OBJECTIVES/GOALS: Contrary to current dogma, prior work has suggested that in humans, SNP-determined endothelial cell reduction in CD39 expression associates with a diminished risk for venous thromboembolism. Our objective was to examine the impact of endothelial cell (EC) CD39 expression on arterial thrombosis that replicates human data. METHODS/STUDY POPULATION: We generated a novel CD39 cell-specific conditional knockout mouse line for endothelial cells (EC-cKO: Tie2-Cre+; cd39flox/flox versus WT: Tie2-Cre-; cd39flox/flox). We validated the knockout of expression of CD39 on EC using FACS analysis and measured EC CD39 activity using the Kinase Glo ATP hydrolysis assay on magnetically sorted EC. We then used a standard FeCl3 carotid injury model to evaluate time to arterial thrombosis in vivo by measuring time to occlusion tracked via a Doppler flow probe on the exposed vessel. RESULTS/ANTICIPATED RESULTS: FACS analysis revealed a specific 97% knockout of CD39 expression on EC (p<0.001) but not on other cells within the vasculature. There was also significant reduction in ATP hydrolysis (81%; p=0.019) in EC-cKO mouse EC versus WT. We next examined the time to arterial thrombosis. EC-specific conditional knockout of CD39 exhibited a significant prolongation in time to thrombosis compared to WT (WT: 8.28 minutes +/- 0.82; EC-cKO: 11.92 minutes +/- 1.34; p=0.024). Analysis of carotid blood-flow revealed that EC-cKO and WT mice had similar baseline blood flow velocity (p=0.51), but after vessel injury with FeCl3, EC-cKO mice exhibited a 16% increase maximal flow velocity relative to baseline compared to WT (p<0.001), as well as a 19% increase at 2-minutes post-injury in comparison to EC-WT mice (p<0.001). DISCUSSION/SIGNIFICANCE: Our findings demonstrate that CD39 activity plays a role in modulating arterial thrombosis and blood-flow regulation within the vasculature. These findings exemplify the therapeutic potential of modulating endothelial CD39 activity, as well as the potential for using SNPs within the gene coding for CD39 as a cardiovascular disease marker.

OBJECTIVES/GOALS: The global incidence of calcific aortic valve disease (CAVD) increased 3.5-fold since 1990. No preventative or therapeutic pharmaceutical therapies exist for CAVD. We will establish the therapeutic potential of osteoclast-derived exosomes though characterization of contents and mechanisms of action to protect against mineralization. METHODS/STUDY POPULATION: Exosomes were purified from conditioned media collected from murine myeloid precursor cells, RAW264.7 (control), and osteoclasts induced to differentiate from RAW264.7 cells (OD). Protein content of exosomes was determined using proteomic analyses. Nucleic acid contents will be identified by sequencing mRNA, miRNA, and DNA. The calcification prevention and reabsorption abilities of control and OD exosomes will be tested using human valvular interstitial cells (VIC) and smooth muscle cell calcification assays and acellular osteologic disc assays, respectively. Comparison between cellular and acellular systems will help identify mechanisms of action, and demonstrate potential therapeutic viability of OD exosomes in preventative vs resorptive treatments. RESULTS/ANTICIPATED RESULTS: OD exosomes, but not control exosomes, prevented calcification in VIC in vitro. OD exosomes contained osteoclast-specific proteins including TRAP, MMP6, cathepsin K, and bone reabsorption factors including V type proton pumps, ATPases, and integrins. These genes are also involved in resorptive activities, and were highly upregulated in OD compared to control exosomes. We anticipate miRNA signatures associated with mineral resorption will also be present. Increased knowledge of exosome cargo will illuminate their mechanism of action and allow future work to engineer increased efficacy. We also anticipate a therapeutic response when OD exosomes are applied after calcification has begun, showing exosomes promote calcium reabsorption. DISCUSSION/SIGNIFICANCE: Establishing therapeutic potential and examining mechanisms of action will pave the way for OD exosomes as a CAVD treatment. Analysis of exosome contents will determine active molecules to be enhanced in future studies. This work will lay a foundation for moving into aortic valve organoid models, which are accepted by the FDA for preclinical trials.

