Objectives/Goals: Bardet-Biedl Syndrome (BBS) is a rare, autosomal recessive syndromic ciliopathy. Obesity in BBS has been attributed to melanocortin 4 receptor (MC4R) pathway in the hypothalamus, which plays a major role in appetite control and energy balance. We aim to understand how BBS proteins play a role in hyperphagia and obesity using zebrafish models. Methods/Study Population: We used a published genetically stable bbs4 mutant zebrafish line to investigate metabolic regulation. Mutants harbor a 5 base pair deletion in bbs4 resulting in loss of gene function. We used behavioral assays to quantify feeding activity, lipid accumulation, and BMI. We monitored expression of appetite regulating and glucose homeostasis genes under fasting and feeding conditions using qRT-PCR in biological triplicate pools of larvae with three technical replicates per experimental condition. Comparative analyses between bbs4 mutants and wildtype (WT) controls were conducted to identify transcriptional and physiological alterations; statistical differences were detected with Dunnett’s multiple comparison test, and p<0.05 was considered significant. Results/Anticipated Results: For the behavioral assays, bbs4 homozygous mutants showed no significant differences compared to WT. Preliminary expression data show a significant decrease in mc4r expression in bbs4 mutants versus WT with concomitant decrease in leptin (lepa/lepb) expression versus WT at hunger initiation. Insulin (insa) showed a 6-fold higher activation and elevated glucagon and gluconeogenesis gene activation for mutants versus WT indicating that bbs4 homozygotes have hyperinsulinism even while fasting. However, in feeding conditions, satiety genes showed no activation in mutants, indicating impaired leptin response. Future studies will elucidate further the interplay between ciliary defects in proximal cellular contexts (i.e., hypothalamus, liver, and pancreas) and whole organism impact of impaired BBS protein. Discussion/Significance of Impact: Our findings indicate that bbs4 mutant zebrafish have preserved ciliary control over appetite and glucose homeostasis, as well as defective leptin-MC4R signaling and fasting-induced hyperinsulinism. Our preclinical model will facilitate the development of treatments for obesity associated with BBS.

Objectives/Goals: Our goal is to characterize how peripheral immune cells from people with HIV (HIV+) and control (HIV-) donors respond to stimulation with lipopolysaccharide from Porphyromonas gingivalis (Pg-LPS) and lipoteichoic acid from Staphylococcus aureus (Sa-LTA) and differentiate how these affect signaling in brain organoids. Methods/Study Population: To characterize peripheral blood mononuclear cells (PBMCs) response to microbial products, HIV+ and HIV– PBMCs were exposed to Pg-LPS or Sa-LTA for 24 or 48 hours. iNOS expression was assessed by western blot. IL-6 was quantified by ELISA, while TNF-α and IL-10 were measured via Ella multiplex. To differentiate how bacterial products directly affect neurons, human brain organoids (hBOs) were stimulated with Pg-LPS or Sa-LTA for 24 hours. To differentiate how bacterial products indirectly affect neurons, hBOs were co-cultured with PBMCs previously exposed to microbial products (primed). TNF-α, IL-6, and IL-10 levels were quantified by ELISA and multiplex. Neural function and reactivity were analyzed by measuring PSD95, synaptophysin, vGLUT1, pERK/tERK, and GFAP expression with western blot. Results/Anticipated Results: Pg-LPS increased TNF-α in HIV– PBMCs only at 48 h (p=0.033); in HIV+ PBMCs, TNF-α increased at 24 h (p=0.026) but declined by 48 h (p=0.011). HIV+ PBMCs secreted IL-6 at 24 h, which decreased by 48 h (p=0.025). IL-10 secretion remained stable in HIV– PBMCs but was elevated at 24 h versus 48 h in HIV+ PBMCs (p=0.006). No changes in iNOS expression were detected. Sa-LTA did not induce notable effects. In hBOs, direct stimulation with Pg-LPS or Sa-LTA did not alter TNF-α and IL-10 levels, nor neuronal markers. IL-6 was undetectable in Pg-LPS-treated hBOs and decreased in Sa-LTA-treated hBOs. Interestingly, hBOs co-cultured with primed PBMCs had higher astrocyte reactivity (p=0.034) than hBOs directly exposed to Pg-LPS 24h, while hBOs co-cultured with untreated PBMCs showed higher levels of neuronal markers. Discussion/Significance of Impact: HIV+ PBMCs exhibit a pro-inflammatory response to Pg-LPS at 24h, counterbalanced by an anti-inflammatory modulation. Oral pathogen products may be driving distinct immune activation dynamics in PWH and in the brain, potentially contributing to neuroinflammatory mechanisms that trigger neuronal dysfunction and cognitive impairment.

