OBJECTIVES/SPECIFIC AIMS: The study aimed at assessing whether M. bovis BCG infection and inflammation exacerbates the development of atherosclerosis in Ldlr-/- mice. METHODS/STUDY POPULATION: Twelve-week old male Ldlr-/- mice (n=10) were infected with M. bovis BCG (0.3–3.0x10^6 colony-forming units (CFUs)) via the intranasal route, to simulate a natural respiratory route of infection. Mice were subsequently fed a western-type diet (WD) containing 21% fat and 0.2% cholesterol for 16 weeks. Age-matched uninfected Ldlr-/- mice (n=10) fed with an identical WD served as controls. Mice were euthanized after 16 weeks of WD to examine atherosclerotic lesions in aortic root sections and en face aorta using Oil Red O staining. Plasma cholesterol and triglyceride levels were measured by enzymatic assays and lipoprotein distribution was assessed using fast protein liquid chromatography. Because of the important role of T cells and monocytes in atherosclerosis development, we assessed these cell subsets in blood using flow cytometry at 8 and 16 weeks. Experiments were conducted in duplicate. We used unpaired Student’s t-test for group comparisons of numeric variables and flow cytometry data. RESULTS/ANTICIPATED RESULTS: M. bovis BCG infection significantly increased atherosclerotic lesions in en face aorta (plaque size per aorta area ratio; 0.15±0.13 vs. 0.06±0.02; P<0.01), but not in the aortic root. There were no significant differences in plasma cholesterol (1,160 mg/dL vs. 1,278 mg/dL; P = 0.36), triglycerides (340 mg/dL vs. 413 mg/dL; P = 0.28), or lipoprotein profiles between infected vs. uninfected mice at 16 weeks. M. bovis BCG increased circulating T lymphocytes (1,490 cells/uL vs. 1,227 cells/uL; P = 0.03) and monocytes (901 cells/uL vs. 414 cells/uL; P<0.01) within 8 weeks post-infection. When we assessed T lymphocyte subsets, M. bovis BCG infection increased total CD4+ T cell counts (556 cells/uL vs. 416 cells/uL; P<0.01) but not CD8+ T cells. No differences in the proportion of CD44+CD25+ activated T lymphocytes were noted between groups. When we assessed monocyte subsets, M. bovis BCG infection increased the numbers of Ly6Chigh (709 cells/uL vs. 362 cells/uL; P<0.01) and Ly6Clow (145 cells/uL vs. 35 cells/uL; P<0.01) monocytes. Infection was associated with an increased proportion of Ly6Clow monocytes at week 8 (17% vs. 8%; P<0.01) and week 16 (19% vs. 5%; P<0.01), compared to uninfected mice. DISCUSSION/SIGNIFICANCE OF IMPACT: M. bovis BCG infection increased the extent of atherosclerosis formation in the aortas of WD-fed hyperlipidemic Ldlr-/- mice after 16 weeks. Lipid profiles were similar between infected and uninfected mice, and therefore do not explain the observed differences in atherosclerosis. Compared to uninfected controls, M. bovis BCG-infected mice exhibited increased CD4+ T cell and monocyte driven inflammation. Interestingly, M. bovis BCG-infected mice had a higher proportion of non-classical Ly6Clow monocytes, suggesting a pro-atherogenic contribution of these cells in our model. Overall, our results support a pathogenic role of mycobacterial infection in atherosclerosis development and ASCVD.

