ABSTRACT IMPACT: This work provides supporting evidence for the development of a novel immunosuppression therapy for transplant patients. OBJECTIVES/GOALS: Our laboratory reported that inhibition of the kinase DNA-PK(cs) in mice delays allogeneic graft rejection in part by mitigating the induction of certain cytokines. We hypothesized that this was due to an inhibition of intracellular signaling programs in T cells and designed studies to identify the mechanism(s) by which this occurs. METHODS/STUDY POPULATION: The immortalized Jurkat T cell line was used to evaluate the effect of the DNA-PK(cs) inhibitor NU7441 on T cell activation by PMA/Ionomycin or PMA/PHA. Mouse primary splenocytes also were used to demonstrate the universality and reproducibility of our observations. Initially, protein mass spectrometry of lysates from untreated and NU7441-treated Jurkat cells identified proteins of interest regulated by DNA-PK(cs) that play a role in T cell activation and cytokine production. CRISPR genome editing was used to validate a potential downstream target of DNA-PK(cs). Western blot, ELISA, and flow cytometry were used to document changes in protein levels with respect to treatments. RESULTS/ANTICIPATED RESULTS: We observed that expression of the transcription factor Egr1 was highly induced after activation but attenuated after treatment with NU7441 in both Jurkat T cells and mouse splenocytes. Phosphorylated serine 301 of Egr1 was identified by mass spectrometry in stimulated cells and fits the kinase consensus sequence for DNA-PK(cs). Both an endogenous CRISPR-generated serine 301 to alanine mutant and expression of a plasmid-based S301A mutant resulted in an unstable form of Egr1 that was barely detectable. In contrast, expression of either a S301 to D or E phospho-mimetic mutant resulted in a stable form of the protein detectable by Western blot. Further evaluation of these mutants and Egr1 phosphorylation is underway to determine the mechanism by which DNA-PK(cs) kinase regulates protein stability. DISCUSSION/SIGNIFICANCE OF FINDINGS: We previously reported a role for DNA-PK(cs) in immunomodulation. We now have evidence that this occurs in part through stabilization of Egr1. We believe this novel finding will lead to uncovering a broader role for DNA-PK(cs) as a mediator of protein stability in T cells and provide support for targeting DNA-PK(cs) in immunosuppression therapy.

Objectives/Goals: In recent years, there has been growing interest in the development of air pollution prediction models, particularly in low- and middle-income countries that are disproportionately impacted by the effects of air pollution. Recent methodological advancements, particularly in machine learning, provide novel opportunities for modeling efforts. Methods/Study Population: We estimate daily ground-level fine particulate matter (PM2.5) concentrations in the Mexico City Metropolitan Area at 1-km2 grids from 2005 to 2023 using a multistage approach. Spatial and temporal predictor variables include data from the moderate resolution imaging spectroradiometer (MODIS), Copernicus Atmosphere Monitoring Service (CAMS), and additional meteorological and land use variables. We employed machine-learning-based approaches (random forest and gradient boosting algorithms) to downscale satellite measurements and incorporate local sources, then utilized a generalized additive model (GAM) to geographically weight predictions from the initial models. Model performance was evaluated using 10-fold cross-validation. Results/Anticipated Results: On average, the random forest, gradient boosting, and GAM models explained 75, 82, and 83% of variations measured in PM2.5 concentrations. PM2.5 levels were generally higher in densely populated urban centers and lower in suburban and rural areas. Important predictors of ground-level PM2.5 included wind (both u and v components), 2-meter mean air temperature, elevation, and the normalized difference vegetation index (NDVI). Discussion/Significance of Impact: Using novel machine learning-based approaches, we developed robust models with fine-scale spatial (1-km2) and temporal (daily) variations of PM2.5 in Mexico City from 2005 to 2023. The predicted PM2.5 concentrations can further advance public health research on air pollution in Mexico City and beyond.

