TY - JOUR A1 - Naseem, Muhammad A1 - Othman, Eman M. A1 - Fathy, Moustafa A1 - Iqbal, Jibran A1 - Howari, Fares M. A1 - AlRemeithi, Fatima A. A1 - Kodandaraman, Geema A1 - Stopper, Helga A1 - Bencurova, Elena A1 - Vlachakis, Dimitrios A1 - Dandekar, Thomas T1 - Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems JF - Scientific Reports N2 - Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions. KW - cytokinins KW - 6-benzylaminopurine Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231317 VL - 10 ER - TY - JOUR A1 - Wölfel, Angela A1 - Sättele, Mathias A1 - Zechmeister, Christina A1 - Nikolaev, Viacheslov O. A1 - Lohse, Martin J. A1 - Boege, Fritz A1 - Jahns, Roland A1 - Boivin-Jahns, Valérie T1 - Unmasking features of the auto-epitope essential for β\(_1\)-adrenoceptor activation by autoantibodies in chronic heart failure JF - ESC Heart Failure N2 - Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine–serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211–214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211–214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF. KW - antibody/autoantibody KW - β1-adrenoceptor/β1-adrenergic receptor KW - chronic heart failure KW - conformational auto-epitope KW - cyclic peptides/cyclopeptides KW - cyclopeptide therapy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235974 VL - 7 IS - 4 ER - TY - JOUR A1 - Dekant, Raphael A1 - Langer, Michael A1 - Lupp, Maria A1 - Adaku Chilaka, Cynthia A1 - Mally, Angela T1 - In vitro and in vivo analysis of ochratoxin A-derived glucuronides and mercapturic acids as biomarkers of exposure JF - Toxins N2 - Ochratoxin A (OTA) is a widespread food contaminant, with exposure estimated to range from 0.64 to 17.79 ng/kg body weight (bw) for average consumers and from 2.40 to 51.69 ng/kg bw per day for high consumers. Current exposure estimates are, however, associated with considerable uncertainty. While biomarker-based approaches may contribute to improved exposure assessment, there is yet insufficient data on urinary metabolites of OTA and their relation to external dose to allow reliable estimates of daily intake. This study was designed to assess potential species differences in phase II biotransformation in vitro and to establish a correlation between urinary OTA-derived glucuronides and mercapturic acids and external exposure in rats in vivo. In vitro analyses of OTA metabolism using the liver S9 of rats, humans, rabbits and minipigs confirmed formation of an OTA glucuronide but provided no evidence for the formation of OTA-derived mercapturic acids to support their use as biomarkers. Similarly, OTA-derived mercapturic acids were not detected in urine of rats repeatedly dosed with OTA, while indirect analysis using enzymatic hydrolysis of the urine samples prior to LC–MS/MS established a linear relationship between urinary glucuronide excretion and OTA exposure. These results support OTA-derived glucuronides but not mercapturic acids as metabolites suitable for biomonitoring. KW - ochratoxin A KW - biomarker of exposure KW - glucuronide KW - mercapturic acid KW - mycotoxin Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245146 SN - 2072-6651 VL - 13 IS - 8 ER - TY - THES A1 - Claßen, Alexandra T1 - The ERK-cascade in the pathophysiology of cardiac hypertrophy T1 - Die ERK-Kaskade in der Pathophysiologie der Herzhypertrophie N2 - ERK1/2 are known key players in the pathophysiology of heart failure, but the members of the ERK cascade, in particular Raf1, can also protect the heart from cell death and ischemic injury. An additional autophosphorylation (ERK1 at Thr208, ERK2 at Thr188) empowers ERK1/2 translocation to the nucleus and phosphorylation of nuclear targets which take part in the development of cardiac hypertrophy. Thereby, targeting this additional phosphorylation is a promising pharmacological approach. In this thesis, an in silico model of ERK cascade in the cardiomyocyte is introduced. The model is a semi-quantitive model and its behavior was tested with different softwares (SQUAD and CellNetAnalyzer). Different phosphorylation states of ERK1/2 as well as different stimuli can be reproduced. The different types of stimuli include hypertrophic as well as non-hypertrophic stimuli. With the introduced in-silico model time courses and synergistic as well as antagonistic receptor stimuli combinations can be predicted. The simulated time courses were experimentally validated. SQUAD was mainly used to make predictions about time courses and thresholds, whereas CNA was used to analyze steady states and feedback loops. Furthermore, new targets of ERK1/2 which partially contribute, also in the formation of cardiac hypertrophy, were identified and the most promising of them were illuminated. Important further targets are Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 and SPIN90. Cardiomyocyte gene expression data sets were analyzed to verify involved components and to find further significantly altered genes after induced hypertrophy with TAC (transverse aortic constriction). Changes in the ultrastructure of the cardiomyocyte are the final result of induced hypertrophy. N2 - ERK1/2 sind bekannte Schlüsselfiguren bei der Entstehung der Herzinsuffizienz. Weitere Komponenten der ERK-Kaskade, insbesondere Raf1, können das Herz jedoch vor Zelltod und ischämischem Schaden schützen. Eine zusätzliche Autophosphorylierung von ERK1 an Thr208 bzw. von ERK2 an Thr188 ermöglicht ERK1/2 die Translokation zum Zellkern und befähigt ERK dort zur Phosphorylierung von nukleosolischen Zielproteinen, welche eine Herzmuskelhypertrophie auslösen. Daher erscheint diese zusätzliche Autophosphorylierung als eine vielversprechende pharmakologische Zielstruktur. In dieser Arbeit wird ein in-silico Modell der ERK-Kaskade im Kardiomyozyten präsentiert. Das Modell ist ein semi-quantitatives Modell und wurde mit den Programmen SQUAD und CellNetAnalyzer getestet. Verschiedene Phosphorylierungs-Zustände von ERK1/2 als auch verschiedene Stimuli (hypertrophe als auch nicht-hypertrophe) können mit dem Modell reproduziert werden. Mit dem präsentierten in-silico Modell können sowohl zeitliche Abläufe als auch synergistische und antagonistische Effekte vorhergesagt werden. Die simulierten zeitlichen Abläufe wurden durch in-vitro Experimente validiert. SQUAD wurde hauptsächlich für die Modellierung von zeitlichen Abläufen und Schwellenwerte genutzt, wohingegen CellNetAnalyzer vor allen Dingen zur Analyse von Fließgleichgewichten und Rückkopplungs-Mechanismen genutzt wurde. Darüberhinaus wurden Zielstrukturen von ERK1/2, welche zusätzlich an der Entstehung der Herzhypertrophie mitwirken, identifiziert. Diese umfassen unter anderem Caspase 8, GAB2, Mxi-2, SMAD2, FHL2 und SPIN90. Gen-Expressions-Datensätze von Kardiomyozyten nach TAC (transverse aortic constriction) wurden analysiert. Diese wurden mit den im Model vorhandenen Strukturen verglichen und signifikant veränderte Expressionslevel wurden identifiziert. Veränderungen der Ultrastruktur des Kardiomyozyten sind das Ergebnis der induzierten Hypertrophie. KW - Herzhypertrophie KW - Systembiologie KW - ERK-cascade KW - ERK-Kaskade KW - cardiac hypertrophy KW - in-silico model KW - In-silico Modell Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229664 ER - TY - THES A1 - Anton, Selma T1 - Characterization of cAMP nanodomains surrounding the human Glucagon-like peptide 1 receptor using FRET-based reporters T1 - Charakterisierung der Rezeptor-assoziierten cAMP Nanodomänen des humanen Glucagon-like peptide 1 Rezeptors mittels FRET-basierter Sensoren N2 - Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely. In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a Förster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA. Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment. N2 - G Protein gekoppelte Rezeptoren (GPCRs) stellen eine große und sehr vielfältige Familie an Membranproteinen dar, deren primäre Funktion die Signalübertragung von extrazellulären Stimuli in intrazelluläre Signale ist. Dank ihrer breiten Expression im gesamten menschlichen Körper regulieren sie unterschiedliche zelluläre Prozesse und damit deren physiologische Funktion, unter anderem die Sinnesempfindung, zelluläre Kommunikation und Neurotransmission. GPCRs stehen im Zusammenhang mit unterschiedlichen Erkrankungen wie Herzinsuffizienz, Krebs, neurologischen Funktionsstörungen und diverser metabolischer Krankheiten, weswegen sie als Ziele („Targets“) zur Behandlung verschiedener Erkrankungen erforscht und genutzt werden. Aufgrund ihrer Expression auf der Zelloberfläche sind sie leicht zugänglich, und die Diversität ihrer Liganden begünstigt zusätzlich ihre Nutzung als pharmakologische Targets. Heutzutage vermitteln bereits 30% aller weltweit zugelassenen Arzneistoffe ihre Wirkung an GPCRs. GPCRs üben ihre Funktion aus, indem sie hauptsächlich an G Proteine binden, welche wiederum die Produktion sogenannter second messenger in Gang setzen. cAMP ist das Hauptsignalmolekül der Rezeptoren, welche an das stimulatorische GS Protein koppeln. cAMP überträgt hunderte ankommende Signale in einer hochspezifischen Weise, indem es an unterschiedliche Effektorproteine bindet, welche sich in bestimmten zellulären Regionen befinden. Dadurch koordiniert dieses Signalmolekül eine Vielzahl zellulärer Prozesse, angefangen bei der Regulierung von Ionenkanalaktivität über die Kontraktilität glatter- und quergestreifter Muskulatur bis hin zur Genexpression, Zellproliferation und Apoptose. Durch die pleiotropen Effekte, welche durch cAMP reguliert werden, stellt sich die Frage, wie GS-gekoppelte Rezeptoren Signalspezifität erreichen, obwohl sie ihre Funktion durch dieses eine Signalmolekül ausführen. Ursprünglich ging man von einer uneingeschränkten Diffusion und dadurch homogenen Verteilung von cAMP in der Zelle aus. Diese Vorstellung ist jedoch nicht mit der Signalisierungsspezifität von GPCRs vereinbar, da unter diesen Umständen cAMP unselektiv all seine Effektorproteine in der gesamten Zelle aktivieren könnte. Daher entstand die Hypothese der cAMP-Kompartimentierung, wobei die Zelle lokal begrenzte Bereiche mit hohen oder niedrigen cAMP Konzentrationen umfassen würde. Jedoch gab es bisher keinerlei Beweise für die Existenz und die molekulare Zusammensetzung mutmaßlicher Domänen. Folglich setzten wir uns als Ziel, hochkonzentrierte cAMP-Kompartimente in der Zelle zu lokalisieren, ihre räumliche Dimension aufzuklären und ihre Rolle zur Realisierung zellulärer Signalisierungsspezifität zu ermitteln. Im Rahmen der vorliegenden Studie setzten wir einen Förster resonance energy transfer (FRET)-basierten cAMP Sensor ein, fusionierten ihn mit dem humanen glucagone-like peptide 1 Rezeptor (hGLP1R) als Prototyp eines GS-koppelnden Rezeptors, um cAMP am Ursprung des Signals zu messen. Mittels dieser Sensoren weisen wir eine Rezeptor-umgebende begrenzte cAMP Domäne nach, welche eine erhöhte cAMP Konzenztration aufweist (Figure ‎3.10). Bei Stimulation des Rezeptors mit GLP1 Konzenztrationen beginnend bei 10 fM entsteht eine Rezeptordomäne mit lokal erhöhten cAMP Konzentrationen, welche getrennt von Plasmamembran und Cytosol ist. Wir zeigen, dass das hGLP1R-Kompartiment geschützt ist vor cAMP Signalen, welche an weiteren, unabhängigen GS-gekoppelten Rezeptoren ihren Ursprung haben (Figure ‎3.11). Um die räumliche Dimension dieser Domäne zu untersuchen, verwendeten wir Nanolinker der Länge 30- und 60 nm als Abstandhalter zwischen Rezeptor und Sensor (Figure ‎3.12) und zeigen dabei, dass sich die Domäne über eine Länge von 60 Nanometern erstreckt, wobei ein abnehmender cAMP-Gradient erkennbar ist. Weiterhin beweisen wir, dass Phosphodiesterasen (PDEs) Schlüsselfaktoren für die Bildung des cAMP-Gradienten um den Rezeptor herum sind, indem sie die Diffusion ins Cytosol beschränken (Figure ‎3.13). Darüber hinaus zeigen wir (Figure ‎3.15), dass Rezeptor-spezifische cAMP Signale PKA-Phosphorylierung in der Rezeptordomäne auslösen und, dass AKAPs elementar für nanodomänen PKA-Aktivität sind, wohingegen die cytosolische PKA-Phosphorylierung unabhängig von AKAP-Targeting der PKA ist (Figure ‎3.16). Zusammenfassend beweisen unsere Ergebnisse die Existenz einer Rezeptor-umgebenden Nanodomäne mit erhöhten cAMP Spiegeln eines GS-gekoppelten Rezeptors. Zeitgleiche Studien in unserer Gruppe zeigen, dass cAMP in der Zelle weitgehend gebunden vorliegt und diffusionslimitiert ist. Dies stellt den Nachweis für eine eingeschränkte Diffusion als molekulare Voraussetzung für die Bildung von Signalkompartimenten dar. Wir gehen davon aus, dass unsere Ergebnisse ein Ausgangspunkt für die Aufklärung von Rezeptoren als