Objectives/Goals: Metallothioneins (MT) are cysteine-rich proteins that bind metals and regulate oxidative stress. MT2A, the most expressed MT gene, is lost in ~70% of High Grade Serous Ovarian Cancer cases. We hypothesize that MT2A loss disrupts mitochondrial function and metabolism, promoting tumor progression. Methods/Study Population: To explore this, MT2A was knocked down (KD) in CAOV3 HGSOC cells via lentiviral shRNA. KD was confirmed transcriptionally via RT-qPCR and functionally using a nuclei-counting assay, which assesses sensitivity to cadmium chloride (CdCl₂), a cytotoxic heavy metal. Mitochondrial membrane potential (MMP) was assessed via flow cytometry, and oxygen consumption rate (OCR) was measured with Resipher. Clonogenic potential was evaluated by soft-agar colony formation assays. Results/Anticipated Results: MT2A KD cells showed a significant decrease in nuclei count in response to CdCl₂. MMP was reduced in MT2A KD cells, indicating compromised mitochondrial integrity. OCR was significantly decreased in MT2A KD cells, suggesting impaired oxidative phosphorylation. These phenotypes are characteristic features of the Warburg effect, which is a hallmark of aggressive cancers. Soft agar assays revealed that MT2A loss significantly enhanced the cell’s ability to multiply adhesion free from a single cell, a phenotype associated with aggressive oncogenic transformation. Discussion/Significance of Impact: In summary, MT2A loss disrupts mitochondrial dynamics and oxidative metabolism while promoting tumor aggressiveness. These findings support our hypothesis that MT2A plays a key role in regulating mitochondrial homeostasis and may influence metabolic adaptation in HGSOC.

Objectives/Goals: Develop a precision hydrogel-organoid model to determine how extracellular matrix (ECM) biomechanics drive the malignant transformation of oral epithelial dysplasia (OED) to oral squamous cell carcinoma (OSCC) and identify mechanoresponsive pathways. Methods/Study Population: Patient-derived oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) organoids will be encapsulated in an interpenetrating network hydrogel engineered with tunable stiffness and viscosity to mimic healthy, dysplastic, and cancerous tissue. Organoid growth, morphology, and expression of mechanosensitive markers will be analyzed by imaging and immunostaining. Single-cell RNA sequencing will identify mechanoresponsive markers across genetically diverse patient lines. Targeted inhibitors of EGFR, YAP, and p53 pathways will text whether blocking these signals can prevent mechanics-driven malignant transformation. Results/Anticipated Results: We anticipate that OED organoids in stiffer matrices similar to OSCC tumor stroma will exhibit increased proliferation, altered morphology, and activation of tumorigenic markers similar to OSCC. scRNA-seq is expected to reveal conserved and patient-specific mechanosensitive pathways. Targeted inhibition of these pathways should mitigate mechanics-driven malignant transformation. These outcomes will define molecular mechanisms linking ECM biomechanics to tumor progression and reveal new therapeutic opportunities for early intervention. Discussion/Significance of Impact: This hydrogel-organoid model links patient-specific genetics and ECM biomechanics to tumor progression, enabling controlled studies of how mechanical cues drive malignancy and therapeutic response in a clinically relevant, precision medicine context.

Objectives/Goals: Acute myeloid leukemia (AML) is an aggressive blood cancer that hides from the immune system, which led to ~140,000 deaths worldwide in 2024. AML often recurs or resists therapy; therefore, our goal is to develop and test TriKE-PACCs, which combine dual tumor antigen targeting with NK cell activation, to overcome antigen escape and heterogeneity in AML. Methods/Study Population: TriKE-PACC molecules were engineered by combining a TriKE [anti-CD16 single-domain antibody (sdAb), an IL-15 moiety, and an antitumor antibody domain single-chain variable fragment (scFv) or an sdAb] with a PACC arm containing a second antigen-binding scFv/sdAb linked to IL-15Rα. TriKE-PACC constructs were produced in Expi293 cells. To identify clinically relevant AML tumor targets, we analyzed the expression of tumor antigens in AML bone marrow samples obtained from 20 patients using flow cytometry. We modeled antigen escape and heterogeneity using CRISPR/Cas9-generated knockout AML lines. TriKE-PACC-mediated NK cell activity was measured via degranulation, cytokine production, and Incucyte-based cytotoxicity assays against AML cell lines and CRISPR knockout models. Results/Anticipated Results: Our examination of AML patient bone marrow samples revealed diverse antigen profiles, highlighting the need for modular TriKE-PACC designs. The dual-targeting TriKE-PACCs enhanced NK cell activation via CD107a expression and the production of IFNγ and TNFα in co-culture assays with AML cell lines. Notably, TriKE-PACCs retained activity against AML models lacking a targeted antigen, demonstrating the ability of the second tumor targeting arm to overcome antigen escape. Furthermore, NK cell-mediated killing was improved against antigen-deficient AML cells when treated with the TriKE-PACCs compared to standard TriKEs. Discussion/Significance of Impact: TriKE-PACCs offer a modular and customizable NK-engaging immunotherapy to address AML heterogeneity and resistance. Our data, collected utilizing primary patient samples, support the use of this platform for the future development of personalized, dual-targeted therapies to enhance the efficacy of AML treatments.

