature. For this purpose platelet aggregation is investigated based on a corresponding gene regulatory network and associated receptors are detected. Furthermore a transition from one to another type of T-helper cell is analyzed in a tumor setting where missing agents are calculated to induce the corresponding switch in vitro. Next a gene regulatory network of a myocardiocyte is investigated where it is shown how the presented framework can be used to compare different treatment strategies with respect to their beneficial effects and side effects quantitatively. Moreover a constitutively activated signaling pathway, which thus causes maleficent effects, is modeled and intervention points with corresponding treatment strategies are determined that steer the gene regulatory network from a pathological expression pattern to physiological one again.}, subject = {Bioinformatik}, language = {en} } @article{MatosMachadoSchartletal.2019, author = {Matos, Isa and Machado, Miguel P. and Schartl, Manfred and Coelho, Maria Manuela}, title = {Allele-specific expression variation at different ploidy levels in Squalius alburnoides}, series = {Scientific Reports}, volume = {9}, journal = {Scientific Reports}, doi = {10.1038/s41598-019-40210-8}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200910}, pages = {3688}, year = {2019}, abstract = {Allopolyploid plants are long known to be subject to a homoeolog expression bias of varying degree. The same phenomenon was only much later suspected to occur also in animals based on studies of single selected genes in an allopolyploid vertebrate, the Iberian fish Squalius alburnoides. Consequently, this species became a good model for understanding the evolution of gene expression regulation in polyploid vertebrates. Here, we analyzed for the first time genome-wide allele-specific expression data from diploid and triploid hybrids of S. alburnoides and compared homoeolog expression profiles of adult livers and of juveniles. Co-expression of alleles from both parental genomic types was observed for the majority of genes, but with marked homoeolog expression bias, suggesting homoeolog specific reshaping of expression level patterns in hybrids. Complete silencing of one allele was also observed irrespective of ploidy level, but not transcriptome wide as previously speculated. Instead, it was found only in a restricted number of genes, particularly ones with functions related to mitochondria and ribosomes. This leads us to hypothesize that allelic silencing may be a way to overcome intergenomic gene expression interaction conflicts, and that homoeolog expression bias may be an important mechanism in the achievement of sustainable genomic interactions, mandatory to the success of allopolyploid systems, as in S. alburnoides.}, language = {en} } @phdthesis{Memmel2019, author = {Memmel, Simon}, title = {Automatisierte Algorithmen zur Analyse der Migration und der strahleninduzierten DNA-Sch{\"a}den humaner Glioblastomzellen nach kombinierter PI3K/mTOR/Hsp90-Inhibierung}, doi = {10.25972/OPUS-18571}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185710}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Das hohe invasive Potential und die starke Resistenz gegen Radio-/Chemotherapie von Glioblastoma multiforme (GBM) Zellen machen sie zu dem t{\"o}dlichsten Tumor ihrer Art. Es ist deshalb von großem Interesse die Grundlagen, welche der Migrationsf{\"a}higkeit und DNA Reparatur zu Grunde liegen, besser zu verstehen. Im ersten Teil dieser Arbeit wurden zwei Algorithmen zur automatischen Analyse der Migration in der Einzelzellverfolgung und im Wundheilungsassay modifiziert. Die Auswertung der Daten konnte automatisch und somit schnell, effektiv und mit geringerem Arbeitsaufwand durchgef{\"u}hrt werden. Mit Hilfe dieser automatischen Algorithmen wurde die Migrationsf{\"a}higkeit von zwei GBM-Zelllinien (DK-MG und SNB19) untersucht. Zus{\"a}tzlich wurde die konfokale Laserscanning- sowie die hochaufl{\"o}sende dSTORM-Fluoreszenzmikroskopie verwendet um die, der Zellbewegung zu Grunde liegende, Struktur des F Aktin und der fokalen Adh{\"a}sionskinase (FAK) aufzul{\"o}sen und darzustellen. Unter Anwendung dieser genannten Methoden sind die Effekte des dualen PI3K/mTOR Inhibitors PI-103 alleine und in Kombination mit dem Hsp90 Inhibitor NVP AUY922 mit und ohne Bestrahlung auf die Bewegung untersucht worden. Es konnte festgestellt werden, dass sich beide Zelllinien deutlich in ihrem migratorischem Potential in vitro unterscheiden und zudem auch markante Unterschiede in ihrer Morphologie aufweisen. Die weniger invasiven DK MG-Zellen besitzen eine polarisierte Zellstruktur, wohingegen SNB19-Zellen sich durch multipolare ungerichtete Bewegung auszeichneten. Zudem wurde die Migration, durch PI3K/mTOR Inhibition mit PI-103 bei den DK-MG-Zellen (p53 wt, PTEN wt), sehr effektiv unterdr{\"u}ckt. Wohingegen sich die SNB19-Zellen (p53 mut, PTEN mut) resistent gegen diesen Inhibitor zeigten. Hsp90 Inhibition offenbarte in beiden Zelllinien einen starken inhibitorischen Effekt auf die Migration der Zellen sowie die Reorganisierung des F Aktinskelettes. In der zweiten H{\"a}lfte dieser Arbeit wurde ein Augenmerk auf die DNA-DSB-Reparatur der GBM Zellen nach ionisierender Strahlung gelegt. Zun{\"a}chst wurde eine automatische Analysesoftware „FocAn-3D" entwickelt, mit dessen Hilfe die DNA Doppelstrangbruchreparaturkinetik untersucht werden sollte. Diese Software erm{\"o}glicht es die gesamten Zellkerne mit ihren γH2AX-Foci in 3D-cLSM-Aufnahmen zu untersuchen. Es konnte somit eine Verbesserung der Genauigkeit in der Ausz{\"a}hlung der γH2AX-Foci erreicht werden, welche 2D beschr{\"a}nkter Software verwehrt bleibt. Mit FocAn-3D konnte der gesamte Verlauf der Induktions- und Abbauphase der γH2AX-Foci in DK MG- und SNB19-Zellen mit einem mathematischen Modell ausgewertet und dargestellt werden. Des Weiteren wurde die Nanometerstruktur von γH2AX- und pDNA-PKcs-Foci mittels hochaufl{\"o}sender dSTORM-Mikroskopie untersucht. Konventionelle Mikroskopiemethoden, begrenzt durch das Beugungslimit und einer Aufl{\"o}sung von ~200 nm, konnten die Nanometerstruktur (<100 nm) der Reparaturfoci bisher nicht darstellen. Mit Hilfe der beugungsunbegrenzten dSTORM-Mikroskopie war es m{\"o}glich in DK MG- und SNB19-Zellen die Nanometerstruktur genannten Reparaturproteine in den Foci mit einer Aufl{\"o}sung von bis zu ~20 nm darzustellen. γH2AX-Foci zeigten sich als eine Verteilung aus einzelnen Untereinheiten („Nanofoci") mit einem Durchmesser von ~45 nm. Dies l{\"a}sst die Vermutung zu, dass es sich hier um die elementare Substruktur der Foci und somit der γH2AX enthaltenen Nukleosome handelt. DNA-PK-Foci wiesen hingegen eine diffusere Verteilung auf. Die in dieser Arbeit ermittelten Unterschiede im Migrationsverhalten der Zellen rechtfertigen eine weitere pr{\"a}klinische Untersuchung der verwendeten Inhibitoren als potentielle Zelltherapeutika f{\"u}r die Behandlung von GBM. Zudem konnte sich dSTORM als machtvolles Hilfsmittel, sowohl zur Analyse der Migration zugrundeliegenden Zytoskelettstruktur und der Effekte der Hsp90 Inhibierung, als auch, der Nanostruktur der DNA-DSB-Reparaturfoci herausstellen. Es ist anzunehmen, dass beugungsunbegrenzte Mikroskopiemethoden sich als bedeutende Werkzeuge in der medizinischen und biologischen Erforschung der DNA-Reparaturmechanismen herausstellen werden. Das in dieser Arbeit entwickelte ImageJ Plugin „FocAn-3D" bewies sich ebenfalls als ein vielversprechendes Werkzeug f{\"u}r die Analyse der Reparaturkinetik. Mit Hilfe von „FocAn-3D" sollte es somit m{\"o}glich sein u.a. den Einfluss gezielter Inhibition auf den zeitlichen Verlauf der Induktion und des Abbaus der DNA-Reparaturmaschinerie genauer zu studieren.}, subject = {Glioblastom}, language = {de} } @phdthesis{Kaymak2019, author = {Kaymak, Irem}, title = {Identification of metabolic liabilities in 3D models of cancer}, doi = {10.25972/OPUS-18154}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181544}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.}, subject = {Tumor}, language = {en} } @phdthesis{KaltdorfgebSchuch2019, author = {Kaltdorf [geb. Schuch], Kristin Verena}, title = {Mikroskopie, Bildverarbeitung und Automatisierung der Analyse von Vesikeln in \(C.