OBJECTIVES/GOALS: Although the approval of immune checkpoint inhibitors (ICIs) revolutionized the treatment of metastatic breast cancer (BC), yet about 30% remain unresponsive. Since the potency of ICIs depends on the efficient presentation of tumor-specific antigens by cancer cells, compounds which increase such presentation could increase efficacy of ICIs. METHODS/STUDY POPULATION: A library of the ester and urethane derivatives of polyether ionophore antibiotic, monensin (MON) has been synthesized. MTT cell viability assays were performed on the panel of human and mouse BC cell lines, and non-cancerous breast epithelial cells to determine IC50 values of MON and its derivatives. Selectivity Indexes were calculated to identify the most selective compounds towards cancer versus non-cancer cells. Major Histocompatibility Complex (MHC) class I and II presentation and Programmed death-ligand 1 (PD-L1) expression have been determined using flow cytometry. Proteins involved in apoptosis, autophagy and immunogenic cell death were identified through immunoblotting. At least three biological replicates have been performed for each experiment. RESULTS/ANTICIPATED RESULTS: MON and several of its derivatives shown activity in nanomolar range against MDA-MB-231 human BC cell line. MON and its most potent derivatives significantly increased MHC class I and II presentation and downregulated the expression of PD-L1 in BC cell lines. DISCUSSION/SIGNIFICANCE: Present findings will lead to the development of new therapeutic approaches that can serve as single agents or be used in combination with existing ICIs for the treatment of metastatic BC. By pushing the boundaries of our understanding and developing new therapies, this research can make an impact in improving outcomes for patients with metastatic BC.

OBJECTIVES/GOALS: Distinguishing indolent from aggressive prostate cancer and early identification of men at risk of developing aggressive, metastatic disease is of great importance. We aim to explore the relationship between N-glycan and collagen composition in prostate tumor tissue and the long-term outcome of the disease. METHODS/STUDY POPULATION: Matrix assisted laser desorption/ionization mass spectrometry can be utilized to characterize N-glycan profiles in formalin fixed paraffin embedded tissues. Collagen may also be characterized using ECM-targeted collagenase MALDI imaging. These approaches were used to analyze prostatectomy samples with different clinical outcomes. Tissue microarrays containing tissues from 75 non-progressors (no evidence of disease; NED) and 50 metastatic cases (MET) were examined. From a combined list of 90 N-glycans and 500 collagenase peptides, the average AUC intensity value for each glycan and collagen peptide was extracted and assessed as a predictor of metastatic progression. Ancestral informative markers were analyzed and polygenic hazard risk scores were generated for samples as well. RESULTS/ANTICIPATED RESULTS: Three N-glycans and three collagen peptides were found to discriminate between NED and MET cases with statistical significance. The best performing N-glycan was Hex6HexNAc6Fuc1 with an AUC of 0.77 (p<0.001). While the best performing collagen peptide was COL1A2 with an AUC of C 0.77 (p<0.001). DISCUSSION/SIGNIFICANCE: Both a collagen peptide and N-glycan were discovered as promising biomarkers to predict metastasis. Future validation studies are needed to confirm biomarker potential and to determine if the addition of these biomarkers can strengthen current genomic classifier’s ability to predict metastatic prostate cancer.

OBJECTIVES/GOALS: RETgene fusions in sarcoma are rare and their impact on pathogenicity is unknown. Malignant peripheral nerve sheath tumors (MPNST) are a deadly, genomically heterogenous soft tissue sarcoma rarely harboring targetable aberrations. We present a case of a CCDC6-RET fusion MPNST sensitive to RET-inhibitor therapy in a xenograft model. METHODS/STUDY POPULATION: Lung tumor tissue was obtained per an approved collection protocol from a 21yo male patient with a spontaneous MPNST harboring an inactivating mutation in NF-1 and a CCDC6-RET gene fusion detected by a commercially available sequencing panel (Signatera). To confirm pathogenicity of the RET fusion, fresh tumor tissue was engrafted into immunocompromised NSG mice in the anterior and posterior flanks, harvested at ~10 weeks, and re-transplanted into bilateral flanks. When tumor diameters reached 0.5-1cm (~4 weeks), mice were randomized into 3 groups (n=6/group) and treated with either vehicle (V) (PBS), the RET-specific inhibitor selpercatinib (S) (20mg/kg twice daily), or the multi-kinase inhibitor cabozantinib (C) (30mg/kg daily) by oral gavage. Mice were monitored weekly for weight and tumor size. RESULTS/ANTICIPATED RESULTS: 92% (33/36) of implanted tumors were evaluable for treatment response. Pre-treatment tumor volumes (mm3) across all three groups were similar (mean/Std Dev – V: 230/111, S: 271/132, C: 230/123). At day 7, tumor growth was significantly inhibited by S and C versus V (ANOVA p < 0.001, post-hoc Tukey’s V vs S p= 0.0178, V vs C p< 0.0001, S vs C p= 0.0005). V-treated tumors increased in volume by 60% while S reduced tumor volume by ~80% and C reduced tumor volume by ~20%. S and C treatments were tolerated well. and S improved survival with 100% of mice alive at day 63 vs 0% in V and C groups. 6 of the 12 implanted tumors treated with S, 50% increased in size after ~6-weeks following a >90% initial tumor reduction in tumor volume. Follow-on molecular studies in S-resistant tumors are ongoing. DISCUSSION/SIGNIFICANCE: Targetable genomic changes in MPNST, especially in RET, are infrequent and often considered stochastic. Our findings suggest that precision medicine approaches pairing genomic sequencing and in vivotesting of target gene pathogenicity may guide treatment planning and novel discovery for rare, difficult to treat sarcomas.

