Revisiting ameloblastin; addressing the EMT-ECM axis above and beyond oral biology.

Ameloblastin (AMBN) is best characterized for its role in dental enamel formation, regulating cell differentiation and mineralization, and cell matrix adhesion. However, AMBN has also been detected in mesenchymal stem cells in addition to bone, blood, and adipose tissue. Using immunofluorescence in a pilot scheme, we identified that AMBN is expressed in different parts of the gastrointestinal (GI) tract. AMBN mRNA and protein detection in several tissues along the length of the GI tract suggests a role for AMBN in the structure and tissue integrity of the extracellular matrix (ECM). Intracellular AMBN expression in subsets of cells indicates a potential alternative role in signaling processes. Of note, our previous functional AMBN promoter analyses had shown that it contains epithelial-mesenchymal transition (EMT) regulatory elements. ΑΜΒΝ is herein presented as a paradigm shift of the possible associations and the spatiotemporal regulation of the ECM regulating the EMT and vice versa, using the example of AMBN expression beyond oral biology.

Solution blow spinning of highly deacetylated chitosan nanofiber scaffolds for dermal wound healing.

Biocompatible fibrous scaffolds based on highly deacetylated chitosan were fabricated using high-throughput solution blow spinning. Scanning electron microscopy analysis revealed that the chitosan nanofiber scaffolds had ultrafine and continuous fibers (300-1200 nm) with highly interconnected porous structures (30-75% porosity), mimicking some aspects of the native extracellular matrix in skin tissue. Post-treatment of as-spun nanofibers with aqueous potassium carbonate solution resulted in a fibrous scaffold with a high chitosan content that retained its fibrous structural integrity for cell culture. Analysis of the mechanical properties of the chitosan nanofiber scaffolds in both dry and wet conditions showed that their strength and durability were sufficient for wound dressing applications. Significantly, the wet scaffold underwent remarkable elastic deformation during stretch such that the elongation at break dramatically increased to up to 44% of its original length, showing wavy fiber morphology near the break site. The culture of normal human dermal fibroblast cells onto scaffolds for 1-14 days demonstrated that the scaffolds were highly compatible and a suitable platform for cell adhesion, viability, and proliferation. Secretion profiles of wound healing-related proteins to the cell culture medium demonstrated that chitosan fibers were a promising scaffold for wound healing applications. Overall, the dense fibrous network with high porosity of the chitosan nanofiber scaffold and their mechanical properties indicate that they could be used to design and fabricate new materials that mimic the epidermis layer of natural skin.

Antifungal activity of well-defined chito-oligosaccharide preparations against medically relevant yeasts.

Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DPn) of 32 and fraction of acetylation (FA) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 µg mL-1) and inhibition patterns showed a time- and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.

Attachment and spatial organisation of human mesenchymal stem cells on poly(ethylene glycol) hydrogels.

Strategies that enable hydrogel substrates to support cell attachment typically incorporate either entire extracellular matrix proteins or synthetic peptide fragments such as the RGD (arginine-glycine-aspartic acid) motif. Previous studies have carefully analysed how material characteristics can affect single cell morphologies. However, the influence of substrate stiffness and ligand presentation on the spatial organisation of human mesenchymal stem cells (hMSCs) have not yet been examined. In this study, we assessed how hMSCs organise themselves on soft (E = 7.4-11.2 kPa) and stiff (E = 27.3-36.8 kPa) poly(ethylene glycol) (PEG) hydrogels with varying concentrations of RGD (0.05-2.5 mM). Our results indicate that hMSCs seeded on soft hydrogels clustered with reduced cell attachment and spreading area, irrespective of RGD concentration and isoform. On stiff hydrogels, in contrast, cells spread with high spatial coverage for RGD concentrations of 0.5 mM or higher. In conclusion, we identified that an interplay of hydrogel stiffness and the availability of cell attachment motifs are important factors in regulating hMSC organisation on PEG hydrogels. Understanding how cells initially interact and colonise the surface of this material is a fundamental prerequisite for the design of controlled platforms for tissue engineering and mechanobiology studies.

Predicting the points of interaction of small molecules in the NF-κB pathway.

BACKGROUND: The similarity property principle has been used extensively in drug discovery to identify small compounds that interact with specific drug targets. Here we show it can be applied to identify the interactions of small molecules within the NF-κB signalling pathway. RESULTS: Clusters that contain compounds with a predominant interaction within the pathway were created, which were then used to predict the interaction of compounds not included in the clustering analysis. CONCLUSIONS: The technique successfully predicted the points of interactions of compounds that are known to interact with the NF-κB pathway. The method was also shown to be successful when compounds for which the interaction points were unknown were included in the clustering analysis.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

BACKGROUND: Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. RESULTS: Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. CONCLUSION: The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.