Objectives/Goals: Our objective in this study was to develop a nanoparticle-based RNA delivery system that reprograms Müller glia toward neuronal fates in human relevant models, enabling localized and transient regeneration without viral vectors. Methods/Study Population: - Results/Anticipated Results: Fabricated nanoparticles encapsulating RNA cargoes were ~110–150 nm in size with polydispersity indices of ~0.2–0.3 and encapsulation efficiencies >95%. Nanoparticles successfully delivered GFP mRNA in both organoid and retinal explant models, showing significant expression of fluorescent reporters in both live cell imaging and in slides prepared using immunohistochemistry. We anticipate that our delivery system will lead to transcription factor expression after delivery of corresponding RNAs with evidence of proliferation and neuronal marker upregulation. We also hypothesize that this approach will enhance neurogenesis in retinal organoids and explants, potentially establishing a platform for non-viral retinal regeneration. Discussion/Significance of Impact: This work demonstrates a clinically translatable, virus-free strategy for retinal repair through targeted RNA delivery. By enabling transient expression of neurogenic factors, nanoparticle-mediated reprogramming presents a scalable and adaptable approach for restoring vision in degenerative retinal diseases.

Objectives/Goals: To assess the safety, tolerability, and adverse effects of adding acetazolamide to temozolomide in newly diagnosed MGMT-methylated malignant glioma, and to explore efficacy, survival outcomes, and Bcl-3 as a potential predictive biomarker. Methods/Study Population: Adults (>18 y) with newly diagnosed, MGMT-methylated, IDH-wildtype glioblastoma, and Karnofsky performance status > 60 receiving standard temozolomide (TMZ) + radiation (RT) were enrolled. Sixty patients (24 initial, 36 subsequent) received acetazolamide (ACZ) twice daily starting on the day of adjuvant TMZ initiation. ACZ began at 250 mg BID for 1 week, then escalated to 500 mg BID, given on days 1–21 of each 28-day cycle (TMZ days 1–5), for up to 6 cycles if not limited by tumor progression/toxicity. Clinical and MRI follow-up occurred every 2 months. Results/Anticipated Results: Preliminary data (initial 24 patients) have shown that no patient had a dose-limiting toxicity. Adverse events were consistent with known sequelae of acetazolamide and TMZ. In the 23 WHO Grade 4 patients, the median overall survival (OS) was 30.1 months, and the median progression-free survival was 16.0 months. The 2-year OS was 60.9%. In total, 37% of the study population had high BCL-3 staining and trended toward shorter OS (17.2 months vs N.R., P=.06) (Riley et al., 2024) All of these factors will be evaluated in a prospective study including 60 patients Discussion/Significance of Impact: Initial data in a smaller cohort showed that adding acetazolamide is safe and tolerable in GBM patients on standard temozolomide. Survival compares favorably to historical data in MGMT-methylated GBM. BCL-3 may be a prognostic biomarker. These findings will be assessed and discussed in a 60-patient prospective study.