OBJECTIVES/GOALS: Research objectives include prototyping a mobile phone application that allows physicians to monitor CD19-directed CAR T-cell therapy patients remotely after discharge. This app will also enable standardized data collection across different centers that provide CAR-T cell therapy and allow for the harmonization of follow-up protocols. METHODS/STUDY POPULATION: Literature review and semi-structured interviews with patients, clinical coordinators, and other experts in the field will be used to determine what parameters must be included in the mobile application prototype to effectively monitor the side effects of CD19-directed CAR T-cell therapy. The mobile phone application will be designed using process mapping to integrate data from self-reporting and wearable technologies, including the Garmin smart watch. Figma will then be used to develop new screens based on an existing patient monitoring app for Allogeneic Stem Cell Transplant follow-up. Finally, a preliminary feasibility study will be conducted to collect feedback on the app prototype from CAR T-cell therapy patients, providers, and stakeholders. RESULTS/ANTICIPATED RESULTS: The anticipated results of this study include an app prototype that will include the functionalities required to monitor patients for adverse effects of CD19-directed CAR T-cell therapy. This will include the parameters that will be recorded or measured using a combination of self-reporting, a reliable body temperature sensor, and the Garmin watch which monitors basic vitals, activity, and sleep. Additional parameters may be added during the stakeholder co-design process. The app prototype will include a physician interface where doctors can monitor their patients and will be alerted if they require further physician assessment. It is expected that the app will provide standardized monitoring of patients when they are discharged from the hospital after receiving CAR T-cell therapy. DISCUSSION/SIGNIFICANCE: This app will allow physicians to monitor patients for general follow-up and adverse effects, including cytokine release syndrome and neurotoxicity. Future studies may utilize this app to develop best practices for harmonizing CAR-T follow-up protocols across Canada.

OBJECTIVES/GOALS: Preeclampsia increases cardiovascular (CV) risk, likely via persistent endothelial dysfunction and angiotensin II type 1 receptor autoantibodies (AT1R-Ab). We aim to assess coronary endothelial function (CEF) and AT1R-Ab levels in postpartum preeclampsia with a hypothesis this mediates CV risk. METHODS/STUDY POPULATION: We prospectively enrolled age and CV risk factor matched postpartum women. Coronary MRI was performed at rest and with isometric handgrip stress, an endothelial dependent stressor. CEF was quantified as % stress-induced change in coronary cross-sectional area (%CSA) and in coronary blood flow (%CBF). AT1R-Ab was measured using a novel antigen capture enzyme-linked immunosorbent assay. RESULTS/ANTICIPATED RESULTS: Women with and without preeclampsia were similar in age (mean 32.7+5.0 years), BMI (mean 28.0+6.3 kg/m2) and race/ethnicity (58% White, 35% Black and 4% Hispanic). %CSA was lower with (-2.1+13.6) vs without preeclampsia (8.8+17.1), p=0.023. %CBF was also lower with (11.3 [-11.8, 25.2]) vs without preeclampsia (25.7 [-0.7, 62.9]), p=0.039. AT1R-Ab was higher among women with preeclampsia (p=0.029) and was inversely associated with %CBF (beta coefficient -4.6 [-8.9, -0.3], p=0.037) but not with %CSA. DISCUSSION/SIGNIFICANCE: Women with preeclampsia have elevated AT1R-Ab and impaired CEF demonstrated by insufficient coronary reserve with exercise. Coronary endothelial dysfunction and dysregulation of the renin-angiotensin pathway likely contribute to long-term CV risk and should be considered for targeted risk reduction.

