Objectives/Goals: Previous studies of nanoparticles in a penetrating spinal cord injury demonstrated their ability to modulate immune responses to reduce secondary injury. This study investigated nanoparticles in contusion injury models, which is more common clinically, to quantify their efficacy as a function of injury severity. Methods/Study Population: Female C57BlJ/6 mice were anesthetized, and a T9/T10 laminectomy was performed to expose the spinal cord. A stereotactic setup with an Infinite Horizon impactor was used to position the spinal cord to receive a mild (30 kdyne), moderate (50 kdyne), or severe (70 kdyne) contusive injury. Within two hours of injury, poly(lactide-co-glycolide) nanoparticles, formed via a single emulsion with sonication protocol, or phosphate-buffered saline (PBS) vehicle were injected intravenously via tail vein injection. Nanoparticles (NPs) or PBS were administered daily for 7 days. Function was evaluated using the Basso Mouse Scale (BMS) test for hind limb function starting 3 days post-injury and repeated weekly thereafter, as well as a horizontal ladder beam test once they had recovered to at least occasional plantar stepping. Results/Anticipated Results: In the moderate injury group, NP-treated mice had significantly higher BMS scores starting 6 weeks post-injury (p=0.009), with the NP-treated mice performing better than control to the end of the study. In the mild injury group, NP-treated mice scored significantly better on BMS starting at 3 days post-injury (p=0.005). This difference persisted through week 4, and then the vehicle-treated mice maintained a slightly lower score through the end of the study. In the severe injury group, no significant difference on BMS scores was observed between groups and animals did not recover enough function to be evaluated via ladder beam. Scoring of the ladder beam test for moderate and mild injury studies is underway. Animals treated with NPs are anticipated to have a significantly higher number of successful steps compared to control. Discussion/Significance of Impact: Spinal cord injury consists of a primary injury and secondary injury caused by inflammation at the injury site. Nanoparticles present an opportunity to reduce secondary injury to improve functional outcomes of individuals with SCI. This study of NPs in a graded severity contusion model further illustrates the feasibility and utility of NPs in SCI.

Objectives/Goals: Global surgical education is largely driven by high-income countries (HICs), with curricula not tailored to the needs of low- and middle-income countries (LMICs). This study assessed country-specific needs for global surgical curricula and used generative AI to develop tailored curricula. Methods/Study Population: A curriculum framework was developed using expert opinion. Using a focused needs assessment survey, we evaluated international medical students’ and trainees’ needs for structured global surgery curricula, covering research, education, data and develop tailored curriculum templates for each country, ensuring alignment with the distinct needs of respective LMIC and HIC respondents. The AI-generated curricula were then compared across countries to identify variations in content and focus areas. Results/Anticipated Results: A total of 145 respondents from 18 countries and 6 continents participated, with 94 from LMICs and 51 from HICs. Four countries [Uganda (n = 31), Nigeria (n = 34), the USA (n = 23), and the UK (n = 23)] had more than 10 respondents, with the creation of a country specific global surgery curriculum. Curricula developed by HIC trainees focused on access to resources and infrastructure, future directions of global surgical research, and the role of medical students and early career development with a decreased focus on the history of global surgery. LMIC country-based curriculum focused on introducing the concepts of global surgery, quantifying the burden and epidemiology of surgical disease and had a greater emphasis on case studies and use cases, with decreased focus on resources and collaboration. Discussion/Significance of Impact: The research introduces a “precision education” approach that could help close the surgical education access gap globally. Further pilot and qualitative studies are necessary to validate the feasibility of AI-generated needs-based curricula.

