OBJECTIVES/SPECIFIC AIMS: This study investigates the process configuration parameters involved in targeted drug delivery to the avian respiratory system. Previously, direct intratracheal aerosol delivery in an avian model using a commercial atomizer was found to result in delivery of a high portion of the total dose into one lung lobe. We hypothesize that controlling process configuration will decrease the asymmetric distribution. METHODS/STUDY POPULATION: A 3D printed model of an avian trachea and mainstream bronchi was constructed to create a representative model for direct instillation of aerosols. Construction of the model respiratory tract included the trachea and the first mainstream bronchi bifurcation to measure left/right (L/R) distribution of aerosol delivered. Both liquid aerosol delivery (LAD) using a commercial atomizer and dry aerosol delivery (DAD) using a custom-built dry powder insufflator device were tested. Two experimental variables were controlled: (1) retraction distance from the carina and (2) centering of device shaft in the lumen of the trachea. Measurement of device efficiency (dose delivered to the 3D model at as fraction of total dose), aerosol delivery efficiency (dose captured at L/R bifurcations as a fraction of total dose), and aerosol lateralization (L/R) was conducted. RESULTS/ANTICIPATED RESULTS: The aerosol delivery efficiency for both LAD and DAD devices [73.9% (95% CI: 68.2–79.2) and 73.4% (95% CI: 55.5–91.3), respectively] did not have an appreciable difference. However, the LAD device had a higher efficiency as compared with the DAD device. The L/R distribution for the DAD device was found to be highly dependent on both retraction distance and shaft centering. Appreciable improvement in the L/R distribution was seen using the DAD device by increasing the retraction distance distal to the carina. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of targeted drug delivery to treat pulmonary pathogens requires a careful design, manufacture, and therapeutic positioning of devices. In particular, clinically relevant animal models and treatment regimes requires a sound understanding of the physical processes controlling aerosol distribution in the respiratory system. By using a simulated respiratory model, many of the physical parameters of drug delivery can be tested before using a live animal model. This is especially important from an animal welfare perspective as well as an animal subject availability aspect.

OBJECTIVES/SPECIFIC AIMS: Costs associated with the treatment of skin diseases accounted for greater than 4% of total US healthcare spending in 2013, an increase of $46 billion (170%) since 2004. Considering the increase in novel treatments and spending, cost-utility analyses (CUAs) may provide a better understanding of costs in dermatology. In this study, we conduct a systematic overview of study quality among CUAs related to dermatology. METHODS/STUDY POPULATION: We queried studies from the Tufts Medical Center Cost-Effectiveness Analysis Registry (www.cearegistry.org), a database supplying information on all peer-reviewed cost-effectiveness analysis through 2014. Database methodology was previously discussed here. We queried studies using keywords from the 24 major skin disease categories (e.g., diseases relating to actinic damage were searched by using “actinic,” “actinic keratosis”). We collected data on study design, reporting methods, and analyzed relevant data stratified by 2 time-periods (1976–2008 and 2009–2014) chosen to encompass a comparable number of studies. RESULTS/ANTICIPATED RESULTS: In total, 42 and 50 studies corresponding to the 2 time-periods were retrieved (representing 14/24 disease categories). Based on the recommended data reporting guidelines for CUAs, study quality remained largely unchanged across the 2 phases. Across the 2 time-periods, a societal perspective was used in 19% and 12% of studies, costs and (quality adjusted life-years) QALYs were discounted in 67% and 72% of studies, a correct (incremental cost-effectiveness ratio) ICER was reported in 67% and 72% of studies, and a sensitivity analysis was included in 88% and 84% of studies, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest the quality of dermatology-related CUAs, as evaluated by recommended data reporting guidelines, to be generally stable during the analyzed time-periods. However, the quality of our results may be limited by the small number of CUAs within dermatology (10/24 disease categories did not have CUAs across any time-period). Moving forward, we encourage researchers within dermatology to pursue additional investigation towards cost-effective practices while adhering closely to recommended quality reporting guidelines for CUAs.