OBJECTIVES/GOALS: Many economic evaluations rely on clinical trial data that may not represent real world populations and intervention effectiveness. We compare risk and cost-effectiveness for the Diabetes Prevention Program (DPP) clinical trial cohort and a real world population eligible for the national DPP to assess the impact of using real world data. METHODS/STUDY POPULATION: To produce real world (US population) representative results, we identified National Health and Nutrition Examination Survey (NHANES) subjects eligible for the national DPP and adjusted projections using survey weights. We used clinical predictive models to estimate individual diabetes risk, and microsimulation to estimate lifetime costs, benefits, and net monetary benefits (NMB) for lifestyle intervention and metformin. We compared results across the DPP clinical trial and NHANES populations. RESULTS/ANTICIPATED RESULTS: Three-year risk of diabetes onset for the DPP trial population (mean of 19.7%, median of 10.3%) exceeded corresponding risk for the NHANES population (mean of 14.6%, median of 4.8%). The proportion of individuals with a three-year diabetes risk < 10% for the DPP trial population (49%) was less than the corresponding proportion for NHANES (67%). Mean NMB for metformin for the DPP trial population ($9,749) exceeded the corresponding value for NHANES ($5,391). The proportion of subjects with negative NMB was 49% for the DPP trial population and 67% for NHANES. Lifestyle intervention had a mean NMB of $34,889 for the DPP trial population and $28,652 for NHANES. Only 20% of the NHANES population eligible for national DPP met inclusion/exclusion criteria for the DPP trial. DISCUSSION/SIGNIFICANCE: Real world populations eligible for the national DPP include a greater proportion of low-risk individuals, and for these people, prevention programs may confer smaller benefits. Technology assessments based on clinical trial data should be revised using real world population and treatment effect data.

OBJECTIVES/GOALS: Glioblastoma (GBM) is the most malignant brain tumor in adults and remains incurable with an average survival of 15 months after diagnosis. There is great need for treatment options without side effects that are devastating to the quality of life for patients. GBM tumors can circumvent cellular damage by upregulating antioxidant production. METHODS/STUDY POPULATION: Highly aggressive tumors tend to exhibit increased oxidative metabolism, and thus rely on a mechanism to eliminate reactive oxygen species (ROS) in order for cells to evade autophagy and cell death. We propose that recurrent GBM cells achieve this is by promoting methionine metabolism, upregulating glutathione production and preventing ROS accumulation. We investigated the expression of AHCY and MAT2A, two key enzymes in the methionine pathway, at the gene and protein level in both GBM and non-GBM tissues. We probed for markers of cell death following pharmacological inhibition and siRNA knockdown, and performed metabolite-mediated rescue experiments. Finally, we evaluated changes in cellular respiration using the Seahorse XFe96 real-time mitochondrial stress test following inhibitor treatment. RESULTS/ANTICIPATED RESULTS: The selective AHCY inhibitor markedly reduced cell viability in different cancer cell types, but significantly reduced cell viability in recurrent GBM cells compared to newly diagnosed GBM (p=.009; MD: -0.828, 95% CI -1.350 to -0.306) and normal astrocytes (p=.073; MD: -0.609, 95% CI -1.305 to 0.085). AHCY and MAT2a protein expression appeared to be higher in GBM cells compared to normal astrocytes, medulloblastoma cells and other cancer cell lines. Genetic knockdown of AHCY and MAT2A demonstrated reduced cell viability, increased Caspase, SOD2, LC3-II and Transferrin receptor expression. Acute treatment with the AHCY inhibitor induced cell death, markedly reduced oxygen consumption rate and ablated spare respiratory capacity in recurrent GBM cells compared to newly diagnosed GBM cells. DISCUSSION/SIGNIFICANCE: Oxidative damage was induced following interference with key methionine pathway enzymes by pharmacological inhibition, while a similar concentration of drug largely preserved normal astrocyte viability. These results point to a novel targetable mechanism of disease progression and expand the realm of treatment options for recurrent GBM.

