OBJECTIVES/GOALS: The purpose of this work is to investigate natural buffering capacity of liver tissue and tumors, to understand and exploit differences for therapy. Using this work, we will determine the concentrations of reagents (acids or bases) used in ablation treatment to optimize treatment by increasing tumor toxicity and minimizing healthy tissue toxicity. METHODS/STUDY POPULATION: For this preliminary study, two methods will be used: benchtop pH experiments ex vivo and non-invasive imaging using acidoCEST MRI in vivo. For ex vivo, two types of tissues will be tested: non-cancerous liver and tumor tissue from HepG2 inoculated mice (n = 10). After mice are euthanized, pH will be measured in tissue homogenates at baseline and then the homogenates will be placed in either acidic (acetic acid) or basic (sodium hydroxide) solutions with varied concentrations (0.5–10M) and time recorded until pH returns to baseline. For in vivo imaging, Mia PaCA-2 flank model mice (n = 10) will be imaged with acidoCEST MRI to quantify pH at baseline. Mice will then be injected intratumorally with (up to 100 μL of) acid or base at increasing concentrations and imaged to quantify pH changes in the tumor. RESULTS/ANTICIPATED RESULTS: For this study, buffering capacity is defined as the concentration threshold for which tissue can buffer pH back to within normal range. Non-cancerous tissue is likely to buffer a wider range of concentrations compared to tumor tissue. From the benchtop experiment, comparison of time-to-buffer will be made for each concentration of acid/base for the two tissue types. AcidoCEST MRI will provide in vivo buffering capacity and potentially demonstrate tumor heterogeneity of buffering capacity. For both experiments, a pH vs. concentration curve for the two tissue types will allow for comparison of ex vivo to in vivo experiments, which will differentiate contributions of local tissue buffering capacity from the full body’s natural bicarbonate buffer system that depends on respiration and blood flow. DISCUSSION/SIGNIFICANCE OF IMPACT: The pH of the body must be maintained within a narrow range. With cancer, impairment in regulation of tumor metabolism causes acidosis, lowering extracellular pH in tumors. It remains unclear if pH plays a role in local recurrence or tumor toxicity. This work will determine if acidoCEST MRI can measure deliberate alteration of pH and how this change affects biology.

OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the amygdala in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecular PSD95-nNOS inhibitor, co-immunoprecipitation, Western blotting, and RNA-sequencing. RESULTS/ANTICIPATED RESULTS: We show that fear conditioning enhances the PSD95-nNOS interaction and that the small-molecule ZL006 inhibits this interaction. Treatment with ZL006 also attenuates rodent cued-fear consolidation and prevents fear-mediated shifts in glutamatergic receptor and current densities in the basolateral amygdala (BLA). With RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored by systemic ZL006 treatment. Network data and gene ontology enrichment analysis with Ingenuity Pathway Analysis and DAVID software found that cell-cell interaction, cognition-related pathways, and insulin-like growth factor binding were significantly altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results reveal novel genetic targets that underlie plasticity of fear-memory circuitry via their contribution of NMDAR-mediated fear consolidation and can inform future strategies for targeting fear related disorders like PTSD. CONFLICT OF INTEREST DESCRIPTION: Anantha Shekhar and Yvonne Lai are co-founders of Anagin, Inc., which is developing some of the related molecules for the treatment of PTSD.

OBJECTIVES/GOALS: Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase, is decreased in diverse models of kidney disease, and Sirt3 activation prevents disease progression in many of those models. We are investigating if pharmacological activation of Sirt3 ameliorates kidney disease in a mouse model of Alport syndrome. METHODS/STUDY POPULATION: Alport syndrome is a hereditary orphan disease arising from a defect in the collagen IV α3α4α5 heterotrimer, a component of the glomerular basement membrane. Male and female Col4a3tm1Dec knockout mice and wild type controls on the 129X1/SvJ background were harvested at 9–10 weeks of age. Serum and urine were collected prior to euthanasia; renal pathology was assessed by histology; and renal cortical mRNA and protein levels were assessed by qRT-PCR and western blot, respectively. Studies are ongoing using dietary administration of a Sirt3 activator, nicotinamide riboside (500 mg/kg/day), in Col4a3 transgenic mice on both the 129X1/SvJ and C57BL/6J backgrounds. RESULTS/ANTICIPATED RESULTS: Col4a3−/− mice have elevated BUN (P < 0.0001, both sexes), serum creatinine (P < 0.001, male; P < 0.0001, female), and urinary albumin-to-creatinine ratio (P < 0.0001, both sexes) compared to Col4a3+/+ controls. On histology, Col4a3−/− mice have extensive renal fibrosis compared to Col4a3+/+ controls. Sirt3 expression is decreased in the renal cortices of Col4a3−/− mice at the mRNA (P < 0.0001, male; trend, P = 0.07, female) and protein levels (P < 0.05, male; P < 0.001, female) compared to Col4a3+/+ controls. All experiments had 5–9 mice per group. Results of the prevention study with nicotinamide riboside, a Sirt3 activator, are unknown at the time of abstract submission. DISCUSSION/SIGNIFICANCE OF IMPACT: Col4a3−/− mice have severe renal impairment and decreased renal cortical expression of Sirt3 at the mRNA and protein levels compared to Col4a3+/+ controls. However, it is unknown at this time if pharmacologically activating Sirt3 prevents this renal decline.

