Differentielle Proteomanalyse und Charakterisierung von Oberflächenproteinen des retinalen Pigmentepithels gesunder und an ERU erkrankter Pferde

Die equine rezidivierende Uveitis (ERU) ist eine sehr häufig auftretende autoimmune Augenerkrankung bei Pferden, welche meist mit dem Verlust der Sehfähigkeit der betroffenen Augen einhergeht. Da die ERU das einzig spontane Tiermodell für die humane autoimmune Uveitis darstellt, ist die Erforschung der zugrundeliegenden Pathomechanismen der ERU nicht nur veterinärmedizinisch, sondern auch für die Humanmedizin von großer Bedeutung. Charakteristisch für die ERU sind der Zusammenbruch der Blut-Retina-Schranke (BRS) und die Infiltration von autoaggressiven T-Lymphozyten in das innere Auge mit anschließender Zerstörung retinaler Strukturen. Beim Pferd wird die BRS, aufgrund der weitestgehend avaskulären Retina, hauptsächlich von der äußeren Komponente der BRS gebildet, dem retinalen Pigmentepithel (RPE). Im physiologischen Zustand stellt das RPE durch feste Zell-Zellverbindungen sowohl eine stabile mechanische, als auch durch seine Fähigkeit, mit Mediatoren des Immunsystems kommunizieren und interagieren zu können, eine effektive immunologische Barriere dar. Die im Verlauf der ERU stattfindenden pathophysiologischen Mechanismen, welche für den Zusammenbruch dieser Barriere verantwortlich sind, konnten bislang nicht ausreichend geklärt werden. Vor allem Änderungen im Expressionsmuster des Zelloberflächenproteoms könnten hierbei aufgrund der ständigen Interaktion und Kommunikation der RPE-Zellen mit ihrer Umgebung eine entscheidende Rolle spielen. Deshalb war es das Ziel dieser Arbeit, differentiell regulierte Zelloberflächen-proteine zwischen gesunden und uveitischen RPE-Zellen zu detektieren, welche maßgeblich an der Pathogenese der ERU beteiligt sein könnten. Um so nah wie möglich die am RPE in vivo stattfindenden physiologischen und pathophysiologischen Prozesse widerspiegeln zu können, wurden RPE-Zelloberflächenproteine von gesunden und an ERU erkrankten Pferden in dieser Studie mittels einer neuartigen in situ Biotinylierungsmethode angereichert und anschließend massenspektrometrisch analysiert. Dabei konnten insgesamt 148 Proteine identifiziert werden, von denen 81,8 % Plasmamembranproteine waren, was deutlich für den Erfolg der neuartigen Anreicherungsmethode sprach. Unter den 148 insgesamt identifizierten Proteinen befanden sich 27 differentiell regulierte Proteine, wovon in uveitischem RPE drei hoch- und 24 herunterreguliert waren. Neben den für RPE-Zellen klassischen Proteinen wie RPE65, Rhodopsin und S-Arrestin konnten auch mehrere Proteine detektiert werden, die unseres Wissens zuvor noch nicht in RPE-Zellen beschrieben wurden, wie der Glukosetransporter 4, Synaptotagmin 1 und Peripherin 2. Funktionell besonders interessant fanden wir die vier Proteine Synaptotagmin 1, Basigin, Collectrin und Perpherin 2, welche alle mit einer verminderten Expression in uveitischem RPE zu finden waren. Interessanterweise ergab sich aus einer Pathway-Analyse für alle vier Proteine eine Beteiligung an „Visual Functions“ und „Immunological Diseases“. Mittels weiterführender Analysen wie der Durchflusszytometrie, der Immunhistologie und der Quantifizierung der Protein-Fluoreszenzintensitäten ist es gelungen die bereits massenspektrometrisch identifizierte verminderte Expression von Synaptotagmin 1, Basigin, Collectrin und Perpherin 2 zu verifizieren und die Proteine näher zu charakterisieren. Die in dieser Arbeit präsentierte neuartige in situ Biotinylierungsmethode zur Anreicherung von Oberflächenproteinen, welche anschließend mittels LC-MS/MS identifiziert wurden, erwies sich als sehr effektive und innovative Methode, um Oberflächenproteine so nah wie möglich in ihrem physiologischen und pathophysiologischen in vivo Vorkommen zu untersuchen. Daher liefert der in dieser Arbeit generierte Datensatz der differentiell regulierten Proteine zwischen gesunden und uveitischen RPE-Zellen eine solide Grundlage für weitere funktionelle Analysen zur Aufklärung der Pathogenese der ERU.Equine recurrent uveitis (ERU) is a highly prevalent autoimmune eye disease in horses, which usually results in blindness of affected eyes. Since ERU is the only spontaneous animal model for human autoimmune uveitis, investigations of underlying pathomechanisms of ERU are not only of high importance for veterinary medicine, but also for human medicine. Characteristic features of ERU are the breakdown of blood-retinal barrier (BRB) and infiltration of autoaggressive T-lymphocytes into the inner eye with subsequent destruction of retinal structures. The BRB of the horse is, due to its widely avascular retina, formed by the outer component of the BRB, the retinal pigment epithelium (RPE). Based on its strong cell-cell connections, the RPE constitutes a solid mechanical barrier and due to its ability to communicate and interact with mediators of the immune system, an effective immunological barrier under physiological conditions, as well. Pathophysiological mechanisms taking place in course of ERU, which are responsible for the breakdown of BRB, were not sufficiently clarified to date. In this context, especially changes of cell surface proteome could play a decisive role due to the continuous communication and interaction of the RPE cells with their environment. Therefore, the goal of this study was to detect differentially expressed cell surface proteins of healthy and uveitic RPE cells, which might crucially contribute to the pathogenesis of ERU. To reflect physiological and pathophysiological processes taking place at the RPE as close as possible to the in vivo situation, cell surface proteins of RPE cells of healthy and ERU diseased horses were captured by a novel in situ biotinylation method and afterwards analyzed by mass spectrometry. Thereby, a total of 148 proteins were identified, of which 81.8 % were plasma membrane proteins, representing the success of this novel enrichment method. 27 of 148 totally identified proteins were differentially expressed. Among these, three proteins were upregulated and 24 proteins were downregulated in uveitic RPE. In addition to proteins typically expressed by RPE cells like RPE65, rhodopsin and S-arrestin, several proteins like glucose transporter 4, synaptotagmin 1 and peripherin 2 were detected, which to our knowledge were not described in RPE cells so far. Of high functional interest to us were four proteins, namely synaptotagmin 1, basigin, collectrin and peripherin 2, which were all downregulated in uveitic RPE cells. Interestingly, a pathway analysis showed involvement of all four proteins in “visual functions” and “immunological diseases”. By performing further analysis such as flow cytometry, immunohistochemistry and quantification of fluorescence intensities, reduced expression of synaptotagmin 1, basigin, collectrin and peripherin 2, which were identified by mass spectrometry, was verified and the proteins were further characterized. In this study, a novel in situ biotinylation method used for cell surface protein enrichment with following identification of these proteins by LC-MS/MS was undertaken. The method proved to be an effective and innovative method to investigate cell surface proteins as closely as possible to their physiological and pathophysiological appearance in vivo. Therefore, the data set of differentially regulated proteins of healthy and uveitic RPE cells, which was generated within the present study, sets a great foundation for further functional analysis for clarifying the pathogenesis of ERU

Differentielle Proteomanalyse und Charakterisierung von Oberflächenproteinen des retinalen Pigmentepithels gesunder und an ERU erkrankter Pferde

Die equine rezidivierende Uveitis (ERU) ist eine sehr häufig auftretende autoimmune Augenerkrankung bei Pferden, welche meist mit dem Verlust der Sehfähigkeit der betroffenen Augen einhergeht. Da die ERU das einzig spontane Tiermodell für die humane autoimmune Uveitis darstellt, ist die Erforschung der zugrundeliegenden Pathomechanismen der ERU nicht nur veterinärmedizinisch, sondern auch für die Humanmedizin von großer Bedeutung. Charakteristisch für die ERU sind der Zusammenbruch der Blut-Retina-Schranke (BRS) und die Infiltration von autoaggressiven T-Lymphozyten in das innere Auge mit anschließender Zerstörung retinaler Strukturen. Beim Pferd wird die BRS, aufgrund der weitestgehend avaskulären Retina, hauptsächlich von der äußeren Komponente der BRS gebildet, dem retinalen Pigmentepithel (RPE). Im physiologischen Zustand stellt das RPE durch feste Zell-Zellverbindungen sowohl eine stabile mechanische, als auch durch seine Fähigkeit, mit Mediatoren des Immunsystems kommunizieren und interagieren zu können, eine effektive immunologische Barriere dar. Die im Verlauf der ERU stattfindenden pathophysiologischen Mechanismen, welche für den Zusammenbruch dieser Barriere verantwortlich sind, konnten bislang nicht ausreichend geklärt werden. Vor allem Änderungen im Expressionsmuster des Zelloberflächenproteoms könnten hierbei aufgrund der ständigen Interaktion und Kommunikation der RPE-Zellen mit ihrer Umgebung eine entscheidende Rolle spielen. Deshalb war es das Ziel dieser Arbeit, differentiell regulierte Zelloberflächen-proteine zwischen gesunden und uveitischen RPE-Zellen zu detektieren, welche maßgeblich an der Pathogenese der ERU beteiligt sein könnten. Um so nah wie möglich die am RPE in vivo stattfindenden physiologischen und pathophysiologischen Prozesse widerspiegeln zu können, wurden RPE-Zelloberflächenproteine von gesunden und an ERU erkrankten Pferden in dieser Studie mittels einer neuartigen in situ Biotinylierungsmethode angereichert und anschließend massenspektrometrisch analysiert. Dabei konnten insgesamt 148 Proteine identifiziert werden, von denen 81,8 % Plasmamembranproteine waren, was deutlich für den Erfolg der neuartigen Anreicherungsmethode sprach. Unter den 148 insgesamt identifizierten Proteinen befanden sich 27 differentiell regulierte Proteine, wovon in uveitischem RPE drei hoch- und 24 herunterreguliert waren. Neben den für RPE-Zellen klassischen Proteinen wie RPE65, Rhodopsin und S-Arrestin konnten auch mehrere Proteine detektiert werden, die unseres Wissens zuvor noch nicht in RPE-Zellen beschrieben wurden, wie der Glukosetransporter 4, Synaptotagmin 1 und Peripherin 2. Funktionell besonders interessant fanden wir die vier Proteine Synaptotagmin 1, Basigin, Collectrin und Perpherin 2, welche alle mit einer verminderten Expression in uveitischem RPE zu finden waren. Interessanterweise ergab sich aus einer Pathway-Analyse für alle vier Proteine eine Beteiligung an „Visual Functions“ und „Immunological Diseases“. Mittels weiterführender Analysen wie der Durchflusszytometrie, der Immunhistologie und der Quantifizierung der Protein-Fluoreszenzintensitäten ist es gelungen die bereits massenspektrometrisch identifizierte verminderte Expression von Synaptotagmin 1, Basigin, Collectrin und Perpherin 2 zu verifizieren und die Proteine näher zu charakterisieren. Die in dieser Arbeit präsentierte neuartige in situ Biotinylierungsmethode zur Anreicherung von Oberflächenproteinen, welche anschließend mittels LC-MS/MS identifiziert wurden, erwies sich als sehr effektive und innovative Methode, um Oberflächenproteine so nah wie möglich in ihrem physiologischen und pathophysiologischen