\) \(elegans\) und anderen biologischen Strukturen}, doi = {10.25972/OPUS-16062}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160621}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Thema dieser Thesis ist die Analyse sekretorischer Vesikelpools auf Ultrastrukturebene in unterschiedlichen biologischen Systemen. Der erste und zweite Teil dieser Arbeit fokussiert sich auf die Analyse synaptischer Vesikelpools in neuromuskul{\"a}ren Endplatten (NME) im Modellorganismus Caenorhabditis elegans. Dazu wurde Hochdruckgefrierung und Gefriersubstitution angewandt, um eine unverz{\"u}gliche Immobilisation der Nematoden und somit eine Fixierung im nahezu nativen Zustand zu gew{\"a}hrleisten. Anschließend wurden dreidimensionale Aufnahmen der NME mittels Elektronentomographie erstellt. Im ersten Teil dieser Arbeit wurden junge adulte, wildtypische C. elegans Hermaphroditen mit Septin-Mutanten verglichen. Um eine umfassende Analyse mit hoher Stichprobenzahl zu erm{\"o}glichen und eine automatisierte L{\"o}sung f{\"u}r {\"a}hnliche Untersuchungen von Vesikelpools bereit zu stellen wurde eine Software namens 3D ART VeSElecT zur automatisierten Vesikelpoolanalyse entwickelt. Die Software besteht aus zwei Makros f{\"u}r ImageJ, eines f{\"u}r die Registrierung der Vesikel und eines zur Charakterisierung. Diese Trennung in zwei separate Schritte erm{\"o}glicht einen manuellen Verbesserungsschritt zum Entfernen falsch positiver Vesikel. Durch einen Vergleich mit manuell ausgewerteten Daten neuromuskul{\"a}rer Endplatten von larvalen Stadien des Modellorganismus Zebrafisch (Danio rerio) konnte erfolgreich die Funktionalit{\"a}t der Software bewiesen werden. Die Analyse der neuromuskul{\"a}ren Endplatten in C. elegans ergab kleinere synaptische Vesikel und dichtere Vesikelpools in den Septin-Mutanten verglichen mit Wildtypen. Im zweiten Teil der Arbeit wurden neuromuskul{\"a}rer Endplatten junger adulter C. elegans Hermaphroditen mit Dauerlarven verglichen. Das Dauerlarvenstadium ist ein spezielles Stadium, welches durch widrige Umweltbedingungen induziert wird und in dem C. elegans {\"u}ber mehrere Monate ohne Nahrungsaufnahme {\"u}berleben kann. Da hier der Vergleich der Abundanz zweier Vesikelarten, der „clear-core"-Vesikel (CCV) und der „dense-core"-Vesikel (DCV), im Fokus stand wurde eine Erweiterung von 3D ART VeSElecT entwickelt, die einen „Machine-Learning"-Algorithmus zur automatisierten Klassifikation der Vesikel integriert. Durch die Analyse konnten kleinere Vesikel, eine erh{\"o}hte Anzahl von „dense-core"-Vesikeln, sowie eine ver{\"a}nderte Lokalisation der DCV in Dauerlarven festgestellt werden. Im dritten Teil dieser Arbeit wurde untersucht ob die f{\"u}r synaptische Vesikelpools konzipierte Software auch zur Analyse sekretorischer Vesikel in Thrombozyten geeignet ist. Dazu wurden zweidimensionale und dreidimensionale Aufnahmen am Transmissionselektronenmikroskop erstellt und verglichen. Die Untersuchung ergab, dass hierf{\"u}r eine neue Methodik entwickelt werden muss, die zwar auf den vorherigen Arbeiten prinzipiell aufbauen kann, aber den besonderen Herausforderungen der Bilderkennung sekretorischer Vesikel aus Thrombozyten gerecht werden muss.}, subject = {Mikroskopie}, language = {de} } @article{PaulsHamaratTrufasuetal.2019, author = {Pauls, Dennis and Hamarat, Yasmin and Trufasu, Luisa and Schendzielorz, Tim M. and Gramlich, Gertrud and Kahnt, J{\"o}rg and Vanselow, Jens and Schlosser, Andreas and Wegener, Christian}, title = {Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate}, series = {European Journal of Neuroscience}, volume = {50}, journal = {European Journal of Neuroscience}, number = {9}, doi = {10.1111/ejn.14516}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204863}, pages = {3502-3519}, year = {2019}, abstract = {Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE ), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD ) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila . We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD -encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila . dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.