OBJECTIVES/SPECIFIC AIMS: Prostate cancer is the second most common cancer in the world for men. For reasons still unclear, aggressive PCa disproportionately affects males of African ancestry (MoAA). Incidence and mortality rates are highest in MoAA as they have consistently shown a 2.3–3.0-fold higher risk of mortality compared with Caucasian men. This aggressiveness of PCa may be due to specific biological factors. Studies have established that microRNAs (miRNAs), essential in post-transcriptional gene regulation, are dysregulated in PCa. miR-1207-3p is encoded at the PVT1 gene locus, which is located downstream of MYC on the 8q24 PCa susceptibility locus. The chromosomal region 8q24 is associated with aggressive PCa. Yet miR-1207-3p in PCa in MoAA has never been investigated. Moreover, studies have shown that PVT1/MYC co-operation is a fundamental feature in all cancers with 8q24 amplification and 98% of the 8q24 amplicons contained concurrent amplification of the MYC and PVT1 loci. Moreover, MYC has been linked to PCa aggressiveness, and has been reported to be downstream of androgen receptor (AR) in some PCa. However, the mechanisms regulating c-MYC have never been studied in MoAA. We have recently demonstrated that miR-1207-3p has prognostic value in PCa, and directly binds to the 3′ UTR of Fibronectin type III domain containing 1 (FNDC1) to regulate a novel FNDC1/fibronectin (FN1)/AR pathway upregulated in metastatic PCa. However, the relevance of this novel and clinically significant miR-1207-3p molecular pathway in PCa in MoAA is unknown. Therefore, we hypothesized that miRNA 1207-3p, encoded at the 8q24 PCa susceptibility locus, is a PCa biomarker with clinical applications in MoAA. Our specific aim was to assess the clinical relevance of miR-1207-3p, FNDC1, FN1, AR, and MYC expression in aggressive PCa in a cohort of West African Black males. METHODS/STUDY POPULATION: Consequently, we aimed to determine the expression profile of miRNA-1207-3p, FNDC1, FN1, AR, and MYC in histologically confirmed normal prostate (n=24), benign prostate (n=44) and malignant prostate tissue (n=29) from prostatectomy or transrectal ultrasound-guided biopsies in patients recruited at the University College Hospital, Ibadan, Nigeria, a sub-Saharan Black African population. In total, 17 patients had tumor tissues with Gleason score ≥8. Tissues were collected in compliance with Institutional Ethics Board approved protocols. RNA extraction, cDNA synthesis, and quantitative-PCR were performed to analyze mRNA expression of miRNA-1207-3p, FNDC1, FN1, AR, and MYC. Statistical analysis were performed using SPSS software. All data were analyzed by the 1-way ANOVA test. Tukey post-hoc test was performed to determine the differences in mean expression between normal and tumor prostate tissues. p<0.05 were considered significant. RESULTS/ANTICIPATED RESULTS: We discovered that miR-1207-3p is significantly underexpressed in prostate tumor tissues [0.09±0.02, 95% CI (0.04, 0.136), p=0.000] in comparison with normal prostate tissue in MoAA [0.92±0.15, 95% CI (0.60, 1.244), p=0.000]. This is the first description of miR-1207-3p differential expression in human clinical PCa in MoAA. In contrast, FNDC1 was significantly overexpressed in prostate tumor tissues [21.93±8.21, 95% CI (4.97, 38.89), p=0.003] in comparison with normal prostate tissues in MoAA [1.57±0.45, 95% CI (0.625, 2.51), p=0.003]. The same positive correlation with advanced disease held true for FN1 [tumor: 13.66±3.53, 95% CI (6.38, 20.93), p=0.000; normal: 1.07±0.235, 95% CI (0.58, 1.56), p=0.000], AR [tumor: 20.49±6.74, 95% CI (6.50, 34.48), p=0.000; normal: 0.94±0.20, 95% CI (0.52, 1.36), p=0.000], and c-MYC [tumor: 33.93±8.43, 95% CI (16.53, 51.33), p=0.000; normal: 1.94±0.36, 95% CI (1.18, 2.70)]. The significantly increased mean expression for FNDC1, FN1, AR, and c-MYC in prostate tumor tissues in comparison with normal prostate tissues indicate that their overexpression is associated with increased risk of cancer progression in MoAA. DISCUSSION/SIGNIFICANCE OF IMPACT: These results show that the underexpression of miR-1207-3p and the overexpression of FNDC1, FN1, AR and MYC is associated with aggressive PCa in MoAA. miR-1207-3p, and it molecular target FNDC1, may be useful biomarkers for prognostication of PCa in MoAA.

