Micromechanical Properties and Structure of the Pericellular Coat of Living Cells Modulated by Nanopatterned Substrates

Das mechanisch stark beanspruchte Knorpelgewebe in Gelenken besteht zum überwiegenden Teil aus einer komplexen extrazellulären Matrix (ECM). Chondrozyten, spezialisierte in der Matrix eingebettete Zellen, erneuern diese fortwährend, um deren Abrieb und Verschleißzu verhindern. Die Zellen werden durch eine mikrometerdicke perizelluläre Matrix (PCC) geschützt, die ein Überleben und ein Teilen der Zellen trotz der hohen mechanischen Belastung ermöglicht. Die PCC ist von entscheidender Bedeutung für eine Vielzahl weiterer biologischer Prozesse, wie der Motilität, der Zellalterung und der Ostheoarthrose. Auf molekularer Ebene ist die Zusammensetzung und Wechselwirkung der verschiedenen PCC-Komponenten gut verstanden: Der überwiegende Teil der PCC besteht aus Wasser und ist damit mit lichtmikroskopischen Methoden nicht detektierbar. Das Rückgrat der PCC wird aus stark hydratisierten Hyaluronsäurepolymeren und daran angebundenen HA-Bindungsproteinen gebildet. Informationen über die mesoskopische Struktur der PCC sind allerdings kaum vorhanden. Diese ist jedoch von fundamentaler Bedeutung für das Verständnis der Kraftübertragung aus dem Knorpelgewebe auf die Zellen sowie zur Aufklärung des Mechanismus, der den Zellen eine aktive Anpassung der PCC ermöglicht Im Rahmen dieser Arbeit wurden daher neue Methoden zur Visualisierung der PCC etabliert, die eine dreidimensionale Darstellung, sowie die mikromechanische Charakterisierung der PCC lebender Zellen ermöglichen. Diese Methoden erlaubten die Untersuchung der dynamischen Anpassung der PCC bei Zellteilung, Motilität und Phagozytose. Die mesoskopische Struktur der PCC konnte von den erhaltenen Messdaten abgeleitet und durch entsprechende Modellsysteme aus endständig angebundenen HA Molekülen unterstützt werden. Darüber hinaus konnte das Wechselspiel von PCC und ECM mit Hilfe von Adhäsionsstudien auf homogenen sowie nanostrukturierten Oberflächen, welche die ECM-Wechselwirkungen kontrollieren, untersucht werden

Tuning RGD Motif and Hyaluronan Density to Study Integrin Binding

Well-controlled surfaces with immobilized substrates enable novel approaches to investigate specific aspects of biological processes related to cell adhesion or motility. A subset of integrins, cellular transmembrane glycoproteins, recognize the evolutionarily conserved tripeptide sequence RGD, and anchor cells to their surrounding proteins as well as mediate bidirectional signaling. In this study, the main question was how co-presentation of hyaluronan (HA), an essential component of the extracellular matrix (ECM), and the RGD motif affect integrin binding. We report a method to prepare self-assembled monolayers on gold surfaces, co-presenting the cell adhesive RGD motif and small HA molecules, to investigate integrin containing proteoliposome binding. This technique enables an independent adjustment of the RGD motif and HA density while maintaining a passivating background: Layer formation and subsequent interactions with αIIbβ3 integrins, which are reconstituted in liposomes, was monitored by label-free quartz crystal microbalance with dissipation monitoring (QCM-D). Exceeding a critical RGD motif density of 40% results in enhanced binding of proteoliposomes. Co-presentation studies with varying HA and constant RGD motif density demonstrate that marginal amounts of HA are sufficient to prevent integrin binding. These findings are of specific importance in relation to cancer cell microenvironments, which show highly enriched HA in the surrounding ECM to reduce adhesion properties

Phenotypic Selection in Ornamental Breeding: It's Better to Have the BLUPs Than to Have the BLUEs

Plant breeders always face the challenge to select the best individuals. Selection methods are required that maximize selection gain based on available data. When several crosses have been made, the BLUP procedure achieves this by combining phenotypic data with information on pedigree relationships via an index, known as family-index selection. The index, estimated based on the intra-class correlation coefficient, exploits the relationship among individuals within a family relative to other families in the population. An intra-class correlation coefficient of one indicates that the individual performance can be fully explained based on the family background, whereas an intra-class correlation coefficient of zero indicates the performance of individuals is independent of the family background. In the case the intra-class correlation coefficient is one, family-index selection is considered. In the case the intra-class correlation coefficient is zero, individual selection is considered. The main difference between individual and family-index selection lies in the adjustment in estimating the individual's effect depending on the intra-class correlation coefficient afforded by the latter. Two examples serve to illustrate the application of the BLUP method. The efficiency of individual and family-index selection was evaluated in terms of the heritability obtained from linear mixed models implementing the selection methods by suitably defining the treatment factor as the sum of individual and family effect. Family-index selection was found to be at least as efficient as individual selection in Dianthus caryophyllus L., except for flower size in standard carnation and vase life in mini carnation for which traits family-index selection outperformed individual selection. Family-index selection was superior to individual selection in Pelargonium zonale in cases when the heritability was low. Hence, the pedigree-based BLUP procedure can enhance selection efficiency in production-related traits in P. zonale or shelf-life related in D. caryophyllus L

