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Spemann Graduate School of Biology and Medicine

Works: 15 works in 15 publications in 1 language and 36 library holdings
Roles: Contributor
Publication Timeline
Most widely held works by Spemann Graduate School of Biology and Medicine
Proteomic analysis of lung metastases in a murine breast cancer model reveals divergent influence of CTSB and CTSL overexpression by Florian Christoph Sigloch( )

1 edition published in 2017 in English and held by 3 WorldCat member libraries worldwide

Abstract: Studies in the MMTV-PyMT (PyMT) breast cancer mouse model have shown a strong influence of the lysosomal cysteine cathepsins B or L on lung metastasis formation. Transgenic expression of human CTSB (tgCTSB) or CTSL (tgCTSL) both led to similar metastatic phenotypes with increased metastatic burden in the PyMT mice. However, recent studies in other tumor models proved marked differences in effects of either cathepsin on the proteome composition. We sought to analyze and compare proteome changes in the metastatic proteome of PyMT mice expressing either tgCTSB or tgCTSL to evaluate similarities and differences in those models.<br><br>Performing an explorative, quantitative proteome comparison based on LC-MS/MS, we identified up to 3,000 proteins from murine lung metastases in three independent biological replicates per genotype. In both cases, when compared to wild-type (WT) mice, we noticed a pronounced impact of transgene cathepsin expression on the metastasis proteome. Highlights include increased moesin, integrin beta 1 and vinexin levels in the tgCTSB dataset and increased saposin and granulin levels in the tgCTSL dataset. Importantly, non-supervised hierarchical clustering clearly separated tgCTSB vs. tgCTSL induced proteome changes.<br><br>In summary, tgCTSB and tgCTSL both display a strong and distinct impact on proteome composition of lung macrometastases in the PyMT model. Our observations suggest that they impact malignant behavior in distinct ways, thus further emphasizing interest into their tumor-contextual functionality
Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization by Melanie Christine Föll( )

1 edition published in 2018 in English and held by 3 WorldCat member libraries worldwide

Abstract: Background<br>Proteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filter aided sample preparation (FASP) protocol is lacking. Furthermore, it is unknown how common histological stainings influence the outcome of the DTR protocol.<br><br>Methods<br>Four single consecutive murine kidney tissue specimens were prepared with the DTR approach or with the FASP protocol using both 10 and 30 k filter devices and analyzed by label-free, quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We compared the different protocols in terms of proteome coverage, relative label-free quantitation, missed cleavages, physicochemical properties and gene ontology term annotations of the proteins. Additionally, we probed compatibility of the DTR protocol for the analysis of common used histological stainings, namely hematoxylin & eosin (H&E), hematoxylin and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissue specimens per condition.<br><br>Results<br>On average, the DTR protocol identified 1841 ± 22 proteins in a single, non-fractionated LC-MS/MS analysis, whereas these numbers were 1857 ± 120 and 1970 ± 28 proteins for the FASP 10 and 30 k protocol. The DTR protocol showed 15% more missed cleavages, which did not adversely affect quantitation and intersample comparability. Hematoxylin or hemalaun staining did not adversely impact the performance of the DTR protocol. A minor perturbation was observed for H&E staining, decreasing overall protein identification by 13%.<br><br>Conclusions<br>In essence, the DTR protocol can keep up with the FASP protocol in terms of qualitative and quantitative reproducibility and performed almost as well in terms of proteome coverage and missed cleavages. We highlight the suitability of the DTR protocol as a viable and straightforward alternative to the FASP protocol for proteomics-based clinical research
Microglia-mediated neuroinflammation and neurotrophic factor-induced protection in the MPTP mouse model of parkinson's disease-lessons from transgenic mice by Venissa Machado( )

1 edition published in 2016 in English and held by 3 WorldCat member libraries worldwide

