Objectives/Goals: Nucleoside transport by ENT2 facilitates transit of the lupus anti-DNA autoantibody Deoxymab into cells and across the blood–brain barrier into brain tumors. This work examines the Deoxymab-nucleoside interactions that contribute to membrane crossing and apply them in brain tumor therapeutics. Methods/Study Population: Deoxymab interactions with individual nucleosides, nucleobases, and pentose sugars are examined by surface plasma resonance (SPR) and cell penetration assays in a panel of cell lines including glioblastoma, breast cancer, and normal breast epithelial cells. Deoxymab-conjugated gold nanoparticles are generated and tested for binding to normal human astrocytes and glioma cells, and the impact of supplemental nucleosides on this binding is determined. Deoxymab-gold nanoparticles are tested for brain tumor localization by systemic and local administration in mice bearing orthotopic glioblastoma brain tumors and enhancement of laser interstitial thermal therapy (LITT) examined. Results/Anticipated Results: Individual nucleosides significantly increase the efficiency of cell penetration by Deoxymab in all cell lines tested. In contrast, component nucleobases and pentose sugars significantly suppress the uptake of the autoantibody into cells. Deoxymab-conjugated gold nanoparticles bind DNA in vitro and to astrocytes in culture and are anticipated will enhance the efficacy if LITT in vivo by associating with DNA released by necrotic tumors and/or by locally administered nucleosides in brain tumor environments and subsequently act as heat sink to amplify LITT impact. Discussion/Significance of Impact: Deoxymab is a DNA-targeting, cell-penetrating autoantibody. These findings establish nucleosides as the components of DNA that promote autoantibody membrane crossing through ENT2 activity and indicate potential for use of Deoxymab-gold nanoparticles in combination with LITT for brain tumor therapy.

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 ALS. Our findings will provide insights for therapeutic strategies, while we also use gene editing to investigate correcting variants in ALS patients. 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 transfected cell’s DNA was sequenced to validate gene editing. To confirm transcriptional adaption, changes in levels of protein and gene expression will be measured via immunoblot and quantification of CHCHD10 and CHCHCD2 from whole cell lysates of the edited cells. Results/Anticipated Results: Utilizing CRISPR-Cas9 tools, we anticipate that CHCHD2 gene can functionally compensate for the loss of function in the CHCHD10 locus through transcriptional adaptation. Additionally, employing single-stranded oligodeoxynucleotides (ssODNs) we aim to accurately correct the genetic aberrations in ALS patient cells, and study the pathomechanisms of aberrant CHCHD10. Discussion/Significance of Impact: The significance of this research lies in its potential to uncover transcriptional adaptation in humans, which could explain why patients with the same genetic variant experience different symptoms. By understanding this mechanism, we could pave the way for novel therapies, especially for CHCHD10-associated ALS.

Objectives/Goals: We operationalized our evaluation using the RE-AIM framework, by defining its dimensions as nodes within a net effect diagram in the form of radar charts to visually display group variation among nodes. This enhanced our ability to measure the reach, effectiveness, and implementation of our efforts in under-resourced Los Angeles communities. Methods/Study Population: We applied the RE-AIM framework to guide the operationalization of evaluation methods, defining the RE-AIM dimensions generally with a focus on reach, effectiveness, and implementation. We developed and defined a standardized scoring system for metrics that contributed to the RE-AIM dimensions of focus, using data from our activities such as health education workshops targeting diverse, under-resourced populations in Los Angeles. Our standardized scoring system ranged from 1 to 5, reflecting the degree of success within each metric/dimension. Scores were mapped in net effect diagrams in the form of radar charts to enable comparative analysis and visualization, highlighting a variety of grouped variables (i.e., language, locations, and adaptation). Results/Anticipated Results: The operationalized and developed scoring system allowed us to standardize assessment across the RE-AIM dimensions, making it possible to visualize our impact through net effect diagrams. These diagrams illustrated variations in reach, effectiveness, and implementation across different community engagement activities stratified by group variables, providing insights into our impact and areas for adjustment. Preliminary results suggest that the net effect diagram effectively captures both broad and nuanced impacts and serves as a viable application of the RE-AIM framework. The use of standardized scoring enhances data comparability and offers a dynamic visual tool for monitoring ongoing and future initiatives while serving as a tool to display and report our impact. Discussion/Significance of Impact: Operationalizing evaluation with the RE-AIM framework and implementing a standardized scoring system allows us to visualize and monitor effectiveness in real time. This system supports data-driven decision-making for our sustainable, impactful community engagement initiatives ultimately contributing to our goal of improving health equity.