in vivo Vorkommen zu untersuchen. Daher liefert der in dieser Arbeit generierte Datensatz der differentiell regulierten Proteine zwischen gesunden und uveitischen RPE-Zellen eine solide Grundlage für weitere funktionelle Analysen zur Aufklärung der Pathogenese der ERU.Equine recurrent uveitis (ERU) is a highly prevalent autoimmune eye disease in horses, which usually results in blindness of affected eyes. Since ERU is the only spontaneous animal model for human autoimmune uveitis, investigations of underlying pathomechanisms of ERU are not only of high importance for veterinary medicine, but also for human medicine. Characteristic features of ERU are the breakdown of blood-retinal barrier (BRB) and infiltration of autoaggressive T-lymphocytes into the inner eye with subsequent destruction of retinal structures. The BRB of the horse is, due to its widely avascular retina, formed by the outer component of the BRB, the retinal pigment epithelium (RPE). Based on its strong cell-cell connections, the RPE constitutes a solid mechanical barrier and due to its ability to communicate and interact with mediators of the immune system, an effective immunological barrier under physiological conditions, as well. Pathophysiological mechanisms taking place in course of ERU, which are responsible for the breakdown of BRB, were not sufficiently clarified to date. In this context, especially changes of cell surface proteome could play a decisive role due to the continuous communication and interaction of the RPE cells with their environment. Therefore, the goal of this study was to detect differentially expressed cell surface proteins of healthy and uveitic RPE cells, which might crucially contribute to the pathogenesis of ERU. To reflect physiological and pathophysiological processes taking place at the RPE as close as possible to the in vivo situation, cell surface proteins of RPE cells of healthy and ERU diseased horses were captured by a novel in situ biotinylation method and afterwards analyzed by mass spectrometry. Thereby, a total of 148 proteins were identified, of which 81.8 % were plasma membrane proteins, representing the success of this novel enrichment method. 27 of 148 totally identified proteins were differentially expressed. Among these, three proteins were upregulated and 24 proteins were downregulated in uveitic RPE. In addition to proteins typically expressed by RPE cells like RPE65, rhodopsin and S-arrestin, several proteins like glucose transporter 4, synaptotagmin 1 and peripherin 2 were detected, which to our knowledge were not described in RPE cells so far. Of high functional interest to us were four proteins, namely synaptotagmin 1, basigin, collectrin and peripherin 2, which were all downregulated in uveitic RPE cells. Interestingly, a pathway analysis showed involvement of all four proteins in “visual functions” and “immunological diseases”. By performing further analysis such as flow cytometry, immunohistochemistry and quantification of fluorescence intensities, reduced expression of synaptotagmin 1, basigin, collectrin and peripherin 2, which were identified by mass spectrometry, was verified and the proteins were further characterized. In this study, a novel in situ biotinylation method used for cell surface protein enrichment with following identification of these proteins by LC-MS/MS was undertaken. The method proved to be an effective and innovative method to investigate cell surface proteins as closely as possible to their physiological and pathophysiological appearance in vivo. Therefore, the data set of differentially regulated proteins of healthy and uveitic RPE cells, which was generated within the present study, sets a great foundation for further functional analysis for clarifying the pathogenesis of ERU