OBJECTIVES/GOALS: There is increasing evidence that cannabidiol (CBD) has promising potential to treat PTSD. However, more research is warranted to fully understand the benefits of CBD for PTSD. This poster will describe the design and methodology of one of the first ever pilot RCTs examining CBD (vs. placebo) combined with prolonged exposure therapy for PTSD. METHODS/STUDY POPULATION: This study is an early Phase II double-blind, pilot RCT. Participants are 24 individuals 18-65 years old who meet DSM-5 criteria for PTSD on the CAPS-5 and were recruited from local hospitals and the community. Individuals complete a standardized baseline assessment with an independent evaluator to assess study eligibility. Participants who meet study inclusion are randomized to 18 days of CBD 250mg (BID) or placebo delivered in combination with 10-sessions Prolonged Exposure (PE) psychotherapy over 2 weeks. Individuals begin medication 3 days prior to beginning PE to ensure steady state. Participants complete self-report and biomarker outcomes at select timepoints during study participation, and are also asked to complete a 1-month follow-up assessment following treatment. RESULTS/ANTICIPATED RESULTS: This aims of this study are to: 1) examine the safety, feasibility, and PTSD symptom reductions associated with the combined intervention; 2) evaluate biomarkers associated with the endocannabinoid system and stress response; 3) determine the association between changes in biomarkers and PTSD symptoms following treatment. It is expected that CBD+PE will be safe and feasible, and that there will be a detectable signal of CBD vs. placebo in the reduction of PTSD symptoms. It is also anticipated that CBD will have higher levels of endocannabinoids and lower stress response levels compared to placebo. Lastly, we expect that greater changes in biomarkers will be associated with lower levels of PTSD severity following treatment. DISCUSSION/SIGNIFICANCE: Although there is growing interest in cannabinoids for psychiatric conditions, such as PTSD, controlled trials are limited and have yet to examine the proposed intervention for PTSD. If successful, this study will enhance the feasibility of a larger, adequately powered RCT to address immediate and long-term improvements for PTSD treatments.

OBJECTIVES/SPECIFIC AIMS: The first aim of the study is to evaluate the accuracy of serum biomarkers of acute GVHD measured after four weeks of corticosteroid therapy to predict 6 month NRM. The second aim of this study is to compare the accuracy of the biomarker algorithm to that of clinical response to corticosteroids after four weeks. The third aim of the study is to develop a novel regression model that uses weekly biomarker measurements over the first month of corticosteroid therapy to predict 6 month NRM. METHODS/STUDY POPULATION:. Patients who received HCT at one of 22 IRB-approved centers and provided blood samples to the Mount Sinai Acute GVHD International Consortium (MAGIC) biorepository and developed GVHD between January 2008 to May 2018 are included in this study. Patients were divided by time into a training set (Jan 2008-Dec 2015, n=233) for model development and a validation set (Jan 2015-May 2018, n=357) to evaluate the predictive performance of the model. The later time of the validation set was chosen deliberately to model contemporaneous GVHD treatment practices. The size of each group was designed so that there would be roughly equal numbers of deaths in both groups. RESULTS/ANTICIPATED RESULTS:. Serum concentrations of GVHD biomarkers after one month of corticosteroid therapy were measured in the validation set, and the predicted probability of NRM ($\hat{\rm p}$) was computed according to the previously published algorithm: $\log[-\log(1 - \hat{\rm p})]=-11.263 + 1.844({\rm logST}2)+ 0.577({\rm logREG}3\alpha)$. The performance of the biomarker algorithm was evaluated by creating receiver operating characteristic (ROC) curves and calculating the area under the curve (AUC) in the validation set. The AUC of the biomarker algorithm was a significantly better predictor of 6 month NRM than clinical response to treatment after four weeks of corticosteroids (0.84 vs. 0.64, p<0.001), which is a clinically relevant improvement in accuracy. To evaluate serial biomarker monitoring, serum biomarker concentrations will be measured weekly at five time points from treatment initiation to one month after corticosteroid therapy. We will use these values in the training set to develop a regression model for 6 month NRM that accounts for repeated biomarker measurements. The performance of this model will be tested in the validation set and the accuracy of the serial biomarker measurements will be compared to the accuracy of measuring biomarkers at the single time point after four weeks of corticosteroid therapy. An AUC improvement of 0.05 would be considered clinically significant. DISCUSSION/SIGNIFICANCE OF IMPACT: Clinical response to treatment after four weeks has been the standard endpoint in GVHD interventional trials for decades. If biomarkers measured at the same time more accurately predict long term mortality, this study would provide the basis for a novel endpoint in GVHD trials and enable more accurate determination of effect size of experimental interventions. An accurate biomarker algorithm will prove useful in guiding immunosuppressive treatment decisions for patients with GVHD. Patients identified by the algorithm as low-risk may benefit from reduced-dose corticosteroid therapy, potentially reducing lethal opportunistic infections. Patients identified as high-risk will be candidates for more intensive immunosuppression or investigational therapies. This precision medicine approach tailors therapy to the individual patient’s biology.