Objectives/Goals: Pharmacogenomic (PGx) testing identifies genetic variations affecting medication response but is not yet in routine clinical whole-genome sequencing (WGS) workflows. We aimed to establish a streamlined bioinformatics pipeline for incorporating PGx reporting into clinical WGS and to determine clinical implications for medication treatment. Methods/Study Population: A PGx profiling pipeline based on existing WGS data was developed, integrating three WGS-based PGx calling tools: Aldy, PyPGx, and Cyrius (CYP2D6 only), to provide genotype calls for 17 key pharmacogenes. The pipeline was validated using WGS data from 70 individuals with diverse backgrounds (36% European, 27% African, 27% Asian, and 10% admixed) from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results were manually reviewed against published data. The validated pipeline was then applied to 144 clinical patients previously screened for neurodevelopmental disorders or suspected hereditary diseases, followed by diplotype-to-phenotype translation and preemptive PGx-guided medication recommendations based on consensus guidelines and FDA labeling for commonly used medications. Results/Anticipated Results: Congruent phenotype call rates for GeT-RM samples were 100% for 13 genes (CFTR, CYP2B6, CYP2C19, CYP2C9, CYP3A4, CYP4F2, DPYD, G6PD, IFNL3, NAT2, NUDT15, TPMT, and VKORC1), 99% for three genes (CYP3A5, SLCO1B1, UGT1A1), and 97% for CYP2D6, indicating strong pipeline performance. Among 144 clinical patients, 99.3% had at least one clinically actionable PGx results relevant to 36 of top 300 medications in the USA across psychotropic, cardiovascular, musculoskeletal, gastrointestinal, and other therapeutic areas. The most prevalent drug–gene interactions involved sertraline and CYP2B6, affecting 49% patients: 41% were intermediate metabolizers who may require slower titration and lower maintenance doses, while 8% poor metabolizers may benefit from a lower starting dose or alternative antidepressants. Discussion/Significance of Impact: Our validated WGS-based PGx profiling pipeline successfully extracted actionable PGx data from clinical WGS. By aligning PGx profiles with guideline-recommended clinical actions, we demonstrated the clinical value of integrating PGx reporting in WGS workflows, improving personalized medication management.

Objectives/Goals: This study examines associations between childhood violence exposure, accelerated biological aging, and adolescent depression using DNA methylation-based epigenetic clocks. Findings aim to identify biomarkers for early detection, guide interventions, and address youth mental health disparities. Methods/Study Population:Data from the Future of Families and Child Wellbeing Study (N = 4,898), a diverse urban U.S. cohort, were analyzed. Childhood violence exposure, assessed using the Parent-Child Conflict Tactics Scale, included measures of physical, emotional, and psychological aggression and neglect. Biological aging at age 15 was evaluated using second-generation epigenetic clocks derived from saliva DNA methylation patterns, while depressive symptoms were measured with the CES-D scale. Multiple linear regression models tested associations between early violence exposure, epigenetic aging, and depressive symptoms, adjusting for socioeconomic status, caregiver mental health, and other key covariates. Results/Anticipated Results: Preliminary results suggest that early violence exposure may be linked to accelerated biological aging and depressive symptoms during adolescence, a critical developmental period. Epigenetic clocks offer an objective method for identifying high-risk youth, complementing mental health evaluations. With further validation and participatory action research, these findings could guide the development of biomarkers for longitudinal testing in school-based screenings and community health programs. These tools aim to be accessible, culturally relevant, and tailored to diverse populations, enhancing early detection, informing personalized interventions, and supporting scalable clinical applications. Discussion/Significance of Impact: This study explores links between early adversity, biological aging, and mental health, advancing understanding of adolescent depression. Epigenetic biomarkers could improve risk detection and guide tailored interventions in schools and community settings, enhancing access and reducing disparities.

