OBJECTIVES/GOALS: Early dysphagia detection reduces risk of pulmonary complications, length of hospital stay, and overall healthcare costs. The biggest limitation for early detection has been the lack of a sensitive, reliable, and noninvasive screening tool. The bedside swallow examination may miss silent aspiration in up to 40% of patients. The objective of this study is to evaluate if acoustic parameters can distinguish normal and wet voice in a canine laryngeal model. Ultimately, our goal is to establish whether the sensitivity of the bedside swallow examination can be augmented with the addition of an acoustic screen in humans. METHODS/STUDY POPULATION: Two excised canine larynges were used for laryngeal phonation simulations under six different conditions over 48 hours. Acoustic recordings were obtained while the larynges were in vibration at the phonation threshold pressure. Phonation was recorded under dry conditions and when the laryngeal introitius was covered with low viscosity glycerin (9.5cP) or high viscosity glycerin (950cP), as well as in adducted and abducted conditions. The latter mimics glottic insufficiency seen in presbylarynx or vocal fold paralysis. RESULTS/ANTICIPATED RESULTS: A total of 112 voice samples were generated and analyzed for pitch, sound pressure level (SPL), % shimmer, % jitter, relative average perturbation (RAP), and noise-to-harmonics using PRAAT software. A multivariate regression model showed that pitch, SPL, % shimmer, % jitter and RAP could significantly predict wetness in abducted conditions only. Could you please add numbers and p values? DISCUSSION/SIGNIFICANCE OF IMPACT: This pilot study indicates that classic acoustic perturbation measures distinguish the dry from the wet larynx only in glottic insufficiency condition in an ex vivo canine laryngeal model. Our next step is to study whether non-linear time series analysis and machine learning can differentiate dry and wet phonation in both adducted and abducted conditions in our animal model. CONFLICT OF INTEREST DESCRIPTION: Dr. Anais Rameau is a co-founder and Chief Executive Officer of MyophonX, a wearable device used to restore speech in patients with limited phonation capacity.

ABSTRACT IMPACT: Investigating the relationship between ICH volume at presentation and cortical atrophy over time may help to explain the phenomenon of cognitive impairment after ICH. OBJECTIVES/GOALS: Non-traumatic intracerebral hemorrhage (ICH) is the most common type of hemorrhagic stroke. ICH causes both mechanical as well as oxidative injury leading to neurologic deterioration. However, the relationship between ICH volume and brain atrophy is not fully understood. To that end, we aimed to investigate this relationship. METHODS/STUDY POPULATION: A retrospective cohort of adult ICH patients over a 10-year study period were studied. Demographic data, ICH cause, location, laterality, and treatment, as well as other data, were collected. DICOM files of CT or MRI at presentation and all subsequent follow-up imaging up to 5-years post-ICH were obtained. Using a novel semi-automated image segmentation tool developed in MATLAB by our group, ICH volumes were segmented. Using T1w MRI data, ipsi- and contralateral brain volumes were segmented using NeuroQuant, a fully automated software for MRI volumetric processing, for both initial and follow-up MRIs. Ipsilateral and global cerebral volume changes over time were calculated using initial and follow-up MRI images. Spearman rank correlation between volume changes and ICH volumes were calculated for each patient. RESULTS/ANTICIPATED RESULTS: 75 spontaneous ICH patients with adequate imaging follow-up and both early and follow-up MRI comparisons were considered. Of these, 14 patients had MRIs adequate for segmentation by NeuroQuant. There was a positive correlation (R^2 = 0.72, p=0.03) between ABC/2, the traditional ICH volume measuring approach, and our semi-automatic segmentation tool, with ABC/2 tending to overestimate ICH volume. There was an average of -6.51% of total brain volume loss with respect to initial brain volume at follow-up. There was a negative correlation between ICH volume and both global (Spearman rank correlation coefficient (R)=-0.714, p=0.004)) and local atrophy (R=-0.785, p=0.0009), meaning that as ICH volume increases, there is greater brain volume loss. DISCUSSION/SIGNIFICANCE OF FINDINGS: Greater ICH volume is associated with greater brain volume loss both ipsilaterally, reflected as encephalomalacia, and globally. These findings are important as encephalomalacia can result in focal neurologic deficit and other neurological symptoms over time, while global brain atrophy is associated with dementia and cognitive decline.