Objectives/Goals: Our team has developed Specialized Hydrogel In Natural Enhancement for Vaginal Health (SHINE-VH), a synthetic mucin-mimetic, to strengthen the cervicovaginal mucus barrier and mitigate microbial colonization to prevent bacterial vaginosis. Methods/Study Population: We formulated SHINE-VH via polymer chemistry and evaluated its efficacy through translational assays. Polymers were synthesized in ultra-high molecular weights and engineered to exhibit hydrophilicity, mucoadhesion, and high viscosity, thus replicating the physical properties of healthy mucus. SHINE-VH readily dissolved in water and was safety-tested using human vaginal (VK2/E6E7) and endocervical (End1/E6E7) epithelial cell lines. Following confirmation of biocompatibility, we characterized its physicochemical properties through shear rheology and sugar permeability. To examine barrier integrity and anti-inflammatory activity, VK2/E6E7 cells were cultured on transwell inserts and pre-treated with SHINE-VH prior to 24-hour exposure to lipopolysaccharide (LPS) (10 µg/mL). Results/Anticipated Results: SHINE-VH maintained a high molecular weight (Mn > 1.0 MDa) and narrow dispersity (Đ = 1.0–1.4), indicating uniform polymer length. The formulation exhibited viscoelastic behavior (η* = 0.1–1 Pa•s) comparable to native vaginal mucus. SHINE-VH supported high biocompatibility across vaginal and endocervical cell lines (> 90% viability), with no tested dose falling below ISO 10993-5 safety thresholds. SHINE-VH enhanced epithelial barrier integrity by restricting LPS translocation (p = 0.03, g = –1.26) and attenuated inflammation through reduced IL-6 secretion (p = 0.009, g = –1.97). Lastly, while blocking toxins, SHINE-VH preserved nutrient exchange, permitting glucose/maltose diffusion (> 90% in 72 h). Collectively, these findings confirm SHINE-VH preserves epithelial health. Discussion/Significance of Impact: Bacterial vaginosis affects ~30% of women globally and contributes to severe gynecologic and obstetric complications. SHINE-VH offers a novel, non-antibiotic approach to BV management by strengthening vaginal mucosal integrity and limiting bacterial invasion, potentially reducing recurrence and improving women’s reproductive health.

Objectives/Goals: This research aims to develop a regulatory and clinical framework that integrates pharmacogenomic testing into opioid prescribing in order to optimize analgesic efficacy, minimize toxicity, and reduce the incidence of serious adverse events in chronic pain and palliative care populations. Methods/Study Population: A structured literature review was conducted using PubMed, Google Scholar, and ClinicalTrials.gov (2010–2025) with the terms “CYP2D6,” “COMT,” “OPRM1,” “opioid,” “pharmacogenomics,” and “precision dosing.” Human studies evaluating genotype–response, adverse events, or dosing variability were included. The FDA REMS database and Federal Register were reviewed to determine how pharmacogenomic testing could be integrated into existing Elements to Assure Safe Use. CPIC and PharmGKB guidelines were consulted to confirm gene–drug clinical relevance, while CMS reimbursement data were analyzed to assess coverage feasibility and implementation barriers. Results/Anticipated Results: Clinical trial “Pharmacogenetics and Pharmacokinetics of Oxycodone to Personalize Post-Operative Pain Management” shows that CYP2D6 genotype significantly alters oxycodone metabolism and analgesic response. Poor metabolizers experience reduced pain relief, while ultra-rapid metabolizers face increased toxicity with codeine, tramadol, and oxycodone. COMT variants affect pain sensitivity and morphine dosing, and OPRM1 A118G influences receptor binding and opioid response. Although opioid REMS programs do not include PGx testing, Vanda v. West-Ward (2018) supports integration, while CMS data show higher reimbursement for multigene panels ~74% than single-gene tests ~43%, reinforcing feasibility. Discussion/Significance of Impact: Adverse drug reactions remain a leading cause of preventable mortality. Integrating pharmacogenomic testing into REMS would advance precision opioid prescribing, reduce toxicity, and improve pain management while creating a scalable model for personalized therapeutics.

Objectives/Goals: We aim to clarify the role of the intestinal tuft cell-acetylcholine (ACh)-muscarinic receptor axis in inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC) and to investigate whether altering tuft cell activity impacts colitis severity and CAC. Methods/Study Population: (1) To characterize tuft cell distribution in intestinal tissues of humans with and without IBD and CAC, we will use immunohistochemistry, RNAscope, and in situ hybridization. (2) In mice treated with dextran sodium sulfate (DSS) to induce colitis, we will use indolepropionic acid and succinate to manipulate intestinal tuft cell numbers. We will derive enteroids from these and control mice and compare tuft cell numbers, ACh synthetic markers, and anti- and pro-inflammatory cytokines. (3) We will compare the effects of altered tuft cell populations on colitis severity and tumor number and size in mice treated with or without DSS/azoxymethane to induce colitis and CAC. Results/Anticipated Results: Intestinal tuft cells are key participants in anti-inflammatory signaling. Hence: (1)compared to tissue from healthy controls, we anticipate finding altered tuft cell distribution and fewer tuft cells in intestinal tissues from humans with IBD and CAC; (2) compared to controls, intestinal enteroids from mice treated with indolepropionic acid or succinate prior to DSS treatment will retain more tuft cells and an augmented anti-inflammatory cytokine signature; (3) mice with augmented tuft cell numbers will exhibit attenuated colitis and CAC. Discussion/Significance of Impact: Interplay between gut immune and epithelial cells, neurons, and the microbiome underlies colitis. Current therapies target only immune cells, with limited efficacy. Studying other players in IBD pathogenesis, like tuft cells, is likely to reveal novel therapeutic targets.