Quelle für Signalkompartimente darstellen, jedoch bedarf es weiterer Studien, um die präzise molekulare Zusammensetzung und die beteiligten Proteine dieser Signaldomäne zu untersuchen. Das Grundverständnis der Signalisierungskaskaden auf molekularer Ebene könnte es uns ermöglichen, die zellulären Reaktionen zu manipulieren, um eine Fehlfunktion der Signalisierung in erkrankten Zellen wiederherzustellen. Da der hGLP1R entscheidend für Aufrechterhaltung ausgeglichener Blutglucosespiegel ist, würde die Erfassung der molekularen Details der kompartimentalisierten Signalübertragung die Feinabstimmung der Rezeptorsignale ermöglichen, um ihn als spezifisches Target zur Behandlung von Diabetes Mellitus einzusetzen. KW - FRET KW - cAMP KW - compartments KW - GPCR Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-190695 ER - TY - JOUR A1 - Wagner, Michael A1 - Sadek, Mirna S. A1 - Dybkova, Nataliya A1 - Mason, Fleur E. A1 - Klehr, Johann A1 - Firneburg, Rebecca A1 - Cachorro, Eleder A1 - Richter, Kurt A1 - Klapproth, Erik A1 - Kuenzel, Stephan R. A1 - Lorenz, Kristina A1 - Heijman, Jordi A1 - Dobrev, Dobromir A1 - El-Armouche, Ali A1 - Sossalla, Samuel A1 - Kämmerer, Susanne T1 - Cellular mechanisms of the anti-arrhythmic effect of cardiac PDE2 overexpression JF - International Journal of Molecular Sciences N2 - Background: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. Methods: Cellular arrhythmia, ion currents, and Ca\(^{2+}\)-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. Results: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in I\(_{CaL}\) seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in I\(_{NaL}\) and Ca\(^{2+}\)-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. Conclusion: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF. KW - PDE2 KW - arrhythmia KW - CaMKII KW - heart failure Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285888 SN - 1422-0067 VL - 22 IS - 9 ER - TY - JOUR A1 - Sedaghat-Hamedani, Farbod A1 - Rebs, Sabine A1 - El-Battrawy, Ibrahim A1 - Chasan, Safak A1 - Krause, Tobias A1 - Haas, Jan A1 - Zhong, Rujia A1 - Liao, Zhenxing A1 - Xu, Qiang A1 - Zhou, Xiaobo A1 - Akin, Ibrahim A1 - Zitron, Edgar A1 - Frey, Norbert A1 - Streckfuss-Bömeke, Katrin A1 - Kayvanpour, Elham T1 - Identification of SCN5a p.C335R variant in a large family with dilated cardiomyopathy and conduction disease JF - International Journal of Molecular Sciences N2 - Introduction: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. Methods: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. Results: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na\(^+\) channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. Conclusion: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype. KW - familial DCM KW - conduction disease KW - SCN5a Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284442 SN - 1422-0067 VL - 22 IS - 23 ER - TY - THES A1 - Schihada, Hannes T1 - Novel optical methods to monitor G-protein-coupled receptor activation in microtiter plates T1 - Neue optische Methoden zur Messung der Aktivierung von G-Protein-gekoppelten Rezeptoren in Mikrotiter-Platten N2 - G-protein-coupled receptors (GPCRs) regulate diverse physiological processes in the human body and represent prime targets in modern drug discovery. Engagement of different ligands to these membrane-embedded proteins evokes distinct receptor conformational rearrangements that facilitate subsequent receptor-mediated signalling and, ultimately, enable cellular adaptation to altered environmental conditions. Since the early 2000s, the technology of resonance energy transfer (RET) has been exploited to assess these conformational receptor dynamics in living cells and real time. However, to date, these conformational GPCR studies are restricted to single-cell microscopic setups, slowing down the discovery of novel GPCR-directed therapeutics. In this work, we present the development of a novel generalizable high-throughput compatible assay for the direct measurement of GPCR activation and deactivation. By screening a variety of energy partners for fluorescence (FRET) and bioluminescence resonance energy transfer (BRET), we identified a highly sensitive design for an α2A-adrenergic receptor conformational biosensor. This biosensor reports the receptor’s conformational change upon ligand binding in a 96-well plate reader format with the highest signal amplitude obtained so far. We demonstrate the capacity of this sensor prototype to faithfully quantify efficacy and potency of GPCR ligands in intact cells and real time. Furthermore, we confirm its universal applicability by cloning and validating five further equivalent GPCR biosensors. To prove the suitability of this new GPCR assay for screening purposes, we measured the well-accepted Z-factor as a parameter for the assay quality. All tested biosensors show excellent Z-factors indicating outstanding assay quality. Furthermore, we demonstrate that this assay provides excellent throughput and presents low rates of erroneous hit identification (false positives and false negatives). Following this phase of assay development, we utilized these biosensors to understand the mechanism and consequences of the postulated modulation of parathyroid hormone receptor 1 (PTHR1) through receptor activity-modifying protein 2 (RAMP2). We found that RAMP2 desensitizes PTHR1, but not the β2-adrenergic receptor (β2AR), for agonist-induced structural changes. This generalizable sensor design offers the first possibility to upscale conformational GPCR studies, which represents the most direct and unbiased approach to monitor receptor activation and deactivation. Therefore, this novel technology provides substantial advantages over currently established methods for GPCR ligand screening. We feel confident that this technology will aid the discovery of novel types of GPCR ligands, help to identify the endogenous ligands of so-called orphan GPCRs and deepen our understanding of the physiological regulation of GPCR function. N2 - Die Klasse der G-protein-gekoppelten Rezeptoren (GPCRs) stellt die größte Familie membranständiger Proteine dar. GPCRs regulieren eine Vielzahl diverser physiologischer Prozesse in eukaryotischen Zellen und kontrollieren so unterschiedliche Zellfunktionen im menschlichen Organismus. Sie stellen die Zelloberflächenrezeptoren für verschiedenartige extrazelluläre Stimuli, wie zum Beispiel Photonen, niedermolekulare chemische Verbindungen, Peptide und Lipide dar. Die Wechselwirkung mit diesen sogenannten Liganden stabilisiert spezifische GPCR-Konformationen. Diese dienen wiederum als Ausgangspunkt für nachgeschaltete intrazelluläre Signalkaskaden, die beispielweise über membranverankerte G-Proteine vermittelt werden können. Während endogene GPCR-Agonisten diese Signalweiterleitung verstärken, können andere Biomoleküle wie Lipide, Ionen oder andersartige Membranproteine die Funktion, und damit die Signalweiterleitung der GPCRs modulieren. Aufgrund ihrer Einbindung in eine Vielzahl physiologischer und pathophysiologischer Prozesse, wurden GPCRs schon früh als Angriffspunkte („Targets“) zur Behandlung verschiedener Erkrankungen erforscht und genutzt. Heutzutage vermitteln etwa 30% aller zugelassenen Arzneistoffe ihre Wirkung über G-protein-gekoppelte Rezeptoren. Dennoch wird das große Potential dieser Rezeptorfamilie als Targets für medikamentöse Behandlungen noch nicht in vollem Umfang ausgeschöpft. Tatsächlich gibt es für mehr als 200 GPCRs, die nicht der olfaktorischen Wahrnehmung dienen, noch keine Arzneistoffe, da wenig über deren Pharmakologie und physiologische Bedeutung bekannt ist. Zudem wird die Entwicklung neuartiger GPCR-Liganden erheblich durch das eingeschränkte Methodenrepertoire beeinträchtigt. Alle derzeit etablierten Techniken zur Identifizierung neuer GPCR-Liganden erfassen entweder den Ligand-GPCR-Bindungsprozess, der keine Informationen über die tatsächliche Aktivität der Verbindung liefert, oder messen weit-nachgeschaltete Signale, wie Änderungen sogenannter „Second-Messenger“-Konzentrationen (meist cAMP oder Calcium) und Reporter-Gen-Expressionslevel. Aufgrund ihrer Entfernung vom eigentlichen Rezeptor-Aktivierungsprozess haben diese Methoden allerdings bedeutende Nachteile und produzieren so häufig Falsch-Positive und Falsch-Negative Ergebnisse. Seit den frühen 2000er wurden GPCR-Konformationssensoren auf Basis von Fluoreszenz-Resonanz-Energie-Transfer (FRET) zur Messung der Ligand-induzierten Rezeptordynamik genutzt. Jedoch wies keiner der bisher entwickelten FRET- oder BRET- (Biolumineszenz-Resonanz-Energie-Transfer) Sensoren ausreichende Signalstärke auf, um im Hochdurchsatz-Screening (HTS) angewendet werden zu können. Die vorliegende Studie beschreibt das erste GPCR-Sensordesign, das aufgrund seiner exzellenten Signalstärke im Hochdurchsatz-Verfahren verwendet werden kann. Wir haben 21 unterschiedliche FRET- und BRET-Sensoren des α2A-adrenergen Rezeptors (α2AAR) getestet und dabei die Kombination der kleinen und hellen Luziferase NanoLuciferase (Nluc) mit dem rot-fluoreszierenden HaloTag-Farbstoff 618 als sensitivstes RET-Paar identifiziert. Der α2AARNluc/Halo(618) Biosensor ermöglicht die Messung der Aktivität und Wirkstärke von α2AAR-Liganden im Mikrotiterplattenformat. Um die universelle Anwendbarkeit dieses Sensordesigns zu prüfen, wurden fünf weitere Nluc/Halo(618)-basierende Sensoren für GPCRs unterschiedlicher Unterfamilien entwickelt. Zudem konnten wir zeigen, dass diese GPCRNluc/Halo(618)-Fusionsproteine weiterhin ihre natürlichen Signalkaskaden in Gang setzen können und damit die biologische Funktionalität dieser Rezeptoren erhalten ist. Außerdem belegt die vorlegende Arbeit, dass diese neue Sensor-Generation zur Messung Ligand-vermittelter Rezeptordynamiken im Hochdurchsatz-Format und zur Untersuchung der GPCR-Regulation durch endogene Modulatoren genutzt werden kann. Zusammenfassend kann gesagt werden, dass wir den ersten HTS-kompatiblen Assay zur Messung der GPCR-Konformationsänderungen entwickelt haben. Diese Biosensoren erlauben die Charakterisierung neuartiger GPCR-Liganden direkt auf der Rezeptorebene und funktionieren damit unabhängig von nachgeschalteter Signalamplifikation oder Überlagerung verschiedener Signalwege, welche die Aussagekraft traditioneller GPCR-Screening-Verfahren häufig beeinträchtigen. Diese Technik kann zur Entdeckung neuartiger GPCR-Arzneistoffe genutzt werden, zu einem besseren Verständnis bisher kaum erforschter Rezeptoren beitragen und der Identifizierung und Charakterisierung potentieller GPCR-Modulatoren dienen. KW - G-Protein gekoppelte Rezeptoren KW - Hochdurchsatz-Screening KW - Förster Resonanz Energie Transfer KW - High-thropughput screening KW - Bioluminescence resonance energy transfer KW - G-protein-coupled receptors KW - Receptor dynamics Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-175415 ER - TY - JOUR A1 - Bauer, Benedikt A1 - Mally, Angela A1 - Liedtke, Daniel T1 - Zebrafish embryos and larvae as alternative animal models for toxicity testing JF - International Journal of Molecular Sciences N2 - Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species. KW - danio rerio KW - alternative methods KW - organ toxicity KW - 3R KW - transgenic animals Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284225 SN - 1422-0067 VL - 22 IS - 24 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Schuele, Carolin A1 - Stopper, Helga T1 - Cell survival after DNA damage in the comet assay JF - Archives of Toxicology N2 - The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided. KW - Cell death and comet assay KW - DNA damage KW - DNA repair Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265339 VL - 95 IS - 12 ER - TY - THES A1 - Kodandaraman, Geema T1 - Influence of insulin-induced oxidative stress in genotoxicity and disease T1 - Einfluss von insulininduziertem oxidativem Stress auf Genotoxitität und Krankheit N2 - Hormones are essential components in the body and their imbalance leads to pathological consequences. T2DM, insulin resistance and obesity are the most commonly occurring lifestyle diseases in the past decade. Also, an increased cancer incidence has been strongly associated with obese and T2DM patients. Therefore, our aim was to study the influence of high insulin levels in accumulating DNA damage in in vitro models and patients, through the induction of oxidative stress. The primary goal of this study was to analyze the genotoxicity induced by the combined action of two endogenous hormones (insulin and adrenaline) with in vitro models, through the induction of micronuclei and to see if they cause an additive increase in