OBJECTIVES/GOALS: Neoantigen vaccine immunotherapies have shown promise in clinical trials, but identifying which peptides to include in a vaccine remains a challenge. We aim to establish that molecular structural features can help predict which neoantigens to target to achieve tumor regression. METHODS/STUDY POPULATION: Proteins were prepared by recombinant expression in E. coli followed by in vitro refolding. Correctly folded proteins were purified by chromatography. Affinities of protein-protein interactions were measured by surface plasmon resonance (SPR) and thermal stabilities of proteins were determined by differential scanning fluorimetry. All experiments were performed at least in triplicate. Protein crystals were obtained by hanging drop vapor diffusion. The protein crystal structures were solved by molecular replacement and underwent several rounds of automated refinement. Molecular dynamics simulations were performed using the AMBER molecular dynamics package. RESULTS/ANTICIPATED RESULTS: A T cell receptor (TCR) expressed by tumor-infiltrating T cells exhibited a 20-fold stronger binding affinity to the neoantigen peptide compared to the self-peptide. X-ray crystal structures of the peptides with the major histocompatibility complex (MHC) protein demonstrated that a non-mutated residue in the peptide samples different positions with the mutation. The difference in conformations of the non-mutated residue was supported by molecular dynamics simulations. Crystal structures of the TCR engaging both peptide/MHCs suggested that the conformation favored by the mutant peptide was crucial for TCR binding. The TCR bound the neoantigen/MHC with faster binding kinetics. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that the mutation impacts the conformation of another residue in the peptide, and this alteration allows for more favorable T cell receptor binding to the neoantigen. This highlights the potential of non-mutated residues in contributing to neoantigen recognition.

OBJECTIVES/GOALS: Our goal is to employ a structure-guided design approach to engineering a safer and more effective variant of the TIL1383I T cell receptor (TCR) currently under study in clinical trials for malignant melanoma METHODS/STUDY POPULATION: Using our unpublished structure of TIL1383I we are in process of designing a panel of TCR variants with the goal of identifying candidates that improve “focus” towards the tyrosinase antigen presented on the MHC class I molecule HLA-A2. RESULTS/ANTICIPATED RESULTS: Structural analysis of TIL1383I revealed key residues, particularly beta-chain residues E97, G101, L102, responsible for engaging the tyrosinase peptide bound to HLA-A2. The crystal structure of TIL1383I in complex with tyrosinase-HLA-A2 also highlighted its uncharacteristic binding geometry and we therefore hypothesize that this binding orientation is associated with the observed CD8 co-receptor independence of TIL1383I. Indeed, functional analysis with TIL1383I-transduced CD8-positive and CD8-negative T cells, transduced T cells expressing a truncated CD8 lacking the intracellular LCK signaling domain, and tyrosinase peptide variants presented by HLA-A2 mutants outline this co-receptor independence. Combined with our interrogation of tyrosinase peptide cross-reactivity via a peptide positional scanning library approach, structure-guided design resulted in the identification of TIL1383I variants with improved binding affinities to the tyrosinase peptide as well as an understanding of structural characteristics that may contribute to TIL1383I’s co-receptor independence.

OBJECTIVES/GOALS: My long-term career objective is to become an established independent researcher focused on understanding and modulating immune responses to biologics in order to enhance their efficacy and understand the underlying mechanisms by which these interact with the immune system. METHODS/STUDY POPULATION: In this study we will evaluate the utility of cyanuric chloride based synthetic lipids in the presentation of peptide epitopes of the gene delivery vector, adeno-associated virus (AAV). The lipopeptide conjugates will be administered to mice via liposomal formulations to assess their ability to induce immune responses by ELISA as compared to mice treated with the AAV. The three-dimensional conformation of the peptides will be evaluated using nuclear magnetic resonance to determine their similarity with the natural conformation that these peptides adapt on the viral surface. Additionally, to assess the translatability of this conjugation strategy, we will test the ability of our lipopeptides to bind to serum antibodies from human subjects. RESULTS/ANTICIPATED RESULTS: We anticipate that peptide presentation using cyanuric chloride lipids will achieve a robust response in mice following immunization. Immunizations with our lipids should induce the production of antibodies targeting AAV, albeit a milder response that the virus itself, given the complexity of viral epitopes. Nuclear magnetic resonance will inform us on how to improve the synthetic lipids to optimize peptide presentation by altering the characteristics of the lipid anchors. Finally, by using human serum to test for the ability of our lipopeptides to bind to antibodies in serum from patients positive for AAV antibodies, we can become informed on whether our strategy has utility in human studies or whether our method is limited to animal models of human disease. DISCUSSION/SIGNIFICANCE OF IMPACT: The current work seeks to develop a strategy to present peptides from a well characterized biologic, AAV, on a liposome surface. Our ultimate purpose is to employ liposomal formulations as decoys that target AAV-specific lymphocytes to improve the in vivo efficacy of AAV.