OBJECTIVES/GOALS: Real-world studies have been gaining momentum in providing evidence of treatment effectiveness and hold great potential for facilitating the drug regulatory process. The U.S. Food and Drug Administration (FDA) has recognized this by providing a framework for using real-world data (RWD) to generate real-world evidence (RWE). The objective of this study is to assess the current level of RWE implementation in clinical studies. METHODS/STUDY POPULATION: Using keywords relevant to RWE, we reviewed studies on drugs, biologics, and medical devices published on PubMed in 2018. Information regarding the therapeutic area of focus, intervention type, study design, primary outcome, and data source was recorded. Further analyses of the three main therapeutic areas of study (oncology, cardiology, and infectious diseases) were performed to determine how RWE was being utilized. In addition, a broad “real-world” search was performed on Clinicaltrials.gov, from which we extracted relevant observational and Phase I, II, II/III, III, III/IV, and IV studies. A supplemental PubMed search was used to evaluate published studies in order to identify which field these trials were concentrated in and the outcome of interest.” RESULTS/ANTICIPATED RESULTS: After application of “real-world” search terms to PubMed, 995 hits were generated and of these, 311 studies were excluded. More than half of the studies were observational and retrospective in nature (64%) with 70% examining drug/biologic outcomes. RWE data sources were largely dominated by medical records and claims data. The primary uses of RWE across oncology, cardiology, and infectious diseases included supporting drug product effectiveness, assessing safety, and evaluating treatment patterns. Of the 207 RWE studies identified on ClinicalTrials.gov, 66 were cancer randomized controlled trials (RCTs), a majority of which were used for post-marketing safety evaluations. Further research will be conducted to determine the precise role of RWE in all studies (e.g. historical comparator, label expansion). DISCUSSION/SIGNIFICANCE OF IMPACT: By examining the use of RWE in regulatory decision making, we can inform stakeholders of the extent to which robust RWE studies complement evidence generated by RCTs. Thought to reflect a product’s performance in a broader and more diverse population, RWE can provide greater insight to clinical trial conduct and ultimately transform patient outcomes.

OBJECTIVES/GOALS: Numerous diseases, including AL amyloidosis, are due to expression of aberrant antibodies. Significant effort has gone into plasma cell toxic therapies with varying degrees of success, but no therapies preventing antibody synthesis have been developed. The goal of this study is to assess BiP targeting to prevent antibody secretion in plasma cells. METHODS/STUDY POPULATION: Using 4 multiple myeloma cell lines (KMS11, RPMI8226, ANBL-6, U266), we knocked down BiP expression with RnaseH dependent siRNA or subA toxin, a bacterial toxin that specifically cleaves BiP, and measured changes in unfolded protein and intracellular light chains by flow cytometry during drug induced ER stress created by the intracellular calcium depleting agent thapsigargin. BiP is the master regulator of the unfolded protein response (UPR), an ER stress pathway important for protein folding. BiP is also an ER resident protein folding chaperone important for proper antibody folding. We hypothesized that BiP downregulation will lead to decreased folded antibody in the cell, increased unfolded antibody and constitutive activation of the UPR. RESULTS/ANTICIPATED RESULTS: 1 to 4 hours after treatment with thapsigargin plus siRNA against BiP, levels of BiP are significantly decreased. The levels of intracellular light chains decrease, and the level of unfolded protein within the cells increases dramatically. Interestingly, in alignment with the UPR literature, 24 hours post treatment, these levels have normalized again in surviving cells. SubA treatment increased BiP expression by 4 hours, contrary to our hypothesis, and minimally increased unfolded proteins and minimally decreased intracellular light chains. We expect that further functional testing of antibody secretion by ELIspot assays will show decreased secretion of antibody with BiP siRNA treatment. Combination therapies with other UPR stressing agents may act synergistically to affect antibody production. DISCUSSION/SIGNIFICANCE: BiP knockdown reduces antibodies and boosts unfolded proteins. SubA toxin ineffectiveness likely stems from increased BiP due to feedback loops. Combining anti-BiP treatments with UPR stressing drugs like bortezomib may halt antibody synthesis and induce cell death. These findings support BiP as a viable drug target for antibody-related diseases.

