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: 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: 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: 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.

OBJECTIVES/GOALS: LSD1 is a histone demethylase important in GBM regulation. Our goal is to design a therapeutic strategy for LSD1 inhibitors to meet clinical needs in GBM. Despite the abundance of LSD1 inhibitors, resistance emerges in GBM mouse models. We aim to understand the relevance of proliferative signaling pathways, such as MAPK, in LSD1 inhibitor resistance. METHODS/STUDY POPULATION: Following LSD1 knockdown in GBM cells, we determined differentially expressed genes using RNA-seq and gene set enrichment analysis (GSEA). Kinase signaling processes enriched for LSD1 expression were identified. Utilizing western blot, we assessed LSD1’s impact on MAPK signaling in patient-derived GBM stem cells (GSCs) and pediatric high-grade glioma cell models. Pharmacological evaluation of LSD1 involved five inhibitor candidates. Additionally, we explored LSD1 inhibition in combination with brain penetrant kinase inhibitors, osimertinib and ulixertinib, directed against the epidermal growth factor receptor (EGFR) and MAPK, respectively. The treatment combinations were assessed at multiple concentrations and analyzed using SynergyFinder. RESULTS/ANTICIPATED RESULTS: Pharmacological LSD1 inhibition after 24 hours induced increased phosphorylated ERK1/2 across multiple glioma cell lines. Concurrent LSD1 and EGFR/MAPK inhibition demonstrated improvedin vitro efficacy compared to individual agents. Notably, the combination of Iadademstat (ORY-1001) and osimertinib demonstrated the highest synergy score of 37.2 using the bliss synergy model in the GSC17s. Furthermore, 11 out of the 12 combination treatments tested had a synergistic relationship, with bliss synergy scores greater than 10. DISCUSSION/SIGNIFICANCE: Our study addresses the pressing need for novel therapeutic strategies in GBM. We leveraged pharmacological tools of LSD1 inhibition to determine how they could be used most effectively, revealing kinase inhibition as a promising strategy with demonstrated in vitro efficacy. Future efforts will focus on validating these findingsin vivo.

OBJECTIVES/GOALS: Motivations and hesitations about participating in genetic research among those at risk of inherited conditions are unclear. We aim to understand perceptions, perspectives, and concerns of these individuals regarding genetic research studies, especially for hard-to-diagnose diseases. METHODS/STUDY POPULATION: Mix method study of 150 Hispanics individuals in Puerto Rico (PR) at risk for in heriting a condition. These individuals, with limited diagnostic data, are attending genetics clinics or invited to a genetics study at the University of Puerto Rico Medical Sciences Campus. Structured surveys and interviews will be conducted. Surveys will gauge general perceptions and feelings toward genetic research, while interviews will provide a deeper understanding of participants’ personal narratives and experiences. All sessions will be recorded, transcribed, and analyzed using NVivo qualitative analysis software. Thematic analysis will be employed to identify recurring themes and sentiments. RESULTS/ANTICIPATED RESULTS: Weanticipatevaried responses: some enthusiastic about genetic research benefits, others having reservations due to privacy, cultural beliefs, or past experiences. A significant portion may express concerns about genetic research’s impact on insurance and potential discrimination. We also expect to uncover systemic challenges that hinder participation among Hispanics living in PR, such as a lack of information or misconceptions about genetic research. This study will overview factors, both encouraging and inhibitory, influencing decisions to join genetic research. Quantitative genetic literacy survey data will undergo descriptive analysis and multivariate logistic regression. DISCUSSION/SIGNIFICANCE: Hispanics in PR exhibit a rich tapestry of genetic variations being a focal point for genetic research. Understanding perceptions is vital among those at risk for inherited conditions. Insights can shape outreach and education strategies, ensuring participants are informed, concerns met, and empowered to make decisions alignined their views.