Objectives/Goals: CYP2D6 is a highly polymorphic gene with significant pharmacogenomic and public health implications. Understanding the population distribution of CYP2D6 variants offers opportunities to enhance awareness of its pharmacological relevance, particularly across different ancestral groups. Methods/Study Population: Using the National Institute of Health All of Us Research Program Data Browser, we identified CYP2D6 alleles with a frequency ≥0.10 in the overall cohort or within ancestral subgroups and classified in ClinVar as having either “drug response” or ”undefined” significance. We assessed alleles with frequency differences ≥0.10 between ancestral subgroups and the overall population to highlight subgroup-specific pharmacogenomic relevance. CYP2D6alleles with ClinVar “undefined” significance classifications were cross-referenced with expert pharmacological resources (e.g., PharmVar) to identify candidates for reclassification as ”drug response” significance variants. Results/Anticipated Results: Among the 89CYP2D6 alleles with ClinVar “drug response” significance classification, 7 of 11 (64%) were more frequent (≥ 0.10) in the East Asian ancestry subgroup compared to the overall population. Among 6,262 CYP2D6 alleles with “undefined” significance classification, 60 showed elevated frequencies (≥ 0.10) in at least one subgroup: Middle Eastern (31/60; 52%), African (22/60; 37%), East Asian (21/60; 35%), and South Asian (4/60; 7%) ancestries. Of these 60 alleles, 16 (27%) map to 41–76, star allele haplotypes that contain at least one sub-allele with “definitive” allele evidence level in PharmVar. Discussion/Significance of Impact: Our findings highlight the differential distribution and potential pharmacological relevance of CYP2D6 alleles across ancestral subgroups. These insights support potential reclassification of certain ”undefined” alleles and highlight the importance of inclusive pharmacogenomic research.

Objectives/Goals: Nutrition status is an overlooked, modifiable driver of total joint arthroplasty (TJA) recovery that can reduce complication rates. Therefore, we will profile preoperative nutrition status in TJA patients using integrated clinical measures, identify metrics that predict postoperative complications, and validate their clinical utility. Methods/Study Population: We will enroll 800 elective hip (THA), knee (TKA), and shoulder (TSA) arthroplasty patients. Assessments will include blood biomarkers (albumin, lymphocytes, vitamin D/A, zinc, HbA1c, lipids), bioelectrical impedance (BIA) for body composition metrics (phase angle, body fat%, skeletal muscle index: SMI, visceral adipose area: VFA), and handgrip strength (muscle quality index: MQI). Biomarkers will inform on deficiencies, immunity, and metabolic health, reflecting surgical readiness. We will compute Onodera’s Prognostic Nutritional Index (OPNI), SMI, and MQI and will evaluate infections, hospitalizations, revisions, and prolonged stay via records and encounters at 3 months and 1 year postop. Analyses will link nutrition metrics to complications to identify the strongest, actionable predictors. Results/Anticipated Results: Among the first 100 arthroplasty participants assessed (THA: 43%, TKA: 49%, TSA: 8%), 56% were male (age 66.3±10.4 yrs). Mean lymphocytes was 1.9±0.6×109/L (normal: 1.0-4.8×109/L). Zinc averaged 59.9±11.6 µg/dL (normal: 60-120 µg/dL) with 47% deficient (50.9±6.6 µg/dL) versus 53% in normal range (66.8±5.0 µg/dL). Average vitamin D was 36.3 ng/mL (sufficient: >30 ng/mL), with 42% of patients low (20.8±5.6 ng/mL) versus 48% sufficient (47.8±15.1 ng/mL). Body composition measures: BMI 30.2±5.9 kg/m2 (2% underweight, 18% normal, 27% overweight, 27% obese 1, 14% obese 2, 6% obese 3); body fat 35.1±9.4%, VFA 155.8±55.3 cm2 (healthy: <100 cm2), SMI 7.9±1.5 kg/m2, phase angle 5.0±0.8°; and MQI 1.4±0.4. Comorbidities: T2DM 18%, HTN 67%, hypercholesterolemia 28%, CVD 37%; and 8% used GLP-1 medications. Discussion/Significance of Impact: These early data show high zinc and vitamin D deficiency (47% and 42%, respectively), obesity (47% class I–III), and substantial comorbidities, underscoring the need for standardized nutrition screening in orthopedics. These findings justify targeted preop interventions and support an NIH R01 focused on reducing complications.