OBJECTIVES/GOALS: The objective of this research was to learn how the oncogenic transcription factor, ERG, is regulated in prostate cancer. If we could learn how ERG is regulated and which genes are important for its oncogenic phenotype in prostate cells, we could design new therapeutic strategies against ERG, which has proven to be difficult to target. METHODS/STUDY POPULATION: We conducted an shRNA screen in prostate cells to determine candidate genes and pathways that are important for ERG function. To validate the findings of the screen, we performed a variety of cell-based functional assays, including trans-well migration, wound healing, and clonogenic survival assays. To further investigate the mechanism between ERG and the genes revealed by the screen, we performed biochemical and molecular biology experiments such as Western blotting and qRT-PCR for protein and mRNA expression, co-immunoprecipitation assays to determine protein-protein interactions, and chromatin immunoprecipitation (ChIP-qPCR) to determine transcription factor binding to DNA sites. RESULTS/ANTICIPATED RESULTS: The screen revealed that genes involved in the toll-like receptor 4 (TLR4) pathway are important for ERG-mediated migration. We tested the effect of a TLR4 inhibitor on ERG function and observed decreased migration and clonogenic survival exclusively in ERG-positive cells. Expression of pMEK and pERG was reduced when TLR4 was inhibited, which suggests a mechanism in which TLR4 upregulates pMEK, leading to the phosphorylation and activation of ERG. This is supported by functional assays in which cells expressing a phosphomimetic ERG are resistant to the TLR4 inhibitor. We demonstrated that ERG drives the transcription of TLR4 and its endogenous ligands HSPA8 and BGN. Therefore, ERG can sensitize the cell to TLR4 activation by increasing the number of receptors as well as providing the ligands needed for stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: This research provides a new therapeutic pathway for treating ERG-positive patients through TLR4 inhibition. This can be beneficial because many patients become resistant to the standard therapy, leaving very few treatment options. TLR4-based therapies could provide an alternative for patients who have developed resistance.

OBJECTIVES/GOALS: High cholesterol is among the major causes of cardiometabolic complications. People with high cholesterol have about twice the risk of heart disease as people with lower levels. Approximately every 40 seconds, an American will have a heart attack. Costs related to Heart Attack exceed 60 Billion USA dollars per year. Renin-Angiotensin-Aldosteron System (RAAS) is implicated in the genesis of coronary heart disease and in the perpetuation of heart failure. Angiotensin-Converting Enzyme inhibitors (ACE-I) have emerged as the treatment of choice for patients with all degrees of heart failure. Many clinical trials (Consensus, 1987; Save 1990) provide the evidence that ACE-I preserves cardiac function, prevents cardiovascular death, myocardial infarction & stroke and limit remodeling after myocardial infarction. However, there are still controversies in cardiology and a debate over cardioprotection is continuing:

• Do ACE Inhibitors have unique properties, beyond their antihypertensive effect?

• Can we protect the heart during hypercholesterolemia?

• In which way hypercholesterolemia affects mitochondria bioenergetics?

• How does ramipril affect mitochondrial bioenergetics during the course of experimental hypercholesterolemia?

Objectives/Goals were: To evaluate the mitochondrial actions of chronically administered ramipril (non-SH-containing ACE inhibitor) in cholesterol-fed rabbits by determining the influence of ramipril on:

• myocardial oxygen consumption (State 4, State 3), Respiratory Control Ratio (RCR), and adenosine diphosphate - oxygen index (ADP/O) and

• oxidative stress biomarkers

METHODS/STUDY POPULATION: Animal treatments. In the course of twelve-week the male Chinchilla rabbits (n = 10/group) received once a day a single dose, in group: A - sunflower oil (control animals); B – atherogenic 2% hypercholesterolemic diet; C - atherogenic diet and ramipril (1 mg/kg) and D - ramipril (1 mg/kg) only. Animals were terminated in accordance with the U.K. “Animals (Scientific Procedures) Act.” Isolation of mitochondria - Mitochondria from rabbit heart were isolated by tissue digestion (trypsin), fractionation and differential centrifugation. Mitochondrial respiratory functional measures (State 4 - Basal; State 3 - ADP-stimulated respiration, RCR and ADP/O) and biochemical markers of oxidative damage (the nitrite level) were measured polarographically (Clark electrode, YSI, USA) and spectrophotometrically, respectively, in isolated heart mitochondrial suspensions. Statistics - All results are reported as means ± SD. Comparisons between ramipril treated and control animals were performed by unpaired t-test or one-way ANOVA with a Tukey adjustment for multiple comparisons. A P value < 0.05 was considered significant for all tests. RESULTS/ANTICIPATED RESULTS: Plasma cholesterol levels: After a period of 12 weeks

• Plasma cholesterol levels in control rabbits (A) were low (1.36 ± 0.23 mmol/l).

• Cholesterol-fed rabbits became hypercholesterolemic and their plasma total cholesterol level was higher even than 10 mmol/l. The level of total cholesterol in the high-cholesterol-diet group was significantly increased compared with the level in the normal-diet group (p < 0.01).

• In the high-cholesterol-diet group treated with ramipril (C), the plasma cholesterol level was not affected by the drug ramipril (10.54 ± 1.31 mmol/l). ACE-I ramipril did not infuence the concentration of total cholesterol.

• Plasma cholesterol levels in group D were low (1.46 ± 0.29 mmol/l).

• Atherogenic 2% cholesterol diet (B) caused a decline in mitochondrial function (in both, State 3 and 4) (−25%).

• Mitochondria from group C animals (treatment with ramipril along with 2% cholesterol diet) exhibited higher State 3 respiratory rates compared with group B.

• Mild inhibition of mitochondrial respiration was recorded in group D, in both respiratory states (V4&V3).

