Background: Chronic kidney disease is a global health burden, and phosphate retention has been linked to adverse outcomes. The impact of dietary phosphate on kidney fibrosis in chronic kidney disease remains uncertain.  Objective: To test whether dietary phosphate intake alters kidney fibrosis in Col4a3 knockout mice, a model of chronic kidney disease.  Methods: Col4a3 knockout and wild type mice were fed low, standard, or high phosphate diets. Kidney fibrosis was assessed by Col1a1 messenger RNA expression relative to Hprt using quantitative polymerase chain reaction. Delta Ct values, calculated as Col1a1 minus Hprt, were compared between groups using the Mann–Whitney test.  Results: Wild type mice had a mean delta Ct of -5.81, while knockout mice on a standard phosphate diet had a mean delta Ct of -1.17. This 4.64 cycle difference corresponds to approximately 25-fold higher Col1a1 expression in knockout versus wild type mice, confirming fibrosis in this chronic kidney disease model. Among knockout mice, Col1a1 expression was similar on standard versus low phosphate diets (-1.17 vs -1.18). High phosphate did not increase expression and was numerically lower than standard phosphate.  Conclusion: Col4a3 knockout mice exhibit markedly increased kidney Col1a1 expression relative to wild type, validating this chronic kidney disease fibrosis model. Dietary phosphate intake, however, did not significantly alter Col1a1 expression in knockout mice, suggesting phosphate is not a major driver of Col1a1 mediated fibrosis in this setting. Ongoing studies with histological endpoints will further define the role of phosphate in chronic kidney disease progression.  

Platinum-resistant non-small cell lung cancer (NSCLC) continues to carry poor prognoses despite advances in targeted- and immunotherapy. Integrin αvβ6 is a cell surface receptor overexpressed in various epithelial cancers including NSCLC. Using patient derived xenograft models of NSCLC, our preliminary studies reveal αvβ6 expression is not only preserved in NSCLC but amplified following treatment with platinum-based chemotherapy. Importantly, we demonstrate αvβ6 expression is elevated in chemotherapy resistant NSCLC. Our goal is to investigate if αvβ6 targeted for radioligand therapy (RLT) is a viable treatment for chemotherapy-resistant NSCLC. Using flow cytometry, we have identified human NSCLC cell lines with high (H358) and low (A549) basal αvβ6 expression that can be used for in vitro and in vivo evaluation of the efficacy of 177Lu-αvβ6 RLT. The peptide, A20FMDV2, specifically binds αvβ6 and will serve as the target binding motif of our RLT agent. Radiolabeling of the A20FMDV2 peptide with Lu-177 achieved >95% radiochemical purity and a specific activity of 187 μCi/nmol, and radioligand cell binding confirmed selective binding to αvβ6+ H358 resulting in a 3-fold higher binding compared to αvβ6-low A549 cells. Future work will include SPECT imaging, biodistribution, and dosimetry studies in NSCLC cell xenografts, PDXs, and chemotherapy-resistant PDXs. Collectively, these efforts aim to establish αvβ6 RLT as a viable therapeutic intervention for platinum resistant NSCLC with broad implications in other chemotherapy-resistant solid tumors. 

Spinocerebellar ataxias are neurodegenerative disorders that progressively impair gait, balance, and coordination affecting approximately 1 in 5,000 people worldwide. There is an emerging need for treatment, yet trials for new therapies are limited by dependence on clinical examination, requiring long study durations and/or large sample sizes to confidently assess treatment effects. Biomarkers reflecting underlying pathology provide a way to assess treatment effects across shorter time scales with increased sensitivity. MicroRNAs (miRNAs) in exosomes (small extracellular vesicles) have emerged as a potential source for such biomarkers with previous sequencing studies demonstrating disease-specific changes in cancer, neurodegenerative disorders (e.g., Alzheimer’s, Parkinson’s), and cardiovascular disease. Isolation of miRNA from exosomes is a complex process, often performed on limited biological material, which may affect downstream analysis. To illustrate this, we analyzed two internal exosome miRNA datasets sequenced from low biomass neuron-specific samples using two commonly-used RNA-sequencing analysis tools (HTSeq and featureCounts) with default parameters, achieving differing results with a substantial number of reads misaligned using both tools. To develop an optimized analysis pipeline for low-biomass exosomal miRNA sequencing data, we evaluated HTSeq and featureCounts parameters using published exosome miRNA sequencing datasets to optimize detection of reads corresponding to the mature miRNA sequence. Ultimately, featureCounts was selected as the preferred tool as its minOverlap parameter allowed for improved detection of partially overlapping reads relative to HTSeq. A minOverlap of 15bp was found to produce maximal inclusion of true miRNA reads. This optimized pipeline is expected to improve accurate miRNA detection from low biomass clinical samples.  