Objectives/Goals: Diabetic kidney disease (DKD) affects 40% of diabetic patients, leading to renal failure, yet the molecular drivers remain elusive. MicroRNAs, noncoding regulators of gene expression, may hold the key. This study aims to identify key miRNAs in DKD, providing crucial insights for early intervention. Methods/Study Population: miRNA sequencing was conducted on kidneys from 8-week old male BTBR wild type and BTBR ob/ob mice. BTBR ob/ob mice lack the hormone leptin and spontaneously develop type 2 diabetes, with morphological renal lesions characteristic of human DKD. Total RNA was extracted from whole kidney sections and processed using the QIAseq miRNA library kit. Sequencing was performed on an Illumina NextSeq 550 platform. GeneGlobe analysis was used to identify differentially expressed miRNA functional pathways, while ingenuity pathway analysis (IPA) was employed to predict master regulators and causal networks involved in DKD. Results/Anticipated Results: miRNA sequencing identified significantly differentially expressed miRNAs (p < 0.05) between 8-week-old BTBR WT and BTBR ob/ob male mice, including miR-34a (-6.86 fold), miR-122 (-5.01 fold), miR-129 (-2.23 fold), miR-142a (+2.78 fold), miR-346 (+4.66 fold), miR-547 (-2.49 fold), miR-592 (+11.81 fold), miR-802 (-6.95 fold), and miR-6539 (-7.93 fold). Qiagen GeneGlobe analysis revealed biological processes potentially targeted by these miRNAs, including endocytosis, phagocytosis, hyperglycemia (p = 7.59e-3), and insulin-dependent diabetes (p = 4.32e-4). IPA predicted activation of RRAS, a small GTPase regulating cell growth and signaling (Z-score +2), with miR-34a and miR-122 targeting MYC, PI3K, and TGF-β in DKD progression in BTBR ob/ob mice. Discussion/Significance of Impact: We identified kidney miRNA expression in BTBR ob/ob mice at a pivotal disease stage. miR-34a, miR-122, and RRAS emerged as key drivers in DKD progression, showing remarkable early biomarker potential. These findings lay the groundwork for early detection and innovative therapies to halt DKD and improve patient outcomes.

Objectives/Goals: Many left hemisphere stroke survivors have a reading disorder (alexia), which is experienced as decreasing well-being. Therapies produce inconsistent results, demonstrating a need for treatment response predictors. We identify neural correlates of a computational model of reading, which may provide biomarkers to improve therapeutic outcomes. Methods/Study Population: Left hemisphere stroke survivors (LHSS) (n  =  52) performed an oral reading task and tests of semantic and phonological processing. Artificial neural network (ANN) models, mapping between orthography (visual word form), phonology (auditory word form), and semantics (word meaning), were trained to read single words at an adult reading level. Stroke was simulated by removing percentages (in 10% intervals) of the connections into and out of semantics, phonology, and the combination thereof. The lesioned model producing the smallest average Euclidean distance over word and pseudoword reading accuracy to each LHSS was selected as the matched model. Two voxelwise lesion-symptom mapping (VLSM) analyses identified the neural correlates of the percent of phonological and semantic links removed in the matched models. Results/Anticipated Results: Model reading was correlated with LHSS reading (high-frequency regular words, r(48) = 0.96; high-frequency irregular words, r(48) = 0.94; low-frequency regular words, r(48) = 0.97); low-frequency irregular words, r(48) = 0.85; all p’s Discussion/Significance of Impact: Our results show that ANN models of reading, when closely matched to LHSS reading performance, directly connect cognitive processes to the brain. Using matched models as a precision medicine framework to predict therapy response or to identify targets for neurostimulation provides a valuable route toward improving poststroke language outcomes.

Objectives/Goals: We have shown that parathyroid hormone-related protein (PTHrP) is enriched at the LIFR promoter in breast cancer cells and inhibits the expression of dormancy-associated genes including LIFR. The objective of this study is to define where all PTHrP binds DNA and identify pathways that are regulated by PTHrP that promote breast cancer colonization of the bone. Methods/Study Population: In this study, we use human estrogen receptor-positive MCF7 breast cancer cells which we and others have reported lie dormant in the bone. MCF7 cells were engineered to express either PTHrP with an HA-tag (MCF7P), or a vector control (MCF7V). We use Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a method of mapping protein-DNA interactions, to define where PTHrP binds DNA. Here, an HA-specific antibody identifies regions of DNA that are bound to PTHrP in MCF7P cells compared to MCFV cells. Next, we perform DNA sequencing and gene set enrichment analysis (GSEA) on genes identified by CUT&RUN to identify pathways that are regulated by PTHrP. These experiments will determine how PTHrP regulates dormancy and breast cancer colonization in the bone. Results/Anticipated Results: We completed IgG (-control), H3K4me3 (+ control), and HA (PTHrP) CUT&RUN on MCF7V and MCF7P cells, and submitted DNA for sequencing. This study will define where PTHrP binds the genome and identify pathways regulated by PTHrP. Previously, through ChIP-qPCR we showed that PTHrP binds the LIFR promoter to repress LIFR expression. Given this result, we expect that PTHrP binds to the promoters of dormancy-associated genes including LIFR in MCF7P cells compared to MCF7V cells. PTHrP may be involved in regulating other processes besides dormancy to induce expansion of breast cancer cells in the bone, so we will use GSEA to identify pathways that are altered in MCF7P cells when PTHrP is over-expressed compared to MCF7V cells. Together, this will define how PTHrP regulates gene expression of bone metastatic breast cancer cells. Discussion/Significance of Impact: This study will unveil mechanisms of metastatic breast cancer expansion in the bone by defining where PTHrP binds the genome to regulate gene expression. These findings will reveal therapeutic vulnerabilities that will be used to target bone-disseminated tumor cells to prevent lethal recurrence.