OBJECTIVES/GOALS: Aging is the biggest risk factor for cancer, yet little is known about cancer immunotherapy effects. Here we investigate melanoma response to αPD-1, αPD-L1 and αPD-L2 in young vs. aged hosts. We look at different immune outcomes as possible mechanism. METHODS/STUDY POPULATION: We tested αPD-1 (100 μg/mouse), αPD-L1 (100 μg/mouse) or αPD-L2 (200 μg/mouse) in aged (18-24 months) and young (3-8 months) mice challenged orthotopically with B16. Tumors and draining lymph nodes (TDLN) were analyzed by flow. Bone marrow-derived DC were generated with GM-CSF. RESULTS/ANTICIPATED RESULTS: We reported that αPD-1 treats young and aged with B16 and αPD-L1 only treats young. aPD-L2 treated B16 in aged but, remarkably, not young, the first anti-cancer single agent immunotherapy exhibiting this property. Efficacy in young (aPD-1, aPD-L1) and aged (aPD-1, aPD-L2) correlated with increased T cell stem cells (TCSC) and total tumor-infiltrating lymphocytes (TIL), but TCSC differed by age and treatment (e.g., distinct CCR2, CXCR5, CXCR3, PD-1 and TIM- expression). Aged expressed significantly more T-cell PD-1 and up to 40-fold more PD-L2 versus young in myeloid and NK cells, and TCSC. Bone marrow-derived DC experiments suggest aged DC are destined for high PD-L2 versus young. DISCUSSION/SIGNIFICANCE OF IMPACT: Treatment differences in aged vs. young could depend on immune checkpoint or TCSC differences. We are now identifying mechanisms for increased PD-L2 and contributions to aPD-L2 efficacy in aged, and testing TCSC effects. Our work can improve cancer immunotherapy in aged hosts and further provide important insights even in young hosts.

OBJECTIVES/SPECIFIC AIMS: The aims of this study are (1) to develop and characterize a novel nonhuman primate model of pneumococcal pneumonia that mimics human disease; and (2) determine whether Streptococcus pneumoniae can: (a) translocate to the heart, (b) cause adverse cardiac events, (c) induce cardiomyocyte death, and (d) lead to scar formation during severe pneumonia in baboons. METHODS/STUDY POPULATION: Six adult baboons (Papio cynocephalus) were surgically tethered to a monitoring system to continuously assess their heart rate, temperature, and electrocardiogram (ECG). A baseline transthoracic echocardiogram, 12-lead ECG, serum troponin-I levels, brain natriuretic peptide, and heart-type fatty acid binding protein (HFABP) levels were obtained before infection and at the end of the experiment to determine cardiovascular damage during pneumococcal pneumonia. Animals were challenged with 108 colony-forming units of S. pneumoniae in the right middle lobe using flexible bronchoscopy. Three baboons were rescued with ampicillin therapy (80 mg/kg/d) after the development of pneumonia. Cardiac damage was confirmed by examination of tissue sections using immunohistochemistry as well as electron and fluorescence microscopy. Western-blots and tissue staining were used to determine the presence of necroptosis (RIP3 and pMLKL) and apoptosis (Caspase-3) in the cardiac tissue. Cytokine and chemokine levels in the heart tissue were determined using Luminex technology. RESULTS/ANTICIPATED RESULTS: Four males (57%) and three (43%) females were challenged. The median age of all baboons was 11 (IQR, 10-19) years old, which corresponds to a middle-aged human. Infected baboons consistently developed severe pneumonia. All animals developed systemic inflammatory response syndrome with tachycardia, tachypnea, fever, and leukocytosis. Infection was characterized by initial leukocytosis followed by severe leukopenia on day 3 postinoculation. Non-specific ischemic alterations by ECG (ST segment and T-wave flattering) and in the premortem echocardiogram were observed. The median (IQR) levels of troponin I and HFABP at the end of the experiment were 3550 ng/mL (1717–5383) and 916.9 ng/mL (520.8–1323), respectively. Severe cardiomyopathy was observed using TEM and H&E stains in animals with severe pneumonia. Necroptosis was detected in cardiomyocytes of infected animals by the presence of pMLKL and RIP3 in cardiac tissues. Signs of cardiac remodeling indicated by disorganized collagen deposition was present in rescued animals but not in the other animals. DISCUSSION/SIGNIFICANCE OF IMPACT: We confirmed that baboons experience cardiac injury during severe pneumococcal pneumonia that is characterized by myocardial invasion, activation of necroptosis, and tissue remodeling in animals rescued by antimicrobial therapy. Cardiac damage by invading pneumococci may explain why adverse cardiac events that occur during and after pneumococcal pneumonia in adult human patients.