OBJECTIVES/GOALS: Illuminate processes and findings of the Environmental Scan of Adaptive Capacity and Preparedness of CTSAs done by the CTSA Working Group. Share challenges, strategies, and lessons learned for CTSAs to build Capacity to address clinical and translational barriers while responding to emergencies. METHODS/STUDY POPULATION: An Environmental Scan approach for searching, collecting, analyzing and using information from diverse sources regarding CTSA hubs experiences during emergency as related to research implementation, translation, support, adaptation, and preparedness: - Triangulating multiple data sources and mixed methods (e.g., literature review, document/RPPR analysis, and expert review); - Secondary analysis of the JCTS COVID-19 Survey of the CTSAs: challenges, lessons learned, and practices that work in various program components/areas; - Using feedback of CTSA professionals from multiple disciplines to enhance our knowledge of emergency preparedness and efficient adaptation to public health and other crises. RESULTS/ANTICIPATED RESULTS: The E-scan approach helped identify challenges, successful practices, and evidence-based strategies for building adaptive capacity and preparedness of CTSAs across various scientific sectors of the translational science spectrum. Some of the findings include: - Roadmaps for the creation of new collaborative research resources (biobanks; data repositories, etc.); - Rapid clinical and research decision making during public health crises; - New community-based research strategies to facilitate communication, research dissemination, and participant recruitment based on existing trust-based networks; - Innovative resource allocation to guarantee continuity of training and research opportunities for trainees. DISCUSSION/SIGNIFICANCE: The Environmental Scan of the Adaptive Capacity of CTSA hubs provides useful knowledge and tools to diverse clinical research stakeholders for mitigating the impact of a disaster via adjusting programs, practices, and processes, and building capacity for effective, emergency-ready and responsive research, training, and community engagement.

OBJECTIVES/SPECIFIC AIMS: The foci of this evaluation is to:. determine if cultural and contextual adaptations identified by community stakeholders via ShARP sessions change research design/implementation/dissemination strategies. examine changes in stakeholder engagement by the research team after the initial ShARP session. METHODS/STUDY POPULATION: CCH staff measured session effectiveness for multiple stakeholders at multiple time points. Researchers and community stakeholders completed surveys to assess process and function at the end of the session. CCH staff follow-up with researcher team members approximately 12-18 months after the session to assess longer term outcomes and changes resulting from stakeholder input gathered at the ShARP. RESULTS/ANTICIPATED RESULTS:. 16 ShARPs sessions were hosted in a three year period. o 4 hosted in Spanish o 2 focused solely on youth populations. 141 stakeholders representing diverse professional backgrounds participated in sessions and represented a cross section of: o Ages: 12- 17 (5%); 18-24 (6%); 25-34 (24%); 35-44 (23%); 45-54 (12%); 55-64 (9%); 65 and older (13%); No Response (8%) o Gender Identities: Female (62%); Male (34%); Transgender (0%); No Response (4%) o Race/Ethnicities: Asian, Pacific Islander (13%); African American/Black (28%); Hispanic/Latino/x (25%); Native Americans, First Nations, American Indian (0%); Caucasian/White (24%); Multiracial (2%); No Response (9%). Feedback from research teams (aggregate of Strongly Agreed/Agreed responses) o ShARP panel was made up of relevant stakeholders (97%) o ShARP session was worthwhile (100%) o Stakeholder input will improve my research project (100%) o I would engage stakeholders in future projects (40%). Feedback from community stakeholders (aggregate of Strongly Agreed/Agreed responses) o ShARP session worthwhile (89%) o I have an increased understanding of research after participating in this session (89%) o Based on the experience, would consider providing input on a research study in the future (90%) o This session was the first time I was asked to provide input on a research study (46%) DISCUSSION/SIGNIFICANCE OF IMPACT: Preliminary results indicate engaging stakeholders in research can provide cultural and contextual adaptations that increase research relevance and feasibility in any phase of research. Conversely, stakeholders indicated an increased understanding of research. This poster will feature stakeholder and researcher perspectives. Increasing dialogues between research teams and community stakeholders can improve research design and relevance. The ShARPs programs aims to increase these types of dialogues which can be especially important for research teams who are unsure of who or how to begin engaging stakeholders in research. Gathering additional data via follow-up interviews will help us better understand the impact this program has on long term stakeholder engagement in research.