OBJECTIVES/SPECIFIC AIMS: The preliminary analysis sought to retrospectively characterize the role of hypocretin receptor 2 (HCRTR2) in the development and prognosis of AD along with associated behavioral measures including smoking, self-reported drinking history, and neuroticism. Given the results in this study along with the paucity of information regarding the functional significance of rs2653349, we intend to comprehensively characterize HCRTR2 using haplotype analyses. We will then identify relationships between our haplotype analysis and IV alcohol self-administration using the Computer-Assisted Infusion System, and phenotypes identified in a sleep study. Furthermore, we aim at identifying functional loci in the hypocretin/orexin system by investigating differential allele expression in the orexin receptors in hippocampus tissue obtained from postmortem human brains. METHODS/STUDY POPULATION: This study examined 1569 European American and African American individuals between 18 and 65 years old, 922 of whom with a current diagnosis of AD. Participants were genotyped for HCRTR2 rs2653349 and ancestry was determined via a genome-wide panel of ancestry informative markers. AD was diagnosed using the Structured Clinical Interviews for DSM-IV (SCID-IV) for psychiatric disorders and recent alcohol use was assessed by 90-day Timeline Follow-back (TLFB) interviews. Smoking was assessed using the Fagerström Test for Nicotine Dependence and neuroticism was measured using the NEO Personality Inventory. RESULTS/ANTICIPATED RESULTS: In European Americans, a significant difference was found in current AD diagnosis between AX carriers and GG carriers (z=−2.390, p=0.017). This relationship remained significant in a logistic regression model controlled for age and gender (R2=0.269, p=0.015). TLFB drinking measures were compared based on the median values to correct for the ceiling effect resulting from the assessment covering the past 90 days. Total drinks (U=8.280, p=0.004), number of drinking days (U=6.983, p=0.008), and average drinks per days (U=7.221, p=0.007) were all noted to significantly differ between the two allele groups among Caucasians. The associations between rs2653349 and total drinks (R2=0.115, p=0.023) and heavy drinking days (R2=0.190, p=0.015) remained significant in linear regressions controlled for age and gender. Furthermore, Caucasian AX carriers had a higher median number of drinking days relative to GG homozygotes among current AD positive subjects (U=6.937, p=0.012) and a lower median number of drinking days among current AD negative subjects (U=4.430, p=0.035). Among Caucasian AD negative subjects, there was a significantly greater frequency of smokers (χ2=3.550, p=0.046). In African American participants, there were no significant differences in AD diagnosis and in measures of AD severity by genotype. African American males diagnosed with current AD had higher rates of smoking in the AX group (χ2=4.969, p=0.017). No significant associations were found between rs2653349 and neuroticism in any of the cohorts analyzed in this sample. DISCUSSION/SIGNIFICANCE OF IMPACT: The results suggest that, among Caucasians, AX carriers have an increased risk to develop AD independently of their age and gender. In addition, among individuals with a diagnosis of AD, AX carriers reported a greater number of drinking days, as measured by the TLFB, suggesting that this polymorphism also exerts an effect on the severity of the disease. This effect on increased alcohol consumption was absent in Caucasian AX carriers without current AD diagnosis. In future analysis, we will explore how different genetic profiles in HCRTR2, and also HCRTR1, may alter the orexin signaling pathway and how such alterations may predispose patients to develop AD and exacerbate AD once it develops.

Objectives/Goals: Community engagement has long been an underutilized strategy when conducting dementia research, especially for the recruitment of high-risk communities such as refugees. We created CHAMP: Championing Healthy Aging in Multicultural Populations Health and Resource Fair, aimed to promote early cognitive screening in aging Black refugees. Methods/Study Population: On August 9, 2025, the Health and Aging from Multicultural Perspectives Lab (HAMPLAB), in partnership with Global Lexington and the Lexington Senior Center, hosted the inaugural CHAMP Health and Resource Fair. Twenty-two vendors participated in the fair, including the Neurology department, pharmaceutical practice department, Bluegrass Lions Diabetes Project, local fire department, and the Alzheimer’s Association. Over 200 Black refugees in Lexington Kentucky and 18 volunteers attended the event. Complimentary services provided at the health and resource fair include health screening (e.g., A1C and blood pressure measurements), fresh food produce, fire safety, and back-to-school supplies. Results/Anticipated Results: Thirty-eight percent of the 55 attendees screened for A1C exhibited elevated levels. Many of these individuals were not previously diagnosed with prediabetes or diabetes and were offered counseling and follow-up. Also, over 100 people were screened for high blood pressure, and 62 eligible middle-older age adults signed up for various dementia research participation. These results indicate the importance of community-based events in which high-risk individuals can be identified and presented with practical next steps. Discussion/Significance of Impact: The CHAMP Health and Resource Fair illustrates the dual benefit of community engagement: advancing public health awareness through immediate health feedback and strengthening recruitment for ongoing research initiatives.

OBJECTIVES/SPECIFIC AIMS: The study aim was to 1) elucidate mechanisms contributing to the evolution of PSCI using a clinically relevant model of diabetes, a major risk factor for stroke and cognitive impairment, and 2) develop angiotensin type 2 receptor (AT2R) agonism as a therapeutic target. METHODS/STUDY POPULATION: Diabetes was induced in male Wistar rats by a HFD & low dose streptozotocin combination. At 12-14 weeks of age a total of 69 control & diabetic rats were subjected to 1 hr middle cerebral artery occlusion (MCAO) or Sham surgery. 3 days post-MCAO, rats that met the pre-set inclusion criteria were administered C21 or saline in drinking water at a dose of 0.12 mg/kg/day Adhesive removal task (ART) & 2-trial Ymaze were utilized to test sensorimotor & cognitive function at baseline as well as 1, 2, 4 and 8 weeks post-stroke. At week 8 post-stroke cell suspensions from freshly harvested brains were analyzed by flow cytometry utilizing antibodies against cell surface markers for M1 (CD11b+/CD45 low/ CD86+/TNFa+), M2 (CD11b+/CD45 low/ CD206+/IL-10+), and residential microglia (CD11b+/CD45+/ TMEM119+). RESULTS/ANTICIPATED RESULTS: Control rats progressively recovered from stroke-induced functional deficits by week 8, while diabetics still remained impaired (P< 0.05). 8 weeks post-MCAO only diabetic rats exhibited a decline in sensorimotor (P< 0.05) and cognitive function (P< 0.05) compared to Shams. Delayed administration of C21 on D2 post-stroke halted the decline and improved sensorimotor (P< 0.05) and cognitive function (P< 0.01). Flow cytometric analyses indicate that 8 post-stroke vehicle diabetics had an elevated M1/M2 ratio within the ipsilateral prefrontal cortex and hippocampus (P< 0.01, 0.01). They also had a larger percentage of non-residential microglia/macrophages, indicative of compromised blood brain barrier (BBB) integrity. Treatment with C21 significantly lowered the M1/M2 ratio (P< 0.05) and improved the BBB integrity. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together this study suggests that the use of comorbid disease models such as diabetes, may allow for more translational evaluations of PSCI. Higher translational relevance may also lead to a higher number of successful clinical trials and more FDA approved stroke therapies. It also suggests that C21 may serve as a potential therapeutic to modulate the development of PSCI.

