Spatial Variation in Young Ovine Cortical Bone Properties.

Significant effort continues to be made to understand whether differences exist in the structural, compositional, and mechanical properties of cortical bone subjected to different strain modes or magnitudes. We evaluated juvenile sheep femora (age = 4 months) from the anterior and posterior quadrants at three points along the diaphysis as a model system for variability in loading. Micro-CT scans (50 micron) were used to measure cortical thickness and mineral density. Three point bending tests were performed to measure the flexural modulus, strength, and post-yield displacement. There was no difference in cortical thickness or density between anterior or posterior quadrants; however, density was consistently higher in the middle diaphysis. Interestingly, bending modulus and strength were higher in anterior quadrants compared to posterior quadrants. Together, our results suggest that there is a differential spatial response of bone in terms of elastic bending modulus and mechanical strength. The origins of this difference may lie within the variation in ongoing mineralization, in combination with the collagen-rich plexiform structure, and whether this is related to strain mode remains to be explored. These data suggest that in young ovine cortical bone, modulation of strength occurs via potentially complex interactions of both mineral and collagen-components that may be different in regions of bone exposed to variable amounts of strain. Further work is needed to confirm the physiological load state of bone during growth to better elucidate the degree to which these variations are a function of the local mechanical environment.

Yeast and Filaments Have Specialized, Independent Activities in a Zebrafish Model of Candida albicans Infection.

Candida albicans dimorphism is a crucial virulence factor during invasive candidiasis infections, which claim the lives of nearly one-half of those afflicted. It has long been believed that filaments drive tissue invasion and yeast mediates bloodstream dissemination, but observation of these activities during infection has been prevented by technical limitations. We used a transparent zebrafish infection model to analyze more comprehensively how C. albicans utilizes shape to disseminate and invade. This model facilitated the use of diverse, complementary strategies to manipulate shape, allowing us to monitor dissemination, invasion, and pathogenesis via intravital imaging of individual fungal cells throughout the host. To control fungal cell shape, we employed three different strategies: gene deletion (efg1Δ/Δ cph1Δ/Δ, eed1Δ/Δ), overexpression of master regulators (NRG1 or UME6), and modulation of the infection temperature (21°C, 28°C, or 33°C). The effects of these orthogonal manipulations were consistent, support the proposed specialized roles of yeast in dissemination and filaments in tissue invasion and pathogenesis, and indicate conserved mechanisms in zebrafish. To test if either morphotype changes the effectiveness of the other, we infected fish with a known mixture of shape-locked strains. Surprisingly, mixed-strain infections were associated with additive, but not synergistic, filament invasion and yeast dissemination. These findings provide the most complete view of morphotype-function relationships for C. albicans to date, revealing independent roles of yeast and filaments during disseminated candidiasis.

Targeted exon skipping with AAV-mediated split adenine base editors.

Techniques for exclusion of exons from mature transcripts have been applied as gene therapies for treating many different diseases. Since exon skipping has been traditionally accomplished using technologies that have a transient effect, it is particularly important to develop new techniques that enable permanent exon skipping. We have recently shown that this can be accomplished using cytidine base editors for permanently disabling the splice acceptor of target exons. We now demonstrate the application of CRISPR-Cas9 adenine deaminase base editors to disrupt the conserved adenine within splice acceptor sites for programmable exon skipping. We also demonstrate that by altering the amino acid sequence of the linker between the adenosine deaminase domain and the Cas9-nickase or by coupling the adenine base editor with a uracil glycosylase inhibitor, the DNA editing efficiency and exon-skipping rates improve significantly. Finally, we developed a split base editor architecture compatible with adeno-associated viral packaging. Collectively, these results represent significant progress toward permanent in vivo exon skipping through base editing and, ultimately, a new modality of gene therapy for the treatment of genetic diseases.

Erratum: Author Correction: Targeted exon skipping with AAV-mediated split adenine base editors.

[This corrects the article DOI: 10.1038/s41421-019-0109-7.].