• In cholestrerol-supplemented hearts myocardial oxygen consumption was markedly reduced (State 4 and State 3) compared to controls. Administration of high-cholesterol diet decreased not only the respiratory activity of rabbit heart mitochondria (RHM), but also the sensitivity of respiratory chain to ADP (ADP/O), while concomitantly caused an increase in nitrite production. Possible explanation: high-fat diet affects the fluidity of mitochondrial membrane – Electron transport chain (ETC) may be damaged, and unable to support high rates of respiration (e.g. substantial cytochrome c could be lost).

• Administration of ACE-I ramipril along with cholesterol diet partially abolish reduction in MQO2 and improved coupling efficiency (ADP/O). Possible explanation: reduced coupling efficiency means the coupling mechanism itself is altered (e.g. the respiratory complexes slip and pump fewer protons than normal and less ATP is produced per oxygen consumed. Ramipril partially improved coupling efficiency and increased the amount of ATP per oxygen consumed.

• RCR - No significant difference between the groups were found. High RCR indicates good function (a high capacity for substrate oxidation and ATP turnover). Low RCR usually indicates dysfunction. However, there is no absolute RCR value that is diagnostic of dysfunctional mitochondria, because values are substrate- and tissue-dependent.

• NO• exerts metabolic control over mitochondrial respiration. Group B: The lowered state 3-respiration in heart mitochondria seems to contribute to the increased NO production, and elevated nitrite level.

• In a system as complex as OXPHOS, conclusions about overall efficiency must involve measurements of: mito membrane potential, proton transport, ATP synthesis and modular kinetic analisys.

OBJECTIVES/GOALS: Recent studies indicate B cells are involved in dermal fibroblast activation and collagen deposition in the skin. However, B cell distribution in epidermal and dermal layers is unknown. Here, We aim to characterize the distribution of B cells residing in normal skin and keloidal scars. METHODS/STUDY POPULATION: One abdominal normal skin sample and two keloid samples (ear and shoulder) were obtained from the University of Colorado Biorepository Core Facility and from the Plastic Surgery Clinics. Five micron sections from formalin-fixed paraffin-embedded samples were prepared for multiplex fluorescence immunohistochemistry by the Human Immunology & Immunotherapy Initiative. We stained for CD20+, CD19+, and DAPI. Slides were imaged using Vectra®3 scanning system from PerkinElmer. Images were analyzed in InForm®Tissue Finder, phenotpr, phenoptrReports by Akoya biosciences. RESULTS/ANTICIPATED RESULTS: We found a significant increase in the percentage of CD20+ and CD19+ B cells in keloid skin compared to normal skin tissue (14.50% and 14.20% vs 6.47% and 7.56% of the total cells), respectively. Interestingly, we found that in the epidermis of keloid skin CD20+ cell were more abundant (14.46%) whereas in the epidermis normal skin CD20+ cells were less predominant (5.14%). In the dermis of keloid skin, CD20+ and CD19+ were in equal proportions (13%) whereas in normal skin CD19+ cells were more predominant (10.44%) compared to CD20+ cells (7.04%). Dual positive B cells, CD19+/CD20+ cells, were more abundant in keloid dermis (11.06%) compared to normal skin dermis (1.24%). DISCUSSION/SIGNIFICANCE OF IMPACT: B cells are involved in fibroblast activation in diseases such as scleroderma and rheumatoid arthritis. With the increase of CD19+/CD20+ B cells in keloids, the role of B cells in keloid pathogenesis warrants further study. CD27 staining may determine if these are activated or follicular B cells.

OBJECTIVES/GOALS: The objective of this study is to define the molecular mechanisms that control survival of malignant stem cells in acute myeloid leukemia (AML). Leukemia stem cells (LSCs) are not effectively eradicated by standard treatment and lead to resistance and relapse, which contribute to poor survival rates. METHODS/STUDY POPULATION: The recently FDA approved venetoclax, a BCL2 inhibitor, with azacitidine, a hypomethylating agent leads to a 70% response rate in AML patients. Analysis of patients treated with this regimen showed direct targeting of LSCs. BCL2 has a non-canonical function in regulation of intracellular calcium. To determine how BCL2 mediated calcium signaling plays a role in LSC biology, we used LSCs isolated from venetoclax/azacitidine (ven/aza) sensitive and resistant patient samples to measure expression of calcium channels via RNA seq. BIO-ID, siRNA, flow cytometry, seahorse assays, calcium measurements and colony assays were used to determine the effects of calcium channel perturbation on LSC biology. RESULTS/ANTICIPATED RESULTS: BCL2 inhibition leads to decreased OXPHOS activity in primary AML specimens. BIO-ID studies revealed cation/metal ion transporters, ER membrane proteins and ER membrane organization as top enriched pathways interacting with BCL2. RNA-seq data showed increased expression of genes involved in calcium influx into the ER in ven/aza sensitive LSCs and increased expression of genes involved in calcium efflux from the ER in ven/aza resistant samples. Ven/Aza resistant LSCs have increased mitochondrial calcium content, consistent with their increased OXPHOS activity as calcium is required for OXPHOS. Perturbation of these channels leads to decreased OXPHOS activity and decreased viability in LSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: We postulate that a deeper understanding of the mechanisms behind ven/aza targeting of LSCs will lead to the development of novel therapies for patients who do not respond to ven/aza. Our data show targeting intracellular calcium signaling could be a viable therapeutic strategy for AML patients.