OBJECTIVES/GOALS: High aortic stiffness is associated with increased cardiovascular morbidity and mortality. The purpose of this work is to demonstrate the potential of our compliant stent-graft design to therapeutically increase aortic compliance over a standard aortic stent-graft. METHODS/STUDY POPULATION: The aorta from a human cadaver will be excised and placed into a pulse duplicator circuit. The stiffness of the system will be estimated using the pulse wave velocity (PWV), which will be calculated using the time delay between pressure measurements at proximal and distal locations in the system. Baseline measurements with the unstented aorta will be compared to two cases: (1) with a standard stent-graft placed, and (2) with our compliant stent-graft prototype in the descending thoracic aorta. PWV is calculated as the distance between the pressure sensors divided by the time delay. Faster PWV is associated with a stiffer vessel, or lower aortic compliance. RESULTS/ANTICIPATED RESULTS: Prior work in vitro showed that the compliant stent-graft reduced peak and pulse pressures compared a standard, rigid stent-graft. We also expect the compliant device to exhibit lower PWV compared to a rigid stent-graft. Depending on the aortic tissue stiffness, the compliant stent-graft could raise or lower PWV compared to no stent. Mean pressure in the compliant case is likely to be slightly higher than the other two cases because the compliant stent-graft’s narrower lumen increases flow resistance. Although mean pressure will be higher, peak pressure should be lower than in the standard stent-graft because the added compliance decreases overall pressure swing between systole and diastole. DISCUSSION/SIGNIFICANCE OF IMPACT: Lower PWV in the compliant stent-graft over the standard stent-graft will indicate its potential to therapeutically lower aortic stiffness in patients needing aortic stenting. Positive outcomes from this study will be a step toward the eventual translation of a compliant stent-graft to clinical use.

OBJECTIVES/SPECIFIC AIMS: One of the key difficulties in predicting allergenic pollen exposures has been a lack of information on source plant location and abundance. However, the increasing availability of spatially explicit data from remote sensing offers new opportunities to create comprehensive inventories of allergenic pollen producing plants. METHODS/STUDY POPULATION: In this study, we use a spatially oriented field survey to map common ragweed (Ambrosia artemisiifolia) in Detroit, MI, USA. We then combine this with remote sensing imagery and LiDAR to predict ragweed presence and potential pollen production across 344 km2 of Detroit. Finally, we compare this with measurements of airborne pollen concentrations collected throughout the city. RESULTS/ANTICIPATED RESULTS: Our initial results show that ragweed is present in ~2% of the city, and its presence and abundance are strongly associated with demolished building (p<0.001). The uneven distribution of ragweed plants across the city leads to substantially higher pollen concentrations in neighborhoods where more buildings have been recently demolished. DISCUSSION/SIGNIFICANCE OF IMPACT: Our approach offers an effective way to quantify allergenic pollen production, airborne concentrations, and exposures across a large metropolitan area. This in turn provides insight on how to best reduce airborne pollen concentrations: in this case, by changing post-demolition land management practices.

OBJECTIVES/GOALS: The primary aim is to evaluate the efficacy of a Mandibular Advancement Device (MAD) vs conservative treatment for adults with non-apneic snoring, as measured by the sleeping partner. The secondary aim is to evaluate the effectiveness of treatment of snoring on the sleeping partner’s sleep quality. METHODS/STUDY POPULATION: We plan to enroll 60 pairs of primary snorers and their sleeping partners in our randomized clinical trial. Snorers will be randomized to either 4 weeks of conservative therapy, consisting of nightly Mometasone nasal rinse, breathe-rite strips, mouth taping, and lateral positional therapy, or 4 weeks of Mandibular Advancement Device therapy (MAD). 30 pairs of snorers and their partners will be in each arm. At follow up the primary outcome measure, the Clinical Global Impression of Improvement Scale (CGI-I), will be assessed by the sleeping partner to evaluate the response to snoring treatment. RESULTS/ANTICIPATED RESULTS: To date, there is no study reporting the rate of response in participants using MAD in Primary Snoring. Due to lack of preliminary data and effect size, we hypothesize that the rate of the responders in the MAD group will be 20% higher than the rate of responders in the active control group based on literature studies and preliminary results. A responder will be classified as someone whose sleeping partner rates on the CGI-I scale that the snoring was much improved or very much improved. MAD has been shown previously to be an effective therapy at treating sleep apnea and reducing snoring, and we anticipate it will continue to be so for patients who do not have sleep apnea. DISCUSSION/SIGNIFICANCE: Snoring is a nearly ubiquitous problem that prevents restful sleep for spouses of snorers, which is known to have detrimental health effects. Yet it does not have scientifically proven treatments. Our study will evaluate these treatments in an effort to improve the health of the sleeping partners.