Fair inclusion of research subjects is necessary to ensure that post-acute sequelae COVID-19 (PASC) research results benefit all members of society. Scientists should conduct research on a broad sample of individuals who represent clinically relevant factors influencing a disease. Without demographic diversity and sociological and environmental variability, research outputs are less likely to apply to different populations and would thus increase health disparities. The goal of this narrative literature review and ethical analysis is to apply fair selection criteria to PASC research studies. We briefly highlight the importance of fair subject selection in translational research and then identify features of PASC, as well as PASC research, that hinder fair inclusion of research participants. We will demonstrate that determining an adequate and representative sample is not simply a matter of ensuring greater diversity; rather, fairness requires a broader evaluation of risks, burdens, and benefits specific to underrepresented populations. We provide recommendations to ensure fair subject selection in PASC research and promote translation toward positive health outcomes for all individuals, including the most vulnerable.

OBJECTIVES/GOALS: Cardiovascular disease (CVD) is the most common cause of death in systemic lupus erythematosus (SLE). Genome-wide association studies have identified single nucleotide polymorphisms linked with CVD risk, but the association with SLE is not well established. We aimed to determine associations using machine learning in a multi-ethnic lupus cohort. METHODS/STUDY POPULATION: We will use data from the established SLE cohort study named Genetic Profile Predicting the Phenotype (PROFILE). PROFILE was constituted in 1998 by combining existing cohorts at multiple sites which are also of defined ethnicity (Hispanics of Mexican ancestry and Puerto Rico, African American, and Caucasian). The cohort consists of 3,118 individuals and the database contains socioeconomic–demographic, clinical, laboratory, and genetic variables. Genetic data consist of 196,524 single nucleotide polymorphisms. To detect risk genes and predict an individual’s SLE risk will design a random forest classifier using SNP genotype data. Logistic regression models will be performed with CVD as the outcome, adjusted for age, sex, ethnicity, disease duration, and traditional and nontraditional risk factors for CVD. RESULTS/ANTICIPATED RESULTS: We expect to find several established and new susceptibility genes associated with CVD. DISCUSSION/SIGNIFICANCE: This approach offers an opportunity to characterize distinct genetic risk factors and the relationship of CVD with SLE. These data may be important in the identification of patients at high risk for such events and may allow the design of preventive strategies which may beneficially have an impact on the morbidity and mortality of SLE patients.

OBJECTIVES/GOALS: Target: Computationally identify the markers of ulcer severity and risk of amputation from datasets that include demographics data, clinical, laboratory data, and medical history over 6000 patients. METHODS/STUDY POPULATION: In this study we will use tables of demographics such as age, gender, and ethnicity/race. Inspired by previous research we’ll include wound age (duration in days), wound size, number of concurrent wounds of any etiology, evidence of bioburden/infection, Wagner grade, being non ambulatory, renal dialysis, renal transplant, peripheral vascular disease, and patient hospitalization. Another table will include laboratory vital signs to include physiological variables such as height, weight, body mass index, pulse rate, blood pressure, respiratory rate, and temperature. We’ll include also social data like smoking status, socio-economic status, housing condition. RESULTS/ANTICIPATED RESULTS: Our project aligns with previous efforts to identify high risk Diabetic Foot Ulcer individuals but also takes a different perspective by collecting and marking clinical data from a subset of patients (e.g., severity, Hispanic versus non-Hispanic) and computationally process these data to provide a tool that can identify DFU severity and high-risk patients. We will obtain samples from Hispanics and non-Hispanics because these two groups are likely to have significant differences in the progression of ulcer severity. The rationale is that by comparing these two groups, we will assess and study the factors that are differentially present. It is our expectation that the proposed project will provide an easy-to-use tool for DFU progression and risk of amputation and contribute to identify high-risk individuals. DISCUSSION/SIGNIFICANCE: Diabetes prevalence estimates in Bexar County, TX exceeds national estimates (15.5% vs. 11.3%) and diagnosed cases are higher among Hispanic adults (13.4%) compared to their non-Hispanic white counterparts (9.5%). Late identification of severe foot ulcers minimizes the likelihood of reducing amputation risk.