OBJECTIVES/GOALS: Ebolavirus disease survivors report persistent, debilitating health concerns dubbed Post-Ebola Syndrome (PES). Attention to PES in young survivors is lacking, we describe PES in pediatric EVD survivors in Eastern Sierra Leone. Additionally, we introduce our proposal investigating differential presentations of PES in pediatric survivors. METHODS/STUDY POPULATION: EVD survivors were enrolled a median of 2.5 years after resolution of disease. Survivors were eligible if listed in a national register maintained by the Sierra Leone Association of Ebola Survivors. Household contacts (HCs) were identified by survivors. Participants were assigned into three comparison groups: pediatric (7-11), adolescent (12-17) and young adult (18-25). A self-reported symptom questionnaire, and a physical exam were conducted. Variables were clustered within organ system and compared across groups. RESULTS/ANTICIPATED RESULTS: Pediatric survivors had lower levels of long-term sequelae compared to adolescents and young adults. Symptoms and abnormal physical exam signs increase with age. Musculoskeletal, psychiatric, ophthalmologic, and GI signs and symptoms were significantly different between groups. Pediatric survivors had significantly more persistent sequelae than age-matched HCs with no history of EVD; particularly within the cardiac/GI (p=.006) and psychiatric/neurological (p=.025) clusters. PES is heterogeneous with respect to age, calling for a deeper understanding of age-based differences. Even the youngest group of survivors experienced significantly more sequelae than HCs, highlighting the elevated symptom burden in these children over their peers. DISCUSSION/SIGNIFICANCE: Understanding mechanistic drivers will ultimately improve targeted treatments for PES. We will characterize symptom groups defining PES in children, determine the relationship between accelerated aging and PES in this population, and test how immune profiles associated with accelerated aging relate to the development of PES in children.

OBJECTIVES/GOALS: Residents of environmental justice (EJ) communities experience significantly higher rates of negative health outcomes associated with poor air quality. Low-cost air sensors may supplement regulatory monitoring to better measure air pollution at local scales, but widespread application of this technology remains limited due to many challenges. METHODS/STUDY POPULATION: To address these obstacles, we designed a training program to equip community and academic research partners with the skills and knowledge to successfully apply low-cost sensors in community-engaged environmental health research. The R esearch Innovations using Sensor Technology in Environmental Justice Communities (RISE Communities) program was established through an NIEHS R25 award in 2022 and has three specific aims: 1) Foster community-academic partnerships through research education, training, and team development activities, 2) Provide technical training in the application of low-cost sensors for indoor, outdoor, and personal air monitoring in EJ communities, and 3) Establish a community of practice to address air quality in communities nationwide. RESULTS/ANTICIPATED RESULTS: We hosted our first cohort in August 2023, training five community-academic research teams in team collaboration, community-engaged research, and technical skills for collecting and analyzing data from PurpleAir sensors. Each team received 12 sensors to take to their home EJ communities to begin their projects. Community of practice development continues through interactive webinars and development of a web-based repository of training videos and discussion board posts. Evaluation data show high participant learning and satisfaction, with mean confidence scores improving on 6/8 metrics. Evaluation data also suggest several areas for improvement such as more time spent in teams for planning and additional opportunities for interaction within the cohort and with program instructors for problem-solving. DISCUSSION/SIGNIFICANCE: Effective training for team-based community-engaged research requires careful planning for team development and study implementation. Longitudinal training and support for the technical aspects of utilizing air sensors is also critical to team success. The RISE Communities program is actively recruiting for future training cohorts.

OBJECTIVES/GOALS: Weaimed to compare subjective and objective sleep in individuals with chronic post-concussive symptoms. We hypothesized an association between self-reported sleep quality and objective sleep parameters, which is different for concussed and control cohorts. METHODS/STUDY POPULATION: 28 individuals with chronic post-concussive symptoms and 13 age-matched controls (no concussion history) completed the ISI, PSQI, PROMIS Depression, Anxiety, Stress and Cognitive questionnaires at enrollment. Objective sleep parameters were obtained for a minimum of 7 days and up to 30 days with a validated sleep monitoring device placed under the subject’s bed (Emfit). For each night, raw activity data per minute were analyzed to determinein-bed, sleep, wake, andout-of-bedtimes. These measures were used to calculate total sleep time (TST), sleep onset latency (SOL), and wake after sleep onset (WASO) for each night. RESULTS/ANTICIPATED RESULTS: Concussed individuals reported worse sleep with PSQI and ISI scores significantly higher than controls. They also showedsignificant associations between PSQI and Dep ression, ISI and Depression, and ISI and Anxiety scores. There was no difference between objective sleep parameters in the concussed and control cohorts (in-bed/sleep/wake/out-of-bed times, TST, SOL, and WASO). Instead, higher PSQI, ISI, Depression, Anxiety, and Stress scores (greater symptom burden) were all associated with later sleep times, where as higher Cognitive scores (greater cognitive function) were associated with earlier sleep times, regardless of group status. DISCUSSION/SIGNIFICANCE: Concussed individuals report worse subjective sleep but no differences to controls when objectively assessing sleep. Depression/anxiety, and not concussion status, determine objective sleep parameters. Psychiatric comorbidities should inform the treatment of post-concussive sleep disturbances.

