OBJECTIVES/GOALS: Our goal is to explore the complex, the non-linear interplay among chronic conditions collectively contributing to a greater detrimental impact on the progression of Alzheimer’s disease (AD) than a single chronic condition alone in individuals with normal cognition, MCI, and AD. METHODS/STUDY POPULATION: We used longitudinal data from National Alzheimer Coordinating Center (n = 41,437) and focused on individuals with normal cognition (n =16,884, mean age (SD) = 70.72 (9.7)). Random forest models were used to predict newly developed MCI or AD from baseline to the most recent visits. We used self-reported baseline data on 50 chronic conditions and comprehensive clinical and demographic information (e.g., age, sex, APOE status, mini-mental status exam (MMSE) scores, education, BMI, and depressive symptoms). A binomial random forest was used to identify significant interactions (with p-values RESULTS/ANTICIPATED RESULTS: Our model demonstrated an AUC of 0.708 and a classification error rate of 25.4%. Variables of importance for predicting MCI or dementia were age, coronary artery bypass, depression, APOE status, smoking, and depressive symptoms. Two-way interactions, such as age X MMSE score, age X depressive symptoms, and age X BMI, were significant. Three-way interactions, including age X depressive symptoms X MMSE score, or depressive symptoms X BMI X MMSE score, were significant. However, when we explored the random forest model using only the chronic condition data, we found an AUC of 0.602 and an error rate of 27.15%. We found that depression, anxiety, hypercholesterolemia, stroke, and the interaction between BMI and anxiety were significant. DISCUSSION/SIGNIFICANCE: Random Forest models indicate that not only known factors including age, baseline cognitive status, and APOE status, but also chronic conditions like depression, anxiety, hypercholesterolemia, and stroke may predict cognitive impairment.

OBJECTIVES/GOALS: The COVID-19 pandemic disrupted HIV care, though it prompted preventive measures for respiratory pathogens, particularly among PWH. We therefore quantified trends in respiratory ADE incidence during vs. before the COVID-19 pandemic to assess effects of these measures on non-COVID-19 illnesses. METHODS/STUDY POPULATION: We included PWH aged ≥18 years in care at the Vanderbilt Comprehensive Care Clinic in Nashville, Tennessee from 2017-2023. Individuals contributed time from the last of March 31, 2017 or clinic enrollment until the first of death, March 31, 2023 (study close), or final clinic visit (if there was no visit ≤12 months before study close). We described respiratory ADE incidences (per 1,000 person-years) in each year of the study; we used Poisson regression with robust variance to estimate the incidence rate ratio (IRR) and 95% confidence interval (CI) for respiratory ADEs in the three years following vs. before the World Health Organization’s pandemic designation for COVID-19 (March 2020). RESULTS/ANTICIPATED RESULTS: Among 4,880 persons contributing 19,510 person-years, 69 (1.4%) developed ≥1 respiratory ADE. Median age at cohort entry was 42.6 (interquartile range [IQR]: 32.1, 52.3) years and at first respiratory ADE was 43.6 (IQR: 36.1, 51.2) years. The overall average respiratory ADE incidence in the pre-pandemic period (March 2017-March 2020) was 4.5 (95% CI: 3.3-6.3) per 1,000 person-years and during the post-pandemic period (April 2020-March 2023) was 4.1 (95% CI: 1.8-9.0) per 1,000 person-years. When accounting for repeated outcomes and annual variation, the modeled respiratory ADE incidence was 10% lower (IRR=0.9, 95% CI: 0.6-1.4) during vs. before the COVID-19 pandemic. DISCUSSION/SIGNIFICANCE: Respiratory ADE incidence dropped 10% following the COVID-19 pandemic declaration, though the confidence interval for this change contains the null. It is plausible that nonpharmaceutical COVID-19 mitigation measures drove a brief but impermanent decline, though further research is needed to assess whether diagnostic biases also played a role.

OBJECTIVES/SPECIFIC AIMS: The study aimed to determine the effects of bilateral frontal active transcranial direct current stimulation (tDCS) at 2 mA for 12 minute Versus sham stimulation on functional connectivity of the working memory network during an fMRI N-Back task. METHODS/STUDY POPULATION: Stimulation was delivered over bilateral frontal dorsolateral prefrontal cortex via and MRI-compatible tDCS device during an fMRI working memory task in healthy older adults in a within-subject design. RESULTS/ANTICIPATED RESULTS: Active stimulation compared with sham resulted in significant increases in functional connectivity in working memory related brain regions during the N-Back task. DISCUSSION/SIGNIFICANCE OF IMPACT: Older adults typically have reduced functional connectivity compared with young adults. Our findings demonstrate that a single session of tDCS can increase functional connectivity of the working memory network in older adults. Based on this mechanism of effect, tDCS may serve as an adjunctive method for interventions aiming to enhance cognitive processes in older adults.