OBJECTIVES/GOALS: To demonstrate safety and limit damage to off-target organs, we will be determining dosimetry parameters through experimentation and Monte Carlo simulations with our brachy therapy applicator designed to improve upon current designs to treat a 3-dimensional volume. METHODS/STUDY POPULATION: Low-cost materials were used to manufacture our High Dose Rate (HDR) applicator and a readily available after loading system was used to load our configuration with a radioactive source. The dosimetry of our device was analyzed using commercially available software and external beam therapy films to generate depth dose profiles and superficial dose distribution. Additionally, we attempt to confirm Task Group No. 43 (TG-43) dosimetry parameters using Monte Carlo simulations for our device. These data were compared with currently available applicators used for intraoperative radiotherapy. RESULTS/ANTICIPATED RESULTS: We anticipate that we will be able to validate dosimetry parameters for our device in preparation for clinical use. We aim to show our dose distributions align well with proposed target volumes while considering the composition and shape of our applicator. We hope to demonstrate that, unlike current applicators, our design is more effective at treating a 3-dimensional target volume. DISCUSSION/SIGNIFICANCE: By 2040, pancreatic cancer will be the second-largest cause of cancer-related deaths. Even with current brachytherapy applicators, 30-40% of pancreatic cancer seems to recur near the surgical site after surgery. By preventing local recurrence, we hope to improve patient outcomes.

OBJECTIVES/GOALS: Our goal is to determine whether microplastic fibers (MPFs) provide signals for dendritic cell-induced Th2 polarization via epithelial-cell-derived thymic stromal lymphopoietin (TSLP). We seek to highlight a potential mechanism for MPF-induced airway toxicity associated with asthma exacerbation. METHODS/STUDY POPULATION: Primary human bronchial epithelial cells (NHBEs) were grown and differentiated at an air-liquid interface. Dyed and undyed polyester MPFs (14x45 µm) generated using a cryomicrotome were delivered to NHBEs through a custom designed mesh-hopper system. After the exposure period (6, 12, 24 hrs), cell viability was assessed using alamarBlue, and RT-qPCR was performed to determine mRNA expression of asthma associated genes (i.e., TSLP, IL-13, IL-33, etc.,) in NHBEs. Bulk mRNA-sequencing followed by bioinformatics will be performed to observe other plausible pathways tweaked by lung cell exposure to MPFs. RESULTS/ANTICIPATED RESULTS: Through gravimetric analysis, it was determined that the mesh-hopper system can achieve delivery efficiencies of at least 85% for as low as 500 fibers. Following exposure, results show polyester MPFs (500 - 1,000 fibers) exposed to NHBEs at multiple time points (6, 12, 24 hrs) did not result in a statistically significant decrease in cell viability. Treatment with 500 undyed MPFs resulted in a slight increase in TSLP expression at 6 hrs that decreased over time, whereas all other treatment groups resulted in TSLP downregulation. Similarly, 500 undyed MPFs resulted in an increase in IL-13 expression at both 6 and 12 hrs with all other treatment groups leading to IL-13 downregulation. We anticipate the RNA-seq results will show pro-inflammatory pathways are highly targeted following NHBE exposure to MPFs. DISCUSSION/SIGNIFICANCE: This study is one of the first to mechanistically assess the impact of MPFs on lung cells while simultaneously addressing the need for a reliable system that delivers MPFs to ALI cultures to better mimic inhalation and avoid inadequate resuspension of particles in liquid medium.

OBJECTIVES/GOALS: Strip craniectomy with orthotic helmet therapy (SCOT) and open cranial vault reconstruction (OCVR) are mainstay surgical treatments for sagittal craniosynostosis. The purpose of this study is to compare long-term morphologic outcomes of the skull, subjective appearance, and perioperative outcomes between patients who underwent SCOT and OCVR. METHODS/STUDY POPULATION: Patients who underwent SCOT or OCVR for isolated, non-syndromic sagittal craniosynostosis with preoperative computerized tomography (CT) imaging were identified at UCSF between 2000 and 2020. Perioperative outcomes were extracted from the medical chart. Anthropometric measurements will be used to assess baseline severity using preoperative CT imaging and postoperative skull morphology using 3D camera imaging. Patient satisfaction surveys will be administered to parents at the time of postoperative 3D imaging. Subjective appearance will be rated among adolescents and craniofacial surgeons using patients’ postoperative 3D imaging. Descriptive statistics will be calculated using Student’s t test and Mann-Whitney U. Chi-square tests and Fisher exact tests will be used to compare categorical outcomes. RESULTS/ANTICIPATED RESULTS: 47 patients were included in the study (18 SCOT and 29 OCVR) with similar follow-up between groups (SCOT 2.3 +/- 1.5, OCVR 2.6 +/- 2.0). There were no significant differences in sex or race. OCVR had longer operative times (p-value 0.0003), higher estimated blood loss (p DISCUSSION/SIGNIFICANCE: SCOT was associated with superior perioperative outcomes compared with OCVR due to its minimally invasive technique. Our results for long-term healing outcomes and subjective appearance may further guide craniofacial surgeons in selecting the most optimal operative technique.