Objectives/Goals: Differences in gait patterns related to post-traumatic (PTOA) and idiopathic (IDIO) knee osteoarthritis (OA) in aging adults is not well understood. Therefore, the purpose of this study was to assess the impact of knee OA phenotype (PTOA vs IDIO) on the knee total joint moment (TJM) during walking in aging adults with and without end-stage knee OA. Methods/Study Population: Six adults with PTOA and six adults with IDIO were tested prior to knee replacement. Data from 12 healthy, asymptomatic adults were used as control data. All subjects underwent 3D gait analysis at a self-selected speed. TJM was calculated as the square root of the sum of the squared planar internal sagittal, frontal, and transverse plane knee moments across the stance phase. The corresponding sagittal, frontal, and transverse plane moments at the peak TJM in the 1st (TJM1) and 2nd (TJM2) half of the stance phase were extracted. A repeated-measures ANOVA was used to assess group differences in biomechanical parameters of the dominant (control group) and surgical (OA groups) limb, while knee pain (VAS: 0 – 10) during the walking task for the PTOA and IDIO groups was compared using an independent t-test (alpha=0.05). Results/Anticipated Results: Both the PTOA and IDIO groups walked at slower speeds compared to the control group (p<0.01). There was a main effect of knee OA phenotype on frontal plane TJM (p=0.003). The PTOA group exhibited a significantly lower frontal plane moment at TJM2 compared to the control (p=0.047) and IDIO (p=0.019) groups. There were no differences in sagittal, frontal, or transverse plane knee loading at TJM1 nor any differences in sagittal and transverse plane knee loading at TJM2 (p > 0.05). In addition, there were no differences in self-reported knee pain during walking between the PTOA and IDIO groups (p=0.25). Discussion/Significance of Impact: People with PTOA exhibit more rapid knee joint degeneration than those with IDIO. Lower frontal plane loading during TJM2 in PTOA may be associated with a more rapid decline in knee muscle function. Further evaluation of knee muscle function in PTOA and IDIO will provide better insight into these differences in gait patterns between PTOA and IDIO.

Objectives/Goals: Define how CKD and iron therapy influence tubular epithelial cell (TEC) iron handling through parallel kidney and urine studies in mice and patients, uncovering cellular signatures that predict injury or recovery and may guide individualized iron therapy in CKD. Methods/Study Population: We used two mouse models of CKD and fresh urine samples from pediatric CKD patients and healthy controls to assess tubular iron handling. Kidney tubular epithelial cells (TECs; CD45−AQP1+) were analyzed by flow cytometry for labile iron pool (LIP) using FerroOrange and sorted into LIP^hi and LIP^low subsets for RNA-seq. Expression of injury markers such as KIM1 and NGAL was used to evaluate tubular stress, linking iron accumulation to epithelial injury and adaptive responses. By combining mouse CKD models with patient urine analysis, we traced how kidney tubular epithelial cells (TECs) manage iron across species. Results/Anticipated Results: In CKD mouse models, tubular epithelial cells (TECs) showed reduced labile iron pool (LIP) and increased ferroptosis, with a smaller population of iron-overloaded cells exhibiting elevated KIM-1 and EMT-related transcriptional changes. Iron chelation lowered LIP in tubular cells and iron therapy exasperated KIM-1 expression. Urinary TECs mirrored tissue findings, with LIP and KIM-1 levels reflecting systemic iron status and CKD severity. In human CKD, urinary LIPHI TECs expressed higher KIM-1 and injury-associated signatures, validating urinary TEC profiling as a noninvasive readout of tubular health. In both species, iron handling and toxicity proceeded differently depending on the nature of the damage (tubular or glomerular), identifying urinary TECs as a noninvasive window into kidney iron toxicity. Discussion/Significance of Impact: Chronic kidney disease affects 10–15% of the world population, and anemia is a common complication. Iron therapy is toxic to kidney cells, contributing to tubular injury. Our studies identify urinary cells as a noninvasive test of renal iron status and link iron handling to injury, enabling safer, personalized therapy to slow kidney damage in patients.

Objectives/Goals: This study aims to uncover immuno-endotypes in sepsis-associated pediatric acute respiratory distress syndrome (SA-PARDS) using single-cell RNA sequencing (scRNA seq) to analyze the immune cell populations of the respiratory tract of intubated pediatric subjects with SA-PARDS. Methods/Study Population: The inclusion criteria are an age of less than 18 years, admission to the PICU, a diagnosis of SA-PARDS, and intubation. Both sepsis and PARDS will be defined using the most recent consensus definitions. Exclusion criteria include an order of limited resuscitation and clinician discretion. A tracheal aspirate and blood sample will be obtained on days 1, 3, and 7. Both samples will be processed for single-cell RNA sequencing via the HIVE platform according to the manufacturer’s protocol. cDNA libraries will be generated and undergo 150 bp sequencing, quality control, and alignment. The Seurat package in R will be used for cell-type annotation and analysis of differential gene expression. Clinical variables, labs, and outcomes will be recorded in REDCap. Results/Anticipated Results: We evaluated five samples from two subjects. After quality control, 31,330 cells remained for analysis. UMAP projections were created, and we annotated cell types. We found that most of the cells were immune cells with very few epithelial cells. There were two groups of neutrophils, which we termed classic and migratory. Classical neutrophils expressed classic neutrophil markers, and migratory neutrophils expressed the classical markers plus increased adhesion markers. We identified a third group of cells that were neutrophil-like, and these were isolated for further analysis. These cells exhibited characteristics of exhausted neutrophils, including downregulation of CXCR1 and CXCR2, and upregulation of PI3 and SLIPI. In each subject, the proportion of exhausted neutrophils increased over time. Discussion/Significance of Impact: We found that in our subjects with SA-PARDS, there were a subset of neutrophils characterized by down-regulation of classical neutrophil markers and up-regulation of neutrophil stress markers, suggested an exhausted phenotype.