Objectives/Goals: To create, train, and evaluate the FAST-PACE (Promoting Academic and Community Engagement) Toolkit that catalyzes academic-community translation science teams during a public health emergency. The toolkit is a road map based on the Research Readiness and Partnership Protocol (R2P2), which was developed from the Flint Water Crisis. Methods/Study Population: A literature review was conducted by the Michigan Institute for Clinical & Health Research Community Engagement (MICHR CE) program and the Community-Based Organization Partners (CBOP), to identify important and common elements in disaster response protocols with a set of key interviews (n = 31) to glean perspectives from community leaders. Key findings were extracted and reviewed to generate guidelines and recommendations for the R2P2 protocol. The co-developed FAST-PACE Toolkit launched its expansion statewide to address emergencies and health disparities of communities in crisis. The iterative process consisted of community report-outs, gathering input from stakeholders, via discussion, and evaluation surveys. The feedback was used to develop, enhance, and tailor the toolkit and training content. Results/Anticipated Results: Data from training (n = 8) of the critical elements of the FAST-PACE Toolkit, which provides guidance for academic and community team members that includes 1) assessing community assets and needs; 2) engaging in clear and bidirectional communication; 3) facilitating transparency and equitable partnering; 4) identifying health equity and justice issues; and 5) conducting the evaluation of research. The training will be disseminated in-person and virtually across the state of Michigan resulting in participants sharing community-identified health issues and social determinants of health to assist MICHR CE to suggest resources to address health impacts. Discussion/Significance of Impact: The FAST-PACE Toolkit borne from the flint water crisis and confounded by other crises used CEnR principles to create a translation science roadmap. It equips communities and collaborating academic institutions across the state to respond to public health crises and fosters equitable translation science partnerships built on respect and trust.

OBJECTIVES/GOALS: Older adults are included in clinical research infrequently compared to their burden of chronic illness. The goal for this study is to learn from older adults about their lived experiences with research and use this knowledge to develop tools and solutions aimed at increasing their inclusion. METHODS/STUDY POPULATION: This study utilized the 5T Model (developed by Duke CTSA) and Community Engagement Studio (CES) (developed by Vanderbilt CTSA) to connect and engage with community experts (older adults and those who work with older adults) in Oregon. Two CES were completed with 14 community experts and 4 investigators interested in including older adults in their studies. Participants took part in a 2-hour facilitated discussion to gain insight from their perspectives on research. The 5T Model was shared with participants and used to guide the discussion and elicit feedback on the model and identify gaps in resources and training needed for investigators to enhance inclusion of older adults in research. RESULTS/ANTICIPATED RESULTS: Trust, relationships, education, and diversity were themes identified across all of the 5Ts. Participants discussed the need for inclusion and diversity within research, with an emphasis on those at the oldest ages, rural populations, and lower socioeconomic status. Participants acknowledged both investigators and participants require more education, with a great need for improving health literacy for research participants. Participants saw trust and relationships as an integral part of older adult inclusion in research, with the relationship being not only that between investigator and participant, but between them and the communities that support older adults, including family members. DISCUSSION/SIGNIFICANCE: This study highlighted the voices of older adult research participants, allowing for participant-informed findings and solution development. Future directions will focus on developing and refining tools and resources for investigators and expanding to other underrepresented populations.