Job satisfaction of midwives working in a labor ward : a repeat measure mixed-methods study

INTRODUCTION: Job satisfaction of midwives is important to prevent skill shortage. Those working in midwife-led models of care work more independently and have more responsibility. No previous study investigated if a self-initiated and self-responsible project could enhance job satisfaction of midwives working in a medical-led maternity unit. The aim of this study was therefore to assess job satisfaction before and after the implementation of such a project. METHODS: This is longitudinal observational study at three time points using quantitative and qualitative methods. A total of 43 midwives working in a Swiss labor ward participated in the online surveys and in the focus group discussions. The surveys comprised questions from validated instruments to assess job satisfaction. Descriptive and multivariable time series analysis were used for quantitative and content analysis for qualitative data. RESULTS: Adjusted predicted scores decreased between t0 and t1, and subsequently increased at t2 without reaching baseline values (e.g. ‘professional support subscales’ between t0 and t1: (0.65; 95% CI: 0.45–0.86 vs 0.26; 95% CI: 0.08–0.45, p=0.005) and between t0 and t2 (0.65; 95% CI: 0.45–0.86 vs 0.29; 95% CI: 0.12–0.47, p=0.004). Focus group discussions revealed four themes: ‘general job satisfaction’, ‘challenges with the implementation’, ‘continuity of care’ and ‘meaning for the mothers’. Midwives perceived the additional tasks as stressors. CONCLUSIONS: The implementation of new projects might enhance work-related stress and consequently have negative impacts on job satisfaction in an early phase. Heads of institutions and policy makers should recognize the needs of support and additional resources for staff when implementing new projects

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation

Vascular surveillance by haptotactic blood platelets in inflammation and infection

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets

EZH2 inactivation in RAS-driven myeloid neoplasms hyperactivates RAS-signaling and increases MEK inhibitor sensitivity.