genomic damage. This is important for multifactorial diseases having high levels of more than one hormone, such as metabolic syndrome and conditions with multiple pathologies (e.g., T2DM along with high stress levels). Furthermore, the combination of insulin and the pharmacological inhibition of the tumor suppressor gene: PTEN, was to be tested in in vitro models for their genotoxic effect and oxidative stress inducing potential. As the tumor suppressor gene: PTEN is downregulated in PTEN associated syndromes and when presented along with T2DM and insulin resistance, this may increase the potential to accumulate genomic damage. The consequences of insulin action were to be further elucidated by following GFP-expressing cells in live cell-imaging to observe the ability of insulin, to induce micronuclei and replicative stress. Finally, the detrimental potential of high insulin levels in obese patients with hyperinsulinemia and pre-diabetes was to be studied by analyzing markers of oxidative stress and genomic damage. In summary, the intention of this work was to understand the effects of high insulin levels in in vitro and in patients to understand its relevance for the development of genomic instability and thus an elevated cancer risk. N2 - In-vitro-Genotoxizitätsstudien mit hohen Konzentrationen von Insulin und die Kombination mit Adrenalin zeigten keinen additiven Anstieg der Mikrokernzahl. Der Insulinrezeptor und der AKT-Signalweg waren in den insulinvermittelten Genomschaden involviert. Die endogenen ROS-Quellen, Mitochondrien und NOX, waren an dem insulinvermittelten DNA-Schaden beteiligt. Hohe Konzentrationen von mitochondrialen ROS alleine, verursacht durch einen Komplex III Mitochondrien-Inhibitor, führten zu Zytotoxizität, aber nicht zu einer Zunahme des Genomschadens. Daher ist die durch das NOX-Enzym vermittelte ROS-Produktion wahrscheinlich der gemeinsame Faktor des genotoxischen Signalweges von Insulin und Adrenalin. Die Überstimulation des NOX-Enzyms führte zu einer Sättigung der zellulären biologischen Effekte und fehlender Additivität bei der Induktion von Genomschaden. Dies könnte jedoch unter physiologischen Bedingungen anders sein, da die Hormonspiegel niedriger sind und die ROS-Quellen nicht durch jedes einzelne der Hormone bereits maximal genutzt und daher erschöpft werden. Damit könnte die Möglichkeit eines additiven Genomschadens in vivo bestehen. Die Rolle des AKT-Signalwegs bei der Insulin-vermittelten genomischen Schädigung ist bereits etabliert und hier wurde nun die Funktion des negativen Regulatorproteins PTEN untersucht. Die Ergebnisse zeigten, dass die PTEN Inhibierung nicht nur zu einer erhöhten Genotoxizität durch MN-Induktion führte, sondern auch zur Beeinträchtigung der mitochondrialen Funktion. Obwohl kein Anstieg von ROS nach PTEN-Inhibierung beobachtet wurde, könnte die mitochondriale Dysfunktion zur metabolischen Imbalance sowie zur Zunahme des Genomschadens führen. Dies könnte insbesondere bei Patienten mit bestimmten PTEN-assoziierten Syndromen und Krebserkrankungen, die eine defekte PTEN-vermittelte Tumorsuppressorfunktion, DNA-Reparaturdefekte und kompromittierte antioxidative Abwehrmechanismen aufweisen, eine wichtige Rolle spielen. Wenn diese Patienten zusätzlich von Hyperinsulinämie betroffen sind, könnte eine Akkumulation von Genomschaden erfolgen und das Risiko zur Krebsentstehung wäre erhöht. Der Mechanismus der Genomschadensinduktion durch Insulin wurde bisher mit einer ROS-vermittelten DNA-Oxidation in Verbindung gebracht, aber noch nicht mit der mitogenen Signalgebung. Bei dieser beschleunigte das mitogene Potential des Insulins die Zellteilung und verursachte einen leichten replikativen Stress. Der milde replikative Stress könnte der Kontrolle durch die mitotischen Checkpoint-Proteine entgehen und zu Chromosomen-Fehlverteilungen und Chromosomenbrüchen führen. Dieser Effekt wurde in der Krebszelllinie Hela in Form von multipolaren Spindeln und Mikronuklei beobachtet und es ist nicht klar ob normale Zellen mit effizienterer Kontrolle dies verhindern könnten. Insgesamt könnte ein durch hohe Insulinspiegel vermittelter Schaden im Kontext anderer Komorbiditäten wie etwa PTEN Syndromen, metabolischem Syndrom oder Adipositas zu einer Akkumulation von DNA-Schäden führen. Schließlich zeigte die Analyse von Proben adipöser Patienten eine Zunahme von DNA-Schaden und oxidativem Stress im Vergleich zu den gesunden Kontrollen. Der Anstieg des DNA-Schadens war am höchsten in der Untergruppe der Patienten mit Insulinresistenz. Hoher Insulinspiegel bedeutet somit ein Risiko vom erhöhten oxidativen Stress und Genomschaden, insbesondere in Kombination mit Komorbiditäten. Erschwert wird das Verständnis dieser multifaktoriellen Zusammenhänge durch das komplexe Zusammenspiel von oxidativem Stress und seiner zellulären Regulation in vielen physiologischen sowie pathophysiologischen Prozessen. Daneben ist es eine Herausforderung, Genomschäden bei den geringen Wirkspiegeln hormoneller Effekte zu detektieren. Weitere Untersuchungen der komplexen Insulin-vermittelten Genomschadenswege werden notwendig sein, um mögliche Risiken der Hyperinsulinämie bei Erkrankungen wie Stoffwechselkrankheiten, Diabetes Typ 2 und Adipositas besser zu charakterisieren. KW - Insulin KW - Genotoxicity KW - Micronucleus Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-242005 ER - TY - JOUR A1 - Bankoglu, Ezgi Eyluel A1 - Stipp, Franzisca A1 - Gerber, Johanna A1 - Seyfried, Florian A1 - Heidland, August A1 - Bahner, Udo A1 - Stopper, Helga T1 - Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay JF - Archives of Toxicology N2 - The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction. KW - human biomonitoring KW - DNA damage KW - DNA repair KW - comet assay KW - blood samples Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265326 VL - 95 IS - 5 ER - TY - JOUR A1 - Weigand, Isabel A1 - Ronchi, Cristina L. A1 - Vanselow, Jens T. A1 - Bathon, Kerstin A1 - Lenz, Kerstin A1 - Herterich, Sabine A1 - Schlosser, Andreas A1 - Kroiss, Matthias A1 - Fassnacht, Martin A1 - Calebiro, Davide A1 - Sbiera, Silviu T1 - PKA Cα subunit mutation triggers caspase-dependent RIIβ subunit degradation via Ser\(^{114}\) phosphorylation JF - Science Advances N2 - Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome. KW - mutation triggers KW - phosphorylation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270445 VL - 7 IS - 8 ER - TY - JOUR A1 - Guth, Sabine A1 - Hüser, Stephanie A1 - Roth, Angelika A1 - Degen, Gisela A1 - Diel, Patrick A1 - Edlund, Karolina A1 - Eisenbrand, Gerhard A1 - Engel, Karl-Heinz A1 - Epe, Bernd A1 - Grune, Tilman A1 - Heinz, Volker A1 - Henle, Thomas A1 - Humpf, Hans-Ulrich A1 - Jäger, Henry A1 - Joost, Hans-Georg A1 - Kulling, Sabine E. A1 - Lampen, Alfonso A1 - Mally, Angela A1 - Marchan, Rosemarie A1 - Marko, Doris A1 - Mühle, Eva A1 - Nitsche, Michael A. A1 - Röhrdanz, Elke A1 - Stadler, Richard A1 - van Thriel, Christoph A1 - Vieths, Stefan A1 - Vogel, Rudi F. A1 - Wascher, Edmund A1 - Watzl, Carsten A1 - Nöthlings, Ute A1 - Hengstler, Jan G. T1 - Contribution to the ongoing discussion on fluoride toxicity JF - Archives of Toxicology N2 - Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries. KW - pharmacology/toxicology KW - occupational medicine/industrial medicine KW - environmental health KW - biomedicine, general Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-307161 SN - 0340-5761 SN - 1432-0738 VL - 95 IS - 7 ER - TY - JOUR A1 - Barile, Frank A. A1 - Berry, Colin A1 - Blaauboer, Bas A1 - Boobis, Alan A1 - Bolt, Herrmann M. A1 - Borgert, Christopher A1 - Dekant, Wolfgang A1 - Dietrich, Daniel A1 - Domingo, Jose L. A1 - Galli, Corrado L. A1 - Gori, Gio Batta A1 - Greim, Helmut A1 - Hengstler, Jan G. A1 - Heslop-Harrison, Pat A1 - Kacew, Sam A1 - Marquardt, Hans A1 - Mally, Angela A1 - Pelkonen, Olavi A1 - Savolainen, Kai A1 - Testai, Emanuela A1 - Tsatsakis, Aristides A1 - Vermeulen, Nico P. T1 - The EU chemicals strategy for sustainability: in support of the BfR position JF - Archives of Toxicology N2 - The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations. KW - pharmacology/toxicology KW - occupational medicine/industrial medicine KW - environmental health KW - biomedicine, general Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-307154 SN - 0340-5761 SN - 1432-0738 VL - 95 IS - 9 ER - TY - THES A1 - Jarzina, Sebastian Oskar T1 - Assessment of systemic toxicity in vitro using the Adverse Outcome Pathway (AOP) concept: nephrotoxicity due to receptor-mediated endocytosis and lysosomal overload and inhibition of mtDNA polymerase-ɣ as case studies T1 - Bewertung der systemischen Toxizität in vitro unter Verwendung des Adverse Outcome Pathway (AOP)-Konzepts: Nephrotoxizität infolge rezeptorvermittelter Endozytose und lysosomaler Überlastung sowie Hemmung der mtDNA-Polymerase-ɣ als Fallstudien N2 - The US National Research Council (NRC) report "Toxicity Testing in the 21st Century: A Vision and a strategy (Tox21)", published in 2007, calls for a complete paradigm shift in tox-icity testing. A central aspect of the proposed strategy includes the transition from apical end-points in in vivo studies to more mechanistically based in vitro tests. To support and facilitate the transition and paradigm shift in toxicity testing, the Adverse Outcome Pathway (AOP) concept is widely recognized as a pragmatic tool. As case studies, the AOP concept was ap-plied in this work to develop AOPs for proximal tubule injuries initiated by Receptor-mediated endocytosis and lysosomal overload and Inhibition of mtDNA polymerase-. These AOPs were used as a mechanistic basis for the development of in vitro assays for each key event (KE). To experimentally support the developed in vitro assays, proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) were treated with model compounds. To measure the dis-turbance of lysosomal function in the AOP – Receptor-mediated endocytosis and lysosomal overload, polymyxin antibiotics (polymyxin B, colistin, polymyxin B nonapeptide) were used as model compounds. Altered expression of lysosomal associated membrane protein 1/2 (LAMP-1/2) (KE1) and cathepsin D release from lysosomes (KE2) were determined by im-munofluorescence, while cytotoxicity (KE3) was measured using the CellTiter-Glo® cell via-bility assay. Importantly, significant differences in polymyxin uptake and susceptibility be-tween cell lines were observed, underlining the importance of in vitro biokinetics to determine an appropriate in vitro point of departure (PoD) for risk assessment. Compared to the in vivo situation, distinct expression of relevant transporters such as megalin and cubilin on mRNA and protein level in the used cell lines (RPTEC/TERT1 and NRK-52E) could not be con-firmed, making integration of quantitative in vitro to in vivo extrapolations (QIVIVE) neces-sary. Integration of QIVIVE by project partners of the University of Utrecht showed an im-provement in the modelled biokinetic data for polymyxin B. To assess the first key event, (KE1) Depletion of mitochondrial DNA, in the AOP – Inhibition of mtDNA polymerase-, a RT-qPCR method was used to determine the mtDNA copy number in cells treated with mod-el compounds (adefovir, cidofovir, tenofovir, adefovir dipivoxil, tenofovir disoproxil fumarate). Mitochondrial toxicity (KE2) was measured by project partners using the high-content imaging technique and MitoTracker® whereas cytotoxicity (KE3) was determined by CellTiter-Glo® cell viability assay. In contrast to the mechanistic hypothesis underlying the AOP – Inhibition of mtDNA polymerase-, treatment with model compounds for 24 h resulted in an increase rather than a decrease in mtDNA copy number (KE1). Only minor effects on mitochondrial toxicity (KE2) and cytotoxicity (KE3) were observed. Treatment of RPT-EC/TERT1 cells for 14 days showed only a slight decrease in mtDNA copy number after treatment with adefovir dipivoxil and tenofovir disoproxil fumarate, underscoring some of the limitations of short-term in vitro systems. To obtain a first estimation for risk assessment based on in vitro data, potential points of departure (PoD) for each KE were calculated from the obtained in vitro data. The most common PoDs were calculated such as the effect concentra-tion at which 10 % or 20_% effect was measured (EC10, EC20), the highest no observed effect concentration (NOEC), the lowest observed effect concentration (LOEC), the benchmark 10 % (lower / upper) concentrations (BMC10, BMCL10, BMCU10) and a modelled non-toxic con-centration (NtC). These PoDs were then compared with serum and tissue concentrations de-termined from in