OBJECTIVES/GOALS: Our overall objective is to develop a directly observable and reproducible method of enhanced blood flow through tumor vessels (i.e. dynamic control) at the time of systemic treatment delivery. Our central hypothesis is that the dynamic control of tumor vessels will improve (1) systemic drug delivery and (2) effector cell trafficking to target tumor. METHODS/STUDY POPULATION: B16 melanoma cells were inoculated into C57BL/6 (B6) mice (male and female) in both regional (hind leg) and systemic (flank) models. Dynamic control consisted of an IV saline bolus (500 ul) and phenylephrine (10 ug). Tumor vessel response was observed in real-time through window chambers using intravital microscopy (IVM). Dynamic control was combined with melphalan (20 mg/ml) either regionally (isolated limb perfusion) or systemically. Outcomes included tumor growth, survival, IHC, and toxicity. Dynamic control will be combined with adoptive transfer of effector T cells. B6 mice will be inoculated with B16/OVA (flank with window chamber) and treated with fluorescently labeled (calcein), OVA-specific CD8+ T cells from OT-1 transgenic mice. IVM, IHC, and flow cytometry will be used to measure T cell trafficking. RESULTS/ANTICIPATED RESULTS: Dynamic control (1) restored blood flow in non-functional tumor vessels and (2) increased and then transiently reversed blood flow in functional vessels. Vessel diameters did not change, suggesting that shunting of systemic blood to the tumor vasculature accounted for the observed changes. Dynamic control augmented tumor responses in our regional therapy model of melanoma. Increases in DNA adduct formation (melphalan mechanism of action) detected by IHC, decreased tumor growth, and increased survival were observed with dynamic control. There was no increased limb toxicity. Similarly, dynamic control augmented responses in our systemic therapy model (decreased tumor growth and improved survival). We anticipate that dynamic control will improve trafficking of effector T cells in the next set of experiments. DISCUSSION/SIGNIFICANCE OF IMPACT: Heterogeneous responses to systemic therapies represent a major gap in current cancer treatment. An essential requirement for any effective therapy is its ability to reach tumor via the tumor-associated vasculature. We have therefore developed an approach to enhance drug delivery (dynamic control), which we also plan to test in clinical trials.

OBJECTIVES/GOALS: Infectious keratitis is a potentially sight-threatening disease. Its epidemiology has been considered on various national and regional levels, which demonstrates unique patterns in patient risk factors, causative organisms, antimicrobial resistance and clinical outcomes. This study will examine the patterns specific to the Rochester, NY area. METHODS/STUDY POPULATION: This project will be a ten-year retrospective study, examining all patients who were diagnosed with infectious keratitis at the University of Rochester Medical Center between 2011 and 2021. The study population is selected from a compiled list of those patients with an ICD diagnosis code including ’keratitis’ in the eRecord that falls within our chosen date range. Participants were excluded if the patient’s physician documented that the keratitis is most likely not infectious in etiology. If eligible for the study, there is documentation of patient demographics, clinical risk factors, clinical course, culture data from corneal scraping and antimicrobial resistance patterns, if available. Once data is collected, it will be analyzed and compared to pre-existing regional and national data. RESULTS/ANTICIPATED RESULTS: Between 2011 and 2021, there were 1652 patients with ICD diagnoses of keratitis at the University of Rochester Medical Center. Of these patients, we anticipate approximately 1,200 to meet our inclusion criteria. Some of the major risk factors for developing infectious keratitis in this population include contact lens use, immunocompromised state (elderly, diseased or iatrogenic) and corneal trauma. Clinical complications include progression to endophthalmitis, need for interventional surgery, or failure to improve on clinical exam after 2 weeks of antimicrobials. Causative organisms are most often bacterial. Certain organisms are associated with specific risk factors while other organisms predict greater risk of complications. We also anticipate patterns of antimicrobial resistance to emerge. DISCUSSION/SIGNIFICANCE: This study will provide insights into the risk factors and clinical course of infectious keratitis according to causative organisms in the Rochester, NY area. This can help us mitigate risk factors and help prognosticate for patients, once diagnosed. Also, patterns of antimicrobial resistance can inform empiric antimicrobial recommendations.

