OBJECTIVES/SPECIFIC AIMS: Immuno-oncology (IO) strategies are promising new approaches for the treatment of a variety of malignancies, including multiple myeloma (MM). Regulatory T cells (Tregs), which suppress effector T cell function, are a limitation to durable IO responses. The transcription factor FOXP3 is critical for the mature Treg phenotype. FOXP3 homodimerization is required for DNA binding and transcriptional activity, and mutations mapping to the dimerization region are associated with IPEX syndrome, resulting in dysfunctional Tregs in humans. We therefore hypothesize that inhibitors of FOXP3 dimerization will repress Treg suppression and enhance the anti-MM activity of IO. METHODS/STUDY POPULATION: To discover FOXP3 dimerization inhibitors, we are modeling FOXP3 homodimerization in vitro. Currently, we are optimizing an ALPHA screen and an ELISA-based dimerization assay using recombinant full length and truncated versions of FOXP3 to discover peptidomimetics that inhibit homodimerization. Induced Tregs expanded from human PBMCs will be treated with lead biologics and functional assays will be performed. RESULTS/ANTICIPATED RESULTS: Here we demonstrate Treg suppression of T cell proliferation and IFN-γ secretion after 5 days of co-culture under basal conditions. Additionally, we developed a MM/T cell co-culture system to measure anti-MM T cell responses and show decreased anti-MM T cell activity in the presence of Tregs. We expect to exploit the assays outlined here to demonstrate defective Treg suppression when FOXP3 dimerization is inhibited. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies support drug discovery efforts that will ultimately improve IO therapies for patients with MM.

OBJECTIVES/GOALS: While most transgender and non-binary (TNB) youth experience improved mental health post-initiating gender-affirming hormones (GAH), some continue to experience persistent, significant depressive symptoms two years post-GAH. Importantly, few studies have examined this issue given the lack of existing longitudinal studies. METHODS/STUDY POPULATION: We aimed to identify intervenable factors predicting persistent clinical depressive symptoms (PD) among TNB youth two years post-initiation of GAH utilizing the Trans Youth Care U.S. Study, an ongoing, multisite, observational study of TNB youth from four major pediatric hospitals across the U.S. We compared TNB youth (ages 12-20 at baseline) with persisting depression symptoms (PD) two-years post-GAH (i.e., PD; N=59) and those youth without (non-PD; n=215). Logistic regression estimated the association between PD and risk (e.g., negative expectations) and protective factors (e.g., parental acceptance, self-efficacy), measured at baseline and longitudinally. A mixed-effects model compared the rate of change of these factors between PD and non-PD youth. Models controlled for birth sex. RESULTS/ANTICIPATED RESULTS: Participants (Mean age=16) identified as transmasculine, then transfeminine, followed by non-binary. PD youth had higher negative expectations at baseline and internalized transphobia by 2-years, while non-PD youth reported greater parental acceptance over 2-years. The odds of PD compared to non-PD decreased with increasing self-efficacy at baseline (OR=0.7, 95% CI:0.5-0.9), whereas negative expectation for the future was associated with increased odds (OR=1.3, 95% CI:0.9,1.8). Moreover, the odds of PD increased 50% with increased rate of change in negative expectations, and odds decreased 50% with increased rate of change in self-efficacy, after adjusting for baseline negative expectations and self-efficacy, respectively. DISCUSSION/SIGNIFICANCE: We identified key intervenable factors for mental health treatment for TNB youth with PD; specifically, negative expectations increased risk for PD while self-efficacy appeared to buffer against PD risk. These findings also support assessment of youth for negative expectations for the future conferring greater risk for later PD.

OBJECTIVES/GOALS: Stigma may be a barrier to care for Latinx children with autism, given lasting effects on individuals and families in other disorders. No study has explored autism stigma and its effect in this group. We explore the Latinx community’s beliefs and stigmatizing attitudes towards and the effect of stigma on Latinx individuals with autism and their family. METHODS/STUDY POPULATION: Approximately 30 Latinx parents of typically-developing children recruited from Portland, Oregon and Birmingham, Alabama will complete a 60-90 minute interview and demographics questionnaire in Spanish. Participants will receive $50 for completion of the study. Using a phenomenological approach, a coding framework will be developed for the qualitative interview data. Structural coding will establish a set of predetermined codes that characterize the data generally. An emergent coding approach will be used to create new codes exploring each predetermined global characterization more precisely. Interviews and coding will cease when thematic saturation is achieved. RESULTS/ANTICIPATED RESULTS: Research exploring perspectives of parents of children with autism has suggested that autism stigma is high in the Latinx population and results in social isolation. However, results regarding developmental disability stigma in the general Latinx population have shown low rates of reported stigma. This suggests: 1.) stigma is not as high as reported by parents of children with autism, 2.) those perpetuating stigma are not reporting it explicitly on quantitative measures, and/or 3.) those perpetuating stigma are not aware that these attitudes are stigmatizing. A qualitative approach allows for a better characterization of this understudied phenomenon and explores the disconnect between the stigmatized and the stigmatizing. DISCUSSION/SIGNIFICANCE: By answering: “How do Latinx community members view autism?” and “How does stigma affect social support?” we open avenues for research, including: utilizing collectivistic culture in linguistically- and culturally-sensitive ways to spread knowledge of autism, provide social support for families, and increase inclusivity.