OBJECTIVES/GOALS: Distinguishing tumor tissue from normal brain parenchyma remains a major challenge during the resection of gliomas, leading to the persistence of tumor cells. This study aims to assess the choline kinase alpha-targeting fluorophore JAS239 as a novel fluorescent agent to intraoperatively visualize gliomas in an orthotopic murine model. METHODS/STUDY POPULATION: The human glioblastoma-derived U87 MG-Luc2 cell line will be intracranially implanted in nude mice and tumor growth will be assessed using bioluminescence imaging. After 14 days, the mice will be treated with either antiangiogenic therapy (10 mg/kg bevacizumab, twice/week) or saline (control). Tumor growth will be monitored until 21-28 days after initial implantation, at which point JAS239 (4.0 mg/kg, 90 min before sacrifice) and Evans Blue (4 ml/kg, 60 min before sacrifice) will be administered. The mice will be sacrificed, and their brains will be harvested and sectioned for near-infrared imaging. The brain sections will be processed for histopathologic analysis, allowing for the correlation of observed fluorescence with the distribution of tumor and comparison of signal-to-background ratios. RESULTS/ANTICIPATED RESULTS: JAS239 is an indocyanine-based choline mimetic (excitation 745 nm, emission 775 nm) that has been shown to cross the blood-tumor barrier (BTB) in rodent glioblastoma studies. PET imaging with choline-based radiotracers like 18F-choline has also been shown to delineate both contrast-enhancing tumor (CET) and non-contrast-enhancing tumor (NCET) regions, supporting the hypothesis that JAS239 will be able to visualize heterogeneous glioma tissue in our mouse model. Evans Blue is a passive dye in the visible light spectrum (excitation 620 nm, emission 680 nm) expected to only fluoresce in CET regions due to the disruption of the BTB. JAS239 is expected to fluoresce in both CET and NCET regions, which will be assessed by the fluorescence in mice treated with bevacizumab (expected to renormalize the BTB and model NCETs). DISCUSSION/SIGNIFICANCE: JAS239 may allow for real-time visualization of heterogeneous glioma tissue, which is important because there are no current intraoperative imaging agents for NCETs. Future research and clinical translation of this class of agents may allow surgeons to maximize the safe resection of gliomas, improving progression-free and overall survival rates.

OBJECTIVES/GOALS: Cognitive dysfunction and/or depressionfollowing ischemic stroke results in loss of independence in daily functioning. The objective of this work is to assess neural correlates of post-stroke cognitive deficits and the effect of left frontal transcranial electrical stimulation on cognitive control and associated brain rhythms. METHODS/STUDY POPULATION: We recorded midfrontal scalp EEG from 15 healthy and 13 participants with stroke while they performed a multi-source interference task (MSIT). The stroke cohort also performed additional MSIT sessions where they received active and sham transcranial direct current stimulation (tDCS) on the left prefrontal cortex (PFC). The EEG was pre-processed to get rid of eye movement and other channel noise artifacts and filtered to retain 0.5-55 Hz components. A Morlet wavelet was used to estimate power in theta (4-8 Hz), alpha (8-15 Hz) and gamma (35-50 Hz) frequency bands over a period of 2 seconds following MSIT image presentation. A generalized linear mixed effects model was used to find effect of group on behavior and EEG oscillations. A GLME was also used to find effects of active tDCS on behavior and EEG. RESULTS/ANTICIPATED RESULTS: We found Group (healthy v stroke) as a significant predictor of both response time (behavior) and conflict evoked theta power in the frontal channels (F1-Fz, F2-Fz). We also found that active tDCS significantly improved MSIT performance as compared to sham, after accounting for cognitive load. Active tDCS also induced low frequency oscillations in frontal EEG channels compared to sham. Preliminary results indicate that mid-frontal theta oscillations are a potential neural correlate of post-stroke cognitive deficit and tDCS of the left PFC might be a promising therapeutic intervention to ameliorate this. DISCUSSION/SIGNIFICANCE: Current therapeutic approaches often do not alleviate post stroke executive dysfunction, hence a better understanding of the brain network changes underlying such deficit can elucidate neural correlates of post stroke cognitive deficit to inform the development of neuromodulation interventions.