OBJECTIVES/GOALS: We designed the Biocascade Exhaled Breath Sampler (BEBS) to characterize viral aerosol shedding among individuals with influenza and other respiratory virus infections. We first aimed to test the BEBS on volunteer COVID-19 cases and report the aerodynamic size distribution of exhaled breath aerosol particles carrying SARS-CoV-2 RNA. METHODS/STUDY POPULATION: From June 15 through December 15, 2022, we recruited 27 PCR-confirmed COVID-19 cases from a college campus and the surrounding community to provide 30-minute breath samples into a well-validated Gesundheit-II (G-II) exhaled breath aerosol sampler. Among these individuals, 17 provided an additional exhaled breath sample into the newly designed BEBS. We quantified samples for viral RNA using reverse transcription digital polymerase chain reaction (RT-dPCR) and determined the viral RNA copies collected within two aerosol size fractions (≤5 µm and >5 µm in diameter) from the G-II, and four aerosol size fractions (<1.15 µm, 1.15–3.2 µm, 3.3–8.2 µm, and >8.2 µm) from the BEBS. RESULTS/ANTICIPATED RESULTS: Individuals with a SARS-CoV-2 Omicron BA.4 or BA.5 infection shed virus in aerosols at an average rate of 7.5x103 RNA copies per 30-minute G-II sample, with 78% of the total RNA in aerosols ≤5 µm in diameter. Among the BEBS samples, 10% of the total viral RNA was detected in aerosols <1.15 µm, 43% in 1.15–3.2 µm, 37% in 3.3–8.2 µm, and 10% in the >8.2 µm size fraction. Based on viral RNA loads, our results indicate that exhaled aerosols ≤3.2 µm contribute the majority of SARS-CoV-2 inhalation exposure. DISCUSSION/SIGNIFICANCE: Our data provide additional evidence that respirable aerosols contribute to the spread of SARS-CoV-2. Thus, our data suggest that mitigation measures designed to reduce infectious aerosol inhalation, such as ventilation and the use of air cleaners and respirators, are needed to control the spread.

OBJECTIVES/GOALS: This project aimed to examine the impacts of biological sex and alexithymia on frontal lobe connectivity in executive functioning (EF)-related neural networks during successful inhibition as a means to index vulnerability for future cognitive decline. METHODS/STUDY POPULATION: Healthy, cognitively intact older adults (n=43, 33 female, Mage=79) completed the 20-item Toronto Alexithymia Scale (TAS-20) and the stop-signal task in this study. We used electroencephalography (EEG) source estimation to investigate EF-related frontal connectivity during successful inhibition in stop-signal task trials. Connectivity was measured in bilateral frontal ROIs relevant to inhibition using time series correlations over the N200 (186-350ms) and P300 (340-616ms) time windows, associated with the inhibitory subprocesses of conflict processing and performance evaluation, respectively. RESULTS/ANTICIPATED RESULTS: Those with higher alexithymia, as measured by the difficulty identifying feelings (DIF) facet of the TAS-20, had lower right anterior cingulate cortex (ACC)-left superior frontal gyrus (SFG) connectivity in the P300 window, suggesting impaired performance evaluation. Further, in females specifically, those with higher DIF had greater right inferior frontal gyrus (rIFG)-bilateral ACC connectivity in the N200 window than those with lower DIF, suggesting greater resources were allocated for conflict processing and inhibition. Right ACC-rIFG connectivity also correlated with better stop accuracy and faster stop-signal reaction time, supporting this network’s role in successful inhibition. DISCUSSION/SIGNIFICANCE: Overall, during successful inhibition, higher DIF was associated with reduced performance monitoring efficiency as well as greater resource allocation for conflict processing during motor stopping in women only. Thus, alexithymia (via DIF) may exacerbate age-related EF dysfunction and risk for future cognitive decline, especially for females.

