Die Signalvermittlung der Rezeptor-Tyrosinkinase Ror2 in mesenchymalen Bindegewebszellen

Distinkte Mutationen in der Rezeptor-Tyrosinkinase Ror2 (Regeneron orphan receptor 2) resultieren u.a. in schweren skelettalen Fehlbildungen wie der dominanten Brachydaktylie Typ B. Trotz des Wissens über die Funktion von Ror2 und die Auswirkungen von Mutationen auf die Embryonalentwicklung in vielen Organismen ist nur sehr wenig über die Signalwege bekannt, in die Ror2 involviert ist. In dieser Arbeit konnten neue Interaktionspartner von Ror2 identifiziert und funktionell charakterisiert werden, was einen besseren Einblick in Aufbau und Funktion von Ror2-abhängigen Signalwegen ermöglicht. Sowohl TAK1 (TGF-beta-aktivierte Kinase 1), ein bedeutender Regulator der Wnt-Signalwege, als auch Bprp (Basic proline-rich protein) interagieren mit den C-terminalen Domänen von Ror2. Die TAK1/Ror2-Interaktion führt dabei zur Phosphorylierung eines TGY-Motivs innerhalb der distalen Serin-/Threonin-reichen Domäne von Ror2. Diese intrazelluläre Ror2-Interaktion mit TAK1 wird durch die extrazelluläre Bindung von Wnt-1, nicht jedoch Wnt-5a, unterdrückt. Parallel konnte in einem Wnt-abhängigen Reporter-System gezeigt werden, dass die Bindung von Wnt-1 an Ror2 zu einer Stimulierung des kanonischen Wnt-Signalweges führt. Die Deletion der C-terminalen Serin-/Threonin- und Prolin-reichen Sequenzen führt zu einer erheblichen Steigerung dieses Effektes und erlaubt daher die Rolle von Ror2 bei verschieden schweren Ausprägungen der Brachydaktylie Typ B auf eine Steigerung des kanonischen Wnt-Signalweges zurückzuführen.Distinct mutations within the receptor tyrosine kinase Ror2 (Regeneron orphan receptor 2) result, amongst others, in brachydactyly type B, a severe autosomal dominant skeletal disorder. Considerable knowledge about Ror2 and its mutations in embryonic development exist but only little is known about the signalling pathways Ror2 is involved in. In this thesis new interacting partners of Ror2 were identified and functionally characterized allowing a deeper insight into the composition and function of Ror2-dependent signalling pathways. Both TAK1 (TGF-beta-activated kinase 1), a crucial regulator of Wnt signaling pathways, and Bprp (basic proline-rich protein) interact with the C-terminal domains of Ror2. TAK1/Ror2 interaction leads to the phosphorylation of a TGY-motif within the distal serine-/threonine-rich domain of Ror2. This intracellular Ror2 interaction with TAK1 is suppressed by extracellular Wnt-1- but not by Wnt-5a-binding. A Wnt-dependent reporter system showed that the association of Wnt-1 with Ror2 leads to the stimulation of the canonical Wnt signaling pathway. Deletion of the C-terminal serine-/threonine- and proline-rich sequences entails a significant increase in canonical Wnt signalling and allows, therefore, the hypothesis that the Ror2-dependent level of canonical Wnt signaling may be responsible for the marked variability of brachydactyly type B phenotypes

Influence of the Available Surface Area and Cell Elasticity on Bacterial Adhesion Forces on Highly Ordered Silicon Nanopillars

[Image: see text] Initial bacterial adhesion to solid surfaces is influenced by a multitude of different factors, e.g., roughness and stiffness, topography on the micro- and nanolevel, as well as chemical composition and wettability. Understanding the specific influences and possible interactive effects of all of these factors individually could lead to guidance on bacterial adhesion and prevention of unfavorable consequences like medically relevant biofilm formation. On this way, the aim of the present study was to identify the specific influence of the available surface area on the adhesion of clinically relevant bacterial strains with different membrane properties: Gram-positive Staphylococcus aureus and Gram-negative Aggregatibacter actinomycetemcomitans. As model surfaces, silicon nanopillar specimens with different spacings were fabricated using electron beam lithography and cryo-based reactive ion etching techniques. Characterization by scanning electron microscopy and contact angle measurement revealed almost defect-free highly ordered nanotopographies only varying in the available surface area. Bacterial adhesion forces to these specimens were quantified by means of single-cell force spectroscopy exploiting an atomic force microscope connected to a microfluidic setup (FluidFM). The nanotopographical features reduced bacterial adhesion strength by reducing the available surface area. In addition, the strain-specific interaction in detail depended on the bacterial cell’s elasticity and deformability as well. Analyzed by confocal laser scanning microscopy, the obtained results on bacterial adhesion forces could be linked to the subsequent biofilm formation on the different topographies. By combining two cutting-edge technologies, it could be demonstrated that the overall bacterial adhesion strength is influenced by both the simple physical interaction with the underlying nanotopography and its available surface area as well as the deformability of the cell