OBJECTIVES/GOALS: Transcriptional adaptation is a phenomenon in which a mutation in one gene leads to the genetic compensation of another homogenous gene. Understanding the mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of rare Amyotrophic lateral sclerosis patient phenotypes. METHODS/STUDY POPULATION: The presence of a premature termination codon triggers transcriptional activation. Therefore, we utilized CRISPR-Cas9 tool to generate a premature termination codon in CHCHD10 gene in multiple types of cells, including induced pluripotent stem cells derived from patient samples with known CHCHD10 mutations causative for Amyotrophic lateral sclerosis. CRISPR-Cas9 tool was delivered via ribonucleoprotein electroporation and transfect cell’s DNA was sequenced to validate gene editing. To confirm transcriptional adaption, changes in levels of protein and gene expression will be measured via immunoblot and quantification of CHCHD10 and CHCHCD2 from whole cells lysates of the edited cells. RESULTS/ANTICIPATED RESULTS: We anticipate that CHCHD2 transcriptional adaptation can functionally compensate for the locus loss of function of CHCHD10. This mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of patient phenotypes. DISCUSSION/SIGNIFICANCE: Our approach would advance discovery science towards by exploring transcriptional adaptation mechanism in humans, which can lead to novel therapies for rare Amyotrophic lateral sclerosis, such as CHCHD10.

OBJECTIVES/GOALS: Irritability, a proneness to anger and frustration, is a transdiagnostic symptom associated with poor mental health outcomes. Levels of irritability vary across development and high-risk trajectories have been observed. This study aims to use machine learning to predict irritability trajectories across the transition to adolescence. METHODS/STUDY POPULATION: Data were from the Adolescent Brain Cognitive Development (ABCD) Study, which is a 10-year longitudinal study that tracks the brain development, cognitive skills, physical health, and psychosocial functioning of a large, national sample starting from preadolescence. The baseline sample consisted of 11,861 9-10-year-old preadolescent youth. Irritability was parent-rated at baseline, 1-year, 2-year, 3-year, and 4-year follow-ups on the Child Behavior Checklist (CBCL) irritability index. Latent class growth analysis (LCGA) was used to determine developmental trajectories of irritability. Two machine learning approaches were applied to develop predictive models of youth irritability developmental trajectories. We used baseline (preadolescent) variables that spanned a wide range of domains. RESULTS/ANTICIPATED RESULTS: Preliminary results fromthe LCGA indicated best support for a four-class model that differentiated growth trajectories in irritability across the transition to adolescence: 1) persistent low irritability (n = 8691, 73.27%), 2) moderate irritability and decreasing (n = 1257, 10.60%), 3) low to moderate irritability and increasing (n = 1295, 10.92%), and 4) chronic high irritability (n = 618, 5.21%). We expect the machine learning analyses to generate predictive models with acceptable accuracy. We hypothesize that the most important predictors in the models will originate from the youth mental health domain, including baseline youth irritability, externalizing symptoms, internalizing symptoms, and oppositional behaviors, and the parent psychopathology domain, particularly parent irritability. DISCUSSION/SIGNIFICANCE: The present study elucidates unique developmental trajectories of irritability and generates predictive models to classify high-risk irritability trajectories using machine learning approaches. Clinicians can use these predictive models to identify at-risk youth and provide early intervention to preadolescents at high risk.

OBJECTIVES/GOALS: Diabetic Kidney Disease (DKD) is a common diabetes complication, often linked to end-stage renal disease in the United States (US). While autophagy and miRNAs are pivotal, miR-451’s specific role remains understudied. Our study explores its renoprotective effects in an accelerated DKD mouse model. METHODS/STUDY POPULATION: We assessed the effect of miR-451 mimic treatment on Diabetic Kidney Disease (DKD) in BTBR ob/ob mice, known for their rapid DKD-like renal lesions. Mice were divided into four groups: WT (wild-type), BTBR ob/ob, WT+miR-451 (wild-type with miR-451 mimic), and BTBR ob/ob+miR-451 (BTBR ob/ob with miR-451 mimic). MiR-451 mimics were administered at 2mg/kg body weight once weekly for three consecutive weeks. We collected spot urine and monitored blood glucose levels at each time point. After the treatment period, mice were euthanized for kidney and blood samples. Western blot analysis assessed autophagy-related protein markers. Statistical analysis included Student’s t-test and ANOVA (p<0.05). RESULTS/ANTICIPATED RESULTS: The study assessed the impact of miR-451 mimic treatment in BTBR ob/ob mice. Albumin:creatinine ratio increased fourfold (p=0.01) in BTBR ob/ob mice at 5 weeks. MiR-451 mimic treatment had no impact on body weight. Blood glucose levels were notably higher in both treated and untreated BTBR ob/ob mice at 12 (425±33.1 mg/dL; p=0.04) and 13 weeks (383±25.3 mg/dL; p=0.007). However, a significant drop occurred from week 13 (554.7±10.8 mg/dL) to week 14 (289±13.3 mg/dL; p=0.0002) in BTBR ob/ob miR-451 treated mice. Western blot analysis in whole kidney homogenates showed a 91% reduction (p=0.02) in YWHAZ, a predicted miR-451 target, in treated BTBR ob/ob mice and a 95% reduction (p=0.01) in WT mice. Furthermore, miR-451 mimic treatment led to a 68% increase (p=0.01) in ATG101 and a 44% increase in Beclin-1 in BTBR ob/ob mice. DISCUSSION/SIGNIFICANCE: The study uncovers miR-451-based interventions as a promising avenue to counter Diabetic Kidney Disease by modulating autophagy, potentially introducing novel therapies for at-risk individuals. However, practical DKD treatments will require further research and rigorous clinical validation to harness the full potential of these insights.