Objectives/Goals: The aging (65+) population is rapidly growing and faces unique health issues exacerbated by immune function decline. However, the driving mechanisms behind this decline are poorly understood. We will use a mouse model to study the relationship between stiffness and cell mobility within the lymph node (LN) as one potential driving mechanism. Methods/Study Population: We will collect inguinal LNs from young (6–8 weeks) and aged (18+ months) female mice and section them for ex vivo analysis, including quantification of proteins in the fibroblastic reticular network (collagen I, collagen III, collagen VI, laminin, and fibronectin) using immunofluorescent staining and Python scripts; analysis of biomechanical properties (elastic modulus, loss modulus, and pore size) using multiple particle tracking (MPT); and migration analysis of adoptively transferred B- and T-cells using live imaging and MATLAB scripts. We will use ANOVA to compare abundance and organization of proteins, biomechanical properties, and cell migration between young and aged LNs, as well as using Pearson’s correlation coefficients to identify relationships between characteristics. Results/Anticipated Results: We hypothesize a positive correlation between fibroblastic reticular network protein deposits, especially collagens, and stiffness (elastic modulus) within murine LNs due to known mechanisms underlying age-related fibrosis. We also hypothesize that areas of increased stiffness (as revealed by MPT) will exhibit decreased cell migration due to physical hindrance to B- and T-cell mobilization. Furthermore, we hypothesize that aged murine LNs will exhibit a significant increase in stiffness and resultant decreased cell mobility when compared to young murine LN, particularly in areas with increased collagen localization. Discussion/Significance of Impact: These studies will elucidate structure–function relationships driving age-associated LN fibrosis and stiffness, and the resultant effect on cell migration, thus clarifying some of the potential driving mechanisms behind immune aging and providing data capable of informing the development of relevant models, therapeutics, and interventions.

Objectives/Goals: The purpose of this study was to examine associations between prenatal family and community exposures and early childhood DNA methylation (DNAm) at genomic regions related to energy regulation or fat deposition. Results could contribute to the understanding of disparities in metabolic health risk among children from disadvantaged communities. Methods/Study Population: This is part of a cross-sectional study of mother–child dyads recruited from a previous RCT with pregnant women in California who had a pre-pregnancy BMI of 25–40. DNA was extracted from saliva of 49 child participants between 1 and 4 years old and is undergoing whole-genome bisulfite sequencing. Outcomes are percent DNAm, both genome-wide and at genomic regions related to energy regulation or fat deposition. Prenatal exposures were measured by the Geospatial Research, Analysis, and Services Program (GRASP) Place & Health burden percentile ranking, based on census tract of residence during pregnancy, with higher percentiles indicating greater burden. We will use regression to model associations of percent DNAm at gene loci of interest on GRASP percentile, controlling for relevant covariates (e.g., age, sex). Results/Anticipated Results: Final data collection, cleaning, and analysis are in progress and on target to be completed in Fall 2025. We will present final results of these analyses, including descriptive characteristics of the sample (e.g., summary and frequency statistics) and overall DNA methylation patterns (e.g., b or M values as appropriate), and whether there is support for our hypotheses that children with greater prenatal family and community exposures will have DNAm in pertinent genomic regions related to energy regulation or fat deposition (e.g., regression parameters and confidence intervals). Discussion/Significance of Impact: Findings will advance knowledge of early-life physiologic effects of exposures during pregnancy. Future clinicians may use this knowledge to earlier identify and treat children at high risk for adverse child metabolic health outcomes such as obesity, and health advocates can use it to promote policies to reduce adverse prenatal environments.