OBJECTIVES/GOALS: Genetic variation in L-type voltage-gated calcium channels, including CaV1.3, is associated with increased risk for psychiatric disorders including bipolar disorder and schizophrenia. Additionally, rare mutations in CaV1.3 have been linked to epilepsy, developmental delay, and autism. Deletion of CaV1.3 in mice is associated with impaired consolidation of contextual fear conditioning. Some studies have also observed affective behavior deficits in CaV1.3-deficient mice, but other studies have not found affective phenotypes, perhaps due to differences in genetic backgrounds, sex ratios, or task protocols. CaV1.3 is important for slow afterhyperpolarization in hippocampal and amygdala neurons, which prevents excessive firing in response to sustained excitatory input, and CaV1.3-deficient amygdala neurons exhibit hyperexcitability and impaired LTP. CaV1.3 is also expressed in the cerebellum, but its functional role there is not well understood. Given its importance in shaping neuronal activity in the hippocampus and amygdala, we hypothesized that loss of CaV1.3 would cause abnormalities in motor learning as well as affective and cognitive behaviors. METHODS/STUDY POPULATION: Wild-type (WT), haploinsufficient (Hap), and knockout (KO) mice were maintained on a congenic C57BL/6NTac genetic background and were subjected to behavioral tasks including open field, rotarod, ErasmusLadder, elevated zero maze, forced swim test, and tail suspension test. Data were analyzed with sexes combined and with sexes separated to assess for sex as a biological variable. Studies were analyzed by one-way ANOVA, two-way ANOVA, or generalized linear mixed model, where appropriate. RESULTS/ANTICIPATED RESULTS: CaV1.3 KO was associated with impaired motor learning in the rotarod task (p < 0.05), as well as impaired associative learning in the ErasmusLadder task (p < 0.01), despite intact locomotor function on both tasks. When examined by sex, the rotarod phenotypes were driven by motor learning impairments in males (both Hap and KO, p < 0.05 and p < 0.01, respectively), whereas the ErasmusLadder associative learning phenotypes were present in both sexes only in the KO condition, consistent with previously reported impairments in CaV1.3-deficient mice in consolidation of contextual fear conditioning. Although KO mice learned the motor aspects of the ErasmusLadder task, they learned more slowly. They also failed to learn start cues, which requires intact associative learning. No differences were observed in overall exploration or locomotor activity in open field or elevated zero maze. Analyses from affective tasks are ongoing. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary studies provide new evidence that CaV1.3 is important for the function of neural circuits involved in motor learning, and concur with previous data showing its involvement in associative learning. Our data differ slightly from previous studies of CaV1.3 in motor learning, which could be attributable to differences in task protocols and/or genetic background. These results highlight the importance of CaV1.3 in a variety of behaviors, which may help explain why variation in CaV1.3 expression and function has pleiotropic effects in humans.

OBJECTIVES/GOALS: High blood-pressure (BP) is a common adverse effect of erythropoietin (EPO) therapy among patients with chronic kidney disease on hemodialysis, and even among otherwise healthy individuals who receive EPO. In human genetics, EPO is associated with not only red blood cell traits, but hypertension (HTN) as well. Currently, there is no vascular gene expression data available in the setting of EPO-induced HTN that may explain precise role of key cellular players in its hypertensive etiology. Our aim is to characterize vascular transcriptome to identify key cellular players in EPO-induced HTN. METHODS/STUDY POPULATION: 10-12 week C57BL/6 male and female mice were randomly divided into two groups 1. Vehicle (0.9% saline-VEH), 2. EPO, (N = 4). VEH and EPO were intraperitoneal administered (EPO 75U/30g, 3 times/week) for 20 days. Blood-pressure was measured non-invasively via tail-cuff plethysmography. We characterized in-vivo transcriptome response of mouse descending aorta to EPO-HTN and vehicle control group by high-throughput RNA-sequencing. RESULTS/ANTICIPATED RESULTS: Systolic blood pressure (SBP) was significantly higher in EPO treatment, compared to vehicle (males and females combined SBP VEH 116.29 ± 6.21, EPO 129.57 ± 4.59, mean ± s.d., adjusted P = 0.0012). Comparison of in-vivo transcriptional differences between vehicle and EPO-treated reveal statistically significant changes in cellular pathways consistent with hypertension such as upregulation of RAS signaling pathway and oxidative stress. In-vitro mouse aortic smooth muscle cells, EPO markedly increased phosphorylated-ERK activity, suggesting increased RAS activity. DISCUSSION/SIGNIFICANCE OF IMPACT: This study highlights the importance of previously unknown vascular key players and advances our understanding of the transcriptional events associated with EPO-induced hypertension.

