Vergleich der Spaltungsprozesse des sezernierten mCLCA3- und des transmembranären mCLCA6-Proteins

CLCA proteins, originally termed chloride channels, calcium activated, have on the one hand been described to play a modulatory role in diseases with secretory dysfunctions, predominantly cystic fibrosis (CF), asthma or chronic obstructive pulmonary disease, and, on the other hand, in cancer. They possess a broad tissue expression pattern including mucous membranes of various organs. Members of the CLCA protein family modulate endogenous chloride conductances in cultured cells in a still elusive way. The cellular processing of CLCA proteins may indicate their role as signalling molecules because CLCA proteins are either fully secreted proteins or possess one single transmembrane domain in the carboxy-terminal subunit, while the amino-terminal subunit undergoes ectodomain shedding. A recently identified HEXXH zinc binding motif indicates that CLCA proteins might act as metalloproteases, implying a putative channel activating function. This study addresses the question of whether CLCA proteins might indeed represent metalloproteases. As all CLCA proteins undergo post-translational cleavage, the cleavage process of murine mCLCA3, a secreted CLCA family member relevant for CF as an example for secretory diseases, was investigated with regard to an autoproteolytic activity. Furthermore, this study includes the comparison of the cleavage processes of a secreted murine CLCA family member with a murine CLCA family member of CF relevant tissues possessing a transmembrane domain. The results were supposed to either corroborate or neglect the hypotheses that CLCA proteins are metalloproteases (hypothesis I) and that the cleavage processes of secreted CLCA proteins differ from those of transmembrane CLCA proteins due to the transmembrane domain (hypothesis II). Therefore, this study will set the stage for investigating the proteolytic role of the putative CLCA proteases in the fields of secretory disorders or cancer. The tissue expression pattern and the role of the secreted murine CLCA family member mCLCA3 in CF mouse models have been intensively studied in the past. To identify a murine CLCA family member with a transmembrane domain in CF relevant tissues, the tissue expression patterns and cellular processing of murine mCLCA5 and mCLCA6 were investigated. The mCLCA5 protein was expressed in keratinizing keratinocytes of stratified squamous epithelium of skin, cervix, stomach and other organs. Though cell culture experiments and computational analyses suggested a transmembrane domain in the carboxy- terminal subunit, this study failed to identify the carboxy-terminal subunit associated with the plasma membrane in immunohistochemical analyses. The mCLCA5 protein was rather associated with keratohyaline granules. In contrast, the mCLCA6 protein was identified at the apical plasma membrane of non-goblet cell enterocytes in both the small and large intestine but in no other organs. In addition, the presence of a transmembrane domain of mCLCA6 was corroborated via acid treatment. Thus, mCLCA6 was used as a murine transmembrane CLCA family member expressed in intestine as a CF relevant tissue and its cleavage process was compared with that of the secreted mCLCA3 expressed in intestinal goblet cells. In metalloproteases, the HEXXH zinc-binding amino acid motif is involved in the catalytic process. Mutation E157Q of the HEXXH motif of mCLCA3 or mCLCA6 abrogated cleavage of both proteins in the endoplasmic reticulum, consistent with the previously reported data for hCLCA1. In contrast to mCLCA3E157Q whose uncleaved precursor was fully secreted similar to the wild- type protein, the precursor molecule of mCLCA6E157Q was cleaved at the plasma membrane instead of the endoplasmic reticulum. Both the cleavage of mCLCA3 in the endoplasmic reticulum and the cleavage of mCLCA6E157Q at the plasma membrane were zinc-dependent. In contrast to mCLCA3, however, which was capable of intermolecular autoproteolytic cleavage, the cleaving agent of mCLCA6E157Q at the plasma membrane remains unidentified. The cleavage may therefore be performed by a metalloprotease or represents an autoproteolytic