OBJECTIVES/GOALS: Flavored tobacco sales restrictions (FTSRs) are implemented to reduce access to flavored tobacco products. We examined the association between seven local FTSRs implemented in 2018/2019 and e-cigarette use among high school students in the Bay Area region of California. METHODS/STUDY POPULATION: We analyzed data from the California Healthy Kids Survey using a difference-in-differences (D-I-D) strategy. We compared pre- and post-policy changes one year after implementation in current tobacco use (e-cigarettes and cigarettes) among students exposed (n=20,832) versus unexposed (n=66,126) to a FTSR. Exposed students attended school in a city with a FTSR. Other outcomes included ever use of e-cigarettes, ever marijuana use in an e-cigarette, and ease of access to e-cigarettes. RESULTS/ANTICIPATED RESULTS: Pre- to post-policy, current tobacco use did not change in exposed students (e-cigarette: 10.5% to 11.1%; cigarette 2.6% to 2.5%) and decreased in unexposed students (e-cigarette: 12.8% to 11.4%; cigarette: 2.2% to 1.7%). FTSRs were not associated with a change in odds of current e-cigarette (adjusted D-I-D OR: 1.25, 95% CI: 0.95, 1.65) or cigarette use (adjusted D-I-D OR: 1.24, 95% CI: 0.94, 1.63), relative to unexposed students. For both exposed and unexposed groups, there was a 54-57% increased odds of reporting ease of access to e-cigarettes and a 29-35% increased odds of ever using marijuana in an e-cigarette. No change was detected for ever e-cigarette use. DISCUSSION/SIGNIFICANCE: Local FTSRs in California were not associated with a decrease in e-cigarette or cigarette use one-year post-implementation. Increased ease of access and marijuana use may be explanatory factors.

OBJECTIVES/GOALS: Study the regulatory role of sodium glucose co-transporter 1 (SGLT1) in cardiomyocytes and the therapeutic potential of sotagliflozin in hypertrophic cardiomyopathy (HCM) by (a) quantifying SGLT1 expression in HCM and (b) examining the impact of sotagliflozin on cardiac mechanics. METHODS/STUDY POPULATION: * Use Western Blot in cardiac tissue from HCM and non-HCM patients and pre-existing RNA seq and proteomics datasets to quantify SGLT1 levels in HCM. Hypothesis: SGLT1 is upregulated in HCM * Determine how SGLT1/2 inhibition by sotagliflozin will affect cardiac mechanics using living myocardial slice (LMS) preparations. A vibratome creates 200um-thick slices from (a) failing HCM heart explants, (b) septal myectomy samples from HCM patients, and (c) nonfailing rejected donor hearts. LMS are mounted on a force transducer and work-loops are stimulated under varying pre- and after-loads. Collecting baseline and post-drug work loops allows each slice to function as its own control. Hypothesis: sotagliflozin will improve diastolic mechanics by reducing stiffness in the end-diastolic pressure-volume relationship RESULTS/ANTICIPATED RESULTS: * Preliminary results from RNA seq data indicate that SLC5A1 mRNA (encoding gene for SGLT1) is significantly decreased in HCM. No proteomics study examined thus far has detected SLC5A1, indicating that overall SGLT1 levels in cardiac tissue are quite low. We will examine SGLT1 levels in our own HCM and non-HCM tissue samples with both mass-spectrometry and Western Blot. * We analyze six slices from each heart and expect 15 donor hearts and 15 HCM hearts/myectomy samples. We visualize the work loop by plotting stress/strain. Stress/strain at mitral valve closure represents exponential end diastolic pressure-volume relationship; Stress/strain at aortic valve closure represents linear end systolic pressure volume relationship. A two-sample paired t-test will compare change in stiffness and elastance. DISCUSSION/SIGNIFICANCE: This project contributes to a growing body of research surrounding the currently unknown cardioprotective mechanism of SGLT 1/2 inhibitors, furthers the technique of using living myocardial slices to study cardiac mechanics, and supports a trial examining sotagliflozin in HCM, for which disease modifying therapy remains a prevailing unmet need.

