Histone H1.0 is an essential component of stress response in cardiac fibroblasts. Depletion of histone H1.0 prevented expression of actin stress fibers and extracellular matrix proteins, and attenuated mechanical behaviors in pathological cardiac fibroblasts. Overtime, this process can lead to fibrosis, excessive scarring of the heart that stiffens muscle tissue and impairs its function. siRNA-mediated depletion of linker histone H1.0 in vivo prevents isoproterenol-induced cardiac fibrosis, suggesting that linker histones direct fibrotic gene expression. To characterize the role of histone H1.0 in vivo for chronic disease models, we developed a mouse model in which H1.0 can be knocked out in cardiac fibroblasts at will using a tamoxifen inducible Cre-lox system. Here, the TCF21 promoter drives Cre recombinase expression in fibroblasts: upon tamoxifen treatment, the modified Cre protein translocates to the nucleus and enacts endonuclease activity at loxP sites. Our approach crossed a mouse with loxP sites in the histone H1.0 gene with the TCF21-driven modified Cre expressing mouse. I validated this model by genotyping the mice (to establish that progeny carry the floxed gene and inducible TCF21-driven Cre) and demonstrating that following tamoxifen treatment, Cre induces histone H1.0 gene disruption and depletes histone H1.0 protein as, measured by western blotting. Validating this model will help elucidate the temporal and cell-type-specific dependence of H1.0 in directing cardiac fibrosis and dysfunction upon injury.

Introduction/Background –The utricle is vestibular organ that detects linear acceleration, contains hair cells (HCs and supporting cells (SCs) forming the vestibular sensory epithelium (VSE). SCs play several functions in the VSE: (a) otoconia secretion, (b) act as HC precursors for regeneration, and (c) provide structural support. However, little is known about the molecular and subcellular organization of SCs. Objective – To investigate the subcellular structure of secretory granules in mouse utricular SCs and identify putative proteins within these granules in the human vestibular utricle. Materials and Methods – I used scanning electron microscopy serial mouse utricle sections to generate 3D reconstructions via VAST Lite and AMIRA software. Protein expression in SC granules was localized with immunofluorescence and confocal microscopy on archival formalin-fixed human utricle sections. Antibodies tested include GFAP and otopetrin-2. Images were acquired using Leica SP8 confocal light sheet microscopy. Results – Three-D (3D) reconstruction revealed, for the first time, the ultrastructural organization of secretory granules in mouse utricular SCs, mitochondria organization around secretory granules are also annotated. Otopetrin and GFAP proteins were localized in the human SCs, and the presence of secretory granules is similar to that found in the mouse SCs. Significance – Insights into SC organization may inform future strategies for HC regeneration and cell survival after ototoxic damage or aging.

Cytosolic 5’ nucleotidase 3 (NT5C3) regulates the nucleoside pool balance and metabolism by catalysing the dephosphorylation of pyrimidine nucleoside monophosphates. Yet there is a lack of knowledge surrounding the function of the gene encoding for NT5C3 in Strongylocentrotus purpuratus. Here, the identity of an unknown S. purpuratus gene is inferred to be NT5C3 using sequence analysis tools such as Blastn, Blastx, Interpro, and Echinobase. Furthermore, the construction of a phylogenetic tree allows for visualization of the evolutionary relationships of NT5C3 in different genera, supporting the gene’s identity. The DNA of NT5C3 is amplified through PCR and cloned by ligating the genes into a plasmid vector for bacterial transformation. Plasmid restriction digests determined cloning success and verified that NT5C3 was correctly ligated into the plasmid vector. Thus, NT5C3 cloning was likely successful. In the future, the DNA of successful clones can be sequenced to confirm the identity of NT5C3. Subsequent storage of the cloned NT5C3 products can be used in future experiments, further analyzing the function of NT5C3 in S. purpuratus.