vivo studies after treatment with therapeutic / supratherapeutic doses of the respective drugs in order to obtain a first estimate of risk based on in vitro data. In addition, AOPs were used to test whether the quantitative key event relationships between key events allow prediction of downstream effects and effects on the adverse outcome (AO) based on measurements of an early key event. Predictions of cytotoxicity from the mathematical rela-tionships showed good concordance with measured cytotoxicity after treatment with colistin and polymyxin b nonapeptide. The work also revealed uncertainties and limitations of the ap-plied strategy, which have a significant impact on the prediction and on a risk assessment based on in vitro results. N2 - Der Bericht des US National Research Council (NRC) „Toxicity Testing in the 21st Century: A Vision and a strategy (Tox21)“, der 2007 veröffentlicht wurde, sieht einen vollständigen Paradigmenwechsel in der Toxizitätsprüfung vor. Ein zentraler Aspekt des Berichts beinhaltet den Übergang von apikal ermittelten Endpunkten für Toxizität in in vivo Studien, zu mehr mechanistisch basierten in vitro Tests. Um den Übergang zu erleichtern und den Paradigmen-wechsel in der Prüfung auf Toxizität zu unterstützen, wird das Adverse Outcome Pathway (AOP) Konzept als pragmatisches Instrument weithin anerkannt. In dieser Arbeit wurde das AOP Konzept angewandt, um neue Ansätze zur Prüfung auf systemische Toxizität zu unter-suchen. Dazu wurden AOPs für proximale Tubulusschäden, die durch lysosomale Überladung und Inhibition der mtDNA Polymerase- initiiert werden, entwickelt. Diese AOPs wurden als mechanistische Grundlage für die Entwicklung von mechanistisch relevanten in vitro Tests für jedes Schlüsselereignis (KE) verwendet. Um die entwickelten in vitro Tests experimentell zu unterstützen, wurden proximale Tubuluszellen aus der Ratte (NRK-52E) und aus dem Men-schen (RPTEC/TERT1) mit Hilfe von Modellsubstanzen behandelt. Zur Messung der Störung der lysosomalen Funktion im AOP – Rezeptor-vermittelte Endozytose und lysosomale Überla-dung wurden Polymyxin-Antibiotika (Polymyxin B, Colistin, Polymyxin B Nonapeptid) als Modellsubstanzen verwendet. Die gestörte Expression des lysosomal assoziierten Membran-proteins 1/2 (LAMP 1/2) (KE1) und die Cathepsin D Freisetzung (KE2) wurden mittels Im-munofluoreszenztechnik bestimmt und die Zytotoxizität (KE3) mittels CellTiter-Glo® Zellvia-bilitätstest gemessen. Zwischen den Zelllinien wurden signifikante Unterschiede in der Auf-nahme von Polymyxinen und der Empfindlichkeit beobachtet, was die Bedeutung der in vitro Biokinetik zur Definition eines geeigneten Ausgangspunktes für die Risikobewertung unter-streicht. Im Vergleich zur in vivo Situation, konnte eine eindeutige Expression von relevanten Trans-portern wie Megalin und Cubilin auf mRNA und Proteinebene in den verwendeten Zelllinien (RPTEC/TERT1 und NRK-52E) nicht gezeigt werden, was eine zusätzliche Integration von quantitativen in vitro zu in vivo Extrapolationen (QIVIVE) unabdingbar macht. Die Integrati-on von QIVIVE durch Projektpartner der Universität Utrecht zeigte eine Verbesserung der modellierten biokinetischen Werte für Polymyxin B. Zur Bestimmung des ersten Schlüsseler-eignisse, (KE1) Depletion von mitochondrialer DNA, im AOP – Hemmung der mitochondria-len DNA Polymerase-, wurde nach Behandlung mit Modellsubstanzen (Adefovir, Cidofovir, Tenofovir, Adefovirdipivoxil, Tenofovirdisoproxil Fumarat) eine RT-qPCR Methode verwen-det, um die mtDNA Kopienzahl zu bestimmen. Die mitochondriale Toxizität (KE2) wurde mittels eines hochauflösenden Bildgebungsverfahrens und MitoTracker® vom Projektpartner des Fraunhofer Institut in Hamburg gemessen, während die Zytotoxizität (KE3) mittels Cel-lTiter-Glo® Zellviabilitätstest ermittelt wurde. Entgegen der mechanistischen Hypothese des AOPs – Hemmung der mitochondrialen DNA Polymerase-, führte eine 24 h Behandlung mit den Modellsubstanzen eher zu einer Erhöhung als zu einer Verringerung der mtDNA-Kopienzahl (KE1). Auch wurden nur geringe Auswirkungen auf die mitochondriale Toxizität (KE2) und Zytotoxizität (KE3) beobachtet. Die Behandlung von RPTEC/TERT1 Zellen über einen Zeitraum von 14 Tagen zeigte eine leichte Abnahme der mtDNA Kopienzahl nach Be-handlung mit Adefovirdipivoxil und Tenofovirdisoproxil Fumarat, was den Bedarf an zeit-aufgelösten Daten und Einschränkungen von kurzfristigen in vitro Systemen unterstreicht. Um eine erste Einschätzung für die Risikobewertung basierend auf in vitro Daten zu erhalten, wurden aus den erhaltenen in vitro Daten für jedes KE mögliche Ausgangspunkte (Points of Departure (PoD)) berechnet. Dazu wurden gängige in vitro PoDs berechnet, wie die Effekt-konzentration, bei der 10 % bzw. 20 % Effekt gemessen wurden (EC10, EC20), die höchste Konzentration ohne Wirkung (no observed effect Konzentration (NOEC)), die niedrigste Konzentration mit beobachteter Wirkung (lowest observed effect Konzentration (LOEC)), die Benchmark 10 % (unterer / obere) Konzentrationen (BMC10, BMCL10, BMCU10) und eine modellierte nicht-toxische Konzentration (NtC). Diese wurden dann mit Serum- bzw. Ge-webskonzentrationen aus in vivo Studien verglichen, die nach Gabe therapeutischer / suprathe-rapeutischer Dosen gemessen wurden. Zusätzlich wurde überprüft, ob es mit Hilfe von quanti-tativen Beziehungen zwischen Schlüsselereignissen möglich ist, basierend auf der Bestimmung früher Schlüsselereignisse nachfolgende Effekte vorherzusagen. Diese Untersuchungen zeig-ten eine gute Korrelation der aus den mathematischen Beziehungen modellierten Daten mit den tatsächlich gemessenen Werten der Zytotoxizität der Modellsubstanzen Colistin und Po-lymyxin B-Nonapeptid. Im Rahmen der Arbeit wurden auch Unsicherheiten und Limitationen der Strategie deutlich, die maßgebliche Auswirkungen auf die Vorhersage und auf die Risiko-bewertung basierend auf in vitro Resultaten haben. KW - Adverse outcome pathway (AOP) KW - Nephrotoxicity KW - In vitro testing KW - QIVIVE KW - Risk Assessment Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-264842 ER - TY - JOUR A1 - Jeanclos, Elisabeth A1 - Schlötzer, Jan A1 - Hadamek, Kerstin A1 - Yuan-Chen, Natalia A1 - Alwahsh, Mohammad A1 - Hollmann, Robert A1 - Fratz, Stefanie A1 - Yesilyurt-Gerhards, Dilan A1 - Frankenbach, Tina A1 - Engelmann, Daria A1 - Keller, Angelika A1 - Kaestner, Alexandra A1 - Schmitz, Werner A1 - Neuenschwander, Martin A1 - Hergenröder, Roland A1 - Sotriffer, Christoph A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Gohla, Antje T1 - Glycolytic flux control by drugging phosphoglycolate phosphatase JF - Nature Communications N2 - Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates. KW - phosphoglycolate phosphatase KW - glycolytic flux control KW - intrinsic metabolism Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300928 VL - 13 IS - 1 ER - TY - JOUR A1 - Mally, Angela A1 - Jarzina, Sebastian T1 - Mapping adverse outcome pathways for kidney injury as a basis for the development of mechanism-based animal-sparing approaches to assessment of nephrotoxicity JF - Frontiers in Toxicology N2 - In line with recent OECD activities on the use of AOPs in developing Integrated Approaches to Testing and Assessment (IATAs), it is expected that systematic mapping of AOPs leading to systemic toxicity may provide a mechanistic framework for the development and implementation of mechanism-based in vitro endpoints. These may form part of an integrated testing strategy to reduce the need for repeated dose toxicity studies. Focusing on kidney and in particular the proximal tubule epithelium as a key target site of chemical-induced injury, the overall aim of this work is to contribute to building a network of AOPs leading to nephrotoxicity. Current mechanistic understanding of kidney injury initiated by 1) inhibition of mitochondrial DNA polymerase γ (mtDNA Polγ), 2) receptor mediated endocytosis and lysosomal overload, and 3) covalent protein binding, which all present fairly well established, common mechanisms by which certain chemicals or drugs may cause nephrotoxicity, is presented and systematically captured in a formal description of AOPs in line with the OECD AOP development programme and in accordance with the harmonized terminology provided by the Collaborative Adverse Outcome Pathway Wiki. The relative level of confidence in the established AOPs is assessed based on evolved Bradford-Hill weight of evidence considerations of biological plausibility, essentiality and empirical support (temporal and dose-response concordance). KW - adverse outcome pathway KW - nephrotoxicity KW - protein alkylation KW - lysosomal disruption KW - mitochondrial DNA polymerase γ Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284405 SN - 2673-3080 VL - 4 ER - TY - JOUR A1 - Maurer, Wiebke A1 - Hartmann, Nico A1 - Argyriou, Loukas A1 - Sossalla, Samuel A1 - Streckfuss-Bömeke, Katrin T1 - Generation of homozygous Na\(_{v}\)1.8 knock-out iPSC lines by CRISPR Cas9 genome editing to investigate a potential new antiarrhythmic strategy JF - Stem Cell Research N2 - The sodium channel Na\(_{v}\)1.8, encoded by SCN10A, is reported to contribute to arrhythmogenesis by inducing the late I\(_{Na}\) and thereby enhanced persistent Na\(^{+}\) current. However, its exact electrophysiological role in cardiomyocytes remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) with a homozygous SCN10A knock-out from a healthy iPSC line by CRISPR Cas9 genome editing. The edited iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro differentiation capacity. The iPSCs are able to differentiate into iPSC-cardiomyocytes, hence making it possible to investigate the role of Na\(_{v}\)1.8 in the heart. KW - arrhythmogenesis KW - cardiomyocytes KW - induced pluripotent stem cells Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300936 VL - 60 ER - TY - THES A1 - Bathe-Peters, Marc T1 - Spectroscopic approaches for the localization and dynamics of β\(_1\)- and β\(_2\)-adrenergic receptors in cardiomyocytes T1 - Spektroskopieansätze zur Bestimmung der Lokalisation und Dynamiken von β\(_1\)- und β\(_2\)-Adrenozeptoren in Kardiomyozyten N2 - In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway. In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes. The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses. N2 - Im Herzen werden der β\(_1\)-adrenerge Rezeptor (AR) und der β\(_2\)-AR, zwei prototypische GPCR, durch die Hormone Adrenalin und Noradrenalin aktiviert. Dabei interagieren beide Rezeptoren mit dem stimulatorischen G\(_s\) Protein, bewirken eine Erhöhung des cyclischen Adenosinmonophosphates (cAMP) und beeinflussen die Kontraktionskraft und Frequenz des Herzens nach einem Stimulus. Jedoch hat die Aktivierung des β\(_1\)-ARs, nicht des β\(_2\)-ARs, auch weitere Effekte, wie z.B. Veränderungen in der Transkription von Genen. Dies wiederum führt zu Spekulationen, wie solch unterschiedliche Effekte von Rezeptoren hervorgerufen werden können, die gleiche Signalwege bedienen. In dieser Arbeit wird untersucht, ob dieses unterschiedliche Verhalten durch eine ungleiche Verteilung dieser beiden Rezeptoren in adulten Kardiomyozyten hervorgerufen werden könnte. Dazu wird die Lokalisation und die Dynamik dieser endogenen Rezeptoren in der Plasmamembran sowie im T-tubulären Netzwerk von intakten adulten Kardiomyozyten, unter Entwicklung und Verwendung hochsensitiver Fluoreszenzspektroskopiemethoden, bestimmt. Dies ermöglicht die örtliche und dynamische Eingrenzung des β\(_2\)-adrenergen Rezeptors unter endogener Expression ausschließlich auf das T-tubuläre Netzwerk. Dementgegen stellt sich heraus, dass sich der β\(_1\)-adrenerge Rezeptor ubiquitär auf der äußeren Membran und den T-Tubuli befindet und diffundiert. In β\(_2\)-AR überexprimierenden transgenen Kardiomyozyten hingegen werden diese Kompartments nicht beibehalten und es findet eine Umverteilung der Rezeptoren, auch unter Einbezug der Zelloberfläche, statt. Diese Daten können stärker darauf hindeuten, dass einige Rezeptorsubtypen sich gezielt und spezifisch bestimmte Zelloberflächen aussuchen, um somit ihre verschiedenen Signale und funktionären Effekte erzeugen zu können. Zu den Techniken, die in dieser Arbeit die Bestimmung der Lokalisation und der Dynamiken der niedrig exprimierten adrenergen Rezeptoren zulassen, gehört die Anwendung von Fluoreszenzspektroskopiemethoden in Kombination mit einem fluoreszierenden β-adrenergen Antagonisten. Weitere Techniken, die im Rahmen dieser Arbeit entwickelt wurden und in weiterführenden Studien aufschlussreiche Erkenntnisse liefern könnten, umfassen die Entwicklung eines Setups aus einer Kombination