OBJECTIVES/GOALS: Childhood psychopathology is a worsening public health crisis leading to negative life outcomes, including self-harm and suicide. Difficulty in self-control as early as 3 years old predicts psychopathology, but the mediating mechanisms of brain function are unknown. Here, we tested one mechanism: functional connectivity (FC) integration. METHODS/STUDY POPULATION: We studied a sample of 204 children [53 F/149 M/2 NC; mean age (SD)=11 years (1.7)] with diverse self-control difficulties (e.g., attention deficit disorder [n=80]; autism spectrum disorders [n=91]). We extracted a general factor of psychopathology (“p-factor”) from the parent-reported Child Behavior Checklist. For participants with high quality fMRI data on 3 self-control tasks (n=79), testing flexibility, working memory, and inhibition, we calculated FC connectomes reflecting a general self-control state, and applied connectome predictive modeling (CPM) to reveal connections predicting overall task impairment. We then measured individual variance in cross-network integration of regions with the most predictive connections and tested for association with p-factor in a multiple linear regression. RESULTS/ANTICIPATED RESULTS: We repeated CPM 1,000 times with 10-fold cross validation to generate a distribution of accuracies for predicted vs. observed task impairment scores (mean r=0.25, permutation p=0.02). Connections selected a maximum of 10,000 times (10 folds * 1,000 repetitions) were strongly predictive of task impairment (r=-0.5, p<0.001), highlighting connectivity of canonical executive networks as well as the default mode network. Regions (n=22) with the top 5% most selected connections were in lateral parietal and frontal cortices and implicated motor control. Between-network integration, operationalized with the graph theory metric participation coefficient, of one of these regions in left posterior superior frontal gyrus significantly predicted p-factor (R2=0.26, F(22,56) = 0.87; B =-0.49, p<0.05). DISCUSSION/SIGNIFICANCE: A portion of dorsolateral prefrontal cortex, associated with executive control, explained individual variance in p-factor. We plan to test alternative predictive models. Identification of such a neuro behavioral mechanism underlying psychopathology may lead to novel intervention targets.

ABSTRACT IMPACT: The results of this research may influence NCCN guidelines on PET/CT surveillance for this population of patients for whom the guidelines are currently vague. OBJECTIVES/GOALS: We aim to quantify and describe the yield of surveillance PET/CT for detecting asymptomatic recurrence of melanoma after primary surgical treatment for stages IIB, IIC, and IIIA. Our goal is to provide evidence to inform appropriate guidelines for scheduling surveillance PET/CT for this population. METHODS/STUDY POPULATION: This is a retrospective study of patients who have undergone a PET/CT at Barnes-Jewish Hospital. Data will be collected in our Research Electronic Data Capture (REDCap) database. The sample size is 158. Data analysis will be explanatory for the yield of imaging and description of false positives and additional unnecessary workup. Survival endpoints will be reported and multivariate analysis with subgroups will be performed for predictors of PET/CT results. Cost-efficiency analysis will be conducted in collaboration with the Center for Health Economics and Policy (CHEP), with emphasis on a patient-oriented perspective. RESULTS/ANTICIPATED RESULTS: To date, we have collected data for 158 patients, with approximately equal numbers of each stage (56 IIB, 54 IIC, 48 IIIA). Due to lack of clear guidelines for this population, there is significant variation of imaging schedules and results between similar patients or groups of patients. This makes it difficult to anticipate results. Based on clinical experience, literature review, and preliminary data, we may anticipate lower yield of routine PET/CT for the detection of asymptomatic recurrence, with frequent false negatives and occasional false positives, in addition to detection and further workup of benign processes. DISCUSSION/SIGNIFICANCE OF FINDINGS: The high-risk stages IIB, IIC, and IIIA currently occupy a gray area where surveillance PET/CT remains controversial. For this group of patients, it is important to weigh the benefits of early detection and risks of false positives, unnecessary workup and anxiety, and cost.