OBJECTIVES/SPECIFIC AIMS: Obesity is a rapidly growing epidemic and long-term interventions aimed to reduce body weight are largely unsuccessful due to an increased drive to eat and a reduced metabolic rate established during weight loss. Previously, our lab demonstrated that exercise has beneficial effects on weight loss maintenance by increasing total energy expenditure above and beyond the cost of an exercise bout and reducing the drive to eat when allowed to eat ad libitum (relapse). We hypothesized that exercise’s ability to counter these obesogenic-impetuses are mediated via improvements in skeletal muscle oxidative capacity, and tested this using a mouse model with augmented oxidative capacity in skeletal muscle. METHODS/STUDY POPULATION: We recapitulated the exercise-induced improvements in oxidative capacity using FVB mice that overexpress lipoprotein lipase in skeletal muscle (mLPL). mLPL and wild type (WT) mice were put through a weight-loss-weight-regain paradigm consisting of a high fat diet challenge for 13 weeks, with a subsequent 1-week calorie-restricted medium fat diet to induce a ~15% weight loss. This newly established weight was maintained for 2 weeks and followed with a 24-hour relapse. Metabolic phenotype was characterized by indirect calorimetry during each phase. At the conclusion of the relapse day, mice were sacrificed and tissues were harvested for molecular analysis. RESULTS/ANTICIPATED RESULTS: During weight loss maintenance, mLPL mice had a higher metabolic rate (p=0.0256) that was predominantly evident in the dark cycle (p=0.0015). Furthermore, this increased metabolic rate was not due to differences in activity (p=0.2877) or resting metabolic rate (p=0.4881). During relapse, mLPL mice ingested less calories and were protected from rapid weight regain (p=0.0235), despite WT mice exhibiting higher metabolic rates during the light cycle (p=0.0421). DISCUSSION/SIGNIFICANCE OF IMPACT: These results highlight the importance of muscular oxidative capacity in preventing a depression in total energy expenditure during weight loss maintenance, and in curbing overfeeding and weight regain during a relapse. Moreover, our data suggest that the thermic effect of food is responsible for the differences in metabolic rate, because no differences were found in activity or resting metabolic rate. Additional studies are warranted to determine the molecular mechanisms driving the ability of oxidative capacity to assist with weight loss maintenance.

ABSTRACT IMPACT: My work focuses on understanding the role of an immunomodulatory pathway in Alzheimer’s disease as a potential therapeutic target. OBJECTIVES/GOALS: My long-term goal is to develop effective drugs to halt the progression of Alzheimer’s disease (AD). The overall objective of this translational work is to study the deregulation of the Lanthionine Synthetase C-like 2 (LANCL2) immunomodulatory pathway in AD. I hypothesize that stimulation of the LANCL2 pathway will inhibit progression of AD. METHODS/STUDY POPULATION: These studies use a transgenic rat model of AD, and a LANCL2-binding drug, BT-11, that crosses the BBB. Tg-AD Fisher 344 rats express human APP with the Swedish mutation and human presenilin-1 with the ? exon 9 mutation. Tg-AD rats develop age-dependent AD-like pathology. Rodent chow containing BT-11 (10mg/Kg bw) was administered orally from 5 months of age (pre-pathology) to 11 months of age. Eight groups of rats (8 rats/group) were included: WT and Tg-AD x 2 sexes x 2 conditions (BT-11 treated and untreated). Rats are analyzed for: (1) Spatial learning and memory with an active place avoidance test. (2) AD pathology including amyloid plaques and activated microglia, with hippocampal immunohistochemistry. (3) LANCL2-signaling and other AD relevant pathways, with hippocampaI western blot and RNAseq analyses. RESULTS/ANTICIPATED RESULTS: I predict that BT-11 will prevent and/or mitigate some aspects of AD pathology displayed by the AD transgenic rats (Tg-AD). Tg-AD rats treated with BT-11 should perform better in the spatial learning and memory tasks, than the untreated Tg-AD rats. I also expect that the immunohistochemical analysis will reveal reduced pathological hallmarks, including amyloid plaques and reactive microglia, in hippocampal tissue of BT-11 treated versus untreated Tg-AD rats. Through western blot and RNAseq analyses, I will establish how BT-11 treatment affects the LANCL2 signaling cascade as well as other AD relevant pathways, in treated and untreated rats from both sexes and both genotypes. DISCUSSION/SIGNIFICANCE OF FINDINGS: My proposed translational research will address the potential of targeting the LANCL2 pathway to improve AD treatment. Discovering the effects of stimulating the LANCL2 pathway on AD pathology is a novel approach in AD drug development. I expect that my studies with BT-11 will positively influence therapeutic outcomes of this devastating condition.