Objectives/Goals: In psychosis spectrum disorders, diffusion tensor imaging studies consistently report reduced fractional anisotropy in the superior longitudinal fasciculus, a major tract linking frontal and parietal cortices, yet the neurochemical underpinnings of these microstructural abnormalities remain poorly understood. Methods/Study Population: We collected diffusion MRI scans and single-voxel PRESS magnetic resonance spectroscopy scans both in parietal and frontal white matter in 48 medication naïve patients and 39 healthy controls. Fractional anisotropy within the superior longitudinal fasciculus was quantified in using TORTOISE and compared with frontal and parietal neurometabolic profiles processed with LC model and partial volume corrected. Results/Anticipated Results: Myoinositol and TNAA were significantly different between patients and controls in both frontal and parietal white matter. Fractional anisotropy of the superior longitudinal fasciculus was significantly different between patients and controls. Both myoinositol and TNAA were associated with superior longitudinal fasciculus fractional anisotropy when modeled independently, but when jointly modeled only TNAA was significantly associated with fractional anisotropy in the parietal white matter while only myoinositol was significant in the frontal white matter. Both parietal and frontal white matter had a significantly greater association between myoinositol and fractional anisotropy than controls. Discussion/Significance of Impact: Disruptions in neuronal integrity and glial dysfunction likely contribute to decreased fractional anisotropy in frontoparietal white matter in first-episode psychosis spectrum disorders. These disturbances are not related to medication effects.

Objectives/Goals: Ovarian cancer is the most lethal gynecologic cancer with emergence of recurrent and resistant disease being frequent. We sought to identify novel ways to effectively kill chemotherapy-resistant forms of ovarian cancer and to prevent recurrence following curative intent with surgical and adjuvant regimens. Methods/Study Population: Our group identified CTPS1 as a novel dependency in triple negative breast cancer (TNBC) and ovarian cancer. We found CTPS1 was highly expressed in breast and ovarian tumor tissues and therapy-resistant models. We demonstrated siRNA knockdown of CTPS1 reduced proliferation in sensitive and resistant cell lines. In collaboration with Step Pharma, who developed the first CTPS1 inhibitor (STP938), we performed genome-wide CRISPR KO screens on STP938-sensitive and -resistant cells. Top synthetic lethal hits identified included TSC1, TSC2, and PPP2R2A. Synergy assays were conducted with STP938 in combination with mTOR inhibitors (Everolimus, Sirolimus, and Temsirolimus) and the PPP2R2A inhibitor LB-100 to evaluate the therapeutic benefit of combination therapies. Results/Anticipated Results: We identified CTPS1 as a novel vulnerability in many ovarian cancer cell line models, including models of chemotherapy and/or PARP inhibitor resistance. The first-in-class CTPS1 inhibitor (STP938) was shown to be a highly effective anticancer agent in sensitive and resistant forms of ovarian cancer, both in vitro and in vivo. Genome-wide CRISPR KO screens identified central components of the mTOR pathway (TSC1 and TSC2), as well as PPP2R2A, as synthetic lethal targets in the setting of STP938 exposure. We anticipate that combinatorial treatment of mTOR inhibitors and PP2R2A inhibitors with STP938 will further enhance efficacy and reduce the risk of recurrence when used in the adjuvant treatment of ovarian cancer patients. Discussion/Significance of Impact: This study will validate synthetic lethal interactions from our CRISPR screen, reveal genes that could be mutated in ovarian cancer patients that may be driving resistance to STP938, and uncover combination therapies that can be used to increase efficacy in ovarian cancer patients treated with STP938.

