OBJECTIVES/GOALS: Estrogen monotherapy in postmenopausal women can reduce kidney function, while dual therapy combining estrogen with a progestin improves renal health. Using the female Long Evans rat as a novel animal model of postmenopausal cardiovascular disease, we found similar results where estrogen worsens renal health while co-administration of medroxyprogesterone acetate (MPA) was protective. MPA cross-activates glucocorticoid receptors (GR), which are targeted clinically for their anti-inflammatory actions. Therefore, our goal was to determine if estrogen monotherapy induces renal damage by increasing inflammation, while dual therapy with MPA opposes inflammation by cross-activating GR. METHODS/STUDY POPULATION: Female Long Evans rats underwent OVX at 11 months of age and received a subcutaneous implant containing E2, E2+MPA or vehicle for 40 days. RESULTS/ANTICIPATED RESULTS: Coadministration of MPA prevented the E2-induced increase in proteinuria (Veh: 0.27 ± 0.07; E2: 3.53 ± 1.16; E2 + MPA: 1.20 ± 0.58 mg/mg creatinine; P = 0.03) and decline in glomerular filtration rate (Veh: 0.51 ± 0.02; E2: 0.24 ± 0.05; E2+MPA: 0.39 ± 0.05 ml/min; P < 0.01). Co-administration of MPA significantly increased renal GR transcript levels compared with E2 alone (Veh: 0.96 ± 0.02; E2: 0.94 ± 0.10; E2+MPA: 1.24 ± 0.04 fold change; P < 0.01). Inflammatory marker COX 2 renal transcript levels were significantly reduced by a similar degree in both mono and dual therapies compared with vehicle (Veh: 1.07 ± 0.06; E2: 0.81 ± 0.04; E2+MPA: 0.81 ± 0.04 fold change; P < 0.01). Neither TNF-alpha and IL-6 mRNA nor urinary beta-microglobulin levels (Veh: 1.71 ± 0.31; E2: 2.88 ± 0.78; E2+MPA: 3.07 ± 1.15 mg/day; ns) were altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results show that the effect of E2 on renal pro-inflammatory markers was not altered by the addition of MPA despite the significant increase in renal GR levels. Therefore, the renoprotective effects of MPA in midlife hormone therapy may be independent of renal GR-mediated changes in the immune profile.

OBJECTIVES/GOALS: Methionine dependence was described 45 years ago as an increased reliance on an exogenous supply of the essential amino acid methionine in most cancer cells compared to normal cells. Methionine depletion, using either synthetic diets or the enzyme methioninase, potentiates the effects of chemotherapy and radiotherapy in tumor-bearing animal models. Two main obstacles prevent methionine dependence from integrating the clinical treatment of cancer. The first is the weight loss associated with methionine depletion therapy, increasing the risk of cachexia in patients. The second is the stubborn absence of a mechanism to explain the inability of cancer cells to adapt to low methionine levels. METHODS/STUDY POPULATION: To address these two obstacles, we are using an immunocompetent murine model of metastatic melanoma to compare the effects of complete methionine deprivation with a moderate, 75-80% methionine restriction similar to the one used to increase lifespan in animal models. In an effort to identify a mechanism of action, we also performed a proteomic screen of two melanoma cell lines divergent for methionine dependence under methionine stress. RESULTS/ANTICIPATED RESULTS: We recently showed that methionine restriction is sufficient to provide gains in treating local and metastatic lesions in vivo, without weight loss. We observed few differences in pathway activation between the two cell lines in response to methionine stress, despite proliferation being cut by half in the methionine dependent cell line. We expect that subcellular translocation events may provide further information on the molecular bases of methionine dependence. DISCUSSION/SIGNIFICANCE OF IMPACT: A moderate restriction in methionine is sufficient to recapitulate the benefits of methionine depletion in cancer, without weight loss. The mechanism behind this effect remains unknown. This work contributes towards the integration of methionine dependence into clinical practice and the discovery of novel drug targets.