process. Interestingly, the delayed cleavage of mCLCA6E157Q does not require membrane association via a transmembrane domain, raising the question of whether the same is true for other transmembrane CLCA or whether it is specific for mCLCA6. The results of this study support the hypothesis that at least secreted CLCA proteins represent metalloproteases because their cleavage is zinc-dependent, abrogated after mutation of the HEXXH motif and they are capable of intermolecular proteolysis, possibly even intramolecular cleavage. The in vivo substrates of CLCA proteases of cluster 1 as well as their role in secretory disorders or cancer remain to be established. Furthermore, the mutant mCLCA6E157Q protein underwent rescue cleavage while the mutant mCLCA3E157Q protein did not. This rescue cleavage was not dependent on the transmembrane domain of the protein. Protein characteristics other than the transmembrane domain might therefore be responsible for the different cleavage processes of the mCLCA3E157Q and the mCLCA6E157Q proteins. These protein characteristics should be addressed in the future. CLCA proteases of cluster 1 may play a role in diseases with secretory dysfunctions including CF as well as in tumor biology. Roles of other metalloproteases in CF include activation of chloride channels, hypersecretion and degradation of mucus. The exact role of CLCA proteases in CF and other diseases requires knowledge on potential substrates and the substrate specificity of CLCA proteases. Investigation of the conserved amino acids at the cleavage site of mCLCA3 could give a first hint towards the substrate specificity of CLCA proteases of cluster 1 and might provide the basis for potential therapeutic interventions in the future.Für CLCA Proteine, ursprünglich als Calcium-aktivierbare Chloridkanäle bezeichnet, wurden modulatorische Funktionen bei verschiedenen Krankheitsbildern mit sekretorischer Dysfunktion wie CF, Asthma und COPD oder auch bei Tumoren beschrieben. Ihr breites Expressionsspektrum umfasst vor allem Schleimhäute in verschiedenen Organen. Zellkulturexperimente wiesen für einige CLCA Vertreter nach, dass sie endogene Calciumaktivierbare Chloridionenströme modulieren. Der genaue Funktionsmechanismus dieser Modulation ist bisher unbekannt, die zelluläre Prozessierung gibt jedoch Hinweise auf eine mögliche Funktion als Signalmoleküle. CLCA Proteine werden entweder vollständig sezerniert oder sind mit einer einzigen Transmembrandomäne in der Plasmamembran verankert, so dass nur die amino- terminale Untereinheit abgegeben wird. Daher ist eine Funktion als eigenständiger Kanal unwahrscheinlich. Eine aktuelle Studie identifizierte ein HEXXH Aminosäuremotiv in CLCA Proteinen. Da dieses HEXXH Motiv vor allem bei Zinkbindenden Metalloproteasen beschrieben wurde, ist eine möglicherweise aktivierende Proteasefunktion von CLCA Proteinen durchaus denkbar. Diese Arbeit beschäftigt sich mit der Frage, ob CLCA Proteine tatsächlich eine Gruppe von Metalloproteasen darstellen. Alle CLCA Proteine werden post- translational gespalten. Dieser proteolytische Prozessierungsschritt wurde in dieser Studie auf eine auto-proteolytische Aktivität von CLCA Proteinen untersucht. Dabei wurden ein sezerniertes und ein transmembranäres CLCA Protein verglichen mit Fokussierung auf CF-relevante murine CLCA Vertreter als Beispiel für eine Rolle von CLCA Proteinen in Dyskrinien. Die Ergebnisse sollten die Hypothese, dass CLCA Proteine Metalloproteasen sind, entweder unterstützen oder widerlegen und somit Möglichkeiten für die gezielte Untersuchung der Rolle von CLCA Proteinen bei Krankheiten wie CF, Asthma, COPD oder Krebs bieten. Das Gewebsexpressionsmuster von sezernierten CLCA Proteinen und besonders die Rolle des murinen mCLCA3 in CF Mausmodellen wurden in der Vergangenheit bereits intensiv erforscht. Zunächst befasste sich diese Studie daher mit der Charakterisierung eines membrangebundenen, murinen CLCA Proteins in CF-relevanten Geweben. Zu diesem Zweck wurden das Expressionsspektrum und die zelluläre Prozessierung der CLCA Vertreter mCLCA5 und mCLCA6 untersucht. Das mCLCA5 Protein wird von Keratinozyten des Stratum granulosum in mehrschichtigen Plattenepithelien exprimiert, ein Zelltyp, der