OBJECTIVES/GOALS: Globally, diabetes affects 537 million people and 15-25% will develop a foot ulcer in their lifetime. Diabetic foot ulcers (DFU) tend to be chronic and non-healing due to the poor wound healing environment, leading to infection or amputation. Our study aims to develop a method to predict and prevent DFU formation. METHODS/STUDY POPULATION: Our preliminary plan is to develop a method to detect high plantar pressures, coupled with the ability to automatically adjust an orthotic device to offload excess pressure. Our current aim is to create a “smart orthotic” which will link with foot mapping technology to automatically offload high pressure areas, reducing the need for a separate clinic visit for orthotic adjustment. We aim to prove that our device will normalize plantar pressure distribution, which will prevent callus and subsequent DFU formation. The current target population includes those with diagnosed diabetes and are ambulatory. RESULTS/ANTICIPATED RESULTS: With our technology, we anticipate normalization of plantar pressure distribution in a more frequent fashion than is currently done. Because annual orthotic fittings, which is current standard of care, do not provide regular enough adjustments to match the rate of diabetic foot structural changes and peak plantar pressure redistribution, our device will address two gaps in management. One, patients will receive near-instantaneous changes in plantar pressure offloading, allowing for near continuous orthotic customization. Secondly, our device would reduce the clinical appointment burden, which would be especially important for patients with multiple medical comorbidities or experience other barriers to accessing healthcare. DISCUSSION/SIGNIFICANCE: While DFUs are commonplace and their complications are well recognized, there still exists a gap in ulcer prevention. Our proposed solution will redistribute pathologic plantar pressures, allow for more frequent monitoring, automatic therapy, and aid in the management of high ulcer risk patients.

OBJECTIVES/GOALS: Compare metabolic health of type 2 diabetics on GLP-1 to those on traditional therapy METHODS/STUDY POPULATION: Outcomes of interest of this study include analyzing GLP-1 agonists on overall metabolic health, focusing primarily on weight loss and ability to wane off insulin without rebounding metabolic health. The data will be collected in a retrospective chart review from medical records of type II diabetics from Children’s of Alabama and will follow patients over two years. The charts have been narrowed to those patients prescribed GLP-1 agonists who have been on the medication for at least one year with consistent visits to the endocrinology clinic.The following data will be collected from the charts:race/ethnicity, date of visit, BMI/weight, A1C, insulin therapy, lipids, LFTs (AST/ALT), and insurance coverage RESULTS/ANTICIPATED RESULTS: With these results, we hope to study the metabolic health effects of GLP-1 agonists on type II diabetes in the pediatric population. It is known that obesity is a risk factor for type II diabetes, and that GLP-1 agonists aid in weight-loss in adults. Further research is needed to see the real world health effects, and with these results we hope to assess if GLP-1 agonist have an affect on metabolic health within the pediatric population by collecting data on values aforementioned. We also hope to compare and contrast the different GLP-1 agonists being used based on adherence, insurance coverage, adverse effects, and patient preference. Currently, only Liraglutide and Exetanide are approved for pediatric type II diabetics. DISCUSSION/SIGNIFICANCE: Insulin use can lead to weight gain; metformin does not aidin weight reductions. If GLP-1 agonists aid in weight loss, it could potentially help slow complications of diabetes in the pediatric population. B-cell function in children declines more rapidly; and, as a result, insulin resistance occurs more rapidly.