Objectives/Goals: The impact of the program on alumni students was measured in a 2023 survey, which assessed key factors and student perspectives on motivation to apply/remain in the program, their engagement activities, how they used the skills acquired in RAP upon graduating, and if they were currently serving in a health profession and/or clinical research. Methods/Study Population: Survey questions were based upon specific components of CTSI-RAP that make it unique. Covered topics related to motivation for participation, meaningful experiences, program effectiveness, future use of RAP knowledge/training, and current career roles in the health professions and/or clinical research. The survey was built and analyzed in REDCap and deployed May–July 2023. The study received exempt certification from the IRB. The survey was sent to 123 alumni from the 2013 to 2021 cohorts. Bounced e-mails were followed up on and two reminder e-mails were sent to initial non-responders. Identifiable demographic information was separated from program evaluation questions for analysis. A subanalysis was performed to determine program impact on students who identified as underserved or disadvantaged. Results/Anticipated Results: Respondents included 82/123 (66.7%) alumni. The survey took approximately 15 minutes. Most of the students 64/82 (78.0%) had 1 year or less research experience. The top three motivating factors for joining the RAP program were gaining clinical research experience, exposure to healthcare settings, and interest in pursuing a healthcare related career. Most alumni rated the overall effectiveness of the RAP program as very or somewhat valuable and the majority felt that the program ranked high or very high among their undergraduate experiences. The program was very influential or influential in defining their long-term plans and goals. Just under half felt that their career aspirations were changed or influenced by the program, which was especially true for those who identified as underserved/disadvantaged. Discussion/Significance of Impact: CTSI-RAP alumni highly value their experience in the program. They have benefitted professionally and are motivated to keep their connection to the program alive. With a decade of clinical research excellence and programming, CTSI-RAP’s impact is well established as a proven model benefiting both students and the clinical research infrastructure.

OBJECTIVES/SPECIFIC AIMS: To develop a robust spatial normalization pipeline for brains of individuals with focal cortical lesions. METHODS/STUDY POPULATION: Individuals with chronic focal cortical lesions from stroke. RESULTS/ANTICIPATED RESULTS: We have developed a robust spatial normalization pipeline for brains of individuals with focal cortical lesions, and demonstrate how the pipeline overcomes obfuscated neuroanatomy to yield both consistent and excellent results. DISCUSSION/SIGNIFICANCE OF IMPACT: Our robust normalization pipeline will enable group analyses of individuals with cortical lesions with greater spatial precision. This greater spatial precision will improve answers to questions about functional localization in the brain, and ultimately allow translation of findings from neuroimaging studies in individuals with cortical lesions to the clinic.

ABSTRACT IMPACT: Demonstrate the role of astrocyte released MMPs in response to pathogenic HIV protein Tat. OBJECTIVES/GOALS: In the presence of the pathogenic HIV protein Tat, astrocytes have been demonstrated to adopt an inflammatory phenotype as well as release extracellular matrix degrading enzymes, MMPs. Our work aims to identify whether MMPs alter perineuronal net integrity and working memory in a mouse model of Tat-induced neuroinflammation. METHODS/STUDY POPULATION: Stereotaxic Injection: C57BL6/J mice were injected bilaterally with HIV-1 IIIB Tat 5ug in 5uL or Vehicle (0.2M KCl, 5mM DTT, 50mM Tris, pH 8.0), into the hippocampus (CA1; -1.9mm AP, ±1.6mm ML, -1.5mm DV from pial surface). All outcome measurements were performed 14-days post injection. Behavior: T-maze was used to assess working memory following Tat exposure. qRT-PCR: TaqMan probes were used according to manufacturer on extracted whole hippocampus mRNA. IF: GFAP and CD68 immunofluorescence was used to determine inflammation post injection. Inhibitory interneurons (parvalbumin positive) and peri-neuronal nets (WFA positive) were quantified. WB: Synaptosomes from whole hippocampi (Syn-PER) were isolated and synaptic excitatory markers were quantified (PSD-95, synaptophysin, GluR2a). RESULTS/ANTICIPATED RESULTS: Tat exposure resulted in impairments in working memory as measured by T-maze alternations and an increase in hippocampal mRNA expression of MMP-13 and IL-1β, indicative of neuroinflammation. We also noted an increase in GFAP+ injection site width 14 days post-Tat injection, suggesting robust gliosis. While there were no changes in the excitatory pre and post synaptic markers we found a significant decrease in the percent of PV+ interneurons with peri-neuronal nets (PNNs) following Tat exposure. Taken together, this preliminary data supports a role for inflammation and PNN integrity in Tat-induced alterations in working memory. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings suggest that Tat contributes to cognitive impairment and that astrogliosis with elevated MMP-13 facilitates the degradation of peri-neuronal nets (PNNs) within the hippocampus. Since PNN degradation can alter neuronal circuitry future studies will focus on Tat-induced changes in hippocampal signaling.