Missense mutation in the tubulin-specific chaperone E (Tbce) gene in the mouse mutant progressive motor neuronopathy, a model of human motoneuron disease

Progressive motor neuronopathy (pmn) mutant mice have been widely used as a model for human motoneuron disease. Mice that are homozygous for the pmn gene defect appear healthy at birth but develop progressive motoneuron disease, resulting in severe skeletal muscle weakness and respiratory failure by postnatal week 3. The disease starts at the motor endplates, and then leads to axonal loss and finally to apoptosis of the corresponding cell bodies. We localized the genetic defect in pmn mice to a missense mutation in the tubulin-specific chaperone E (Tbce) gene on mouse chromosome 13. The human orthologue maps to chromosome 1q42.3. The Tbce gene encodes a protein (cofactor E) that is essential for the formation of primary α-tubulin and β-tubulin heterodimeric complexes. Isolated motoneurons from pmn mutant mice exhibit shorter axons and axonal swelling with irregularly structured β-tubulin and tau immunoreactivity. Thus, the pmn gene mutation provides the first genetic evidence that alterations in tubulin assembly lead to retrograde degeneration of motor axons, ultimately resulting in motoneuron cell death

Interface Immobilization Chemistry of cRGD-based Peptides Regulates Integrin Mediated Cell Adhesion

The interaction of specifi c surface receptors of the integrin family with different extracellular matrix-based ligands is of utmost importance for the cellular adhesion process. A ligand consists of an integrin-binding group, here cyclic RGDfX, a spacer molecule that lifts the integrin-binding group from the surface and a surface anchoring group. c(-RGDfX-) peptides are bound to gold nanoparticle structured surfaces via polyproline, polyethylene glycol or aminohexanoic acid containing spacers of different lengths. Although keeping the integrin-binding c(-RGDfX-) peptides constant for all compounds, changes of the ligand´s spacer chemistry and length reveal signifi cant differences in cell adhesion activation and focal adhesion formation. Polyproline-based peptides demonstrate improved cell adhesion kinetics and focal adhesion formation compared with common aminohexanoic acid or polyethylene glycol spacers. Binding activity can additionally be improved by applying ligands with two head groups, inducing a multimeric effect. This study gives insights into spacer-based differences in integrin-driven cell adhesion processes and remarkably highlights the polyproline-based spacers as suitable ligand-presenting templates for surface functionalization.Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicada

Effect of Co-presentation of Adhesive Ligands and Short Hyaluronan on Lymphendothelial Cells

Controlled activation of lymphangiogenesis through functional biomaterials represents a promising approach to support wound healing after surgical procedures, yet remains a challenge. In a synthetic biological approach, we therefore set out to mimic the basal microenvironment of human primary dermal lymphatic endothelial cells (LECs) during lymphangiogenesis. As the extracellular matrix component hyaluronan (HA) regulates lymphangiogenesis, we designed a bifunctional surface in which adhesive peptide ligands and short HA oligosaccharides (sHA) tethered to nanoparticles are copresented to the basal side of LECs in a controlled, concentration-dependent manner. Exposure of LECs to sHA in solution to mimic luminal stimulation of the cells did not result in modified metabolic activity. However, LECs grown on the bifunctional adhesive surfaces showed a biphasic change in metabolic activity, with increased metabolic activity being observed in response to increasing nanoparticle densities up to a maximum of 540 particles/μm2. Thus, interfaces that concomitantly present adhesive ligands and sHA can stimulate LEC metabolism and might be able to trigger lymphangiogenesis

Shotgun lipidomics of liver and brain tissue of Alzheimer's disease model mice treated with acitretin

Alzheimer’s disease (AD) is a very frequent neurodegenerative disorder characterized by an accumulation of amyloid-β (Aβ). Acitretin, a retinoid-derivative and approved treatment for Psoriasis vulgaris, increases non-amyloidogenic Amyloid-Precursor-Protein-(APP)-processing, prevents Aβ-production and elicits cognitive improvement in AD mouse models. As an unintended side effect, acitretin could result in hyperlipidemia. Here, we analyzed the impact of acitretin on the lipidome in brain and liver tissue in the 5xFAD mouse-model. In line with literature, triglycerides were increased in liver accompanied by increased PCaa, plasmalogens and acyl-carnitines, whereas SM-species were decreased. In brain, these effects were partially enhanced or similar but also inverted. While for SM and plasmalogens similar effects were found, PCaa, TAG and acyl-carnitines showed an inverse effect in both tissues. Our findings emphasize, that potential pharmaceuticals to treat AD should be carefully monitored with respect to lipid-homeostasis because APP-processing itself modulates lipid-metabolism and medication might result in further and unexpected changes. Moreover, deducing effects of brain lipid-homeostasis from results obtained for other tissues should be considered cautiously. With respect to acitretin, the increase in brain plasmalogens might display a further positive probability in AD-treatment, while other results, such as decreased SM, indicate the need of medical surveillance for treated patients