OBJECTIVES/GOALS: Renal vascular resistance (RVR) is the opposition to blood flow by renal arteries. At the population level, dietary salt increases RVR and blood pressure (BP), which are associated with cardiovascular disease. Recent data indicate exogenous ketones may offset adverse cardiorenal effects of salt. METHODS/STUDY POPULATION: Our registered clinical trial (NCT05545501) is a double-blinded, placebo-controlled, crossover study. Participants are being randomized to three 10-day conditions: A) control; B) high salt; C) high salt and ketone supplementation. Ten participants are enrolled (target 30 participants). Renal blood velocity (RBV) in the renal and segmental arteries will be measured in the decubitus position using Doppler ultrasound during a 3-minute baseline and 3-minute cold pressor test. We will measure brachial BP with an automated oscillometric BP monitor. RVR will be calculated as mean BP divided by RBV. Statistical analyses will include ANOVA and correlations with α set at ≤ 0.05. RESULTS/ANTICIPATED RESULTS: We anticipate attenuated RBV and increased BP during the cold pressor test, particularly following high salt loading, leading to greater RVR. We hypothesize ketone supplementation will attenuate the high salt induced increase in RVR during the cold pressor test. In addition to RVR we will examine renal vascular conductance which is the ease with which blood flows through arteries, calculated as RBV divided by mean BP. Additional hemodynamics such as heart rate and systolic and diastolic BP will be reported and correlated with primary outcomes. DISCUSSION/SIGNIFICANCE: Dietary salt plays a role in hypertension, cardiovascular disease, and chronic kidney disease, which are leading causes of death. Ketone supplementation may be a promising approach to counteract the detrimental effects of high dietary salt and improve cardiovascular health in adults.

OBJECTIVES/GOALS: The study aims to explore the role of microglial behavior in cognitive impairment associated with vascular dementia (VaD) and long COVID. Using immunohistochemistry (IHC) and quantitative PCR (qPCR), we will assess the expression of Iba-1, a microglial activation marker, in subjects with VaD and SARS-CoV-2 infection. METHODS/STUDY POPULATION: Out of 48 female C57BL/6 mice, 24 had surgical intervention in the form of bilateral carotid artery stenosis (BCAS) for experimental induction of vascular dementia. After 2 weeks, 12 BCAS and 12 non-BCAS were infected with 1E4 PFU of mouse-adapted 10 (MA10) strain of SARS-CoV-2. 2 weeks post-infection, 4 weeks post-operatively, all animals were euthanized and tissues were processed for cDNA and histology. Immunofluorescence and RT-qPCR used to quantify microglia via Iba-1, BBB integrity via claudin-5 as well as occludin, GFAP, and integrin a5. RESULTS/ANTICIPATED RESULTS: We anticipate observing distinct patterns of microglial behavior in subjects with vascular dementia (VaD) and those with long COVID. Through immunohistochemistry (IHC), we expect to see increased Iba-1 expression, indicative of microglial activation. Quantitative PCR (qPCR) will likely corroborate these findings, showing elevated levels of Iba-1 mRNA. Lastly, we anticipate that the data will reveal interactions between microglia and the blood-brain barrier (BBB). These interactions could provide insights into how microglial behavior influences BBB integrity and, consequently, cognitive function in VaD and long COVID. DISCUSSION/SIGNIFICANCE: This study aims to clarify the role of microglia in cognitive decline linked to vascular dementia and long COVID. By categorizing patients based on microglial activation, we can better tailor treatments. The findings could lead to targeted therapies that address cognitive impairment in these conditions.

OBJECTIVES/GOALS: 48,000,000 people in the U.S. have hearing loss, negatively impacting quality of life and work. Unveiling axon guidance for auditory type II spiral ganglia neurons (SGNs) will aid development of new therapies. I study the role of Eph/Ephrin and planar cell polarity (PCP) signaling during type II SGN turning and outer hair cell (OHC) innervation. METHODS/STUDY POPULATION: This quantitative study was conducted on Efna3 and Vangl2 null mice possessing Neurog1CreERT2 and R26RtdTomato mutations. Spontaneous Cre activity within the Neurogenin1CreERT2 line causes recombination and expression of fluorescent Rosa26 Reporter (R26R)tdTomatoin a restricted number of SGNs, including type IIs. Together, these lines permit SGN sparse labeling. Immunostaining and confocal imaging were used to analyze dsRed in Efna3 and Vangl2 mice and quantify type II SGN turning. In combination, Imaris 3D renderings were used to quantify type II SGN turning, branching, navigation features and temporal effects of EPHRIN-A3-Fc on type IIs via cochlear cultures (a gain-of-function manipulation). For both sexes, 5-6 cochleae per genotype were analyzed and compared by t-test to wildtype (WT) controls. RESULTS/ANTICIPATED RESULTS: Efna3 nulls showed a small rise in type II SGNs incorrectly turning toward the apex at an error rate of 16.0% compared to WTs (n=6; p=0.05). P0 Efna3 nulls had reduced branch number compared to WTs, 4.1 and 7.2, respectively (n=129; p=<0.0001), suggesting EPHRIN-A3 acts as a positive growth cue. In cochlear cultures, EPHRIN-A3-Fc led to type II SGN collapse at E15.5, indicating a repulsive function. However, at P0, EPHRIN-A3-Fc treatment led to type II SGNs with elevated branch numbers compared to Control-Fc treatment: 18.1 and 11.4, respectively (n=116; p=<0.0001). This indicates a positive growth function. At E16.5, EPHRIN-A3 protein immunoreactivity on Deiters’ and pillar cells appears reduced in Vangl2 nulls compared to WT cochleae, suggesting that EPHRIN-A3 acts downstream of PCP signaling. DISCUSSION/SIGNIFICANCE: Results suggest that Eph/Ephrin signaling acts downstream of PCP signaling to mediate type II SGN guidance and EPHRIN-A3 switches its mode of activation. The clinical implications of these findings are that therapeutics targeting EPHRIN-A3 and/or VANGL2 in their given pathways could stimulate new OHC innervation following auditory damage.