aus Weitfeld- und Konfokalmikroskopie und die Implementierung eines stabilen Autofokus mit Hilfe einer elektrisch veränderbaren Linse. KW - G-Protein gekoppelte Rezeptoren KW - Beta-Adrenozeptor KW - Kardiomyozyt KW - Fluoreszenzmikroskopie KW - Fluoreszenzkorrelationsspektroskopie KW - Fluorescence KW - Fluorescence Microscopy KW - G Protein-Coupled Receptor KW - Autofocus KW - Microscopy KW - Beta-Adrenergic Receptor KW - Cardiomyocyte KW - Fluorescence Correlation Spectroscopy KW - FCS KW - GPCR Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-258126 ER - TY - JOUR A1 - Lorenz, Kristina A1 - Rosner, Marsha Rich T1 - Harnessing RKIP to combat heart disease and cancer JF - Cancers N2 - Cancer and heart disease are leading causes of morbidity and mortality worldwide. These diseases have common risk factors, common molecular signaling pathways that are central to their pathogenesis, and even some disease phenotypes that are interdependent. Thus, a detailed understanding of common regulators is critical for the development of new and synergistic therapeutic strategies. The Raf kinase inhibitory protein (RKIP) is a regulator of the cellular kinome that functions to maintain cellular robustness and prevent the progression of diseases including heart disease and cancer. Two of the key signaling pathways controlled by RKIP are the β-adrenergic receptor (βAR) signaling to protein kinase A (PKA), particularly in the heart, and the MAP kinase cascade Raf/MEK/ERK1/2 that regulates multiple diseases. The goal of this review is to discuss how we can leverage RKIP to suppress cancer without incurring deleterious effects on the heart. Specifically, we discuss: (1) How RKIP functions to either suppress or activate βAR (PKA) and ERK1/2 signaling; (2) How we can prevent cancer-promoting kinase signaling while at the same time avoiding cardiotoxicity. KW - RKIP KW - ERK1/2 KW - PKA KW - βAR KW - heart failure KW - cancer Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262185 SN - 2072-6694 VL - 14 IS - 4 ER - TY - JOUR A1 - Schanbacher, Constanze A1 - Bieber, Michael A1 - Reinders, Yvonne A1 - Cherpokova, Deya A1 - Teichert, Christina A1 - Nieswandt, Bernhard A1 - Sickmann, Albert A1 - Kleinschnitz, Christoph A1 - Langhauser, Friederike A1 - Lorenz, Kristina T1 - ERK1/2 activity is critical for the outcome of ischemic stroke JF - International Journal of Molecular Sciences N2 - Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2\(^{wt}\)) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood–brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIP\(^{wt}\)) and its phosphorylation-deficient mutant RKIP\(^{S153A}\), known inhibitors of the ERK1/2 signaling cascade. RKIP\(^{wt}\) and RKIP\(^{S153A}\) attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke. KW - ERK1/2 KW - tMCAO KW - ischemic stroke KW - RKIP Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-283991 SN - 1422-0067 VL - 23 IS - 2 ER - TY - THES A1 - İşbilir, Ali T1 - Localization and Trafficking of CXCR4 and CXCR7 T1 - Lokalisation und Verteilung von CXCR4 und CXCR7 N2 - G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins, and are the master components that translate extracellular stimulus into intracellular signaling, which in turn modulates key physiological and pathophysiological processes. Research within the last three decades suggests that many GPCRs can form complexes with each other via mechanisms that are yet unexplored. Despite a number of functional evidence in favor of GPCR dimers and oligomers, the existence of such complexes remains controversial, as different methods suggest diverse quaternary organizations for individual receptors. Among various methods, high resolution fluorescence microscopy and imagebased fluorescence spectroscopy are state-of-the-art tools to quantify membrane protein oligomerization with high precision. This thesis work describes the use of single molecule fluorescence microscopy and implementation of two confocal microscopy based fluorescence fluctuation spectroscopy based methods for characterizing the quaternary organization of two class A GPCRs that are important clinical targets: the C-X-C type chemokine receptor 4 (CXCR4) and 7 (CXCR7), or recently named as the atypical chemokine receptor 3 (ACKR3). The first part of the results describe that CXCR4 protomers are mainly organized as monomeric entities that can form transient dimers at very low expression levels allowing single molecule resolution. The second part describes the establishment and use of spatial and temporal brightness methods that are based on fluorescence fluctuation spectroscopy. Results from this part suggests that ACKR3 forms clusters and surface localized monomers, while CXCR4 forms increasing amount of dimers as a function of receptor density in cells. Moreover, CXCR4 dimerization can be modulated by its ligands as well as receptor conformations in distinct manners. Further results suggest that antagonists of CXCR4 display distinct binding modes, and the binding mode influences the oligomerization and the basal activity of the receptor: While the ligands that bind to a “minor” subpocket suppress both dimerization and constitutive activity, ligands that bind to a distinct, “major” subpocket only act as neutral antagonists on the receptor, and do not modulate neither the quaternary organization nor the basal signaling of CXCR4. Together, these results link CXCR4 dimerization to its density and to its activity, which may represent a new strategy to target CXCR4. N2 - G protein-gekoppelte Rezeptoren (GPCRs) bilden die größte Klasse der Membranproteine und sind entscheidend an der Übersetzung extrazellulärer Reize in intrazelluläre Signale beteiligt, welche wiederum unzählige physiologische und pathophysiologische Prozesse regulieren. Die Forschungsergebnisse der letzten drei Jahrzehnte deutet darauf hin, dass viele GPCRs mittels noch weitgehend unbekannter Mechanismen miteinander Komplexe bilden können. Trotz vielfältiger Beobachtungen, die für die funktionelle Relevanz von GPCR-Dimeren und -Oligomeren sprechen, ist deren Existenz dennoch weiterhin umstritten, vor allem da verschiedene Methoden auf unterschiedliche quaternäre Anordnungen derselben Rezeptoren hinweisen. Von den derzeit verfügbaren Methoden zur genauen Untersuchung der GPCR Dimerisierung/-Oligomerisierung, stellen die hochauflösende Fluoreszenzmikroskopie sowie die bildbasierte Fluoreszenzspektroskopie die Techniken der Wahl dar. Die hier vorliegende Arbeit beschreibt die Anwendung der Einzelmolekül Fluoreszenzmikroskopie sowie zweier konfokalmikroskopischer Methoden zur Messung der Fluoreszenzfluktuation, mit deren Hilfe die quaternäre Anordnung zweier klinisch hochattraktiver Klasse A GPCRs untersucht wurde: der C-X-C Typ Chemokinrezeptoren 4 (CXCR4) und 7 (CXCR7), letzterer auch bekannt als atypischer Chemokinrezeptor 3 (ACKR3). Der erste Teil der Ergebnisse legt anhand Untersuchungen an einzelnen Molekülen dar, dass CXCR4 überwiegend in Form monomerer Einheiten auftritt, die bei sehr geringen Expressionsleveln kurzlebige Dimere bilden können. Der zweite Teil beschreibt die Etablierung und Anwendung räumlicher und zeitlicher Brillanzmethoden, die auf der spektroskopischen Untersuchung der Fluoreszenzfluktuation beruhen. Die Ergebnisse dieses Abschnitts deuten darauf hin, dass ACKR3 sowohl in Form beständiger Rezeptor-Cluster, und monomere Einheit an der Oberfläche lebender Zellen auftritt. CXCR4 ist bei zunehmender Rezeptordichte hingegen vermehrt in Form von Dimeren zu finden. Zudem kann die Dimerisierung von CXCR4 von dessen Liganden, als auch von der drei dimensionalen Anordnung der Rezeptorteilstrukturen (Rezeptorkonformation)auf unterschiedliche Weise reguliert werden. Die weiteren Ergebnisse legen nahe, dass Antagonisten auf unterschiedliche Weise an CXCR4 binden können und dass der jeweilige Bindungsmodus entscheidend für den Einfluss des Liganden auf Oligomerisierung und basale Aktivität von CXCR4 ist: Während Liganden, die an eine kleinere Untertasche des Rezeptors binden, sowohl die Dimerisierung als auch die Basalaktivität unterdrücken, fungieren Verbindungen, die an eine andere, größere Untertasche binden, lediglich als neutrale Antagonisten und zeigen keinerlei Einfluss auf die quaternäre Anordnung und basale Aktivität von CXCR4. Zusammenfassend verknüpfen diese Ergebnisse CXCR4-Dimerisierung mit der Rezeptordichte in Zellen und seiner Aktivität, was die Grundlage für neue Strategien zur phamakologischen Modulation von CXCR4 darstellen könnte. KW - G-Protein gekoppelter Rezeptor KW - GPCR KW - Receptor KW - Chemokine KW - oligomerization KW - CXCR4 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-249378 ER - TY - JOUR A1 - Spinaci, Andrea A1 - Lambertucci, Catia A1 - Buccioni, Michela A1 - Dal Ben, Diego A1 - Graiff, Claudia A1 - Barbalace, Maria Cristina A1 - Hrelia, Silvana A1 - Angeloni, Cristina A1 - Tayebati, Seyed Khosrow A1 - Ubaldi, Massimo A1 - Masi, Alessio A1 - Klotz, Karl-Norbert A1 - Volpini, Rosaria A1 - Marucci, Gabriella T1 - A\(_{2A}\) adenosine receptor antagonists: are triazolotriazine and purine scaffolds interchangeable? JF - Molecules N2 - The A\(_{2A}\) adenosine receptor (A\(_{2A}\)AR) is one of the four subtypes activated by nucleoside adenosine, and the molecules able to selectively counteract its action are attractive tools for neurodegenerative disorders. In order to find novel A\(_{2A}\)AR ligands, two series of compounds based on purine and triazolotriazine scaffolds were synthesized and tested at ARs. Compound 13 was also tested in an in vitro model of neuroinflammation. Some compounds were found to possess high affinity for A\(_{2A}\)AR, and it was observed that compound 13 exerted anti-inflammatory properties in microglial cells. Molecular modeling studies results were in good agreement with the binding affinity data and underlined that triazolotriazine and purine scaffolds are interchangeable only when 5- and 2-positions of the triazolotriazine moiety (corresponding to the purine 2- and 8-positions) are substituted. KW - A\(_{2A}\) adenosine receptor antagonist KW - purine derivatives KW - triazolotriazine derivatives KW - anti-Parkinson agents KW - anti-inflammatory agents KW - molecular modeling Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-270618 SN - 1420-3049 VL - 27 IS - 8 ER - TY - JOUR A1 - Jarzina, Sebastian A1 - Di Fiore, Stefano A1 - Ellinger, Bernhard A1 - Reiser, Pia A1 - Frank, Sabrina A1 - Glaser, Markus A1 - Wu, Jiaqing A1 - Taverne, Femke J. A1 - Kramer, Nynke I. A1 - Mally, Angela T1 - Application of the adverse outcome pathway concept to in vitro nephrotoxicity assessment: kidney injury due to receptor-mediated endocytosis and lysosomal overload as a case study JF - Frontiers in Toxicology N2 - Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling. KW - adverse outcome pathway (AOP) KW - nephrotoxicity KW - QIVIVE KW - risk assessment KW - key event relationship KW - In vitro toxicity testing Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284796 SN - 2673-3080 VL - 4 ER - TY - JOUR A1 - Klenk, Christoph A1 - Hommers, Leif A1 - Lohse, Martin J. T1 - Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor JF - Frontiers in Endocrinology N2 - Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling. KW - GPCRs KW - parathyroid hormone 1 receptor KW - matrix metalloproteinase KW - ectodomain cleavage KW - biased signaling Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262055 SN - 1664-2392 VL - 13 ER - TY - THES A1 - Bertelsmann, Dietmar T1 - Analysis of the Frequency of Kidney Toxicity in Preclinical Safety Studies using the eTOX Database T1 - Analyse der Häufigkeit von Nierentoxizität in präklinischen Sicherheitsstudien unter Verwendung der eTOX-Datenbank N2 - This research aimed to obtain reliable data on the frequency of different types of renal toxicity findings in 28-day oral gavage studies in Wistar rats, their consistency across species and study duration, as well as the correlation between histopathological endpoints and routinely used clinical chemistry parameters indicative of kidney injury. Analysis of renal histopathological findings was carried out through extraction of information from the IMI eTOX database. Spontaneous renal histopathological findings in 28-day oral gavage studies in control Wistar rats and beagle dogs confirmed tubular basophilia and renal dilation as the most frequent incidental findings in controls, whereas necrosis and glomerulosclerosis were not identified at all or only rarely as a background lesion. Histopathological evidence of necrosis and glomerulosclerosis was associated with changes