OBJECTIVES/SPECIFIC AIMS: Specialized pro-resolving lipid mediators (SPM) actively counter proinflammatory cascades. A deficit of SPMs is one possible mechanism through which inflammation leads to the development of atherosclerotic disease. The purpose of this study is to characterize the profiles of intermediates of SPM synthesis pathways and end-product SPMs in the plasma of patients with peripheral artery disease (PAD). METHODS/STUDY POPULATION: A cross-sectional sample of 52 patients with PAD was recruited at the San Francisco Veterans Affairs Medical Center. PAD was defined as the presence of claudication symptoms and an ankle-brachial index <0.9, or a history of revascularization for claudication. Patients were excluded if they were taking immunosuppressive medications, had a severe acute illness (infection, surgery, illness, critical limb ischemia) within the last 30 days, or had severe hepatic, renal, or nonvascular inflammatory disease. Intermediates of SPM synthesis pathways and end-product SPMs were measured in plasma samples of patients by liquid chromatography-tandem mass spectrometry. RESULTS/ANTICIPATED RESULTS: The average age of the cohort was 69±6.3 and patient comorbidities reflected common comorbidities associated with PAD (hypertension 96%, hyperlipidemia 87%, diabetes mellitus 42%, coronary artery disease 34%). Rutherford categories, measurements of PAD symptom severity, ranged from 0 to III (0 10%, I 40%, II 27%, III 23%). Three EPA products were measured: 18-hydroxyeicosapentaenoic acid (18-HEPE), resolvin E1 (RvE1), and resolvin E2 (RvE2). 18-HEPE, an intermediate of SPM synthesis, was detectable in the plasma of every patient (median: 105 pg/mL, IQR: 54.9–195), whereas the SPM end-products, RvE1 and RvE2, were only detectable in 6 and 10 patients, respectively. In total, 7 DHA products were measured: 14-hydroxydocosahexaenoic acid (14-HDHA), 17-HDHA, resolvin D1 (RvD1), resolvin D2 (RvD2), protectin D1, protectin DX, and maresin 1. The intermediates 14-HDHA (median: 6546 pg/mL, IQR: 3329–12061) and 17-HDHA (median: 644 pg/mL, IQR: 340–1056) were detectable in the plasma of every patient. However, the end-products RvD1, RvD2, protectin D1, protecin DX, and maresin 1 were identified in less than half of the cohort. DISCUSSION/SIGNIFICANCE OF IMPACT: We report the presence of several intermediates of SPM synthesis pathways (18-HEPE, 14-HDHA, and 17-HDHA) in every patient but the presence of SPM end-products in only a limited portion of the cohort. These results suggest that some patients with PAD may have a deficit of SPMs. Further investigation is required to better understand the role of SPMs and mediators of resolution of inflammation in PAD.

OBJECTIVES/GOALS: The 2020-2021 Galveston County Youth Risk Survey continues past efforts to characterize behavioral risks for local youth, identify disproportionate risks among groups, and provide the data needed for action and intervention to improve the health and safety of our youth in Galveston, Texas. METHODS/STUDY POPULATION: While the survey is based on the CDC Youth Risk Behavior Surveillance System (YRBSS), there was community need for an expanded survey and thus a workgroup was formed to create the Galveston Youth Risk Survey, including members from UTMBs Institute for Translational Sciences, a number of other UTMB departments and Centers, the Research, Education, and Community Health (REACH) Coalition, several school districts, Teen Health Clinic, and a variety of other community health organizations with vested interest. The survey was administered in November 2020 via ScanTron and REDCap to two local high schools with populations greater than 2,000. CDC guidelines for administration of the YRBSS were followed. RESULTS/ANTICIPATED RESULTS: A total of 2,428 students completed the survey from GISD and DISD. The survey provided insight into the following categories, with mental health emerging as a pressing issue, specifically stress and depression, particularly among females. All results are reported in several contexts, including comparisons by gender, grade, and ethnicity, a comparison to state and national statistics when available, as well as risk trends from previous surveys. Demographics Safety, including driving, violence, and bullying Mental Health: stress, depression, suicidal ideation, and ACEs Human Trafficking Substance Use, including smoking, alcohol, marijuana, and other drug use Sexual Behaviors, including contraceptive use Body Weight and Body Image Health Conditions Home Life: Support and Security DISCUSSION/SIGNIFICANCE: The report has been presented to the school districts, immediate stakeholders, REACH membership, and the general public. Several presentations have been given to groups to report the findings. Workgroups will now be formed to address the needs of our students, as well as a possible follow-up survey to look at data specific to COVID-19 and mental health.