Objectives/Goals: Immune checkpoint inhibitors (IO) have dramatically improved survival outcomes in patients with metastatic melanoma. Still, many patients do not respond these treatments, and others may experience harmful adverse events (irAEs). Thus, there an unmet need for biomarkers for real-time monitoring and management of patients exposed to IO therapies. Methods/Study Population: Serial serum samples were collected from patients with BRAFV600-mutant metastatic melanoma treated with ipilimumab/nivolumab (IO, n = 14) or dabrafenib/trametinib (TT, n = 10). Methylated cell-free DNA (cfDNA) was isolated and sequenced using enzymatic methyl-seq. We develop a robust computational pipeline to identify the top 250 cell-type specific regions of differential methylation (DMRs) across 24 cell-types. Using these differentially methylated regions, a deconvolution tool was developed to determine the abundance of cell type-specific cfDNA in patient serum, and changes in abundance were tracked over treatment time-course to assess response treatment and identify signals of adverse events. Results/Anticipated Results: We demonstrated improved precision in DMR detection evidenced by a higher area under the receiver operator characteristic curve (AUROC) of 0.85 on average. Pathway and functional annotation analysis revealed melanocyte-specific methylation marker regions regulated genes related to melanocyte development and differentiation, including MITF, SOX9/10, and FOXD3. We show these regions are conserved through the transformation to malignant melanoma, indicating melanocyte cfDNA abundance can be used as a marker for tumor burden. We characterize the dynamics of melanocyte-derived cfDNA over the course of treatment in responders and nonresponders to both IO and TT. We observe that changes in concentrations of cfDNA from other cell types correlate with clinically observed irAE-mediated damage to normal tissue. Discussion/Significance of Impact: We demonstrated the utility of decoding the origins of cfDNA fragments obtained from serial liquid biopsy samples. Using cell-specific methylation marks, we identified a signature from the primary melanoma to assess response to treatment, while also obtaining a signal from other tissues throughout the body to monitor immune related adverse events.

Objectives/Goals: Investigating the B-cell acute lymphoblastic leukemia (B-ALL)-associated germline SNP rs7090445, located in intron 3 of ARID5B, which is more frequently observed in individuals of Hispanic/Latino descent. Investigating the mechanisms behind this inherited single nucleotide polymorphisms (SNP) that may contribute to the higher incidence of B-ALL in this population. Methods/Study Population: Specific Aim 1: We hypothesize ARID5B SNP rs7090445 disrupts intrinsic enhancer function. Identification of critical DNA looping events impacted by ARID5B variants using Capture C. Affinity purification-mass spectrometry to identify potential ARID5B transcription mediators. Specific Aim 2: We hypothesize the B-ALL-associated SNP leads to a partial human B-cell differentiation block. Utilize Cas9-mediated homology-directed repair to create ARID5B SNP in primary human hematopoietic stem cells. Gene-edited HSCs will be differentiated into B cells using an ex vivo system. Fluorescence-activated cell sorting to sort our pool of cells into stages of B-cell development spectrum. Amplicon sequencing and variant allele frequency of rs7090445 SNP to evaluate its impact on B-cell development. Results/Anticipated Results: This proposal is conceptually innovative as it seeks to understand the mechanism by which the B-ALL-associated SNP rs7090445 in intron 3 of ARID5B disrupts enhancer function and investigates its impact on human B-cell development. Future research will investigate a tumor-suppressive role of ARID5B and whether it constitutes a “first-hit” of leukemogenesis. Discussion/Significance of Impact: Successful completion of this research will elucidate the critical role of the B-ALL-associated ARID5B SNP rs7090445 in human B-cell development and leukemogenesis. As this SNP is more prevalent in Hispanic/Latino populations, it will also provide crucial insights into the genetic factors behind the elevated incidence of B-ALL.