OBJECTIVES/SPECIFIC AIMS: To investigate the role of platelet factor-4 (PF4) in B cell differentiation and develop strategies to better modulate B cell differentiation in vitro and in vivo. METHODS/STUDY POPULATION: We use tissue culture and flow cytometry to examine the role of PF4 in B cell differentiation. We use wild type (WT) and PF4−/− mice on a C57Bl6/J background. PF4−/− mice have reduced in vivo B cell differentiation responses. RESULTS/ANTICIPATED RESULTS: We anticipate that our studies will demonstrate that PF4 promotes B cell differentiation in the bone marrow microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: The significance of this project may be valuable in developing efficient methods and strategies to increase or limit B cell numbers in vitro and in human disease.

OBJECTIVES/SPECIFIC AIMS: Middle ear volume (MEV) is a clinically relevant parameter in the treatment of many common conditions including otitis media, tinnitus, and conductive hearing loss. A growing number of studies have shifted from using tympanometry to 3-dimensional volume reconstruction (3DVR) to calculate MEV; however, MEV values between these methodologies have never before been directly compared. Here, our objective is to characterize agreement between MEV measurement methods across disease states and middle ear sizes. METHODS/STUDY POPULATION: Middle ears were identified from 36 patients ranging 18–89 years of age who underwent tympanometry testing during preoperative workup for tympanic membrane (TM) perforation, up to 1 month prior to a standard-of-care temporal bone computed tomography (CT) between October 15, 2005 and October 15, 2015. MEV values calculated by both tympanometry and 3DVR were analyzed for agreement using Bland and Altman plots. A correction factor was calculated where ear canal volumes were available for contralateral middle ears without TM perforation (n=12), and was applied to a second Bland and Altman plot in the corresponding patient subgroup. MEV agreement was characterized across MEV quartiles (1=smallest; 4=largest) and across increasing states of middle ear disease using Kruskal-Wallis and Wilcoxon testing with Bonferonni correction. RESULTS/ANTICIPATED RESULTS: A Bland Altman plot demonstrated significant disagreement of MEV differences as compared to a priori clinical thresholds. Absolute MEV difference was significantly greater in the average MEV fourth to first quartile (p=0.0024), fourth to second quartile (p=0.0024), third to first quartile (p=0.0048), and third to second quartile (p=0.048). Absolute MEV difference was not significantly different across varying states of middle ear disease (p=0.44). DISCUSSION/SIGNIFICANCE OF IMPACT: Statistically evident and clinically significant disagreement was demonstrated across tympanometric and 3DVR MEV estimates. This lack of agreement was most pronounced at higher average MEV and was persistent yet not appreciably different across varying severities of middle ear disease. These findings may limit the generalizability of studies of the middle ear that differ in MEV estimation methodology, particularly in pathophysiological states where MEV is increased.

OBJECTIVES/SPECIFIC AIMS: Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies to the keratinocyte adhesion protein desmoglein 3. Several other autoimmune diseases have defective B cell tolerance checkpoints, resulting in the accumulation of self-reactive and polyreactive B cells. METHODS/STUDY POPULATION: The present work aims to determine whether PV patients develop normal tolerance to self-antigens other than desmoglein 3, as a potential “first hit” in the development of autoimmunity. We use FACS to isolate single B cells at 4 developmental stages from 8 PV patients. We perform single-cell RT-PCR to amplify each B cell receptor, produce monoclonal antibodies, and screen these for autoreactivity using ELISA/IF to several self-antigens. At each B cell stage, we compare the frequencies of self-reactive and polyreactive B cells to those found in healthy controls. RESULTS/ANTICIPATED RESULTS: We anticipate similar frequencies between PV patients and controls, suggesting that the B cell repertoire in PV patients develops normally at early checkpoints. DISCUSSION/SIGNIFICANCE OF IMPACT: The absence of generalized reactivity would distinguish PV from other autoimmune diseases and would show that PV arises from a specific break in tolerance to a single self-antigen (desmoglein 3) during late B cell maturation. Such a result would further support PV as an ideal candidate for targeted immunotherapy.