für CF keine bedeutsame Rolle spielt. Obgleich in Zellkulturexperimenten mit transfizierten HEK293 Zellen beide Untereinheiten den Golgi Apparat passieren, wird nur das amino-terminale Spaltprodukt von mCLCA5 abgegeben. Eine Lokalisation der carboxy-terminalen Untereinheit an der Plasmamembran von Keratinozyten konnte jedoch nicht nachgewiesen werden, in diesen Zellen ist die carboxyterminale Untereinheit assoziiert mit keratohyalinen Granula. Eine Transmembrandomäne erscheint somit in vitro nicht eindeutig. Das mCLCA6 Protein befindet sich in der apikalen Plasmamembran von Enterozyten des Dick- und Dünndarms, es ist in den Krypten des Dickdarms mit dem CFTR Protein colokalisiert. Eine Transmembrandomäne in der carboxy-terminalen Untereinheit konnte mittels Acid Release nachgewiesen werden. Das mCLCA6 Protein ist somit der vielversprechendere Kandidat für eine Modulation bei CF und auch aufgrund der bewiesenen Transmembrandomäne besser für die geplante Studie geeignet. Daher lag der Fokus dieser Studie im Folgenden auf dem Vergleich des Spaltungsprozesses von mCLCA3, dem sezernierten CLCA Protein aus Becherzellen, und mCLCA6, dem transmembranären CLCA Protein aus Nicht-Becherzell Enterozyten. Die Spaltung von mCLCA3 und mCLCA6 findet im endoplasmatischen Retikulum statt. Beide Proteine tragen das HEXXH Motiv, und nach Mutation E157Q dieses HEXXH Motives bleibt die Spaltung aus. Das mutierte mCLCA3E157Q Protein passiert dann den Golgi Apparat und wird als komplex glykosyliertes, ungespaltenes Protein in den Überstand sezerniert. Im Gegensatz dazu wird das mutierte mCLCA6E157Q Protein zwar ebenfalls durch den Golgi Apparat transportiert, es gelangt jedoch an die Plasmamembran, wird dort mittels Transmembrandomäne verankert und gespalten, so dass nur die amino-terminale Untereinheit abgegeben wird. Die Spaltung an der Plasmamembran ist zinkabhängig, es handelt sich entweder um eine verspätete Selbstspaltung des Proteins oder um Proteolyse durch eine Metalloprotease. Der Spaltungsmechanismus ist jedoch nicht abhängig von der Verankerung des mCLCA6E157Q Proteins in der Plasmamembran. Auch ein sezerniertes Trunkat, dem die Transmembrandomäne fehlt, wurde nach der E157Q Mutation noch gespalten. Offensichtlich spielen andere Faktoren bei der unterschiedlichen Prozessierung von mCLCA6 eine Rolle. Dies wirft die Frage auf, ob eine Unterteilung von CLCA Proteinen in sezernierte Proteine und membranständige Proteine ausreichend ist oder ob eine andersartige Unterteilung möglich und sinnvoll wäre. Das mCLCA3 Protein spaltet sich im endoplasmatischen Retikulum mittels intermolekularer Auto-Proteolyse in Abhängigkeit von Zink. Die Spaltung erfolgt zwischen Aminosäure R695 und A696. Das mCLCA3 Protein ist somit in der Lage, ein anderes Protein zu erkennen und zu spalten. Nachgewiesen wurde dies zwar bisher nur für das mCLCA3 Protein selbst, aber zukünftige Studien werden sich mit der Identifizierung von Substraten der CLCA Proteine befassen. Neben der Fähigkeit zur intermolekularen Autoproteolyse sind die Zinkabhängigkeit des Spaltungsprozesses und das katalytisch aktive HEXXH Motiv Hinweise, die die Hypothese untermauern, dass CLCA Proteine des Cluster 1 tatsächlich Metalloproteasen sind. Für die anderen CLCA Cluster spricht zwar bisher nichts gegen eine Metalloproteaseaktivität, der endgültige Beweis muss jedoch noch erbracht werden. CLCA Proteasen des Cluster 1 könnten sowohl bei Krankheiten mit sekretorischer Dysfunktion wie Cystischer Fibrose als auch bei Tumoren eine Rolle spielen. Beispielsweise wurde für andere Proteasen bei CF eine Rolle sowohl bei der Aktivierung von Chloridkanälen als auch bei der Hypersekretion von Mucus oder sogar beim Abbau des vermehrten Mucus propagiert. Die genaue Rolle von CLCA Proteasen bei den genannten Krankheiten erfordert Kenntnisse der Substrate und der Substratspezifität dieser Proteasen. Einen ersten Hinweis auf die Substratspezifität kann die Untersuchung der konservierten Aminosäuren der CLCA Spaltstelle liefern. Damit wird die Erforschung der modulatorischen Funktion von CLCA Proteasen des Cluster 1 bei den genannten Krankheitsfeldern möglich sein und somit die Basis für zukünftige therapeutische Anwendungsgebiete darstellen