OBJECTIVES/GOALS: Increases in anxiety and depression during adolescence may be related to increased biological reactivity to negative social feedback (i.e., social threat sensitivity). Our goal was to identify biomarkers of social threat sensitivity, which may provide unique etiological insight to inform early detection and intervention efforts. METHODS/STUDY POPULATION: Adolescents aged 12-14 (N=84; 55% female; 80% White; 69% annual family income <$70,000) were recruited. Youth viewed a series of happy, neutral, and angry faces while eye-tracking and electroencephalogram (EEG) data were recorded to capture cognitive and neural markers of sensitivity to social threat (i.e., an angry face). Fixation time and time to disengage from angry faces were derived from eye-tracking and event-related potentials were derived from EEG, which index rapid attention capture (P1), attention selection and discrimination (N170), and cognitive control (N2). Adolescents also completed a social stress task and provided salivary cortisol samples to assess endocrine reactivity. Social anxiety and depressive symptoms were self-reported concurrently and one year later. RESULTS/ANTICIPATED RESULTS: Latency to disengage from threatening faces was associated with lower N2 amplitudes (indexing poor cognitive control; r= -.24, p = .03) and higher concurrent social anxiety (r = .28, p = .01). Higher N170 amplitudes, reflecting attentional selection and discrimination in favor of threatening faces, predicted increases in depressive symptoms one year later (b= .88, p = .02). No other neurophysiological measures were associated with each other or with concurrent or prospective symptomatology. DISCUSSION/SIGNIFICANCE: Eye-tracking and EEG measures indexing difficulty disengaging from social threat and poor cognitive control may be biomarkers of social anxiety, which could be utilized as novel intervention targets. High N170 amplitudes to social threat, derived from EEG, may have clinical utility as a susceptibility/risk biomarker for depressive symptoms.

OBJECTIVES/GOALS: Prematurity and perinatal brain injury are known risk factors for strabismus. In this study, we sought to understand the link between neonatal neuroimaging measures in very preterm infants and the emergence of strabismus later in life. Study findings may inform if neonatal brain MRI could serve as a prognostic tool for this visual disorder. METHODS/STUDY POPULATION: This study draws from a longitudinal cohort of very preterm infants (VPT, < 30 weeks gestation, range 23 – 29 weeks) who underwent an MRI scan at 36 to 43 weeks postmenstrual age (PMA). Anatomic and diffusion MRI data were collected for each child . A subset of thirty-three patients in this cohort had records of an eye exam, which were reviewed for a history of strabismus. Patients with MRI scans demonstrating cystic periventricular leukomalacia or grade III/IV intraventricular hemorrhage were classified as having brain injury. Clinical variables with a known association to strabismus or diffusion metrics were included in a multivariable logistic regression model. Diffusion tractography metrics were screened for association with strabismus on univariable analysis prior to inclusion in the regression model. RESULTS/ANTICIPATED RESULTS: A total of 17/33 (51.5%) patients developed strabismus. A logistic regression model including gestational age, PMA at MRI, retinopathy of prematurity (ROP) stage, brain injury, and fractional anisotropy of the right optic radiation was significant at the .001 level according to the chi-square statistic. The model predicted 88% of responses correctly. Each decrease of 0.01 in the fractional anisotropy of the right optic radiation increased the odds of strabismus by a factor of 1.5 (95% CI 1.03 – 2.06; p = .03). Patients with brain injury had 15.8 times higher odds of strabismus (95% CI 1.1 – 216.5; p = .04). Gestational age (OR 1.7; 95% CI 0.9 – 3.3; p = .1) and stage of ROP (OR 0.6; 95% CI 0.2 – 2.0; p = .4) were not significant predictors of strabismus in the multivariable model. DISCUSSION/SIGNIFICANCE: Our findings suggest that strabismus in VPT patients may be related to specific changes in brain structure in the neonatal period. The identified association between neonatal optic radiation microstructure and strabismus supports the possibility of using brain MRI in very preterm infants to prognosticate visual and ocular morbidity.

