New York Esophageal Squamous Cell Carcinoma-1 (NY-ESO-1) is a cancer-testis antigen expressed in various tumors and is an attractive target for cancer immunotherapy. While T-cell receptors (TCRs) that recognize NY-ESO-1 have been developed and used in adoptive T cell therapies, little is known about B cell receptors (BCRs) that can recognize NY-ESO-1. Our lab has shown that B cells can cross-present NY-ESO-1 protein to CD8+ T cells and promote their proliferation and cytotoxicity in vitro. We and others have found that cancer patients whose tumors express NY-ESO-1 have detectable anti-NY-ESO-1 antibodies in their serum, which suggests the presence of memory B cells specific for NY-ESO-1. Indeed, my data demonstrated that B cells derived from melanoma patients’ PBMCs show positive uptake of NY-ESO-1 protein, while those from healthy donors show little to no uptake. Based on this, we sorted patient-derived B cells with NY-ESO-1 uptake to prepare for BCR sequencing. This project aims to design a BCR construct that enables B cells to recognize NY-ESO-1 for improved cross-presentation. The goal is to use these sequences to engineer a BCR construct, transduce B cells, and evaluate their ability to activate NY-ESO-1-specific CD4+ and CD8+ T cells in vitro. Discovering NY-ESO-1-specific BCRs could lead to new ways to promote T cell activation, potentially enhancing cancer immunotherapy strategies by leveraging B cells as antigen-presenting cells.

In purple sea urchin, Strongylocentrotus purpuratus, transcription activator BRG1 is part of an ATP-dependent chromatin remodeling complex, the SWI/SNF complex. BRG1 plays a crucial role in chromatin accessibility and RNA polymerase II binding. This experiment aimed to determine the identity of the BRG1 sequence through gene cloning and sequence analysis. The identity of the sequence was analyzed using DNA sequencing software, including BLAST, InterPro, and Echinobase, a genomic database specific to echinoderms. Phylogenetic analysis was also performed to determine and visualize the evolutionary relationship between the sea urchin BRG1 sequence and the BRG1 genetic sequence of other species. The gene cloning portion of the experiment was done through PCR amplification and bacterial transformation of the amplified and ligated sequence. Colony PCR, restriction digest, and sequencing were then performed to confirm the success of the cloning experiment. The experiment's results were inconclusive on the success of the BRG1 sequence cloning. Further experiments, including another colony PCR and restriction digest, should be performed to confirm the success of cloning. DNA sequencing should also be performed to determine whether the cloned sequence matches the expected sequence for purple sea urchin BRG1. This will open the door for future experiments to determine the role and function of BRG1 in purple sea urchins through specific targeting of this sequence in the S. purpuratus genome.

Fibroblast growth factor receptor 3 (fgfr3) is a protein that plays a key role in regulating bone growth during skeletal development by controlling ossification of bone from cartilage. Disruptions of fgfr3 are known to result in bone conditions such as Achondroplasia, a common type of short-limb dwarfism. This study aims to determine the identity of the unknown portion of the sea urchin genome and clone the gene in bacterial cells. Gene identity was ascertained through bioinformatic tools such as BLASTx, InterPro, and Echinobase, while phylogenetic analysis further supported its identification. The gene was then amplified through PCR, ligated into a plasmid vector, and transformed into E. coli cells. Subsequently, colony PCR, restriction digest, and Sanger sequencing were used to verify cloning success. The unknown gene was characterized as putative fgfr3 using bioinformatic tools and phylogenetic analysis. The gene cloning of fgfr3 was unsuccessful, as the presence of an fgfr3 insert could not be identified in the colony PCR or the restriction digest. Future studies would look towards reattempting the cloning experiment with improved methodologies, as the initial attempt was unsuccessful. A long-term study would aim to characterize the functional role of fgf3 in sea urchin development, such as during embryogenesis.

The aim of this study was to find the identity of an unknown purple sea urchin gene from a portion of its coding region, and clone it into a plasmid vector. Methods included an initial database research (using BLAST, InterPro, and Echinobase), identifying the gene as phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha. This was followed by subsequent rounds of PCR, gel electrophoresis, and product purification of the sample. A vector ligation was then performed, followed by bacterial transformation using competent cells. The experiment was finalized with a colony PCR, inoculation, plasmid DNA isolation, and a restriction digest. A last round of gel electrophoresis analysis allowed for identification of an insert band size similar (667 base pairs) to the expected gene size (693 base pairs). A phylogenetic analysis was performed for the gene and the sample was sent for sequencing, which confirmed the gene identity and indicated that the cloning experiment was a success. The confirmed gene may now be further investigated in terms of its importance and function in the purple sea urchin.

Lipid Droplets (LDs) are organelles present in the cytoplasm across various cell types. Their primary role involves storing free fatty acids (FFAs) for subsequent use in many metabolic pathways. The goal of the study is to investigate whether changes in LD dynamics impact satellite cells (SC) differentiation and the ability to fuse. We targeted enzymes involved in LD formation and breakdown by using specific inhibitors: Atglistatin (ATGLin) to increase the number of LDs, and A922500 and PF-06424439 (DGATin) to inhibit LD formation. We found that ATGLin significantly decreased myoblast fusion by 79% (p < 0.001) at 3 hours and 38% (p < 0.001) at 5 hours, and their ability to grow when compared to control (36% MHC+ area vs 27%, p < 0.0001). Incubation of SCs with DGATin resulted in an increase of myosin heavy chain (MHC) positive area by 18% when compared to the control (p < 0.05). DGATin, Linoleic Acid (LA), and Ferrostatin-1 partially reversed the negative effects of ATGLin. Those data indicate that LD dynamics may play an important role in myogenic progression. The observed results likely arise from an augmented cellular demand for FFAs during periods of heightened growth and the capability of Ferrostatin-1 to prevent ferroptosis, cell death due to lipid peroxidation. The exact mechanism underlying this relationship for the myogenic progression requires further research. This data might help develop an understanding about muscle stem cell biology and new therapies targeting conditions of reduced muscle regeneration capacity.