Objectives/Goals: This pilot study aims to assess the implementation of the DataDay app in memory clinics for patients with MCI or dementia, focusing on usability, user satisfaction, and impact on health outcomes. We seek to identify barriers and facilitators to implementation and evaluate its effect on reducing unnecessary hospital stays. Methods/Study Population: This mixed-methods study will involve 50 participants, 25 diads of patients with MCI or mild-to-moderate dementia and their caregivers from the community. Participants will use DataDay for 12 weeks, receiving reminders to log daily activities such as nutrition, mood, cognition, and physical activity. Baseline demographic data will be collected from self-reported surveys. Participants will receive training on app use, with follow-up interviews at 4, 8, and 12 weeks to gather feedback. Quantitative data analysis will include repeated measures analysis of variance to compare pre- and post-intervention outcomes, such as medication use and ER visits. Thematic analysis will be conducted on interview transcripts to understand user experiences. Results/Anticipated Results: We anticipate the study will demonstrate the feasibility of the DataDay app for self-management in individuals with MCI or dementia. Expected outcomes include improved medication adherence, reduced emergency room visits, and increased user engagement with daily health monitoring. Qualitative feedback is expected to highlight user satisfaction with the app’s reminders and ease of integration into daily routine. We also expect potential challenges to be identified such as initial learning difficulties and technology-related frustration. The data will help refine the app for better usability and inform strategies for widespread implementation in memory assessment clinics. Discussion/Significance of Impact: The study will provide insights into the practicality of implementing DataDay in memory clinics. The results will highlight necessary adjustments and provide key factors for successful adoption in other clinics. DataDay aims to allow individuals with MCI or dementia to manage their condition at home and enhance their quality of life.

OBJECTIVES/SPECIFIC AIMS: To compare methods of ascertaining prevalence for adrenal incidentalomas METHODS/STUDY POPULATION: Retrospective electronic medical record study using Looking Glass Clinical Analytics (Streamline Health, Atlanta, GA, USA) at an urban university medical center. All patients with CT or MR imaging of the abdomen between 1997 and 2014 were identified. Patients with a documented diagnosis (ICD-9 code or problem list) for any history of adrenal disease were excluded. The prevalence of adrenal incidentalomas was ascertained by 2 different detection strategies: (1) documented diagnosis of adrenal incidentaloma or (2) imaging reports containing in the same sentence “adrenal” and “nodule*,” “adenoma*,” or “mass*,” and not containing “no” and “adrenal” in the same sentence. Adrenal pathology surprise was further established in the second approach by excluding patients having previously undergone adrenal lab testing (cortisol, aldosterone, catecholamines, adrenocorticotropic hormone, renin) or having been registered in the cancer registry for any cancer excluding superficial skin cancers. RESULTS/ANTICIPATED RESULTS: In total, 194,624 individuals were identified in our initial search, from which 1056 were excluded for past adrenal disease (Table 1). Detection by the documented diagnosis method yielded 1578 cases (0.8%), compared with 13,697 cases (7.1%) by the imaging report method (Figure 1). Further restricting detection to true “Adrenal Surprise” by excluding those with any past adrenal lab testing and cancer history yielded 10,568 cases (6.1%). Validation studies for the 7.1% prevalence with 100 records revealed an adrenal incidentaloma positive predictive value (PPV) of 98%. When restricted to size ≥1 cm the PPV was 84%. DISCUSSION/SIGNIFICANCE OF IMPACT: Comparing our first strategy using documented diagnoses as criterion for incidentaloma as used in a recent paper by Lopez D (Annals of Internal Medicine 2016; 165: 533–542), we found a prevalence of 0.8% in our population similar to her 0.6%. However, when searching at the level of radiology report text, we found a prevalence ten-fold greater at 7.1%. Therefore, adrenal incidentalomas are more robustly identified by searching radiologic reports.