OBJECTIVES/SPECIFIC AIMS: Cancer clinical trials (CCTs) are vital tools in the advancement of cancer prevention and treatment. Yet, only 3%–5% of eligible patients enroll in CCTs. Low participation can be attributed, in part, to poor communication as well as a lack of awareness and understanding about CCTs. In order to increase participation in trials, interventions should foster meaningful communication about cancer prevention and CCTs, especially between medical professionals and members of the community. Community forums provide a channel to communicate about cancer with members public and to educate prospective participants about CCTs. Thus, our goal was to evaluate the efficacy of hosting community forums about cancer in order to educate the public and influence perceptions of CCT participation. METHODS/STUDY POPULATION: During the Spring of 2016, participants (n=51) who attended a community forum about CCTs completed a pretest and post-test survey assessing their understanding and perceptions of CCTs. RESULTS/ANTICIPATED RESULTS: Results from the pretest to post-test survey revealed a significant positive increase (p=0.01) in participants’ attitudes toward cancer clinical research as well as marginally significant increases in participants’ perceived subjective norms (p=0.06) about participating in CCTs and the perceived personal relevance (p=0.06) of clinical research participation pretest and post-test. DISCUSSION/SIGNIFICANCE OF IMPACT: Findings suggest that community forums about cancer and CCTs could lead to an increased awareness and understanding of CCTs among members of the population and could be useful channels for cancer interventions.

Objectives/Goals: Idiopathic inflammatory myopathies (IIMs) are autoimmune diseases influenced by genetic and environmental factors. This study aims to explore infection patterns preceding IIM onset by applying temporal data mining and machine learning to deidentified patient records and corroborate results from molecular analysis. Methods/Study Population: The dataset used in this work was extracted from TriNetX with a focus on patients who have IIM. Risks for developing the outcomes were assessed using case–control cohorts. For each participant, information was extracted about diagnosis code, date of infection, and study visit in which the infection was reported. This data were then temporally encoded and used to generate sequence files for each of the outcomes. Unsupervised temporal machine learning was then preformed on these files to detect frequent subsequences of infections. Python library scikit-learn was used to perform the unsupervised machine learning with k-means clustering. Results/Anticipated Results: The results of this study identify infections associated with the onset of IIM by analyzing temporal infection patterns. Frequent sequences of infections uncovered, with specific patterns linked to different cohorts, offer insights into the etiology of IIM. Common and cohort-specific infection sequences will help validate existing research and provide new avenues for exploring the disease mechanisms. The findings will highlight significant infection patterns, which will inform our understanding of IIM onset across various patient populations. Discussion/Significance of Impact: The results will provide key insights into pre-symptomatic infection sequences related to IIM onset, enhancing understanding of its etiology and pathogenesis. These findings may aid in developing more precise screening methods for early detection and confirm previous results from analyzing immune signatures of infections in IIM.

OBJECTIVES/SPECIFIC AIMS: Characterize the expression kinetics of HIV-1 Envelope and their relationship to virus production at the cellular level. METHODS/STUDY POPULATION: In vitro and ex vivo laboratory analyses. RESULTS/ANTICIPATED RESULTS: Initial studies addressing the kinetics of cell surface. Envelope (Env) expression reveal that Env expression to peaks on day 2 post infection. Next steps include a series of experiments to compare the kinetics of Env cell surface expression with broadly neutralizing antibody (bNAb)-mediated ADCC and the characterization of virus production kinetics in this same context. To be maximally effective, ADCC elimination of infected cells should occur before peak Env expression. DISCUSSION/SIGNIFICANCE OF IMPACT: Potent bNAbs to HIV-1 recognize vulnerable sites on the HIV-1 Envelope (Env) protein and are of great clinical interest due to their potential use in the prevention and treatment of HIV-1 infection. Their effectiveness depends not only on the neutralization of viral infectivity, but also on the elimination of productively infected cells via antibody-dependent cellular cytotoxicity (ADCC). On a cellular level, ADCC dynamics are determined by the timing and level of Env expression on the surface of HIV-infected cells. This study aims to delineate the expression kinetics of HIV-1 Envelope and their relationship to virus production. We expect that it will provide new insights into the utility of bNAb-mediated ADCC in treating and possibly curing HIV-1 infection; therefore results might have substantial impact on future HIV treatment strategies.