OBJECTIVES/GOALS: This study seeks to culture and characterize mucus-degrading microbes from the microbiome of inflammatory bowel disease (IBD) patients. METHODS/STUDY POPULATION: Stool will be collected from IBD patients and healthy first-degree relatives, then enriched for mucin-degrading microbes through growth on porcine rectal mucin. Dilution plating in both liquid and solid culture formats will be employed to isolate strains capable of growth on mucin. Cultures that are positive for mucin degradation will be identified with 16S rRNA sequencing; unique isolates will be genome sequenced and transcriptionally profiled on simple monosaccharides and mucin in order to identify putative mucin-degrading genes. The abundance of novel enzymes, pathways, and microbes will be compared in healthy and IBD patient populations using existing datasets in the literature. RESULTS/ANTICIPATED RESULTS: We expect to isolate previously uncultured mucin-degrading microbes, which will likely include new strains and possibly new species of bacteria. Through the transcriptomic characterization of mucin-degrading pathways, we will expand the lexicon of known mucin-degrading enzymes and pathways used by bacteria in the human colon. We expect mucin-degrading microbes to be more abundant and active in IBD patients compared to healthy controls. DISCUSSION/SIGNIFICANCE OF IMPACT: There is no cure for IBD and treatment relies heavily on suppressing a patient’s immune system. This research seeks to understand the contribution of the gut microbiota in the pathogenesis of IBD, which may lead to future therapeutic targets.

OBJECTIVES/GOALS: The host immune response during sepsis is now recognized to have anti-inflammatory pathophysiology. We aim to determine whether mitochondrial dysfunction of leukocytes predicts which critically ill septic patients develop immune paralysis and to identify differences in cellular metabolites between patients with and without immune paralysis. METHODS/STUDY POPULATION: Critically ill septic and control adult patients were recruited from one of 6 ICUs in a single-center tertiary care academic hospital. After enrollment, peripheral blood mononuclear cells (PBMCs) were isolated from a tube of whole blood on day 0-1 after ICU admission. Flow cytometry to quantify monocyte HLA-DR was performed to determine whether patients were immune paralyzed or not. Mitochondrial functional assays of PBMCs were performed with inhibitors of the electron transport chain to assess for differences in oxidative phosphorylation and glycolysis utilization. Metabolic profiling of cell pellets was performed to evaluate for specific metabolites and pathways associated with immune paralyzed patients. RESULTS/ANTICIPATED RESULTS: A total of 101 patients were recruited, including 62 control and 39 septic patients. 81 patients had immune paralysis status available for analysis. 52% of all recruited subjects were immune paralyzed. Of these, 58% were controls and 75% were septic. Immune paralyzed septic and control patients showed features of reduced utilization of oxidative phosphorylation (ox phos) including reduced basal respiration, ATP production and maximal respiration compared with non-immune paralyzed septic and control patients. Immune paralyzed septic patients showed diminished glycolysis utilization compared with septic non-immune paralyzed patients. Finally, cellular kynurenine and quinolinate levels were low in both immune paralyzed control and septic patients compared with non-immune paralyzed patients. DISCUSSION/SIGNIFICANCE: The PBMCs of immune paralyzed septic patients show evidence of mitochondrial dysfunction, with reduced ox phos and glycolysis utilization. Low levels of kynurenine and quinolinate, metabolite precursors to NAD+, in immune paralyzed patients may signal key deficiencies and targetable therapeutic avenues for reversal of an immune paralyzed state.