OBJECTIVES/SPECIFIC AIMS: A major limitation of cardiac stem cell transplantation following myocardial infarction (MI) is poor retention of cells in the ischemic microenvironment. Our study aims to better understand and promote the survival and differentiation of human cardiosphere-derived cells (hCDCs) in anoxia, a feature of infarcted myocardium. METHODS/STUDY POPULATION: We previously demonstrated that TGFβ1 and heparin-containing hydrogels (TH-hydrogel) can promote murine CDC survival. In this study, hCDCs were incubated in either normoxia or anoxia for 8 hours with and without TH-hydrogel. In addition, hCDCs without TH-hydrogel were assessed in 16 hours of anoxia. Following incubation, hCDCs were assayed for viability using calcein dye and immunostained for CD31, a marker of endothelial differentiation. RESULTS/ANTICIPATED RESULTS: hCDCs incubated for 8 hours in anoxia in both models equally demonstrated increased survival up to 30% when compared with cells incubated in normoxia. However, in contrast to hCDCs alone, hCDCs with TH-hydrogel additionally demonstrated increased differentiation into endothelial cells in both anoxia and normoxia. We found that hCDCs alone were able to upregulate CD31 only when subjected to 16 hours of anoxia. DISCUSSION/SIGNIFICANCE OF IMPACT: We demonstrate a new, previously unknown response of hCDCs to anoxia. This induces increased viability and differentiation of hCDCs into endothelial cells. The differentiation in anoxia was time dependent and could be expedited with use of TH-hydrogel. Anoxic preconditioning of hCDCs together with the TH-hydrogel system may improve the therapeutic potential of stem cell transplantation following MI.

ABSTRACT IMPACT: The first-line chemotherapies used to treat leishmaniasis are highly toxic intravenous antimonials yet drug resistance has begun to develop, causing the use of oral treatment options with high price tags; there is a strong need for new, safe, and effective chemotherapeutic agents to treat leishmaniasis. OBJECTIVES/GOALS: This study was conducted in order to identify novel chemical compounds that exhibit anti-leishmanial activity and to further characterize their efficacy and toxicity in in vitro and in vivo systems in the hopes of future chemotherapeutic developments. METHODS/STUDY POPULATION: A total of 28 unique 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP) compounds were synthesized and anti-leishmanial efficacy and host cell toxicity were determined using L. donovani mCherry-expressing amastigotes and THP-1 macrophages. Additional pharmacokinetic analyses of a potent 1,4-DAPP compound were conducted, revealing a potential metabolite structure. A select group of the novel compounds were screened in a cutaneous leishmaniasis (CL) murine model using L. major mCherry-expressing parasites and female Balb/C mice. The treatment consisted of 10 intralesional injections of compound over a period of 4 weeks, while lesion growth was monitored via fluorescence and manual measurements. RESULTS/ANTICIPATED RESULTS: Four experimental compounds had IC50 values less than 5 micromolar, providing similar anti-leishmanial activity to Miltefosine. Compound 9279817 had a clearance almost twice the rate of normal hepatic blood flow and had a relatively high volumes of distribution, indicating this compound is rapidly cleared and distributes into tissues. In vitro rat liver microsome assays suggest a rapid metabolism of 9279817 and MS/MS results suggest this metabolite is most likely formed via oxidation of the sulfur on the lower aryl ring. This sulfoxide metabolite has similar efficacy as the parent compound and does not exhibit toxicity in vitro. Three of the experimental compounds behaved similarly to the antimony positive control in the murine CL model. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study revealed a novel structural class of compounds that have anti-leishmanial activity. Experiments show compounds with similar efficacy to Miltefosine while having significantly less cytotoxicity, suggesting that the 1,4-DAPP structural class could be further developed as a potential chemotherapeutic.