Objectives/Goals: The aim of the study is to identify resistance factors for substance use (i.e., factors that explicitly help to avoid or reduce drug use). Identification of resistance factors could inform strategies that seek to reduce the prevalence of substance use and related disorders. Methods/Study Population: Adult twins aged 30–70 years were recruited from the Mid-Atlantic Twin Registry. A mixed-method approach, group concept mapping, was used to identify factors influencing participants to resist using substances. Approximately 155 participants produced 97 statements reflecting substance use resistance factors. Hierarchical cluster analysis and multidimensional scaling assessed how participants sorted and rated statements for their lifetime and current importance. Factor analysis was used to reduce data dimensionality. Reliability analyses were conducted to identify a subset of statements anticipated to consistently represent each cluster. Results were shared with participants to assess accuracy with their experiences. Results/Anticipated Results: Participants sorted 97 statements into 9 thematic clusters: (1) Controlling Personal, Negative Consequences; (2) Concern About Health and Well-being; (3) Lack of Desire; (4) Outside Influences; (5) Social Norms and My Reputation; (6) Career and Legal Impacts, (7) Avoiding Harm to Family and Relationships; (8) Preserving Family Relationships; and (9) Family and Friends Impact on Me. Participants consistently identified health concerns as an important substance use resistance factor. The statements will be further reduced to represent a smaller subset for future use as a scale to measure exposure to resistance factors. Discussion/Significance of Impact: Health concerns related to substance use were identified as an important resistance factor. This has been supported by research on smoking cessation and implemented in smoking prevention campaigns. Therefore, prioritizing health-related outcomes in prevention may be important to reduce substance use prevalence.

Objectives/Goals: To evaluate the impact of social determinants of health (SDOH), specifically socioeconomic status and medical insurance coverage, on access to mental health services for adults aged 60 and older served by Federally Qualified Health Centers (FQHCs) in Puerto Rico. Methods/Study Population: A secondary retrospective analysis of electronic health records from FQHCs in Puerto Rico will be conducted to examine the relationship between SDOH needs and mental health service utilization among adults aged 60 years and older receiving primary care. SDOH data will be collected using the PRAPARE® tool, assessing factors such as socioeconomic status, insurance coverage, and emotional support, with a focus on identifying unmet needs. Multivariable and logistic regression models, using Stata v.17, will be employed to evaluate correlations between these SDOH factors and mental health service utilization, adjusting for age, gender, and mental health conditions. This analysis aims to quantify the impact of SDOH on access to mental health services and elucidate key barriers to care for older adults in Puerto Rico. Results/Anticipated Results: We anticipate that lower socioeconomic status, lack of supplemental insurance, and inadequate family support will be strongly associated with the underutilization of mental health services among older adults in Puerto Rico. These disparities are expected to be more pronounced in individuals with limited income, weaker family networks, leading to significant gaps in access to necessary mental health care. Barriers such as healthcare insecurity, financial hardship, and fragmented support systems will likely emerge as major obstacles. By including patients from both rural and urban regions, the study will capture the distinct challenges each population faces, enhancing the contextual relevance of the findings to broader populations, ultimately informing policy and developing intervention strategies. Discussion/Significance of Impact: The findings will provide crucial insights for developing targeted interventions to enhance mental health care access for older adults in Puerto Rico. These results will inform policy development and public health strategies, addressing disparities and promoting equitable care in underserved populations.

Objectives/Goals: As the aging population increases, maintaining cognitive and physical health becomes crucial. Executive functions (EF), including reaction time, sustained attention, and spatial memory, are essential for daily life and independence in older adults but tend to decline with age, especially in Alzheimer’s disease (AD) patients. Methods/Study Population: Physical balance (PB) impairments further exacerbate this decline, affecting the quality of life and independence. Balance training (BT) emerges as a potential non-pharmacological intervention to enhance EF. This study aims to 1) assess the association between PB impairments and EF deterioration in older adults with AD; 2) evaluate the impact of BT on EF enhancement in this population; and 3) explore changes in brain-derived neurotrophic factor (BDNF) biomarkers before and after physical activity related to BT. This pilot study employs a cross-sectional design with older Hispanic adults (>65 years) in Puerto Rico, comparing a control group without BT intervention to an experimental group with BT intervention. Both groups will be assessed for EF and BDNF biomarkers pre- and post-physical activity. Results/Anticipated Results: Expected outcomes include identifying significant correlations between PB impairment and diminished EF, demonstrating measurable EF improvements following BT, and evidencing sustained BDNF release post-BT despite PB impairments. By understanding the biological mechanisms linking BT and cognitive improvements, particularly the role of BDNF, this research could inform future strategies to mitigate cognitive decline in AD patients through targeted physical interventions. Discussion/Significance of Impact: The findings of this study could provide valuable insights into the development of new preventive interventions, enhancing the quality of life and independence for older adults.