OBJECTIVES/SPECIFIC AIMS: Müller cells, radial glial cells of the retina, are the principal repository of xanthophyll pigment (lutein, zeaxanthin, meso-zeaxanthin), which are modifiable by diet and visible clinically by autofluorescence imaging. To understand the structural basis of xanthophyll visualization in vivo, we used 3-dimensional electron microscopic reconstruction of Müller cells surrounding one cone in a healthy human fovea. METHODS/STUDY POPULATION: From a 21-year-old male organ donor, dissected retinas were rejuvenated by oxygenated Ames medium then fixed in 4% glutaraldehyde. A tissue block 3.5 mm2 centered on the fovea was prepared for Automated Tape Ultramicrotomy (Kasthuri et al., Cell 162: 648–661, 2015). From 1462 serial 65 nm horizontal sections, an area ~250×250 μm was imaged at 6 nm xy resolution. Images were stitched and aligned. TrackEM software on a pen display was used to trace, reconstruct, and display cone #5 (of 186) and its contacting Müller cells. RESULTS/ANTICIPATED RESULTS: Cone 5 is ensheathed by 2 types of Müller cells, outer and inner (Dacey, ARVO, 2016). The outer cell is first seen at the external limiting membrane (ELM) between cones 5 and 17. Moving inward from the ELM, it tightly wraps around cone 5’s fiber in a C-shape profile for 78 µm. This Müller cell also intermittently projects to neighboring cones, 2 of which were close to cone 5 at the ELM. As cone 5’s axon approaches the pedicle, it contorts into a corkscrew. The outer cell fluidly molds to this changing shape. At this level, this Müller cell doubles in volume to encompass not only cone 5, but also cone 17 and another Müller cell. In the final 17 µm of the block the Müller cell’s volume quickly dissipates as it sends a small projection towards the internal limiting membrane, eventually encasing an OFF midget bipolar cell also associated with cone 5. In contrast to this outer cell, an inner Müller cell adjoining cone 5 spans only 19 µm, interacting directly with cone 5 and the outer cell for 3.9 µm. DISCUSSION/SIGNIFICANCE OF IMPACT: Neural-glial relationships in a human fovea are visible through 3-dimensional volume EM. The volume of Müller cells in the fovea was impressive, consistent with a pivotal role in the health of cone photoreceptors and xanthophyll homeostasis. It is possible that individual glia also ensheath the post-receptoral neurons in a cone-driven circuit, supporting the concept that xanthophylls contribute to neural efficiency in vision.

OBJECTIVES/GOALS: Our overarching aim is to examine, in an African American population, cognitive, affective, and neurophysiological processes, as well as risk-taking behavior, in response to racial stigma cues. While accounting for individual differences, we aim to see how these processes and drug-cue reactivity are impacted or altered by exposure to racial cues. METHODS/STUDY POPULATION: Participants will be African Americans between 18 and 25 years of age, equally distributed across genders. We will recruit 75 participants in order to have adequate power to conduct our intended analyses—particularly pertaining to individual differences in risk behavior outcomes. Participants will be asked to complete demographic and self-report questionnaires. Participants will also be asked to complete computerized tasks while their physiological responses (heart rate, skin conductance, and electroencephalographic (EEG) data) are recorded. The tasks are as follow: resting, gambling, go/no-go, picture viewing (positive, negative, and neutral images), and a drug cue image set. These tasks will be repeated after the participant views a racial stigma image set to evaluate the impact of discrimination. RESULTS/ANTICIPATED RESULTS: Data from 18 participants has been collected. Data will be periodically preprocessed and validated (e.g., 1 participant was removed due to data recording errors, so the current valid Nis 17). Generally, we anticipate that behaviors and neural activity will be modulated across all tasks after viewing the racial stigma image set. Specifically, (a) cognitive and affective processing of singular events of racial stigma may indicate a stress response, (b) modulation from chronic experiences of racial stigma render neural systems increasingly sensitive to stigma cues, and thereby less equipped to regulate stress response, (c) the impact of these processes on altering risk behavior (may increase such behaviors), and (d) the impact of these modulations on altering drug-cue reactivity (may amplify reactivity). DISCUSSION/SIGNIFICANCE: The study will identify factors that contribute to stress and risk behavior among African Americans. A substantial gap continues to exist regarding the nature of risk behavior among African Americans, despite the fact that African Americans represent a health disparity population with unique vulnerabilities to health-relevant risk behavior.

OBJECTIVES/SPECIFIC AIMS: Although two Ribeye protein isoforms have been identified in zebrafish, information about the identities of their variants is incomplete. This study aims to identify and characterize both of the Ribeye isoforms and their splice variants. METHODS/STUDY POPULATION: Immunohistochemistry was performed on the retina and neuromasts of zebrafish larva and adults.Ribeye expression was analyzed by western blot. Ribeye proteins will be separated, isolated and identified by mass spectrophotometry. RESULTS/ANTICIPATED RESULTS: Immunohistochemistry performed on larval and adult zebrafish retinas revealed the expression of Ribeye A in the inner and outer plexiform layers. Ribeye B was likewise expressed in both plexiform layers in larval zebrafish, but more pronounced expression in the outer plexiform layer in the adult zebrafish retina. Immunohistochemical experiments also demonstrated the co-expression of both Ribeye isoforms in the hair cells of both larval and adult neuromasts. Analysis of Ribeye expression by western blot showed the presence of more than the three previously identified variants. Current experiments are being conducted to characterize the additional Ribeye variants. We expect to identify the residual Ribeye protein as a result of this analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: This study is necessary in order to gain a clear understanding of Ribeye expression in zebrafish tissues. Doing so will enable us to target this protein for gene editing to address outstanding questions about the mechanisms that govern ribbon synapse function. Synapse and synapse-associated proteins are involved in a wide-array of diseases that arise as a result of their dysfunction (e.g. blindness, deafness, bradycardia, autism spectrum disorders, and schizophrenia). Thus, it is important for us to identify the shared and distinct mechanisms that give rise to diseases associated with synaptic dysregulation. Such information could provide the basis for novel therapeutic interventions for synaptic disorders.