OBJECTIVES/GOALS: The preclinical stage of Alzheimer disease (AD) is a clinically silent period that can be detected through neuroimaging and biofluid biomarkers. The goal of this study was to determine whether performance of complex daily tasks is associated with plasma biomarkers of brain amyloidosis or neuroaxonal injury in cognitively normal (CN) older adults. METHODS/STUDY POPULATION: This is a cross-sectional analysis of an ongoing longitudinal cohort study. CN older adults performed three complex daily tasks (shopping, checkbook balancing, medication management) from the Performance Assessment of Self-Care Skills in their home. Tasks were scored for independence, with more assistance required indicating worse performance. Participants had a plasma sample obtained within two years of completing the tasks. Plasma amyloid (Aβ42 and Aβ40) were evaluated by high precision immunoprecipitation mass spectrometry assays and neurofilament light (NfL) was measured with single molecule array (Simoa) assays. Nonparametric partial correlations were used to quantify the associations between task performance and plasma AD biomarkers, controlling for age and gender. RESULTS/ANTICIPATED RESULTS: 105 CN participants (mean age 74.7 years, 55% female, 88% white) were included. After controlling for age and gender, worse performance of complex daily tasks (more assistance required) was associated with increased plasma NfL (Spearman’s: 0.23, p=0.04) but not plasma Aβ42/Aβ40. DISCUSSION/SIGNIFICANCE: This study suggests that worse performance of complex daily tasks in CN older adults may be associated with increased plasma NfL a marker of neuroaxonal injury, but not with plasma amyloid. These findings could lead to a better understanding of clinical changes that may occur prior to the onset of noticeable memory symptoms in AD or related dementias.

OBJECTIVES/GOALS: Cadmium is a widespread neurotoxic metal pollutant; however prior study results of Cd and later-life cognition are mixed. We investigated association of urinary cadmium on Alzheimer’s Disease (AD) mortality risk, accounting for key co-pollutants smoking and lead, in the presence of competing risks. METHODS/STUDY POPULATION: We included 5692 persons, 60 years old from the 1998-2018 National Health and Nutrition Examination Survey. Underlying cause of death was determined via linked 1999-2019 National Death Index data. Urinary cadmium (UCD) reflects prolonged exposure and was adjusted for creatinine. We used multiple imputation (5 iterations) to recapture substantial model observation drop-out (N=782). We used three Cox proportional hazard models to estimate hazard ratios (HR) and 95% confidence interval (CI) per unit increase in UCD and time to AD mortality: a competing risks model, a survey-weighted model, and a baseline model including neither, all adjusted for demographic characteristics, lead, and smoking. RESULTS/ANTICIPATED RESULTS: Follow-up ranged from 0 to 20.8 years (mean 8.2 years), with a total of 1,987 individuals deceased (14,232 person-years at-risk), including 88 individuals dying from AD. Mean UCD was 0.50 μg/g creatinine (standard error=0.01). In baseline and survey-weighted models fully adjusted for NHANES cycle, poverty income ratio, age, race/ethnicity, sex, marital status, education, smoking status, and blood lead levels, a per unit increase in UCD was associated with approximately twice the rate of AD mortality (baseline model HR: 1.92, 95% CI: 1.28, 2.89; survey-weighted model HR: 2.08, 95% CI: 1.42, 3.06). In the competing risks model, this association was attenuated (HR: 1.58, 95% CI 1.06 2.36). DISCUSSION/SIGNIFICANCE: Our study finds chronic cadmium exposure associated with AD mortality even after accounting for competing risks of AD mortality and confounding effects of cigarette smoking and lead exposure, strengthening the evidence that long-term cadmium exposure adversely affects later-life cognitive health.