Objectives/Goals: Pediatric obesity is a public health issue. Many youths in lifestyle obesity treatments do not improve their weight. Sequential Multiple Assignment Randomized Trial (SMART) study designs can support slow responders (e.g., youth who do not initially lose weight). SMARTs have limited use in pediatric obesity and lack data-driven adaptive rules. Methods/Study Population: This study leveraged an ongoing 6-month family-based health behavior pediatric obesity treatment for children and their parents. Parents with obesity and their children (6–11years) with overweight/obesity (body mass index >85th%ile) who lived in rural areas and were a part of the larger pediatric obesity treatment trial were invited to this study. During the treatment, parent/child dyads were asked to complete additional surveys across nine time points (weeks 1, 2, 3, 4, 6, 8, 12, 16, and 20). Surveys assessed the child’s physical activity, diet, and pain, along with the family environment and parental stress. The child’s anthropometrics were measured each month. Results/Anticipated Results: Data collection will be completed in December 2025. Anticipated results will include analyses to identify responders (i.e., children who reduce their weight outcomes [body mass index (BMI) z-score, BMI percentile, or weight]) and slow responders (i.e., children who do not reduce their weight outcomes) to the pediatric obesity treatment; based on preliminary work, we expect ~50% of the sample to be responders. Once responders/slow responders are identified, survey data will be used to define the adaptive rules, including the tailoring variable (e.g., physical activity or junk foods, or early-response weight outcomes) and decision time point for second randomization. Discussion/Significance of Impact: Data from this study will lay the groundwork for developing data-driven criteria for SMART rules (tailoring variable and decision time point for second randomization). This, in turn, will support the development of adaptive interventions to identify and support the many youths who are slow responders to lifestyle pediatric obesity treatments.

Objectives/Goals: This study aims to identify socioeconomic, clinical, and behavioral risk factors of non-adherence for adults with hypertension and evaluate implications for future treatment strategies. Methods/Study Population: We will conduct a systematic review following PRISMA guidelines. Eligible studies are peer-reviewed RCTs and observational designs assessing factors associated with antihypertensive medication adherence among adults (≥18 years) with hypertension living in North America or Europe. Studies must report adherence as the primary outcome. Studies will be excluded if they are not peer-reviewed, original research articles, published in a non-English language, reviews or meta-analyses, or studies of an intervention aimed at improving adherence. Abstract and title screening, full paper review, and data extraction will be performed in Covidence by two reviewers with conflicts resolved by consensus. Results/Anticipated Results: A total of 5660 records were imported for screening after duplicate removal. Following title and abstract screening, 4990 studies were excluded as irrelevant, leaving 670 records for full-text review. Of 611 full texts reviewed, 491 studies were excluded and 39 are pending conflict resolution. Currently, 119 studies are marked for data extraction. Data to be extracted will include socioeconomic, demographic, clinical, and behavioral determinants of antihypertensive medication adherence. Quality assurance will involve dual independent extraction. Preliminary trends suggest wide variability in study designs and adherence definitions across North American and European populations. Discussion/Significance of Impact: Findings will clarify key predictors of antihypertensive medication non-adherence and highlight gaps in evidence. Results will guide future adherence-improving strategies and inform design of simplified regimens, digital tools, and population-specific hypertension management policies.

Objectives/Goals: The endocannabinoid (eCB) system is integral to pain perception and may have antinociceptive effects. This study is the first to examine sex differences in eCB levels and their associations with pain and risk factors that may contribute to the development or exacerbation of pain symptoms, such as sleep disturbances. Methods/Study Population: Data were analyzed from the Adolescent Brain Cognitive Development StudySM, which included a substudy to measure circulating blood eCB concentrations in a subset of participants (n=403, mean age=12.56±1.05 years, 46.4% female). Covariates included age, race and ethnicity, puberty, sex, BMI, fasting state, and recent exercise. Pain was assessed on day of the blood draw (0–10 scale), and sleep disturbances were measured by the number of nighttime awakenings. Five eCB and eCB-like molecules were examined: anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and 2-oleoylglycerol (2-OG). Results/Anticipated Results: There was a significant sex difference in 2-AG concentrations (β=-5.36, CI [-8.53–2.18], p-adj=0.005), with female youth having lower concentrations than males. Because of this sex difference, all other analyses were sex-stratified. There was no association between pain on the day of the blood draw and eCB concentrations in either sex. Male youth alone exhibited a positive correlation between nighttime awakenings and concentrations of AEA (β=0.62, CI [0.26–0.99], p =0.001) and OEA (β=0.14, CI [0.065–0.21], p<0.001). Discussion/Significance of Impact: These findings suggest there may be sex-specific relationships with the eCB system and risk factors for pain. Future analyses will leverage longitudinal eCB measurements and examine how changes in eCB may relate to pain and sleep problems during adolescence.

