As gene-editing technology has improved, scientists have become increasingly focused on identifying specific targetable genes that may contribute to disease. Previous publications have examined the role of E2F3, a transcription factor responsible for regulating the transition from G1 to S phase. Dysfunction of the E2F3 transcription factor is potentially responsible for the development of cancer. To further investigate the role of E2F3 in disease development, we seek to identify and clone E2F3 in Strongylocentrotus purpuratus, a key model organism for development. Identification of the E2F3 gene as unknown DNA 6753 was completed through BLAST, InterPro, and phylogenetic analysis. DNA was amplified through PCR and further amplified through bacterial transformation. Validation of the size and sequence of the cloned insert was completed through colony PCR, DNA sequencing, and plasmid isolation. Despite successful transformation, colony PCR and restriction enzyme digestion were shown to be unsuccessful in detecting and cloning E2F3. This work enables future studies to manipulate E2F3 in Stronylocentrotus purpuratus and gain insights into the role of E2F3 in development.

Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) is a genetic syndrome characterized by a loss of fumarate hydratase (FH), a key enzyme of the tricarboxylic acid cycle. Clinically, HLRCC drives benign, painful cutaneous and uterine leiomyomas (tumors) and aggressive renal cell carcinoma. Previous research shows FH-loss results in profound metabolic rewiring, forcing tumor cells to rely on an alternative purine salvage pathway for survival and proliferation. This project seeks to further characterize these findings by investigating whether FH-deficient cells exhibit selective dependence on specific purines that can be exploited through precision nutrition interventions. A key step in trialing a precision nutrition intervention is establishing reliable in vivo FH-deficient models using viral-mediated Cre-loxP recombination. A surgical approach was performed to inject Cre-containing virus into the mouse kidney, followed by PCR-based genotyping to assess recombination three months post-operatively. Genotyping results did not demonstrate meaningful Cre-mediated recombination. These findings highlight challenges to establishing robust, spontaneous in vivo models to study HLRCC. Future work will focus on exploring alternative viral delivery systems, such as AAV8, as well as exploring bioorthogonal approaches via inducible, kidney-specific Cre models (e.g., SLC34A1-CreERT2). Establishing a spontaneous FH-deficient RCC model will be paramount to furthering our studies of precision metabolic targeting strategies in HLRCC.

Gene regulatory networks (GRNs) consist of genes represented as nodes and gene interactions as edges between nodes, and provide a means to model the effect of gene interactions across a cell. They have the potential to enable prediction of cellular responses to perturbations, but require robust experimental validation before they can reliably inform predictions. In this study, we developed methods to evaluate the accuracy of GRN models relative to real perturbation data. We determined a ground truth of perturbation effects using expression data from CRISPRi gene knockdowns. Then, using PIDC (Partial Information Decomposition and Context) applied to unperturbed single-cell RNA sequencing data, we inferred the network of interactions between genes. Knockdown effects were predicted from the network by assuming effects would be localized within node neighborhoods. By comparing these predictions to the observed effects, we calculated metrics representing the likelihood of accurate predictions. We tested various cutoff values for network parameters to select optimal thresholds for information retention. Using these, we visualized the network as a graph to gain insight into network structure. We compared short-range connections in the graph model against significant perturbed gene pairs from the data to ensure the network agreed with observed reality. Finally, we explore strategies to further improve model accuracy, including implementing network directionality and verifying consistency with assumptions about biological networks.

