Lipid nanoparticles (LNPs) are a leading platform for mRNA delivery but require low-temperature storage to maintain stability, limiting the ease of distribution. Lyophilization has the potential to improve storage and transport; however, maintaining nanoparticle structure and functional delivery, particularly for antibody-conjugated LNPs, remains a challenge. In this study, we developed and evaluated an optimized lyophilization and reconstitution workflow for both conventional and antibody-conjugated LNPs. mRNA-loaded LNPs were functionalized with targeting antibodies via click chemistry, including CD3-directed constructs for T cell delivery. Formulations were lyophilized in the presence of sucrose-based lyoprotectants (5% and 10% w/v), rehydrated, and characterized for particle size, mRNA encapsulation, and delivery efficiency in Jurkat T cells. Post-lyophilization re-concentration strategies were also assessed to restore effective dosing concentrations. Lyophilized LNPs retained close to 100% transfection efficiency with minimal cytotoxicity. Antibody-conjugated LNPs demonstrated improved gene expression relative to non-targeted controls, and this improvement was maintained following lyophilization. While a reduction in mean fluorescence intensity was observed after lyophilization, the relative performance advantage of antibody-functionalized formulations persisted.

Wound healing, a complex, multi-phase process that restores tissue integrity after injury, spans 4 stages (hemostasis, inflammation, proliferation, and remodeling) with fibroblasts playing important roles in tissue organization and remodeling. Despite the clinical importance of chronic, non-healing wounds, the cellular decision-making processes governing effective repair are not fully understood. Quiescence is a reversible and actively maintained non-dividing cell state that enables cells to pause and subsequently re-engage proliferation when needed, such as during wound healing. Using standard and customized computational approaches to study a time-course human single-cell RNA-seq (scRNA-seq) dataset of skin wounds, we found that proportions of quiescent and proliferating fibroblast populations change across the wound healing stages, with concomitant changes in the activities of state-specific transcription factors (TFs). The early stage was enriched for quiescent TFs, followed by proliferative TFs in intermediate stages, and a combination of both in the late stage. In contrast, our analysis of scRNA-seq chronic wound (venous ulcers) data revealed a higher proportion of proliferating fibroblasts with TF activities resembling intermediate to late healing stages. These findings provide insight into fibroblast regulatory behavior and lay a foundation for future studies on how disrupted quiescence dynamics contribute to impaired/chronic wound healing.

Under nutrient limitation, yeast undergo sporulation, completing meiosis to generate four haploid spores from a starting diploid. The common S288C-derived laboratory strains have low sporulation efficiency due to polymorphisms in three meiotic regulator genes, RME1, MKT1, and TAO3. A recently developed plasmid, pSPObooster, restores efficient sporulation by supplying corrected alleles for RME1 and MKT1, making the lab strains amenable to large scale genetic studies requiring sporulation. This project extends the pSPObooster by adding to it mating-type-specific promoters to drive drug-resistance markers, enabling drug-based selection of haploid progeny with desired mating type after meiosis. Selectable markers, both for drugs and auxotrophies, were used to confirm transformation and mating steps. The modified pSPObooster cassette was transformed into our yeast strain of interest and then mated with other yeast strains in our library. This diploid was then sporulated to evaluate pSPOboosters effectiveness. This work establishes a scalable genetic tool that improves sporulation efficiency while adding selective recovery of progeny with a single mating type. This tool will be used for high-throughput genetic mapping of natural variants affecting phenotypes and screening variant effects in large mutagenic libraries transformed into collections of laboratory yeast gene knockout strains.

Histone lactylation (Kla) is a novel epigenetic modification that regulates gene expression. Increased lactate dehydrogenase (LDH) activity has been shown to accelerate hair cycle entry, but the underlying mechanism remains unknown. We hypothesized that lactate generated by LDH is converted into lactyl-CoA and used for histone lactylation, altering gene expression to promote hair follicle stem cell (HFSC) activation. Understanding this connection could provide mechanistic insight into how metabolic pathways influence adult stem cell behavior. We characterized Kla dynamics in mouse cohorts at distinct metabolic states, including mitochondrial pyruvate carrier inhibitor UK5099-treated aged mice and LDH knockouts, utilizing immunostaining on follicles at various hair cycle stages. Across all cohorts, Kla levels were highest in the hair germ and dermal papilla, key signaling hubs, suggesting lactate-mediated regulation of HFSC activation. In contrast, Kla marks were lower in the bulge region, despite its glycolytic HFSC population. Aged mice showed a global reduction in Kla marks, which were partially restored in UK5099-treated mice. Additionally, LDH knockout tumors exhibited differential expression of Kla marks (H3K9 and H3K18). Future aims will inhibit lactyl-CoA synthetase to investigate the effect of disrupting lactylation on hair cycle progression. Together, these experiments work to solidify histone lactylation as the mechanistic link between metabolic regulation and HFSC activation.

The gradual breakdown of myelin in the aging brain contributes to impaired neural signaling and is a key feature of neurodegenerative diseases such as Alzheimer’s disease. As myelin degrades, debris accumulates and places increasing stress on microglia, the brain’s immune cells responsible for clearance. Over time, this burden contributes to chronic inflammation and cellular dysfunction. Notably, aged microglia often exhibit characteristics of cellular senescence, but it remains unclear whether they are truly senescent or adopt a senescence-like inflammatory state. This study investigates whether myelin debris directly induces bona fide cellular senescence or drives persistent inflammation. I hypothesize that exposure to myelin debris induces true senescence in macrophage-lineage cells. Using mouse bone marrow-derived macrophages (BMDMs), I will compare myelin-treated cells to irradiation-induced senescent and untreated controls. Senescence and inflammatory profiles will be evaluated using quantitative PCR and senescence-associated β-galactosidase staining. By distinguishing true senescence from inflammatory activation, this work will inform whether senolytic therapies may be effective in targeting microglial dysfunction. These findings will help determine whether targeting senescent cells is a viable strategy for modulating neuroinflammation and disease progression.