OBJECTIVES/GOALS: The spectrum of disease caused by SARS-CoV-2 ranges from asymptomatic detection to severe illness, with varying presentations by age. Therefore, we aimed to compare the clinical characteristics between children and adults with SARS-CoV-2. METHODS/STUDY POPULATION: From March 20, 2020, to August 18, 2021, we conducted SARS-CoV-2 surveillance in individuals from metropolitan Nashville, TN. Children with multisystem inflammatory syndrome were excluded. Analyses were restricted to individuals with SARS-CoV-2 infection confirmed by detection of viral RNA in nasal specimens using reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and/or by detection of serum IgG to the SARS-CoV-2 spike and nucleocapsid proteins using enzyme-linked immunosorbent assay (ELISA). Those with negative RT-qPCR results, but a positive ELISA within 4-6 weeks of symptom onset, were classified as SARS-CoV-2 positive. Clinical characteristics between children and adults were compared with Pearson’s chi square. Illness duration was compared using Kaplan Meier estimators. RESULTS/ANTICIPATED RESULTS: Overall, 426/826 (49%) individuals (children: 57 [13%); adults: 369 [87%]) were SARS-CoV-2 positive, with median ages of 12 and 41 years, respectively. Most individuals were female (54%) and white, non-Hispanic (79%). Compared to adults, children were more likely to be asymptomatic (children: 16% vs. adults: 5%; p=0.001). In contrast, symptomatic adults were more likely to report cough (71% vs. 56%), wheezing (21% vs. 8%), shortness of breath (45% vs. 19%), ageusia (67% vs. 23%), and anosmia (64% vs 27%) than symptomatic children (p<0.05). Mean illness duration was shorter in children than adults: 7 days (95% CI: 5.1, 8.9) vs. 14 days (95% CI: 12.4,15.0), respectively. A total of 5% (18/352) of adults reported symptoms lasting ? 4 weeks (range: 31-96 days), whereas all symptoms in children resolved by 31 days. DISCUSSION/SIGNIFICANCE: Overall, children with SARS-CoV-2 present with a shorter and milder disease course compared to adults. Further studies are needed to understand SARS-CoV-2 illness severity across the lifespan.

OBJECTIVES/SPECIFIC AIMS: Our research hypothesis is that resting state fMRI (rsfMRI) data can be used to identify regions of the brain which are associated with cognitive decline in patients – thereby providing a tool by which to characterize AD progression in patients. METHODS/STUDY POPULATION: We used data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) to analyze Mini-Mental State Examination (MMSE) questionnaire scores from 14 patients diagnosed with AD at two measurement occasions. RsfMRI data was available at the first of these occasions for these patients. These rsfMRI data were summarized into 264 node-based graph theory measures of clustering coefficient and eigenvector centrality. To address our research hypothesis, we modeled changes in patient MMSE scores over time as a function of these rsfMRI data, controlling for relevant confounding factors. This model accounted for the high-dimensionality of our predictor data, the longitudinal nature of the outcome, and our desire to identify a subset of regions in the brain most associated with the MMSE outcome. RESULTS/ANTICIPATED RESULTS: The use of either the clustering coefficient or eigenvector centrality rsfMRI predictors in modeling MMSE scores for patients over time resulted in the identification of different subsets of brain regions associated with cognitive decline. This suggests that these predictors capture different information on patient propensity for cognitive decline. Further work is warranted to validate these results on a larger sample of ADNI patients. DISCUSSION/SIGNIFICANCE OF IMPACT: We conclude that different rsfMRI graph theory measures capture different aspects of cognitive function and decline in patients, which could be a future consideration in clinical practice.

OBJECTIVES/GOALS: Congenital anomalies (CAs) affect 3% of live births, yet the cause of 80% of CAs is unknown and for the 20% with an identified cause, variability in penetrance suggests additional risk drivers exist. Our method for identifying and categorizing CAs in electronic health record (EHR) linked biobank databases can expand and improve CA etiologic research. METHODS/STUDY POPULATION: We identified individuals with CAs in three groups: 1. Those with at least one CA 2. Those with multiple CAs (MCA), those with two or more ‘major’ CAs, and 3. Those with CAs in a specific organ system. We also created a novel quantitative approach, using phenome-wide association studies (pheWAS), for determining CA-associated genetic disease billing codes in order to separate individuals that have a known genetic cause for their CAs from those with idiopathic CAs. We updated CA phecodes, aggregates of clinical billing codes, which we used to identify CA cases in Vanderbilt’s EHR-linked biobank database, BioVU. We create a new phecode, ‘All CAs’, for researchers to quickly identify all individuals with at least one CA. We evaluate the definition of MCA using pheWAS analyses to compare ‘minor’ vs ‘major’ CA. RESULTS/ANTICIPATED RESULTS: The new CA phecode nomenclature includes 5.8 times more codes for CAs compared with the previous version (365 vs 56), improving granularity. 85 (19.7%) CA-associated genetic disease billing codes were identified through literature review. PheWAS analyses revealed an additional 16 (3.7%) genetic disease billing codes with one or more significant (p< 2.75 x10-5) association with CA-related phecodes. Identifying CA-associated genetic disease billing codes allows researchers to differentiate between idiopathic CAs and those that have a known genetic cause. PheWAS analyses of individuals with previously considered “minor” CAs showed many associated severe health problems, revealing that the differentiation between “minor” vs “major” CAs when identifying individuals with MCA in the EHR is arbitrary. DISCUSSION/SIGNIFICANCE: Our CA identification method is scalable for the growing number of EHR-linked biobanks. Differentiating between idiopathic CAs from those with known causes will increase power in studies discovering additional genetic drivers of CAs. Our novel method allows for expansion and acceleration of CA epidemiological research in EHR-linked biobank data.