OBJECTIVES/SPECIFIC AIMS: Retinitis pigmentosa (RP), also known as night blindness, is an incurable disease which affects ~1 in 4000 individuals globally. Since there are no effective treatment options for RP, the goal of this project is to identify novel drug treatments that can prevent or slow the disease progression. To this end, we optimized a behavioral assay, visual-motor-response (VMR) assay, to investigate rod function (Ganzen et al., ARVO, 2017; Ganzen et al., IJMS, 2017). This was done utilizing a transgenic zebrafish RP model expressing human rhodopsin with the Q344X mutation. In this study, we used this model to perform a proof-of-concept screen for drugs which may improve the vision of the larvae. METHODS/STUDY POPULATION: To screen for beneficial drugs, the SCREEN-WELL® REDOX library was chosen for screening. This library was selected to identify a compound that may alleviate any excessive oxidative stress in the diseased retina. The Q344X zebrafish line suffers from significant rod degeneration by 7 days postfertilization (dpf) and displayed deficits in VMR under scotopic conditions (Ganzen et al., ARVO, 2017). The Q344X larvae were drug treated beginning at 5 dpf at 10 μM. Compounds that were toxic at this concentration were retested at 1 μM. The 5 dpf stage was chosen as most of the rods are intact, and these concentrations were chosen to optimize the drug effect based on similar studies. Hits were identified by assays that provided a robust and reproducible enhancement in the Q344X VMR. The retinae of any drug hits were dissected from larvae crossed with a rod EGFP reporter line and whole-mounted to analyze rod survival via fluorescence. To determine if drug effects were exerted through the retina, eyeless chokh mutant zebrafish were exposed to the drug and tested with the same assay. RESULTS/ANTICIPATED RESULTS: Of the 84 compounds tested, we identified 1 drug that ameliorated the VMR of the Q344X scotopic VMR. Eyeless chokh mutant zebrafish larvae did not exhibit the same VMR when treated with the same drug. Histological analysis suggested increased rod survival in the drug-treated retina of Q344X mutants. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate that the vision of the Q344X zebrafish was improved via this beneficial drug treatment. Since eyeless chokh larvae did not respond to the same treatment, the drug likely mediated its positive effects through the Q344X retina, likely by improving rod survival. Together, our results have identified a beneficial drug that may treat RP.

OBJECTIVES/GOALS: All IN for Health has initiated social media engagement strategies to create bi-directional communication around improved health and research literacy with the public. In addition, the All IN for Healths community-led advisory board, comprised of diverse individuals and organizations, will provide insight on research design and dissemination. METHODS/STUDY POPULATION: The Indiana Clinical and Translational Sciences Institute (CTSI) has committed to enhancing the publics engagement efforts in the All IN for Health program. This program was established in 2017 to improve the health of Indiana residents through the promotion of health resources and opportunities to participate in research. In 2021, the Indiana CTSI decided to integrate public engagement more formally. An official collaboration with the Indiana CTSIs Community Health Partnerships program was created, a digital engagement specialist was hired, and several new community and population health advisory board members were welcomed for quarterly meetings representing the rural and urban areas of the state, faith-based communities, health departments, and underrepresented groups in research. RESULTS/ANTICIPATED RESULTS: A social media campaign, informed by the All IN for Health advisory board was launched in November 2021, and the advisory board has been meeting on a quarterly basis. All IN for Healths established priority area is currently COVID-19 vaccination uptake, and our prioritized audiences are underserved and under resourced in urban and rural communities, and minoritized populations. All IN for Health has posted approximately 20 times per month on topics including COVID-19, Alzeheimers Disease, Diabetes, fitness, mental health, and research studies with a goal to increase followers and engagements by 20%. The hope is that this engagement will also result in an increase in the monthly e-newsletter subscribers and study volunteer registry participants. DISCUSSION/SIGNIFICANCE: All IN for Health has initiated new public engagement efforts to increase the value of the services offered to Indiana residents. Through consultation and co-design with members of the public and dissemination of research opportunities All IN for Health will be better suited to improve health and research literacy.