OBJECTIVES/GOALS: The overall goal of this study was to determine the effect of early life stress (ELS) on the intestinal CD4+ T cell immune compartment, at homeostasis and after induction of experimental Inflammatory Bowel Disease (IBD). METHODS/STUDY POPULATION: We used a mouse model of ELS, maternal separation with early weaning (MSEW). We used IL-10 reporter mice to enable analysis of IL-10-producing cells. Mice were examined on postnatal day 28 to determine the impact of ELS on gut regulatory T cells. Plasma levels of corticosterone (rodent stress response hormone) was determined by ELISA. Colitis was induced in MSEW and normal rear (NR) mice via intraperitoneal injection of α-IL-10R every 5 days until day 15. Mice were euthanized on days 20 and 30. Colonic tissue sections were stained for histological analysis. Remaining tissue was further processed for flow cytometric analysis of CD4+ T cells and innate lymphoid cells. RESULTS/ANTICIPATED RESULTS: Plasma corticosterone was elevated in MSEW mice compared to their NR counterparts at 4 weeks of age. We observed that the MSEW stress protocol does not affect the baseline colonic CD4+ T cell or innate lymphoid cell populations. There was a reduction in the intestinal CD4+ T cells and regulatory T cells on day 20 in α-IL-10R MSEW mice compared to NR counterparts. This difference disappeared by day 30. Histological scoring showed no difference in disease severity between α-IL-10R treated MSEW and NR mice on day 20. However, on day 30, when α-IL-10R NR mice are recovering from colitis, MSEW mice showed persistent histological inflammation, mainly attributable to sustained epithelial damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that ELS prolongs intestinal inflammation and impairs epithelial repair. Future studies will focus on elucidating the mechanisms responsible for ELS-dependent impairment of mucosal repair in experimental colitis.

Objectives/Goals: This study evaluates the role of visual machine learning algorithms (VMLA) in automating a predictive model of central sarcopenia in geriatric trauma patients based on the psoas:lumbar vertebral index (PLVI) and trauma-specific frailty index (TSFI). Methods/Study Population: 150 trauma patients seen at Jon Michael Moore Trauma Center within J.W Ruby Memorial Hospital in rural West Virginia were included in this investigation across the life spectrum. The VMLA was trained on their standard of care trauma panoramic CT scans. Five expert reviewers segmented bilateral psoas muscles and the L4 vertebrae of each CT image at one slice inferior to the posterior elements of the L4 vertebrae. The data were read into a U-net convoluted neural network as ground truth. Labels were preprocessed to focus on the regions of interest and standardized into four classes: right psoas, left psoas, L4 vertebrae, and background. Performance was evaluated using accuracy, Dice coefficient, and F1 score. Results/Anticipated Results: Between our expert reviewer segmentations, we had significant inter-reader reliability with a Kappa greater than 0.8 and a mean standard deviation of the PLVI of 0.10mm^2. Preliminary VMLA testing on a subset of 70 patients yielded a validation accuracy of 88.5%, a Dice coefficient of 0.86, and an F1 score of 0.87 after 20 epochs. There was a moderate interclass correlation between PLVI and TSFI even though the TSFI lacks sensitivity. In fact, the PLVI is a more accurate predictor of frailty in trauma patients based on various outcome measures such as corrected length of stay. Our ongoing efforts are centered around improving the VMLA. Discussion/Significance of Impact: Our VMLA outperforms the current clinical standard, TSFI. Integration of our VMLA into the clinical workflow has the potential to revolutionize geriatric trauma care by providing rapid, accurate, identification of high-risk frail patients.

OBJECTIVES/GOALS: The objective was to examine the American Heart Association’s (AHA) Essential Eight metrics of cardiovascular (CV) health among Black and White adults with peripheral arterial disease (PAD) collected via validated surveys and medical records. Each metric was examined in association with available social determinants of health (SDoH) factors. METHODS/STUDY POPULATION: This observational study completed data collection through surveys and medical record review. Validated surveys were used to collect Essential Eight metrics of diet, physical activity, sleep, and smoking status. Medical records were used to collect data on body mass index, blood lipids, blood glucose, and blood pressure. Participants with a diagnosis of lower extremity PAD, ability to complete surveys, and provided informed written consent were eligible. Equal numbers of Black and White participants were enrolled. Essential Eight metrics were used to calculate CV health scores for each participant. Scores were examined for association with SDoH factors and by race using Student’s T-test or ANOVA for continuous variables or Chi-Square tests for categorical variables. RESULTS/ANTICIPATED RESULTS: A total of 50 participants will be enrolled, with the expected majority being men and half self-reporting as Black individuals. Worse SDoH is expected to be associated with lower CV health metrics, including lower levels of physical activity and higher levels of saturated fatty food consumption. Higher levels of blood lipids, blood glucose, and blood pressure are expected to be associated with worse SDoH factors. We expect this association to be attenuated by rates of CV medications, such as statin therapy, antiglycemic medications, and antihypertensive medications. No effect modification by rurality is expected, although our projected sample size is small and may impact the ability to examine this interaction. DISCUSSION/SIGNIFICANCE: Black Americans, particularly in the Deep South, are at elevated risk for PAD and critical limb ischemic events, such as amputation. Understanding CV health metrics and SDoH characteristics among adults with PAD is essential to reduce disparities in care and provide valuable information for those at highest risk for complications.