Objectives/Goals: Highlight the importance of community engagement: Showcase how the involvement of Promotoras de Salud is critical for fostering trust and encouraging participation in clinical trials. Cultural relevance and adaptation: Underline the importance of cultural and contextual relevance in developing and refining clinical research tools. Methods/Study Population: The theater test, an interactive evaluation approach akin to a dress rehearsal in theater, was conducted with approximately 60 Promotoras de Salud at a community center near the US-Mexico border. The Promotoras were divided into four groups, each focusing on one domain of the toolkit and facilitated discussions provided critical feedback on the materials and methods. A community engagement liaison with the University of New Mexico Health Sciences Center played a key role in introducing the EXPLORE team to these community leaders, leveraging long-standing relationships that predate this project. Results/Anticipated Results: Post-testing evaluations showed that 97% of the Promotoras were likely to encourage clinical trials in their communities, and 86% saw significant benefits for their community members. The Promotoras provided key insights and recommendations to enhance the toolkit’s cultural and contextual relevance. The community engagement liaison created a bilingual infographic to share these insights, which was presented at a Promotoras meeting, fostering meaningful discussion about clinical trials. Discussion/Significance of Impact: This project underscores the importance of community voices in research, transforming feedback into actionable insights for public health. Engaging Promotoras through theater testing validated the EXPLORE Toolkit and strengthened ties between clinical research and communities impacted by the opioid crisis.

OBJECTIVES/SPECIFIC AIMS: The serotonin receptor 6 (5-HT6) is a potential therapeutic target given its distribution in brain regions that are important in depression, anxiety, and cognition. This study sought to investigate the effects of age on 5-HT6 receptor availability using 11C GSK215083, a PET ligand with affinity for 5-HT6 in the striatum and 5-HT2A in the cortex. METHODS/STUDY POPULATION: In total, 28 healthy male subjects (age range: 23–52 years) were scanned with 11C-GSK215083 on the HR+PET scanner. Time-activity curves in regions-of-interest were fitted with multilinear analysis-1 method. Binding potentials (BPND) were calculated using cerebellum as the reference region and corrected for partial volume effects. RESULTS/ANTICIPATED RESULTS: In 5-HT6 rich areas, regional 11C-GSK215083 displayed a negative correlation between BPND and age in the caudate (r=−0.41, p=0.03) (14% change per decade), and putamen (r=−0.30, p=0.04) (11% change per decade), but not in the ventral striatum and pallidum. Negative correlation with age was also seen in cortical regions (r=−0.41, p=0.03) (7% change per decade), consistent with the literature on 5-HT2A availability. DISCUSSION/SIGNIFICANCE OF IMPACT: This is the first in vivo study in humans to examine the effect of age on 5-HT6 receptor availability. The study demonstrated a significant age-related decline in 5-HT6 availability (BPND) in the caudate and putamen.