OBJECTIVES/GOALS: We study the association of adaptive decision-making, motor planning, and neuromuscular constraints, in children with hemiplegia. We examine how children scale motor decisions to body mechanics and the distance of a target while reaching in sitting/standing, and if they can recalibrate motor decisions to sudden changes in body mechanics. METHODS/STUDY POPULATION: Forty-five 6-12 year-olds with hemiplegia and 45 age/gender matched typically developing controls participate in clinical tests (i.e. balance, visual perceptual skills, etc.) and 3 experiments. Children “reach to tap” toward a target while sitting with both preferred and not preferred arms under three conditions: regular elbow extension siting and standing and elbow extension range reduced by 50% via a splint while sitting. Trials are easy, ambiguous, and difficult. Motor decisions are compared to abilities and motion sensors (IMUs) worn at wrist, arm, sternum and lumbar area, record biomechanical strategies children use under different decisions. Synchronized video analysis presents biomechanical strategies under different decisions. RESULTS/ANTICIPATED RESULTS: Data collection is still underway. A mixed models analysis is used to compare 2 (group: hemiplegic/typically developing) X 2 (arms: healthy/impaired & dominant/non dominant) X 3 (difficulty levels) the children’s decisions. Functional analysis is used to capture biomechanical strategies children use under different decisions and levels of difficulty. Exploration strategies are recorded relative to levels of difficulty. We will also compute correlations between affordance thresholds for all children and measures of sensation, range of motion, cognition and balance (in each posture). Lastly, a secondary analysis will compare behaviors of children with left/right hemisphere lesions, as they differ in spatial abilities. Preliminary results show that children with hemiplegia make errors with both their affected and unaffected side. DISCUSSION/SIGNIFICANCE OF IMPACT: Motor deficits in children with hemiplegia are the primary focus of treatments. Motor learning interventions focus on biomechanical deficits. Results from these studies expand the focus to planning and cognitive control issues underlying motor deficits.

ABSTRACT IMPACT: This work describes, for the first time, the methylome in patients with T-LGLL, focusing on the IL-15 promoter, and clearly demonstrates that 5-azacytidine decreases IL-15 production leading to T-LGLL cell death. These results form the basis a translational clinical trial in T-LGLL that will begin accrual in 2021 OBJECTIVES/GOALS: T-LGLL is an incurable leukemia with few treatment options driven by overexpression of IL-15. Our objective is to characterize the methylation status of the IL-15 promoter in T-LGLL patients and evaluate the potential use of 5-azacytidine (5-aza) in a translational trial by studying the effect of 5-aza in vitro on IL-15 levels, and the IL-15 promoter. METHODS/STUDY POPULATION: We sorted T-LGLL patient (n=3) and normal donor (ND) samples (n=3) for CD3+/CD8+/CD5-/dim for T-LGLL immunophenotype. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing and determined differential methylation and gene expression using 1-way ANOVA analysis. To determine the functional significance of differential methylation, we evaluated MOTN-1 T-LGLL cell viability in vitro with 5-aza at increasing concentrations. Next, we evaluated IL-15 gene expression in MOTN-1 cells treated with 5-Aza versus MOTN-1 with control using western immunoblot. Finally, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control. RESULTS/ANTICIPATED RESULTS: There was significant differential methylation (P= 0.0178) and expression (P =0.0059) in T-LGLL patients vs ND. These data revealed significant differential hypermethylation of gene promoters, including an increase in DNA methylation of the IL-15 promoter in T-LGLL cells vs ND. In MOTN-1 cells treated in vitro with 5-Aza at 24 and 48 hours, a dose-dependent decrease in the viability of T-LGLL cells was observed, from 100% to 49.5%, p=0.037. Further, a marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (p=0.0001). Finally, a decrease in cell viability was observed utilizing the IL-15 inhibitor IBI-15 vs control. These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. DISCUSSION/SIGNIFICANCE OF FINDINGS: Hypermethylation of the IL-15 promoter and subsequent increase in IL-15 is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce T-LGLL cell death. Based on these results, a phase I trial will be conducted using CC-486 (oral 5-Aza) in T-LGLL.