OBJECTIVES/GOALS: Reveal common immune mechanisms in dysregulated age-related lobular involution (ARLI) and post-partum lobular involution (PPLI) to understand their link to increased breast cancer risk, challenging the traditional view of their distinctiveness. Ultimately, to improve breast cancer risk assessment and personalized prevention METHODS/STUDY POPULATION: The Mayo Clinic Benign Breast Disease (BBD) cohort comprises of ~20,000 women with benign biopsies, including ~1000 women with sequential benign biopsies. Lobular involution (LI) status was assessed by selecting perimenopausal women, ages 45-55, with sequential biopsies, comparing acini number and lobule size between initial and subsequent biopsies. NanoString IO360/ BC360 RNA profiling identified differentially expressed genes associated with dysregulated LI. Using multiplex immunofluorescence (mIF), I'll analyze and spatially map immune biomarkers related to dysregulated ARLI and PPLI in BBD tissue from perimenopausal women who did or did not go on to develop breast cancer, assessing the commonality of ARLI and PPLI markers and exploring their potential as risk biomarkers for breast cancer. RESULTS/ANTICIPATED RESULTS: Preliminary findings link patients who display dysregulated ARLI with an increased breast cancer risk and identify vital PPLI biomarkers in perimenopausal women. I expect the biopsies of women who developed post-menopausal breast cancer (PMBC) and post-partum breast cancer (PPBC) to exhibit elevated levels of dysregulated ARLI immune biomarkers and PPLI biomarkers. Spatially mapping these markers promises to provide a more comprehensive understanding of their interactions, potentially revealing common immunological pathways. These findings could transform our current paradigm of ARLI and PPLI as distinct processes and demonstrate their interconnection in shaping breast cancer risk. DISCUSSION/SIGNIFICANCE: PMBC and PPBC dominate majority of breast cancer cases. Both involve activation of the understudied process of lobular involution, which has been shown to have pro-tumorigenic traits. Elucidating these mechanisms will aid more efficient risk stratification and personalized prevention to reduce incidence and mortality of breast cancer.

OBJECTIVES/GOALS: Burst suppression is a neurophysiological marker associated with severe hypoxic-ischemic injury following cardiac arrest. The goal of this study is to identify the anatomical regions of the brain associated with burst suppression post-cardiac arrest. METHODS/STUDY POPULATION: 86 comatose patients post-cardiac arrest admitted to the neurological-ICU from Massachusetts General Hospital and Brigham and Women’s Hospital were included in this study. EEG data after return of spontaneous circulation were preprocessed and artifact was rejected. Burst segments were extracted for source localization analysis from epochs with burst suppression. Four bursts for each patients were manually selected. The source of the bursts were obtained using the Champagne algorithm and mapped on the Desikan-Killiany atlas. The source for each burst was defined as any region of interest (ROI) with power > = 75th percentile relative to all ROIs. The power of the bursts at each source was correlated with the burden of brain injury measured using apparent diffusion coefficient (ADC) per ROI. RESULTS/ANTICIPATED RESULTS: 48 (56%) patients had burst suppression. 5 (10.4%) of patients with burst suppression were independent at the time of hospital discharge. Preliminary analyses was performed on 6 patients (24 bursts in total). ROI’s determined to be sources in a majority of the burst (>=13) were bilateral superior frontal, rostral middle frontal, parstriangularis precentral, superior parietal, inferior parietal, right post central, superior temporal, lateral occipital, and left middle temporal ROI. A lower mean ADC intensity was associated with a higher EEG power in the bilateral superior frontal (r = -0.80, p < 0.0001; r = -0.677, p < 0.001, respectively), left superior parietal (r = -0.53, p = 0.009), left middle temporal (r = -0.43, p = 0.042) ROI. DISCUSSION/SIGNIFICANCE: The source of bursts in patients post-cardiac arrest experiencing burst suppression is not well defined. This study will improve our understanding of how burst suppression is a measure of cortical injury, how it may relate to the burden of injury found on ADC imaging, and patient outcomes.