The unknown gene 4245 of the Strongylocentrotus purpuratus species was identified as Netrin-1 through DNA sequence analysis. Netrin-1, whose function in the organism is involved with its development as the gene plays a role in axon guidance, cell migration during development, promotes bone remodeling, and the derivation of adipose-derived stem cells. The gene's DNA sequence was compared against the BLASTN and BLASTX search algorithms, both of which yielded a top hit identified as Netrin-1-like. InterPro further confirmed the identification by establishing the gene protein family of Laminin/Netrin Extracellular Matrix and its multiple domains, LAMININ_NTER, Lameg_3, and EGF_lam. The gene was then cloned and amplified by PCR, and once the resulting product was confirmed to be the correct size by gel electrophoresis, it was then purified. The purified product was then inserted into a plasmid, which was then transformed into E. coli, resulting in successful colonies. These colonies should be representative of the successful cloning of the Netrin-1 gene, DNA sequence analysis is still required as a way to confirm this. The identification of Netrin-1 and its successful cloning can enable experiments such as CRISPR that will help us better understand the role the gene plays in the development of Strongylocentrotus purpuratus and in other organisms that have this gene in their genomes.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the gastrointestinal tract. It is characterized by tissue damage to the epithelial lining, bloody stool, weight loss, and other systemic symptoms. In this study, we evaluated the therapeutic potential of MXC-017, a novel vimentin-targeting compound developed in our laboratory. IBD was modeled using dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice, with comparisons made among three experimental groups: sham control, DSS control, and DSS + MXC-017 treatment. Disease progression was monitored over a two-week period. Compared to the sham control group, DSS-treated mice exhibited significant weight loss, elevated disease activity index (DAI), colon shortening, splenomegaly, and increased expression of pro-inflammatory cytokines (TNFα, IL-1β, IL-6, IL-10), along with marked epithelial damage. Treatment with MXC-017 mitigated most of these effects, including reducing weight loss, lowering DAI scores, preserving colon length, and decreasing the expression of TNFα, IL-1β, IL-10. Histological analysis further demonstrated improved preservation of epithelial integrity in the DSS + MXC-017 group. However, splenomegaly persisted despite treatment. These findings suggest that targeting vimentin with MXC-017 mitigates key features of DSS-induced colitis and highlights its potential as a therapeutic intervention for IBD.

Strongylocentrotus purpuratus, or the purple sea urchin, contains genes whose sequences have yet to be identified. By identifying these genes, they can be cloned and utilized in future research projects to understand molecular mechanisms within the sea urchin. In this project, unknown gene 1774 was identified and cloned. Its identity was determined using BLAST, InterPro, and Echinobase. To confirm these results, phylogenetic analysis was performed to compare the gene with its orthologs and paralogs. The gene was cloned through PCR, plasmid ligation, and plasmid transformation into E. coli cells. Cloning success was verified using colony PCR, miniprep, and restriction digest, followed by Sanger sequencing analysis to confirm the gene sequence. From the BLAST and Echinobase searches, the unknown gene was identified as being the Neuronal Acetylcholine Receptor Subunit Alpha-3 (Chrna3). InterPro analysis revealed that the majority of the protein is matched by the ligand-gated ion channel family. This gene is mainly expressed in the Peripheral Nervous System (PNS). Cloning success remained inconclusive for gene 1774. Future experiments redoing miniprep, restriction digest, and Sanger sequencing are necessary to confirm the cloning success of this gene. By identifying and cloning the Chrna3 gene, future studies can examine its functions within the PNS of S. purpuratus.

Hematopoiesis, the process of blood component production, is essential to life. However, most of the regulatory pathways that coordinate this process remain undiscovered. This experiment examines hematopoiesis through the lens of the Drosophila lymph gland. It has been established that the lymph gland intermediate zone contains multipotent progenitors, but genes regulating their maturation into hemocytes remain unclear. To investigate this, histone deacetylase 1 (HDAC1) and nucleoporin 154kD (Nup154) genes were knocked down using driver system-controlled RNA interference, and the subsequent effects on hematopoiesis were analyzed with fluorescent microscopy. The results indicate that HDAC1 and Nup154 play crucial roles in hematopoiesis progression and lymph gland morphology.

During early embryonic development of the cell, Epiblast stem cells( EpiSCs) are in a prime pluripotent state and are able to retain the ability to differentiate into multiple lineages. Zfp281 is a transcriptional factor that regulates pluripotency during embryonic development. WNT signaling is a communication pathway in regulating cell proliferation and differentiation. The relationship between Zfp281 and WNT signaling in regulating EpiSC remains underexplored. This study investigates the relationship between transcription factor Zfp281 and WNT signaling in maintaining pluripotency and promoting mesoderm differentiation in EpiSCs. EpiSCs were treated with FA control, dTAG to degrade Zfp281, XAV939 to inhibit WNT signaling, and combined dTAG and XAV939 treatment. Cell proliferation and viability were assessed with cell count, gene expression analysis using qPCR, protein detection via Western blot, and localization through immunostaining. Loss of Zfp281 reduced cell growth and decreased expression of pluripotency associated proteins like SOX2, indicating its role in maintaining the primed state. In contrast, inhibition of WNT signaling significantly reduced expression of mesoderm markers, including WNT8a and OTX2. This shows the importance of WNT signaling in driving differentiation. In conclusion, these results indicate that Zfp281 supports pluripotent identity, while WNT signaling assists in promoting mesoderm markers. This study highlights a regulating balance between self-renewal and differentiation in EpiSC fates.