OBJECTIVES/GOALS: To determine whether cardioprotective effects observed in individuals taking dietary supplementation with eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid, are realized by altering platelet function, and if these effects are mediated through the 12-lipoxygenase derived metabolite, 12-hydroxyeicosapentaenoic acid (12-HEPE). METHODS/STUDY POPULATION: Washed platelets or platelet rich plasma from healthy human donors were treated with EPA and 12-HEPE to assess their ability to inhibit platelet activation. Platelets were stimulated with agonists targeting different steps of the hemostatic response to vascular injury. Platelet aggregation, dense granule secretion, surface expression of integrin αIIbβ3 and P-selectin, and clot retraction were analyzed. To assess signaling through Gαs-GPCRs and protein kinase A activity, phosphorylation of vasodilator-stimulated phosphoprotein (VASP) was examined via western blot following treatment with EPA or 12-HEPE. RESULTS/ANTICIPATED RESULTS: EPA and 12-HEPE dose-dependently inhibit both collagen and thrombin-induced platelet aggregation. Furthermore, 12-HEPE more potently attenuates dense granule secretion and surface expression of platelet activation markers, integrin αIIbβ3 and P-selectin, in comparison to EPA. Plasma treated with EPA delayed thrombin-induced clot retraction, while 12-HEPE had no effect. Additionally, treatment with 12-HEPE increases phosphorylation of VASP, suggesting it could signal through the activation of the eicosanoid Gαs-GPCRs. DISCUSSION/SIGNIFICANCE: Here, we show for the first time that EPA directly inhibits platelet activation through its 12-LOX metabolite, 12-HEPE. These findings provide further insight into the mechanisms underlying the cardioprotective effects of EPA. A better understanding of current PUFA supplementations can inform treatment and prevention of cardiovascular diseases.

OBJECTIVES/GOALS: We prioritize Chlamydia’s public health impact, aim to develop rVCG-MECA for practical use, study robust immunity for effective strategies, and assess animal models for human vaccination adaptation. Our work highlights rVCG-MECA’s translational significance in public health. METHODS/STUDY POPULATION: Female Mice C57BL/6J mice (N=8) were immunized intramuscularly(IM) and intranasally(IN) and boosted twice, two weeks apart, with rVCG-MECA, once with live Chlamydia (C. trachomatis serovar D elementary bodies) and PBS. Specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined from chlamydia shedding following the transcervical challenge. Additionally, Chlamydia-specific cytokine (IFN-γ and IL-4) production by splenic and ILN T cells was assessed after 16 weeks RESULTS/ANTICIPATED RESULTS: Immunization with rVCG-MECA via intramuscular and intranasal routes triggered notable humoral responses in systemic and mucosal tissues. Intramuscular vaccination produced higher IgG2c levels in both tissues, while intranasal vaccination led to elevated IgA levels in mucosal tissues. rVCG-MECA-immunized mice exhibited significantly higher IFN-γ (Th1) secretion compared to IL-4 (Th2), with intramuscular immunization showing the highest IFN-γ levels. These findings anticipate robust immune responses, promising protection against Chlamydia, particularly through the intra muscular route. Overall, our results support rVCG-MECA as a promising Chlamydia vaccine, aligned with public health goals. DISCUSSION/SIGNIFICANCE: This study suggests that IM and IN immunization with rVCG-MECA induces immune effectors such as IFN-gamma and IgG2c that mediate chlamydial clearance in the genetical tract.

OBJECTIVES/GOALS: Artificial intelligence (AI) depends on quality machine learning (ML) algorithms constructed with high-quality training data. This TL1 trainee project develops a disease-agnostic computable phenotype framework for ML algorithm construction, modeling male puberty as a case example. METHODS/STUDY POPULATION: A computable phenotype of male puberty was constructed to answer the question: “Does early pubertal timing increase the risk of developing type II diabetes (T2D) in males?” A computable phenotype of males < 18 years old was created in the TriNetX© Diamond Network utilizing Boolean operator data queries. TriNetX© contains patient electronic health record information (ICD-10 diagnoses, anthropometric measures). An exploratory analysis of patient counts reflecting various computable phenotypes allowed for outcome (T2D) comparison of males diagnosed with precocious puberty (E30.1, ICD code for early pubertal timing) to those without, controlling for body mass index (BMI). RESULTS/ANTICIPATED RESULTS: Subjects (n=12,996,132) displayed the following computable phenotype: Male, < 18 years old, without ever having a BMI documented >85th percentile. Males diagnosed with precocious puberty (E30.1) were 6.89 times more likely to develop T2D when aged 14-18 years old than those without (OR 6.89, 95% CI: 5.17-9.19, p<0.0001). Next steps involve training a ML model on each computable phenotype groupings’ health data, with anticipated results identifying underlying salient pathophysiologic variables. A generalized computable phenotype approach is further developed to: 1) explore clinical questions in large databases like TriNetX©, and 2) model disease development with AI/ML algorithm construction. DISCUSSION/SIGNIFICANCE: Computed phenotypes reveal males with precocious puberty may have increased T2D risk. Next steps utilize subject data to train an AI/ML algorithm, model development to identify salient pathophysiologic variables, and synthesize a generalized AI/ML developmental research framework for dissemination.