OBJECTIVES/GOALS: a) Explore topics related to AMC CRP job titles, descriptions, and pre-requisites for hire b) Describe impact of COVID-19 on the AMC CRP workforce c) Discuss opportunities for improving diversity in the CRP workforce d) Discuss opportunities to enhance institutional staffing culture to retain CRP workforce METHODS/STUDY POPULATION: Qualitative data from a series of workshop breakout sessions and open-text survey materials focusing on AMC CRP recruitment, retention and diversity were analyzed to inform content and recommendations for clinical research job titles and descriptions, pre-requisites, diversity, and current needs. RESULTS/ANTICIPATED RESULTS: While certain institutions have established competency-based frameworks for job descriptions and career ladders, standardization remains generally lacking across CTSA hubs. Significant hiring needs have reached exponential proportions across hubs, unable to meet current and projected clinical research goals. Data confirmed an urgent need for closing gaps in clinical research workforce at AMCs, especially for improving diversity and equity of personnel. Improved collaboration with human resource departments, engagement with principal investigators, and overcoming both organizational and resource challenges were suggested strategies, as well as pipeline development via outreach to universities, community colleges, and high schools to raise awareness of the professional pathways for CRPs. DISCUSSION/SIGNIFICANCE: Based on input from 130 CRP leaders at 38 CTSA hubs and 4 IDeA sites evaluating data from 23 breakout transcripts and ~92 surveys from the Collaborative Conversations Unmeeting, new opportunities emerged during the analysis. The findings will be summarized in a 2022 Synergy manuscript including best practice benchmarking recommendations.

OBJECTIVES/GOALS: Retinal inflammation caused by the activation of resident macrophages (microglia) during diabetes exacerbates glial cell dysfunction, resulting in neuronal loss. The goal is to use rAAV gene therapy to deliver neuronal-derived fractalkine (FKN), minimizing inflammation and vascular damage in the diabetic retina. METHODS/STUDY POPULATION: The human microglial receptor (CX3CR1) binds to FKN, a protein that is expressed on neuronal membranes (mFKN), and undergoes constitutive cleavage to release a soluble domain (sFKN). Deficiencies in CX3CR1 or FKN showed increased microglial activation and elevated retinal pathology. To understand the mechanism by which mFKN and sFKN regulate microglia function, recombinant adeno-associated viruses (rAAVs) expressing mFKN or sFKN were delivered to intact retinas during diabetes. Markers of neuronal loss, vascular damage, and inflammation were analyzed. We hypothesize that the administration of rAAV-sFKN but not rAAV-mFKN will prevent vascular and neuronal damage, and improve visual function. RESULTS/ANTICIPATED RESULTS: rAAV-sFKN minimized microglial activation, blood vessel rupture, fibrinogen deposition, and prevented neuronal loss, compared to mice treated with rAAV-mFKN in a mouse model of diabetic retinopathy (DR). rAAV-sFKN treated mice showed improved visual acuity using a two-choice discrimination task through learning-based behavior. rAAV-sFKN treatment correlated with the success rate of the mice finding the reward based on their ability to distinguish visual cues. Future studies will test the effects of rAAV-sFKN and rAAV-mFKN on microglia inflammatory cytokine release, optic nerve damage and synaptic neurotransmission, peripheral immune responses, and transcriptomic changes in microglia during diabetes. DISCUSSION/SIGNIFICANCE: Current therapies for DR are ineffective in restoring vision. rAAVs-sFKN delivery appears to act as a neuroprotective approach in the diabetic retina. sFKN serves as an alternative pathway to implement translational and therapeutic approaches, minimizing pathology and improving visual function.

OBJECTIVES/GOALS: Acetaminophen (Tylenol, APAP) toxicity has been well documented and well explored over the last 50 years. However, there has been no investigation into identification of specific metabolites that can predict which patients will have adverse reactions to therapeutic doses of APAP. METHODS/STUDY POPULATION: 205 subjects recruited from the Denver, CO community received the highest recommended daily dosing of APAP, 4 grams, for 16 days. Subjects were grouped by 1) alanine aminotransferase (ALT) at any monitored time point above 60units/L (n = 20) vs 2) no increase in ALT at any time point (n = 185). Blood was collected at days 0, 4, 7, 16, and 31. Samples were run on ultra-high performance liquid chromatography mass spectrometry with 27 heavy-labeled standards for metabolites documented to be associated with APAP metabolism. Data will be analyzed to look for significant changes in metabolite and demographic variable expressions using t-tests, chi square and logistic regression, as appropriate. RESULTS/ANTICIPATED RESULTS: It is expected that there will be greater elevations of conjugated non-toxic APAP metabolites (APAP-glucuronide, APAP-sulfate) in subjects whose ALT did not elevate because of successful hepatoprotection. Conjugated APAP metabolites are expected to only be present in samples taken after APAP therapy initiation confirming exposure as compared to being predictive of toxic response. Increases in lactate and cysteine in pre-exposure samples would allow for prediction of APAP toxicity as they are expected to have increased expression in subjects whose ALT became elevated which is indicative of increased hepatic damage due to oxidative damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Identification of metabolites and/or demographic factors associated with toxic response to APAP prior to administration could advise APAP recommendations. Quantification of post-APAP administration metabolites would identify extent of successful hepatoprotective mechanisms.