Funder: MUG (within the PhD program Molecular Medicine)Funder: Austrian Society of Internal Medicine (Joseph-Skoda Award), Austrian Science Fund (grant P32783), Austrian Society of Hematology and Medical Oncology (Clinical Research Grant) and MEFOgrazFunder: Austrian Science Fund (grant P 31430-458 B26) and Leukämiehilfe SteiermarkFunder: Austrian Society of Internal Medicine (Joseph-Skoda Award)and Leukämiehilfe Steiermar

Analog, digital - Opposition oder Kontinuum? : zur Theorie und Geschichte einer Unterscheidung

Forschungsprojekt gefördert durch die DFGDie Opposition der 'neuen digitalen' zu den 'alten analogen' Medien findet sich in Werbung, Popkultur, Wirtschaft, Politik und Wissenschaft. Offenbar hat sich die Unterscheidung analog/digital zur paradigmatischen Leitdifferenz des späten 20. und frühen 21. Jahrhunderts entwickelt. Doch was bedeutet 'analog' bzw. 'digital' in verschiedenen Kontexten genau und gibt es nicht auch Übergänge zwischen beiden Formen? Wann taucht die Unterscheidung auf und in welchem Zusammenhang? Indem sich die Anthologie mit diesen und anderen Fragen aus verschiedenen Perspektiven beschäftigt, räumt sie ein erhebliches Forschungsdefizit nicht nur in den Medienwissenschaften aus

With increasing site quality asymmetric competition and mortality reduces Scots pine (Pinus sylvestris L.) stand structuring across Europe

Heterogeneity of structure can increase mechanical stability, stress resistance and resilience, biodiversity and many other functions and services of forest stands. That is why many silvicultural measures aim at enhancing structural diversity. However, the effectiveness and potential of structuring may depend on the site conditions. Here, we revealed how the stand structure is determined by site quality and results from site-dependent partitioning of growth and mortality among the trees. We based our study on 90 mature, even-aged, fully stocked monocultures of Scots pine (Pines sylvestris L.) sampled in 21 countries along a productivity gradient across Europe. A mini-simulation study further analyzed the site-dependency of the interplay between growth and mortality and the resulting stand structure. The overarching hypothesis was that the stand structure changes with site quality and results from the site-dependent asymmetry of competition and mortality.First, we show that Scots pine stands structure across Europe become more homogeneous with increasing site quality. The coefficient of variation and Gini coefficient of stem diameter and tree height continuously decreased, whereas Stand Density Index and stand basal area increased with site index.Second, we reveal a site-dependency of the growth distribution among the trees and the mortality. With increasing site index, the asymmetry of both competition and growth distribution increased and suggested, at first glance, an increase in stand heterogeneity. However, with increasing site index, mortality eliminates mainly small instead of all-sized trees, cancels the size variation and reduces the structural heterogeneity.Third, we modelled the site-dependent interplay between growth partitioning and mortality. By scenario runs for different site conditions, we can show how the site-dependent structure at the stand level emerges from the asymmetric competition and mortality at the tree level and how the interplay changes with increasing site quality across Europe.Our most interesting finding was that the growth partitioning became more asymmetric and structuring with increasing site quality, but that the mortality eliminated predominantly small trees, reduced their size variation and thus reversed the impact of site quality on the structure. Finally, the reverse effects of mode of growth partitioning and mortality on the stand structure resulted in the highest size variation on poor sites and decreased structural heterogeneity with increasing site quality. Since our results indicate where heterogeneous structures need silviculture interventions and where they emerge naturally, we conclude that these findings may improve system understanding and modelling and guide forest management aiming at structurally rich forests