OBJECTIVES/GOALS: The primary research goal was to identify brain alterations reliably associated with obesity using coordinate-based meta-analysis. A secondary goal was to compare brain alterations in metabolically healthy (MHO) and unhealthy (MUO) obesity. METHODS/STUDY POPULATION: Source data were peer-reviewed studies reporting locations of gray-matter alterations in group-average, case-control contrasts (obese vs. non-obese) cohorts, performed in a whole-brain, voxel-wise manner. Both voxel-based morphometry and voxel-based physiology studies were included. Three coordinate-based meta-analyses were performed: Pooled (MUO + MHO), MHO, and MUO. RESULTS/ANTICIPATED RESULTS: Thirty-two studies reporting a total of 50 case-control contrasts (MHO, 23; MUO, 27) met inclusion criteria, representing 3,368 participants (obese, 1,781; non-obese, 1587). The pooled analysis yielded 8 cerebral foci (3 nuclear, 5 cortical) in regions implicated in reward-seeking, cognitive, and interoceptive behaviors. MHO yielded 7 cerebral foci (4 nuclear, 3 cortical), partially overlapping Pooled results, with similar behavioral loadings. The MUO pattern was distinct, with 3 cerebellar and 1 occipital foci. DISCUSSION/SIGNIFICANCE: Brain alterations occurred reliably in obesity. The dominant pattern (Pooled & MHO) involved cerebral reward-system circuits, evident even in metabolically healthy obesity. Cerebellar alterations occurred exclusively in metabolically unhealthy obesity, a pattern previously reported in metabolic syndrome.

OBJECTIVES/GOALS: Many left hemisphere stroke survivors have a reading disorder (alexia), which is experienced as decreasing well-being. Therapies produce inconsistent results, demonstrating a need for treatment response predictors. We are investigating neural correlates of reading computational models to identify biomarkers to improve therapeutic outcomes. METHODS/STUDY POPULATION: Artificial neural network models of reading, mapping between orthography (visual word form), phonology (auditory word form), and semantics (word meaning), are trained to read single words at a healthy, adult capacity. The models are independently damaged to reflect the individual orthography-to-semantics, semantics-to-phonology, and orthography-to-phonology deficits of a sample of left hemisphere stroke survivors (n = 85). These deficits are measured with cognitive tests assessing the intactness of mappings between representations. Model damage is enacted by removing percentages of the connections between representations. For each type of deficit, the percentages of links removed are entered into a voxel-based lesion symptom mapping analysis to identify areas of cortex associated with that mapping. RESULTS/ANTICIPATED RESULTS: We anticipate that the neural correlates of model layers will be localized to a mostly left-lateralized network. Increased damage to the links between semantics and phonology in the model will likely be related to lesions involving the left posterior superior temporal sulcus and inferior frontal gyrus (IFG). Damaged orthography-to-semantic links will be related to the left fusiform gyrus (FG) and IFG. Finally, damage to the orthography-to-phonology links will be related to the left FG and superior temporal gyrus. DISCUSSION/SIGNIFICANCE: Mapping components of language models onto the brain will improve our understanding of the neural networks supporting language processing. Identifying these neural correlates may also produce biomarkers that can be used in predicting reading impairment at the acute stage or optimizing therapy in the chronic stage of stroke.

