Quadruplex structures of muscle gene promoter sequences enhance in vivo MyoD-dependent gene expression

Gene promoters are enriched in guanine clusters that potentially fold into quadruplex structures. Such quadruplexes were implicated in the regulation of gene expression, plausibly by interacting with transcription factors. We showed previously that homodimers of the myogenic transcription factor MyoD bound in vitro most tightly bimolecular quadruplexes of promoter sequences of muscle-specific genes. By contrast, MyoD-E47 heterodimers formed tighter complexes with d(CANNTG) E-box motifs that govern muscle gene expression. Here, we show that DNA quadruplexes enhance in vivo MyoD and E-box-driven expression of a firefly luciferase (FL) reporter gene. HEK293 cells were transfected with FL expressing p4RTK-FL vector alone or together with MyoD expressing pEMSV-MyoD plasmid, with quadruplexes of α7 integrin or sarcomeric mitochondrial creatine kinase (sMtCK) muscle gene promoters or with a combination thereof. Whereas MyoD elevated by ∼10-fold the levels of FL mRNA and protein, the DNA quadruplexes by themselves did not affect FL expression. However, together with MyoD, quadruplex DNA increased by ∼35-fold the amounts of FL mRNA and protein. Without affecting its expression, DNA quadruplexes bound MyoD in the cells. Based on these results, we propose models for the regulation of muscle gene transcription by direct interaction of MyoD with promoter quadruplex structures

Bovine pericardial extracellular matrix niche modulates human aortic endothelial cell phenotype and function.

Xenogeneic biomaterials contain biologically relevant extracellular matrix (ECM) composition and organization, making them potentially ideal surgical grafts and tissue engineering scaffolds. Defining the effect of ECM niche (e.g., basement membrane vs. non-basement membrane) on repopulating cell phenotype and function has important implications for use of xenogeneic biomaterials, particularly in vascular applications. We aim to understand how serous (i.e., basement membrane) versus fibrous (i.e., non-basement membrane) ECM niche of antigen-removed bovine pericardium (AR-BP) scaffolds influence human aortic endothelial cell (hAEC) adhesion, growth, phenotype, inflammatory response and laminin production. At low and moderate seeding densities hAEC proliferation was significantly increased on the serous side. Similarly, ECM niche modulated cellular morphology, with serous side seeding resulting in a more rounded aspect ratio and intact endothelial layer formation. At moderate seeding densities, hAEC production of human laminin was enhanced following serous seeding. Finally, inflammatory marker and pro-inflammatory cytokine expression decreased following long-term cell growth regardless of seeding side. This work demonstrates that at low and moderate seeding densities AR-BP sidedness significantly impacts endothelial cell growth, morphology, human laminin production, and inflammatory state. These findings suggest that ECM niche has a role in modulating response of repopulating recipient cells toward AR-BP scaffolds for vascular applications

Label-Free Assessment of Collagenase Digestion on Bovine Pericardium Properties by Fluorescence Lifetime Imaging

The extracellular matrix architecture of bovine pericardium (BP) has distinct biochemical and biomechanical properties that make it a useful biomaterial in the field of regenerative medicine. Collagen represents the dominant structural protein of BP and is therefore intimately associated with the properties of this biomaterial. Enzymatic degradation of collagen molecules is critical for extracellular matrix turnover, remodeling and ultimately tissue regeneration. We present a quantitative, label-free and non-destructive method for monitoring changes in biochemical and biomechanical properties of BP during tissue degradation, based on multi-spectral fluorescence lifetime imaging (ms-FLIm). Strong correlations of fluorescence intensity ratio and average fluorescence lifetime were identified with collagen content, Young's Modulus and Ultimate tensile strength during collagenase degradation, indicating the potential of optically monitoring collagen degradation using ms-FLIm. The obtained results demonstrate the value of ms-FLIm to assess the quality of biomaterials in situ for applications in regenerative medicine

Synthetic cells with self-activating optogenetic proteins communicate with natural cells

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells’ capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues