Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is both an oncofetal RNA-binding protein and an N6-methyladenosine or m6A reader of any changes made to the RNA. The discovery of RNA methylases and methylation-related RNA-binding proteins, known as readers, helped establish the basis of the epitranscriptome hypothesis, connecting mRNA modifications to post-translational regulation of genes, and underlaying IGF2BP3's posited relationship with m6A and leukemogenesis. By conducting metabolic profiling of various leukemic cell lines, including those with IGF2BP3 knockouts, we sought to identify how IGF2BP3 affected metabolism, specifically changes in one-carbon metabolism and downstream glycolytic pathways. We found that IGF2BP3 affected the synthesis of S-adenosyl methionine (SAM), a substrate for cellular methylation reactions, via the action of the enzyme MAT2B or methionine adenosyltransferase complex, which controls the reaction for SAM production. IGF2BP3 was additionally found to result in decreased m6A concentration on mRNA related to metabolic pathways, a change in patterns that suggests that IGF2BP3, beyond its role as an RNA-binding protein, could serve as a writer of the epitranscription in leukemia cells. This helped establish a connection between oncogenic metabolism and post-translational modifications on RNA, since oncogenic changes in the gene were maintained by the m6A methylation near the 3’-untranslated regions near the stop codon.

Heterozygous germline mutations in PTEN are associated with autism spectrum disorder (ASD) and macrocephaly, yet their functional impact remains unclear. While complete PTEN loss increases neural proliferation and brain size, heterozygous mutations may act through dominant negative mechanisms. This study investigates whether patient-derived PTEN mutations (p.H93Y, p.G132D) result in partial or complete loss of function in human neurodevelopment. Using induced pluripotent stem cells (iPSCs) from affected individuals, we generated isogenic CRISPR-corrected controls and PTEN knockout lines. We assessed PI3K–AKT–mTOR pathway activity via Western blot and examined cerebral organoid growth as a model of early brain development. Preliminary results show that complete PTEN loss increases AKT phosphorylation, while heterozygous mutations do not significantly alter AKT activation compared to controls. No conditions showed increased downstream mTORC1 signaling, suggesting potential compensatory mechanisms. Organoid growth analyses are ongoing. These findings suggest that heterozygous PTEN mutations may not function as dominant negatives at the level of AKT signaling. This work advances understanding of how PTEN dysregulation contributes to altered neurodevelopment in ASD and may inform future therapeutic strategies targeting this pathway.

Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disorder affecting reproductive-aged women worldwide. Despite its prevalence, the disease etiology is not fully understood. While genome-wide association studies (GWAS) pinpoint risk variants, the genetic mechanisms driving PCOS remain unclear due to limited reliable cell models. Primary human granulosa cells are biologically relevant but exhibit low viability in culture. To address this gap, I aim to validate and characterize the KGN cell line, an immortalized human granulosa tumor-derived model. To establish its reproducibility, I optimized culture conditions, determining a 1:5 passage ratio optimal for growth, reaching 80% confluency within four days. Images of KGN cultures confirmed the expected spindle-like shape. Samples were verified mycoplasma-free by PCR, and freeze-thaw tests demonstrated high post-cryopreservation viability. Additionally, I extracted high-purity RNA to validate baseline expression of key steroidogenic (CYP19A1, STAR) and folliculogenesis (FOXL2, FSHR, INHA) genes. Preliminary PCR primer validation using standard and melting curves demonstrated strong expression for FOXL2 and STAR, while optimization for additional primers is still ongoing. Future work will utilize RT-qPCR to quantify gene expression and indirect immunofluorescence to verify target protein localization. Validating the reproducibility of the KGN line will provide our lab a system to uncover the functional roles of non-coding PCOS variants.

The intestinal epithelium undergoes rapid turnover, yet it must maintain stable communication with the gut microbiome and nervous system. Using a fate-mapping approach, we identified a previously unrecognized population of long-lived, serotonin-producing enterochromaffin (EC) cells in the colon, termed persistent colonic EC (pcEC) cells. Through EdU incorporation assays, organoid cultures, and an injury-repair model, we demonstrate that pcECs are non-proliferative and lack regenerative capacity. Instead, pcECs represent a terminally differentiated population characterized by a distinct molecular profile that distinguishes them from canonical EC cell subtypes. Notably, depletion of the microbiota with antibiotics significantly increased pcEC cell abundance, suggesting that microbial cues regulate their population dynamics. Functional analysis using ex vivo calcium imaging revealed that, unlike canonical EC cells that respond to noxious stimuli, pcECs preferentially respond to basal luminal cues under homeostatic conditions. Consistent with a role in gut-brain communication, ~50% of distal colon pcECs directly contact gut-extrinsic sensory nerve fibers. Together, these findings identify pcECs as a specialized sensory population that supports persistent gut-brain signaling despite rapid epithelial turnover. Future studies will define how pcECs influence neural circuits and physiological responses. This work provides a foundation for targeting sensory epithelial-neural circuits in gastrointestinal disease.

Pluripotent stem cell differentiation is a complex process through which stem cells develop into specialized types, traditionally due to transcriptional changes specific to each germ lineage. However, metabolites and metabolic enzymes also have a major role in influencing differentiation patterns. Their localization within the cell can be a determinant to their function, as recent studies have shown the presence of tricarboxylic acid (TCA) cycle enzymes acting within nuclear processes. In order to further understand the function of nuclear proteins, we plan on studying the composition of pluripotent stem cell nuclei, the nuclear changes that occur during differentiation, and the quantification of metabolites within the nucleus. To accomplish this, we developed a model based on prior research that improves nuclear isolation for metabolomics analyses. Human pluripotent stem cells were transfected with a custom vector that contains proteins which will label mitochondria and nuclei with specific epitope tags. Protein isolation was then conducted using magnetically labeled beads conjugated to each tag, which resulted in enrichment of mitochondrial and nuclear proteins separately, affirming our model for organelle isolation. We plan on using this methodology to measure and quantify nuclear metabolites throughout differentiation to understand the importance of metabolic localization on function and regulation.