Objectives/Goals: Head and neck cancer (HNC) patients frequently experience cachexia (≥5% weight loss) that goes underrecognized in clinical practice. We assessed whether weights in electronic health records (EHR) were frequent and sustained enough to characterize weight-loss magnitude and timing, and to quantify cachexia underdiagnosis and diagnostic delay. Methods/Study Population: Patients were identified in the Emory Healthcare EHR (2016–2019) using ICD-10 codes for head and neck malignancies. Those with ≥2 codes within 90 days were eligible cases; the index date was the first code of the qualifying pair. Weights were extracted for all encounters six months before through one year after the index date; implausible values were removed. Feasibility analyses included all eligible patients while cachexia analyses were limited to those with ≥3 post-index weights. Baseline weight was the median pre-index value or the closest post-index value ≤14 days. Underdiagnosis was defined as ≥5% weight loss from baseline without a cachexia-related ICD code recorded 90 days prior to, or any time after, the index date: cachexia (R64), abnormal weight loss (R63.4), muscle wasting (M62.5x), or anorexia (R63.0). Results/Anticipated Results: EHR data demonstrated high feasibility for longitudinal analyses: among 561 HNC patients, the median number of weight measurements in the first year was 18 (IQR 8–27) with a median interval of 7 days (IQR 2–14). Among 499 patients analyzed for cachexia, the median max weight loss was 11.2% (IQR 4.9–18.2), occurring a median of 5 months after the index date (IQR 2.4–8.4). Overall, 373 patients (75%) lost ≥5% of baseline weight, yet cachexia-related ICD codes were present in only 113 (23%). Among uncoded patients, 274 met the ≥5% weight-based criterion, thus 55% of HNC patients were at risk for underdiagnosis. The median time to cachexia coding was 5.4 months after the index date (IQR 2.5–14.4). In 38% of HNC cachexia patients, coding occurred more than 6 months after ≥5% weight loss, indicating a meaningful diagnostic delay. Discussion/Significance of Impact: EHR data provide sufficient density and cadence to track cancer-related weight loss with precision and reveal that cachexia often goes unrecognized despite clear, measurable weight loss. These findings reveal a gap between data visibility and clinical action and support EHR-based monitoring to enable earlier recognition and timely care.

OBJECTIVES/SPECIFIC AIMS: MDSCs are potent suppressors of T cell function, and have been recently found to be implicated in skin diseases driven by T cell dysregulation. However, the function of MDSCs in CLE is poorly understood. We sought to characterize the MDSC population in the peripheral blood of DLE patients and evaluate their ability to suppress autologous T cells. METHODS/STUDY POPULATION: All patients were recruited through the UT Southwestern Cutaneous Lupus Registry. PBMCs from 32 CLE patients and 16 age-matched and gender-matched controls were analyzed using flow cytometry. Monocytic MDSCs were identified by the phenotype of CD14+ HLA-DR neg/low. Furthermore, autologous MDSCs and T cells were purified from CLE PBMCs (n=4) and co-cultured at different ratios of these cells. T cell function was measured by secretion of IFN-γ by ELISA. RESULTS/ANTICIPATED RESULTS: Monocytic MDSCs in CLE PBMCs (median: 2.04%, IQR: 0.67%–5.07%) were significantly higher compared with healthy control PBMCs (median: 0.5%, IQR: 0.1%–1.07%, p=0.002). Although not significant on subset analysis, patients with CLE limited to the head and neck had the highest levels of MDSCs. CLE MDSCs (n=4) were found to suppress autologous activated T-cells in a dose-dependent manner. DISCUSSION/SIGNIFICANCE OF IMPACT: In this cross-sectional study of patients of the UT Southwestern Cutaneous Lupus Registry, we observed differences in the levels of MDSCs among PBMCs of CLE patients Versus healthy controls. CLE patients had significantly higher levels of MDSCs, which could be explained by the presence of an inflammatory state in this group. Furthermore, CLE MDSCs were able to suppress autologous T cells, showing that these cells are functionally patent in CLE blood. Their up-regulation in CLE blood may represent the body’s response to limiting disease severity, since most patients had mild disease activity.