OBJECTIVES/GOALS: Natural killer (NK) cells are a potential cancer therapeutic but expanding NK cells efficiently in vitro is difficult. Natural killer cell deficiency (NKD), a primary immune deficiency affecting only NK cells, is caused by defects in several DNA replication proteins. By studying NKD we will achieve better NK cell in vitro differentiation. METHODS/STUDY POPULATION: One patient with NKD has a compound heterozygous mutation in the essential DNA replication protein MCM10. We hypothesize that in individuals with NKD, dramatic telomere erosion from abnormal DNA replication leads to premature senescence and the loss of NK cells. To test our hypothesis, we will knockout one allele of MCM10 or over express MCM10 in NK cells isolated from blood. We will then monitor telomere length, expansion and cytotoxic activity of these NK cells. To understand the role of MCM10 in early stages of NK cell development we will deplete MCM10 in induced pluripotent stem cells and differentiate these cells into NK cells. During this differentiation we will monitor progression through NK cell developmental stages as well as telomere length and senescence markers. RESULTS/ANTICIPATED RESULTS: Telomeres insulate chromosomes and induce permanent growth arrest (senescence) when they are critically short. We have demonstrated that depletion of a DNA replication protein causes telomere erosion and increases senescence markers. NK cells have shorter telomeres and lower telomerase expression than other immune cells. We predict, this relatively poor telomere maintenance sensitizes NK cells to telomere loss upon depletion of replication proteins. During in vitro differentiation, we expect NK cell precursors to undergo premature senescence secondary to telomere shortening. Furthermore, we expect supplementation of DNA replication proteins will enhance NK cell expansion and maturation. DISCUSSION/SIGNIFICANCE OF IMPACT: NKD patients have provided the scientific community with clues as to what proteins NK cells rely on for their development. This project aims not only to understand why these proteins are critical, but to harness that information for cellular anti-cancer therapeutics.

OBJECTIVES/GOALS: The role of the vaginal microbiome (VM) in HIV disease progression is poorly understood. We examined VMs of HIV+ Elite Controllers (ECs) and HIV+ Long-Term Non-Progressors (LTNPs) compared to controls: HIV-positive antiretroviral (ARV) treated (HIV+ATs) and HIV-negative women in the Women’s Interagency HIV Study (DC/Chicago/Atlanta sites). METHODS/STUDY POPULATION: VMs were surveyed via both V3/V4 region of 16S rRNA gene amplicon sequencing and metagenomics sequencing in 67 women across 4 study groups: 1) LTNPs (CD4 >500 cells/mL for 5+ years without ARVs) (n = 7) and 2) ECs (HIV RNA <80 copies/mL for 2+ years without ARVs) (n = 8), matched with 3) HIV+ ATs (on ARVs for ≥1 year with CD4 increase ≥100 cells/mm3) (n = 34), and 4) HIV- women (n = 18). Metagenomes were characterized from specimens collected at two time points: 1) vaginal swabs collected 2016-2017 (n = 62) and 2) cervicovaginal lavage collected 2002-2016 (n = 35; DC/Chicago only). We used VIRGO (human vaginal non-redundant gene catalog), a newly developed referencing framework to comprehensively catalog VM gene content, taxonomy and functions. RESULTS/ANTICIPATED RESULTS: Women were 89% African American with a mean age of 46 years (SD 8.8). The most prevalent species were Gardnerella vaginalis (predominant in 34%), Lactobacillus iners (predominant in 21%), and L. crispatus (predominant in 14%). 90% of LTNP and 45% of EC samples were Lactobacillus-dominant vs. 28% of HIV- and 30% of HIV+ATs. L. crispatus and L. iners in ECs/LTNPs had significantly different gene content and greater gene richness vs. controls. G. vaginalis-predominant communities were found in 66% of HIV- and 68% of HIV+ATs, compared to 46% of EC and 0% of LTNP. The G. vaginalis strains present in EC/LTNP also showed significantly lower gene richness and different gene content vs. controls. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest unique VM communities among EC/LTNP, and led us to hypothesize that differential regulation of vaginal immunity drives the observed differences. The similarity between VMs of HIV- and HIV+ATs warrants further study. Larger longitudinal VM studies are needed to assess associated functional pathways and understand the etiology of VM association with HIV progression. CONFLICT OF INTEREST DESCRIPTION: The authors have no conflicts of interest to disclose.

OBJECTIVES/GOALS: In the crowded environment of the intestine, selected commensal bacteria and enteric viruses interact. The biological significance of this interaction, in either normal or pathological condition is not known. To study this interaction, we are developing a physiologically relevant model of an human intestinal epithelium. METHODS/STUDY POPULATION: Intestinal biopsies (ileum region) and fecal samples of 6 healthy and 6 active Crohn’s patients are being collected to derive human intestinal enteroid (HIE) lines. 2D-polarized HIE will be first characterized with studies of epithelial permeability, tight junctions and cell type composition, and co-cultured with matching fecal samples. The (co-)cultures will be then infected with human norovirus (HNoV), our model enteric virus, and infection will be quantified by RT-qPCR. In addition, the interaction of HNoV with bacteria derived from healthy or Crohn’s will be determined quantitatively by flow cytometry (viral tagging) and qualitatively by 16S sequencing of the total versus HNoV-bound bacterial species. RESULTS/ANTICIPATED RESULTS: Crohn’s patients are characterized by a microbiome dysbiosis and, in particular, by a high abundance of Enterobacteriacae. HNoV interacts with Enterobacter cloacae, and interestingly, HNoV infection is associated with exacerbation and reactivation of Crohn’s disease. By re-creating the intestinal milieu of healthy and Crohn’s patients, we expect that the kinetics of infection by HNoV will be higher in Crohn’s as compared to healthy volunteers. In addition, by studying the composition of the HNoV-bound bacterial component of Crohn’s versus heathy volunteers, we will be able to identify the contribution of selected bacteria to the expected increase of infection. DISCUSSION/SIGNIFICANCE OF IMPACT: With this study, we will fill the gap of knowledge on the importance of commensal bacteria and enteric virus interactions in healthy and diseased condition. This new knowledge will be paramount for the identification of novel strategies to combat highly prevalent virus infections.