OBJECTIVES/GOALS: Chronic kidney disease (CKD) affects nearly 40% of adults with diabetes. Our objective is to assess trends in risk factor control and use of 2022 ADA and KDIGO guideline-recommended medications. METHODS/STUDY POPULATION: Using data from 1988 to 2020 from the National Health and Nutrition Examination Survey, we will examine trends in sociodemographic risk factors and glycemic, blood pressure, and lipid control among adults with CKD and diabetes. Glycemic control will be defined as a hemoglobin A1c (HbA1c)<7%, blood pressure control will be examined at cutoffs of 130/80 and 120/80 mmHg, and lipid control will be defined as a fasting triglyceride level ≥150 mg/dL, a low-density lipoprotein (LDL) <100 mg/dL without atherosclerotic cardiovascular disease (ASCVD), or LDL<70 mg/dL if they have ASCVD. We will assess changes in the use of commonly used antidiabetic, antihypertensive, and lipid-lowering medications. RESULTS/ANTICIPATED RESULTS: We hypothesize that from 1988-2020, blood pressure control has improved while glycemic and lipid control has not improved among adults with diabetes and CKD. We expect decreases in sulfonylurea use and increases in DPP-4 inhibitor, metformin, ACE inhibitor, angiotensin receptor blocker, statin, and insulin use over time among those with diabetes and CKD. In addition, there is likely a significant gap between those who are eligible to use newer medications like SGLT2 inhibitors or GLP-1 receptor agonists and who are currently using them within this subpopulation. DISCUSSION/SIGNIFICANCE: This study will examine adherence to guideline-recommended management and identify gaps in care for adults with CKD and diabetes, which may inform how best to optimize medication use for cardiorenal protection in this high-risk patient population.

OBJECTIVES/GOALS: We recently identified a CA-MRSA strain in Brooklyn, New York (USA300-BKV) causing an outbreak of severe skin infections in predominantly healthy children. The evolution of USA300-BKV included acquisition of a novel prophage, and our objective is to identify the prophage-encoded gene(s) and mechanism responsible for increased bacterial virulence. METHODS/STUDY POPULATION: We deleted candidate genes from a novel mosaic block of phage-encoded genes in USA300-BKV that have been shown to enhance virulence in a murine skin infection model. Deletion mutants and complemented clones will be evaluated in vivo to identify culprit genes and determine the effect of lineage-specific genetic variation on the phenotype. Complementary studies include a comprehensive characterization of phage and bacterial genes expressed during lysogeny in vitro using RNA sequencing (RNA-Seq), and in vivo using a targeted approach focusing on known bacterial virulence and phage lytic pathways as well as candidate genes identified by in vitro studies. RESULTS/ANTICIPATED RESULTS: Comparison of otherwise isogenic lab strains showed that the mosaic block of phage genes present in USA300-BKV enhance skin abscess size in mice, confirming previous results. As this region of the phage, named mΦ11, does not contain known toxin genes, we hypothesize that mΦ11 modulates expression of bacterial host genes to enhance virulence. Thus, transcriptional profiles of CA-MRSA containing mΦ11 and selected deletion mutants are expected to reveal changes in known or novel virulence factors compared to controls. Candidate regulators specific to the mosaic block include an adenine methyltransferase linked to changes in global gene expression of other bacterial species. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results will broaden scientific understanding of phage-bacterial interactions and determine the mechanisms by which phage impact virulence independent from toxin gene carriage. Identification of phage-encoded gene(s) enhancing CA-MRSA contagion will inform surveillance efforts and identify novel therapeutic targets.