}, language = {en} } @article{DechaudVolffSchartletal.2019, author = {Dechaud, Corentin and Volff, Jean-Nicolas and Schartl, Manfred and Naville, Magali}, title = {Sex and the TEs: transposable elements in sexual development and function in animals}, series = {Mobile DNA}, volume = {10}, journal = {Mobile DNA}, doi = {10.1186/s13100-019-0185-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202510}, pages = {42}, year = {2019}, abstract = {Transposable elements are endogenous DNA sequences able to integrate into and multiply within genomes. They constitute a major source of genetic innovations, as they can not only rearrange genomes but also spread ready-to-use regulatory sequences able to modify host gene expression, and even can give birth to new host genes. As their evolutionary success depends on their vertical transmission, transposable elements are intrinsically linked to reproduction. In organisms with sexual reproduction, this implies that transposable elements have to manifest their transpositional activity in germ cells or their progenitors. The control of sexual development and function can be very versatile, and several studies have demonstrated the implication of transposable elements in the evolution of sex. In this review, we report the functional and evolutionary relationships between transposable elements and sexual reproduction in animals. In particular, we highlight how transposable elements can influence expression of sexual development genes, and how, reciprocally, they are tightly controlled in gonads. We also review how transposable elements contribute to the organization, expression and evolution of sexual development genes and sex chromosomes. This underscores the intricate co-evolution between host functions and transposable elements, which regularly shift from a parasitic to a domesticated status useful to the host.}, language = {en} } @article{SbieraKunzWeigandetal.2019, author = {Sbiera, Silviu and Kunz, Meik and Weigand, Isabel and Deutschbein, Timo and Dandekar, Thomas and Fassnacht, Martin}, title = {The new genetic landscape of Cushing's disease: deubiquitinases in the spotlight}, series = {Cancers}, volume = {11}, journal = {Cancers}, number = {11}, issn = {2072-6694}, doi = {10.3390/cancers11111761}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-193194}, pages = {1761}, year = {2019}, abstract = {Cushing's disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD's genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5\%) and USP48 (13.3\%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5\% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5\% and 7\%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways.}, language = {en} } @article{OttoSchmidtKastneretal.2019, author = {Otto, C. and Schmidt, S. and Kastner, C. and Denk, S. and Kettler, J. and M{\"u}ller, N. and Germer, C.T. and Wolf, E. and Gallant, P. and Wiegering, A.}, title = {Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells}, series = {Neoplasia}, volume = {21}, journal = {Neoplasia}, number = {11}, doi = {10.1016/j.neo.2019.10.003}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202451}, pages = {1110-1120}, year = {2019}, abstract = {The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.}, language = {en} } @article{DrescherKleinSchmittetal.2019, author = {Drescher, Nora and Klein, Alexandra-Maria and Schmitt, Thomas and Leonhardt, Sara Diana}, title = {A clue on bee glue: New insight into the sources and factors driving resin intake in honeybees (Apis mellifera)}, series = {PLoS ONE}, volume = {14}, journal = {PLoS ONE}, number = {2}, doi = {10.1371/journal.pone.0210594}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200935}, pages = {e0210594}, year = {2019}, abstract = {Honeybees (Apis mellifera) are threatened by numerous pathogens and parasites. To prevent infections they apply cooperative behavioral defenses, such as allo-grooming and hygiene, or they use antimicrobial plant resin. Resin is a chemically complex and highly variable mixture of many bioactive compounds. Bees collect the sticky