OBJECTIVES/GOALS: 1) Investigate the network level interactions of attention and learning during an attention network task (ANT) and an implicit learning contextual cueing (CC) task. 2) Assess the effect attention rehabilitation strategies have on behavioral and neural responses pre/post-attentional intervention. METHODS/STUDY POPULATION: This study involves refractory epilepsy patients with implanted intracranial electrodes and moderate-to-severe traumatic brain injury (m/sTBI) survivors. In epileptic patients, we will identify connectivity of cortical regions via the ANT, which probes components of attention (alerting, orienting, and executive control) and a CC task that probes implicit learning. We hypothesize that modulation of attention and learning can be seen at the network level. In TBI we will assess improvement following two attention rehabilitation paradigms behaviorally; and use our results from epileptic patients to guide measurement of treatment-related neuroplastic change via scalp electroencephalography. RESULTS/ANTICIPATED RESULTS: When the proposed objectives are complete, we expect to determine how the implicit learning rate in m/sTBI changes as a result of both direct attention and metacognitive-strategy training, and discern the neuroanatomical networks associated with attention and implicit learning based on connectivity results. We expect to identify intracranial regions and networks that exhibit modulatory effects associated with attention and implicit learning. Additionally, we anticipate that deficits in attention will be mitigated following training and hypothesize that implicit learning rate will improve in TBI patients as a result of both attentional rehabilitation paradigms. DISCUSSION/SIGNIFICANCE OF IMPACT: Characterizing intracranial activity in epilepsy patients will give electrophysiology data unattainable in TBI patients. This intracranial perspective will enable us to propose mechanisms of action that may result from our interventions and enable critique of current rehabilitation treatments.

OBJECTIVES/GOALS: Vaginal ring delivery of antiretroviral drugs may provide protection against acquisition of HIV-1 when used as pre-exposure prophylaxis. As part of a randomized placebo-controlled safety trial of a tenofovir disoproxil fumarate (TDF) intravaginal ring (IVR), we assessed product acceptability through surveys of 17 women after continuous ring use. METHODS/STUDY POPULATION: Sexually active, HIV negative women between the ages of 18 and 45 were enrolled to investigate the safety and pharmacokinetics of three months of continuous TDF IVR use. The study was designed to include 40 US participants randomly assigned (3:1) to a TDF or placebo IVR. Twelve were randomized to TDF and five were assigned to the placebo group before the study was electively discontinued due to development of vaginal ulcerations in eight women in the TDF group. Acceptability data regarding TDF and placebo ring use was gathered via self-administered, computer-based questionnaires at the one- and three-month study visits. Participants were asked about overall attitudes and feelings regarding the TDF and placebo IVR, vaginal changes associated with ring use, and their experiences with ring use during menses and with sex. RESULTS/ANTICIPATED RESULTS: The mean age of participants was 30 years (range 18 - 42). Sixteen of 17 (94%) participants completed all study questions at both visits. When asked about ring likeability at one-month, 12 of 16 (75%) women reported overall liking the ring, including 5 of 8 (63%) who developed ulcerations. Vaginal changes described during ring use included 8 participants who indicated that the “vagina was wetter” and 2 who reported that the “vagina was drier.” Additionally, 10 of 12 (83%) who had their period during the first month of the study were not bothered by ring use during menses, and 11 of 16 (69%) stated that the ring was not bothersome with use during sex. When asked at the three-month visit, most reported that they would prefer to wear the ring rather than use a condom during sex, however, condom use was low at baseline in this population. DISCUSSION/SIGNIFICANCE OF IMPACT: Despite unanticipated ulcers, the IVRs were acceptable, especially when used with menses and during sex. Regardless of the group assigned or vaginal changes experienced, and even amongst those who developed ulcerations, the women had positive attitudes towards the ring, which is promising for future use of vaginal rings as a method for HIV prevention.