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/SPECIFIC AIMS: 1) Determine the mutational landscape, including translocation, mutations and mutational signatures as well as copy number variations of pPCL and identify significant differences to non pPCL MM. 2) Determine whether genetic changes pertinent to pPCL could be explored as therapeutic targets to improve the dismal prognosis of this patient population. METHODS/STUDY POPULATION: Samples from overall 19 pPCL patients that presented to the Myeloma Center, UAMS between 2000-2018 were used for this study. We performed gene expression profiling (GEP; Affymetrix U133 Plus 2.0) of matched circulating peripheral PCs and bone marrow (BM) PCs from 13 patients. Whole exome sequencing (WES) was performed on purified CD138+ PCs from BM aspirates from 19 pPCL patients with a median depth of 61x. CD34+ sorted cells, taken at the time of stem cell harvest from the same 19 patients, were used as controls. Translocations and mutations were called using Manta and Strelka and annotated as previously reported. Copy number was determined by Sequenza. RESULTS/ANTICIPATED RESULTS: 1) GEP from the BM and circulating peripheral PCs showed that the expression patterns of the two samples from each individual clustered together, indicating that circulating PCs and BM PCs in pPCL result from the same clone and are biologically clearly related. 2) The clinical characteristics from the patient cohort used for WES analysis were as follows: median age was 58 years (range 36–77), females accounted for 74% (14/19), an elevated creatinine level was found in 78% (14/18) and an elevated LDH level in 71% (10/14). All patients presented with an ISS stage of III. Median OS of the whole dataset was poor at 22 months, which is consistent with OS from previously reported pPCL cohorts. 3) Primary Immunoglobulin translocations were common and identified in 63% (12/19) of patients, including MAF translocations, which are known to carry high risk in 42% (8/19) of patients [t(14;16), 32% and t(14;20), 10%] followed by t(11;14) (16%) and t(4;14) (10%). Furthermore, 32% (6/19) of patients had at least one MYC translocation, which are known to play a crucial role in disease progression. 4) The mutational burden of pPCL consisted of a median of 98 non-silent mutations per sample, suggesting that the mutational landscape of pPCL is highly complex and harbors more coding mutations than non-pPCL MM. 5) Driver mutations, that previously have been described in non-pPCL MM showed a different prevalence and distribution in pPCL, including KRAS and TP53 with 47% (9/19) and 37% (7/19) affected patients respectively compared to 21% and 5% in non-PCL MM. PIK3CA (5%), PRDM1 (10%), EP300 (10%) and NF1 (10%) were also enriched in the pPCL group compared to previously reported cases in non-pPCL MM. 6) Biallelic inactivation of TP53 – a feature of Double Hit myeloma - was found in 6/19 (32%) samples, indicating a predominance of high risk genomic features compared to non-pPCL MM. Furthermore, analysis of mutational signatures in pPCL showed that aberrant APOBEC activity was highly prevalent only in patients with a MAF translocation, but not in other translocation groups. DISCUSSION/SIGNIFICANCE OF IMPACT: In conclusion we present one of the first WES datasets on pPCL with the largest patient cohort reported to date and show that pPCL is a highly complex disease. The aggressive disease behavior can, at least in part, be explained by a high prevalence of MAF and MYC translocations, TP53 and KRAS mutations as well as bi-allelic inactivation of TP53. It is of interest that only KRAS but not NRAS mutations are highly enriched in pPCL. From all highly prevalent genomic alterations in pPCL, only KRAS mutations offer a potential for already available therapeutically targeting with MEK inhibitors, which should be further explored.

OBJECTIVES/SPECIFIC AIMS: The overall goal of this project is to understand the genetic and clinical differences in POAG that specifically increase risk in individuals of African genetic ancestry. We will approach this goal by completing the following objectives: (i) localize a genetic signal that accounts for the significantly increased risk for primary open-angle glaucoma in African Americans and (ii) utilize electronic health records (EHR) data to expand our understanding of risk to incorporate endophenotypes of glaucoma and other clinically recorded variables that may influence disease risk. METHODS/STUDY POPULATION: We will genotype at least 200 available African American samples with glaucoma on the Illumina Infinium® Expanded Multi-Ethnic Genotyping Array (MEGAEX) and perform admixture mapping. We will then access EHR data to expand our analysis beyond glaucoma to encompass other relevant risk modifiers captured in the clinical record. RESULTS/ANTICIPATED RESULTS: We anticipate localizing a genetic signal or signals that may account for the increased POAG risk in African Americans. Our calculations indicate that we have ~81% power to detect association at a LOD score of 2 and a risk ratio of 2. Thus, we are well-powered to detect a true signal at this modest level of association DISCUSSION/SIGNIFICANCE OF IMPACT: This project will not only help to achieve precision medicine by filling in the gaps in knowledge regarding glaucoma in African Americans, but it will also address health disparities and aid in the realization of the full potential of “big data” so that all of these elements can be incorporated into a better understanding of health disparities.

OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and commonprimary tumor type in the central nervous system across all age groups; affectedadults have a life expectancy of just 14 months. As glioma cells invade thesurrounding normal parenchyma they remodel the composition and ultrastructure ofthe surrounding extracellular matrix (ECM), suggesting that the native (i.e.,“normal”) microenvironment is not ideal for their survivaland proliferation. Recent reports describe suppressive and/or lethaleffects of mammalian ECM hydrogels derived from normal (nonneoplastic) sourcesupon various cancer types. ECM-based bioscaffolds placed at sites of neoplastictissue resection in humans have never been reported to facilitate cancerrecurrence. The objective of the present research is to evaluate mammalian ECMas a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECMhydrogels from porcine dermis, small intestine, and urinary bladder wereproduced as described previously. Primary glioma cells were graciously suppliedby Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered throughATCC. Cells were plated onto tissue culture plastic at~60% confluence and allowed to attach for 24 hours beforetreatment. The saline-soluble fraction (SSF) of ECM was obtained by mixinglyophilized, comminuted ECM with 0.9% saline for 24 hours thenfiltering the resulting mixture through a 10 kDa molecular weight cutoff column.All assays and kits were followed according to the manufacturer’sinstructions. Cell viability was measured via MTT assay(Vybrant® MTT Cell Proliferation Assay, Invitrogen)and by live/dead staining(LIVE/DEAD® Cell Imaging Kit, Invitrogen). Timelapse videos were created by taking images every 20 minutes for 18 hours(phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagentwas ordered from Biotium. Temozolomide was ordered through Abmole. All in vivowork was conducted according to protocols approved by the University ofPittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS:ECM hydrogels derived from porcine dermis, small intestine, or urinary bladderall decreased the viability of primary glioma cells in vitro, with urinarybladder extracellular matrix (UBM) having the most dramatic effects. The SSF ofUBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, wassufficient to recapitulate this detrimental effect upon neoplastic cells invitro and was used for the remainder of the experiments described herein. In acell viability assay normalized to the media treatment, non-neoplastic CHME5 andN1E-115 cells scored 103% and 114% after 48 hours whentreated with UBM-SSF and 2 primary high-grade glioma cell types scored17% and 30.5% with UBM-SSF (n=2).Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence ofUBM-SSF for 18 hours while most primary glioma cells shriveled and died withinthis time. Darkfield time-lapse video of wells containing Nucview dye,fluorescent upon cleavage by active caspase-3, confirmed that within 12 hoursmost primary glioma cells underwent apoptosis while CHME5 and HFF did not. Inculture with primary astrocytes, high grade primary glioma cells, and U-87 MGglioma cells for 24 hours, UBM-SSF was found to significantly increase thepopulation of primary astrocytes compared with media(p<0.05) while decreasing the 2 glioma cell types toapproximately one-third as many cells as the media control(p<0.0001). A dose-response of temozolomide from 0to 10,000 μM showed that when treating 2 non-neoplastic cell types(CHME5 and HFF) and 2 types of primary glioma cell there was no difference insurvivability at any concentration. Contrasted to this, a dose-response ofUBM-SSF from 350 to 7000 μg/mL showed that thenon-neoplastic cells survived significantly better than the glioma cells atconcentrations of 875 μg/mL and upward(p<0.05). In preliminary animal experiments, largeprimary glioma tumors in the flanks of athymic nude mice were resected andreplaced with either UBM SSF or Matrigel (an ECM product of neoplastic cellorigin). After 7 days the resection sites with UBM-SSF had little tumor regrowthif any compared with the dramatic recurrence seen in the Matrigel injectionsites (n=2). In a separate survival study comparing PBS to UBM-SSFinjections in the flank-resection model, all animals given PBS had to besacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSFwere sacrificed at 15, 24, and 39 days (n=3), indicating a moderateincrease in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OFIMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZin 2005, the standard of care for patients with glioblastoma multiforme has notimproved. These findings indicate that non-neoplastic ECM contains potentbioactive regulators capable of abrogating malignancy. Our in vitro data suggestthese molecules appear to have no deleterious effect on non-neoplastic cellswhile specifically inducing apoptosis in glioma cells. Our in vivo data suggestthat these molecules may be useful in delaying glioma recurrence, thus resultingin extended lifespan. Delivering soluble fractions of ECM to a tumor site mayrepresent a novel approach to glioma therapy, sidestepping traditional cytotoxictherapies in favor of utilizing putative endogenous anti-tumor pathways.

Persistent barriers exist to engage rural providers in research and training. Provider shortages exacerbate these challenges, leading to a scarcity of time and limiting motivation to participate in research. We present application of an innovative engagement model to increase rural primary care provider participation in research. Using our Community Engagement and Research Core, we demonstrate that fundamental principles of training and expertise, attention to efficiency and multitasking, and commitment to community are important for addressing provider recruitment barriers. We encourage other Clinical and Translational Science Centers to provide similar services to their local investigators to enhance provider engagement in research.

A clinical and translational scientist (CTS) often seeks to increase their knowledge of statistical topics to effectively conduct biomedical research studies. A common method for obtaining this knowledge is through existing online educational materials that are suggested by a biostatistical collaborator or identified by the CTS. However, the volume of available educational materials on diverse statistical topics makes the task of identifying high-quality educational resources at an appropriate level challenging and time consuming for CTSs and collaborative biostatisticians. In response to these challenges, the Biostats4You website was created, where existing online educational materials for a variety of statistical topics are vetted to identify those most appropriate for CTSs. In this manuscript, we describe the resource review process, provide information about statistical topics and resources currently available, and make recommendations for how CTSs and collaborative biostatisticians can utilize the Biostats4You website to improve training, mentoring, and collaborative research practices.