Objectives/Goals: This review examined if sleep duration is associated with established Alzheimer’s disease (AD) fluid biomarkers, such as amyloid-β peptides (Aβ40 and Aβ42), total-tau (t-tau), phosphorylated tau (p-tau181 and p-tau217), neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP). Methods/Study Population: We searched PubMed, CINAHL, and SCOPUS through September 15, 2024, using keywords and appropriate subject headings related to AD, fluid biomarkers, and sleep. The search was developed and conducted in collaboration with a medical librarian. We also searched Google Scholar and screened the reference lists of relevant reviews. Two independent reviewers screened 1,657 peer-reviewed articles, of which 21 met the inclusion criteria (14 with biomarkers measured in cerebrospinal fluid [CSF] and 7 in blood). Two review authors independently extracted study details from included articles using a standardized data extraction template. Results/Anticipated Results: Sample sizes ranged from 18 to 4,712 participants. Sleep duration was assessed using self-reported measures in 8 studies and objective measures in 13. For the 14 studies using CSF biomarkers, lower Aβ42 (3/14), Aβ40 (1/14), or the ratio (1/14) were associated with either short or long sleep duration; t-tau (3/14) and p-tau181 (4/14) levels were mostly associated with short sleep. For the 7 blood-based biomarker studies, Aβ42 (2/7), Aβ40 (2/7), and the ratio (3/7) had mixed results with either short or long sleep. T-tau (1/7) and p-tau181 (1/7) levels were associated with long sleep; NfL (2/7) was associated with both short and long sleep. Six studies reported nonlinear relationships, with both short and long sleep associated with unfavorable biomarker profiles. None of the studies investigated p-tau 217 or GFAP. Discussion/Significance of Impact: Our results suggest that the relationship between sleep duration and AD fluid biomarkers is very complex, and it highlights the importance of sleep in AD risk assessment and prevention. The inconsistency in findings stresses the need for standardized study design and measurement methods to clarify causality and inform clinical guidelines.

Objectives/Goals: Aim 1: Assess the correlation between Tuft cell T2R functionality and Th2 sinonasal inflammation, disease burden, and post-surgical outcomes in AERD patients. Aim 2: Determine if hyperfunctional Tuft cell T2Rs enhance denatonium-stimulated inflammatory responses in nasal epithelial air–liquid interface (ALI) cultures from AERD patients. Methods/Study Population: Aim 1: We will conduct a prospective cohort study with 82 AERD patients. Taste sensitivity to denatonium (DB), serving as a proxy for tuft cell T2R functionality, will be assessed using a validated 13-point scale, and correlations with clinical outcomes – SNOT-22, histopathologic, CT, and endoscopic scores – will be analyzed using linear regression. Aim 2: Sinonasal epithelial cells will be collected from AERD patients either hyper- or hyposensitive to DB and from healthy controls. We will establish ALI cultures and expose them to varying DB concentrations. Secretions will be analyzed for antimicrobial peptide release via bacterial kill assays and for IL-25 and β-defensin 2 via ELISA. Tuft cell frequency and baseline IL-25 mRNA expression will be assessed using immunofluorescence and quantitative real-time polymerase chain reaction, respectively. Results/Anticipated Results: We expect that higher DB taste sensitivity in AERD patients will correlate with worse clinical outcomes, reflected by elevated 6-month postoperative SNOT-22 scores, indicating increased symptoms. Additionally, we anticipate that preoperative Lund-MacKay and Lund-Kennedy scores, along with histopathological metrics, will be worse in DB-hypersensitive patients, establishing a link between taste sensitivity and disease burden. In vitro, we predict that AERD patients with DB hypersensitivity will demonstrate significantly higher IL-25 and β-defensin 2 secretion and reduced bacterial colonies in kill assays. We also expect increased tuft-cell frequency and baseline IL-25 mRNA in AERD-derived cultures compared to healthy controls, highlighting T2R functionality’s role in AERD pathogenesis. Discussion/Significance of Impact: This project aims to investigate a putative new role for Tuft cells in AERD pathogenesis by correlating Tuft cell T2R functionality with outcomes in AERD patients and with inflammatory response in vitro. Findings could lead to predictive clinical taste tests and future genotyping studies to identify T2R polymorphisms correlated with AERD severity.