Abstract: Parkinson's disease (PD) is a neurodegenerative disease characterised by histopathological and biochemical manifestations such as loss of midbrain dopaminergic (DA) neurons and decrease in dopamine levels accompanied by a concomitant neuroinflammatory response in the affected brain regions. Over the past decades, the use of toxin-based animal models has been crucial to elucidate disease pathophysiology, and to develop therapeutic approaches aimed to alleviate its motor symptoms. Analyses of transgenic mice deficient for cytokines, chemokine as well as neurotrophic factors and their respective receptors in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have broadened the current knowledge of neuroinflammation and neurotrophic support. Here, we provide a comprehensive review that summarises the contribution of microglia-mediated neuroinflammation in MPTP-induced neurodegeneration. Moreover, we highlight the contribution of neurotrophic factors as endogenous and/or exogenous molecules to slow the progression of midbrain dopaminergic (mDA) neurons and further discuss the potential of combined therapeutic approaches employing neuroinflammation modifying agents and neurotrophic factors
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1 edition published in 2015 in English and held by 3 WorldCat member libraries worldwide

Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation by Fraua Christina Ferlemann( )

1 edition published in 2017 in English and held by 3 WorldCat member libraries worldwide

Abstract: Neuroblastoma is the most common extra-cranial solid tumor in children. Its broad spectrum of clinical outcomes reflects the underlying inherent cellular heterogeneity. As current treatments often do not lead to tumor eradication, there is a need to better define therapy-resistant neuroblastoma and to identify new modulatory molecules. To this end, we performed the first comprehensive flow cytometric characterization of surface molecule expression in neuroblastoma cell lines. Exploiting an established clustering algorithm (SPADE) for unbiased visualization of cellular subsets, we conducted a multiwell screen for small molecule modulators of neuroblastoma phenotype. In addition to SH-SY5Y cells, the SH-EP, BE(2)-M17 and Kelly lines were included in follow-up analysis as in vitro models of neuroblastoma. A combinatorial detection of glycoprotein epitopes (CD15, CD24, CD44, CD57, TrkA) and the chemokine receptor CXCR4 (CD184) enabled the quantitative identification of SPADE-defined clusters differentially responding to small molecules. Exposure to bone morphogenetic protein (BMP)-4 was found to enhance a TrkAhigh/CD15−/CD184− neuroblastoma cellular subset, accompanied by a reduction in doublecortin-positive neuroblasts and of NMYC protein expression in SH-SY5Y cells. Beyond yielding novel marker candidates for studying neuroblastoma pathology, our approach may provide tools for improved pharmacological screens towards developing novel avenues of neuroblastoma diagnosis and treatment
The feat of packaging eight unique genome segments by Sebastian Giese( )

1 edition published in 2016 in English and held by 3 WorldCat member libraries worldwide

Abstract: Influenza A viruses (IAVs) harbor a segmented RNA genome that is organized into eight distinct viral ribonucleoprotein (vRNP) complexes. Although a segmented genome may be a major advantage to adapt to new host environments, it comes at the cost of a highly sophisticated genome packaging mechanism. Newly synthesized vRNPs conquer the cellular endosomal recycling machinery to access the viral budding site at the plasma membrane. Genome packaging sequences unique to each RNA genome segment are thought to be key determinants ensuring the assembly and incorporation of eight distinct vRNPs into progeny viral particles. Recent studies using advanced fluorescence microscopy techniques suggest the formation of vRNP sub-bundles (comprising less than eight vRNPs) during their transport on recycling endosomes. The formation of such sub-bundles might be required for efficient packaging of a bundle of eight different genomes segments at the budding site, further highlighting the complexity of IAV genome packaging
Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis by Gizem Turaç( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

Axitinib and sorafenib are potent in tyrosine kinase inhibitor resistant chronic myeloid leukemia cells by Sebastian Halbach( )

1 edition published in 2016 in English and held by 2 WorldCat member libraries worldwide

Acetylation of human TCF4 (TCF7L2) proteins attenuates inhibition by the HBP1 repressor and induces a conformational change in the TCF4::DNA complex by Susanne Claudia Elfert( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