Objectives/Goals: Conventional magnetic resonance imaging (MRI) methods are unable to visualize bone comparable to computed tomography (CT). Radiation therapy can result in post-treatment complications, such as osteoradionecrosis (ORN). We aim to integrate black bone (BB) MRI into routine follow-up MRI scans as an alternative to CT to monitor the development of ORN. Methods/Study Population: We assess the CT, T2-weighted (T2W), and BB MRI scans of 21 head and neck cancer patients without active ORN. All images are acquired within 4 weeks of each other to minimize anatomical variability. The mandibles are manually segmented on each image, and the MR images are registered to the CT to map the CT contour onto the MR images for comparison. Image segmentation evaluation metrics, including dice similarity coefficient (DSC), mean distance to agreement (MDA), and Hausdorff distance (HD), assess for spatial differences between each MRI sequence, using CT as reference standard. DSC measures contour volume, while MDA and HD assess the differences in contour boundaries, with HD sensitive to outliers. We employ a Wilcoxon signed-rank test using a significance value of p≤0.05 to assess for statistical significance. Results/Anticipated Results: Using metrics widely accepted in biomedical research, we assess the performance of BB to visualize mandible against its conventional T2W MRI counterpart when compared to CT. Black bone has a significantly different DSC (median=0.878, IQR=0.0397) than T2W (median=0.806, IQR=0.0876; p<0.0001), indicating high volumetric overlap between contours on BB and CT. Black bone has a significantly different MDA (median=0.0731mm, IQR=0.0227mm) than T2W (median=0.119mm, IQR=0.0697; p<0.0001), indicating high agreement between contour boundaries on BB and CT. Black bone has a significantly different HD (median=0.649mm, IQR=0.211mm) than T2W (median=1.80, IQR=0.933; p<0.0001), indicating a submillimeter difference between contour boundaries, including outliers, on BB and CT. Discussion/Significance of Impact: Our results exhibit BB as a promising non-ionizing alternative to CT for bony visualization compared to conventional MRI. BB has potential to detect early anatomical changes in bone, providing a standardized assessment of ORN. This can improve our dose–response understanding of post-treatment toxicities, refining radiation treatment planning.

Objectives/Goals: The Never-in-mitosis A-related (NEK) kinases are a family of 11 poorly explored kinases that play varying roles in cellular processes. Aberrant NEK activity is implicated in various human diseases, including cancer. We hypothesize that NEK9 drives triple-negative breast cancer (TNBC) motility. Methods/Study Population: Baseline NEK9 expression was determined across five breast cancer and three non-breast cancer cell lines by immunoblot. We expanded the analysis to six additional TNBC cell lines. To determine the impact of NEK9 on TNBC biology, we evaluated the effects of NEK9 pharmacological inhibition with novel inhibitor BA 03-53-11 (BA) at 0µM, 0.5µM, 1µM, and 2µM. We treated TNBC cell lines MDA-MB-231, BT-549, HS578T, and TNBC patient-derived cell line TUBcX-4IC and performed functional assays to assess impacts on cell motility (scratch assay), viability (crystal violet staining), and proliferation (Ki-67 staining). Results/Anticipated Results: NEK9 levels were elevated in the TNBC cell line compared to other breast cancer subtypes, osteosarcoma, and non-cancerous cells. Additionally, NEK9 protein expression was variable across TNBC cell lines. While pharmacological NEK9 inhibition did not significantly alter TNBC cell viability or proliferation, we observed that NEK9 inhibition suppressed cell motility in TNBC cells compared to vehicle control. Taken together, these preliminary results support a role for NEK9 in regulating TNBC progression. Our ongoing studies aim to test NEK9-mediated motility and proliferation in additional TNBC cell lines and evaluate NEK9 as a therapeutic target for this aggressive breast cancer subtype. Discussion/Significance of Impact: TNBC is an aggressive form of breast cancer that lacks targeted therapies. Because aberrant kinase activity underpins oncogenic transformation, kinase inhibitors have emerged as ideal therapeutic agents. Elucidation of NEK9 function in TNBC is essential for the development of novel therapeutics.

Objectives/Goals: Youth with painful disorders of gut–brain interaction (DGBIs) report school-related challenges: stigma from school staff and poorer performance. The present study explored how stigma from school staff correlates with poorer school functioning through internal locus of control and pain interference. Methods/Study Population: Participants included 133 youth aged 8–18 years (Mage= 13.8). Youth completed a series of validated self-report questionnaires assessing pain-related stigma from school personnel, pain interference (i.e., the degree to which pain prevents engagement in daily activities), health-related locus of control (LOC – the belief that one’s health is determined by their own actions), and school functioning. A sequential mediation analysis was conducted using 95% bootstrapped confidence intervals to ascertain the indirect effect of school-based stigma on school functioning through internal LOC and pain interference. An alpha level of 0.05 was determined a priori. Results/Anticipated Results: School-based stigma was significantly correlated with poorer school functioning, even after controlling for internal LOC and pain interference, β = -0.22, p = .003. Evidence suggests a partial mediation model, such that school-based stigma is indirectly associated with poorer school functioning via internal LOC and pain interference (β = -0.028, 95% CI [-0.069, -0.002]). More specifically, greater school-based stigma correlates with a greater internal LOC (β = 0.19, p= .03). In turn, a greater internal LOC correlates with greater pain interference (β = 0.32, p<.001). Finally, greater pain interference correlates with poorer school functioning (β = -0.46, p< .001). Discussion/Significance of Impact: Stigma from teachers and nurses may cause youth with DGBIs to feel more responsible for their pain. Consequently, as they internalize this stigma, their pain may present a greater barrier to engagement in daily activities, including participation in school activities.