OBJECTIVES/SPECIFIC AIMS: Children born with a single ventricle congenital heart defect requires three invasive open-heart surgeries in the first three years of life. The third operation, the Fontan procedure, includes connection of the vena cava (VC) to the pulmonary artery (PA) using a bio-inert conduit to reduce work required by the right ventricle (RV). While this operation greatly extends the lives of HLHS patients, the Fontan circuit eventually fails, and the only solution is a scarcely available donor heart. This failed circuit is explained by the “Fontan paradox” where central venous pressures build up over time, causing increased systemic resistance and congestion. The absence of the sub-pulmonary ventricle leads to abnormal hemodynamics associated with life-threatening complications. We believe that decreasing central venous pressures through the use of a tissue engineered contractile, patient specific conduit will decrease the amount and severity of complications caused by the “Fontan paradox.” We will use amniotic fluid derived induced pluripotent stem cells (AF-iPSCs) differentiated into cardiomyocytes (CMs) to generate flow within a biodegradable conduit. Additionally, AF-iPSC will be differentiated into structural support cells (SSCs), including cardiac fibroblasts and epicardium. Several studies suggest advanced contraction and structure of CMs in specific ratios with SSCs, particularly mouse and human fetal fibroblasts. In combination, these cells have shown advanced tissue organization and function through mechanically and electrically aligned junctions. This allows them to have a magnitude higher contractile force than CMs alone, making them ideal for increasing pressure within a tissue engineered construct. This poster focuses on the differentiation and selection of SSCs. METHODS/STUDY POPULATION: AF-iPSCs differentiation began at roughly 80% confluency. Mesoderm formation occurred via WNT pathway modulation by supplementing RPMI+insulin media with 0.5 ng/mL BMP4 at day 0, followed by 3 ng/mL BMP4, 2 ng/mL Activin A, and 5 ng/mL BFGF for four days. Then, RPMI+insulin media was supplemented with 10 ng/mL of BMP4 until day fifteen for epicardial formation. Cells were lifted to induce epithelial-to-mesenchymal transition (EMT) and RPMI-insulin media was supplemented with 10 ng/mL BFGF for cardiac fibroblasts. They were then harvested and characterized using immunofluorescence. Planned experiments include RT-qPCR for further characterization of cardiac fibroblasts. Additionally, a fibroblast isolation plating technique will be utilized to obtain cardiac fibroblast from AF-iPSC CMs and AF-iPSC epicardium. Commercially obtained human cardiac fibroblasts will be utilized as a control for all studies. RESULTS/ANTICIPATED RESULTS: Immunofluorescence (IF) revealed positive expression of vimentin and α-SMA indicating a fibroblast and vascular smooth muscle phenotype after supplementation with 10 ng/mL of BMP4 after EMT induction. It is expected that IF of epicardial formation at day 15 will show positive expression of WT1, a well-known epicardial marker. We also suspect RT-qPCR will reveal high expression of cardiac fibroblast specific markers COL1A1, PDGFA, TCF21, and THSB1. We expect to yield a higher number of cardiac fibroblast from the small molecule AF-iPSC differentiation compared to a timed plating technique of AF-iPSC CMs and AF-iPSC epicardium (separately plated). Results will be quantified and compared using the aforementioned techniques. DISCUSSION/SIGNIFICANCE OF IMPACT: Discussion/significance of impact: Although fibroblasts make up a large portion of cells in the heart and greatly enhance CM function, they are poorly characterized in the literature and not easily obtained. This study will provide an efficiency comparison on the best method for acquiring cardiac fibroblast for cardiac tissue engineering applications as we move forward with translational cardiac pediatric research.

OBJECTIVES/SPECIFIC AIMS: Obstructive lung disease following particulate matter (PM) exposure is a major health concern. Coexisting metabolic syndrome (MetSyn) often occurs. Receptor for advanced glycation end products (RAGE) is highly expressed in the lung, is a strong predictor of FEV1, and is a key mediator of MetSyn. To determine if the loss of RAGE protects from the persistence of effects of particulate associated lung injury in a murine model. METHODS/STUDY POPULATION: Wild type (WT) and RAGE knockout (RKO) mice were exposed to 100 μg of PM (WTC-Aggregate, PM53) or PBS control by oropharyngeal aspiration. Lung function, methacholine challenge, and bronchoalveolar lavage (BAL) were quantified 28 days after PM exposure using flexiVent (Scireq Montreal, QC). BAL was obtained and cell differentials, cytokines and transcription factors were assayed. Bio-volume to airspace ratio and mean chord length were measured (Image J and Adobe Photoshop). RESULTS/ANTICIPATED RESULTS: WT mice were hyper-reactive to methacholine compared with their PBS controls 28 days after a single exposure to PM. They recovered from increased neutrophilia, loss of FEV, decreased compliance, and increased resistance, which were previously observed 24-hours after exposure. RKO were not hyper-reactive when compared with their own PBS controls. Lung histology shows persistence of loss of alveolar space in WT mice exposed to PM after 28 days. Area fraction was significantly higher in PM exposed WT mice after 28 days which was not significant after 24 hours. Mean chord length was significantly shorter for PM exposed at both time points for WT mice. The relative expression of phosphorylated to total CREB and ERK1/2 proteins was lower in RKO PM exposed mice compared with WT PM while STAT3 expression was lower in WT PM compared with WT PBS. PM induced a lower fold change of total proteins from the PBS controls in RKO for CREB, p38, ERK1/2, STAT3, and STAT5. JNK and p70S6k total proteins expressed a decreased fold change in WT PM exposed mice compared with WT PBS controls. DISCUSSION/SIGNIFICANCE OF IMPACT: A single dose of PM can produce persistent airway hyper-reactivity after 28 days of exposure. This PM induced injury is alleviated in the absence of RAGE, similar to what was seen at 24 hours. Inhibiting RAGE may be key to limiting the persistent inflammatory effects of high intensity PM exposure.