OBJECTIVES/GOALS: Measuring the area of skin involvement in chronic graft-versus-host disease (cGVHD) relies on costly, time-consuming manual assessment, with high disagreement among experts (>20%). Our published AI method, trained on labeled 3D photos, showed promise for delineating affected areas. We aim to improve its performance using unlabeled 2D photos. METHODS/STUDY POPULATION: Our published AI model (baseline) was trained on 360 labeled photos of 36 cGVHD patients,from a 3D camera with calibrated distance and lighting.Our gold standard labels were contours around affected skin, marked by a trained expert. A second unlabeledcohort of 974 standard 2D photos of 8 cGVHD patients was used to improve the baseline model. First the baseline model predicted affected areas on the unlabeled photos. Photos with good predictions were added to the training set with their AI-predicted labels. The model was then re-trained with the expanded labeled set. Models were successively trained with more AI labels until performance stopped improving. AI performance was assessed on a test set of 20 photos from 20 patients unseen during training, labeled by 4 experts to improve accuracy. RESULTS/ANTICIPATED RESULTS: Model performance was calculated by comparing against the gold standard labels on the test set. To quantify the spatial overlap of labeled areas the Dice coefficient was used (0 is no overlap, 1 is complete agreement), where higher values are better. To estimate clinical error we used surface area error (Error), where lower values are better. On the test set, the baseline model had a median Dice of 0.57 [interquartile range: 0.39 – 0.82] and Error of 57.6% [20.2 – 103.3%]. Re-training with additional AI-predicted labels from 8 new patients, the model yielded a median Dice of 0.60 [0.35 – 0.80] and Error of 50% [12.5 – 103.8%]. This approach is being expanded to a further 300 unlabeled patients, where we anticipate significant improvements to AI performance and consistency. DISCUSSION/SIGNIFICANCE: Evaluating AI models in standard photos could provide a consistent method of assessing and tracking cutaneous cGVHD and relieve the burden of costly expert assessment. A reliable automated AI tool would provide a meaningful improvement to the current standard of manual assessment and could be easily applied to large patient cohorts.

OBJECTIVES/GOALS: An age-dependent restitution defect in our neonatal pig intestinal ischemia model is rescued by unknown factors within homogenized mucosa of weaned pigs. A postnatally maturing network of enteric glia regulates the epithelial barrier, so we aim to show rescue is due to replacement of glial factors. METHODS/STUDY POPULATION: Jejunal tissues from suckling or weaned pigs were assessed by RNAseq and processed for immunofluorescent histology and 3-D volume imaging. Jejunal ischemia was surgically induced in weaned pigs and injured mucosa was recovered ex vivo with or without the glial inhibitor fluoroacetate (FA) while monitoring transepithelial electrical resistance (TER). RESULTS/ANTICIPATED RESULTS: Ingenuity Pathways Analysis of RNAseq data revealed significant suppression of numerous pathways critical for epithelial wound healing in suckling pigs (Z-score <−2 for of nine key pathways). Volume imaging studies confirmed lower density (P≤0.05) and complexity of the subepithelial glial network in suckling pigs. Treatment with FA inhibited TER recovery (P<0.0001) and restitution (P<0.05) in weaned pigs, mimicking the suckling pig phenotype and supporting glia as an important regulator of restitution in our model. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings provide important evidence that a developing glial network may be critical to the postnatal development of intestinal barrier repair mechanisms. Ongoing work will explore glial-epithelial interactions in vitro to further define postnatal development of barrier repair.

ABSTRACT IMPACT: This study characterizes interactions between human limbic circuitry and ventral temporal cortex using single pulse electrical stimulation, which may inform emerging stimulation therapies for epilepsy. OBJECTIVES/GOALS: The goal of electrical brain stimulation treatment is to modulate brain network function. However, stimulation inputs to different brain sites alter the network in a variety of ways. This study examines that variability by characterizing responses in a target region while stimulating multiple other brain sites. METHODS/STUDY POPULATION: We measured voltages in intracranial EEG in 6 patients who had electrodes implanted for epilepsy monitoring. We stimulated pairs of electrodes at multiple sites in the brain with a single pulse every 5 to 7 s and measured the resulting corticocortical evoked potential (CCEP) responses in the ventral temporal cortex (VTC). Using a novel clustering method, we uncovered sets of distinct canonical response shapes from the 20 to 500 ms post-stimulation period. This allowed us to group stimulation sites that evoked similar responses. We then related each group to high frequency, broadband, changes in spectral power as a reflection of local neuronal activity. RESULTS/ANTICIPATED RESULTS: We found that the VTC receives strong inputs specifically from the amygdala and hippocampus, both in terms of amplitude and broadband spectral power change. However, inputs from the hippocampus produced a different canonical shape than those from the amygdala. We also observed that VTC responses to inputs from the insula clustered in shape with those from the amygdala. These clustering patterns were consistent across subjects, although the actual shapes of the clusters showed variability. We further observed that some shapes were more associated with increases in overall neuronal activity than others, as reflected by broadband spectral power change. DISCUSSION/SIGNIFICANCE OF FINDINGS: Stimulation of connected sites may drive excitability at the target region in ways that are described by sets of full-time-course responses. By capturing their shapes, we can begin to decipher canonical input types at the circuit level. This approach might identify how stimulation inputs can be tailored to therapy while mitigating adverse effects.