in clinical chemistry parameters in 28-day oral gavage Wistar rat studies. Necrosis was frequently accompanied by a statistically significant rise in serum creatinine and serum urea, whereas serum albumin was frequently found to decrease statistically significantly in treatment groups in which necrosis was recorded. In contrast to necrosis, glomerulosclerosis was not associated with statistically significant changes in serum creatinine and urea in any of the 28-day oral gavage Wistar rat treatment groups, but appears to be best reflected by a pattern of statistically significantly lowered serum albumin and serum protein together with a statistically significant increase in serum cholesterol. As might have been expected based on the high background incidences of tubular basophilia and dilation, no consistent changes in any of the clinical chemistry parameters were evident in animals in which renal lesions were con� fined to renal tubular basophilia or dilation. In summary, the routinely provided clinical chemistry parameters are rather insensitive - novel kidney biomarkers such as Cystatin C, β-trace protein and Kidney injury molecule 1 should further be evaluated and integrated into routine preclinical and clinical practice. However, evaluation of clinical chemistry data was limited by the lack of individual animal data. Even though an extensive amount of preclinical studies is accessible through the eTOX database, comparison of consistency across time was limited by the limited number of shorter- and longer term studies conducted with the compounds identified as causing renal histopathological changes within a 28- day study in rats. A high consistency across time for both treatment-related tubular basophilia and treatment-related dilation cannot be confirmed for either of the two effects as these two findings were both induced only rarely in studies over a different treatment-duration other than 28 days after administration of the compounds which provoked the respective effect in a 28-day study. For the finding of necrosis consistency across time was low with the exception of “AZ_GGA_200002321”, in which renal papillary necrosis was identified consist� ently throughout different treatment durations (2, 4, 26, 104 weeks). No shorter and longer-term studies were available for the compounds identified as causing glomerulosclerosis within a 28-day study in rats. No consistent findings of the selected histopathological endpoints were identified in any of the corresponding 28-day oral gavage beagle dog studies after treatment with the identical compounds, which caused the respective ef� fect after 28-day treatment in rats. However, in the overwhelming majority of cases, beagle dogs were administered lower doses in these studies in compar� ison to the corresponding 28-day Wistar rat studies. Searching the eTOX database yielded no 28-day oral gavage studies in Wistar and Wistar Han rats in which accumulation of hyaline droplets, tubular atrophy or hyperplasia was recorded. Only one 28-day oral gavage Wistar rat study was identified with the histopathological result of neutrophilic inflammation. Consequently, evaluation of these four renal findings in relation to clinical chemistry parameters and consistency across time and species cannot be made. In summary, this work contributes knowledge through mining and evaluating the eTOX database on a variety of specific renal endpoints that frequently occur after administration of trial substances in 28-day oral gavage studies in Wistar rats in the field of preclinical toxicity with specific focus on their frequency relation to background findings, as well as consistency across time and species. Targeted statistical evaluation of in vivo data within joint research ventures such as the eTOX project, presents an enormous opportunity for an innovative future way of aiding preclinical research towards a more efficient research in the preclinical stage of drug development. This could be achieved through the aug� mentation of methodological strategies and possibly novel software tools in order to predict in vivo toxicology of new molecular entities by means of information that is already available before early stages of the drug development pipeline begin. N2 - Diese Arbeit zielte darauf ab, verlässliche Daten über die Häufigkeit verschiedener Arten von Nierentoxizitätsbefunden in 28-tägigen oralen Sondenstudien an Wistar-Ratten zu erhalten. Untersucht wurde weiterhin die Konsistenz der Toxizitätsbefunde unterschiedlicher Spezies und Studiendauer sowie die Korrelation zwischen histopathologischen Endpunkten und routinemäßig verwendeten klinisch-chemischen Parametern, die auf eine Nierenschädigung hinweisen. Die Analyse der histopathologischen Nierenbefunde wurde durch Extraktion von Informationen aus der IMI eTOX-Datenbank durchgeführt. Spontane renale histopathologische Befunde in 28-tägigen oralen Sondenstudien an Wistar-Ratten und Beagles bestätigten tubuläre Basophilie und renale Dilatation als häufigste Nebenbefunde bei den Kontrolltieren, während Nekrose und Glomerulosklerose gar nicht oder nur selten als Hintergrundläsion identifiziert wurden. Der histopathologische Nachweis von Nekrose und Glomerulosklerose war mit Änderungen der klinisch-chemischen Parameter in 28-tägigen Wistar-Rattenstudien mit oraler Sonde verbunden. Nekrose ging häufig mit einem statistisch signifikanten Anstieg von Serumkreatinin und Serumharnstoff einher, während Serumalbumin in Behandlungsgruppen, in denen Nekrose aufgezeichnet wurde, häufig statistisch signifikant abnahm. Im Gegensatz zur Nekrose war Glomerulosklerose in keiner der 28-tägigen Wistar-Ratten-Behandlungsgruppen mit oraler Sonde mit statistisch signifikanten Veränderungen von Serumkreatinin und Harnstoff assoziiert, sondern scheint sich am besten in einem Muster von statistisch signifikant erniedrigtem Serumalbumin und Serumprotein zusammen mit einem statistisch signifikanten Anstieg des Serumcholesterins widerzuspiegeln. Wie aufgrund der hohen Hintergrundinzidenzen von tubulärer Basophilie und Dilatation zu erwarten war, waren bei Tieren, bei denen Nierenläsionen auf renale tubuläre Basophilie oder Dilatation beschränkt waren, keine konsistenten Änderungen der klinisch-chemischen Parameter erkennbar. Zusammenfassend sind die routinemäßig bereitgestellten klinisch-chemischen Parameter eher unempfindlich - neuartige Nieren-Biomarker wie „Cystatin C“, „β-trace protein“ und „Kidney injury molecule 1“ sollten weiter evaluiert und in die routinemäßige präklinische und klinische Praxis integriert werden. Die Auswertung der Daten zur klinischen Chemie war jedoch durch das Fehlen individueller Tierdaten begrenzt. Trotz der umfangreichen Anzahl an präklinischen Studien in der eTOX-Datenbank wurde der zeitliche Vergleich der Konsistenz durch die begrenzte Anzahl von Kurz- und Langzeitstudien eingeschränkt, welche mit denselben Substanzen durchgeführt wurden, die innerhalb einer 28-Tage-Studie an Ratten als Verursacher von renalen histopathologischen Veränderungen identifiziert wurden. Eine hohe zeitliche Konsistenz sowohl für die behandlungsbedingte tubuläre Basophilie und Dilatation kann für keinen der beiden Effekte bestätigt werden, da diese beiden Befunde nur selten in Studien über eine andere Behandlungsdauer als 28 Tage nach Verabreichung derselben Substanzen, die den jeweiligen Effekt in einer 28-Tage-Studie hervorriefen, induziert wurden. Für den Befund der Nekrose war die zeitliche Konsistenz gering. Eine Ausnahme stellte Substanz "AZ_GGA_200002321" dar, bei der über verschiedene Behandlungsdauern (2, 4, 26, 104 Wochen) hinweg konstant renale papilläre Nekrose festgestellt wurde. Für die Substanzen, die in einer 28-Tage-Studie an Ratten als glomeruloskleroseauslösend identifiziert wurden, waren keine Kurz- und Langzeitstudien verfügbar. In keiner der korrespondierenden 28-Tage-Studien an Beagles mit oraler Sonde wurden konsistente Befunde der ausgewählten histopathologischen Endpunkte nach Behandlung mit den identischen Verbindungen, die den jeweiligen Effekt nach 28-tägiger Behandlung in Ratten verursachten, festgestellt. In der überwiegenden Mehrheit der Fälle wurden den Beagles in diesen Studien im Vergleich zu den entsprechenden 28-Tage-Wistar-Rattenstudien niedrigere Dosen verabreicht. In der eTOX-Datenbank konnten keine 28-tägigen oralen Sondenstudien an Wistar- und Wistar-Han-Ratten gefunden werden, in denen eine Akkumulation von hyalinen Tröpfchen, tubuläre Atrophie oder Hyperplasie aufgezeichet wurde. Nur eine 28-tägige Wistar-Rattenstudie wurde mit dem histopathologischen Ergebnis einer neutrophilen Entzündung identifiziert. Folglich kann eine Bewertung dieser vier Nierenbefunde in Bezug auf klinische Chemie und Konsistenz über Zeit und Spezies nicht vorgenommen werden. Insgesamt zeigt dieser Arbeit, dass eine gezielte statistische Auswertung von in vivo-Daten im Rahmen von Forschungsverbünden wie dem eTOX-Projekt eine enorme Chance bietet, die präklinische Forschung in Zukunft auf dem Weg zu einer effizienteren Forschung in der präklinischen Phase der Arzneimittelentwicklung zu unterstützen. Dies könnte außerdem durch die Erweiterung methodischer Strategien und möglicherweise neuartiger Software-Tools erreicht werden, um die In-vivo-Toxikologie neuer molekularer Entitäten mit Hilfe von Informationen vorherzusagen, die bereits vor Beginn der Arzneimittelentwicklungspipeline verfügbar sind. KW - renal toxicity KW - etox database KW - rats KW - toxicity Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-257104 ER - TY - JOUR A1 - Chilaka, Cynthia Adaku A1 - Obidiegwu, Jude Ejikeme A1 - Chilaka, Augusta Chinenye A1 - Atanda, Olusegun Oladimeji A1 - Mally, Angela T1 - Mycotoxin regulatory status in Africa: a decade of weak institutional efforts JF - Toxins N2 - Food safety problems are a major hindrance to achieving food security, trade, and healthy living in Africa. Fungi and their secondary metabolites, known as mycotoxins, represent an important concern in this regard. Attempts such as agricultural, storage, and processing practices, and creation of awareness to tackle the menace of fungi and mycotoxins have yielded measurable outcomes especially in developed countries, where there are comprehensive mycotoxin legislations and enforcement schemes. Conversely, most African countries do not have mycotoxin regulatory limits and even when available, are only applied for international trade. Factors such as food insecurity, public ignorance, climate change, poor infrastructure, poor research funding, incorrect prioritization of resources, and nonchalant attitudes that exist among governmental organisations and other stakeholders further complicate the situation. In the present review, we discuss the status of mycotoxin regulation in Africa, with emphasis on the impact of weak mycotoxin legislations and enforcement on African trade, agriculture, and health. Furthermore, we discuss the factors limiting the establishment and control of mycotoxins in the region. KW - fungi KW - mycotoxin KW - legislation KW - food safety KW - food security Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-278941 SN - 2072-6651 VL - 14 IS - 7 ER - TY - JOUR A1 - Tolstik, Elen A1 - Ali, Nairveen A1 - Guo, Shuxia A1 - Ebersbach, Paul A1 - Möllmann, Dorothe A1 - Arias-Loza, Paula A1 - Dierks, Johann A1 - Schuler, Irina A1 - Freier, Erik A1 - Debus, Jörg A1 - Baba, Hideo A. A1 - Nordbeck, Peter A1 - Bocklitz, Thomas A1 - Lorenz, Kristina T1 - CARS imaging advances early diagnosis of cardiac manifestation of Fabry disease JF - International Journal of Molecular Sciences N2 - Vibrational spectroscopy can detect characteristic biomolecular signatures and thus has the potential to support diagnostics. Fabry disease (FD) is a lipid disorder disease that leads to accumulations of globotriaosylceramide in different organs, including the heart, which is particularly critical for the patient’s prognosis. Effective treatment options are available if initiated at early disease stages, but many patients are late- or under-diagnosed. Since Coherent anti-Stokes Raman (CARS) imaging has a high sensitivity for lipid/protein shifts, we applied CARS as a diagnostic tool to assess cardiac FD manifestation in an FD mouse model. CARS measurements combined with multivariate data analysis, including image preprocessing followed by image clustering and data-driven modeling, allowed for differentiation between FD and control groups. Indeed, CARS identified shifts of lipid/protein content between the two groups in cardiac tissue visually and by subsequent automated bioinformatic discrimination with a mean sensitivity of 