OBJECTIVES/GOALS: Although gut fungi have been implicated in the immunopathogenesis of inflammatory bowel disease, the fungal microbiome has not been deeply explored across endo-histologic activity and treatment-exposure in ulcerative colitis. METHODS/STUDY POPULATION: Our retrospective cohort was derived from the Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease. We evaluated the fungal composition of fecal samples from 98 ulcerative colitis patients across endoscopic activity (n=43), endo-histologic activity (n=41), and biologic-exposure (n=98). Across all subgroups, we assessed fungal diversity and differential abundance of specific taxonomic groups. RESULTS/ANTICIPATED RESULTS: We identified 504 unique fungal amplicon sequence variants across the cohort of 98 patients, dominated by phylum Ascomycota. Compared to endoscopic remission, patients with endoscopic activity had an increased global fungus load (p DISCUSSION/SIGNIFICANCE: Endoscopic inflammation in ulcerative colitis is associated with altered fungal diversity driven by expansion of Saccharomyces and Candida compared to remission. The role of these fungal taxa as potential biomarkers and targets for personalized approaches to therapeutics in ulcerative colitis should be evaluated.

Objectives/Goals: Common mamographic findings, breast arterial calcifications (BAC), are a sex-specific biomarker for cardiovascular disease in women. While less common, BAC is frequently encountered on mammograms in males. This study aims to investigate BAC and its association with cardiovascular risk factors in men. Methods/Study Population: A retrospective single-institution study of male patients with mammography studies was performed between September 1, 2018, and September 3, 2025. Diagnostic mammographic images of male patients from a high-volume breast imaging facility were evaluated for the presence of BAC. BAC severity will be assigned a score based on the Canadian Society of Breast Imaging’s grading system. Unique ICD-10 diagnosis codes from the electronic medical record that have been established as independent risk factors for cardiovascular disease will also be reviewed. Male patients who have a prior history of myocardial infarction, coronary revascularization, stroke, heart failure, peripheral vascular disease, breast cancer, dementia, or chronic dialysis/renal transplant were excluded. Results/Anticipated Results: The study is in process of pulling male mammograms from our institution. Current volume of male mammograms at the institution is about 160 male mammograms performed per month. Therefore, we anticipate having around 13,600 male mammograms to analyze the BAC presence. Anticipated results include that patients with independent cardiovascular risk factors such as increased systolic blood pressure, increased LDL levels, and current smokers will have a higher prevalence of BAC presence. Additionally, those with a presence of BAC will be older compared to those that have an absence of BAC on mammography. If BAC is shown to be a significant risk factor for the development of cardiovascular disease, perhaps a mammogram might be recommended in men purely for cardiovascular risk assessment. Discussion/Significance of Impact: A significant association between the presence of BAC and CVD risk factors may provide a supplementary assessment of CVD risk among men, offering a novel tool for risk stratification. Implementing a standardized BAC reporting model may allow for earlier identification of asymptomatic patients at high risk for adverse cardiac events.