OBJECTIVES/GOALS: To demonstrate a successful example of clinical and translational research at a busy veterinary teaching hospital and highlight a collaborative effort in Comparative Oncology between the University of Florida’s (UF) Colleges of Medicine and Veterinary Medicine. METHODS/STUDY POPULATION: The UF College of Veterinary Medicine (CVM) is a full-time teaching hospital with multiple departments actively recruiting patients for clinical trials. These departments include but are not limited to Oncology, Internal Medicine, Dermatology, Cardiology, and Emergency and Critical Care. The Oncology department collaborates with the doctors at the UF Health Cancer Center (UFHCC) as part of a Comparative Oncology Initiative, which has many ongoing canine and feline trials focusing on immunotherapy. RESULTS/ANTICIPATED RESULTS: As of August 2023, there are 60 clinical trials actively recruiting and enrolling patients at the UF CVM. 57% of these trials are interventional studies, while the other 43% are observational studies. The UFHCC Comparative Oncology Initiative has successfully completed one clinical trial focusing on canine gliomas; has 4 clinical trials that are actively recruiting patients, and 6 trials that are opening for enrollment in the near future. These studies focus on osteosarcoma, melanoma, and squamous cell carcinoma. It is anticipated that with continued successful collaborations, more clinical trials will be possible, and new treatment options will become available for not only veterinary patients but human patients as well. DISCUSSION/SIGNIFICANCE: Clinical and translational research is an important part of veterinary medicine to further patient care. Due to ongoing collaborative efforts, not only veterinary patients but also human patients will benefit from the research being conducted at the UF CVM.

ABSTRACT IMPACT: Recognizing Interdisciplinary Collaborative Research in Promotion and Tenure Processes OBJECTIVES/GOALS: Academic institutions have traditionally focused on individual achievements for promotion. We present our effort on identifying and measuring attitudes on promotion and tenure (PT) criteria that values and rewards interdisciplinary research (IR). We have developed a toolkit to facilitate the recognition of IR in PT processes. METHODS/STUDY POPULATION: Our group reviewed appointment, promotion and tenure (APT) policies from each of the six Health Science Schools and the College of Engineering at the University of Washington (UW) to assess language of objective criteria and attributes of IR to guide APT committees in the evaluation of interdisciplinary researchers. We surveyed faculty about their attitudes relating to IR within the context of promotion and tenure. Interviews of department chairs and administrators about institutional policies and infrastructure that supports or inhibits IR, and current best practices, were conducted. We have developed toolkits for junior faculty, department chairs, external reviewers, and APT committees to facilitate rewarding IR at promotion. RESULTS/ANTICIPATED RESULTS: Review of APT policies found criteria that recognizes IR for APT in three schools. 118 faculty responded to the survey (44% Professor, 26% Associate, and 37% eligible for APT committees). The majority of faculty reported they were currently conducting IR (95%), considered IR important (98%), and believed the UW faculty code should encourage IR (85%). Although a vast majority considered their units supportive of IR (>80%), less than half (43%) reported that their APT criteria provided examples that included participation in IR. Our survey also found that APT committees were challenged about best practices to reward IR, APT external reviewers struggle to evaluate individual vs team contributions, and individual faculty are challenged to describe contributions for APT within context of an interdisciplinary team. DISCUSSION/SIGNIFICANCE OF FINDINGS: IR is conducted and valued by UW faculty; however, current structures, policies, and APT code do not facilitate IR for promotion and tenure. We have developed a toolkit for promotion-eligible faculty, chairs, external reviewers, and APT committees to facilitate IR. Our goal is to modify UW faculty code and unit APT criteria to recognize and reward IR.

OBJECTIVES/SPECIFIC AIMS: The aim objective of this research work is the study of neurocognitive endophenotypes in the new classifications of mental disorders. METHODS/STUDY POPULATION: Neuropsychological tests,such as the UPSIT test were used. The population was composed by a sample of patients with a diagnosis of schizophrenia RESULTS/ANTICIPATED RESULTS: Olfactory discrimination is a potencial neurocognitive endophenotype in the study of schizophrenia research DISCUSSION/SIGNIFICANCE OF IMPACT: The aim impact of this research work is the study of Dimensional classifications of mental disorders.

OBJECTIVES/GOALS: The goal of this study is to investigate the potential independent relationship between epicardial adipose tissue (EAT) and cardiometabolic health in youth-onset type 2 diabetes (T2D) and explore changes in EAT as a potential mediator of changes in cardiometabolic health in response to vertical sleeve gastrectomy (VSG). METHODS/STUDY POPULATION: We will assess glycemic control, insulin sensitivity and secretion in youth with T2D before and 3 months after VSG. Fasting labs, anthropometrics, and a 4-hour, frequently sampled liquid mixed meal tolerance test (45g carbohydrates, 14g fat, and 14g protein) were performed. Calculations included glucose, insulin, and GLP-1 area under the curve (AUC), Matsuda Index, HOMA-IR, and oral disposition index (DI). These cardiometabolic outcomes will then be assessed for associations between total EAT volume, measured from cardiac MRI. RESULTS/ANTICIPATED RESULTS: Previous studies have shown that individuals with obesity have higher EAT than lean controls, and adults with T2D have even higher EAT than obese controls. Therefore, we anticipate that our participants will have higher volume of EAT than what has been reported in the literature and that they will have worsening cardiometabolic outcomes without MBS. Our anticipated results will include: Weight and BMI, hemoglobin A1c, diabetes medications, Matsuda Index, HOMA-IR, DI, and glucose and insulin AUC during an MMTT. Cardiac MRI's are being analyzed and will give total EAT volume and will be analyzed for correlations with the cardiometabolic outcomes of body composition, aortic stiffness, blood pressure, cardiac structure and function, as well as lipid panel and insulin sensitivity. DISCUSSION/SIGNIFICANCE: This study is the first to specifically assess EAT in adolescents with T2D. The assessment of EAT will be done with gold-standard MRI and correlated with cardiometabolic health assessed by gold-standard methods. Together, the results will give insight into EAT as a potential independent cardiometabolic risk factor in adolescents undergoing VSG.

OBJECTIVES/GOALS: Studies to improve uptake of Chronic Obstructive Pulmonary Disease Clinical Practice Guidelines (COPD CPG) have yielded inconsistent results. We hypothesized that using implementation science would facilitate rigorous site ‘diagnosis’, and promote effective contextual tailoring of COPD CPG, while piloting the use of telehealth for this. METHODS/STUDY POPULATION: The study was conducted in two Veterans Affairs primary care clinics located in a small sized city. A detailed formative evaluation was conducted using key informant interviews (with VA staff and veterans with COPD who received care at this location) and quantitative data. Multidisciplinary stakeholder group was engaged and strategies to address the determinants identified through the previous step were identified. Telehealth was strongly encouraged as the primary modality for implementing the COPD CPG and we are collecting pilot data on this. Tele-Facilitation, used as the meta-strategy was employed in conjunction with other strategies such as develop/distribute educational materials, tailor strategies, change record systems and revise professional roles. RESULTS/ANTICIPATED RESULTS: Primary Care at the VA is provided by Patient Aligned Care Teams (PACT-teams), where each team consists of multiple health professionals to provide collaborative care to the patient. Discussions with the multidisciplinary stakeholder team suggested that any implementation effort primarily focused on physician and nursing efforts was unlikely to succeed due to competing demands. A pharmacy-centric model that allowed for the PACT-team clinical pharmacist to address most of the COPD CPG (inhaler technique education/assessment, inhaler choice optimization, COPD-specific patient education, spirometry use, smoking and immunization) was developed and implemented with incorporation of telehealth (video visits and telephone). We will present pilot implementation outcomes using RE-AIM framework elements. DISCUSSION/SIGNIFICANCE: This use of implementation science to implement COPD CPG and novel use of telehealth has enormous potential for impact. Increasing reach/adoption by targeting primary care practices can help permeate quality care to the underserved population. This data will allow us to explore generalizability through wider scale implementation studies.

OBJECTIVES/GOALS: The detection of liver fibrotic changes at an early and reversible stage is essential to prevent its progression to end-stage cirrhosis and hepatocellular carcinoma. Liver biopsy, which is the current gold standard for fibrosis assessment, is accompanied by several complications due to its invasive nature in addition to sampling errors and reader variability. In this study, we evaluate the use of quantitative parameters extracted from hybrid ultrasound and photoacoustic imaging to detect and monitor fibrotic changes in a DEN rat model. METHODS/STUDY POPULATION: Liver fibrotic changes were induced in 34 Wistar male rats by oral administration of Diethylnitrosamine (DEN) for 12 weeks. 22 rats were imaged with B-mode ultrasound at 3 different time points (baseline, 10 weeks and 13 weeks) for monitoring liver texture changes. Texture features studied included tissue echointensity (liver brightness normalized to kidney brightness) and tissue heterogeneity. 12 rats were imaged with photoacoustic imaging at 4 time points (baseline, 5 wks, 10 wks, and 13 wks) to look at changes in tissue oxygenation. Hemoglobin oxygen saturation (sO2A) and hemoglobin concentration (HbT) in the right and left lobes of the liver were measured. 8 rats were used as controls. Liver tissue samples were obtained following 13 weeks from DEN start time for METAVIR histopathology staging of fibrosis. RESULTS/ANTICIPATED RESULTS: Texture features studied showed an increase with time in DEN rats. Normalized echointensity increased from 0.28 ± 0.06 at baseline to 0.46 ± 0.10 at 10 weeks (p < 0.0005) and 0.53 ± 0.15 at 13 weeks in DEN rats (p < 0.0005). In the control rats, echointensity remained at an average of 0.25 ± 0.05 (p = 0.31). Tissue heterogeneity increased over time in the DEN-exposed rats from a baseline of 208.7 ± 58.3 to 344.6 ± 52.9 at 10 weeks (p < 0.0005) and 376.8 ± 54.9 at 13 weeks (p = 0.06) however it stayed constant at 225.7 ± 37.6 in control rats (p = 0.58). The quantitative analyses of the photoacoustic signals showed that blood oxygen saturation significantly increased with time. At 5 weeks sO2AvT increased by 53.83 % (± 0.25), and HbT by 35.31 % (± 0.07). Following 10 weeks of DEN; sO2AvT by 92.04 % (± 0.29), and HbT by 55.24 % (± 0.1). All increases were significant p < 0.05. In the 13th week, however, the values of all of these parameters were lower than those in the 10th week, however, the decrease was statistically insignificant. DISCUSSION/SIGNIFICANCE OF IMPACT: Quantitative features from B-mode ultrasound and photoacoustic imaging consistently increased over time corresponding to hepatic damage, inflammation and fibrosis progressed. The use of this hybrid imaging method in clinical practice can help meet the significant need for noninvasive assessment of liver fibrosis.

ABSTRACT IMPACT: This analysis helps disentangle various paths to translational collaboration, with implications for departmental capacity building and support. OBJECTIVES/GOALS: Studies that bridge research collaboration networks are cross-disciplinary and translational. We explored the characteristics of researchers and their collaboration patterns in bridging research grants at University of Rochester School of Medicine and Dentistry. METHODS/STUDY POPULATION: the database of sponsored research grants from 2011 to 2018, obtained from an internal University database was transformed into a two-mode network of grant-to-investigator. Grants at 90th percentile and above of normalized two-mode betweenness centrality were defined as ‘bridging grants’. For each grant we extracted the gender, academic rank, academic degree, affiliating department, and centrality-status (being at 75th percentile of degree centrality in one-mode collaboration network) of the Principal Investigator (PI), as well as the number of co-investigators (CI) and the existence of central actor(s) in the research team. RESULTS/ANTICIPATED RESULTS: Out of 2491 sponsored grants, 250 were ‘bridging grants’. The significant predictors of bridging were centrality of PI, existence of central CI(s), PI holding PhD, and larger number of CIs. The PI’s academic rank (being full professor) and gender were not significant predictors. Among bridging grants 79 included both central PI and CIs (central actors group) and 60 included no central actor on the team. In the latter group, more PIs were clinical faculty and fewer were full professors. Network analysis of affiliating departments showed that Medicine was the prominent actor in the central actors group, while the network of no-central actor group was more fragmented with Neurology as central. DISCUSSION/SIGNIFICANCE OF FINDINGS: Widely recognized researchers are more likely to collaborate with each other in bridging studies possibly marginalizing less experienced peers. Bridging grants led by less central researchers, often clinician-scientists, may thrive where supportive culture and departmental facilities exist.

Objectives/Goals: This work aims to identify functional brain networks that differentiate opioid use disorder (OUD) subjects from healthy controls (HC) using machine learning (ML) analysis of resting-state fMRI (rs-fMRI). We investigate the default mode network (DMN), salience network (SN), and executive control network (ECN), as well as demographic features. Methods/Study Population: This work uses high-resolution rs-fMRI data from a National Institute on Drug Abuse study (IRB #HM20023630) with 31 OUD and 45 HC subjects. We extract rs-fMRI blood oxygenation level-dependent (BOLD) features from the DMN, SN, and ECN. The Boruta ML algorithm identifies statistically significant features and brain activity mapping visualizes regions of heightened neural activity for OUD. We conduct fivefold cross-validation classification experiments (OUD vs. HC) to assess the discriminative power of functional network features with and without incorporating demographic features. Demographic features are ranked based on ML classification importance. Follow-up Boruta analysis is performed to study the medial prefrontal cortex (mPFC), posterior cingulate cortex, and temporoparietal junctions in the DMN. Results/Anticipated Results: Boruta ML analysis identifies the DMN as the most salient functional network for differentiating OUD from HC, with 33% of DMN features found significant (p < 0.05), compared to 10% and 0% for the SN and ECN, respectively. The Boruta ML algorithm identifies age and education as the most significant demographic features. Brain activity mapping shows heightened neural activity in the DMN for OUD. The DMN exhibits the greatest discriminative power, with a mean AUC of 69.74%, compared to 47.14% and 54.15% for the SN and ECN, respectively. Fusing DMN BOLD features with the most important demographic features improves the mean AUC to 80.91% and the F1 score to 73.97%. Follow-up Boruta analysis highlights the mPFC as the most important functional hub within the DMN, with 65% significant features. Discussion/Significance of Impact: Our study enhances the understanding of OUD neurobiology, identifying the DMN as the most significant network using ML rs-fMRI BOLD feature analysis. Ethnicity, education, and age rank are the most important demographic features and the mPFC emerges as a key functional hub for OUD. Future research can build on these findings to inform treatment of OUD.

OBJECTIVES/GOALS: Black women participate in the least amount of physical activity in the U.S., and determining methods to increase motivation and consistent exercise are warranted. Purpose: This study assessed differences in psychological and behavioral outcomes in young Black women in a culturally-tailored and theory-based resistance exercise (RE) study. METHODS/STUDY POPULATION: Women (M= 22.7 ± 3.6y) were randomized to the standard exercise group (SEG; n= 6) or motivational exercise group (MEG; n= 8), and completed 10 weeks of RE with a Black woman trainer and 11 weeks of unsupervised RE. The MEG discussed and received text messages about exercise education, self-regulation, autonomy, and competence. Motivation was measured by the Behavioral Regulation in Exercise Questionnaire-3, the Physical Activity Self-Regulation Scale-12 measured self-regulation, and the Basic Psychological Needs in Exercise Scale measured competence, autonomy, and relatedness. Adherence was calculated as # of completed/total sessions, and retention was the percent of women who completed ≥2 days/week of unsupervised RE. An ANOVA and Bonferroni post hoc analyses were used to determine significant findings. RESULTS/ANTICIPATED RESULTS: Significant time effects were found for intrinsic motivation (Λ = .691, F [11, 2]= 17.494, p< .001), basic psychological needs (Λ = .951, F [11, 2]= 22.691, p< .001), and self-regulation (Λ = .881, F [10, 2]= 40.942, p< .001), but no main interactions. Significant increases occurred from pre-testing to 3-mo follow-up for intrinsic motivation (pre: 1.80 ± .90 vs 3mo: 2.71 ± .63, p= .002), competence (pre: 1.98 ± .93 vs 3mo: 3.55±.81, p< .001), autonomy (pre: 2.14 ± .75 vs 3mo: 3.81 ± .74, p< .001) relatedness (pre: 2.78±1.60 vs 3mo: 4.32 ± .58, p< .001), and self-regulation (pre: 18.4 ± 2.88 vs 3mo: 33.79 ± 3.11, p< .001). Adherence rates for both groups were 93%. Retention rates were 33% for SEG and 25% for MEG. MEG had 38% complete RE 1 day/week opposed to none in SEG. DISCUSSION/SIGNIFICANCE: Ten weeks of culturally-tailored, supervised RE showed efficacy in significantly increasing motivation and behavioral practices to help sustain exercise. Future research should further explore strategies to use during unsupervised training to help increase exercise adherence in young Black women.