Abstract: The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As<br>nuclear effectors of Wnt/ß-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us to investigate whether vertebrate TCF/LEF proteins are subject to acetylation. Through co-expression with p300 and CBP and subsequent analyses using mass spectrometry and immunodetection with anti-acetyl-lysine antibodies we show that TCF4 can be acetylated at lysine K150 by CBP. K150<br>acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of ß-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K150 acetylation we substituted K150 with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/ß-catenin-responsive promoter regions did not indicate a general role of K150<br>acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K150<br>R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K150 using a bacterial expression system or amino acid substitutions at K150 alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K150<br>acetylation leads to a conformational change that may also represent the mechanism whereby acetylated TCF4E acquires resistance against HBP1. In summary, TCF4 not only recruits acetyltransferases but is also a substrate for these enzymes. The fact that acetylation affects only a subset of TCF4 splice variants and is mediated preferentially by CBP suggests that the conditional acetylation of TCF4E is a novel regulatory mechanism that diversifies the transcriptional output of Wnt/ß-catenin signaling in response to changing intracellular signaling milieus
Alterations of Gab2 signalling complexes in imatinib and dasatinib treated chronic myeloid leukaemia cells by Sebastian Halbach( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

Abstract: Background:<br>The Gab2 docking protein acts as an important signal amplifier downstream of various growth factor receptors and Bcr-Abl, the driver of chronic myeloid leukaemia (CML). Despite the success of Bcr-Abl tyrosine kinase inhibitors (TKI) in the therapy of CML, TKI-resistance remains an unsolved problem in the clinic. We have recently shown that Gab2 signalling counteracts the efficacy of four distinct Bcr-Abl inhibitors. In the course of that project, we noticed that two clinically relevant drugs, imatinib and dasatinib, provoke distinct alterations in the electrophoretic mobility of Gab2, its signalling output and protein interactions. As the signalling potential of the docking protein is highly modulated by its phosphorylation status, we set out to obtain more insights into the impact of TKIs on Gab2 phosphorylation.<br>Findings:<br>Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS), we show now that imatinib and dasatinib provoke distinct effects on the phosphorylation status and interactome of Gab2. This study identifies several new phosphorylation sites on Gab2 and confirms many sites previously known from other experimental systems. At equimolar concentrations, dasatinib is more effective in preventing Gab2 tyrosine and serine/threonine phosphorylation than imatinib. It also affects the phosphorylation status of more residues than imatinib. In addition, we also identify novel components of the Gab2 signalling complex, such as casein kinases, stathmins and PIP1 as well as known interaction partners whose association with Gab2 is disrupted by imatinib and/or dasatinib.<br>Conclusions:<br>By using MS-based proteomics, we have identified new and confirmed known phosphorylation sites and interaction partners of Gab2, which may play an important role in the regulation of this docking protein. Given the growing importance of Gab2 in several tumour entities we expect that our results will help to understand the complex regulation of Gab2 and how this docking protein can contribute to malignancy
Non-directional radial intercalation dominates deep cell behavior during zebrafish epiboly by Robert Bensch( )

1 edition published in 2013 in English and held by 2 WorldCat member libraries worldwide

Abstract: Epiboly is the first coordinated cell movement in most vertebrates and marks the onset of gastrulation. During zebrafish epiboly, enveloping layer (EVL) and deep cells spread over the vegetal yolk mass with a concomitant thinning of the deep cell layer. A prevailing model suggests that deep cell radial intercalations directed towards the EVL would drive deep cell epiboly. To test this model, we have globally recorded 3D cell trajectories for zebrafish blastomeres between sphere and 50% epiboly stages, and developed an image analysis framework to determine intercalation events, intercalation directionality, and migration speed for cells at specific positions within the embryo. This framework uses Voronoi diagrams to compute cell-to-cell contact areas, defines a feature-based spatio-temporal model for intercalation events and fits an anatomical coordinate system to the recorded datasets. We further investigate whether epiboly defects in MZspg mutant embryos devoid of Pou5f1/Oct4 may be caused by changes in intercalation behavior. In wild-type and mutant embryos, intercalations orthogonal to the EVL occur with no directional bias towards or away from the EVL, suggesting that there are no directional cues that would direct intercalations towards the EVL. Further, we find that intercalation direction is independent of the previous intercalation history of individual deep cells, arguing against cues that would program specific intrinsic directed migration behaviors. Our data support a dynamic model in which deep cells during epiboly migrate into space opening between the EVL and the yolk syncytial layer. Genetic programs determining cell motility may control deep cell dynamic behavior and epiboly progress
A novel murine model of myeloproliferative disorders generated by overexpression of the transcription factor NF-E2 by Kai Björn Kaufmann( )

1 edition published in 2012 in English and held by 2 WorldCat member libraries worldwide