Objectives/Goals: Diabetes mellitus (DM) increases cardiovascular (CV) mortality risk in chronic kidney disease (CKD). It is unknown if patients with CKD and DM have a greater risk of impaired CV functional capacity compared to those without DM. We investigated CV functional capacity alterations in CKD patients with and without DM using cardiopulmonary exercise testing (CPET). Methods/Study Population: We performed a cross-sectional study of data from the Cardiorespiratory Fitness in Individuals with CKD in Indiana (FIT-INDY) cohort. FIT-INDY is an ambulatory referral cohort consisting of patients within the Indiana University Health system. All individuals included in this analysis had been diagnosed with CKD and underwent CPET as part of their medical standard of care. CPET was used to assess peak oxygen uptake (VO₂Peak), the gold standard measure of CV functional capacity. Participants were stratified into two groups: CKD+DM (n=53) or CKD (n=63). Group comparisons were performed using a t-test, Mann–Whitney test, or Fisher’s exact test. Adjusted analyses were completed using multiple linear regression. Results/Anticipated Results: The CKD+DM group had a higher prevalence of males, higher body mass index, heart failure, use of beta-blockers, SGLT2 inhibitors, and GLP1-agonsits (all p’s < 0.05). Age, race, and estimated glomerular filtration rate were similar between groups (all p’s ≥ 0.05). CPET demonstrated greater CV functional impairment in the CKD+DM group for VO2Peak [CKD+DM = 11.5 ± 4.1 mL/kg/min, CKD = 14.4 ± 5.2 mL/kg/min], VO2AT [CKD+DM = 7.7 (6.2 - 9.4) mL/kg/min, CKD = 8.8 (7.1 -10.9) mL/kg/min], and peak HR [CKD+DM = 107 (91 - 123.2) bpm, CKD = 116 (99.2 - 131.2) bpm; all p’s < 0.05]. After adjusting for sex, left ventricular ejection fraction, and medications, significance remained for VO2Peak and VO2AT, but not peak HR. Echocardiography findings were similar between groups before and after adjusting (all p’s ≥ 0.05). Discussion/Significance of Impact: CPET revealed worse CV impairment in patients with coexisting CKD and diabetes that were not evident on resting echocardiographic assessment. These findings suggest that CPET could be utilized as a diagnostic tool to risk-stratify this patient population.

Objectives/Goals: This study addresses mobility disability in the aging Deep South population by: 1) identifying protective movement patterns against obesity-related decline and 2) providing clinicians evidence-based, comprehensive exercise recommendations to combat the region’s disproportionate burden of mobility disability. Methods/Study Population: We will recruit 60 participants: young adults (n=20, 18–35 years), older sedentary adults (n=20, 65+ years), and older lifelong runners (n=20, 65+ years, 10+ years consistent activity). Participants will undergo 3D gait analysis using motion capture and force plates across multiple walking speeds and dual-task conditions. We will measure novel speed-invariant collision dynamics (H1/K1 joint powers) alongside traditional propulsive measures (A2/H3). This approach eliminates speed confounds that have plagued aging research, allowing true assessment of whether lifelong exercise preserves youthful movement patterns and cognitive-motor integration. Novel, quantitative measures like this are critical for helping populations facing high obesity rates and manual labor demands. Results/Anticipated Results: We hypothesize older runners will demonstrate 1) smaller dual-task costs during cognitive challenge, suggesting maintained cognitive-motor integration and 2) more efficient ankle–hip power distribution, reflecting preservation of energy-efficient movement strategies. These findings will establish which biomechanical qualities are most protective against age-related decline and may reveal relationships between superior movement patterns and overall health status. Given the Deep South’s unique convergence of obesity, chronic disease, and limited healthcare access, identifying sustainable interventions like running that preserve mobility is critical for reducing regional health disparities. Discussion/Significance of Impact: Speed-invariant biomarkers enable early detection of movement dysfunction before disability manifests and understanding how biomechanical health relates to common comorbidities allows more comprehensive, targeted interventions addressing the region’s obesity and chronic disease burden.