ABSTRACT IMPACT: Circadian disruption is known to cause significant human pathology but has not been evaluated in pancreas cancer carcinogenesis; through understanding how disruption of circadian rhythms can lead to pancreas cancer development and spread, preventive and therapeutic strategies can be devised. OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer due to early spread and poor response to therapy. Identifying factors driving PDAC growth could lead to new therapeutic strategies. Thus, we evaluated the extent to which circadian rhythm disruption, a factor strongly associated with cancer formation, contributes to PDAC pathogenesis. METHODS/STUDY POPULATION: To achieve the objective, we evaluated mice with pancreas lineage Kras-mutation (KC mice), which are predisposed to develop the full spectrum of pancreas cancer precursor lesions (pancreatic intra-epithelial neoplasia or PANIN-1, 2, 3) and PDAC. We subjected KC mice to a light-dark phase shift protocol known to induce circadian disruption (KCCD, n = 18), and another group to standard lighting conditions (KCNC, n = 31), with equal numbers of males and females in each group. The mice were allowed access to food and water ad libitum until sacrifice at age 9 months. Histopathologic evaluation of the pancreas was then performed to assess for pancreatic inflammation, pancreatic precursor lesions (PANIN) and PDAC. Fisher’s Exact Test was used to evaluate differences in incidence. RESULTS/ANTICIPATED RESULTS: As expected, both groups of mice demonstrated 100% incidence of chronic pancreatitis and PANIN-1 (low-grade precursor lesion) at age 9 months. This is consistent with the KC phenotype. However, the KCCD mice demonstrated a significant increase in acute pancreatic inflammation (61.1% vs 19.4%, p = 0.005) compared to KCNC mice. Furthermore, intermediate grade precursor lesions (PANIN-2) were also significantly increase in the KCCD mice (38.9% vs 6.5%, p = 0.006). Incidence of high-grade precursor lesions (PANIN-3, or carcinoma in situ: 22.2% vs 9.7%) and PDAC (27% vs 19%) were also increased, but these were not statistically significant. These results are notable given the established progression from higher grade premalignant PANIN lesions (PANIN-2, PANIN-3) to PDAC. DISCUSSION/SIGNIFICANCE OF FINDINGS: Insight into how circadian disruption leads to increased PANIN-2 formation and increase in acute inflammation may be advantageous for understanding circadian disruption in PDAC carcinogenesis. The circadian clock is present in immune cells and disruption can induce immune dysregulation. This mechanism will be evaluated in follow up studies.

OBJECTIVES/GOALS: Our concept of reciprocal innovation (RI) supports global health (GH) research partnerships that address shared health challenges for mutual benefit in both high and low- and middle-income (LMIC) settings. To advance this GH approach, the Indiana CTSI launched a RI program building on longstanding global health partnerships in East Africa METHODS/STUDY POPULATION: A core component of the program is annual RI workshops to promote reciprocal approaches in GH, identify priority areas for reciprocal research, and link investigators and stakeholders across settings. The first meeting in 2019 was in-person and focused on identifying health priority areas from the perspective of Indiana stakeholders. The second meeting was held virtually and focused on priority areas in East Africa. The third meeting focused on shared priority areas and discussing potential RI research projects. Agenda sessions include (1) presenting successful examples of funded RI projects; (2) breakout groups to share proposal ideas in preparation for the RI grants program; (3) building partnerships with colleagues in similar fields RESULTS/ANTICIPATED RESULTS: As of 2021, three RI workshops have been held with an average of 60 attendees at each workshop. Participants identified several overlapping priority areas for research and RI in Indiana and East Africa, including research in chronic disease, substance abuse, infant and maternal health, and access to healthcare. A Global Health Innovation Exchange of RI projects was created to support connections between locally- and globally-focused investigators. The repository is used to share updates on project progress, outcomes, and published materials. Workshops have also been used to explore a reciprocal innovation virtual platform to facilitate and foster more regular collaborations between globally and locally-focused investigators and pursue research projects on shared health challenges for mutual benefit DISCUSSION/SIGNIFICANCE: The collaboration at the stakeholder meetings set the foundation for continued partnership building, strong proposals for RI grants, and dissemination and translation of successful RI projects. To leverage momentum from the meetings, we are building a virtual RI platform to connect PIs across multiple CTSAs and increase the footprint of RI efforts