Objectives/Goals: This study aims to explore transcriptional adaptation, where mutations in one gene trigger compensatory changes in related genes, and how this affects the variability in clinical manifestations of amyotrophic lateral sclerosis (ALS). Methods/Study Population: The presence of a premature termination codon triggers transcriptional activation. Therefore, we utilized CRISPR-Cas9 tool to generate a premature termination codon in CHCHD10 gene in multiple types of cells, including induced pluripotent stem cells derived from patient samples with known CHCHD10 mutations causative for amyotrophic lateral sclerosis. CRISPR-Cas9 tool was delivered via ribonucleoprotein electroporation, and transfect cell’s DNA were sequenced to validate gene editing. To confirm transcriptional adaptation, protein and gene expression changes will be assessed by immunoblotting and quantification of multiple target loci from whole-cell lysates of edited cells. Results/Anticipated Results: By employing CRISPR-Cas9-mediated editing, we hypothesize that transcriptional adaptation will enable the adaptive gene to functionally compensate for the loss of function in the gene of interest harboring a premature termination codon. Discussion/Significance of Impact: The significance of this research lies in its potential to identify novel genes capable of triggering transcriptional adaptation in humans. Understanding this mechanism could pave the way for new therapeutic strategies, particularly for CHCHD10-associated ALS.

Objectives/Goals: The primary objective of this study is to test whether NAD+ modulation lowers mitochondrial oxidative stress to restore endothelial nitric-oxide signaling and limit microvascular remodeling in diabetes in murine and 3D tissue-engineered models. The goal is to define NAD-responsive pathways and candidate blood biomarkers of microvascular injury. Methods/Study Population: Use db/db diabetic mice to measure effects of NAD+ modulation on capillary density, perfusion, endothelial nitric oxide (NO) signaling, and mitochondrial reactive oxygen species (ROS) by histology, immunohistochemistry (IHC), qPCR/immunoblot, and functional assays. Build a 3D microfluidic model of diabetic retinal capillary disease from human endothelial/pericyte co-cultures with a retina-on-a-chip model; induce diabetic stress (hyperglycemia + inflammatory cytokines) and apply an NAD+ modulation to map dose–responses for barrier integrity, ROS, NO, and morphology. Analyze publicly available transcriptomic datasets from people with and without diabetic microvascular disease to identify oxidative-stress signatures and candidate circulating biomarkers. Results/Anticipated Results: Expect increasing NAD+ availability to reduce mitochondrial ROS and inflammatory signaling, increase SIRT3/SOD2 activity, improve NO bioavailability, and prevent capillary rarefaction and leak in db/db mice. In the microfluidic model, anticipate a rescue of barrier function, lumen stability, and branching with NAD+, defining an effective dose window. Cross-platform analyses should converge on NAD+-responsive pathways (redox, mitochondrial metabolism, endothelial junctions) and nominate blood biomarkers that correlate with microvascular status. Together, results will provide actionable preclinical evidence for NAD+ modulation as a therapeutic strategy. Discussion/Significance of Impact: By targeting a shared driver, mitochondrial oxidative stress, this work could deliver a mechanism-based therapy and practical biomarkers for diabetic microvascular disease, accelerating translation from bench models to patient-focused trials and improving cardiovascular outcomes.

Objectives/Goals: Our objective is to develop and validate a human iPSC-derived 3D osteosarcoma (3D-iOSA) model that replicates chromosomal instability (CIN), metastasis, and chemoresistance. The model will serve as a platform to evaluate chemotherapy and immune cell therapeutics, offering improved predictive fidelity compared to 2D and spheroid systems. Methods/Study Population: Human iPSCs were engineered by CRISPR/Cas9-mediated knock out of TP53 and RB1, differentiated to mesenchymal stem cells with overexpression of cMYC ± hRAS. Cells were embedded in hyaluronan and osteogenically differentiated to generate 3D organoids (3D-iOSA), with osteoblastic commitment confirmed by RUNX2 and BGLAP immunohistochemistry (IHC). Tumorigenic and metastatic potential was assessed via subcutaneous implantation into immunodeficient mice, followed by histopathology, IHC, RNA sequencing, and 5X whole genome sequencing (WGS). MAP (methotrexate, doxorubicin, and cisplatin) chemotherapy response and chimeric antigen receptor (CAR)-immune cell cytotoxicity were compared across 2D and 3D systems to evaluate chemoresistance, immune evasion, and translational fidelity of the 3D-iOSA model. Results/Anticipated Results: 3D-iOSA exhibited osteogenic lineage commitment with RUNX2 and BGLAP expression and disorganized, anaplastic morphology correlated with mutational burden. Tumors displayed greater histologic complexity than 2D-derived tumors, with pulmonary metastases in 50% of primary and 70% of secondary recipients. WGS revealed recurrent copy number alterations and subclonal variation, indicating ongoing CIN. MAP half-maximal inhibitory concentration values were 106-260× higher in 3D-iOSA organoids than 2D cultures, increasing with genetic complexity. B7H3 CAR-iPSC NK-cell and B7H3/HER2 dual-CAR γδ T-cell screening showed system-dependent efficacy, with reduced cytotoxicity in 3D-iOSA organoids versus 2D and spheroid cultures, highlighting 3D-iOSA superior predictive power for immunotherapy evaluation. Discussion/Significance of Impact: 3D-iOSA is the first human OSA model to replicate CIN-driven evolution, chemoresistance, and metastasis. It distinguishes differential immune therapy responses with greater predictive power than 2D or spheroid models, providing a scalable, physiologically relevant platform to accelerate targeted therapy development.