Efficient three-material PLIC interface positioning on unstructured polyhedral meshes

This paper introduces an efficient algorithm for the sequential positioning (or nested dissection) of two planar interfaces in an arbitrary polyhedron, such that, after each truncation, the respectively remaining polyhedron admits a prescribed volume. This task, among others, is frequently encountered in the numerical simulation of three-phase flows when resorting to the geometric Volume-of-Fluid method. For two-phase flows, the recent work of Kromer & Bothe (doi.org/10.1016/j.jcp.2021.110776) addresses the positioning of a single plane by combining an implicit bracketing of the sought position with up to third-order derivatives of the volume fraction. An analogous application of their highly efficient root-finding scheme to three-material configurations requires computing the volume of a twice truncated arbitrary polyhedron. The present manuscript achieves this by recursive application of the Gaussian divergence theorem in appropriate form, which allows to compute the volume as a sum of quantities associated to the faces of the original polyhedron. With a suitable choice of the coordinate origin, accounting for the sequential character of the truncation, the volume parametrization becomes co-moving with respect to the planes. This eliminates the necessity to establish topological connectivity and tetrahedron decomposition after each truncation. After a detailed mathematical description of the concept, we conduct a series of carefully designed numerical experiments to assess the performance in terms of polyhedron truncations. The high efficiency of the two-phase positioning persists for sequential application, thereby being robust with respect to input data and possible intersection topologies. In comparison to an existing decomposition-based approach, the number of truncations was reduced by up to an order of magnitude

Development of a robot-human-interface using an RGBD camera

Against the backdrop of global economic development, industrial assembly in Germany is in a state of change, prompted by the country’s high-wage business environment. This article describes the need for new technologies to the increase flexibility and security regarding human-robot cooperation. Furthermore, a possible model for managing this transition is described on detail, involving the use of an RGBD camera from Microsoft. With this camera, it should be possible to detect state and position changes of people in a human-robot workstation and consequently adapt the movements of the robot. Overall, the essential aim of this paper is to suggest ways to increase economic efficiency within assembly processes along with increasing security

Treatment of Focal Cartilage Defects in Minipigs with Zonal Chondrocyte/Mesenchymal Progenitor Cell Constructs

Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (_CT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Preclinical Testing of New Hydrogel Materials for Cartilage Repair: Overcoming Fixation Issues in a Large Animal Model

Reinforced hydrogels represent a promising strategy for tissue engineering of articular cartilage. They can recreate mechanical and biological characteristics of native articular cartilage and promote cartilage regeneration in combination with mesenchymal stromal cells. One of the limitations of in vivo models for testing the outcome of tissue engineering approaches is implant fixation. The high mechanical stress within the knee joint, as well as the concave and convex cartilage surfaces, makes fixation of reinforced hydrogel challenging. Methods. Different fixation methods for full-thickness chondral defects in minipigs such as fibrin glue, BioGlue®, covering, and direct suturing of nonenforced and enforced constructs were compared. Because of insufficient fixation in chondral defects, superficial osteochondral defects in the femoral trochlea, as well as the femoral condyle, were examined using press-fit fixation. Two different hydrogels (starPEG and PAGE) were compared by 3D-micro-CT (μCT) analysis as well as histological analysis. Results. Our results showed fixation of below 50% for all methods in chondral defects. A superficial osteochondral defect of 1 mm depth was necessary for long-term fixation of a polycaprolactone (PCL)-reinforced hydrogel construct. Press-fit fixation seems to be adapted for a reliable fixation of 95% without confounding effects of glue or suture material. Despite the good integration of our constructs, especially in the starPEG group, visible bone lysis was detected in micro-CT analysis. There was no significant difference between the two hydrogels (starPEG and PAGE) and empty control defects regarding regeneration tissue and cell integration. However, in the starPEG group, more cell-containing hydrogel fragments were found within the defect area. Conclusion. Press-fit fixation in a superficial osteochondral defect in the medial trochlear groove of adult minipigs is a promising fixation method for reinforced hydrogels. To avoid bone lysis, future approaches should focus on multilayered constructs recreating the zonal cartilage as well as the calcified cartilage and the subchondral bone plate

Self-reported changes in sleep patterns and behavior in children and adolescents during COVID-19

Abstract The COVID-19 pandemic and lockdowns worldwide forced children and adolescents to change and adapt their lives to an unprecedented situation. Using an online survey, we investigated whether they showed changes in sleep quality and other related factors due to this event. Between February 21st, 2021 and April 19th, 2021, a total of 2,290 Austrian children and adolescents (6–18 years) reported their sleep habits and quality of sleep as well as physical activity, daylight exposure and usage of media devices during and, retrospectively, before the pandemic. Results showed an overall delay of sleep and wake times. Almost twice as many respondents reported having sleeping problems during the pandemic as compared to before, with insomnia, nightmares and daytime sleepiness being the most prevalent problems. Furthermore, sleeping problems and poor quality of sleep correlated positively with COVID-19 related anxiety. Lastly, results showed a change from regular to irregular bedtimes during COVID-19, higher napping rates, a strong to very strong decrease in physical activity and daylight exposure, as well as a high to very high increase in media consumption. We conclude that the increase in sleeping problems in children and adolescent during COVID-19 is concerning. Thus, health promoting measures and programs should be implemented and enforced