Objectives/Goals: The UCLA Clinical and Translational Science Institute (CTSI) training programs have been optimized by clinical research experts since 2013. They inculcate an interest in clinical and translational research careers. The acquisition of new skillsets and early exposure to potential career opportunities often influence lifetime decision-making. Methods/Study Population: The first program, in 2013, was the CTSI Research Associates Program (CTSI-RAP), which exposes undergraduate students to clinical research opportunities. RAP students are now mentoring high school students in the Mentoring and Advocacy in Teaching Clinical and Health-Related Research (MATCH) program. The Fiat Lux seminar is a research course designed to allow freshman students to explore diverse interests. The Leveraging Amazing Undergraduates in Clinical Research at UCLA Health (LAUNCH) program continues the workforce development pipeline by recruiting and training recent graduates to enter clinical research study coordinator careers. Each of these programs has their own stellar track records in terms of high interest and satisfaction and are assessed by annual evaluations from stakeholders. Results/Anticipated Results: CTSI-RAP is in its 11th year and a recent 10-year impact survey demonstrated the value of the program to students and their career decision-making especially those who are underserved and/or disadvantaged. The MATCH program arose from the interest of RAP students to mentor STEM high school students from local disadvantaged schools and is now in its 4th year across the state. The Fiat Lux freshman seminar began with a clinical research essentials emphasis, followed by an FDA/regulatory focus, and is now evolving to highlighting specific innovative areas of research with this year’s course spotlighting Cellular & Gene Therapy/Regenerative Medicine. LAUNCH is now in its third year, having been inspired by graduating RAP students wishing to continue in clinical research and feedback from their own focus groups. Discussion/Significance of Impact: The UCLA CTSI has supported these highly successful workforce development pipeline programs, which have had a demonstrated impact on students and the overall institutional clinical research infrastructure. Their stellar reputations generate high interest at UCLA and serve as model programs for implementation at other academic medical centers.

There is an urgent need for vaccines to the 2019 coronavirus (COVID19; SARS-CoV-2). Vaccine development may not be straightforward, due to antibody-dependent enhancement (ADE). Antibodies against viral surface proteins can, in some cases, increase infection severity by ADE. This phenomenon occurs in SARS-CoV-1, MERS, HIV, Zika, and dengue virus infection and vaccination. Lack of high-affinity anti-SARS-CoV-2 IgG in children may explain the decreased severity of infection in these groups. Here, we discuss the evidence for ADE in the context of SARS-CoV-2 infection and how to address this potential translational barrier to vaccine development, convalescent plasma, and targeted monoclonal antibody therapies.

Temporal challenges are not only contextual in nature but manifest internally in teams when members enter the team with different temporal orientations (e.g., time urgency and pacing style). Researchers have demonstrated that temporal diversity has important implications for key team outcomes (performance, timeliness, and team conflict) across a range of samples and countries. Unfortunately, the practical implications of this research have yet to be unpacked. We respond to this need by developing an approach to translate temporal diversity research studies into actionable, evidence-based team interventions. Because journal articles are often deficient on actionable steps, whereas practitioner-friendly outlets tend to be deficient on scientific rigor, incorporating both criteria necessitates merging these literatures. Specifically, we delineate four main steps: (1) identify significant moderators, (2) match the moderators to scientifically based interventions, (3) design intervention tools with specific, actionable procedures, and (4) evaluate the effectiveness of the intervention tools by designing research studies. We believe the process we outline to marry actionable and evidence-based benchmarks is applicable to other research domains in team science beyond temporal research. It is our hope that this research will be a catalyst for further exploration of interventions that can help team members navigate temporal differences.

