Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error

Molecular and biophysical mechanisms regulating hypertrophic differentiation in chondrocytes and mesenchymal stem cells

Chondrocyte hypertrophy is one of the key physiological processes involved in the longitudinal growth of long bones, yet the regulation of hypertrophy is also becoming increasingly relevant for clinical application of mesenchymal stem cells (MSCs) and screening for drugs to treat hypertrophic osteoarthritis. The extraordinary cell volume increase during hypertrophy is accompanied by an up-regulation of collagen X, matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF), all which are targets of the runt-related transcription factor 2 (Runx2). Many pathways, including parathyroid hormone-related protein (PTHrP)/Indian Hedgehog, Wingless/Int (Wnt)/β-catenin, and transforming growth factor beta (TGF-β)/Sma and Mad Related Family (Smad) pathways, can regulate hypertrophy, but factors as diverse as hypoxia, co-culture, epigenetics and biomaterial composition can also potently affect Runx2 expression. Control of hypertrophic differentiation can be exploited both for cartilage repair, where a stable phenotype is desired, but also in bone regeneration, where hypertrophic cartilage could act as a template for endochondral bone formation. We hope this review will motivate the design of novel engineered microenvironments for skeletal regeneration applications

Correlating cell architecture with osteogenesis: first steps towards live single cell monitoring

Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC) being the primary sites of adhesion. They consist of a large number of clustered transmembrane proteins (integrins). FC integrins connect the cell cytoskeleton with the cell substratum. It has been demonstrated that cell spreading increases osteoblast differentiation in pre-osteoblastic progenitors. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g., bone-specific alkaline phosphatase (bALP)) and late genes (e.g., osteocalcin (OC)). In the present study we have used immunohistochemistry and RT-PCR to determine osteogenic differentiation of human bone cells (HBC). For online monitoring, fluorescently-tagged actin and vinculin were used for transfection of HBCs. Transfection of HBCs with an OC promoter gene construct allowed us to online monitor the gradual process of osteogenesis. We found distinct changes in cell architecture upon osteogenic differentiation thus providing evidence for the connection between cell shape and functional state

Self-assembly of glycerol monooleate with the antimicrobial peptide LL-37: a molecular dynamics study

Over the past decade, the rapid increase in the incidence of antibiotic-resistant bacteria has promoted research towards alternative therapeutics such as antimicrobial peptides (AMPs), but their biodegradability limits their application. Encapsulation into nanocarriers based on the self-assembly of surfactant-like lipids is emerging as a promising strategy for the improvement of AMPs' stability and their protection against degradation when in biological media. An in-depth understanding of the interactions between the structure-forming lipids and AMPs is required for the design of nanocarriers. This in silico study, demonstrates the self-assembly of the amphiphilic lipid glycerol monooleate (GMO) with the antimicrobial peptide LL-37 into nanocarriers on the molecular scale. Molecular dynamics (MD) simulations show the formation of direct micelles, with either one or two interacting LL-37, and vesicles in this two- component system in agreement with experimental results from small-angle X- ray scattering studies. The hydrophobic contacts between LL-37 and GMOs in water appear responsible for the formation of these nanoparticles. The results also suggest that the enhanced antimicrobial efficiency of LL-37 in these nanocarriers that was previously observed experimentally can be explained by the availability of its side chains with charged amino acids, an increase of the electrostatic interaction and a decrease of the peptide's conformational entropy upon interacting with GMO. The results of this study contribute to the fundamental understanding of lipid–AMP interactions and may guide the comprehensive design of lipid-based self-assembled nanocarriers for antimicrobial peptides