Human Papillomavirus is responsible for hundreds of thousands of deaths annually and a leading cause of malignancy, yet limited post-infection treatments exist and resulting metastatic cancers have poor outcomes. Adoptive cell transfer using transgenic TCRs against constitutively expressed E6 and E7 antigens is a promising approach to combating HPV malignancies. Specifically, however, transgenic HSC engraftment is positioned to confer far superior outcomes to traditional peripheral T cell-based therapies—due to improved persistence and sustained activity among effector populations.    Vectors for E6 and E7-specific TCRs under the MND promoter successfully transduce PBMCs to generate high construct expression and an antigen-specific lysis of HPV+ tumor cell lines. However, engraftment of MND-TCR-transduced HSCs in mice led to poor reconstitution of construct-expressing cells among peripheral T-cell populations.    Considering the strength of the MND promoter and detectable integration of vector among mature T-cells, it’s likely that MND-driven constructs overexpress TCR, causing negative selection during thymus maturation. Therefore, comparatively-tame EF1a and PGK promoters may serve as superior regulators of TCR expression, preventing negative selection during HSC development while maintaining effector function in mature T cells.    Restriction site cloning in plasmids replaced MND promoters upstream of E6 and E7-specific TCRs with EF1a and PGK alternatives. The four newly-generated constructs were used to produce lentiviral vectors for future transduction of PBMC and HSC populations. Ultimately, after measuring TCR expression in transduced cells as well as capacity for an antigen-specific effector response in T-cells, an optimal promoter will be selected for use in an HSC-based TCR therapy against HPV-induced malignancy.

Cancer is fundamentally a disease of dysregulated cellular proliferation. Altered lipid metabolism is emerging as a key feature of tumors, likely to support the increased membrane and organelle biogenesis in rapidly dividing cancer cells. Previous studies have defined the lipidome during individual stages of the cell cycle. Furthermore, the conventional synchronization method of limiting nutrients alters the cell’s regulatory pathways governing lipid metabolism, prompting an increase in lipid synthesis for cell survival independent of their proliferative need. To address this gap in our knowledge, we propose a comprehensive study of cellular lipid composition during the entire cell cycle. Initially, cells will be synchronized using the CDK4 inhibitor Palbociclib and then released. Shotgun lipidomics will be performed as cells move through each stage of the cell cycle. In parallel, 13C-glucose stable isotope enrichment studies using gas chromatography-mass spectrometry and mathematical modeling of isotopologues will be performed to define the contribution of cholesterol and fatty acid synthesis and import to the lipidome across the cell cycle. Finally, we will assess the impact of pharmacological inhibition of lipid synthesis on the lipidome during different phases of the cell cycle. We anticipate dynamic changes in the synthesis of lipids, and consequently, the cellular lipidome as cells progress through the cell cycle. This study has broad implications for the role of lipids in cell biology, and could identify new therapeutic targets to decrease cancer proliferation and survival. 

Over the past decade, biomolecular condensates have emerged as a central element of many cellular processes. These membraneless organelles, formed by liquid-liquid phase separation (LLPS) of RNA, protein, and other biomolecules, carry out numerous functions including molecular transport within living cells, particularly within cytoplasm. Among them, several endogenous condensates are shown to interact with the cytoskeleton of the cell and transport in a certain direction that is mainly driven by motor proteins. The positioning and spatiotemporal location of these condensates are of paramount importance in a healthy cell, failure of which can lead to serious malfunctions and damages to cells and overall behavior of the body. Here, we introduced ribonucleoprotein (RNP) condensates, artificially expressed in mammalian cells, to investigate active transport mechanisms and spatial localization of biomolecules within cytosol. We designed a system that consists of two parts: a microtubule motor protein fused with MS2 coating protein (MCP) and a phase-separating, single-stranded RNA motif, termed nanostar, that incorporates the MS2 domain. Upon expression of both parts in the cell, we expect that the RNA nanostars containing MS2, bind and recruit the motor proteins via fused MCP and form RNP condensates. Thanks to the presence of motor proteins, these condensates can form either around the nucleus at centrosome or at the periphery of the cell, depending on the type of motor protein used that can be either plus- or minus-end. Our preliminary results demonstrate that both parts of our system can successfully be expressed in cells and form expected nanostructures.  

Bile acids are liver-synthesized molecules essential for lipid absorption and metabolic  signaling. The elimination of excess cholesterol depends on bile acid synthesis, as bile acids  are produced from cholesterol. In the classical pathway, cholesterol 7-α hydroxylase (CYP7A1)  catalyzes the first and rate-limiting step, while sterol 12-α hydroxylase (CYP8B1) is necessary  for producing cholic acid. This bile acid acts as a feedback regulator, causing downregulation of  bile acid synthesis when levels are high. Because the Tarling-Vallim laboratory is studying the  intricacies of bile acid synthesis in vitro, I aimed to disrupt CYP8B1 in human embryonic kidney  293T (HEK293T) cells using CRISPR gene editing. I designed guide RNAs targeting human  CYP8B1 and completed all steps of cloning. I confirmed the CRISPR plasmid sequences  through restriction digest and Sanger sequencing, followed by maxiprep to ensure sufficient  plasmid stock. To validate the plasmids for gene disruption, I co-transfected HEK293T cells with  the CRISPR plasmids and a construct expressing human CYP8B1. Transfection efficiency was  confirmed with additional transfection of green fluorescent protein (GFP), which I visualized  using a fluorescence microscope, and total protein was quantified by bicinchoninic acid (BCA)  assay. By Western blot analysis, CYP8B1 protein was significantly reduced or absent in  samples treated with CRISPR compared to controls, demonstrating successful disruption of  CYP8B1. Future generation of human liver cell lines disrupted for this protein will provide a  molecular platform for studying regulation of bile acid synthesis in metabolic disease models.

Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer, most patients with LUAD are diagnosed at an advanced stage, where prognosis is poor. Progression is influenced by metabolic and epigenetic alterations, yet the interplay between these processes remains incompletely understood. This study investigates the effects of alpha-ketoglutarate (AKG), a key tricarboxylic acid cycle metabolite and epigenetic cofactor, on histone methylation and signaling pathways relevant to liver biology. The larger aim is to connect Kras cancer with systemic effects of AKG, one of which is through the liver. Using a murine model system stratified by KRAS mutation status, age, and sex, we assessed global epigenetic changes via Western blot analysis of H3K4me3 and H3K27me3, alongside evaluation of mTOR pathway activity through p-S6(Ser235/236), p-4EBP1, and p-P70 phosphorylation  in the liver. mTOR is one of the most critical hepatic signaling pathways. AKG treatment altered histone methylation patterns, with responses observed across genotypic and age groups, suggesting context-dependent epigenetic modulation. Concurrently, AKG influenced downstream mTOR signaling, suggesting potential cross-talk between metabolic inputs and signaling cascades. These findings highlight AKG’s capacity to reshape the epigenetic and signaling landscape in the liver, and further investigation into its role in cancer and into metabolic interventions for systemic modulation of cancer is ongoing.