OBJECTIVES/GOALS: Vanishing White Matter Disease (VWM), is a childhood neurodegenerative leukodystrophy that presents with motor deficits, neurologic decline, and seizures leading to death.There are no treatments. Herein we investigate adeno-associated virus serotype 9 (AAV9) gene addition therapy for VWM. METHODS/STUDY POPULATION: To serve as a baseline for disease correction, we characterized the severe VWM Eif2b5I98M murine model with clinically relevant readouts including motor function, gait mapping and myelin loss through magnetic resonance imaging (MRI). Molecular characterization through the identification of biomarkers was also investigated. To provide targeted disease correction, we designed four gene replacement constructs to drive the rapeutic EIF2B5 expression in astrocytes—a critical cell type for VWM pathology. We are currently evaluating our AAV vectors in two murine VWM models, Eif2b5R191H and Eif2b5I98M, and are monitoring disease progression using traditional and clinically relevant readouts. RESULTS/ANTICIPATED RESULTS: The I98M mice display significant mobility loss, ataxic gait, and demyelination. Molecular characterization also indicates that the integrated stress response is significantly dysregulated, supporting the classic VWM phenotype. Our previous biodistribution study confirmed our ability to efficiently target astrocytes using varying iterations—including one novel—of the glial fibrillary acidic protein (GFAP) promoter. Our data suggests that targeting astrocytes with gene addition delays disease onset, partially rescues motor function, and attenuates myelin loss. Survival of the AAV9-gfaABC(1)D-EIF2B5 treated I98M mice is also significantly increased (p<0.0001), currently with a 2-fold extension in life expectancy. DISCUSSION/SIGNIFICANCE: Overall, we anticipate emergence of a lead astrocyte-targeted gene therapy candidate in which the data will be strengthened through the evaluation of clinically relevant measures in two murine models of disease, allowing fortimely translation to the clinic.

OBJECTIVES/GOALS: The goal of this study is twofold: To develop a method for an ex vivo hypofibrinolytic control and second to analyze patterns in standard and recently developed clinical coagulation assays for the detection of hypofibrinolytic states. METHODS/STUDY POPULATION: We analyzed blood samples from healthy patients first under normal conditions and then laced with human recombinant PAI-1 under three different concentrations. We then analyzed both samples using standard clinical assays (PT, aPTT, D-dimer, Fibrinogen), thromboelastography point-of-care tests (Hemosoncs- Quantra system), and with research assays of clot size and aggregation. Our previous research of diagnostic errors showed the patient group with the highest overall risk of these non-identifiable thrombotic complications was post-menopausal women with chronic diseases. We therefore focused our patient population to healthy post-menopausal women who were not using hormone replacement therapy. RESULTS/ANTICIPATED RESULTS: Research assays showed PAI-1 significantly increased clot size and aggregation. Preliminary results of clinical assays showed no detectable difference in hypofibrinolytic samples at any concentration. We anticipate ongoing testing will show similar results. Results on Quantra tests showed much larger differences between control and hypofibrinolysis samples, and we anticipate ongoing testing will achieving statistical significance. It is still unknown whether the mean value for hypofibrinolysis samples on the Quantra Clot Stability assay will be outside of the “normal” reference range. We theorize that this may be due to hypofibrinolytic changes in the overall structure and core density of the clots. DISCUSSION/SIGNIFICANCE: Cellular stress stimulates a concomitant activation of inflammation and coagulation, including decreased fibrinolysis. Unfortunately, current clinical assays do not assess clot breakdown. This connection would account for the increased rate of thrombosis in patients with chronic inflammation without detectable results on clinical tests.