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus that establishes persistent latent infection with the potential to reactivate and cause severe illness. During latency, the viral genome (episome) avoids detection by replicating and segregating into daughter cells along with the cell's genome. We hypothesized that Latency-Associated Nuclear Antigen (LANA) modulates the expression of critical cell-cycle regulators and checkpoints to preserve the episome in latent KSHV infection. The objective of this project was to optimize synchronization of latently infected and wild-type HEK293T cells to early S-phase using a double thymidine block. To initiate synchronization, cells were incubated with 2mM thymidine for 16 hours. Subsequently, the thymidine-containing medium was removed, and the cells were washed with growth medium. The cells were then released into fresh medium for 8 hours. Cells were again treated with 2mM thymidine for an extended 24 hours. Following the second block, cells were washed, released, and harvested at defined time points post-release. Western blotting indicated that infected HEK293T cells exhibited lower expression of CDT1, a licensing factor for DNA replication. Additionally, the upregulation of Geminin, a marker of S-phase entry, was delayed in latently infected cells compared to wild-type controls. These findings suggested that latent KSHV infection alters the regulation and timing of cell-cycle progression. Characterizing the structural and functional role of LANA in cell-cycle regulation will address critical gaps in understanding KSHV episome maintenance during latency. This study aims to elucidate how oncogenic herpesviruses impact the host cell cycle, potentially guiding the development of targeted therapeutics. 

Aging is associated with systemic and chronic inflammation with progressive decline in function at the molecular, cellular, and organ levels. The decline of muscle function with age is characterized by reduced muscle mass and impaired regenerative capacity, leading to overall reductions in motility. Skeletal muscle has remarkable regenerative capacities under normal conditions. Acute muscle injury triggers cycles of activation, proliferation and differentiation of muscle stem cells (MuSCs), the key cell type that repairs damaged muscle tissue  and maintains healthy muscle. With age, these processes become dysregulated, and the overall ability for MuSC to activate and respond to acute injury is diminished. Previous work has shown overexpression of the epigenetic reprogramming factors Oct4, Sox2, Klf4, c-Myc(OSKM) leads to fundamental change in aged skeletal muscle to that of a younger phenotype. RNA sequencing and proteomics data using a skeletal muscle specific epigenetic reprogramming revealed the upregulation of the IL-17a signaling pathway with age and a reversal of phenotype through the induction of OSKM.  In vitro addition of recombinant IL-17a hindered muscle stem cell activation and proliferation, reduced cellular senescence, and altered heterochromatin organization in both muscle stem cells and muscle fibers. These results indicate that the upregulation of IL-17a with age could be a driver of muscle dysfunction over time and that timely blockade of signaling may play a beneficial role in muscle regeneration in aged individuals.     

Genome-wide association studies (GWAS) have identified thousands of SNPs associated with complex traits and diseases. Polygenic risk score (PRS) methods leverage these findings to improve risk prediction. However, many existing PRS approaches are limited to additive genetic effects and are sensitive to modeling choices, leading to biased and inconsistent results. To address these limitations, our team propose the Adaptively Boosting Polygenic Risk Scores (AB-PRS) method, which iteratively updates and selects variants to recover signals missed or biased in initial PRS estimation. By building on a pre-trained PRS, AB-PRS adaptively incorporates additional variants and refines effect size estimates. We evaluated AB-PRS using both simulated datasets and a real-world cohort from the Microbiome and Insulin Longitudinal Evaluation Study (MILES). The AB-PRS method enhanced predictive performance and uncovered a diabetes-associated SNP, rs2908286, which was not present in the pre-trained PRS summary statistics. These findings highlight AB-PRS as a flexible and robust framework for advancing PRS construction.