Community engagement is important for reaching vulnerable populations in the coronavirus disease 2019 (COVID-19) pandemic. A risk communication framework was implemented by a community-engaged research (CEnR) partnership in Southeast Minnesota to address COVID-19 prevention, testing, and socioeconomic impacts. Bidirectional communication between Communication Leaders and community members within their social networks was used by the partnership to refine messages, leverage resources, and advise policy makers. Over 14 days, messages were delivered by 24 Communication Leaders in 6 languages across 9 electronic platforms to 9882 individuals within their networks. CEnR partnerships may effectively implement crisis and emergency risk communication to vulnerable populations in a pandemic.

Community-engaged research (CEnR) is now an established research approach. The current research seeks to pilot the systematic and automated identification and categorization of CEnR to facilitate longitudinal tracking using administrative data. We inductively analyzed and manually coded a sample of Institutional Review Board (IRB) protocols. Comparing the variety of partnered relationships in practice with established conceptual classification systems, we developed five categories of partnership: Non-CEnR, Instrumental, Academic-led, Cooperative, and Reciprocal. The coded protocols were used to train a deep-learning algorithm using natural language processing to categorize research. We compared the results to data from three questions added to the IRB application to identify whether studies had a community partner and the type of engagement planned. The preliminary results show that the algorithm is potentially more likely to categorize studies as CEnR compared to investigator-recorded data and to categorize studies at a higher level of engagement. With this approach, universities could use administrative data to inform strategic planning, address progress in meeting community needs, and coordinate efforts across programs and departments. As scholars and technical experts improve the algorithm’s accuracy, universities and research institutions could implement standardized reporting features to track broader trends and accomplishments.

COVID-19 reinforced the need for effective leadership and administration within Clinical and Translational Science Award (CTSA) program hubs in response to a public health crisis. The speed, scale, and persistent evolution of the pandemic forced CTSA hubs to act quickly and remain nimble. The switch to virtual environments paired with supporting program operations, while ensuring the safety and well-being of their team, highlight the critical support role provided by leadership and administration. The pandemic also illustrated the value of emergency planning in supporting organizations’ ability to quickly pivot and adapt. Lessons learned from the pandemic and from other cases of adaptive capacity and preparedness can aid program hubs in promoting and sustaining the overall capabilities of their organizations to prepare for future events.

OBJECTIVES/SPECIFIC AIMS: Our objective is to study the role of soluble adenylyl cyclase in the melanocyte regulation of pigment in response to ultraviolet radiation. Melanocytes are specialized cells that produce melanin in organelles called melanosomes, and melanin determines the pigmentation of hair and skin. cAMP is a master regulator of pigmentation and transmembrane class of adenylyl cyclases are essential for expression of important enzymes involved in melanogenesis. However, pigmentation is also controlled by melanosomal pH, which regulates melanogenesis, tyrosinase activity, and melanosome maturation. The relationship between melanosomal pH and cAMP has been elusive. Soluble adenylyl cyclase is a noncanonical source of cAMP that is not responsive to G proteins but rather functions as a pH sensor. We recently demonstrated that loss of soluble adenylyl cyclase (sAC) activity leads to increased melanosomal pH as well as increased pigmentation in cells and hair. We expanded our research to investigate the role of sAC in the intrinsic response of melanocytes to ultraviolet radiation. METHODS/STUDY POPULATION: We utilized sACfl/fl (wild type) and sACKO mouse melanocytes and compared their change in pigmentation in response to ultraviolet radiation. Melanin was used as a measure of pigmentation. We irradiated these cells at differing doses of UVB (0, 1, 2, or 3 mJ/cm2) daily for 3 days. After UVB treatment, cells were observed and the surviving cell numbers were determined. Cells were then analyzed for melanin content using spectroscopy. RESULTS/ANTICIPATED RESULTS: We found that while both sACfl/fl and sACKO cells had increased melanin content in response to UVB, the melanin content of sACKO cells increased more compared with sACfl/fl cells (p=0.001 at daily dose of 3 mJ/cm2). In addition, sACKO cells required less UVB dose to induce a response. We also observed that sACKO cells show increased cell death compared with sACfl/fl cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Although both sACfl/fl and sACKO cells can induce melanin production in response to UV, our results suggest that sACKO cells are more sensitive. We believe that this increased response in sACKO cells is due to increased melanosomal pH. In addition, sACKO cells show increased cell death, suggesting that sAC is important in the damage response secondary to UV exposure. UV plays a wide range of roles in skin biology such as contributing to cancer risk and pigmentation. Since pigmentation is essential for the protection of the skin from UV insult, further investigation of possible mechanisms in which sAC can influence pigmentation in response to UV is warranted.

