New insights into the fundamental role of topological constraints as a determinant of two-way junction conformation

Recent studies have shown that topological constraints encoded at the RNA secondary structure level involving basic steric and stereochemical forces can significantly restrict the orientations sampled by helices across two-way RNA junctions. Here, we formulate these topological constraints in greater quantitative detail and use this topological framework to rationalize long-standing but poorly understood observations regarding the basic behavior of RNA two-way junctions. Notably, we show that the asymmetric nature of the A-form helix and the finite length of a bulge provide a physical basis for the experimentally observed directionality and bulge-length amplitude dependence of bulge induced inter-helical bends. We also find that the topologically allowed space can be modulated by variations in sequence, particularly with the addition of non-canonical GU base pairs at the junction, and, surprisingly, by the length of the 5′ and 3′ helices. A survey of two-way RNA junctions in the protein data bank confirms that junction residues have a strong preference to adopt looped-in, non-canonically base-paired conformations, providing a route for extending our bulge-directed framework to internal loop motifs and implying a simplified link between secondary and tertiary structure. Finally, our results uncover a new simple mechanism for coupling junction-induced topological constraints with tertiary interactions

Secretion of angiogenic proteins by human multipotent mesenchymal stromal cells and their clinical potential in the treatment of avascular osteonecrosis.

Osteonecrosis is a frequent complication after treatment for childhood leukemia and other steroid-based therapies. The success rate of core decompression surgery is limited. Therefore, we evaluated relevant biological characteristics of human multipotent mesenchymal stromal cells (MSCs) in vitro. MSCs cultured under low-oxygen tensions showed decreased proliferation and differentiation into bone. However, these MSCs secreted significant amounts of vascular endothelial-derived factor in the presence of interferon-c. These in vitro results with potential effects on neovascularization and bone regeneration as well as findings in animal models prompted us to treat five patients with steroid-induced osteonecrosis of the femur by core decompression surgery and instillation of expanded autologous MSCs. Within 3 weeks of culture, sufficient numbers of MSCs were generated using animal protein-free culture conditions. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization. Application of MSCs during core decompression was feasible and safe. Median follow-up is 16 months and the patients in this pilot study reported clinical improvement. Formation of mineralized bone in the osteonecrotic cavity was proven by computed tomography. Taken together, MSCs display biological properties that may add to the efficiency of surgical treatment in osteonecrosis and should be evaluated in larger patient cohorts