90–96%. Of note, this genotype differentiation was successful at a very early time point during disease development when only kidneys are visibly affected by globotriaosylceramide depositions. Altogether, the sensitivity of CARS combined with multivariate analysis allows reliable diagnostic support of early FD organ manifestation and may thus improve diagnosis, prognosis, and possibly therapeutic monitoring of FD. KW - coherent anti-Stokes Raman scattering (CARS) microscopy KW - Raman micro-spectroscopy KW - cardiovascular diseases KW - Fabry Disease (FD) KW - Gb3 and lyso-Gb3 biomarkers KW - multivariate data analysis KW - immunohistochemistry Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284427 SN - 1422-0067 VL - 23 IS - 10 ER - TY - JOUR A1 - Gmach, Philipp A1 - Bathe-Peters, Marc A1 - Telugu, Narasimha A1 - Miller, Duncan C. A1 - Annibale, Paolo T1 - Fluorescence spectroscopy of low-level endogenous β-adrenergic receptor expression at the plasma membrane of differentiating human iPSC-derived cardiomyocytes JF - International Journal of Molecular Sciences N2 - The potential of human-induced pluripotent stem cells (hiPSCs) to be differentiated into cardiomyocytes (CMs) mimicking adult CMs functional morphology, marker genes and signaling characteristics has been investigated since over a decade. The evolution of the membrane localization of CM-specific G protein-coupled receptors throughout differentiation has received, however, only limited attention to date. We employ here advanced fluorescent spectroscopy, namely linescan Fluorescence Correlation Spectroscopy (FCS), to observe how the plasma membrane abundance of the β\(_1\)- and β\(_2\)-adrenergic receptors (β\(_{1/2}\)-ARs), labelled using a bright and photostable fluorescent antagonist, evolves during the long-term monolayer culture of hiPSC-derived CMs. We compare it to the kinetics of observed mRNA levels in wildtype (WT) hiPSCs and in two CRISPR/Cas9 knock-in clones. We conduct these observations against the backdrop of our recent report of cell-to-cell expression variability, as well as of the subcellular localization heterogeneity of β-ARs in adult CMs. KW - GPCR KW - β-adrenergic receptors KW - hiPSC-CM KW - cardiomyocyte KW - fluorescence correlation spectroscopy KW - FCS KW - fluorescence KW - CRISPR/Cas9 KW - differentiation Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288277 SN - 1422-0067 VL - 23 IS - 18 ER - TY - JOUR A1 - Djelić, Ninoslav A1 - Borozan, Sunčica A1 - Dimitrijević-Srećković, Vesna A1 - Pajović, Nevena A1 - Mirilović, Milorad A1 - Stopper, Helga A1 - Stanimirović, Zoran T1 - Oxidative stress and DNA damage in peripheral blood mononuclear cells from normal, obese, prediabetic and diabetic persons exposed to thyroid hormone in vitro JF - International Journal of Molecular Sciences N2 - Diabetes, a chronic group of medical disorders characterized byhyperglycemia, has become a global pandemic. Some hormones may influence the course and outcome of diabetes, especially if they potentiate the formation of reactive oxygen species (ROS). There is a close relationship between thyroid disorders and diabetes. The main objective of this investigation was to find out whether peripheral blood mononuclear cells (PBMCs) are more prone to DNA damage by triiodothyronine (T\(_3\)) (0.1, 1 and 10 μM) at various stages of progression through diabetes (obese, prediabetics, and type 2 diabetes mellitus—T2DM persons). In addition, some biochemical parameters of oxidative stress (catalase-CAT, thiobarbituric acid reactive substances—TBARS) and lactate dehydrogenase (LDH) were evaluated. PBMCs from prediabetic and diabetic patients exhibited increased sensitivity for T\(_3\) regarding elevated level of DNA damage, inhibition of catalase, and increase of TBARS and LDH. PBMCs from obese patients reacted in the same manner, except for DNA damage. The results of this study should contribute to a better understanding of the role of thyroid hormones in the progression of T2DM. KW - diabetes KW - oxidative stress KW - DNA damage KW - lymphocytes KW - thyroid hormone Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285988 SN - 1422-0067 VL - 23 IS - 16 ER - TY - JOUR A1 - Sedaghat-Hamedani, Farbod A1 - Rebs, Sabine A1 - Kayvanpour, Elham A1 - Zhu, Chenchen A1 - Amr, Ali A1 - Müller, Marion A1 - Haas, Jan A1 - Wu, Jingyan A1 - Steinmetz, Lars M. A1 - Ehlermann, Philipp A1 - Streckfuss-Bömeke, Katrin A1 - Frey, Norbert A1 - Meder, Benjamin T1 - Genotype complements the phenotype: identification of the pathogenicity of an LMNA splice variant by nanopore long-read sequencing in a large DCM family JF - International Journal of Molecular Sciences N2 - Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20–40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5′ splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies. KW - familial DCM KW - laminopathy KW - long-read sequencing KW - nanopore KW - induced pluripotent stem cell cardiomyocytes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290415 SN - 1422-0067 VL - 23 IS - 20 ER - TY - JOUR A1 - Bittner, Nataly A1 - Boon, Andy A1 - Delbanco, Evert H. A1 - Walter, Christof A1 - Mally, Angela T1 - Assessment of aromatic amides in printed food contact materials: analysis of potential cleavage to primary aromatic amines during simulated passage through the gastrointestinal tract JF - Archives of Toxicology N2 - Recent analyses conducted by German official food control reported detection of the aromatic amides N-(2,4-dimethylphenyl)acetamide (NDPA), N-acetoacetyl-m-xylidine (NAAX) and 3-hydroxy-2-naphthanilide (Naphthol AS) in cold water extracts from certain food contact materials made from paper or cardboard, including paper straws, paper napkins, and cupcake liners. Because aromatic amides may be cleaved to potentially genotoxic primary amines upon oral intake, these findings raise concern that transfer of NDPA, NAAX and Naphthol AS from food contact materials into food may present a risk to human health. The aim of the present work was to assess the stability of NDPA, NAAX and Naphthol AS and potential cleavage to 2,4-dimethylaniline (2,4-DMA) and aniline during simulated passage through the gastrointestinal tract using static in vitro digestion models. Using the digestion model established by the National Institute for Public Health and the Environment (RIVM, Bilthoven, NL) and a protocol recommended by the European Food Safety Authority, potential hydrolysis of the aromatic amides to the respective aromatic amines was assessed by LC–MS/MS following incubation of the aromatic amides with digestive fluid simulants. Time-dependent hydrolysis of NDPA and NAAX resulting in formation of the primary aromatic amine 2,4-DMA was consistently observed in both models. The highest rate of cleavage of NDPA and NAAX was recorded following 4 h incubation with 0.07 M HCl as gastric-juice simulant, and amounted to 0.21% and 0.053%, respectively. Incubation of Naphthol AS with digestive fluid simulants did not give rise to an increase in the concentration of aniline above the background that resulted from the presence of aniline as an impurity of the test compound. Considering the lack of evidence for aniline formation from Naphthol AS and the extremely low rate of hydrolysis of the amide bonds of NDPA and NAAX during simulated passage through the gastrointestinal tract that gives rise to only very minor amounts of the potentially mutagenic and/or carcinogenic aromatic amine 2,4-DMA, risk assessment based on assumption of 100% cleavage to the primary aromatic amines would appear to overestimate health risks related to the presence of aromatic amides in food contact materials. KW - aromatic amides KW - primary aromatic amine KW - food contact materials KW - simulated digestion Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324697 VL - 96 IS - 5 ER - TY - THES A1 - Nemec, Katarina T1 - Modulation of parathyroid hormone 1 receptor (PTH1R) signaling by receptor activity-modifying proteins (RAMPs) T1 - Regulierung der Signalübertragung des Parathormon 1-Rezeptors (PTH1R) durch Rezeptoraktivitäts-modifizierende Proteine (RAMPs) N2 - The receptor activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that interact with several G protein-coupled receptors (GPCRs), the largest and pharmacologically most important family of cell surface receptors. RAMPs can regulate GPCR function in terms of ligand-binding, G-protein coupling, downstream signaling, trafficking, and recycling. The integrity of their interactions translates to many physiological functions or pathological conditions. Regardless of numerous reports on its essential importance for cell biology and pivotal role in (patho-)physiology, the molecular mechanism of how RAMPs modulate GPCR activation remained largely elusive. This work presents new insights that add to the common understanding of the allosteric regulation of receptor activation and will help interpret how accessory proteins - RAMPs - modulate activation dynamics and how this affects the fundamental aspects of cellular signaling. Using a prototypical class B GPCR, the parathyroid hormone 1 receptor (PTH1R) in the form of advanced genetically encoded optical biosensors, I examined RAMP's impact on the PTH1R activation and signaling in intact cells. A panel of single-cell FRET and confocal microscopy experiments as well canonical and non-canonical functional assays were performed to get a holistic picture of the signaling initiation and transduction of that clinically and therapeutically relevant GPCR. Finally, structural modeling was performed to add molecular mechanistic details to that novel art of modulation. I describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique pre-activated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity and kinetics of cAMP accumulation. Additionally, RAMP2 increases PTH- and PTHrP-triggered β-arrestin2 recruitment to PTH1R and modulates cytosolic ERK1/2 phosphorylation. Structural homology modeling shows that structural motifs governing GPCR-RAMP interaction originate in allosteric hotspots and rationalize functional modulation. Moreover, to interpret the broader role of RAMP's modulation in GPCRs pharmacology, different fluorescent tools to investigate RAMP's spatial organization were developed, and novel conformational biosensors for class B GPCRs were engineered. Lastly, a high throughput assay is proposed and prototyped to expand the repertoire of RAMPs or other membrane protein interactors. These data uncover the critical role of RAMPs in GPCR activation and signaling and set up a novel platform for studying GPCR modulation. Furthermore, these insights may provide a new venue for precise modulation of GPCR function and advanced drug design. N2 - G Protein-gekoppelte Rezeptoren (GPCRs) bilden die größte und pharmakologisch wichtigste Familie von Zelloberflächenrezeptoren, die zahlreiche (patho-)physiologische Prozesse im menschlichen Körper steuern. GPCRs übertragen während des Rezeptoraktivierungsprozesses extrazelluläre Signale in das Zellinnere, wo durch die extrazelluläre Stimulation Konformationsänderungen des Rezeptorkerns auslöst und die Bindung intrazellulärer Bindungspartner – G Proteine, G Protein-gekoppelte Rezeptorkinase und Arrestine - ermöglicht. Es handelt sich also um einen kritischen Prozess in der Signaltransduktion, der durch einige endogene Moleküle wie Ionen, Lipide oder andere Proteine moduliert werden kann und Auswirkungen auf nachgeschaltete Signalkaskaden hat. GPCRs bilden gewebeabhängige Oligomere mit ihren interagierenden Partnern, Rezeptor-Aktivitäts-modifizierende Proteinen (RAMPs), ubiquitär exprimierten Membranproteinen. Bekannt ist, dass sie die Ligandenbindung, die G- Protein-Kopplung, die nachgeschaltete Signalisierung, das Trafficking und das Recycling einiger GPCRs modulieren. Ihre Rolle im kritischsten Prozess der Signaltransduktion - der Rezeptoraktivierung - wurde jedoch nur begrenzt erforscht. Anhand des physiologisch und therapeutisch wichtigen Parathormon-Rezeptors (PTH1R), einem GPCR der Klasse B, wurden die Modulationseffekte von RAMPs auf den Prozess der Rezeptoraktivierung und ihre Folgen für die nachgeschaltete Signalübertragung analysiert. Hierzu wurden verschiedene optische Biosensoren zur Messung der Aktivierung des PTH1R und seiner Signalkaskade entwickelt und in verschiedenen Versuchsanordnungen eingesetzt, mit dem Ziel einen holistischen Blick auf die Interaktion zwischen PTH1R und RAMPs und ihre funktionellen Auswirkungen zu erhalten. Die Interaktion zwischen PTH1R und RAMPs erwies sich als besonders ausgeprägt für RAMP2, und RAMP2 zeigte eine