ABSTRACT IMPACT: Cholecystokinin-B Receptor -Mediates Growth of Hepatocellular Carcinoma with the use proglumide. Proglumide is a non-selective antagonistic drug therefore, strategies that block signaling at the CCK-BR may provide to be a novel therapeutic option for Hepatocellular Carcinoma treatment OBJECTIVES/GOALS: Cholecystokinin (CCK)and gastrin mediate the growth of Hepatocellular Carcinoma (HCC) through CCK-R and interruption of this signaling pathway could decrease HCC. CCK-Receptors are overexpressed in HCC and proliferation may be mediated through CCK-B. Blockade of the CCK-BR with proglumide decreased both growth in vitro and tumor growth in vivo. METHODS/STUDY POPULATION: RNA was extracted from murine Hepa1-6, RIL-175 and human HepG2 cells and was evaluated by qRT-PCR for expression of CCK-AR, CCK-BR and gastrin. CCK-R protein expression was analyzed by flow cytometry. HCC cells were treated in vitro with CCK peptide, the CCK-AR antagonist or the CCK-BR antagonist. Proliferation of selective CCK-R KO cells was compared to that of wild-type cells. To determine the effect of a CCK-R antagonist on tumor growth in vivo two cohorts of mice bearing subcutaneous Hepa1-6 or RIL-175 HCC tumors were treated with an oral bioavailable CCK-R antagonist proglumide or untreated water for 3-4 weeks. The mice bearing Hepa1-6 tumors were placed on a high-fat diet to raise blood CCK levels. Mice bearing RIL-175 tumors were fed standard chow to determine if proglumide could block autocrine growth by gastrin. RESULTS/ANTICIPATED RESULTS: The mRNA expression of CCK-AR, CCK-BR and gastrin were increased 80-90-fold in all HCC cell lines compared to that of normal liver. CCK-BRs were detected on >85% of the cells by flow cytometry. CCK peptide (1nM) stimulated HCC growth in vitro in both wild-type cells and in CCK-AR KO cells but not in CCK-BR KO cells. CCK-BR antagonist blocked CCK-stimulated growth in vitro but the CCK-AR antagonist did not, suggesting that the CCK-BR was responsible for mediating proliferation. In vivo tumor growth was significantly reduced with proglumide treatment by 70% (p<0.05) in Hepa1-6 and by 73% (p<0.001) in RIL-75 tumors, respectively. DISCUSSION/SIGNIFICANCE OF FINDINGS: CCK-Rs are overexpressed in HCC and proliferation appears to be mediated through the CCK-BR. Downregulation with CRISPR Cas9 or blockade of the CCK-BR with an antagonist decreases growth in vitro and proglumide therapy decreases tumor growth in vivo. Strategies that block signaling at the CCK-BR maybe a novel therapeutic option for HCC treatment.

ABSTRACT IMPACT: This work may lead to new treatments for crystalline nephropathies. OBJECTIVES/GOALS: This study investigated obeticholic acid (OCALIVA ®) as a potential treatment for 2,8-dihydroxyadenine (2,8-DHA) nephropathy using a mouse model. The treatment was investigated in both sexes at two timepoints. METHODS/STUDY POPULATION: Male and female C57BL/6J mice (12 weeks of age) were fed chow (Research Diets D19120401i) or chow admixed with adenine (0.2% w/w) ad lib for either 3.5 or 7 weeks. Mice were treated with either vehicle (corn oil) or obeticholic acid (10 mg/kg BW) by gavage 5 days per week. Each of the 16 combinations of sex/diet/timepoint/treatment groups had an n = 6 (96 mice in total). Food and body weights were measured twice per week, and 24-hour urines were collected prior to euthanasia. Serum and organs were collected and processed for biochemical and histopathological analyses. RESULTS/ANTICIPATED RESULTS: At both the 3.5-week and 7-week timepoints, dietary adenine robustly increased BUN and serum creatinine compared to control diet in vehicle-treated male and female mice (P < .01, all comparisons). At the 3.5-week timepoint, obeticholic acid reduced BUN in male (P < .05) but not female adenine mice. Obeticholic acid did not affect serum creatinine at this timepoint. At the 7-week timepoint, obeticholic acid reduced BUN in female (P < .05) but not male adenine mice. At the 7-week timepoint, obeticholic acid reduced serum creatinine in both male (P < .05) and female (P < .01) mice. Biochemical and histopathological analyses are ongoing, and we anticipate that the results will agree with the serum chemistries. DISCUSSION/SIGNIFICANCE OF FINDINGS: Obeticholic acid is FDA-approved for primary biliary cholangitis, and it is in clinical trials for several other hepatobiliary diseases. Although currently untested in humans, it is nephroprotective in many preclinical models of kidney disease. This study is the first to investigate obeticholic acid in a model of crystalline nephropathy.

