The inner membrane complex (IMC) in apicomplexan parasites is a conserved membrane-cytoskeletal system beneath the plasma membrane that modulates parasite replication and motility. In Toxoplasma gondii, the IMC includes fourteen intermediate filament-like proteins called alveolins which are characterized by a conserved alveolin domain and play roles in reinforcing the organelle and assisting in daughter cell assembly. Here we identify two alveolin subcomplexes: IMC7 and IMC12 form an interdependent complex exclusive to maternal parasites, while IMC3, IMC6, and IMC10 form a complex enriched in the daughter IMC. Utilizing CRISPR/Cas9 gene knockout and pairwise yeast-two-hybrid (Y2H) assays, we demonstrate direct binding between IMC7 and IMC12. Previous work indicated IMC3 and IMC6 interact via their alveolin domains; our Y2H analysis further reveals an interaction between IMC10 and both IMC3 and IMC6. AlphaFold prediction suggests that the alveolins utilize beta sheets formed via the alveolin domain for interaction and filament formation. Surprisingly however, truncation studies of IMC10 show that the minimal functional region surprisingly lies outside the alveolin domain in the C-terminus of the protein, which includes a conserved valine-rich region that appears to be sufficient for tethering to the IMC and function. In addition, the alveolin domain of IMC10 alone cannot target properly to the IMC. These findings make IMC10 the first alveolin to function without its alveolin domain.

Volumetric muscle loss (VML) injury removes large portions of skeletal muscle and overwhelms its natural regenerative capacity. Muscle stem cells normally preserve muscle integrity alongside a supportive niche. This multicellular environment is lost in VML, impairing regeneration with subsequent fibrosis and chronic functional deficit. Skeletal muscle progenitor cells (SMPCs) cultured in vitro may restore muscle and the niche but are embryonic-like and lack robust regeneration in published protocols. A 2D differentiation protocol from the Pyle Lab directs human pluripotent stem cells into PAX7+ early fetal-like SMPCs. Maturation improves with supportive cells, but spatial multicellular organization may be needed to progress further. We hypothesize 3D culture systems will enhance SMPC maturity by better recapitulating native skeletal muscle. In promoting crosstalk between cell types, 3D organoid cultures may improve muscle fiber development and neuromuscular junction formation. Over 60 days, we performed RT-qPCR and immunofluorescence to compare cell populations and organization between 2D and 3D. Similar supportive cell types were in both; however, extended culture duration and spatial organization in 3D may improve contractility maintenance and produce more mature muscle fibers and neuromuscular structures. This work aims to determine if 3D culture more effectively recapitulates native skeletal muscle architecture and function, advancing SMPC maturation toward adult-like for cell-based therapies targeting injuries like VML.

It has been shown that proteolytic destruction of SARS-CoV-2 virions by the immune system liberates peptide fragments that contribute to inflammation. Additionally, there is evidence of proteasomal cleavage producing peptides with antimicrobial properties in healthy cells. The AMP-similarity distribution of the human peptidome, scored by distance from an SVM classification boundary, remains stable across tissues and organ systems in the absence of non-host peptides, and preliminary analysis indicates that elevated AMP-like fragment abundance correlates with inflammatory potential. We propose that inflammatory effects are characterized by a shift in this distribution toward higher AMP-like peptide content resulting from viral cleavage. Currently, no well-defined criterion exists to identify the level of AMP-like cleavage potential from the viral peptidome that characterizes inflammatory effects, leaving open the question of which coronaviruses and viruses at large will cause inflammation. This project employs data-driven and computational methods to analyze viral and host peptide data to develop such a criterion.

Macrophages are central regulators of T cell responses, shaping proliferation, differentiation, and activation across diverse immune contexts. Previous studies have suggested that naïve macrophages suppress T cell proliferation and activation. However, using an alternative experimental design involving macrophage pre-activation followed by co-culture, we instead observed a robust pro-proliferative effect of naïve macrophages on T cells. This discrepancy suggests that previously reported suppressive effects may arise from activation-dependent experimental conditions. To define the scope of this effect, we examined whether macrophage-driven proliferation was restricted to specific T cell subsets. Naïve macrophages promoted proliferation across both CD4⁺ and CD8⁺ compartments, including both naïve and memory populations. We next assessed functional consequences of macrophage co-culture. After 14 days, T cells co-cultured with naïve macrophages exhibited increased intracellular granzyme B levels and, upon PMA restimulation, produced higher levels of TNF compared to non–co-cultured controls. These findings indicate that naïve macrophages not only enhance T cell expansion but also potentiate their effector function. Together, our results challenge the prevailing view of naïve macrophages as suppressive and instead support a model in which they promote both T cell proliferation and functional activation, with important implications for immune regulation in health and disease.

Targeted anti-cancer therapies often produce unpredictable responses, creating a clinical need for reliable pre-treatment biomarkers. Variation in mitochondrial DNA (mtDNA) has emerged as a potential contributor to treatment response, but the functional consequences of mtDNA sequences in influencing therapy resistance are not well understood. Studies of mtDNA variation may reveal new mechanisms of tumor evolution and identify opportunities to overcome therapeutic resistance. Here, we used a tumor organoid–immune cell co-culture model to examine how mtDNA affects response to immune-checkpoint blockade (ICB) therapy, testing both spheroid and basement membrane embedded approaches. Tumor cells were engineered to harbor defined homoplasmic mtDNA mutations using MitoPunch, generating stable, isogenic lines that isolate mitochondrial genetic effects. Following mtDNA sequence validation, we will treat these mtDNA-engineered tumor organoids in co-culture with peripheral blood mononuclear cells (PBMCs) and ICB therapy, followed by functional readouts of treatment efficacy and tumor cell viability. Our results aim to uncover the contribution of mtDNA to tumor treatment response and resistance, while laying the groundwork for future in vivo studies.