spezifische allosterische Modulation der PTH1R-Konformation, sowohl im basalen als auch im Liganden- aktivierten Zustand. Ein einzigartiger voraktivierter oder (meta-stabiler) Zustand ermöglichte eine schnellere Rezeptoraktivierung auf Liganden-spezifische Weise. Außerdem beeinflusste RAMP2 die G Protein- und Nicht-G Protein-vermittelte Signalübertragung indem es die PTH-vermittelte Gi3-Signalempfindlichkeit und die Kinetik der cAMP-Akkumulation modulierte. Weiterhin erhöhte RAMP2 die Menge der β-Arrestin2-Rekrutierung an PTH1R auf Liganden-spezifische Weise. Dies könnte mit einer erhöhten zytosolischen ERK-Menge zusammenhängen, die hat sich von der nukleären ERK-Phosphorylierung unterscheidet. Um einen molekularen Mechanismus für die vorgestellten Ergebnisse vorzuschlagen, wurden mehrere strukturelle Modelle entwickelt und analysiert. Diese Arbeit liefert den Beweis, dass RAMP die GPCR-Aktivierung mit funktionellen Auswirkungen auf die zelluläre Signalübertragung reguliert. Die Ergebnisse sollten im Zusammenhang mit zellspezifischen Koexpressionsmustern interpretiert werden und können zur Entwicklung von fortschrittlichen Therapeutika positiv beitragen. Da GPCRs praktisch alle Zellfunktionen koordinieren und seit jeher wichtigen Angriffspunkten für Medikamente sind, tragen die vorgestellten Erkenntnisse zum universellen Verständnis der molekularen Mechanismen bei, die den menschlichen Körper orchestrieren. KW - G-Protein gekoppelter Rezeptor KW - GPCR KW - RAMP KW - PTH1R KW - FRET KW - BRET KW - pharmacology KW - Fluoreszenz-Resonanz-Energie-Transfer KW - Förster Resonanz Energie Transfer Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-288588 ER - TY - JOUR A1 - Nwogha, Jeremiah S. A1 - Abtew, Wosene G. A1 - Raveendran, Muthurajan A1 - Oselebe, Happiness O. A1 - Obidiegwu, Jude E. A1 - Chilaka, Cynthia A. A1 - Amirtham, Damodarasamy D. T1 - Role of non-structural sugar metabolism in regulating tuber dormancy in white yam (Dioscorea rotundata) JF - Agriculture N2 - Changes in sugar composition occur continuously in plant tissues at different developmental stages. Tuber dormancy induction, stability, and breaking are very critical developmental transitions in yam crop production. Prolonged tuber dormancy after physiological maturity has constituted a great challenge in yam genetic improvement and productivity. In the present study, biochemical profiling of non-structural sugar in yam tubers during dormancy was performed to determine the role of non-structural sugar in yam tuber dormancy regulation. Two genotypes of the white yam species, one local genotype (Obiaoturugo) and one improved genotype (TDr1100873), were used for this study. Tubers were sampled at 42, 56, 87, 101, 115, and 143 days after physiological maturity (DAPM). Obiaoturugo exhibited a short dormant phenotype and sprouted at 101-DAPM, whereas TDr1100873 exhibited a long dormant phenotype and sprouted at 143-DAPM. Significant metabolic changes were observed in non-structural sugar parameters, dry matter, and moisture content in Obiaoturugo from 56-DAPM, whereas in TDr1100873, significant metabolic changes were observed from 101-DAPM. It was observed that the onset of these metabolic changes occurred at a point when the tubers of both genotypes exhibited a dry matter content of 60%, indicating that a dry matter content of 60% might be a critical threshold for white yam tuber sprouting. Non-reducing sugars increased by 9–10-fold during sprouting in both genotypes, which indicates their key role in tuber dormancy regulation in white yam. This result implicates that some key sugar metabolites can be targeted for dormancy manipulation of the yam crop. KW - sugars KW - metabolism KW - yam KW - tuber KW - genotypes KW - dormancy KW - regulation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304486 SN - 2077-0472 VL - 13 IS - 2 ER - TY - JOUR A1 - Hadi, Naji Said Aboud A1 - Bankoglu, Ezgi Eyluel A1 - Stopper, Helga T1 - Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells JF - Archives of Toxicology N2 - Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells. KW - co-culture KW - micronuclei KW - mitotic disturbance KW - cytochrome P450s KW - membrane transporters KW - pyrrolizidine alkaloids Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-324708 VL - 97 IS - 1 ER - TY - JOUR A1 - Schanbacher, Constanze A1 - Hermanns, Heike M. A1 - Lorenz, Kristina A1 - Wajant, Harald A1 - Lang, Isabell T1 - Complement 1q/tumor necrosis factor-related proteins (CTRPs): structure, receptors and signaling JF - Biomedicines N2 - Adiponectin and the other 15 members of the complement 1q (C1q)/tumor necrosis factor (TNF)-related protein (CTRP) family are secreted proteins composed of an N-terminal variable domain followed by a stalk region and a characteristic C-terminal trimerizing globular C1q (gC1q) domain originally identified in the subunits of the complement protein C1q. We performed a basic PubMed literature search for articles mentioning the various CTRPs or their receptors in the abstract or title. In this narrative review, we briefly summarize the biology of CTRPs and focus then on the structure, receptors and major signaling pathways of CTRPs. Analyses of CTRP knockout mice and CTRP transgenic mice gave overwhelming evidence for the relevance of the anti-inflammatory and insulin-sensitizing effects of CTRPs in autoimmune diseases, obesity, atherosclerosis and cardiac dysfunction. CTRPs form homo- and heterotypic trimers and oligomers which can have different activities. The receptors of some CTRPs are unknown and some receptors are redundantly targeted by several CTRPs. The way in which CTRPs activate their receptors to trigger downstream signaling pathways is largely unknown. CTRPs and their receptors are considered as promising therapeutic targets but their translational usage is still hampered by the limited knowledge of CTRP redundancy and CTRP signal transduction. KW - adiponectin KW - AMPK KW - C1q/TNF related protein (CTRP) KW - inflammation KW - metabolism Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304136 SN - 2227-9059 VL - 11 IS - 2 ER - TY - JOUR A1 - Rebs, Sabine A1 - Streckfuss-Bömeke, Katrin T1 - How can we use stem cell-derived cardiomyocytes to understand the involvement of energetic metabolism in alterations of cardiac function? JF - Frontiers in Molecular Medicine N2 - Mutations in the mitochondrial-DNA or mitochondria related nuclear-encoded-DNA lead to various multisystemic disorders collectively termed mitochondrial diseases. One in three cases of mitochondrial disease affects the heart muscle, which is called mitochondrial cardiomyopathy (MCM) and is associated with hypertrophic, dilated, and noncompact cardiomyopathy. The heart is an organ with high energy demand, and mitochondria occupy 30%–40% of its cardiomyocyte-cell volume. Mitochondrial dysfunction leads to energy depletion and has detrimental effects on cardiac performance. However, disease development and progression in the context of mitochondrial and nuclear DNA mutations, remains incompletely understood. The system of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) is an excellent platform to study MCM since the unique genetic identity to their donors enables a robust recapitulation of the predicted phenotypes in a dish on a patient-specific level. Here, we focus on recent insights into MCM studied by patient-specific iPSC-CM and further discuss research gaps and advances in metabolic maturation of iPSC-CM, which is crucial for the study of mitochondrial dysfunction and to develop novel therapeutic strategies. KW - mitochondrial cardiomyopathy KW - iPSC-cardiomyocytes KW - maturation strategies KW - Barth syndrome KW - Friedreich’s ataxia KW - lysosomal storage disorders Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-327344 VL - 3 ER - TY - JOUR A1 - Hartmann, Nico A1 - Knierim, Maria A1 - Maurer, Wiebke A1 - Dybkova, Nataliya A1 - Hasenfuß, Gerd A1 - Sossalla, Samuel A1 - Streckfuss-Bömeke, Katrin T1 - Molecular and functional relevance of Na\(_V\)1.8-induced atrial arrhythmogenic triggers in a human SCN10A knock-out stem cell model JF - International Journal of Molecular Sciences N2 - In heart failure and atrial fibrillation, a persistent Na\(^+\) current (I\(_{NaL}\)) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that Na\(_V\)1.8 contributes to arrhythmogenesis by inducing a I\(_{NaL}\). Genome-wide association studies indicate that mutations in the SCN10A gene (Na\(_V\)1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these Na\(_V\)1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the I\(_{NaL}\) and action potential duration. Ca\(^{2+}\) measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca\(^{2+}\) leak. The I\(_{NaL}\) was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of Na\(_V\)1.8. No effects on atrial APD\(_{90}\) were detected in any groups. Both SCN10A KO and specific blockers of Na\(_V\)1.8 led to decreased Ca\(^{2+}\) spark frequency and a significant reduction of arrhythmogenic Ca\(^{2+}\) waves. Our experiments demonstrate that Na\(_V\)1.8 contributes to I\(_{NaL}\) formation in human atrial CMs and that Na\(_V\)1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore Na\(_V\)1.8 could be a new target for antiarrhythmic strategies. KW - Na\(_V\)1.8 KW - iPSC-cardiomyocytes KW - late Na\(^+\) current (I\(_{NaL}\)) KW - CRISPR Cas9 Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-362708 SN - 1422-0067 VL - 24 IS - 12 ER - TY - JOUR A1 - Eberl, Hanna A1 - Rebs, Sabine A1 - Hoppe, Stefanie A1 - Sedaghat-Hamedani, Farbod A1 - Kayvanpour, Elham A1 - Meder, Benjamin A1 - Streckfuss-Bömeke, Katrin T1 - Generation of an RBM20-mutation-associated left-ventricular non-compaction cardiomyopathy iPSC line (UMGi255-A) into a DCM genetic background to investigate monogenetic cardiomyopathies JF - Stem Cell Research N2 - RBM20 mutations account for 3 % of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient. KW - cell biology KW - developmental biology KW - general medicine KW - RBM20 mutations KW - DCM genetic background KW - monogenetic cardiomyopathies Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350565 SN - 1873-5061 VL - 74 ER - TY - JOUR A1 - Janz, Anna A1 - Walz, Katharina A1 - Cirnu, Alexandra A1 - Surjanto, Jessica A1 - Urlaub, Daniela A1 - Leskien, Miriam A1 - Kohlhaas, Michael A1 - Nickel, Alexander A1 - Brand, Theresa A1 - Nose, Naoko A1 - Wörsdörfer, Philipp A1 - Wagner, Nicole A1 - Higuchi, Takahiro A1 - Maack, Christoph A1 - Dudek, Jan A1 - Lorenz, Kristina A1 - Klopocki, Eva A1 - Ergün, Süleyman A1 - Duff, Henry J. A1 - Gerull, Brenda T1 - Mutations in DNAJC19 cause altered mitochondrial structure and increased mitochondrial respiration in human iPSC-derived cardiomyocytes JF - Molecular Metabolism N2 - Highlights • Loss of DNAJC19's DnaJ domain disrupts cardiac mitochondrial structure, leading to abnormal cristae formation in iPSC-CMs. • Impaired mitochondrial structures lead to an increased mitochondrial respiration, ROS and an elevated membrane potential. • Mutant iPSC-CMs show sarcomere dysfunction and a trend to more arrhythmias, resembling DCMA-associated cardiomyopathy. Background Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy. Methods We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca\(^{2+}\) kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tv\(_{HeLa}\)). Results Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca\(^{2+}\) concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation. Conclusions Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca\(^{2+}\) kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy. KW - cell biology KW - molecular biology KW - dilated cardiomyopathy with ataxia KW - genetics KW - metabolism KW - mitochondria KW - OXPHOS KW - ROS KW - contractility Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350393 SN - 2212-8778 VL - 79 ER - TY - INPR A1 - Brenner, Marian A1 - Zink, Christoph A1 - Witzinger, Linda A1 - Keller, Angelika A1 - Hadamek, Kerstin A1 - Bothe, Sebastian A1 - Neuenschwander, Martin A1 - Villmann, Carmen A1 - von Kries, Jens Peter A1 - Schindelin, Hermann A1 - Jeanclos, Elisabeth A1 - Gohla, Antje T1 - 7,8-Dihydroxyflavone is a direct inhibitor of pyridoxal phosphatase T2 - eLife N2 - Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small molecule screening, protein crystallography and biolayer interferometry, we discover and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain. KW - 7,8-dihydroxyflavone (7,8-DHF) KW - pyridoxal phosphatase (PDXP) KW - vitamin B6 KW - PDXP inhibitors Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350446 ER -