OBJECTIVES/GOALS: Due to clinical advances, cancer survivors are living longer but have an increased risk of a second primary cancer (SPC). This cross-sectional study aims to examine SPC prevalence in immigrant women and compare healthcare use (HCU) and cancer screening in immigrants with SPC versus (1) immigrants with a single cancer and (2) US-born women with SPC. METHODS/STUDY POPULATION: The study population will include adult women with breast/gynecologic primary cancer (PC) from the 2005, 2008, 2010, 2013, and 2015 National Health Interview Survey. First-generation immigrant or US-born status will be defined by region of birth. SPC includes diagnosis with a second cancer type ≥1 year after the initial PC diagnosis. We will compare the prevalence of ≥1 SPC in immigrant and US-born women. To evaluate HCU and cancer screening differences, we will assess sociodemographic and socioeconomic factors, risk behaviors, length of US residence, and citizenship status with descriptive statistics. In regression analyses, we will compare number of provider visits and cancer screening rates in immigrant women with SPC versus immigrants with PC alone and US-born women with SPC after matching by age and PC type. RESULTS/ANTICIPATED RESULTS: Disparities in cancer diagnosis, quality of care, receipt of recommended treatment, and screening rates among immigrants in the US are well documented. Therefore, we hypothesize that immigrant cancer survivors will have similar or higher rates of SPC compared to women born in the US with variations based on health status. We further hypothesize that immigrants with SPC will report lower rates of HCU after diagnosis of their first cancer and cancer screening compared to US-born women. However, we expect that immigrants with SPC will report similar or higher rates of HCU and cancer screening compared to immigrant women with PC alone. DISCUSSION/SIGNIFICANCE: To our knowledge, this study will be the first to describe SPC among immigrant cancer survivors in the US. This research will inform interventions to improve cancer care delivery and ultimately reduce SPC in immigrants with cancer.

OBJECTIVES/SPECIFIC AIMS: The aims of this study are 2-fold: (1) to determine if maternal schistosomiasis affects maternal immunity to tetanus and/or transplacental transfer of antitetanus toxoid (TT) immunoglobulin G (IgG) from mother to infant and (2) determine the influence of maternal schistosomiasis on infant BCG vaccine immunogenicity. METHODS/STUDY POPULATION: The study will utilize blood samples from a historic cohort of 100 mother-infant pairs from Kisumu, Kenya, a schistosomiasis-endemic area. For the first aim, we will evaluate maternal schistosomal circulating anodic antigen, which has improved sensitivity and specificity to detect active schistosomiasis from serum, and antisoluble egg antigen IgG positivity compared with quantitative maternal anti-TT IgG at delivery and anti-TT IgG cord blood to maternal blood ratio (cord:maternal ratio). For the second aim, we will evaluate association between maternal schistosomiasis as detected by circulating anodic antigen and antisoluble egg antigen IgG at delivery and infant BCG-specific Th1-cytokine positive CD4+ cells at 10 weeks following BCG vaccination at birth. RESULTS/ANTICIPATED RESULTS: We hypothesize that active maternal schistosomiasis will be associated with decreased maternal anti-TT IgG and reduced efficiency of transplacental transfer, as measured by infant cord blood to maternal blood ratio of anti-TT IgG. We also expect that maternal schistosomiasis will be associated with decreased infant immunogenicity to BCG vaccine. DISCUSSION/SIGNIFICANCE OF IMPACT: This is a formative study on infant vaccine immunity using laboratory methodology not previously applied. Understanding infant immunity in the setting of maternal schistosomiasis will inform vaccination strategies and tailor vaccine development in schistosome-endemic areas such as Kenya, where neither TB nor neonatal tetanus have been eradicated. Additionally, our results will inform public health policies to consider integration of antischistosomal agents in antenatal care.

OBJECTIVES/GOALS: Translating Research into Practice (TRIP), a hybrid implementation pragmatic clinical trial and CTSA collaboration, aims to implement a standardized breast cancer patient navigation protocol across five sites in Boston, MA. The goal of this study was to assess individual and institutional barriers and facilitators to implementing this protocol. METHODS/STUDY POPULATION: From November 2019 to August 2020, researchers conducted ethnographic observations of Patient Navigators (PN) at three of the five participating sites. Each PN at each site was observed for two, four-hour blocks by researchers trained in ethnographic research. Observers took notes using TRIPs 11 Step Protocol as a guide, which includes identifying patients at risk for delays in care, screening and referring patients to resources for health-related social needs, and tracking patients across the care continuum. Fieldnotes were uploaded into Dedoose and coded deductively by four researchers using a comparison and consensus approach. Researchers analyzed the data to identify barriers and facilitators to both implementing each protocol step and maximizing navigations ability to promote health equity. RESULTS/ANTICIPATED RESULTS: Across all sites, PNs faced barriers to adhering to the TRIP Protocol due to practical workflow constraints including their level of engagement across the cancer care continuum. Although there are other staff members who engage in navigation activities, navigation is often viewed solely as the responsibility of the PN. Operationalizing navigation as a person rather than a process creates confusion around the role, and PNs are often seen as a catchall position when other staff do not know how to help a patient. The time that PNs spend on tasks unrelated to core navigation activities described in the TRIP Protocol prevents PNs from navigating patients most at risk for delays in care. A lack of continuity across the care continuum can create role confusion for the PNs. DISCUSSION/SIGNIFICANCE: Patient Navigation can promote health equity; however, any task that pulls PNs away from navigating patients most at risk for delays in care diminishes this potential. PNs abilities to enact the TRIP protocol, which they saw as valuable, is circumscribed by the extent to which navigation is operationalized as a process within the institution.

Objectives/Goals: To address the manual, time-consuming processes of validating IRB compliance and ensuring the secure delivery of i2b2 data, this project automates compliance checks, streamlines Protected Health Information (PHI) access, and provides timely, secure data availability while reducing administrative burdens and non-compliance risks. Methods/Study Population: This project enhances the i2b2 application to automate compliance processes and facilitate secure data delivery through integration with REDCap. By linking i2b2 with the IRB system, the application performs automatic compliance checks for project requests, verifying GCP and HIPAA certifications, only allowing the release of IRB-approved PHI variables, safeguarding against unauthorized data access. Manual signatures confirm non-automated compliance processes. Once verified, the application automatically creates a REDCap project, assigns user access, and securely delivers data, ensuring compliance with HIPAA regulations. Results/Anticipated Results: The automated system successfully streamlined IRB compliance checks and data delivery for i2b2 requests. Validation of certifications like GCP and HIPAA, now occurs automatically, significantly reducing the risk of non-compliance. Personnel access to data is limited to IRB-approved PHI, ensuring data security and adherence to institutional standards. The integration with REDCap has reduced manual processes, cutting data request processing time to approximately 30 minutes. Researchers and administrative staff experienced a notable decrease in administrative burden, with faster, more efficient access to approved data while maintaining full compliance with IRB and HIPAA regulations. Discussion/Significance of Impact: The lessons learned can be adapted by institutions to improve compliance efficiency and reduce administrative overhead. Implementing similar automation of certification checks and data delivery, sites can enhance data security, minimize errors, and ensure faster, compliant access to research data.

OBJECTIVES/GOALS: This study was conducted in order to identify novel chemical compounds that exhibit anti-leishmanial activity and to further characterize their efficacy and toxicity in in vitro and in vivo systems in the hopes of future chemotherapeutic developments. METHODS/STUDY POPULATION: We developed a novel, target-free fluorometric high-throughput screen (HTS) to identify small molecules with anti-leishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVERset-EXP library cassette #5 yielded 210 compounds that killed 80% of parasites. One hundred nine (109) molecular scaffolds were represented within the hit compounds, including the 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP). A total of 27 novel 1,4-DAPP compounds were synthesized and anti-leishmanial efficacy and host cell toxicity was determined using L. donovani mCherry expressing amastigotes and THP-1 macrophages. Additional pharmacokinetic analyses of a potent 1,4-DAPP compound were conducted. RESULTS/ANTICIPATED RESULTS: Four experimental compounds had IC50 values less than 5μM, providing similar anti-leishmanial activity to miltefosine. Compound 9279817 had a clearance almost twice the rate of normal hepatic blood flow and had a relatively high volume of distribution, indicating this compound is rapidly cleared and distributes into tissues. in vitro rat liver microsome assays suggest a rapid metabolism of 9279817, and MS/MS results suggest this metabolite is most likely formed via oxidation of the sulfur on the lower aromatic ring. DISCUSSION/SIGNIFICANCE OF IMPACT: This study revealed a novel structural class of compounds that have anti-leishmanial activity. in vitro experiments show compounds with similar efficacy as miltefosine while having significantly less toxicity, suggesting that this class could be further developed as a potential chemotherapeutic.

OBJECTIVES/GOALS: Children with Down syndrome are at increased risk of respiratory diseases including asthma. Prenatal antibiotic exposure has been shown to be associated with the development of childhood asthma. We aim to estimate the association between prenatal antibiotic exposure and childhood asthma among children with Down syndrome. METHODS/STUDY POPULATION: We conducted a retrospective cohort study of mother-child dyads of children with Down syndrome who were born 1995-2013. Both children and mothers were continuously enrolled in the Tennessee Medicaid Program (TennCare). Prenatal antibiotic exposure was measured using mother’s prescription fill records. Childhood asthma was defined between age 4.5-6 years by asthma-related healthcare encounters and asthma-specific medication fills. We assessed the association between prenatal antibiotic exposure and childhood asthma among children with Down syndrome using modified Poisson regression adjusting for maternal age, race, residence, education, marital status, smoking during pregnancy, maternal asthma status, delivery method, number of siblings, and children’s sex. RESULTS/ANTICIPATED RESULTS: Among 346 mother-child dyads of children with Down syndrome, 273 (78.9%) children were exposed prenatally to antibiotics and 104 (30.0%) had asthma by age 4.5-6 years. Among those who were exposed to at least one course, the median antibiotic course equaled 2 (interquartile range: 1-4). Prenatal antibiotic exposure was associated with a 20% increase in risk of childhood asthma in the unadjusted analysis (risk ratio [RR] 1.20, 95% confidence interval [CI] 0.78, 1.83) and a 26% increase in risk after adjustment (adjusted RR 1.26, 95% CI 0.79, 2.01). DISCUSSION/SIGNIFICANCE: In our study population, the majority of children with Down syndrome were exposed to antibiotics prenatally and the prevalence of asthma was high. Prenatal antibiotic exposure was associated with an increased risk of childhood asthma among children with Down syndrome; however, this increase was not statistically significant.

Objectives/Goals: Endometrial cancer is one of the few cancers that has both a rising incidence and mortality rate. Molecular classification is becoming more important for the management of endometrial cancer but the ability to translate this into clinical practice remains constrained. Our goal is to use AI to predict the molecular subtype from histopathology slides. Methods/Study Population: We utilized the open source endometrial cancer datasets from The Cancer Genome Atlas (TCGA) (N = 387) and Cancer Proteomics Transcriptomic Tumor Analysis Consortium (CPTAC) (N = 135) to develop and train a vision transformer AI model. We used a proprietary cohort of patients (N = 548) for external validation. Whole slide images (WSI) and molecular subtype data were collected. Subtypes include POLE ultramutated (POLE), microsatellite instability (MSI-H), copy-number low (CNV-L), and copy-number high (CNV-H). WSI were preprocessed, and features were extracted. Modified STAMP protocol was used in training, utilizing a pretrained foundation transformer model (Virchow2). Cross-validation of the TCGA was used for initial training, followed by testing on the CPTAC dataset and validation on our proprietary cohort. Results/Anticipated Results: Fivefold cross-validation of the TCGA database (60% training, 20% testing, and 20% validation) developed a best overall model with a mean AUC of 0.74 (POLE 0.78, MSI-H 0.76, CNV-H 0.86, CNV-L 0.77). Overall precision 0.58, recall 0.55. CNV-H was the subtype with the most accurate prediction. CPTAC holdout testing revealed moderately high AUC (POLE 0.63, MSI-H 0.62, CNV-H 0.98, and CNV-L 0.76). Overall precision 0.54 and recall 0.58. Again, CNV-H was the most accurate prediction. Validation on our proprietary cohort revealed a drop in performance with overall mixed results by AUC (POLE 0.50, MSI-H 0.69, CNV-H 0.78, and CNV-L 0.61). Overall precision 0.57, recall 0.45. Again, CNV-H with the most accurate prediction but F1 score dropped from 0.77 in the CPTAC to 0.47 on validation. POLE was the least accurate prediction subtype. Discussion/Significance of Impact: The CNV-H subtype demonstrated robust performance, suggesting the model effectively captures the features associated with this subtype. CNV-L had moderate performance. MSI-H and POLE were notably lower. WSI-based AI models show translational potential for subtype prediction in the management of endometrial cancer but more work is necessary.