OBJECTIVES/SPECIFIC AIMS: Allergic asthma is a chronic lung disease driven by inappropriate inflammatory responses against inhaled allergens. Neuropilin-2 (NRP2) is a pleiotropic transmembrane receptor expressed in the lung, but its role in allergic airway inflammation is unknown. Here, we characterized NRP2 expression in lung immune cells and investigated the effects of NRP2 deficiency on airway inflammation. METHODS/STUDY POPULATION: NRP2 expression by lung immune cells from NRP2 reporter mice was determined by flow cytometry. NRP2 expression by human alveolar macrophages (AM) from healthy individuals was determined by mRNA analysis and flow cytometry. Airway inflammation in NRP2-deficient mice was assessed by bronchoalveolar lavage (BAL) cytology and inflammatory gene expression in lung tissue. RESULTS/ANTICIPATED RESULTS: NRP2 expression in lung immune cells was negligible under steady-state conditions. In contrast, inhalational exposure to lipopolysaccharide (LPS) adjuvant dramatically induced NRP2 expression in AM, as 63.3% of AM from LPS-treated mice were NRP2+ compared with 1.5% of AM from control mice. Ex vivo treatment of human AM with LPS resulted in a 1.5-fold and 2.6-fold increase in NRP2 mRNA and surface protein expression, respectively. Compared to littermate controls, NRP2-deficient mice had greater numbers of BAL leukocytes and increased lung expression of the T helper type 2 cytokines IL-4 and IL-5. Furthermore, NRP2 deficiency resulted in stochastic development of allergic airway inflammation, as spontaneous airway eosinophilia was detected in 25% (2/8) of NRP2-deficient mice compared with 0% (0/8) of littermate controls. DISCUSSION/SIGNIFICANCE OF IMPACT: NRP2 is expressed by activated human and murine AM and suppresses the spontaneous development of allergic airway inflammation. These findings suggest that NRP2 may play a key role in allergic asthma pathogenesis, and could prove to be an important therapeutic target in patients with asthma and other allergic diseases.

OBJECTIVES/SPECIFIC AIMS: This study aims to test the hypothesis that bacterial biotransformation of chemotherapeutics promotes gut microbial diversity by enhancing persistence of drug-sensitive taxa. METHODS/STUDY POPULATION: The impacts of doxorubicin on a model community of gut bacteria was investigated in vitro in anaerobic batch culture. The synthetic community was composed of specific members predicted by genomic analysis to be sensitive to the therapeutic (i.e., Clostridium innocuum, Lactobacillus sp.), resistant via putative biotransformation (i.e., Escherichia coli, Klebsiella pneumoniae), or resistant via putative efflux (i.e., Enterococcus faecalis). Bacterial growth was monitored in monocultures by measuring OD600 and standard plate counts, and in mixed cultures by strain-targeted qPCR. Doxorubicin concentration was detected via absorbance assay. RESULTS/ANTICIPATED RESULTS: Strains with predicted resistance to doxorubicin by drug biotransformation significantly lowered concentrations of the drug in culture media. In contrast, E. faecalis proved resistant without evidence of drug transformation. Predicted sensitive strains were growth-repressed by the doxorubicin, but able to grow in spent media where biotransformation had occurred. However, they remained growth-repressed in spent media from E. faecalis where drug transformation had not been observed. Bacterial growth kinetics in mixed batch culture were dependent on starting bacterial concentrations and timing of drug exposure. DISCUSSION/SIGNIFICANCE OF IMPACT: This work will be extended to model microbial community responses to doxorubicin as a factor of microbial interactions and extent of drug transformation prior its exposure to sensitive strains. The resulting model will have translational implications for mitigating health risks during pediatric cancer treatment.

OBJECTIVES/SPECIFIC AIMS: Focal cartilage injuries of the knee joint are common and present a treatment challenge due to minimal intrinsic repair. Cartilage tissue engineering techniques currently used in clinical practice are expensive, cumbersome, and often ineffective in patients with mechanical or medical comorbidities. To address these issues, we developed an acellular nanofibrous scaffold with encapsulated growth factors designed to enhanced articular cartilage repair. Our goal is to evaluate this technology in vitro and pilot a large animal model for eventual translation into human subjects. METHODS/STUDY POPULATION: Hyaluronic acid (HA, 65 kDa) will be methacrylated (~40% modification, MeHA) and conjugated with cell-adhesive (RGD) groups. A solution of 4% wt/vol MeHA, 2% wt/vol polyethylene oxide (900 kDa), 0.05% wt/vol Irgacure 2959, and 0.005% wt/vol stromal cell-derived factor-1α (SDF-1α) and/or transforming growth factor-β3 (TGF-β3) will be prepared in ddH2O. The solution will be electrospun onto a rotating mandrel to achieve a dry scaffold thickness of 0.5 mm. The scaffold matt will be UV cross-linked and 5 mm-diameter samples will be cut out. Four groups of scaffolds will be prepared: MeHA, MeHA+SDF, MeHA+TGF, MeHA+SDF+TGF. All groups will be evaluated for fiber diameter, swell thickness, equilibrium compressive modulus, degradation rate, and growth factor release rate over 4 weeks (n=10). Scaffolds will also be seeded with juvenile porcine MSCs (5×104) in 200 μL of medium incubated for 24 hours. Seeded scaffolds will be evaluated for equilibrium compressive modulus, cell infiltration, and chondrogenesis at 4 and 8 weeks (n=10). Scaffolds will then be evaluated in a juvenile Yucatan minipig cartilage defect model. In total, 6 animals will undergo bilateral knee surgery to create four 4 mm-diameter full-thickness cartilage defects in each trochlear grove. All defects will receive microfracture to release marrow elements. Each knee will receive 2 scaffolds of the same group (replicates) with paired microfracture controls, resulting in a sample size of 3. Animals will be sacrificed at 12 weeks and defects will be evaluated via non-destructive indentation testing for mechanical properties, microCT for defect fill and subchondral bone morphology, and histology for ICRS II Visual Histological Assessment Scoring. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown reliable replication of electrospun MeHA scaffolds. We anticipate cross-linking density to correlate positively with compressive modulus, and negatively with swell thickness, degradation rate, and growth factor release rate. We anticipate the addition of SDF-1α and TGF-β3 to increase cell infiltration and chondrogenesis, respectively, within seeded scaffolds. Similarly, we expect minipig defects treated with growth factor-releasing scaffolds to show greater mechanical properties, defect fill, and ICRS II score compared with MeHA scaffolds without growth factor. DISCUSSION/SIGNIFICANCE OF IMPACT: This study has the potential to show how an HA-based cell-free scaffold can be augmented with 2 growth factors that act synergistically to improve cartilage repair in a large animal model. This technology would improve upon the cell-free scaffolds already used clinically for autologous matrix-induced chondrogenesis and is directly translatable.