OBJECTIVES/SPECIFIC AIMS: The aims of this study are threefold. Firstly, we are examining the effects of increased in vitro afterload (a proxy for hypertension) on human induced pluripotent stem cell cardiomyocyte (hiPSC-CM) response to sunitinib in a durable and dynamic cardiac microtissue culture system. Secondly, we are exploring effects of repeat exposure and recovery of both sunitinib and afterload throughout the lifetime of the hiPSC-CM microtissue. Finally, we are assessing methods to prevent and treat sunitinib induced cardiotoxicity. Primary outcomes for this study are commonly utilized metrics of cardiotoxicity: degree of caspase activation, electrophysiology benchmarks for minimum voltage threshold and maximum capture rate, and microtissue force generation. METHODS/STUDY POPULATION: HiPSC-CMs are cultured and matured as 3D cardiac microtissues (CMTs) on a microtissue array. After maturation, cells are exposed to sunitinib doses of 0µM, 0.5µM, 1µM or 5µM for 12 hours. Concurrently with sunitinib dosing, increases in microtissue array stiffness are created with application of an external magnetic field. Afterload spring constants are fixed at pre-determined physiologic values ranging from 0.5µN/µm, to 5µN/µm. For Aim 1: Half of the CMTs are harvested at 8 hours after sunitinib dosing to conduct the caspase 3/7 assay, and the remainder are examined for 3 days following drug exposure to track temporal changes in electrophysiology and force generation. For Aim 2: After CMT maturation, 12-hour exposures to sunitinib are repeated three times at a fixed dose, with doses separated by one week. Concurrently with sunitinib dosing, increases or decreases in microtissue stiffness are created by changing the strength of an applied external magnetic field to create “ramp up” or “ramp down” stiffness conditions. Caspase assay and contractility metrics are measured at each timepoint. For Aim 3: Experimental conditions are conducted as described in Aim 1. Prior to the introduction of sunitinib, either carvedilol or an AMP-kinase activator is added to the CMT culture media at physiologic concentrations. Primary outcomes are examined as in Aim 1. RESULTS/ANTICIPATED RESULTS: Aim 1: We hypothesize that increases in microtissue afterload, synchronized with sunitinib exposure will augment sunitinib toxicity in cardiomyocytes resulting in elevations of caspase 3/7 activity and minimum voltage capture as well as decreases in maximum capture rate and maximum force generation. Aim 2: We hypothesize that repeat exposures to both sunitinib and to increases in afterload will augment sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Additionally, we hypothesize that decreases in afterload will decrease effective sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. Aim 3: We hypothesize that exposure to an AMP-kinase activator but not carvedilol will decrease the effects of sunitinib toxicity in CMTs via the primary outcomes mentioned in Aim 1. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of small molecule, targeted chemotherapeutic agents is increasingly common. Many of these agents cause cardiotoxic side effects, the mechanisms of which are incompletely understood. Our lab has developed a novel 3D tissue engineering platform capable of supporting durable in vitro cardiac microtissues that experience dynamic alterations in their biomechanical load. By using this platform to examine the cardiotoxic effects of sunitinib, insight into treatment and prevention of this common problem will be developed.

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.

ABSTRACT IMPACT: Examining lipid immunity for Mycobacterium tuberculosis in a translatable Guinea pig model may serve as a critical foundation for the creation of an efficacious human lipid based vaccine against tuberculosis. OBJECTIVES/GOALS: CD1 is a group of glycoproteins on antigen-presenting cells (APCs) that present lipid antigens to T cells. Mycobacterium tuberculosis (Mtb) has a lipid-rich cell wall which is essential for the pathogenesis of tuberculosis. Our goal is to determine the frequency, phenotypes, and functionality of CD1 T cells against Mtb using the guinea pig model. METHODS/STUDY POPULATION: Guinea pigs serve as the best translational model for CD1 immunology as they have both group 1 and group 2 CD1 complexes, comparable to human CD1. We performed ex-vivo and in-vivo experiments to analyze lipid antigen-specific CD1 T cell responses with Mtb infection. Assays to detect lipid-specific CD1 T cell activation include cellular proliferation, cytotoxicity assays, and interferon-gamma (IFNγ) release assay (Elispot) using both synthetic and Mtb-derived lipids. We isolated and characterized CD1 T cells using tetramerized CD1 complexes loaded with specific Mtb lipids. Spatial interaction between lipid loaded CD1 APCs with CD1 T cells were demonstrated by immunohistochemistry (IHC). Lastly, we will investigate the impact of lipid-based immunology via knockdown and overexpression of CD1 complexes. RESULTS/ANTICIPATED RESULTS: The cytotoxicity assay demonstrated that the CD1b1 and CD1b3 complexes play roles in the presentation of Mtb lipids, specifically glucose monomycolate, and mycolic acid, as noted by T cell killing of fibroblasts that express specific CD1 complexes that can present Mtb lipids. Similarly, cellular proliferation exhibited lipid specific T cell proliferation. IFNγ production by the stimulated CD1-restricted T cells (Elispot) was weak indicating CD1 T cells may not produce IFNγ. IHC successfully showed CD1 APCs in lungs and spleens of infected guinea pigs. It is anticipated that knocking out CD1 expression will lead to impaired immunity, and increase severity of disease as noted by pathologic lesions/bacterial burden, and systemic spread; in contrast, CD1 enhancement will limit the severity of tuberculosis. DISCUSSION/SIGNIFICANCE OF FINDINGS: We characterized CD1 T cells in infected guinea pigs at the tissue level, demonstrating Mtb lipid immunology. As a result, we laid the groundwork for investigating whether augmenting lipid immunity in the guinea pig model will enhance immunity against tuberculosis. Fruition of such work may lead to the development of effective tuberculosis vaccines.