material from different plant species and use it for nest construction and protection. Despite its importance for colony health, comparatively little is known about the precise origins and variability in resin spectra collected by honeybees. To identify the botanical resin sources of A. mellifera in Western Europe we chemically compared resin loads of individual foragers and tree resins. We further examined the resin intake of 25 colonies from five different apiaries to assess the effect of location on variation in the spectra of collected resin. Across all colonies and apiaries, seven distinct resin types were categorized according to their color and chemical composition. Matches between bee-collected resin and tree resin indicated that bees used poplar (Populus balsamifera, P. x canadensis), birch (Betula alba), horse chestnut (Aesculus hippocastanum) and coniferous trees (either Picea abies or Pinus sylvestris) as resin sources. Our data reveal that honeybees collect a comparatively broad and variable spectrum of resin sources, thus assuring protection against a variety of antagonists sensitive to different resins and/or compounds. We further unravel distinct preferences for specific resins and resin chemotypes, indicating that honeybees selectively search for bioactive resin compounds.}, language = {en} } @article{LechermeierZimmerLueffeetal.2019, author = {Lechermeier, Carina G. and Zimmer, Frederic and L{\"u}ffe, Teresa M. and Lesch, Klaus-Peter and Romanos, Marcel and Lillesaar, Christina and Drepper, Carsten}, title = {Transcript analysis of zebrafish GLUT3 genes, slc2a3a and slc2a3b, define overlapping as well as distinct expression domains in the zebrafish (Danio rerio) central nervous system}, series = {Frontiers in Molecular Neuroscience}, volume = {12}, journal = {Frontiers in Molecular Neuroscience}, number = {199}, doi = {10.3389/fnmol.2019.00199}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201797}, year = {2019}, abstract = {The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington's and Alzheimer's diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain disorders.}, language = {en} } @article{CecilGentschevAdelfingeretal.2019, author = {Cecil, Alexander and Gentschev, Ivaylo and Adelfinger, Marion and Dandekar, Thomas and Szalay, Aladar A.}, title = {Vaccinia virus injected human tumors: oncolytic virus efficiency predicted by antigen profiling analysis fitted boolean models}, series = {Bioengineered}, volume = {10}, journal = {Bioengineered}, number = {1}, doi = {10.1080/21655979.2019.1622220}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200507}, pages = {190-196}, year = {2019}, abstract = {Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients.}, language = {en} } @article{DerakhshaniKurzJaptoketal.2019, author = {Derakhshani, Shaghayegh and Kurz, Andreas and Japtok, Lukasz and Schumacher, Fabian and Pilgram, Lisa and Steinke, Maria and Kleuser, Burkhard and Sauer, Markus and Schneider-Schaulies, Sibylle and Avota, Elita}, title = {Measles virus infection fosters dendritic cell motility in a 3D environment to enhance transmission to target cells in the respiratory epithelium}, series = {Frontiers in Immunology}, volume = {10}, journal = {Frontiers in Immunology}, number = {1294}, doi = {10.3389/fimmu.2019.01294}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201818}, year = {2019}, abstract = {Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit.}, language = {en} } @article{PaponovDindas Krol etal.2019, author = {Paponov, Ivan A. and Dindas , Julian and Kr{\´o}l , Elżbieta and Friz, Tatyana and Budnyk, Vadym and Teale, William and Paponov, Martina and Hedrich , Rainer and Palme, Klaus}, title = {Auxin-Induced plasma membrane depolarization is regulated by Auxin transport and not by AUXIN BINDING PROTEIN1}, series = {Frontiers in Plant Science}, volume = {9}, journal = {Frontiers in Plant Science}, issn = {1664-462X}, doi = {10.3389/fpls.2018.01953}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195914}, year = {2019}, abstract = {Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling.}, language = {en} }