Comparative effects of N-cadherin protein and peptide fragments on mesenchymal stem cell mechanotransduction and paracrine function.

The recent interest in exploiting cadherin-derived fragments to mimic intercellular adhesion in engineered hybrid biomaterials raises questions about which cadherin constructs effectively mimic cadherin interactions. This study compared the biophysical properties of and signaling initiated by three different, immobilized N-cadherin-derived fragments, in order to identify a minimal construct that mimics intercellular adhesion in biomaterials. Specifically, we compared: i) the full N-cadherin extracellular region with all five ectodomains (EC1-5), ii) the first two ectodomains (EC1-2) of N-cadherin, and iii) a peptide containing the histidine-alanine-valine-aspartic acid-valine (HAVDI) sequence in the first extracellular domain. Comparisons of the binding kinetics and affinities between each of these ligands and N-cadherin expressed on mesenchymal stem cells (MSCs) revealed quantitative differences. Nevertheless, MSCs exhibited similar, rigidity-dependent spreading and traction forces when cultured on gels displaying any of these N-cadherin ligands. There were, however, differences in cell signaling and secretory activities. MSCs cultured on the full N-cadherin extracellular domain (EC1-5) exhibited stiffness-dependent changes in nuclear YAP/TAZ localization and significantly higher secretion of vascular endothelial growth factor and insulin growth factor 1, compared to cells cultured on hydrogels displaying either EC1-2 or the HAVDI peptide. The increased paracrine secretion also enhanced myogenic differentiation. These findings reveal functional differences between N-cadherin derived ligands important for the design of biomaterials that mimic intercellular adhesion.

Diagnosis and Treatment of Mitochondrial Myopathies.

Mitochondrial myopathies are progressive muscle conditions caused primarily by the impairment of oxidative phosphorylation (OXPHOS) in the mitochondria. This causes a deficit in energy production in the form of adenosine triphosphate (ATP), particularly in skeletal muscle. The diagnosis of mitochondrial myopathy is reliant on the combination of numerous techniques including traditional histochemical, immunohistochemical, and biochemical testing combined with the fast-emerging molecular genetic techniques, namely next-generation sequencing (NGS). This has allowed for the diagnosis to become more effective in terms of determining causative or novel genes. However, there are currently no effective or disease-modifying treatments available for the vast majority of patients with mitochondrial myopathies. Existing therapeutic options focus on the symptomatic management of disease manifestations. An increasing number of clinical trials have investigated the therapeutic effects of various vitamins, cofactors, and small molecules, though these trials have failed to show definitive outcome measures for clinical practice thus far. In addition, new molecular strategies, specifically mtZFNs and mtTALENs, that cause beneficial heteroplasmic shifts in cell lines harboring varying pathogenic mtDNA mutations offer hope for the future. Moreover, recent developments in the reproductive options for patients with mitochondrial myopathies mean that for some families, the possibility of preventing transmission of the mutation to the next generation is now possible.

Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency.

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease.