OBJECTIVES/GOALS: The goal of this study is to describe the implementation of a formal research mentoring training program at Miami CTSI in collaboration with University of Colorado CTSI, to build effective mentor-mentee relationships that help build successful research careers for junior faculty. METHODS/STUDY POPULATION: The Miami CTSI Research Mentoring Training is a three-part series for early stage faculty and their mentors to strengthen mentoring skills and build successful relationships. The curriculum uses evidence-based strategies and is modeled after University of Colorado CTSIs CO-Mentor Training Program. The pilot training was conducted as a train-the-trainer program with subsequent workshops led by Miami CTSI faculty. The program has mentee-only, mentor-only, and joint sessions covering topics such as networking skills, managing financial aspects of an academic career, and career mapping. To assess program effectiveness, pre/post surveys were conducted and follow up surveys are planned. Data collected will assess participant diversity, research productivity, and mentoring relationship status. RESULTS/ANTICIPATED RESULTS: A total of 49 mentor-mentee teams from 18 departments participated in the training over two cohorts. An upcoming third cohort will be included in the analysis. Overall, 100% mentors and 86% mentees reported the overall value of the program as positive. Post-training, mentees reported an increase in confidence in creating career development plans and articulating career goals. Mentors reported an increase in confidence in providing guidance tailored to mentees needs. Mentees reported improvement in their knowledge of developing personal narratives and handling financial aspects of research; both mentors and mentees reported improvement in insights into achieving work-life balance. Follow up survey results will provide insight into the evolution of the mentoring relationships. DISCUSSION/SIGNIFICANCE: Miami CTSIs mentoring training demonstrated successful participation and positive feedback from mentors and mentees and is poised to become a critical component of the Miami CTSIs research career development pipeline.

OBJECTIVES/SPECIFIC AIMS: The overall objective of this study is to develop a novel, clinically-relevant, image-guided mouse model for radiation-induced cardiotoxicity, which can be used to gain insight into clinically-targetable, pathophysiologic mechanisms of cardiac injury in thoracic radiotherapy patients. METHODS/STUDY POPULATION: Photon or sham radiation will be administered at differential doses to a defined portion of the heart and/or lungs of C57BL/6 female mice using micro-CT visualization of the heart with Xstrahl’s MuriSlice Software applied to the Small Animal Radiation Research Platform (SARRP). Cardiac and lung segments from a subset of mice will be harvested at specific time points for confirmation of radiation targeting, local apoptosis assessment, and evaluation of fibrosis and vascular tissue morphology. Quantitative echocardiography, myocardial 18F-fluorodeoxyglucose positron emission tomography computed tomography (18F-FDG PET/CT), and myocardial perfusion imaging (MPI) with Technicium-99 (Tc-99) sestamibi will be implemented to identify sensitive imaging measures of cardiac injury and asses myocardial mechanics, inflammation, and perfusion deficits, respectively. Concurrently, a multiparametric analysis will be conducted to identify novel, circulating biomarkers of cardiotoxicity. RESULTS/ANTICIPATED RESULTS: We hypothesize that a clinically-relevant mouse model can be generated by the in situ, focal irradiation of a portion of heart and/or lung tissue segments, and can be used to elucidate molecular mechanisms of radiation-induced cardiac damage. We anticipate time-dependent and dose-dependent, focal histopathologic changes in the mouse heart, with cardiac fibrosis development, vascular damage, and cellular apoptosis in irradiated mice. Additionally, we anticipate that our mouse model of focal heart irradiation will reveal radiologic and biochemical changes that can be used to characterize and predict radiation-induced cardiac injury. Specifically, we expect our quantitative echocardiography, FDG-PET, and MPI parameters to identify and characterize cardiac damage that topographically matches histopathological analysis, and expect levels of select biochemical markers to differentially vary with time. DISCUSSION/SIGNIFICANCE OF IMPACT: Our mouse model of radiation-induced cardiotoxicity has the potential to shift current preclinical research paradigms to more closely mimic the radiation plans most commonly administered in clinical practice. The primary technologic innovation to be developed here is the use of the SARRP to deliver image-guided, in situ, focal radiation to a defined portion of the mouse heart. From a conceptual perspective, we propose a novel approach for phenotyping radiation-induced cardiac damage in patients undergoing chest radiation therapy, integrating sensitive radiomic and biochemical markers into a predictive model of cardiotoxicity.

OBJECTIVES/GOALS: Nearly all thoracic aortic aneurysm patients suffer from hypertension leading to elevated wall tension and abnormal extracellular matrix remodeling. PTSD patients have higher blood pressure both at rest and in response to stimuli. Although stress is associated with cardiovascular disease, the exact mechanism linking the two is still unknown. METHODS/STUDY POPULATION: Adult C57BL/6 mice underwent a PTSD induction protocol consisting of inescapable foot shock followed by single prolonged stress. The mice were assessed incrementally for their PTSD-like phenotype using specific behavioral tests chosen to assess for each of the human criteria of PTSD according to the DSM-V. Tail cuff blood pressure measurements were taken serially throughout the 16-week protocol. At terminal study, thoracic aortic diameter measurements were obtained through digital microscopy and plasma was harvested for cytokine analysis. Thoracic aortic aneurysms (TAA) were induced through periadventitial application of a calcium chloride solution on the descending thoracic aorta in BPH/2J and BPN/3J adult mice. The thoracic aortic diameter was measured at terminal study through digital microscopy. RESULTS/ANTICIPATED RESULTS: Using our PTSD-like mouse model we have demonstrated that PTSD-like mice have significantly higher systolic blood pressure following a reminder of the traumatic event than control mice recapitulating the human phenotype. They also had increased plasma proinflammatory cytokines and larger thoracic aortic diameters than control mice. Although the increased thoracic aortic diameter is not an aneurysm, it suggests ECM remodeling is occurring predisposing the aorta to aneurysm formation. Finally, we have shown that in neurogenic hypertensive mice, TAA formation was accelerated by 12 weeks with roughly 70% dilation at 4 weeks post-TAA induction surgery as compared to roughly a 20% dilation in control mice. DISCUSSION/SIGNIFICANCE: Altogether, these studies reinforce the link between stress and TAA development, and our mouse model will allow for the underlying mechanism to be elucidated. Better understanding of the mechanism linking PTSD and TAA will allow for the creation of novel therapeutics to treat PTSD symptoms while also delaying TAA progression.

ABSTRACT IMPACT: The key to advancing precision medicine is to deepen our understanding of drug modes-of-action (MOA). This project aims to develop a novel method for predicting MOA of potential drug compounds, providing an experimental and computational platform for more efficient drug discovery. OBJECTIVES/GOALS: To develop (1) a targeted CRISPR-Cas9 chemical-genetic screen approach, and (2) a computational method to predict drug mode-of-action from chemical-genetic interaction profiles. METHODS/STUDY POPULATION: Screening drugs against a gene deletion library can identify knockouts that modulate drug sensitivity. These chemical-genetic interaction (CGI) screens can be performed in human cell lines using a pooled lentiviral CRISPR-Cas9 approach to assess drug sensitivity/resistance of single-gene knockouts across the human genome. A targeted, rather than genome-wide, library can enable scaling these screens across many drugs.

CGI profiles can be derived from phenotypic screen readouts. These profiles are analogous to genetic interaction (GI) profiles, which represent sensitivity/resistance of gene knockouts to a second gene knockout rather than a drug. To computationally predict a drug’s genetic target, we leverage the property that a drug’s CGI profile will be similar to its target’s GI profile. RESULTS/ANTICIPATED RESULTS: Five proof-of-principle screens will be conducted with compounds that have existing genome-wide profiles and well-characterized MOA. I will generate CGI profiles for these five compounds and identify genes that are drug-sensitizers or drug-suppressors. I will then evaluate whether targeted library screens can recapitulate the CGIs found in genome-wide screens. Finally, I will develop a computational tool to integrate these CGI profiles with GI profiles (derived from another project) to predict gene-level and bioprocess-level drug targets. These predictions (from both targeted and genome-wide profiles) will be benchmarked against a drug-target and drug-bioprocess standard. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work will develop a scalable, targeted chemical-genetic screen approach to discovering how putative therapeutics work. The targeted screen workflow provides a method for higher-throughput drug screening. The computational pipeline provides a powerful tool for exploring the MOA of uncharacterized drugs or repurposing FDA-approved drugs.

OBJECTIVES/SPECIFIC AIMS: Aim 1: To compare frozen MOM to fresh MOM over time as an agent to inoculate DHM and measure the enrichment of commensal microbes and their beneficial bioactive components similar to MOM. Hypothesis: Frozen or fresh MOM inoculated in DHM will produce similar microbial content to MOM over time allowing for the production of beneficial bacterial compounds that may contribute to host immune response. Aim 2: To determine the effect of MOM storage (fresh vs frozen) on the expansion of bioactive components from live microbiota in DHM. Hypothesis: Both fresh and frozen MOM will produce similar results when inoculated into DHM to restore the microbial content (including their bioactive components) similar to each MOM sample. Aim 3: To compare the microbiome found in a mother’s MOM to the microbiome in her infant’s stool. Hypothesis: The mother/infant pair will share a common microbiome between the mother’s MOM and her infant’s stool. METHODS/STUDY POPULATION: Subjects will include 12 pump-dependent mothers of infants born < 34 weeks gestation admitted to the University of Florida Health Shands Hospital, Neonatal Intensive Care Unit (NICU). Inclusion criteria consists of mothers expressing over 100 ml of MOM per day, producing at least 45 ml of MOM at an expression session, at least 18 years of age, and speak English. Mothers are excluded if they have taken antibiotics within 3 days of sample collection, are HIV+, or delivered an infant who has a chromosomal abnormality or is severely ill. An expressed MOM sample will be collected and divided into two fractions: (A) fresh and (B) frozen at -20C for 24 h. The fresh fraction (A) will be processed immediately while the frozen fraction (B) will be processed after 24 h. Each MOM will be inoculated in DHM at dilutions of 10% and 30% and incubated at different time points: 0 h (T0), 2 h (T2), 4 h (T4) at 37°C. At each time point, total viable cell counts as well as microbiome analysis through 16S ribosomal shotgun sequencing will be performed and compared for differences. Bacteria isolated from each MOM will be saved, identified through 16S ribosomal sequencing and grown in culture for future studies. Fecal samples from each corresponding infant will be collected within 48 h after collection of the MOM sample. Stools will be homogenized and subjected to DNA extraction to perform 16S ribosomal shotgun sequencing. Microbiome analysis will be conducted, compared between fecal samples as well as with the microbiome of the MOM. RESULTS/ANTICIPATED RESULTS: Study is ongoing. We anticipate similar results with fresh or frozen MOM to that of a previous pilot study, where enriched microbiota similar to MOM was found when fresh MOM was inoculated and incubated in DHM. The microbiome analysis of the infant fecal samples may illustrate the influence that the microbiome of the MOM may have on the development of the infants’ gastrointestinal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: The purpose of this study is to provide evidence on the ability, timing, and efficacy of inoculating DHM with fresh and frozen MOM. Study results will inform future studies to support the implementation of an inoculation procedural protocol to be used in clinical practice and human milk banking. The description of the MOM microbiome, as well as the gastrointestinal microbiome, will expand scientific knowledge on the role breast milk has on the origins of health and disease.

OBJECTIVES/GOALS: Mammals require iron for hemoglobin, respiration, immunity and as cofactor in enzymes. But free iron is toxic from the production of reactive oxygen species. Ferroportin is the sole exporter of cellular iron and it crucially determines cellular and systemic iron levels. Labile iron must be tightly regulated. This requires structural understanding. METHODS/STUDY POPULATION: We built structure of human ferroportin (FPN1) using the ab ignition prediction approaches of Rosetta/Robetta and by comparative modeling with distance restraints in MODELLER. Templates selected were from solute carrier protein families of distantly related orthologs and homologs including a proton coupled peptide transporter (PDB ID: 4IKV) and the bacterial iron transporter in outward-open and inward-open states, (PDB ID: 5AYM, 5AYO). Each model was validated by experimental mass spectrometry data. The energy minimized structural model was inserted into a lipid bilayer, placed in a rectangular simulation box, covered with TIP3P water solvent balanced with counterions and conditioned. Finally, we carried out 350 nanoseconds molecular dynamics simulations. RESULTS/ANTICIPATED RESULTS: Our first model of FPN1 (571aa), using Rosetta/Robetta ab initio approach, resembles the structure of the proton-dependent transporter, POT and consists of 12 transmembrane helices. The membrane spanning helices veer away from the orientation in the structure of 4IKV. The alternate model using MODELLER and the method of satisfaction of constraints, returned one template, the structure of Bdellovibrio bacteriovorus iron (Fe2+) transporter homolog (5AYN, 440aa) with sequence identity of 19%. Aligning FPN1 on the template sequence incorporating structural information revealed better conservation (29%). This model also comprises 12 transmembrane helices in two bundles separated by a large intracellular loop. The iron binding site predicted in both models match the structures of distant bacterial homologs. DISCUSSION/SIGNIFICANCE OF IMPACT: We are using these experimentally verified structures and functional data to answer questions about the mechanism of ferroportin iron transport, structural dynamics and the significance of mutations in ferroportin seen in different populations, especially the Q248H mutation found in Africans and black Americans with moderate to high prevalence.

OBJECTIVES/GOALS: Targeting the V-domain immunoglobulin suppressor of T cell activation (VISTA) signaling pathway has been suggested as a promising approach for overcoming resistance to current immune checkpoint therapies in advanced cancer. This review will synthesize the rapidly-expanding literature on VISTA protein expression on prognosis in various cancers. METHODS/STUDY POPULATION: To determine the prognostic significance of high VISTA expression across treatment-naïve malignant tumors, a systematic review and meta-analysis will be performed of published cohort studies which measured VISTA protein expression on solid tumors. Primary and secondary outcome endpoints of overall survival (OS) and disease-specific survival (DSS) will be compared across cohort studies using a random effects model to calculate pooled hazard ratios (HRs) for each time-to-event end point with 95% CIs. For articles that only provide Kaplan-Meier (KM) curves, the Engauge Digitizer software will be used to measure the time and survival probability coordinates on the KM curves to estimate the HRs. Correlations of VISTA expression and clinicopathological characteristics will be evaluated by pooled risk ratios. RESULTS/ANTICIPATED RESULTS: A search of 4 electronic databases including Pubmed, Embase, Web of Science and Cochrane resulted in 5578 publications of which 66 containing a broad spectrum of malignant solid tumors will undergo full-text review for study inclusion. Tumor types most represented with at least 3 articles include lung, pancreas, skin, head & neck, colorectal, mesothelioma, cervix, soft tissue, breast, liver and ovarian. Our working hypothesis is that the pooled HR for high VISTA expression on overall survival will be approaching 1.0 given conflicting reports across the cancer literature. Risk of bias will be assessed across studies. Quantifications of heterogeneity will be assessed by visual exploration of forest plots as well as by multiple statistical metrics including the Q statistic and the I²coefficient. DISCUSSION/SIGNIFICANCE: The results of this systematic review and meta-analysis will provide a more comprehensive understanding of VISTA’s prognostic role both across all malignant tumors and for subgroups of similar tumor types which may impact the types of tumors and tumor microenvironments selected for early trials of anti-VISTA therapy.

OBJECTIVES/GOALS: Evaluate the use of IC to improve stroke survivors’ capacity for reactive stepping and adapt their gait cycles in response to a difficult walking environment. We hypothesize that IC will improve stroke survivors’ protective stepping response via improvements in muscle activation and motor learning METHODS/STUDY POPULATION: Stroke survivors have an impaired capacity for protective stepping. Decreased paretic muscle activation results in increased reaction time and reduced force generation. Ischemic conditioning (IC) is a vascular stimulus which improves motor performance in chronic stroke. It is performed by delivering transient, intermittent bouts of ischemia to a limb. It has been demonstrated that IC increases muscle activation post-stroke. 9 chronic stroke survivors completed 3 testing sessions and 7 intervention sessions. Participants walked on an instrumented treadmill and were perturbed unilaterally every step at the waist via a cable pulley system. Kinetic and kinematic data were collected. Step width was measured as the difference in position of the heel markers at the instant of heel strike in the frontal plane. RESULTS/ANTICIPATED RESULTS: After one and seven sessions of IC, controls did not alter their responses from baseline testing, but stroke survivors increased their step width by an average of 15% and 23% respectively. DISCUSSION/SIGNIFICANCE: Ischemic conditioning may be a useful intervention to improve stroke survivors’ ability to adapt their paretic foot placement in response to lateral perturbations during gait. Interventions which optimize muscle activation and neural adaptation could significantly improve balance post-stroke.

Recent reports on the burden of cardiovascular disease (CVD) in the USA indicate that despite significant declines in CVD mortality in the late 20th century, this decline is now decelerating and may be worsened by inequalities in health care. Social factors contribute to most of the cardiovascular health disparities documented to date. Hispanics/Latinos and African-Americans share a higher prevalence of cardiovascular risk factors and experience higher rates of poverty and social stressors than non-Hispanic Whites. We propose that the use of social and behavioral data beyond basic and sometimes loose identifiers of race/ethnicity, educational attainment, and occupation would inform clinical practice and greatly facilitate the provision of adequate guidance and support to patients regarding continuity of care, adherence to medications and treatment plans, and engagement of participants in future research. This perspective briefly highlights factors deemed to be critical for the advancement of Hispanic/Latino health and delineates pathways toward future applications.

OBJECTIVES/GOALS: The New Jersey Alliance for Clinical & Translational Science conducted an observational study of current clinical research training to establish a comprehensive infrastructure. The goal was to develop a cross training catalogue of available resources for trainees to fill gaps in their Clinical Research training. METHODS/STUDY POPULATION: The focus of this observational study is the evaluation of (1) available clinical research training programs, (2) training areas aligned with the Joint Task Force competency domains, (3) specific target audiences who utilize trainings offered nationally, and (4) availability of resource toolboxes. This assessment was extracted by clicking on each of the hubs Education & Training and Resources tabs. The determination of the training areas and competency skills that aligned with the JTFs eight domains was confirmed by reviewing the course description on each website. RESULTS/ANTICIPATED RESULTS: CTSA hub website analysis of each hub revealed that each CTSA hub region has their own regional focus on training areas targeted for specific audiences. The validation of the NJ ACTS Education and Offering Inventory study allowed authors to categorize available training programs which significantly showed that Basic Training Programs targeted 22% of Undergraduate Students, Community Engagement Training Programs targeted 15% of Early Career Faculty, Experiential Training targeted 20% of Early Career Faculty and Undergraduate Students, Scholarly & Certificate Training targeted 20.5% of Graduate Students, and Graduate/Post Graduate Programs targeted 22.5% of Graduate Students and KL2 scholars. DISCUSSION/SIGNIFICANCE: This information helps build the infrastructure of a National Cross-Training Resource Platform in aid to encourage trainees to take advantage of available trainings across the CTSA Consortium.

OBJECTIVES/GOALS: Human placentation requires complex coordination between maternal and fetal cell types but remains incompletely understood. We hypothesize that uterine natural killer (uNK) cells, an immune cell type that increases in abundance during the implantation window, is essential for appropriate implantation and placentation. METHODS/STUDY POPULATION: We plan to examine stromal cell (SC) decidualization, spiral artery remodeling, and EVT invasion, processes vital for early pregnancy establishment, in the presence or absence of secretory phase uNK cells. Fetal extravillous trophoblasts (EVTs) will be isolated from first trimester pregnancy tissue; maternal SCs, endothelial cells (ECs) and uNK cells will be obtained from secretory phase uterine tissue. SCs will be placed in monoculture and coculture with uNK cells and prolactin will be measured to evaluate decidualization. To study EVT invasion, we will utilize our novel “implantation-on-a-chip” device to determine how addition of uNK cells affects EVT migration through a collagen-matrigel matrix. In this system, we will also examine spiral artery remodeling with or without uNK cells via TUNEL staining. RESULTS/ANTICIPATED RESULTS: We anticipate that uNK cell addition to SCs will lead to a significant increase in SC prolactin levels, suggesting a role of uNK cells in endometrial decidualization. In vitro, we expect the addition of uNK cells will increase EC apoptosis and promote EVT invasion. DISCUSSION/SIGNIFICANCE OF IMPACT: Although decidual NK cells are known to participate in placentation, the role of pre-pregnancy uNK cells is unknown. uNK cell involvement in processes important for the earliest stages of pregnancy would provide a potential marker for abnormal placentation and offer avenues for intervention to decrease placentation associated perinatal morbidity.

OBJECTIVES/SPECIFIC AIMS: Thoracic endovascular aortic repair (TEVAR) is more effective in remodeling the dissected aorta in acute versus chronic type B aortic dissection (TBAD). It has been hypothesized that this is due to differences in dissection flap biomechanical and structural properties but has not been confirmed in explanted human aortic tissue. We aimed to characterize and compare differences in tissue biomechanics and microstructure between acute and chronic dissection flaps that may underlie these findings. METHODS/STUDY POPULATION: Dissection flaps were obtained at time of operative repair for patients presenting for open aortic replacement to treat acute type A (ACUTE, n=7) or chronic type B (CHRONIC, n=7) aortic dissection. Given that the current treatment modality for acute complicated TBAD is TEVAR, it was not feasible to acquire acute TBAD flaps for analysis. Tissues were cryopreserved and subjected to biaxial tensile testing in the circumferential and longitudinal directions. Stiffness was quantified by the tangent modulus (TM) in the low and high linear regions of the compiled equibiaxial response curves for each cohort. Extensibility was defined as the intersection of the fitted line from the high linear region with the x-axis, and the degree of anisotropy (DA) was defined as the mean absolute percentage error of the strains in both directions. Flap architecture and collagen fiber organization were also compared between groups using two-photon microscopy. RESULTS/ANTICIPATED RESULTS: Average age of dissection flaps were 3.4±3.4 days in ACUTE and 1,868.7±1,354.0 days in CHRONIC (p=0.011). There were no differences in age, co-morbidities, maximum aortic diameter, and aortic wall thickness. ACUTE exhibited an anisotropic stress-strain response with increased extensibility longitudinally than circumferentially (0.18 vs. 0.09, p=0.022, DA=0.67) while CHRONIC demonstrated an isotropic response with similar extensibility in either direction (0.11 vs. 0.12, p=0.606, DA=0.26). CHRONIC and ACUTE had comparable stiffness in the circumferential direction (TMlow 439.92 vs. 541.08, p=0.729, and TMhigh 1585.19 kPa vs. 1869.35 kPa, p=0.817). In the longitudinal direction, CHRONIC was significantly stiffer than ACUTE (TMhigh 8347.61 kPa vs. 1201.34 kPa, p=0.049) (FIGURE). Microscopy corroborated these findings with greater collagen fiber organization circumferentially than longitudinally in ACUTE and increasing fibrosis, collagen predominance, and straightening of collagen fibers in CHRONIC. DISCUSSION/SIGNIFICANCE OF IMPACT: Compared to ACUTE, CHRONIC exhibited loss of anisotropy with increased tissue stiffness in the longitudinal direction. Increased dissection flap fibrosis and decreased compliance may explain the worse outcomes for aortic remodeling after TEVAR in chronic TBAD. This study offers biomechanical support for early TEVAR in the acute phase of uncomplicated TBAD.

OBJECTIVES/GOALS: Donor hearts are transported in cold storage (CS) and undergo ischemia-reperfusion injury (IRI) when transplanted. IRI injures microvascular endothelial cells (EC), heightens the immune response, and has been associated with increased autophagy. We aim to understand the changes in autophagy during CS and IRI and its impact on EC immunogenicity. METHODS/STUDY POPULATION: To study autophagy changes during IRI, immunoblotting for autophagy markers was performed in mouse cardiac ECs (MCECs) lysates. MCECs were in a cold preservation solution in a hypoxic chamber for 6 hours(h) and warm conditions with culture medium for 24 h. MCECs, under standard conditions, served as controls. Secreted interferon-gamma (IFN-γ) levels were quantified via ELISA to study autophagy and EC immunogenicity. MCEC-sensitized CD8+ T-cells were isolated from C57BL/6 spleens and co-cultured with MCECs pre-treated for 16 h with rapamycin or starvation, autophagy inducers, or chloroquine, an autophagy inhibitor under normal or IRI conditions. MCECs without any treatment served as controls. RESULTS/ANTICIPATED RESULTS: To determine autophagy levels in IRI, immunoblotting of MCEC lysates revealed a significant increase (P<0.01) in the established autophagy marker, LC3, at early time points post-reperfusion compared to NT conditions, indicating more autophagosome formation during CS and IRI. To assess the role of autophagy in EC immunogenicity, the co-culture experiment revealed that autophagy induction in MCECs under NT and HCS conditions with rapamycin had a 74.9-fold and 51.5-fold reduction of IFN-γ (pg/mL), resepectively, compared to the non-treated controls. In contrast, autophagy inhibition in MCECs with chloroquine resulted in 1.82-fold increase of IFN-γ compared to untreated controls. This suggests a protective role of autophagy in ECs during IRI. DISCUSSION/SIGNIFICANCE: We observed that autophagy may be protective during IRI by mitigating EC immunogenicity. Thus, pharmacologically modulating microvascular EC autophagy in donor hearts prior to transplantation may mitigate insults incurred during CS and IRI.

ABSTRACT IMPACT: This work identifies host immune perturbations in sepsis mortality that suggests targets for a precision medicine paradigm in the host response to infection. OBJECTIVES/GOALS: To compare the early whole blood transcriptome during sepsis between 30-day survivors and non-survivors in the Intensive Care Unit (ICU), and to evaluate for pathway enrichment that might explain sepsis lethality. METHODS/STUDY POPULATION: We enrolled 162 sepsis patients in the first 24 hours of ICU admission, particularly targeting individuals requiring vasopressors. Peripheral whole blood was collected in PAXgene vacutainers. Isolated RNA was analyzed with Affymetrix Human Genome ST 2.0 microarray. Differential gene expression was performed with Bioconductor/R/limma using log2 fold-change +/-0.6 as a threshold for differential expression, and a Benjamini-Hochberg adjusted p-value <0.05 to declare significance. Functional gene enrichment was performed using the Gene Ontology (GO) database with PANTHER overrepresentation test (Fisher’s Exact) on all transcripts with adjusted p-value <0.05. Pathways analysis was performed with the Reactome Project using the raw fold change and significance data to identify dysregulated pathways. RESULTS/ANTICIPATED RESULTS: There were 58 non-survivors (36% mortality). We identified 39 genes as differentially expressed between sepsis non-survivors and survivors; 31 were upregulated in non-survivors and 8 had reduced expression. Several of the most overexpressed transcripts are neutrophil-specific, including LCN, MPO, OLF4M4, DEFA3, and DEFA4. A functional gene overrepresentation test further supports this finding, as the most enriched gene ontologies were neutrophil-mediated killing, neutrophil cytotoxicity, neutrophil extravasation, and respiratory burst, all demonstrating higher than 10-fold enrichment and FDR < 0.02. Pathway analysis of the peripheral blood transcriptome was notable for immune response derangement, specifically downregulation of both innate and adaptive immune pathways (FDR < 0.00001). DISCUSSION/SIGNIFICANCE OF FINDINGS: We identified increased expression of neutrophil-related genes in sepsis non-survivors, replicating candidates previously identified in pediatric sepsis mortality and ARDS. These immune perturbations in sepsis mortality may represent key targets for eventually employing precision medicine strategies in sepsis.

OBJECTIVES/GOALS: Our lab previously identified a population of vascular smooth muscle (SMC)-derived progenitor cells (AdvSca1-SM) which expand robustly in response to disease and can differentiate into multiple cell types. We now aim to define the role of these AdvSca1-SM cells in atherosclerotic plaque progression. METHODS/STUDY POPULATION: Goal one uses SMC lineage tracing mice and a model of atherosclerosis to track reprogramming of SMCs to AdvSca1-SM cells in the setting of disease. Arteries are analyzed using flow cytometry and immunofluorescence to quantify changes in number of mature SMCs and AdvSca1-SM cells. Goal two uses AdvSca1-SM lineage tracing mice with high cholesterol-induced atherosclerosis and plaque neovascularization. Arteries are analyzed to quantify expansion of AdvSca1-SM cells, subsequent re-differentiation into mature SMC, endothelial cells, or macrophages, and contribution to plaque neovascularization. Mechanistic findings from both goals are being investigated in diseased human coronary arteries. RESULTS/ANTICIPATED RESULTS: Flow cytometry from SMC lineage tracing mice revealed a 7- to 13-fold expansion of AdvSca1-SM cells in carotid arteries (p<0.001) and aortas (p = 0.03) after 6 weeks of western diet; no differences in macrophage numbers were observed. Additional SMC and AdvSca1-SM cell lineage tracing mice are on atherogenic diets to assess early and advanced atherosclerosis. We predict that AdvSca1-SM cells will contribute to macrophage accumulation as well as plaque neovascularization in the setting of severe atherosclerosis. Translational relevance of mechanisms driving SMC reprogramming and AdvSca1-SM cell contribution to plaque progression are being applied to studies of diseased human coronary arteries. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggest a role for AdvSca1-SM cells in atherosclerosis. Ongoing work will clarify the mechanisms driving plaque-associated AdvSca1-SM expansion and define the ultimate fates of these cells. In vivo modulation of this process could provide the basis for future anti-atherosclerotic therapies. CONFLICT OF INTEREST DESCRIPTION: AD - CCTSI TOTTS TL1TR002533; SL - 18POST34030397 from the American Heart Association; AJ – no conflicts; KS - 1F31HL147393 from the National Heart, Lung, and Blood Institute, NIH; MM – no conflicts; RT – no conflicts; KSM – no conflicts; RAN - R01CA236222 from the National Cancer Institute, NIH, and 2018-03 from the Lungevity Foundation; and MCMW-E - R01 HL121877 from the National Heart, Lung, and Blood Institute, NIH, and 25A8679 from the Chernowitz Foundation.

OBJECTIVES/GOALS: Anti-cancer therapies, such as chemotherapy, can induce senescence. Senescent cells may produce factors that can promote tumor progression. In this study, we will investigate the effect of senolytics and anti-cancer treatment on fibroblasts, which are a part of the tumor microenvironment, and patient-derived colorectal cancer organoids. METHODS/STUDY POPULATION: We will induce senescence in fibroblast lines via irradation. Induction of senescence will be confirmed by monitoring SASP production, changes in morphology and proliferation rates, and senescence-associated b-galactosidase activity. To investigate the efficacy of senolytics on senescence-induced fibroblasts and CRC tumor organoids, we will creat a dose response curve and calculate IC50 values for proliferating fibroblast, senescent fibroblasts and CRC organoids. To identify the synergistic effects of anti-cancer and senolytic compounds, including Navitoclax and Dasatinib, on fibroblasts and CRC organoids, we will create dose matrixes using senolytics at concentrations that were shown to have senolytic activity and drugs from an anti-cancer library. RESULTS/ANTICIPATED RESULTS: If senescence is induced in the fibroblast lines, we expect to see no changes in confluency over 4 days, the morphology will change from a thin, spindly shape to a flattened shape, and senescence-associated b-galactosidase activity will be observed. After the fibroblast lines are treated with potential senolytic compounds, we would expect to see decreased viability in the senescence-induced fibroblast lines when compared to proliferating fibroblast lines. We predict that the viability of CRC organoid lines will slightly decrease at high concentrations of the senolytic due to overall toxicity. We expect that the senolytic and anti-cancer compounds will have a synergistic effect. Senolytic activity could reduce the senescent cell population that was developed in response to anti-cancer therapy. DISCUSSION/SIGNIFICANCE: There is an increased interest in identifying compounds that selectively promote apoptosis in senescent cells. This study uses a cell-based approach to validate senolytic activity of compounds with senolytic potential in senescence-induced fibroblast lines and investigates the synergistic effects of senolytics and anti-cancer compounds on CRC.

OBJECTIVES/GOALS: Use an easily accessible medium to educate life science researchers and academic innovators interested in the commercialization of academic research at the University of Michigan (UM). METHODS/STUDY POPULATION: Life science research investigators and academic innovators interested in research commercialization and technology development from across the state of Michigan were invited to attend the Idea to Impact: The Translation & Commercialization of Academic Research webinar series, presented by Fast Forward Medical Innovation at the University of Michigan. The webinar series outlined the significance and critical milestones of developing novel therapeutics, medical devices, diagnostics, and digital health innovations, as well as essential collaborations with industry partners to translate a research-based idea into a product of impact. RESULTS/ANTICIPATED RESULTS: 113 investigators and innovators from 28 different institutions, organizations, and companies, registered for the webinar series. Results (N=24) of an evaluation immediately following each webinar revealed that 100% of respondents strongly agreed or agreed that the series was effective in helping them to identify and describe commercialization resources, including funding, education, and mentorship, available at the University of Michigan and within the state. Participants stated that they “loved the practical information” “shared” and that the series was a “great overview that inspired a lot more questions.” The Fast Forward Medical Innovation team was then able to consult with participants to connect them with additional resources. DISCUSSION/SIGNIFICANCE: The data suggests that easily accessible and digestible commercialization education can make navigating the academic entrepreneurial ecosystem easier for investigators and innovators. The recorded webinar series, Idea to Impact: The Translation & Commercialization of Academic Research, serves this purpose.