OBJECTIVES/GOALS: Total-body symptom surveys among patients with idiopathic orthostatic intolerance (OI) at our referral center suggest that non-conventional OI symptoms, such as pelvic pain, impact quality of life. We seek to identify additional common yet unconventional symptoms reported during clinic visits to improve targeted symptom management. METHODS/STUDY POPULATION: Pelvic pain symptom surveys were completed by 178 patients age 18 and over with a chief complaint of OI. Pelvic pain prevalence was assessed using the International Pelvic Pain Society (IPPS) and Pelvic Congestion Syndrome (PCS) surveys. Expanding on this work, surveys will be distributed to a population with the same inclusion criteria but with a broader symptom scope, chosen based on patient reports during clinical encounters—including presence of migraines, cold digits (Raynaud’s phenomenon), anxiety and depression. The Migraine Disability Assessment Test (MIDAS), modified Assessment of Systemic Sclerosis–Associated Raynaud’s Phenomenon (ASRAP), Generalized Anxiety Disorder-7 (GAD-7), and Patient Health Questionnaire-9 (PHQ-9) surveys, respectively, will be used to obtain symptom prevalence. RESULTS/ANTICIPATED RESULTS: Of the pelvic pain survey participants, pelvic pain was endorsed by 144/178 (80.9%) of respondents. Prevalence of the additional surveyed symptoms—migraines, cold digits, anxiety, and depression—will similarly be assessed. Given that in our referral clinic we have observed a trend of patients reporting these additional symptoms, we anticipate that at least some of them will be prevalent in a majority of the patient population formally surveyed, similar to the trend observed with pelvic pain prevalence. In particular, we anticipate many patients will report significant migraines, as migraine pain severity on a scale of 0-10 with 0 being no migraine pain and 10 being the most severe migraine pain imaginable is one of the elements of the IPSS survey, and 106/178 (59.6%) reported migraine pain of 5 or higher. DISCUSSION/SIGNIFICANCE: Novel treatment approaches for OI are needed, as lifestyle management is the current treatment paradigm. Several patients reporting pelvic pain have undergone targeted workup and subsequent symptomatic treatment that has improved their quality of life. Other targeted symptom approaches to prevalent symptoms could have the same effect.

OBJECTIVES/GOALS: University faculty partner with Community Consultants (CC) to: 1) identify health concerns of Chicagos South Side residents, 2) provide information regarding resources that address community health concerns, and 3) disseminate the model across the Institute for Translational Medicines (ITM) institutional partners. METHODS/STUDY POPULATION: CCs met twice monthly with University faculty and staff to identify CGR topics, delivery format, and audience development strategies. Faculty from the University of Chicago and community experts presented on identified health topics. Traditionally held in a community setting, CGR moved to a virtual format due to the pandemic. Recent topics covered included issues around the impact of COVID-19 on African American and LatinX communities and vaccine hesitancy. Audience members were surveyed at the end of each session and provides information on the quality and impact of the content. Twice during the season, partners debriefed on the effectiveness of the partnership and program. A plan was developed to deploy CGR across the ITM partners. RESULTS/ANTICIPATED RESULTS: Since 2010, there have been 63 CGRs, and over 5370 attendees. To date, 95% of audience members report that the content of CGR is of value and would recommend it to others. Audience members surveyed report knowledge gains about the topics presented, satisfaction with venues and speakers, and support the continuation of the series. Despite having to move to a virtual format due to the COVID-19 pandemic, audience members continue to attend CGR and rate the program as valuable and something that they would recommend to others. CCs report commitment to the partnership and CGR. CGR served as a connector for audience members to COVID-19 resources such as grief support groups and financial assistance. Loyola University, an ITM partner, implemented the CGR model, with more planning to do the same. DISCUSSION/SIGNIFICANCE: CGR is effective at providing health information in a community setting. CGRs success is due to the commitment of its partners and its ability to respond to community needs. During the pandemic, attendance at each virtual CGR remained steady and audience feedback remained positive. CGR continues to serve as a connector to valuable resources.

OBJECTIVES/SPECIFIC AIMS: Consequently, we have decided to evaluate the presence of single nucleotide polymorphism (SNP) previously associated with AF on a European-descent population in an attempt to first identify the most common loci present in the PRH population and then search for specific PRH SNP associated with AF. METHODS/STUDY POPULATION: A secondary analysis of a Puerto Rican population sample (n = 120) from The Pharmacogenetics of Warfarin in Puerto Ricans Study will be performed. We will implement data from the 1000 genome project to establish a control group of healthy PRH population. Will evaluate the presence of 111 known single nucleotide polymorphisms associated with AF in Europeans and determine the frequency in PRH population sample, and validate predictability of such SNPs. Using admixture informatic markers (AIM) analysis will determine the percentage of admixture by Yoruba, Native American and Iberic-European. Statistical analysis will include the use of the Pearson Product-Moment Coefficient correlation analysis and multivariate linear regression. For admixture will use Maximum Likelihood Estimation and Markov Chain Monte Carlo models. RESULTS/ANTICIPATED RESULTS: A higher frequency of AF associated European single nucleotide polymorphisms, and an overall higher percentage of European admixture will be associated with atrial fibrillation in Puerto Rican Hispanic patients. DISCUSSION/SIGNIFICANCE OF IMPACT: Our contributions here are expected to be the elucidation of European ancestry as a risk factor for AF. These contributions will be significant because it can provide a robust scientific basis for larger GWAS studies in the Puerto Rican community and further narrow down the mechanism specific to this population. Research in this subject could lead to early identification of patients with high risk of developing atrial fibrillation and further decrease incidence and disease burden in the PRH population. Puerto Rican Hispanics have an exclusive genetic admixture that makes for an appealing research subject that could deliver unique results.