OBJECTIVES/GOALS: This study investigates whether localization of high mobility group box 1 (HMGB1) controls inflammatory signaling and DNA damage response in human keratinocytes, the cell of origin for squamous cell carcinoma (SCC). SCC is especially metastatic in chronic wounds, burns, and in patients with recessive dystrophic epidermolysis bullosa (RDEB). METHODS/STUDY POPULATION: We used CRISPR/Cas9 gene-editing to knock out HMGB1 in a keratinocyte line, p16INK4a-negative keratinocytes immortalized by ectopic hTERT expression (N/TERT-2G [46,XY]). Following gene editing, clonal keratinocyte populations were screened for knockout by PCR followed by TIDE analysis (Tracking of Indels by Decomposition) to identify indels that would result in a frameshift mutation. Total cell lysates for each clonal population were analyzed by immunoblot and immunofluorescence for HMGB1 protein. These cells will be used to assay for DNA damage sensitivity in the presence of genotoxic agents (etoposide, ultraviolet radiation, γ-irradiation). A lentiviral vector will then be used to express mutant forms of HMGB1 that localize to the nucleus (C23/45S) or cytoplasm (C106S) and DNA damage assays repeated. RESULTS/ANTICIPATED RESULTS: We have confirmed by sequencing, immunoblot, and immunofluorescence that HMGB1 is knocked out in a clonal population of N/TERT-2G human keratinocyte cells. We anticipate that cells with a complete absence of HMGB1 will have high sensitivity to DNA damaging agents, but little change in inflammatory signaling. We also expect that cells expressing mutant HMGB1 that localizes exclusively to the cytoplasm will demonstrate an increased sensitivity to DNA damage relative to wild-type controls, while mutant HMGB1 that localizes exclusively to the nucleus will be protected from DNA damage caused by exposure to genotoxic agents. DISCUSSION/SIGNIFICANCE: HMGB1 is a nuclear protein and damage associated molecular pattern that is elevated systemically in patients with RDEB, many of whom will go on to develop metastatic squamous cell carcinoma. The experiments described here investigate whether HMGB1 plays a mechanistic role in skin carcinogenesis via regulation of the DNA damage response.

The recipients of NIH’s Clinical and Translational Science Awards (CTSA) have worked for over a decade to build informatics infrastructure in support of clinical and translational research. This infrastructure has proved invaluable for supporting responses to the current COVID-19 pandemic through direct patient care, clinical decision support, training researchers and practitioners, as well as public health surveillance and clinical research to levels that could not have been accomplished without the years of ground-laying work by the CTSAs. In this paper, we provide a perspective on our COVID-19 work and present relevant results of a survey of CTSA sites to broaden our understanding of the key features of their informatics programs, the informatics-related challenges they have experienced under COVID-19, and some of the innovations and solutions they developed in response to the pandemic. Responses demonstrated increased reliance by healthcare providers and researchers on access to electronic health record (EHR) data, both for local needs and for sharing with other institutions and national consortia. The initial work of the CTSAs on data capture, standards, interchange, and sharing policies all contributed to solutions, best illustrated by the creation, in record time, of a national clinical data repository in the National COVID-19 Cohort Collaborative (N3C). The survey data support seven recommendations for areas of informatics and public health investment and further study to support clinical and translational research in the post-COVID-19 era.