Abstract: The molecular pathophysiology of myeloproliferative neoplasms (MPNs) remains poorly understood. Based on the observation that the transcription factor NF-E2 is often overexpressed in MPN patients, independent of the presence of other molecular aberrations, we generated mice expressing an NF-E2 transgene in hematopoietic cells. These mice exhibit many features of MPNs, including thrombocytosis, leukocytosis, Epo-independent colony formation, characteristic bone marrow histology, expansion of stem and progenitor compartments, and spontaneous transformation to acute myeloid leukemia. The MPN phenotype is transplantable to secondary recipient mice. NF-E2 can alter histone modifications, and NF-E2 transgenic mice show hypoacetylation of histone H3. Treatment of mice with the histone deacetylase inhibitor (HDAC-I) vorinostat restored physiological levels of histone H3 acetylation, decreased NF-E2 expression, and normalized platelet numbers. Similarly,<br>MPN patients treated with an HDAC-I exhibited a decrease in NF-E2 expression. These data establish a role for NF-E2 in the pathophysiology of MPNs and provide a molecular rationale for investigating epigenetic alterations as novel targets for rationally<br>designed MPN therapies
Stress fibers, autophagy and necrosis by persistent exposure to PM2.5 from biomass combustion by Regina Dornhof( )

1 edition published in 2017 in English and held by 2 WorldCat member libraries worldwide

Abstract: Fine particulate matter (PM2.5) can adversely affect human health. Emissions from residential energy sources have the largest impact on premature mortality globally, but their pathological and molecular implications on cellular physiology are still elusive. In the present study potential molecular consequences were investigated during long-term exposure of human bronchial epithelial BEAS-2B cells to PM2.5, collected from a biomass power plant. Initially, we observed that PM2.5 did not affect cellular survival or proliferation. However, it triggered an activation of the stress response p38 MAPK which, along with RhoA GTPase and HSP27, mediated morphological changes in BEAS-2B cells, including actin cytoskeletal rearrangements and paracellular gap formation. The p38 inhibitor SB203580 prevented phosphorylation of HSP27 and ameliorated morphological changes. During an intermediate phase of long-term exposure, PM2.5 triggered proliferative regression and activation of an adaptive stress response necessary to maintain energy homeostasis, including AMPK, repression of translational elongation, and autophagy. Finally, accumulation of intracellular PM2.5 promoted lysosomal destabilization and cell death, which was dependent on lysosomal hydrolases and p38 MAPK, but not on the inflammasome and pyroptosis. TEM images revealed formation of protrusions and cellular internalization of PM2.5, induction of autophagosomes, amphisomes, autophagosome-lysosomal fusion, multiple compartmental fusion, lysosomal burst, swollen mitochondria and finally necrosis. In consequence, persistent exposure to PM2.5 may impair epithelial barriers and reduce regenerative capacity. Hence, our results contribute to a better understanding of PM-associated lung and systemic diseases on the basis of molecular events
Exometabolom analysis of breast cancer cell lines: metabolic signature by Lucas Willmann( )

1 edition published in 2015 in English and held by 2 WorldCat member libraries worldwide

Abstract: Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach
Initial assembly steps of a translocase for folded proteins by Anne-Sophie Blümmel( )

1 edition published in 2015 in English and held by 2 WorldCat member libraries worldwide

Abstract: The so-called Tat (twin-arginine translocation) system transports completely folded proteins across cellular membranes of archaea, prokaryotes and plant chloroplasts. Tat-directed proteins are distinguished by a conserved twin-arginine (RR-) motif in their signal sequences. Many Tat systems are based on the membrane proteins TatA, TatB and TatC, of which TatB and TatC are known to cooperate in binding RR-signal peptides and to form higher-order oligomeric structures. We have now elucidated the fine architecture of TatBC oligomers assembled to form closed intramembrane substrate-binding cavities. The identification of distinct homonymous and heteronymous contacts between TatB and TatC suggest that TatB monomers coalesce into dome-like TatB structures that are surrounded by outer rings of TatC monomers. We also show that these TatBC complexes are approached by TatA protomers through their N-termini, which thereby establish contacts with TatB and membrane-inserted RR-precursors
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Albert-Ludwigs-Universität Freiburg Spemann Graduate School of Biology and Medicine


English (15)