Objectives/Goals: Lowering of FUS with antisense oligonucleotide (ASO) has shown promise in a subtype of amyotrophic lateral sclerosis (FUS-ALS). As inflammation is associated with ALS progression, we characterized immune trajectories in FUS-ALS patients treated with FUS ASO to understand the relationship inflammation may have with treatment response. Methods/Study Population: Twelve participants (10 with a diagnosis of ALS, 1 symptomatic carrier, and 1 asymptomatic carrier) received monthly intrathecal administrations of FUS ASO (i.e., jacifusen). Motor function was measured using the ALS Functional Rating Scale-Revised. Axonal injury was measured by cerebrospinal fluid (CSF) neurofilament light chain (NfL) concentration. 10x Genomics single-cell RNA sequencing (scRNA-seq) with T- cell receptor (TCR) sequencing was performed on paired peripheral blood mononuclear cells (PBMCs) and CSF cells. Proteomic analysis was conducted using the Olink Explore HT platform. Complementary analyses included spectral flow cytometry validation and immunohistochemical staining of post-mortem lumbar spinal cord. Results/Anticipated Results: We observed a proportional shift starting at 6 months, whereby T-cell proportions decreased, and myeloid cell proportions increased over treatment. Decreases in T-cell proportions over treatment were not accompanied by increased CD8+ T-cell infiltration in the ventral horn of the lumbar spinal cord. Differential expression revealed long-term, chronic activation and lysosomal dysregulation in myeloid cells. While CSF monocyte proportions were positively associated with CSF NfL concentration, positive associations with ALSFRS-R score were observed only in PBMC T-cell proportions, particularly cytotoxic subclusters. Proteomic analysis also highlighted a shift toward long-term innate immune cell activation associated with reduced ALSFRS-R score, accompanied by decreases in CSF lysosomal proteases. Discussion/Significance of Impact: These findings provide insight into the immune dynamics of FUS-ALS in the context of CNS FUS knockdown, as well as their potential relationship with clinical outcomes. Further, the long-term shift toward monocyte activation and persistent lysosomal dysregulation signature outside the CNS highlights potential avenues for combination therapy.

Objectives/Goals: Medical therapies to halt progression of aortic stenosis (AS) are now being studied; however, the sensitivity of transthoracic echocardiography (TTE) measures to detect disease progression remain unclear. We will evaluate alternative TTE-derived measures of AS severity as potential markers of disease progression to improve trial efficiency. Methods/Study Population: We will conduct a retrospective cohort study of adults with mild or moderate aortic stenosis who had TTE imaging at Tufts Medical Center from 2012 to 2024. Two cohorts will be examined: 1) a reproducibility cohort of patients with studies less than 2 months apart to assess measurement consistency; and 2) a progression cohort with studies more than 6 months apart to evaluate disease progression over time. Echocardiographic parameters including peak velocity, mean pressure gradient, and aortic valve area will be used. We will use recurrent quantitative measurements to evaluate reproducibility, variability, and signal-to-noise characteristics and to model implications for efficient clinical trial design. Analysis will use RStudio. Results/Anticipated Results: Overall there are 2,668 patients with progressive AS. Median age at first TTE is 76 (68–83) years. 1,120 (42%) are female. The reproducibility cohort includes 991 patients (1,728 paired TTEs, median 10 days (0–16) apart), and the progression cohort includes 323 patients (323 paired TTEs, median 504 days (394–750) apart). Additional demographic and comorbidity data will be obtained. Recurrent measurements will quantify reproducibility, variability, and signal-to-noise characteristics. Anticipated results include 1) identification of the most reliable measures for tracking progression, 2) characterization of measurement variability, and 3) modeling of implications for efficient clinical trial design and evidence-based patient management. Discussion/Significance of Impact: Interest in therapies to halt aortic stenosis progression is growing. Establishing consensus on the most reproducible and practical imaging endpoints is essential. By quantifying real-world variability, we aim to optimize trial design so that effective treatments can be studied and improve outcomes.