OBJECTIVES/GOALS: The glucocorticoid receptor (GR) is a ubiquitous steroid hormone receptor that is emerging as a mediator of breast cancer metastasis. We aim to better understand the biology associated with phospho-GR species in TNBC and their contribution to tumor progression. METHODS/STUDY POPULATION: To better understand how p-S134 GR may impact TNBC cell biology, we probed GR regulation by soluble factors that are rich within the tumor microenvironment (TME), such as TGFβ. TNBC cells harboring endogenous wild-type or S134A-GR species were created by CRISPR/Cas knock-in and subjected to in vitro assays of advanced cancer behavior. RNA-Seq was employed to identify pS134-GR target genes that are uniquely regulated by TGFβ in the absence of exogenously added GR ligands. Direct regulation of selected TGFβ-induced pS134-GR target genes was validated accordingly. Bioinformatics tools were used to probe publicly available TNBC patient data sets for expression of a pS134-GR 24-gene signature. RESULTS/ANTICIPATED RESULTS: In the absence of GR ligands, GR is transcriptionally activated via p38-MAPK-dependent phosphorylation of Ser134 upon exposure of TNBC cells to TME-derived agents (TGFβ, HGF). The ligand-independent pS134-GR transcriptome primarily encompasses gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TGFβ-induced TNBC cell migration, anchorage-independent growth in soft-agar, and tumorsphere formation, an in vitro readout of breast cancer stemness properties. Finally, a 24-gene pSer134-GR-dependent signature induced by TGFβ1 predicts shortened survival in breast cancer. We expect to find similar results using an in-house tissue microarray. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospho-S134-GR is a critical downstream mediator of p38 MAPK signaling and TNBC migration, survival, and stemness properties. Our studies define GR as a required effector of TGFβ1 signaling and nominate pS134-GR as a biomarker of elevated risk of breast cancer dissemination.

Objectives/Goals: The objective of this study is to explore strategies for AI-physician collaboration in diagnosing acute respiratory distress syndrome (ARDS) using chest X-rays. By comparing the diagnostic accuracy of different AI deployment methods, the study aims to identify optimal strategies that leverage both AI and physician expertise to improve outcomes. Methods/Study Population: The study analyzed 414 frontal chest X-rays from 115 patients hospitalized between August 15 and October 2, 2017, at the University of Michigan. Each X-ray was reviewed by six physicians for ARDS presence and diagnostic confidence. We developed a deep learning AI model for detecting ARDS and explored the strengths, weaknesses, and blind spots of both physicians and AI systems to inform optimal system deployment. We then investigated several AI-physician collaboration strategies, including: 1) AI-aided physician: physicians interpret chest X-rays first and defer to the AI model if uncertain, 2) physician-aided AI: the AI model interprets chest X-rays first and defers to a physician if uncertain, and 3) AI model and physician interpreting chest X-rays separately and then averaging their interpretations. Results/Anticipated Results: While the AI model (84.7% accuracy) had higher accuracy than physicians (80.8%), we found evidence that AI and physician expertise are complementary. When physicians lacked confidence in a chest X-ray’s interpretation, the AI model had higher accuracy. Conversely, in cases of AI uncertainty, physicians were more accurate. The AI excelled with easier cases, while physicians were better with difficult cases, defined as those where at least two physicians disagreed with the majority label. Collaboration strategies tested include AI-aided physician (82.4%), physician-aided AI (86.9%), and averaging interpretations (86%). The physician-aided AI approach had the highest accuracy, could off-load the human expert workload on the reading of up to 79% chest X-rays, allowing physicians to focus on challenging cases. Discussion/Significance of Impact: This study shows AI and physicians complement each other in ARDS diagnosis, improving accuracy when combined. A physician-aided AI strategy, where the AI defers to physicians when uncertain, proved most effective. Implementing AI-physician collaborations in clinical settings could enhance ARDS care, especially in low-resource environments.

OBJECTIVES/GOALS: The project investigates the role that resiliency may play within individual, interpersonal, social, and structural contexts in protecting against acute lethal stimulant (meth/amphetamine or cocaine) toxicity. Identifying preventative factors is crucial in developing and implementing risk reduction strategies for people who use stimulants. METHODS/STUDY POPULATION: This is a qualitative study involving in-depth interviews via questionnaire assessing resiliency factors among persons living in San Francisco who have used primarily either meth/amphetamine or cocaine for at least 5 years. The interviews will be coded for salient and recurrent themes and analyzed for code frequency, cooccurrences, clustering of themes and representative excerpts to highlight emergent themes as well as stressors and resilience factors at multiple levels. We aim to assess for substance use patterns, multiple domains of resiliency, medical and psychiatric complaints, and risk reduction strategies. We will recruit participants to match recent decedents from acute stimulant toxicity in various domains including salient demographic information and neighborhood characteristics. RESULTS/ANTICIPATED RESULTS: The anticipated results include a qualitative interview guide for living persons using stimulants in San Francisco to be used to gain insight into the community, illustrate participants’ substance use practices, and allow for better characterization of several discrete resiliency factors that have protected the participants and other community members from suffering lethal stimulant toxicity. We expect to identify individual components (e.g. use patterns, use of harm reduction supplies), interpersonal/social factors (e.g. drug using network, friendships, community connection), and structural influences (e.g. access to care, safe use sites, house and economic stability) that all play a role in resiliency against lethal stimulant toxicity. DISCUSSION/SIGNIFICANCE: Stimulant use is common, along with rising deaths involving stimulants in urban counties and in smaller rural/non-metro counties which are disproportionately affected, posing a public health challenge. We will find discrete, modifiable risk and resiliency factors that can be manipulated to minimize the chances of outcomes like overdose and death.