OBJECTIVES/GOALS: To determine the impact of regional anesthesia (RA) on rates of breast cancer recurrence, breast cancer specific mortality, post mastectomy pain syndrome, and chronic opioid use among patients who received regional anesthesia during primary breast cancer surgery. METHODS/STUDY POPULATION: Study population: Patients who underwent primary breast cancer surgery at Emory University and among a national patient cohort. Methods: This is a retrospective analysis of patients who underwent primary breast cancer surgery. Invasive breast cancer specific survival & breast cancer-specific survival will be estimated using Kaplan-Meier method and compared among patient groups. Cox proportional hazards will be utilized to estimate the unadjusted & adjusted risk of breast cancer recurrence and breast cancer-specific mortality between the two groups. RESULTS/ANTICIPATED RESULTS: We will present on our work comparing rates of breast cancer recurrence among patients who received regional anesthesia compared to patients who did not. DISCUSSION/SIGNIFICANCE: This study aims to establish the effect of regional anesthesia on breast cancer recurrence,breast cancer specific mortality, post mastectomy pain syndrome, and chronic opioid use following primary breast cancer surgery.

OBJECTIVES/GOALS: Impaired vascular function, a subclinical marker of cardiovascular disease, has been identified in prediabetes. Dietary nitrate supplementation has been shown to improve vascular function. However, this has not been studied in prediabetes. The purpose was to determine the effects of dietary nitrate on vascular function in prediabetes. METHODS/STUDY POPULATION: Five individuals with prediabetes (4 men, 1 woman; 55 ± 17 yr; HbA1c = 5.8 ± 0.2) participated in a double-blind, placebo-controlled, repeated measures study. Participants were randomly assigned to a 3-day nitrate supplementation (nitrate-rich beetroot juice, 12.9 mmol, 140 mL), or a placebo supplementation (nitrate-depleted beetroot juice, 0.05 mmol, 140 mL). Following supplementation, participants reported to the lab for measures of vascular function in the lower limb. Doppler ultrasonography was used to measure flow-mediated dilation (FMD) and post-occlusive reactive hyperemia (RH) of the superficial femoral artery in response to a 5-min bout of leg ischemia. RESULTS/ANTICIPATED RESULTS: FMD did not differ between the nitrate-rich (2.87 ± 2.01%) and placebo (2.24 ± 1.69%) conditions (p = 0.48; d = 0.35). Furthermore, peak RH did not differ between the nitrate-rich (1503 ± 443 ml/min) and placebo (1762 ± 414 ml/min) conditions (p = 0.36; d = 0.46). DISCUSSION/SIGNIFICANCE: These preliminary results suggest that dietary nitrate supplementation in the form of beetroot juice does not improve vascular function in individuals with prediabetes.

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.

OBJECTIVES/GOALS: Negative emotions (NE) play a pivotal role in addiction-related processes, including tobacco lapse during a quit attempt. Some NEs (e.g., shame, guilt) are posited to lead to a spiraling effect, whereby lapse predicts increased NEs leading to further lapse. This study goal is to examine associations between NEs and lapse. METHODS/STUDY POPULATION: This study examined associations between tobacco lapse and 13 distinct NEs among people who use tobacco and are trying to quit in two tobacco cessation studies. In Study 1, 220 adult (ages 18-74) cigarette users who identified as Black (50% female) participated in a 14-day study where ecological momentary assessment (with assessments approximately every 4 hours) was used to assess emotions and lapse in real-time and real-world settings. In Study 2, 288 adult (ages 18-71) cigarette users who were low socioeconomic status (51% White, 14% Black, 10% Hispanic, 49% female) participated in a 14-day study with the same study protocol as Study 1. Between and lagged within-person associations testing links between distinct NEs and lapse were examined with multilevel modeling with logistic links for binary outcomes. RESULTS/ANTICIPATED RESULTS: Results from Study 1 suggested that at the between-person level, disgust (OR =1.22, CI: 1.05, 1.42), nervousness (OR=1.23, CI:1.05,1.43), guilt (OR=1.40, CI: 1.16,1.69), and sadness (OR=1.18, CI:1.02,1.36) were predictive of higher odds of lapse, and at the within-person level, shame (OR=1.23, CI:1.04,1.45) was associated with higher odds of lapse. Results from Study 2 were similar and suggested that at the between-person level, disgust (OR=1.35, CI: 1.16, 1.56) and guilt (OR=1.88, CI:1.07,3.30), and at the within-person level, shame (OR =1.31, CI:1.10,1.55), were associated with higher odds of lapse. DISCUSSION/SIGNIFICANCE: The present study uses real-time, real-world data to demonstrate the role of distinct NEs on momentary tobacco lapse and helps elucidate specific NE that hinder the ability to abstain from tobacco use during a quit attempt. Results suggest that disgust, guilt, and shame play consistent roles in predicting lapse among diverse samples of tobacco users.