OBJECTIVES/GOALS: Antibiotic treatment sets the stage for intestinal domination by Candida albicanswhich is necessary for development of invasive disease, but the resources driving this bloom remain poorly defined. We sought to determine these factors in order to design novel prophylaxis strategies for reducing gastrointestinal (GI) colonization. METHODS/STUDY POPULATION: We initially developed a generalizable framework, termed metabolic footprinting to determine the metabolites C. albicanspreferentially uses in the mouse GI tract. After identifying the metabolites C. albicansutilizes, we usedin vitro growth assays in the presence and absence of oxygen to validate out metabolomics findings. We next determined if a probiotic E. coli that utilizes oxygen would reduce C. albicanscolonization compared to a mutant E. coli that could not respire oxygen. Finding that oxygen was a necessary resource, we utilized germ-free mice to determine if Clostridiaspp. known to reduce GI oxygen would prevent C. albicanscolonization. Lastly, we sought to see if 5-aminosalicylic acid (5-ASA) could prevent C. albicanscolonization. RESULTS/ANTICIPATED RESULTS: We found that C. albicans preferentially utilizes simple carbohydrates including fructo-oligosaccharides (e.g., 1-kestose), disaccharides (e.g., β-gentiobiose), and alcoholic sugars (e.g., sorbitol) and is able to grow in vitro on minimal media supplemented with either of these nutrients. However, in the hypoxic environment that is found in the “healthy” colon, C. albicans cannot utilize these nutrients. We next found that pre-colonization in a mouse model with a probiotic E. coli significantly reduced C. albicanscolonization, but the mutant E. coli had no effect on colonization. We next showed that Clostridia supplementation restored GI hypoxia and reduced C. albicanscolonization. Remarkably, we found that 5-ASA significantly reduced GI colonization of C. albicans. DISCUSSION/SIGNIFICANCE: We have shown that C. albicans requires oxygen to colonize the GI tract. Importantly, we found that 5-ASA can prevent an antibiotic mediated bloom of C. albicans by restoring GI hypoxia, which warrants additional studies to determine if 5-ASA can be used as an adjunctive prophylactic treatment in high risk patients.

OBJECTIVES/GOALS: Our aims are to 1) describe changes in thumb Carpometacarpal (CMC1) joint stability following an 8-week clinic-based dynamic stability exercise program using computerized tomography (CAT) and 2) to evaluate the agreement between ultrasound and CAT (reference standard) when quantifying thumb CMC stability. METHODS/STUDY POPULATION: Aim 1: We have enrolled 13/49 participants in a prospective pre-post interventional study of an 8-week clinic-based occupational therapy dynamic stability program. The primary outcome will be change in stability (thumb metacarpal subluxation in mm) when forcefully loading the thumb as per CAT from pre-treatment to post-treatment at 9 weeks. Aim 2: Same 49 participants are undergoing a one-time ultrasound during baseline assessment. Agreement of ultrasound and CAT measurements (thumb metacarpal subluxation in mm) will be assessed by the Bland-Altman method. RESULTS/ANTICIPATED RESULTS: Exercise is a first-line treatment of CMC1 OA yet there is insufficient evidence to support this. Progression of CMC1 OA is characterized by altered joint mechanics. Joint replacement surgery may reduce pain but often worsens thumb mechanics and overall hand function. This study is the first to test the sustained biomechanical effects of non-invasive thumb exercises. Should these benefits exist, this will further support exercise as a first-tier intervention. Should ultrasound be a suitable proxy for CAT, therapists/physicians could monitor thumb CMC mechanics in response to treatment without risk of radiation exposure. We anticipate 1) a statistically significant reduction in thumb CMC subluxation at 9 weeks follow up and 2) high agreement between sonographic and CAT measures of thumb stability. DISCUSSION/SIGNIFICANCE: This study will lay the foundation for future work and may offer critical support for the use of a non-pharmacological and non-surgical approach as first-line treatment of a highly disabling disease. Future study should include controlled trials where hand function, activity limitation, disease progression, and costs are the outcomes in interest.

OBJECTIVES/GOALS: Neonatal hypoglycemia is seen in 65% of maternally diabetic pregnancies, and can lead to severe neurological damage. Neonatal glycemia may also be an indicator of placental function in these pregnancies. The purpose of this study is to identify patterns of neonatal glycemia, and associated endothelial dysfunction, by maternal diabetes subtype. METHODS/STUDY POPULATION: Pregnancies with maternal Type 1 (T1DM), Type 2 (T2DM), and gestational diabetes mellitus (GDM) are being enrolled. Maternal hemoglobin A1c (HbA1c) and umbilical cord insulin/glucose are being collected from 20 pregnancies in each group, 10 of which also undergo placental/umbilical cord tissue collection. Following delivery, neonatal blood glucose levels are also collected every 3-4 hours (4+ measurements) to determine rate of glycemic change. Linear regression modeling will be used to determine associations with placental and umbilical endothelial RNA expression, umbilical cord insulin levels, and maternal HbA1c within each diabetic subtype and between normoglycemic and hypoglycemic neonates. Endothelial gene expression will be compared using paired t-tests with Benjamini-Hochberg correction. RESULTS/ANTICIPATED RESULTS: Thus far, 5 T1DM, 10 T2DM, and 13 GDM samples have been collected. Gestational age at delivery and birth weight were similar between groups (38.1 ± 1.05 weeks; 3.6 ± 0.59 kilograms) and delivery method is evenly distributed (Cesarean section or vaginal delivery). Currently, with limited cohort size, no association is evident between maternal HbA1c and umbilical cord glucose/insulin (p=0.114) or neonatal hypoglycemia diagnosis (p=0.674) when controlled for gestational age and infant birthweight. We hypothesize that, with pending analyses, maternal HbA1c and umbilical cord insulin levels will correlate negatively with the rate of neonatal glycemic change, and positively with the level of inflammatory and angiogenic transcription identified in placental and umbilical endothelium. DISCUSSION/SIGNIFICANCE: Characterization of postnatal glucose control is key to prognosis and risk stratification of infants of diabetic mothers. Understanding placental response to glucose, as well as sequela in the fetal endothelium, is also critical to understanding the pathogenesis of neonatal hypoglycemia and other adverse outcomes of diabetic pregnancy.