Objectives/Goals: Test whether early childhood attachment security relates to epigenetic aging during adolescence, evaluate consistency across clocks, and examine heterogeneity for targeting. Methods/Study Population: Adolescence is a high-stakes period for stress biology. DNA methylation clocks condense CpG information into indices of cellular and system-level aging that relate to stress load and risk profiles. This study examines a population-based cohort with saliva DNA methylation at ages 9 and 15 and 5 clocks. Prespecified longitudinal regression and ANCOVA assess associations and incremental value beyond age 9 clocks. Models adjust for prespecified social and technical covariates; covariate missingness is handled with multiple imputation. False discovery rate control is applied within each wave. Sensitivity checks vary covariates and scaling. Results/Anticipated Results: Findings show directionally consistent protective signals linking early attachment security to youth DNA-based aging. The pattern concentrates by age 9, guiding precision child health efforts toward early relational supports and low-burden, saliva-based screening that is feasible in diverse community and clinical settings. Discussion/Significance of Impact: Results support a pathway to intervention. Attachment screening paired with saliva methylation clocks can identify who benefits from relational supports, when to intervene, and how to set decision thresholds. The approach fits pediatric workflows, guides stratification in attachment-focused trials, and is scalable to external validation.

Objectives/Goals: Anemia occurs universally among preterm infants who are also at risk of severe retinopathy of prematurity (ROP), a vision-threatening morbidity. We aim to determine the effect of anemia and its treatment with iron on the anatomical and molecular development of retinopathy and subsequent retinal function. Methods/Study Population: Oxygen-induced retinopathy (OIR) was induced in rat pups per the well-validated 50/10 OIR model, through 14 days of cyclic hyperoxia (50% FiO2) and hypoxia (10% FiO2) followed by recovery in normoxia. Anemia was induced by phlebotomy from postnatal day (P) 3 to P20, to a target hematocrit of 18%. Half of the pups were housed with a dam receiving standard iron chow (40 mg/kg) and half with a dam receiving high iron chow (400 mg/kg). Rat pups were euthanized at P20 during the neovascular phase of OIR. Retinal vascular morphometry (percent avascular and neovascular areas) was compared between groups by two-way ANOVA. Future outcome measures include retinal single-cell sequencing, flow cytometry, and molecular measures. Results/Anticipated Results: Anticipated Results: We anticipate that iron deficiency anemia will reduce the severity of OIR and that high dose iron treatment will increase it. We anticipate that high-dose iron treatment will induce oxidative stress, induce inflammation, and reduce the hypoxia-like effect of iron deficiency and angiogenic markers. Discussion/Significance of Impact: Upon completion, the results of this study can inform clinicians regarding preclinical effects of isolated anemia and treatment with iron on the development of retinopathy. This research has the opportunity to improve visual outcomes in preterm infants by improving clinical care of this population in the neonatal intensive care unit.

Objectives/Goals: Maternal diabetes is associated with increased inflammation during pregnancy, which may elevate the risk of motor, cognitive, and language delays in their offspring. Our objective is to characterize the neurodevelopmental outcomes at 12 months of age in children born to diabetic mothers using two validated assessment tools. Methods/Study Population: We assessed 54 children across three groups: type 1 diabetes mellitus (DM) (T1DM n = 6), type 2 DM (T2DM n = 24), and control (n=24). Neurodevelopment was assessed using two standardized tools: the Ages and Stages Questionnaire 3rd edition (ASQ-3), a parent questionnaire, and the Bayley Scales of Infant and Toddler Development, 4th edition (BSID-IV). The ASQ-3 is composed of five domains: Communication, Gross Motor, Fine Motor, Problem Solving, and Personal-Social. The BSID-IV is also composed of five domains: Cognitive, Language (Receptive and Expressive), Motor (Gross and Fine), Social-Emotional, and Adaptive Behavior. For this study, we assessed only the Cognitive and Language domains of the BSID-IV. We used the Cochran–Mantel–Haenszel (CMH) Mean Scores test to compare the mean scores across the three groups. Results/Anticipated Results: The ASQ-3 showed no statistically significant differences between groups across all five domains. However, several children showed delays or were at risk for delays in multiple domains: Communication: (1 [1.85%]; 4 [16%] delayed), Gross Motor: (4 [7.4%] delayed), Fine Motor: (13 [24.1%] at risk; 7 [12.9%] delayed), Problem Solving: (9 [16.7%] at risk; 11 (20.3%) delayed, and Personal-Social: 8 [14.8%] at risk, 10 (20%) delayed). Similarly, the BSID-IV showed no statistically significant group differences in Cognition and Language. However, below-average performance was observed in both domains. For Cognition, 14 children (25.9%) scored below average (T1DM:1 [1.85%], T2DM: 7 [12.9%], Con = 6 [11.11%]). For Language, 12 children (22.22%) (T1DM = 1 [1.85%], T2DM = 4 [7.4%], and Con = 7 [12.9%]). Discussion/Significance of Impact: Children from all groups had delays in fine motor, problem-solving, personal-social, cognitive, and language development. These findings suggest that maternal diabetes may influence neurodevelopment. We suggest that these children have continued neurodevelopmental follow-up with their pediatrician to improve these delays.

