The relationship between differences in genotypes, and unique phenotypes is the bedrock of genetics. Recently, dogs have been identified as an invaluable model organism for advancing genomic analyses. Different dog breeds exhibit drastic and distinct morphologies from each other. Meanwhile, dogs within the same breed tend to look very similar. Additionally, Much of the morphological variation observed in different dog breeds has been shown to be the result of a small number of genetic differences. Thus, understanding how very small changes within the dog genome result in massive physical differences can help us utilize these animals for genetic studies better. To further this understanding, we, with the mentorship of my PI, Dr. Matteo Pellegrini, have expanded and optimized a computational pipeline that predicts a dog's breed based on the single-nucleotide polymorphisms (SNPs) present in its genome. This was done by attaining whole-genome sequencing data from public databases, aligning genomic data to the CanFam4 reference genome. A computational tool, SCOPE, was then used to infer admixture proportions based on genome-wide data and assess the efficacy of the developed pipeline. We were able to accurately separate dog breeds into clusters based on the SNPs detected in their genome. Furthermore, using SCOPE, we were able to predict the breed of a dog based on the SNPs detected in their genome.

NF2-related schwannomatosis (NF2-SWN) is a rare genetic condition caused by mutations in the NF2 tumor suppressor gene, coding for the merlin protein. The hallmark manifestation is bilateral vestibular schwannomas, but patients also develop meningiomas, ependymomas, and peripheral neuropathy. Few germline mouse models with Nf2 mutations have been developed but none display spontaneous biallelic loss of merlin and development of schwannomas. Thus, novel animal models that faithfully recapitulate NF2-SWN-associated phenotypes are needed. Ossabaw minipigs are physiologically more similar to humans than mice, including high genetic conservation, making them an ideal model organism. Using hematoxylin-eosin and immunohistochemistry staining techniques, we describe here the histological characteristics of the first minipig model carrying a NF2 germline mutation and developing NF2-SWN phenotypes. Primary findings include a cerebellopontine angle tumor and schwannoma tumorlets in the peripheral nerves. Additional relevant phenotypes include the widespread presence of onion bulb-like lesions in the sciatic nerves, containing concentric layers of collagen deposits. Similar lesions have been linked to neuropathy in NF2-SWN patients and mouse models, a phenotype that can lead to muscle wasting and in rare cases death. Thus, this new model provides a valuable pre-clinical platform and facilitates the investigation of NF2-SWN-specific manifestations in a large organism.

Cranial defect reconstruction is performed in 13.8 million surgeries annually, yet current approaches for reconstruction are limited by donor site morbidity and high complication rates. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds offer a promising alternative for skull regeneration, but tracking human mesenchymal stem cells (hMSCs) within these materials remains challenging. Functionalizing MC-GAG with polyethylene glycol arginine-glycine-aspartic acid (PEG-RGD) peptides may enhance hMSC adhesion by providing integrin-binding sites, while labeling hMSCs with poly-L-lysine–conjugated gold nanoparticles (PLL-AuNPs) may enable high-resolution imaging without impairing osteogenesis. This study evaluated whether PEG-RGD functionalization improves hMSC proliferation and migration and whether PLL-AuNP labeling enables visualization of osteogenesis. hMSCs were labeled with PLL-AuNPs and seeded on MC-GAG or PEG-RGD-MC-GAG scaffolds. Proliferation was measured using WST-8 assays, migration via trans-well assay and DAPI quantification, and osteogenesis using Alizarin Red staining. Immunofluorescence confocal imaging visualized cell morphology and distribution. PEG-RGD-MC-GAG scaffolds significantly increased hMSC proliferation and migration compared to MC-GAG (p < 0.05). PLL-AuNP labeling produced comparable calcium deposition to unlabeled cells, indicating potential for tagging. Together, this approach offers a promising platform for enhanced cranial bone regeneration and real-time cellular tracking.

Cystic fibrosis (CF) is a chronic monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, making it an ideal target for gene therapy. Our group recently developed a lipid nanoparticle (LNP) platform to effectively deliver CRISPR/Cas9 editing reagents designed for site-specific knock-in of a corrected copy of CFTR at the endogenous locus in a human bronchial epithelial cell line, allowing mutation-agnostic functional correction of CFTR. However, low rates of homology directed repair (HDR)-dependent gene insertion remain a challenge. Thus, we explored direct upregulation of HDR via overexpression of cyclin B1 which activates cyclin-dependent kinase 1 (CDK1) to promote activity of end resection proteins that commit the cell to HDR. A messenger RNA (mRNA) construct coding for cyclin B1 and a green fluorescent protein (GFP) reporter was produced and packaged into our previously optimized LNP formulation. After confirming GFP fluorescence, cyclin-B1-GFP mRNA was co-encapsulated with CRISPR/Cas9 editing reagents, leading to a 2-fold increase in knock-in efficiency over a control formulation with an equivalent mass of GFP mRNA. Optimization of cyclin B1 mRNA dosage levels and timing, as well as exploration of co-delivery with CDK1 mRNA, will be necessary to confirm viability for overall improved gene insertion. If successful, this work would be a major step towards a definitive cure for CF and more broadly improving insertion-based gene therapy options for genetic diseases.

Throughout development, embryonic stem cells adopt different fates based on genetic and environmental cues. The cell cycle has been shown to play a role in regulating these fate decisions, but the mechanisms facilitating this regulation are poorly understood. We have shown that perturbations of key cell cycle regulators cul-1 and cdc-25.1 impair gut fate in C. elegans embryos. On a molecular level, the perturbations cause abnormal spatial patterning of a key intestinal fate specifying protein, END-1, and dysregulation of a key upstream Wnt pathway member, WRM-1, in the nucleus of the endodermal precursor cell, E. Although WRM-1 nuclear export is identified as the mechanism governing WRM-1/POP-1 asymmetry and endodermal fate establishment between E and its mesodermal sister, MS, there is a lack of molecular understanding of how CDKs modify nuclear WRM-1 levels in E and MS cells. Therefore, we will examine the effect of CDK disruption on the attenuation of WRM-1 nuclear export. We are analyzing WRM-1 behaviors from time-lapse images of WRM-1GFP embryos treated with CDK inhibitors. RNAi treatments targeting cyclins will also be used to identify the exact Cyclin/Cdk complex responsible for facilitating WRM-1 nuclear export in E. Understanding how the cell cycle affects fate decisions would potentially help improve stem cell technology by revealing how cell cycle dynamics affect the stochasticity of somatic cell reprogramming.