Given the general lack of knowledge and practical use of many genes found in the genome of Strongylocentrotus purpuratus, this experiment set out to determine if the gene Fos-related antigen 2-like could be cloned into Escherichia coli cells. This gene is a component of the AP-1 transcription factor complex involved in proliferation, apoptosis, and differentiation, and has ties to cancer and autoimmune disease progression. This procedure was accomplished through the use of BLAST, Echinobase, and InterPro database searches for identification of the gene, which returned a gene identity of Fos-related antigen 2-like, a part of the Fos transcription factor family. A phylogenetic analysis was completed, illustrating a close relation to similar genes in other species, further confirming gene identity. Then, polymerase chain reaction and purification of the gene, ligation to a plasmid vector, and chemical transformation of the vector into E. coli cells were completed. Successful cloning was verified with a restriction digest and sequencing. A restriction digest showed one colony with an insert of the expected size. Sequencing of these plasmids revealed an insert percent identity of over 99%. As such, the Fos-related antigen 2-like gene was shown to be successfully cloned into E. coli cells. This allows these samples to be used in future procedures, such as in situ hybridizations or cre-lox-based knockout of the gene to determine localization and phenotypic expression, respectively.

Immune checkpoint inhibitor (ICI) therapy is commonly used to reduce tumor growth by over-activating immune cell activity. However, ICI therapy has resulted in off-target inflammatory effects in various organs, leaving patients with poorly understood prognoses when treated with ICIs. Here, we attempt to characterize the inflammatory phenotypes that present in DBA/2J mice when treated with anti CTLA-4 and anti PD-1 combination ICI therapy. Our findings indicate an inadequate cardiac inflammatory response demonstrated by minimal impairment of cardiac function measurements such as diastolic volume and ejection fraction, and minor cardiac fibrosis and T cell infiltration observed in DBA/2J mice when treated with combination ICI therapy. As a result, DBA/2J mice may display resistance to combination ICI therapy, and future studies should consider using mouse models that are made more susceptible to enhanced immune responses following ICI therapy. To understand the ICI-induced cardiac injury pathway and better characterize inflammatory phenotypes, genetic differences and additional experimental strategies can be applied. This can include utilizing an alternative mouse strain or inducing an accelerated immune response to establish potent cardiac inflammation and elucidate phenotypic manifestation of ICI-induced cardiac inflammation.

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.

Mitochondrial dysfunction is implicated in a range of human diseases, including Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS). Efficient generation of induced pluripotent stem cells (iPSCs) from patient-derived fibroblasts offers a powerful platform for disease modeling, but the introduction of mitochondrial mutations can hinder reprogramming. This study aimed to improve iPSC reprogramming efficiency in Mitopunch-engineered wild-type (WTP), MELAS, and Mitopunch-engineered MELAS (MELAS P) cell lines by modulating chromatin accessibility via histone deacetylase (HDAC) inhibition. Initial trials using titrated concentrations of valproic acid (VPA) and trichostatin A (TSA) led to severe cytotoxicity post-transduction, highlighting the need for protocol refinement. In this revised approach, reduced drug concentrations, extended reprogramming periods, and modified culture conditions significantly improved cell viability and colony formation. While TSA remained cytotoxic, VPA-treated cells exhibited robust proliferation and the emergence of iPSC colonies with characteristic morphology. Although genotypic confirmation via sequencing is still pending, these preliminary findings support the use of lower-dose HDAC inhibition as a promising strategy for enhancing reprogramming in mitochondrial disease models, advancing efforts toward patient-specific iPSC generation for therapeutic applications.

Late clinical toxicity due to radiosensitivity can present a significant challenge for patients undergoing radiation therapy, as it results in severe long-term adverse health effects. In this study, we predict that differences in gene expression in lymphoblastoid cell lines (LCLs) after radiation can be used to understand the genetic and biological mechanisms leading to late clinical toxicity in patients with known radiation outcomes. LCLs offer a stable and renewable model for gene expression with a greater potential for an individualized assessment of response to radiation. A total of 39 cell lines from patients with known outcomes were exposed to ionizing radiation, with samples collected 0, 6, 24, and 48 hours after irradiation to analyze differences in gene expression. The anticipated results have the potential to allow insight into the early biologic and genetic mechanisms of late clinically meaningful toxicity to radiation therapy. This information could enable future targeted interventions to prevent radiation toxicity for those at risk.