OBJECTIVES/GOALS: CASK-related microcephaly with pontine and cerebellar hypoplasia (MICPCH) is a rare X-linked neurodevelopmental disorder caused by mutations in calcium/calmodulin-dependent serine protein kinase (CASK). We aim to rescue CASK expression via an CRISPR/dCas9 epigenetic therapeutic and create iPSC-based CASK relevant in vitro model systems. METHODS/STUDY POPULATION: As females have two X-chromosomes, disease causing mutations present with a 50/50 expression of mutant and wildtype, due to the mosaicism caused by random X-chromosome inactivation (XCI). This project will adapt an established CRISPR/dCas9 epigenetic approach to rescue expression from the silenced, wild-type CASK allele. We aim to accomplish this through testing different dCas9 orthologues and a guide RNA screen targeting the CASK promoter. Constructs will be tested for optimal targeting efficacy in vitro and assessed via RT-qPCR. Additionally, epigenetic modifications from our approach will be analyzed through bisulfite sequencing. We also aim to apply this epigenetic rescue technology in disease relevant cell lines and eventually in engineered patient mutation iPSC-derived neurons. RESULTS/ANTICIPATED RESULTS: Our results show the ability to target CASK and assess gene expression changes with CRISPR/dCas9 paired with an epigenetic modifier and transcriptional activator. Additionally, our fibroblast model with nonpathogenic single nucleotide polymorphisms within CASKallow for allele specific analysis of our targeted reactivation. We anticipate that following an increase of CASK expression, there would be a decrease in region specific promoter methylation. Further, with the identification of clinically described disease-causing point mutations that result in a loss of function of CASK protein, induction of the mutant sequence onto a healthy cell background will result in a similar reduction of CASK protein in our cell model. DISCUSSION/SIGNIFICANCE: This project will demonstrate the first therapeutic avenue for CASK-related MICPCH, and the potential to utilize targeted X-reactivation as a platform approach for X-linked disorders. Further, investigation of smaller dCas9 orthologues prepares our approach for future translational applications such as packaging into AAV for delivery.

OBJECTIVES/GOALS: To demonstrate a successful example of clinical and translational research at a busy veterinary teaching hospital and highlight a collaborative effort in Comparative Oncology between the University of Florida’s (UF) Colleges of Medicine and Veterinary Medicine. METHODS/STUDY POPULATION: The UF College of Veterinary Medicine (CVM) is a full-time teaching hospital with multiple departments actively recruiting patients for clinical trials. These departments include but are not limited to Oncology, Internal Medicine, Dermatology, Cardiology, and Emergency and Critical Care. The Oncology department collaborates with the doctors at the UF Health Cancer Center (UFHCC) as part of a Comparative Oncology Initiative, which has many ongoing canine and feline trials focusing on immunotherapy. RESULTS/ANTICIPATED RESULTS: As of August 2023, there are 60 clinical trials actively recruiting and enrolling patients at the UF CVM. 57% of these trials are interventional studies, while the other 43% are observational studies. The UFHCC Comparative Oncology Initiative has successfully completed one clinical trial focusing on canine gliomas; has 4 clinical trials that are actively recruiting patients, and 6 trials that are opening for enrollment in the near future. These studies focus on osteosarcoma, melanoma, and squamous cell carcinoma. It is anticipated that with continued successful collaborations, more clinical trials will be possible, and new treatment options will become available for not only veterinary patients but human patients as well. DISCUSSION/SIGNIFICANCE: Clinical and translational research is an important part of veterinary medicine to further patient care. Due to ongoing collaborative efforts, not only veterinary patients but also human patients will benefit from the research being conducted at the UF CVM.