Spines, skeletons and the strong law of large numbers for superdiffusions

Consider a supercritical superdiffusion (X t ) t≥0 on a domain D⊆R d with branching mechanism (x,z)↦−β(x)z+α(x)z 2 +∫ (0,∞) (e −zy −1+zy)Π(x,dy). The skeleton decomposition provides a pathwise description of the process in terms of immigration along a branching particle diffusion. We use this decomposition to derive the strong law of large numbers (SLLN) for a wide class of superdiffusions from the corresponding result for branching particle diffusions. That is, we show that for suitable test functions f and starting measures μ , ⟨f,X t ⟩P μ [⟨f,X t ⟩] →W ∞ P μ -almost surely as t→∞, where W ∞ is a finite, non-deterministic random variable characterized as a martingale limit. Our method is based on skeleton and spine techniques and offers structural insights into the driving force behind the SLLN for superdiffusions. The result covers many of the key examples of interest and, in particular, proves a conjecture by Fleischmann and Swart [Stochastic Process. Appl. 106 (2003) 141–165] for the super-Wright–Fisher diffusion. <br/

PH-responsive release of chlorhexidine from modified nanoporous silica nanoparticles for dental applications

A pH-sensitive stimulus-response system for controlled drug release was prepared by modifying nanoporous silica nanoparticles (NPSNPs) with poly(4-vinylpyridine) using a bismaleimide as linker. At physiological pH values, the polymer serves as gate keeper blocking the pore openings to prevent the release of cargo molecules. At acidic pH values as they can occur during a bacterial infection, the polymer strains become protonated and straighten up due to electrostatic repulsion. The pores are opened and the cargo is released. The drug chlorhexidine was loaded into the pores because of its excellent antibacterial properties and low tendency to form resistances. The release was performed in PBS and diluted hydrochloric acid, respectively. The results showed a considerably higher release in acidic media compared to neutral solvents. Reversibility of this pH-dependent release was established. In vitro tests proved good cytocompatibility of the prepared nanoparticles. Antibacterial activity tests with Streptococcus mutans and Staphylococcus aureus revealed promising perspectives of the release system for biofilm prevention. The developed polymer-modified silica nanoparticles can serve as an efficient controlled drug release system for long-term delivery in biomedical applications, such as in treatment of biofilm-associated infections, and could, for example, be used as medical implant coating or as components in dental composite materials

Cell type-specific adhesion and migration on laser-structured opaque surfaces

Cytocompatibility is essential for implant approval. However, initial in vitro screenings mainly include the quantity of adherent immortalized cells and cytotoxicity. Other vital parameters, such as cell migration and an in-depth understanding of the interaction between native tissue cells and implant surfaces, are rarely considered. We investigated different laser-fabricated spike structures using primary and immortalized cell lines of fibroblasts and osteoblasts and included quantification of the cell area, aspect ratio, and focal adhesions. Furthermore, we examined the three-dimensional cell interactions with spike topographies and developed a tailored migration assay for long-term monitoring on opaque materials. While fibroblasts and osteoblasts on small spikes retained their normal morphology, cells on medium and large spikes sank into the structures, affecting the composition of the cytoskeleton and thereby changing cell shape. Up to 14 days, migration appeared stronger on small spikes, probably as a consequence of adequate focal adhesion formation and an intact cytoskeleton, whereas human primary cells revealed differences in comparison to immortalized cell lines. The use of primary cells, analysis of the cell–implant structure interaction as well as cell migration might strengthen the evaluation of cytocompatibility and thereby improve the validity regarding the putative in vivo performance of implant material. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Adhesion Forces of Oral Bacteria to Titanium and the Correlation with Biophysical Cellular Characteristics

Bacterial adhesion to dental implants is the onset for the development of pathological biofilms. Reliable characterization of this initial process is the basis towards the development of anti-biofilm strategies. In the present study, single-cell force spectroscopy (SCFS), by means of an atomic force microscope connected to a microfluidic pressure control system (FluidFM), was used to comparably measure adhesion forces of different oral bacteria within a similar experimental setup to the common implant material titanium. The bacteria selected belong to different ecological niches in oral biofilms: the commensal pioneers Streptococcus oralis and Actinomyces naeslundii; secondary colonizer Veillonella dispar; and the late colonizing pathogens Porphyromonas gingivalis as well as fimbriated and non-fimbriated Aggregatibacter actinomycetemcomitans. The results showed highest values for early colonizing pioneer species, strengthening the link between adhesion forces and bacteria’s role in oral biofilm development. Additionally, the correlation between biophysical cellular characteristics and SCFS results across species was analyzed. Here, distinct correlations between electrostatically driven maximum adhesion force, bacterial surface elasticity and surface charge as well as single-molecule attachment points, stretching capability and metabolic activity, could be identified. Therefore, this study provides a step towards the detailed understanding of oral bacteria initial adhesion and could support the development of infection-resistant implant materials in future