Objectives/Goals: To identify population-specific and ancestry-informed genetic loci for blood pressure traits, characterize X chromosome associations, and evaluate evidence for evolutionary pressures influencing blood pressure risk across ancestries, which may be used downstream clinically for personalized medicine and drug target identification. Methods/Study Population: We conducted multi-ancestry and ancestry-stratified genome-wide association studies (GWAS) of systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse pressure (PP) across >2.5M individuals (40% non-European) from 16 cohorts and biobanks. Analyses were adjusted for age, age², sex, body mass index, and the first 10 principal components. Fixed-effects meta-analyses were followed by SuSiEx cross-population fine-mapping to identify ancestry-specific loci. We evaluated genetic differentiation using the fixation index (FST) and allele frequency directionality using the sign test to infer potential evolutionary pressures. Results/Anticipated Results: Multi-ancestry meta-analyses identified 2,406 significant independent loci for SBP (1,594 novel), 2,358 DBP (1,509 novel), and 2,312 PP (1,432 novel). Chromosome X analyses revealed 8 SBP, 80 DBP, and 8 PP loci. SuSiEx fine-mapping identified ancestry-specific signals, including a pulse pressure SNP near CTSL (Asian-specific) and a DBP SNP in CACNA1D (African-specific). Sign tests indicated enrichment of DBP risk alleles when comparing African and Asian populations and when comparing Admixed American and Asian populations (p < 0.01), supported by elevated FST values for variants with larger effect sizes. These findings suggest that ancestry-specific selection may influence blood pressure regulation. Discussion/Significance of Impact: This is the largest and most ancestrally diverse blood pressure genetics study to date and one of few to include X chromosome analyses, which enabled discovery of thousands of novel and population-specific loci and revealed evolutionary and biological insights that may advance precision medicine for hypertension.

Objectives/Goals: Our goal is to improve health outcomes for sepsis survivors experiencing immune-paralysis. We propose a functional diagnostic tool to identify at-risk patients and a novel therapeutic called interleukin-7 to boost the patient’s T-cell-mediated immunity against infection. Methods/Study Population: This multi-center research study recruits healthy adult volunteers and adult septic patients in the intensive care unit to consent to giving blood draws. Blood draws containing immune cells are assessed for functionality through our diagnostic tool. Interleukin-7, a therapeutic candidate to improve immune cell activity, is assessed by flow cytometry, ELISA, and bulk RNA sequencing. Results/Anticipated Results: Using IL-7 during T-cell stimulation in the ELISpot assay increased the number of T-cells producing protective effector protein IFNgamma. Statistical analysis was performed using Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons test with a P-value<0.001. Flow cytometry confirms IL-7 treatment increased the frequency of T-cells to produce IFNgamma and preferentially impacts memory T-cells in sepsis patients. Bulk mRNA sequencing demonstrated increased differentially expressed mRNA transcripts FDR<.001 related to cell survival and T-cell-mediated effector molecules with IL-7-treated cells. IL-7 improved effector protein production was confirmed by a multiplex assay. Discussion/Significance of Impact: There are no diagnostic tools to identify immunocompromised patients or therapies to reverse the immune suppression. Our ELISpot diagnostic tool identifies at-risk immune-impaired patients. Furthermore, our results demonstrate interleukin-7 as a potential therapeutic to improve immune activity.

Objectives/Goals: To investigate how distinct KRAS mutations (G12D, G12V, and G12R) drive distinct molecular and phenotypic programs in pancreatic tumorigenesis, linking early lineage and signaling differences to clinical patterns such as stage at diagnosis, nodal status, and survival to ultimately inform allele-specific therapies. Methods/Study Population: We analyzed clinical cohorts (MSK-IMPACT n=1,360; COMPASS n=100) and conducted lineage-tracing studies in mice carrying KRAS-G12D, KRAS-G12V, or KRAS-G12R alleles under inflammatory, genetic, and pharmacologic perturbations. Spatial transcriptomic and proteogenomic profiling of resected PDAC tissues characterized EMT and immune signaling states. Functional studies assessed EGFR–PI3K/AKT–RAC1/VAV1 signaling, including RAC1 inhibition and constitutive AKT activation, to reveal allele-specific susceptibilities. Results/Anticipated Results: KRAS-G12D drove robust lineage plasticity, enhancer remodeling, and early neoplastic progression. KRAS-G12R/V showed limited EMT and increased inflammatory signatures, with impaired EGFR–RAC1/VAV1 activation restricting lineage reversion. Constitutive AKT activation rescued the tumor-initiating defect of KRAS-G12R in vivo, pinpointing a downstream signaling bottleneck, which could be exploited therapeutically. Spatial and transcriptomic profiling of resected human PDAC revealed that KRAS-G12R tumors were more often early-stage, node-negative, and linked to improved survival relative to KRAS-G12D. Discussion/Significance of Impact: Integrating patient and mouse data reveals a mechanistic hierarchy among KRAS alleles that governs lineage fate, signaling reliance, and clinical course, pointing to opportunities for allele-tailored therapeutic strategies in pancreatic cancer.