OBJECTIVES/GOALS: In young women, there is significant symptomatic overlap among lower urinary tract conditions, including bladder and pelvic pain, leading to misdiagnosis and delayed care. The epidemiology of pelvic pain suggests a microbial involvement, but previous studies have not definitively identified specific bacteria associated with pain diagnoses. METHODS/STUDY POPULATION: We examined urinary bacterial associations with specific symptom clusters, not diagnoses. Catheterized urinary samples were obtained from 78 pre-menopausal controls and cases with bladder and pelvic pain. 16S next-generation sequencing (NGS) characterized urinary microbial populations; validated questionnaires quantified symptom type and severity. K means unsupervised clustering analysis of NGS data assigned subjects to urotypes based on the urinary bacterial community state types. Quantitative PCR (qPCR) confirmed the NGS results and provided objective concentrations for critical taxa. Linear regression analysis confirmed the associations of bacterial concentrations and specific symptoms. RESULTS/ANTICIPATED RESULTS: In a pilot study of 35 reproductive-age women with a variety of complaints NGS revealed four urotypes that correlated with symptomatology. Isolated urgency incontinence was rare; the majority of subjects with symptoms complained of genitourinary pain. Bladder-specific pain (worse with filling, relieved by voiding) was associated with Lactobacillus iners. Asymptomatic patients almost universally had a non-iners, Lactobacillus-predominant microbiota. Vaginal and urethral pain unrelated to voiding correlated with increasing Enterobacteriaceae, primarily Escherichia coli. Detection of these species by qPCR in a validation population (n = 43) was highly predictive of each phenotype (P < 0.00001). Pathologic bacteria were associated with the severity of specific pain symptoms. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results implicate a microbial role in genitourinary pain. We describe clinically-useful bacterial biomarkers for specific pelvic and bladder pain phenotypes. This objective, rapid, and inexpensive testing to classify bladder and pelvic pain would allow more accurate diagnosis and improve treatment. CONFLICT OF INTEREST DESCRIPTION: Dr. Anger is an expert witness for Boston Scientific. Dr. Eilber is an investigator and expert witness for Boston Scientific, an investigator for Aquinox, and a consultant for Boston Scientific and Allergan. Dr. Ackerman is an expert witness for Cynosure.

OBJECTIVES/GOALS: Nicotinamide adenine dinucleotide (NAD) plays essential roles in energy metabolism and cell signaling pathways. NAD functions as a coenzyme by accepting electrons during glycolysis and the TCA cycle and subsequently donates them to complex I of the electron transport chain providing the driving force for ATP production. NAD also acts as a co-substrate for several classes of enzymes, including sirtuin deacetylases. Both NAD and the enzyme that is rate limiting for synthesis, Nicotinamide phosphoribosyltransferase (Nampt), are depleted in the failing heart, concurrent with hyperacetylation and mitochondrial dysfunction. Moreover, treatment with NAD precursors reduced cardiac injury in several heart failure models. However, NAD precursors may have systemic effects, and it remains unproven whether depletion of myocardial NAD is causative or merely correlative for the onset and progression of heart failure. METHODS/STUDY POPULATION: To test this, we generated a cardiac-specific tamoxifen-inducible (αMHC-MerCreMer) model for deletion of Nampt (Nampt cKO) in cardiomyocytes. Adult mice were administered tamoxifen for 5 days leading to deletion of Nampt, resulting in a 72% reduction in myocardial NAD after two-weeks. RESULTS/ANTICIPATED RESULTS: Echocardiography revealed that Nampt cKO mice displayed a significant reduction in left ventricular (LV) contractility as well as cardiac hypertrophy. Despite the further loss of NAD, the majority of animals survived to 8 weeks of age before experiencing sudden deaths resulting in significant mortality over the next several weeks. Remarkably, we observed only a slight increase in acetylation of mitochondrial proteins, and cardiac mitochondria isolated from Nampt-null mice even at 8 weeks displayed a normal or higher oxygen consumption rate. We found that mitochondrial NAD levels were preferentially maintained and depleted at a slower rate compared to those in bulk tissue. DISCUSSION/SIGNIFICANCE OF IMPACT: While mild depletion of cardiac NAD has been reported in heart failure, our data indicate that the heart can adapt to much more severe loss of NAD prior to the loss of viability.

OBJECTIVES/GOALS: Epilepsy with myoclonic-atonic seizures (EMAS) is a childhood onset epilepsy disorder characterized by seizures with sudden loss of posture, or drop seizures. Our objective was to use short-read genome sequencing in 40 EMAS trios to better understand variants contributing to the development of EMAS. METHODS/STUDY POPULATION: Eligibility for the cohort included a potential diagnosis of EMAS by child neurology faculty at Children’s Hospital Colorado. Exclusion criteria included lack of drop seizures upon chart review or structural abnormality on MRI. Some individuals had prior genetic testing and priority for genome sequencing was given to individuals without clear genetic diagnosis based on previous testing. We analyzed single nucleotide variants (SNVs), small insertions and deletions (INDELs), and larger structural variants (SVs) from trio genomes and determined those that were likely contributory based on standardized American College of Medical Genetics (ACMG) criteria. RESULTS/ANTICIPATED RESULTS: Our initial analysis focused on variants in coding regions of known epilepsy-associated genes. We identified pathogenic or likely pathogenic variants in 6 different individuals involving 6 unique genes. Of these, 5 are de novo SNVs or INDELs and 1 is a de novo SV. One of these involve a de novo heterozygous variant in an X-linked gene (ARHGEF9) in a female individual. We hypothesize the skewed X-inactivation may result in primarily expression of the pathogenic variant. We anticipate identifying additional candidate variants in coding regions of genes previously not associated with EMAS or pediatric epilepsies as well as in noncoding regions of the genome. DISCUSSION/SIGNIFICANCE OF IMPACT: Despite the genetic heterogeneity of EMAS, our initial analysis identified de novo pathogenic or likely pathogenic variants in 15% (6/40) of our cohort. As the cost continues to decline, short read genome sequencing represents a promising diagnostic tool for EMAS and other pediatric onset epilepsy syndromes. CONFLICT OF INTEREST DESCRIPTION: The authors have no conflicts of interest to disclose. SD has consulted for Upsher-Smith, Biomarin and Neurogene on an unrelated subject matter. GLC holds a research collaborative grant with Stoke therapeutics on unrelated subject matter.

OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized in vitro septal-hippocampal co-culture system modified from previous published protocols. Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons in the co-culture attained up to ~5-7%, depending on conditions such as embryo age at dissection or ratio of septal to hippocampal cells. We also report on cholinergic synapse structure by examining postsynaptic markers (excitatory and inhibitory) and staining for nicotinic acetylcholine receptor subunits. DISCUSSION/SIGNIFICANCE OF IMPACT: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.

OBJECTIVES/GOALS: Vascularized composite allotransplantation (VCA) restores devastating soft tissue injuries. However, graft viability may be compromised during ischemia time, thus preservation techniques continue to evolve. Here we summarize our preliminary findings from preservation techniques utilizing hyperosmolar extracellular solution (HES) and electric stimulation in a 6 hour ex-vivo perfusion model. METHODS/STUDY POPULATION: A published, MGH rodent hindlimb ex-vivo perfusion model was utilized for this project. Three baseline control elements were taken to compare our results including: a baseline muscle biopsy, harvested hindlimb preserved on ice(4°C) in static cold storage (SCS), and 6 hour perfusion (SNMP) were used to compare results of our four aims. The four aims are shown in Table 1. HES was composed of muscle media and the addition of mannitol until 3 concentrations were made: 300, 500, and 800 mOsm concentrations. In aim 4, the perfusate composition was changed to test a hyper-oncotic purfusate. After 6 hours a muscle biopsy was taken to analyze energy cofactors via liquid chromatography-mass spectrometry, referred to as energy charge. Weight gain (edema), lactate levels, oxygen consumption and energy charge (EC) were used as markers for muscle tissue viability. RESULTS/ANTICIPATED RESULTS: In Aim 1, the higher osmolarity of HES indirectly reduced weight gain but consequentially reduced the EC below 5% when compared to SCS control group. We next incorporate HES into the perfusion model, Aim 2, and noticed a diminution in weight gain. The 500 mOsm group had substantial improvement in EC, lactate production and improved oxygen exchange when compared to a controls: fresh muscle biopsy and SNMP. In Aim 3, after a 6 hour perfusion with the addition of electric stimulation, graft edema improved by 10%, EC improved by 23% and O2 dissociation was highest of all 4 aims. Consequentially, due to muscular contraction the lactate levels were highest. In Aim 4, using a hyper-oncotic perfusate, edema reduced the most during the 6 hour perfusion but revealed lower EC and similar lactate/O2 results. However when left on for 24 hours, edema was significantly higher with lactate build up, EC improved with time as well as O2 dissociation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we have shown our preliminary results comparing our known ex-vivo perfusion model to multiple hypothesis to improve VCA graft viability. These preservation techniques demonstrate promising results but further studies are ongoing to confirm this encouraging outcome.

OBJECTIVES/GOALS: Utilize polymer-based fiber scaffolds and machine learning methods applied to patient biomarker data to enhance and personalize T cell expansion and production for T cell therapy in chronic lymphocytic leukemia. METHODS/STUDY POPULATION: Scaffolds are 1) generated from a co-polymer blend of PDMS and PCL with controlled fiber diameters and pore size, 2) coated with activating antibodies to CD3 and CD28, and 3) used to stimulate T cells from both healthy donors and CLL patients. CLL patients have pre-annotated mutation burdens and clinical biomarkers. T cell populations will be analyzed for exhaustion markers and phenotypes before, during, and after expansion. Cell functionality will be measured by cytokine secretion, cell cycle analysis, and fold expansion, with respect to platform parameters, and analyzed with inputs of disease markers and exhaustion profile of isolated T cells using regression and random forest classifiers. RESULTS/ANTICIPATED RESULTS: We previously showed that engineering the mechanical rigidity of activating substrates can enhance and rescue T cell expansion from exhausted populations. Now we aim to study a broader range of compositions and geometry of scaffolds with respect to capacity to expand CLL T cells. Preliminary data with fiber diameters ranging from 300 nm to 6 um confirm the effect of geometry in modulating expansion. A biorepository of T cells from 80 CLL patients have been isolated concurrently. Anticipated results include correlating exhaustion profile of T cells with clinical biomarkers and identifying markers associated with expansion on panel of platform parameters. DISCUSSION/SIGNIFICANCE OF IMPACT: T cell therapy has shown particular promise in treating blood cancers, yet significant percentage of T cells isolated from patients undergoing treatments are unresponsive to activation. A powerful tool is to predict if and how patient T cells can be robustly expanded on a personalized approach.

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.