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

OBJECTIVES/GOALS: Patient-specific definition of extent of surgical excision is foundational to the safety offered by computer assisted interventions. Consequently, this study aims to develop a pipeline for automated segmentation of bone removed during cortical mastoidectomy, a technically complex otologic surgery. METHODS/STUDY POPULATION: A simulator, previously developed in our lab, allows fully immersive simulation of mastoidectomy using segmented temporal bones generated from CT data. Using the simulator, one attending surgeon will perform three trials of mastoidectomy on 20 different temporal bones. From the simulator we will obtain data on the volume of bone removed for a specific anatomy, averaged between trials. No new U-net (nnU-net), an open-source three-dimensional segmentation network, will then be trained to predict the volume of bone removed using segmented pre-operative CT imaging. Segmentation accuracy will be evaluated with the Dice coefficient, modified Hausdorff distance (mHD), sensitivity and specificity. RESULTS/ANTICIPATED RESULTS: We expect the mean pairwise Dice coefficient to be high indicating relative similarity of volume removed between trials. Moreover, we predict that following five-fold cross-validation the best model will result in a Dice coefficient, mHD, sensitivity, and specificity indicative of volume removed predictions consistent with surgeon-generated data. Finally, given that network training will penalize overlap of the predicted excised bone segment and previously segmented anatomic structures, we expect that no critical anatomical structures will be marked as tissue removed. DISCUSSION/SIGNIFICANCE: We hope to show that deep learning architectures can accurately predict bone removed during mastoidectomy. These predictions can be used for preoperative planning, as clinical endpoints in surgical simulators, or be used in conjunction with surgical robots, all ultimately improving patient safety.

OBJECTIVES/GOALS: Current long bone fracture standard of care uses inert metal intramedullary nails (IMN), 10x stiffer than femur cortex. Consequent “stress-shielding” bone loss sees >5% of patients needing revision surgery. To improve nonunion healing, we develop automated design optimization methods for biodegradable Mg alloy IMNs to control local reloading. METHODS/STUDY POPULATION: Finite element analysis (FEA) is performed on 3D bone-IMN representations to establish this study’s baseline strain states for existing inert IMN geometries within QCT-informed femoral models under simulated biomechanical loading. FEA with Mg alloy properties for same IMN designs simulate transient IMN material loss through discrete time-step models with experimental in vivo Mg corrosion rates and strain-based bone density evolution using remodeling algorithms from literature. Transient stability and strength metrics, fracture zone stress profiles under gradual reloading and manufacturing constraints are formulated through gradient-based sensitivity analysis into a topology optimization framework (TOF) incorporating a reaction-diffusion degradation model to generate IMN topologies. RESULTS/ANTICIPATED RESULTS: TOF designs for Mg alloy IMNs with transient allowable strength constraints, using safety factors to prevent IMN failure, demonstrate higher compliance than standard inert IMNs with mechanical properties closer to native cortical bone. The biodegradation model within the TOF, informed by corrosion behavior from bone-IMN FEA study, predicts how potential design evolutions affect transient strain states of the system. Thus, local fracture region stress states are controlled by the algorithm optimizing for desirable transient stiffness profiles based on a minimum variance objective of fracture zone stress compared to a target bone stress profile. Optimized IMNs with porous, high surface area features achieve 50% decrease in IMN stiffness over 6 months recovery time and complete in vivo degradation in 24 months. DISCUSSION/SIGNIFICANCE: Our TOF reduces “stress-shielding” effects via design for controlled IMN biodegradation to gradually increase fracture zone loading, stimulating remodeling and reducing current risk of post-operative fracture and surgical removal in ~15k cases/yr. in the U.S. In vitro mechanical and in vivo clinical testing is required to validate design results.