OBJECTIVES/GOALS: The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long-term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long-term outcome. METHODS/STUDY POPULATION: Expression of Apolipoprotein E4 predisposes stroke patients to poor long-term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low-density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes. LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell-surface TNF receptor (TNFR1) in non-astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke. We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral-mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. RESULTS/ANTICIPATED RESULTS: Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will elucidate how astrocyte-LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long-term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke. CONFLICT OF INTEREST DESCRIPTION: NA.

OBJECTIVES/GOALS: The primary objective of this study was to apply design sprint methodology to develop a proactive outreach tool prototype for patients with chronic obstructive pulmonary disease (COPD). METHODS/STUDY POPULATION: We utilized a 3-day process to align our team and key stakeholders behind answering the following question: “how might we empower COPD patients to understand their healthcare information, make decisions in partnership with their providers, and more easily manage their daily health?” On Day 1, we focused on understanding and defining the problem, and mapping the patient experience. On Day 2, we quickly brainstormed potential solutions, sketched our top ideas, and listed the solutions’ inherent assumptions. On Day 3, we created a prototype of our top solution and storyboarded each step of the prototype experience to review its potential usability and comprehensibility with patients. RESULTS/ANTICIPATED RESULTS: At the end of the design sprint, our team developed a prototype centered around personalized communication between COPD patients and providers. The prototype focuses on augmenting the current transitional care management (TCM) workflow in the post-discharge period. We are working to further develop the prototype prior to formal testing with care coordinators and patients. We anticipate that our prototype will assist in automating the current TCM workflow and facilitate contact with more patients post-discharge. DISCUSSION/SIGNIFICANCE OF IMPACT: Contact with patients is currently challenging due limited resources and the time sensitive nature of the TCM requirements. Automated patient outreach may be especially effective in engaging patients on a large scale, while also minimizing time and resources needed from healthcare staff.

OBJECTIVES/GOALS: While the current management of single ventricle repairs has drastically prolonged life expectancy, the repair fails over time primarily through pathologic inflammation and fibrosis. Our goal is to demonstrate that cardio-omentopexy can decrease inflammation and fibrosis in swine after cryoinjury. METHODS/STUDY POPULATION: A cryoinjury is created using a liquid nitrogen cooled probe to the right ventricle of 20kg swine. In half the groups the omentum is attached to the heart over the area of the injury. The swine are recovered and monitored for 4 or 8 weeks at which time they are euthanized. The injured area is evaluated via histological and immunohistochemical testing for markers of inflammation and scarring including collagen type, scar area, macrophage activity. RESULTS/ANTICIPATED RESULTS: We anticipate that the addition of omentopexy to cryoinjury will decrease scar area, fibrosis and markers of chronic inflammation. Additionally, we expect an increase in myocytes in the area of injury. We expect that this will occur through the anti-inflammatory and protective mechanism of the omentum. DISCUSSION/SIGNIFICANCE: Cardio-omentopexy, if able to decrease fibrosis and preserve myocytes, may provide a useful adjunct to the treatment of single ventricle repair by prolonging the longevity of the repair. Additionally, as these repairs often require a ventriculotomy, decreasing the operative scar may preserve myocardial function.

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/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/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: 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/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.

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.

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.