ABSTRACT IMPACT: Reducing methionine levels has repeatedly been shown to reduce cancer growth in vivo, while at the same time increasing lifespan in healthy animals. However, the mechanisms behind the beneficial effects of methionine restriction are currently unknown. OBJECTIVES/GOALS: We hypothesized that comparing the response of a cancer cell line to depletion of the amino acids methionine and cysteine would give us insight into the critical role of these two closely related amino acids in cancer, and help advance methionine restriction on the translational science spectrum. METHODS/STUDY POPULATION: We used the human melanoma cell line A101D to analyze the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion in vitro. We measured proliferation/viability, gene expression patterns, and glutathione levels. We also assessed ferroptotic versus apoptotic cell death. We then used a normal human fibroblast cell line to compare responses. RESULTS/ANTICIPATED RESULTS: The transcription factors ATF4 and NRF2 were activated by all three tested conditions in melanoma cells. Glutathione levels were decreased by ˜40% in cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Inhibiting ferroptotic cell death partially rescued proliferation in cysteine-depleted but not in methionine-depleted cells. Almost 70% of cells grown in methionine-depleted media stained positive for Annexin V, an indicator of apoptosis, compared to only 20% of cells grown in cysteine-depleted media. In normal cells, ferrostatin recued proliferation/viability to 86% of control levels in cysteine-depleted cells. Ferrostatin did not affect methionine-depleted normal cells. DISCUSSION/SIGNIFICANCE OF FINDINGS: These results indicate that methionine depletion leads to apoptosis, while cysteine depletion leads to ferroptosis. We found overlapping pathways activated by methionine and cysteine depletion at the gene expression levels, but divergences in cell death pathways ultimately activated.

OBJECTIVES/SPECIFIC AIMS: High-sensitivity diagnostics for early infant diagnosis (EID) of HIV at the point of care (POC) are not widely available. Lateral flow immunoassays (LFA) can detect HIV-p24, but are not sensitive enough in practice. With improvements, LFA are a compelling platform for POC in EID. We used functionalized magnetic beads and immunocomplex dissociation to improve sensitivity of HIV-p24 LFA. Here, we evaluate the utility for LFA to quantitatively report HIV-p24 concentration and estimate HIV viral load. Using purified p24 protein and virion constructs, we determined the limits of detection for HIV-p24 using LFA rapid tests. Using measurements from HIV-p24 ELISA, laboratory-developed RT-qPCR, droplet digital PCR, and gold standard clinical viral load, we further characterized the relationship between HIV-p24 concentration, HIV genomic RNA, and LFA test line signal. METHODS/STUDY POPULATION: We measured HIV-p24 concentration by ELISA (R&D Systems) and LFA (Alere Determine HIV-1/2 Ab/Ag Combo). An LFA reader instrument was used to image test lines and measure test line signal on the LFA. HIV viral loads were measured using RT-qPCR and droplet digital RT-PCR protocols adapted in our lab. We obtained gold standard viral load measurements using the Roche Cobas TaqMan system at Vanderbilt University Medical Center. Data analysis was performed using Prism 7 and Stata 14. RESULTS/ANTICIPATED RESULTS: LFA test line signal increases in a predictable, dose-dependent manner and correlates with concentration of purified HIV-p24 with a linear range between 50 and 1000 pg/mL (Spearman r=1; p=0.0004). We compared p24 concentration (ELISA). We evaluated the utility of LFA to quantify HIV-p24 from virions suspended in human plasma, which increased the limit of detection for HIV-p24 to 100 pg/mL and shifted the linear range 100–10,000 pg/mL (Spearman r=0.77; p<0.001). To evaluate the relationship between HIV-p24 concentration and concentration of HIV RNA, we employed 3 molecular techniques. The LFA is capable of detecting HIV-p24 concentrations that correspond to a range of viral loads between 653,000 and 1655 copies of viral RNA/mL. DISCUSSION/SIGNIFICANCE OF IMPACT: Our preliminary results are very promising, indicating that commercially available LFA can quantitatively measure HIV-p24 concentration to low levels. When coupled with our analysis of the relationship between HIV-p24 concentration and HIV RNA concentration, LFA may be a potential platform allowing us to estimate HIV viral burden at clinically relevant levels. Our next steps will be to evaluate this relationship in primary, clinical specimens in collaboration with the Tennessee Center for AIDS Research. We will incorporate technologies to improve the sensitivity of these LFA and evaluate their performance in field settings in Zambia. Our findings are broadly applicable for use in HIV care and treatment programs and early infant diagnosis programs around the world.