OBJECTIVES/GOALS: Lung Ultrasound Congestion Score (LUS-CS) is a proposed measure for guiding treatment in acute heart failure (AHF). An emergency department (ED) pilot trial of LUS-guided diuresis showed reduced LUS-CS at 48 hours but no difference at hospital discharge or for clinical outcomes. We hypothesized total change in LUS-CS would predict adverse outcomes. METHODS/STUDY POPULATION: This was a post-hoc secondary analysis of the BLUSHED-AHF trial. BLUSHED-AHF was a pilot trial in which AHF patients were randomized to a LUS-guided diuresis strategy vs. usual care in the ED. The intervention was stopped after the ED course (i.e. during hospitalization). BLUSHED-AHF was designed for the intervention to target absolute values of LUS-CS over time, rather than change in LUS-CS from each patient’s baseline. We fit a cox regression model for a primary outcome of death or AHF rehospitalization, with total (ED to Hospital Discharge) change in LUS-CS as the primary predictor, adjusted for the Get-With-The-Guidelines heart failure risk score (GWTG). Survival curves were plotted, and hazard ratios calculated. RESULTS/ANTICIPATED RESULTS: 128 patients in BLUSHED-AHF were analyzed. Greater reduction in LUS-CS from ED to hospital discharge predicted event-free survival (HR = 0.74 for each 20 unit reduction in LUS-CS, 95%CI 0.56-0.99). This effect did not vary by hospitalization length or ED disposition. There was a significant interaction between change in LUS-CS and GWTG score (p DISCUSSION/SIGNIFICANCE: LUS-CS total change, and not absolute values, predict adverse events in LUS-guided diuresis. Post-ED cessation of the intervention in BLUSHED-AHF may have precluded opportunity for clinical benefit. Future trials should run the entire hospital course, target change from baseline, and consider patient selection by AHF severity and initial LUS-CS.

OBJECTIVES/SPECIFIC AIMS: Recent advances in microneedle technology have enabled practical, in vivo dermal interstitial fluid (ISF) sampling. These minimally-invasive techniques allow for collection of ISF without damage to adjacent tissues and do not rely on blister formation. Initial reports of extracellular vesicle (EV) isolation from dermal ISF and paired blood samples suggest that EVs may be more abundant in ISF. Analysis of ISF-derived EVs may allow for more detailed study of intercellular communication at the tissue level, particularly in acute inflammatory conditions. The objective of this study is to describe the isolation and initial characterization of interstitial fluid-derived exosomes. METHODS/STUDY POPULATION: We apply electron microscopy, nanoparticle tracking analysis (NTA), immuochemical, and sequencing methods to describe and distinguish the EV content of interstitial fluid. We include apparently healthy adult human subjects with no active skin disease. We also study immunocompetent, CD-hairless rats to demonstrate the generalizability of the methods. RESULTS/ANTICIPATED RESULTS: We successfully isolated EVs from human and rat interstitial fluid using commercially available precipitation methods. The EVs were initially characterized using UV/Vis spectroscopy, electron microscopy, and NTA. While the study is ongoing, initial results suggest that the concentration and size distribution of EVs differs significantly between blood fractions and ISF. Further immunochemical and sequencing characterization is ongoing. DISCUSSION/SIGNIFICANCE OF IMPACT: We present here the initial characterization of EVs isolated from dermal interstitial fluid. This appears to be the first report of EV characterization using ISF collection methods that do not perturb adjacent tissues (such as with blister or microdialysis methods). The present study lays a foundation for further examination of ISF-derived EVs in acute inflammatory disease such as cellulitis or infectious neuritis. This may enable minimally invasive diagnostics and new research tools to understand intercellular communication in living organisms with increased spatial and temporal resolution.