Objectives/Goals: Our goal is to determine whether insufficient Pumilio2 (Pum2) leads to neurological disorders. We identified predominantly young patients with mutations in PUM2 who suffer from developmental delays, motor incoordination, seizures, and so on. We will utilize Pum2 null mice to identify behavioral issues and neural degeneration. Methods/Study Population: We conducted several neurobehavioral assays with the Pum2 mutant mice (an estimated 40 mice) throughout their development. These assays measure learning and memory, motor coordination, spatial recognition, hyperactivity, and social cues. In addition to these experiments, we conducted immunofluorescence and Nissl staining to characterize brain morphology and Purkinje cells. Additionally, we collected patient-derived fibroblasts for qPCR and Western blot to measure the mRNA and protein levels of PUM2 in the different variants. Using both eCLIP and RNA sequencing, we will determine and rank targets bound by Pum2 in the murine brain. Results/Anticipated Results: At 12 weeks, we discovered Pum2 mutant mice exhibit neurological phenotypes that worsen with age development, such as motor incoordination, muscle atrophy, hyperactivity, impulsiveness, and compulsiveness. We observed a decrease in dendritic density in the Purkinje cells at 10 weeks of age, but no neural degeneration. However, we observed a reduction in dendritic density and aggressive Purkinje cell degeneration much later in adulthood. We obtained patient-derived fibroblasts to conduct Western and qPCR. We found that the missense mutations lead to a decrease in PUM2 protein and mRNA levels. These findings exhibit phenotypes similar to those observed in PUM2 patients, suggesting that insufficient levels of PUM2 lead to neurodevelopmental disorders and ataxia. Discussion/Significance of Impact: Pumilio2 is an RNA-binding protein from the PUF family that either promotes mRNA degradation or inhibits translation. It controls dendritic development and synaptic activity. PUM2 may be a novel ataxia gene that manifests neurodevelopmental problems.

Objectives/Goals: To discover novel phenogroups of atrial fibrillation (AF) from a real-world claims database using a multi-algorithm unsupervised machine learning (ML) approach and to reveal clinical factors associated with each phenogroup using supervised ML. We aim to establish clinically meaningful AF phenogroups to advance precision medicine. Methods/Study Population: We will leverage the Merative MarketScan databases (2016–2024) of real-world claims data with an estimated >600,000 AF patients. Four unsupervised ML clustering techniques will be employed to identify phenogroups: k-means, Gaussian mixture models, agglomerative, and HDBSCAN. Following cluster generation, the algorithms will be evaluated through internal validation via the Davies–Bouldin Index which calculates cluster compactness and separation, and the Adjusted Rand Index to determine stability between algorithms. Then, supervised ML via SuperLearner, which will include advanced algorithms (e.g., gradient boosting machines and support vector machines), will identify clinical factors associated with each phenogroup and will be evaluated using k-fold cross-validation and Shapley values. Results/Anticipated Results: We anticipate that the multi-algorithm approach will yield robust, distinct, and clinically meaningful AF phenogroups. SuperLearner will confirm the key clinical characteristics driving membership in each group (e.g., hypertension and heart failure). These data-driven classifications will provide a robust foundation for subsequent translational analyses. We will perform time-to-event analyses to assess the differential risks of stroke, coronary artery disease and myocardial infarction, heart failure, and rates of healthcare utilization across phenogroups. Next, we will employ the target trial framework to evaluate the comparative effectiveness of anticoagulant therapies on stroke incidence across phenogroups. Discussion/Significance of Impact: This work demonstrates a highly rigorous, multi-algorithm approach to characterize a large, real-world database of AF patients. By constructing novel AF phenogroups, we aim to move beyond the limitations of current AF classifications and advance precision medicine by providing the foundation for individualized therapeutic strategies.