Objectives/Goals: EEG neurofeedback is a promising neuromodulation approach for improving mental health outcomes but lacks standardized, neurophysiologically-grounded treatment protocols. This study uses high-density EEG (hdEEG) analyses to identify neural network patterns that inform development of evidence-based pediatric neurofeedback protocols. Methods/Study Population: This translational neuroscience project leverages resting-state hdEEG data and behavioral assessments from 600 six-year-old children in the Beginnings study (Arkansas Children’s Research Institute; Andres, NCT00616395). Analyses will identify functional and effective connectivity patterns across major brain networks – Default Mode (DMN), Salience (SN), and Central Executive (CEN) – using graph theoretical metrics, phase-locking values, and multivariate analyses. Identified network patterns will be linked with neuropsychological measures, including the Child Behavior Checklist (CBCL), to inform development of standardized neurofeedback protocol recommendations grounded in developmental neuroscience. Results/Anticipated Results: We anticipate identifying distinct patterns of functional connectivity that predict variation in children’s emotional and behavioral functioning. Findings are expected to highlight network patterns – particularly within emotion regulation and executive control circuits – that can guide neurofeedback protocol design. Anticipated products include neurophysiologically informed recommendations for standardized frequency bands, target networks, and protocol parameters to support future clinical trial development. This project represents a key T1 translational step toward establishing a neuroscience-based framework for EEG neurofeedback treatment protocols. Discussion/Significance of Impact: By integrating neurophysiological and behavioral data to guide protocol development, this work addresses a critical translational gap in the EEG neurofeedback literature. Findings will establish a foundation for standardized, evidence-based neurofeedback interventions that could improve accessibility and efficacy in clinical settings.

OBJECTIVES/GOALS: The goal was to understand the effectiveness of a novel clinical trial educational module and a corresponding initiative designed and disseminated by the Southern California Clinical and Translational Science Institute (SC-CTSI) to increase the quality of clinical trials conducted in academia. METHODS/STUDY POPULATION: The CRCs (Clinical Research Coordinators) for the initiative are asked to complete the online training. Possible study protocols are picked to be monitored by the CRCs. The monitor is instructed to study the protocol extensively and prepare for their monitoring visit. The trained monitor from the initiative then reaches out to the CRC of the study that is to be monitored and carries out the monitoring visit. Afterwards, the monitor sends initiative personnel the monitoring report, which is evaluated to see if the monitor checked everything they should have during the visit. The PI of the study is contacted with highlights from the monitoring report and improvements that they can make. RESULTS/ANTICIPATED RESULTS: The first study monitored was a site of a large NIH-sponsored study where the consent forms were signed electronically. It was found that the monitor could not access the consent forms. Therefore, the monitor could not do source data verification. The PI of the study said that they would be raising this issue with the NIH. During the monitoring visit of the second study chosen for the initiative, patient binders were specifically examined for informed consent and source documentation completeness. The charts of patients were also reviewed. The only deviation found was a missing signature in the Investigator Site File. For the last two studies, data will be reported. DISCUSSION/SIGNIFICANCE OF IMPACT: Monitors were not only able to monitor efficiently, but also able to point out deficiencies in the monitoring practices of large studies. This model could be expanded to other academic institutions to establish quality management systems to ensure data integrity and subject protection.