OBJECTIVES/GOALS: Our objective is to develop a patient-friendly application addressing the progression of cognitive impairments in multiple sclerosis (MS) patients. This initiative aims to augment individualized care and precision management of a major MS comorbidity by generating a cognitive health brain map for each patient. METHODS/STUDY POPULATION: Using the UAMS COMS Database, featuring high-resolution multi-contrast MRIs, and a comprehensive clinical, behavioral, and demographic dataset, we are developing a hierarchical learning-based software tool to compute maps correlating brain structure-function and individual cognitive function. Our MRI analysis employs a three-compartment model (NNLS>0.96). Functional scores are defined by individualized accuracy during the modified information processing speed task (e.g., m-SDMT). We utilize a Bayesian classifier with explicit Pearson’s correlation for tissue classification (BF10>100) to compute an index of the likelihood of correlation with cognitive impairment throughout brain tissue. RESULTS/ANTICIPATED RESULTS: This approach allows us to reveal subtle cognitive changes and their potential links to myelin integrity, offering vital insights into disease progression and management. The m-SDMT strongly correlates with the standard SDMT (r=0.79, p<0.001), confirming reliability as a cognitive assessment tool in clinical and research contexts. Analysis of the COMS dataset emphasized insights into the role of fine myelin structure in MS patients' cognitive functionality. Our findings heightened the pivotal significance of myelin integrity in preserving cognitive abilities and identify disruptions in myelin synthesis and homeostasis as primary contributors to cognitive decline. This discovery stresses the critical role that specialized brain pathways, influenced by myelin integrity, play in the pathology of MS. DISCUSSION/SIGNIFICANCE: This development bridges advanced neuroimaging techniques with practical clinical applications, emphasizing the nuanced role of myelin integrity in MS-related cognitive deficits. Our findings advocate for a multidisciplinary approach to MS management, demanding collaborative workforce development and education in translational science.

OBJECTIVES/GOALS: The objective of our research is to define unique molecular and immune markers in benign breast tissue to better identify women at risk of node-positive breast cancer (BC). The goal of the work is to improve individualized risk assessment, to guide targeted prevention and screening recommendations, and to reduce disease incidence and mortality. METHODS/STUDY POPULATION: From the Mayo Clinic’s Benign Breast Disease (BBD) cohort, we matched women who developed node-positive breast cancer after a BBD biopsy (cases; n=42) with women who remained cancer-free (controls; n=37), considering patient age and biopsy date. We used NanoString gene expression profiling to identify differentially expressed genes (DEGs) between cases and controls. We optimized a multiplex immunofluorescence (mIF) approach to simultaneously detect multiple markers within single FFPE tissue slides to correlate cells expressing DEGs in relation to innate and adaptive immune effectors. We used tissue segmentation, cell phenotyping, and spatial relationships to define molecular and immune differences between cases and controls. RESULTS/ANTICIPATED RESULTS: We discovered higher gene expression levels of IRF8 (interferon regulatory factor 8, a factor involved in immune cell differentiation) in controls as compared to cases (p = 0.0024) and found that IRF8 expression is also associated with longer cancer onset times among cases (p = 0.0012). Our pilot mIF experiments revealed higher frequencies of CD4+, CD8+, CD68+, CD20+ and CD11c+ cells in controls compared to cases. We predict that higher IRF8 expression and increased frequencies of immune cells in BBD biopsies indicate a proactive immune environment that may act to prevent cancer development. Furthermore, we predict that our analyses of the spatial localization of these markers by mIF may offer further predictive insight. DISCUSSION/SIGNIFICANCE: Deciphering the relationship between immune alterations in BBD and risk of node positive BC has the potential to improve individualized risk prediction. These insights will foster improved surveillance and informed screening and prevention, ultimately reducing BC incidence and mortality.