Leipzig-Berlin-Erklärung zu NFDI-Querschnittsthemen der Infrastrukturentwicklung

Für den wissenschaftsgeleiteten Aufbau der Nationalen Forschungsdaten-Infrastruktur (NFDI) muss sich die Infrastruktur gemeinsam mit der Forschung weiterentwickeln. Die dafür notwendigen, wechselseitigen Abstimmungen müssen auf Basis tragfähiger Prozesse und Strukturen sichergestellt werden. Themen, die für mehrere Fachkonsortien relevant sind, müssen im Sinne einer nachhaltigen Funktionalität kooperativ und über einzelne Konsortien hinweg bearbeitet werden. Dieses Dokument identifiziert solche Querschnittsthemen und Wege zu ihrer Bearbeitung in der NFDI. Um diese Herausforderung abgestimmt zu adressieren, hat sich die Mehrzahl der Fachkonsortien im Sommer 2019 auf die “Berlin Declaration on NFDI Cross-Cutting Topics” verständigt. Auf einer gemeinsamen Veranstaltung am 25. Februar 2020 in Berlin haben sich Vertreterinnen und Vertreter von Fachkonsortien und Querschnittsinitiativen erneut über die Handlungsfelder der NFDI-übergreifenden Infrastrukturentwicklung ausgetauscht. Dabei haben Fachkonsortien und Querschnittsinitiativen vier modellhafte Vorschläge erarbeitet, um diese Handlungsfelder zu erweitern und im Rahmen der NFDI belastbar und nachhaltig umzusetzen. Diese „Leipzig-Berlin-Erklärung zu NFDI-Querschnittsthemen der Infrastrukturentwicklung“ dient als Diskussionsimpuls und richtet sich an alle Konsortien und am Aufbau der NFDI Beteiligten, sowie diejenigen Fachgruppen, die näher mit Forschungsdatenmanagement befasst sind. Mit der Unterzeichnung dieser Erklärung bestätigen die 27 Konsortien, dass sie gemeinschaftlich und im Einklang mit dem Direktorat und den Gremien der NFDI die benannten Querschnittsthemen und Handlungsfelder weiterentwickeln und im Sinne einer NFDI bearbeiten werden

Visualization of Inflammation in Experimental Colitis by Magnetic Resonance Imaging Using Very Small Superparamagnetic Iron Oxide Particles

Inflammatory bowel diseases (IBD) comprise mainly ulcerative colitis (UC) and Crohn´s disease (CD). Both forms present with a chronic inflammation of the (gastro) intestinal tract, which induces excessive changes in the composition of the associated extracellular matrix (ECM). In UC, the inflammation is limited to the colon, whereas it can occur throughout the entire gastrointestinal tract in CD. Tools for early diagnosis of IBD are still very limited and highly invasive and measures for standardized evaluation of structural changes are scarce. To investigate an efficient non-invasive way of diagnosing intestinal inflammation and early changes of the ECM, very small superparamagnetic iron oxide nanoparticles (VSOPs) in magnetic resonance imaging (MRI) were applied in two mouse models of experimental colitis: the dextran sulfate sodium (DSS)-induced colitis and the transfer model of colitis. For further validation of ECM changes and inflammation, tissue sections were analyzed by immunohistochemistry. For in depth ex-vivo investigation of VSOPs localization within the tissue, Europium-doped VSOPs served to visualize the contrast agent by imaging mass cytometry (IMC). VSOPs accumulation in the inflamed colon wall of DSS-induced colitis mice was visualized in T2* weighted MRI scans. Components of the ECM, especially the hyaluronic acid content, were found to influence VSOPs binding. Using IMC, co-localization of VSOPs with macrophages and endothelial cells in colon tissue was shown. In contrast to the DSS model, colonic inflammation could not be visualized with VSOP-enhanced MRI in transfer colitis. VSOPs present a potential contrast agent for contrast-enhanced MRI to detect intestinal inflammation in mice at an early stage and in a less invasive manner depending on hyaluronic acid content

ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells

The low-density lipoprotein receptor (LDLR) doesn’t directly bind AP-1B; however, it relies on this clathrin adaptor for basolateral exocytosis. Identification of the autosomal recessive hypercholesterolemia protein (ARH) as a link between LDLR and AP-1B explains this apparent discrepancy and provides a model for how other endocytic proteins may contribute to endosomal recycling