OBJECTIVES/GOALS: Aiming to better understand the molecular pathogenesis of familial partial lipodystrophy (PL), we initiated whole-exome sequencing for our patients with PL syndromes. A novel variant of early B cell factor 2 (EBF2) was identified. Here we report the biological impact of a novel truncating EBF2 variant. METHODS/STUDY POPULATION: Using 3T3-L1 and human primary subcutaneous preadipocytes, we performed loss-of-function and gene rescue experiments. All cells were cultured in DMEM with 10% bovine calf serum (Invitrogen) at 5% CO2. After lentivirus transfection, cells were grown to confluence and then exposed to adipogenesis induction media containing dexamethasone (0.25µM), insulin (1µg/ml) and isobutyl methylxanthine (0.5 mM). Total RNA was extracted using RNeasy Mini Kit (Qiagen) and cDNA was synthesized using IScript (Bio-Rad). Real-time qPCR was performed using TaqMan probes for Pparg and Fabp4, two key adipogenesis markers. RESULTS/ANTICIPATED RESULTS: Patient was found to carry a heterozygous nonsense mutation in exon 6 of EBF2, causing the premature termination of the protein at amino acid position 165. Adipogenesis was significantly suppressed in 3T3L1 cells when endogenous Ebf2 was suppressed with siRNA and lentiviral shRNA. Adipocytes with suppressed Ebf2 expression showed marked reduction of intracellular lipid content and Pparg and Fabp4 expression (>80% reduction). With lentiviral gene transfer, EBF2 fully rescued adipogenic potential, whereas the truncated variant EBF2 did not. Of note, 3T3-L1 cells transfected with the EBF2 variant displayed impaired adipogenesis, suggesting a dominant-negative effect of the EBF2 variant on adipogenesis. We confirmed the dominant effect of the EBF2 variant in human adipocyte differentiation. DISCUSSION/SIGNIFICANCE: Our data suggest that EBF2 is indispensable for adipogenesis. The loss of function and dominant-negative effect of the truncating variant of EBF2 likely plays a pathogenic role in PL. Whole exome sequencing of PL patients and ex-vivo functional analysis help identify novel gene variants and better understand the molecular pathogenesis of PL.

Objectives/Goals: We aim to enhance risk prediction in kidney transplantation outcomes by improving models of peptide antigen presentation of mismatched HLA molecules. HLA-derived peptides presented by HLA Class II to T-cells can activate an immune response, ultimately leading to graft failure. We aim to improve peptide prediction by modeling antigen processing. Methods/Study Population: T-cell epitope models for HLA mismatching struggle to predict which peptides are presented because antigen processing by proteases is not well modeled. We model antigen processing of HLA Class II proteins using 3D HLA structures (crystallography data) to create an HLA-specific antigen processing likelihood (APL) model. APL uses conformational stability measurements such as b-factor, COREX, solvent accessible surface area, and sequence entropy to predict cleavage sites from proteolysis. We will integrate APL into a T-cell epitope prediction tool for HLA-derived peptides based on donor and recipient HLA genotypes. Finally, we will associate the risk of graft failure with counts of these peptides derived from APL-integrated prediction models using a historical kidney transplant cohort from 2000 to 2023. Results/Anticipated Results: We expect that applying APL could reduce false-positive peptide binders influencing risk prediction scores. We anticipate improved peptide prediction accuracy compared to existing tools such as NetMHCIIPan, which assumes all possible peptides are equally likely to emerge from antigen processing. NetMHCIIPan is currently used by PIRCHE-II HLA mismatch risk algorithm. We expect that merging antigen processing (APL) and peptide-binding (NetMHCIIPan) models into a unified model would enhance risk stratification for graft failure. Current risk stratification still leads to poor outcomes post-transplant, especially for minority population groups. Our model can identify an alternative pool of well-matched donors and has the potential to improve equity for non-White minority candidates. Discussion/Significance of Impact: Improving the understanding of how HLA matching contributes to kidney transplant outcomes can better stratify risks for kidney transplant recipients, enable personalized treatment, and ultimately improve outcomes for those undergoing kidney transplantation to treat renal diseases.