Objectives/Goals: To support the growing number of clinical research professionals (CRP) working in behavioral and social science contexts, relevant formative assessments of research skills are needed. This study examines the development of an objective formative assessment designed to assess skills relevant to conducting social and behavioral research. Methods/Study Population: A multidisciplinary group at the University of Michigan was convened to develop the assessment. Case studies depicting clinical and translational research conducted in behavioral and social science contexts were used to measure proficiency in seven of the eight ECRPTQ competencies: Scientific Concepts & Research Design, Ethical & Participant Safety Concerns, Clinical Trial Operations (Good Clinical Practice), Study & Site Management, Data Management & Informatics, Leadership & Professionalism, Communication, and Teamwork & Team Science. Three difficulty levels of questions were developed: basic, intermediate, and advanced. To reinforce knowledge and skill development, the assessment was designed to give respondents formative feedback after responding to each question. Results/Anticipated Results: A preliminary “pre-pilot” test was conducted with three postdoctoral scholars to ensure that the assessment items were understandable. The assessment was then pilot tested with a larger group of 40 clinical research professionals (CRP) to test for the clarity and difficulty level of the items. A smaller group of 20 of these CRPs agreed to participate in focus groups to obtain feedback on their user experience. Data regarding years of experience as a CRP, types of studies engaged with, and information regarding professional certification were collected and used in the analyses. Demographic data collected were not connected to user responses. Results of the pilot test and focus groups were used to revise the questions on the final version of the assessment. Discussion/Significance of Impact: To our knowledge, this is the first objective assessment of research skills for CRP working in behavioral and social science contexts. We will discuss how other institutions can use this instrument to evaluate the training needs of their social and behavioral research workforce.

Objectives/Goals: Despite the acknowledgment of post-Ebola syndrome (PES), young EVD survivors have received little attention. The mechanistic drivers and long-term consequences of PES and EVD early in life are unknown. We aim to define PES presentations in pediatric EVD survivors and propose potential mechanistic factors contributing to PES in young people. Methods/Study Population: Here we focus on physical health outcomes in an ongoing cohort study assessing mental and physical health in pediatric EVD survivors (age Results/Anticipated Results: 671 participants were enrolled between 2021 and 2022 (Infected: n = 226, Affected: n = 207, and Control: n = 238). Groups were similar in sex distribution (52.7%, 54.0%, and 53.8% female, respectively) and mean age, although the Infected group was slightly older (14.6 y) than the Affected (13.5 y) and Control groups (14.1 y), a difference unlikely to be clinically significant. Notably, the EVD Infected group exhibited a higher burden of symptoms, with significant findings in cardiac, MSK, ophthalmologic, and “ear, nose, and throat” systems. Principal component analysis showed differential patterns of sequelae across the groups, primarily defined by MSK. Discussion/Significance of Impact: PES is heterogeneous in pediatric EVD survivors. EVD Affected children exhibit a similar yet distinct pattern of clinical sequelae indicating ecological factors impact sequelae and raising questions about the mechanistic drivers of PES in children. Potential mechanisms include inflammation or accelerated aging and immune dysfunction.

