Voltage-Dependent Anion Channel 2 (VDAC2) is a mitochondrial outer membrane protein that regulates mitochondrial Ca2+ uptake. VDAC2 has been shown to impact heart rhythm and heart development, but its precise role in these processes is not fully characterized. Homozygous VDAC2 mutant zebrafish embryos show reduced cell numbers in trabeculae in early stages, which lead to thin ventricle walls in later development. RNA sequencing data analysis showed that in mutant fish, glycolysis and TCA cycle genes are downregulated. This result suggests that VDAC2-mediated calcium signaling regulates cardiac metabolic pathways to drive cardiac development. To verify this interaction, both wildtype and VDAC2 mutant embryos were treated with dichloroacetate (DCA), which increases TCA cycle activity and oxidative phosphorylation. DCA treatment increased ventricular cardiomyocyte numbers in trabeculae, validating our hypothesis. Further research is needed to identify whether this interaction between calcium signaling and metabolism specifically impacts cardiomyocyte proliferation versus other potential causes of decreases in cardiomyocytes. These results help characterize key pathways in cardiac structural development, providing insights into potential therapeutic targets for human conditions related to abnormal heart development.

The SARS-CoV-2 genome contains functionally important sites, such as the frameshifting stimulation element (FSE) and the transcription regulatory sequences (TRS), that serve as potential oligonucleotide drug targets. The FSE regulates the expression of vital non-structural proteins and TRS controls discontinuous transcription of sub-genomic RNAs making them attractive targets for inhibition. We designed antisense oligonucleotides with nucleotides that could potentially form tertiary interactions with structured regions in these targets (3D-ASOs) to promote target binding and improve inhibition. Two lead 3D-ASOs, SBD1 and PRF3p, were identified as potent inhibitors of SARS-CoV-2 replication. Further qRT-PCR assays confirmed the strong inhibitory activity of 3D-ASOs against SARS-CoV-2, with PRF3p showing particularly high efficacy. Dosage-dependent assays revealed EC50 values for PRF3p and SBD1, emphasizing their effectiveness at lower concentrations. Furthermore, a SBD1 variant (SBD1-T15A) which was optimized for the consensus TRS conformation had improved antiviral activity. Low cytotoxicity was seen in cell viability assays with SBD1 and PRF3p, as well as some of their variants. Further characterization of the structure-activity relationship of the most potent ASO, PRF3p, through comparison with variants that lacked certain tertiary interactions, revealed similar activity regardless of tertiary interactions. This research reveals specific sites within these conserved structured regions that could be targeted in SARS-CoV-2.

This abstract has been withheld from publication.

Ascorbate, otherwise known as vitamin C, serves as an essential micronutrient and metabolite due to its reducing properties. This ability helps regulate hepatic function and circulating iron homeostasis, and has been associated with the attenuation of liver disease. A pathway of focus involving ascorbate’s reducing ability is the iron-dependent hypoxia-inducible factor (HIF) pathway, implicated in liver fibrosis. Ascorbate does this by reducing the iron cofactor of prolyl hydroxylase domain proteins to the ferrous state, thereby maintaining enzyme activity to prevent the accumulation of HIF-1alpha protein involved in progressive fibrosis. Thus, this project focuses on investigating the effects of ascorbate on the progression of metabolic dysfunction-associated fatty liver disease (MAFLD), such as the accumulation of fibrosis markers and lobular inflammation, to gain insight into ascorbate’s potential as a dietary therapeutic for MAFLD. To accomplish this, a mouse model was established in which mice fed a high-fat diet or a control diet expressing a non-functional transgene at the L-gulonolactone oxidase locus, an intermediate in ascorbate biosynthesis, were given different levels of ascorbate treatment. Liver tissues were then collected for RNA and protein extraction, followed by quantitative polymerase chain reaction and Western blotting, which showed increased collagen expression in high-ascorbate-treated mice. Altogether, this project aims to determine ascorbate’s effects on MAFLD and limit liver fibrosis.

Collagen turnover is important for maintaining tissue homeostasis; however, the cellular mechanisms regulating its uptake are not entirely understood. This study investigates how disruptions in the non-canonical autophagy pathway affect collagen internalization, focusing on differences between wild-type and ATG16L1-E230 mutant (E230) mouse embryonic fibroblasts (MEFs). Wild-type and E230 MEFs were cultured and plated in proliferating and contact-inhibited conditions. The MEFs were treated with varying concentrations of a fluorescently labeled collagen (DQ-Collagen). Collagen uptake was quantified using flow cytometry and analyzed to assess differences in internalization between genotypes and conditions. These methods allowed for a controlled comparison of uptake efficiency and offered insight into the specific pathways involved. Results showed that wild-type cells exhibited greater collagen uptake than E230 mutant cells, suggesting that the E230 mutation disrupts a mechanism required for effective collagen internalization. This reduction may indicate deficiencies in non-canonical autophagy-related pathways involved in collagen processing. These findings support the conclusion that the pathway affected by the E230 mutation plays a critical role in collagen uptake. Understanding this mechanism has broader implications for wound healing, where collagen remodeling is crucial for proper tissue repair and regeneration.