Chemotherapien und neue zielgerichtete Substanzen können kardiale Nebenwirkungen induzieren. Allerdings stützt sich das aktuelle Wissen vorwiegend auf Daten aus klinischen Studien mit dem Fokus auf akuter Kardiotoxizität. Da in den meisten onkologischen Studien kardiovaskuläre Erkrankungen als Ausschlusskriterium gelten, gibt es kaum Daten zu „real-life“ Patientenkollektiven.
Human pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs. bFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation. Our results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs. Collectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.
Background The membrane bound G-protein coupled bile acid receptor TGR5 is expressed in liver epithelial cells. Its activation through bile acids (BAs) triggers secretion, proliferation and anti-apoptotic effects in normal cholangiocytes and CCA cell lines. TGR5 was found to be overexpressed in human cholangiocarcinoma tissue (CCA) and cell lines generated from CCAs. Especially secondary bile acids play a role in the development of different malignant tumors in the gastrointestinal tract, including liver. Whether TGR5 activation also promotes invasiveness and metastasis development of CCA is unclear and aim of this work. Furthermore, TGR5 and the bile acid receptor S1PR2 show partly overlapping signalling functions in biliary epithelial cells. To date, it is unclear whether there is a potential crosstalk of both receptors.
Abstract Background KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. Methods Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS. Results Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and mitosis defects. These damaging effects were correlated with increased cell death. Conclusion These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.
Background and Aims In human adults, and animals, the Apelin-APJ ligand-receptor system is emerging as having a role in the pathogenesis of cardiovascular function and heart failure. The aim was to investigate expression, and regulation by oxygen, of the Apelin APJ receptor (APJ) in myocardium obtained from children undergoing corrective surgery with cardiopulmonary bypass for repair of congenital heart defects. Methods Western blotting and Real-time PCR were used to determine if APJ was expressed in the infant myocardium, if expression was influenced by the duration of myocardial ischemia and if any relationship existed between APJ expression and early post-operative outcome. The next aim was to determine if there was a difference in mRNA expression of APJ in myocardium from cyanotic patients compared with acyanotic patients and if re-perfusing myocardium in vitro with either hypoxic, normoxic or hyperoxic oxygen affected APJ mRNA expression. Results APJ was expressed in all myocardial samples and myocardium exposed to longer durations of ischemia and cardioplegia expressed higher levels of APJ (p<0.05). There was a significant correlation between APJ expression in myocardium resected after 10 min with both oxygen extraction ratio (p = 0.021, rho = −0.523) and mixed venous oxygen saturation (p = 0.028, rho 0.52). This association did not exist for myocardium collected before 10 min. There was no difference in APJ expression between cyanotic and acyanotic patients. No difference was found in APJ expression whether re-perfused with low, normal or high oxygen. Conclusions Changes in APJ expression were observed during cardiopulmonary bypass in children and the reasons for this require further investigation.
Abstract Background KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. Methods Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP, which served as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA based assay in vitro , complex assembly by Co-immunoprecipitation and localization by microscopy in cellulo . Results Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. KDM6A truncations displayed changes in complex assemblies: affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nucleolar protein Nucleophosmin. Furthermore, we observed a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and hence mitosis defects. Conclusion These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A as well as at a dominant negative effect of KDM6A truncation variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.
"Spaceflight associated neuro-ocular syndrome" (SANS) represents a challenging health condition in modern space medicine. Forty-eight percent of astronauts are diagnosed with SANS after long-term space missions. The pathophysiological mechanism seems to be multifactorial, and yet remains unknown. In this proof-of-concept study we plan to investigate retinal microcirculatory changes in weightlessness and aim to identify their role in the development of SANS.Healthy individuals will take part in a parabolic flight campaign, which recreates fractioned total weightlessness periods. The airplane is specifically equipped, and designed for the execution of parabolic flight maneuvers and scientific research in microgravity. Retinal microcirculation will be assessed with a modified fundus camera, which allows dynamic vessel analysis. We will additionally measure intra-ocular pressure and hemodynamic changes during each phase of the flight. Blood samples will be analyzed at baseline, one hour and 24 hours after exposure to weightlessness.This pilot study aims to investigate the feasibility of retinal microcirculation assessment during varying gravity. Results of this study may generate insights whether venous stasis in the eye, surrogated by the dilatation of retinal vessels and increase in intraocular pressure as signs of venous insufficiency, may potentially contribute to the development of SANS.