Objectives/Goals: The goal of this proposal is to better understand how informing African Americans of their genetic risk affects their behavior as part of a cardiovascular disease (CVD) risk reduction intervention. Aim 1: To determine the effect of genetic risk knowledge on CVD health behavior. Aim 2: To determine the effect of genetic risk knowledge on secondary variables. Methods/Study Population: Method: Fifty participants from the Baton Rouge metropolitan area will be recruited. Participants must be African American adults over the age of 18. Potential participants will be recruited using community-based efforts that have been successful in recruiting this population specifically. Participants will be randomized into one of two groups. Genetically Unblinded Group (GU) will be “genetically unblinded” after baseline orientation. Genetically Blinded Group (GB) will be “genetically blinded” until the end of the study. This study design ensures that we can measure the impact of knowledge of genetic risk on participant behavior. Results/Anticipated Results: Baseline participants’ characteristics (body mass index, blood glucose, and cholesterol) will be summarized by intervention group, with counts and percentages for categorical variables and means and 95% confidence intervals for continuous variables. Primary Outcomes: Attendance in intervention sessions will be counted across groups. Effect on genetic risk knowledge will be determined via comparing the difference between the increased healthy lifestyle behaviors at endpoint between Genetically Unblinded (Cases) and Genetically Blinded Groups (Control). Secondary and Tertiary Outcomes: Mean change in secondary outcomes in the GU group will be compared against the mean change in the GB group. Participant’s survey responses and changes in physical measurement from baseline to endpoint will be observed. Discussion/Significance of Impact: This study empowers African Americans in Baton Rouge by providing genetic risk knowledge for cardiovascular disease. By addressing social determinants of health, it promotes behavior change, improves health outcomes, and fosters trust, potentially reducing health disparities and advancing health equity.

Objectives/Goals: An association has been found between maternal diabetes and neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD), attention-deficit/hyperactivity disorder (ADHD), and intellectual disabilities. Our objective is to observe the effects of type 2 diabetes mellitus (T2DM) on the neurodevelopment of infants. Methods/Study Population: A prospective study was performed on thirty infants who were evaluated using four neonatal neurodevelopmental assessments. Thirteen of these infants were from mothers with T2DM and seventeen of them were from mothers without diabetes. We used the Hammersmith Neonatal Neurological Examination (HNNE), Dubowitz exam, The Capute Scales, and The General Movement Assessment (GMA) to assess the neurodevelopment of these infants. HNNE assesses posture, reflexes, tone, and movement of infants. The Dubowitz exam is used to measure tone and reflexes to get an estimated gestational age (GA) of the infant. The Capute Scales has two subsections, one measures language (receptive and expressive) and the other measures visual-motor development. Lastly, the GMA was used to observe the general movements of the infant. Results/Anticipated Results: Cochran–Mantel–Haenszel determined between-group differences. HNNE, Dubowitz, and the Capute Scales had no significant difference between groups. HNNE results: 4 T2DM below cutoff; 9 controls below cutoff. Discussion/Significance of Impact: Offspring of T2DM mothers show a risk of motor delays in infancy and later language/cognition delays. Offspring of T2DM mothers should be followed due to the risk of motor delays. Early intervention could mitigate delays. This would be the first novel use of these four tools to evaluate 1-month-olds.

Objectives/Goals: This study investigates the contribution of the stool secretome (the soluble factors secreted by microbes into extracellular space) to in vitro α-synuclein (αSyn) oligomerization using stool cultures from patients with multiple system atrophy (MSA), a rare neurodegenerative disease hallmarked by pathologic αSyn aggregates. Methods/Study Population: Stool samples from MSA patients (n = 20), household controls (n = 20), and healthy controls (n = 20) will be cultured using an adapted dilution-to-extinction approach. The goal is to reduce microbial complexity progressively to produce random secretome combinations that may affect αSyn oligomerization differentially. The original inoculant and dilutions will be cultured anaerobically to collect conditioned media (CM) enriched with microbial secretomes. CM will be used to expose a fluorescence resonance energy transfer (FRET) biosensor assay and a Gaussia luciferase protein complementation assay – both modified to quantify αSyn-αSyn interaction indicating oligomerization. Any CM-altering αSyn oligomerization will undergo multiomic characterization to identify potential causative agent(s). Results/Anticipated Results: Specific microbe-produced molecules from the literature are anticipated to modulate αSyn oligomerization, identified by targeted, reductionist studies that selected and tested separately single microbial factors on αSyn aggregation in vitro and in vivo. From these studies, lipopolysaccharide and bacterial amyloid protein are expected to increase αSyn oligomerization, while short-chain fatty acids, such as butyrate, are expected to interfere with and decrease oligomerization. As a complementary systemic approach, this study’s agnostic methods involving MSA stool culture combined with the proposed dilution-to-extinction method are expected to identify additional MSA stool secretome components modulating αSyn oligomerization that might otherwise be missed in earlier reductionist approaches. Discussion/Significance of Impact: Completion of this reverse-translational work will aid in discovering MSA stool secretome components modulating αSyn oligomerization. Identification of specific factors contributing to pathologic αSyn behavior might set the stage for patient screenings for identified stool markers and could lead to microbiome-based interventions for MSA.