OBJECTIVES/GOALS: Suicide is a growing public health problem with the rate of suicide increasing 33% since 1999. Physicians are not immune to this growing problem. Physicians represent a unique population that has been understudied with respect to suicide. The aim of the study is to investigate risk factors unique to physicians compared to the general population. METHODS/STUDY POPULATION: Using data from the National Violent Death Reporting System, a nationwide CDC database which aggregates information on violent deaths, we extracted demographic and circumstantial data on 172,135 suicide decedents ≥ 25 years old in participating states from 2003-2017. Of these, we found complete information regarding demographics, occupation, and circumstance for 160,159 suicide decedents. We separated suicide decedents by physician-status and compared 795 physicians to 159,364 non-physician decedents using chi-squared test. We then used multivariate logistic regression to examine differences in suicide method and circumstance by physician-status status, controlling for age, sex, and race. RESULTS/ANTICIPATED RESULTS: Compared to non-physicians, physicians were more likely to be male (84.5% vs 77.3%, p<0.001) and older (45.1% ≥65 vs. 17.8%, p<0.001). Controlling for demographics, physicians were less likely to complete suicide by firearm (aOR=0.60, 95%CI=0.51-0.71) but were more likely to suicide by overdose (aOR=1.41, 95%CI=1.13-1.77) or cutting (aOR=2.81, 95%CI=2.03-3.88). Physicians were more likely to have job related stressors (aOR=2.24, 95%Cl=1.83-2.74) and legal problems (aOR=2.34, 95%Cl=1.70-3.21). Physicians were also more likely to leave a suicide note (aOR=1.48, 95%Cl=1.26-1.73) but were less likely to be intoxicated on alcohol at time of death (aOR=0.76, 95%Cl=0.62-0.93). Physicians were no different than non-physicians in terms of financial or relational stressors. DISCUSSION/SIGNIFICANCE: Physicians are more likely to be male and older. Given their medical training, overdose and cutting may be more accessible and lethal methods for physicians. Physicians are more likely to leave a suicide note and less likely to be intoxicated, which may imply less impulsivity. Job stressors and legal problems may also contribute to physician suicide.

ABSTRACT IMPACT: Optimization of primary septal-hippocampal co-cultures facilitates studying central cholinergic synapse formation and dysfunction OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates the study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized in vitro septal-hippocampal co-culture system modified from previously published protocols. Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons attained in the co-culture is ˜5-7%. The size of these cholinergic neurons is strikingly similar to that reported for BFCNs in the intact brain (mean 30μm, range 18-43μm). All sampled cholinergic neurons (28/28 neurons) also expressed molecular machinery necessary for GABA release. Staining for a cholinergic postsynaptic marker shows that 63% of the contacts made with are synaptic. DISCUSSION/SIGNIFICANCE OF FINDINGS: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.

OBJECTIVES/GOALS: Multiple induction agents can facilitate rapid sequence intubation (RSI) in management of refractory status epilepticus (rSE), many which have anti-seizure properties. We examine the anti-seizure efficacy and safety of induction agents used during RSI in the management of rSE. METHODS/STUDY POPULATION: We conducted a single-center retrospective review of patients admitted to the neuro-ICU intubated for management of rSE. Propofol, ketamine and benzodiazepines were considered anti-seizure medication (ASMs), etomidate was not. Patients were treated with propofol or midazolam following intubation. Our primary outcome was clinical or electrographic recurrence of SE within 12 hours of intubation. Exploratory outcomes included time to recover command following, duration of mechanical ventilation (MV) and complications related to intubation. We used multivariable logistical regression to evaluate outcomes between patients induced with ASMs and etomidate. A Fisher exact test was used to compare rSE cessation in a subset of patients with continuous electroencephalography (cEEG) at the time of intubation. RESULTS/ANTICIPATED RESULTS: We identified 149 induced for RSI in management of rSE: 88 patients intubated using ASMs (propofol,n=56; ketamine,n=14; benzodiazepines,n=18) and 61 patients intubated with etomidate. Forty-one patients had recurrence (29.9% ASMS, 24.6% etomidate). The induction agent was not associated with recurrence of SE, time to command following, or duration of MV. Twenty-seven patients had cEEG monitoring at the time of intubation. Sixteen of the 22 patients induced with ASMs had cessation of rSE with induction, while 1 of 5 intubated with etomidate had cessation (Fisher exact test, p=0.047). There were 34 patients with post-induction hypotension (22.9% ASMs, 22.9% etomidate (Fisher exact test, p=1)). DISCUSSION/SIGNIFICANCE: Induction with an anti-seizure medication during intubation was more likely to halt rSE, but did not decrease the likelihood of clinical or electrographic recurrence of rSE and may not affect time to recovery of command following or duration of MV.