Skeletal muscle alterations in tachycardia-induced heart failure are linked to deficient natriuretic peptide signalling and are attenuated by RAS-/NEP-inhibition

Background Heart failure induced cachexia is highly prevalent. Insights into disease progression are lacking. Methods Early state of left ventricular dysfunction (ELVD) and symptomatic systolic heart failure (HF) were both induced in rabbits by tachypacing. Tissue of limb muscle (LM) was subjected to histologic assessment. For unbiased characterisation of early and late myopathy, a proteomic approach followed by computational pathway-analyses was performed and combined with pathway-focused gene expression analyses. Specimen of thoracic diaphragm (TD) served as control for inactivity-induced skeletal muscle alterations. In a subsequent study, inhibition of the renin-angiotensin-system and neprilysin (RAS-/NEP) was compared to placebo. Results HF was accompanied by loss of protein content (8.7±0.4% vs. 7.0±0.5%, mean±SEM, control vs. HF, p<0.01) and a slow-to-fast fibre type switch, establishing hallmarks of cachexia. In ELVD, the enzymatic set-up of LM and TD shifted to a catabolic state. A disturbed malate-aspartate shuttle went well with increased enzymes of glycolysis, forming the enzymatic basis for enforced anoxic energy regeneration. The histological findings and the pathway analysis of metabolic results drew the picture of suppressed PGC-1α signalling, linked to the natriuretic peptide system. In HF, natriuretic peptide signalling was desensitised, as confirmed by an increase in the ratio of serum BNP to tissue cGMP (57.0±18.6pg/ml/nM/ml vs. 165.8±16.76pg/ml/nM/ml, p<0.05) and a reduced expression of natriuretic peptide receptor-A. In HF, combined RAS-/NEP-inhibition prevented from loss in protein content (8.7±0.3% vs. 6.0±0.6% vs. 8.3±0.9%, Baseline vs. HF-Placebo vs. HF-RAS/NEP, p<0.05 Baseline vs. HF-Placebo, p = 0.7 Baseline vs. HF-RAS/NEP). Conclusions Tachypacing-induced heart failure entails a generalised myopathy, preceding systolic dysfunction. The characterisation of “pre-cachectic” state and its progression is feasible. Early enzymatic alterations of LM depict a catabolic state, rendering LM prone to futile substrate metabolism. A combined RAS-/NEP-inhibition ameliorates cardiac-induced myopathy independent of systolic function, which could be linked to stabilised natriuretic peptide/cGMP/PGC-1α signalling

Transarterial chemoembolization for hepatocellular carcinoma: development and external validation of the Munich-TACE score

Background: Allocation of patients with hepatocellular carcinoma (HCC) to the adequate therapy is determined by both tumor burden and liver function. The Barcelona Clinic Liver Cancer (BCLC) staging system and therapeutic algorithm recommends transarterial chemoembolization (TACE) based on the best evidence available to patients with intermediate-stage HCC (BCLC-B). However, many centers also treat subgroups of patients outside these recommendations and with more advanced disease by TACE. The purpose of this study was to identify prognostic factors in a TACE cohort, including BCLC-B patients, as well as patients treated outside of BCLC-B, to test the prognostic capabilities of published staging systems and to optimize prognostication for TACE patients.Patients and Methods: A cohort of 186 first-line TACE patients was analyzed. Independent prognostic factors were identified and used to construct the Munich-TACE score (M-TACE). M-TACE was tested against established staging systems (including BCLC and two recently published TACE-specific scores) and a ranking using concordance index and Akaike Information Criterion was performed. Finally, an external validation in an independent TACE cohort (n=71) was conducted.Results: Bilirubin, Quick/international normalized ratio, C-reactive protein, creatinine, -feto protein, and tumor extension were identified as independent prognostic factors and used to construct M-TACE. M-TACE identifies three distinct subgroups (P<0.0001) with median survival times of 35.2, 16.9, and 8.6 months, respectively. Compared with established staging systems, M-TACE showed the best prognostic capabilities in both cohorts of patients (cohort 1: c-index, 0.71;Akaike Information Criterion: 1276;cohort 2: c-index, 0.754).Conclusion: We identified independent risk factors for patients treated with TACE. The newly constructed M-TACE score is superior to established staging systems and might prove helpful to identify patients who are most suitable for TACE