OBJECTIVES/GOALS: To explore the severity of posttraumatic stress disorder (PTSD) symptoms in association with hippocampal and amygdala volumes in ICU survivors. We hypothesize that the severity of posttraumatic stress symptoms in ICU survivors is associated with lower volumes of both the hippocampus and amygdala. METHODS/STUDY POPULATION: Secondary analysis of the VISIONS study, a prospective sub-study of the BRAIN-ICU cohort, which included survivors of critical illness. Patients were screened for preexisting PTSD before discharge. The PTSD Checklist Specific (PCL-S) was used at 3 and 12 months to evaluate the ICU as a traumatic experience. A score of >30, indicated significant symptoms of PTSD. A Philips Achieva 3T MRI scanner was used to scan patients at both discharge and 3-month follow-up. To compare median brain volumes at discharge and 3 months for those with and without significant PTSD symptomatology (PCL-S ≥30) at 3 and 12 months, we used a Kruskal-Wallis (KW) equality-of-populations rank test. RESULTS/ANTICIPATED RESULTS: The median age for our sample was 58.5 (52.6, 63.7). One-third of the sample was female, and 90% were Caucasian. Fifty-seven percent of individuals (N = 12) had at least one prior mental health diagnosis, with two having a prior history of PTSD. One third of individuals experienced delirium during their critical illness. At 3-month follow up, there were three patients with PTSD symptomatology and one at 12-month follow up. Median brain volumes (hippocampus or amygdala) did not differ between individuals with or without PTSD symptomatology at either 3 or 12 months (p-values for all tests >0.05). DISCUSSION/SIGNIFICANCE OF IMPACT: Although our study did not reveal significant differences in brain volumes between PTSD patients and non-PTSD patients, sample size is a major limitation and larger scale studies should be undertaken to elucidate possible neurobiological markers of PTSD in ICU survivors. CONFLICT OF INTEREST DESCRIPTION: Dr. Wilson would like to acknowledge salary support from the Vanderbilt Faculty Research Scholars Program (1KL2TR002245), HL111111 and GM120484. Drs. Ely and Jackson as well as Mrs. Kiehl all receive funding for their time working on this investigation from AG035117 and HL111111. Dr. Ely would additionally like to acknowledge salary support from the Tennessee Valley Healthcare System Geriatric Research Education and Clinical Center (GRECC). Dr. Ely will also disclose additional funding for his time from AG027472 and having received honoraria from Orion and Hospira for CME activity; he does not hold stock or consultant relationships with those companies. The authors would like to acknowledge the following: this work was conducted in part using the resources of the Center for Computational Imaging at Vanderbilt University Institute of Imaging Science and the Advanced Computing Center for Research and Education at Vanderbilt University, Nashville, TN, and study data were collected and managed using REDCap electronic data capture tools hosted at Vanderbilt University.

OBJECTIVES/GOALS: To reduce hospitalizations, health care systems are studying ways of improving social determinants of health (SDoH) in patients with chronic disease such as diabetes (DM). Our goal was to better characterize the SDoH of a cohort of DM patients by using socio-economic information from census data. METHODS/STUDY POPULATION: Our study population included DM patients seen in primary care practices of a large health care system in 2013-2017. We integrated socio-economic status (SES) information from the American Factfinder to data extracted from the electronic health record (EHR). Addresses for the cohort were geocoded using ArcMap to obtain the census tract information for median income, poverty status, educational level, and supplemental food benefits using American Community Survey 5-Year estimates. We used multivariable logistic regression to calculate odds ratio (OR) and 95% confidence intervals [], with 3+ comorbidities as the dependent variable and demographic and SES variables as independent variables. RESULTS/ANTICIPATED RESULTS: Our study population included 13,782 patients: 53% were female, 65% white, 28% Black, 27% were on Medicare, 3% on Medicaid, median age was 60, 53% had 3+ comorbidities. Median income was $66,243, poverty level 6%, receiving food benefits 8%, no high school degree 8%, and bachelor’s degree or higher 30%. After evaluating collinearity, our multivariable analysis showed that patients with 3+ comorbidities were more likely to have income < $52,000 (lower quartile) versus $84,001 (upper quartile), OR = 1.2 [1.0-1.4]; be female, OR = 1.6 [1.4-1.7]; divorced or widowed versus married, OR = 1.5 [1.3-1.7], 1.4 [1.3-1.6]; and be on Medicare, Medicaid or both, OR = 2.4 [2.2-2.6], 2.2 [1.8-2.6], 6.0 [4.5-8.3]. DISCUSSION/SIGNIFICANCE OF IMPACT: Census tract-based SES could provide invaluable information to health care providers when associated to the EHR. We found that median income, which is not collected in the EHR, was significantly associated with a higher burden of disease. Census tract SES could serve as a proxy for evaluating SDoH.

OBJECTIVES/GOALS: Trichomonas vaginalis (TV) has a prevalence of 26% in Baltimore and is associated with preterm delivery (PTD). Yet screening and treatment of TV is not advised due to conflicting data on harms. Our goal is to investigate the association between asymptomatic TV treatment and PTD. METHODS/STUDY POPULATION: This is a retrospective cohort study of women who delivered a child at The Johns Hopkins Hospital between 7/1/16 – 11/19/19. Exclusion criteria included multiple gestation, stillborn, miscarriage, diabetes, hypertension/ preeclampsia, HIV, and history of PTD. Chart review and ICD-10 diagnosis codes were used to collect data on demographics, STI test results, lab encounter diagnoses, STI treatment during pregnancy, and labor encounter diagnoses. Preliminary analysis for crude incidence of PTD in asymptomatic and symptomatic women treated for TV was performed using TriNetx, a global research network compiling all de-identified data within the Hopkins system. RESULTS/ANTICIPATED RESULTS: Three hundred and eighty women were tested for TV, 240 (63%) were asymptomatic and 140 (37%) women were symptomatic. Mean ages were 26 (SD:5) and 26 (SD:5) years, respectively. Black women comprised 87% of the asymptomatic cohort and 93% of the symptomatic cohort. Women of Hispanic ethnicity were represented by 4% of the asymptomatic cohort and 7% of the symptomatic cohort. Crude incidence of PTD was 4.1% among asymptomatic women and 7.1% among symptomatic women. Incidence ratio comparing asymptomatic PTD incidence to symptomatic PTD incidence was 0.58 with 95% CI (0.22, 1.56). DISCUSSION/SIGNIFICANCE OF IMPACT: Preliminary data from our study suggests there is no difference in PTD between asymptomatic and symptomatic women treated for TV. Future steps include multiple linear regression using a larger dataset. These preliminary data suggest TV should be considered for screening during pregnancy.