OBJECTIVES/GOALS: Metabolic syndrome (MetS), defined as a cluster of cardiometabolic disease risk factors, is seldom coded using the ICD-10 system in electronic medical records (EMR). The goal of this study was to use EMR to construct MetS with a digital phenotype to amplify the pool of patients available to assess risk for neurocognitive disease in this population. METHODS/STUDY POPULATION: A digital phenotype using the EMR platform TriNetX (n=38 million patients between age 50 and 80) was created by clustering codes for the individual components of MetS (insulin resistance, hypertension, dyslipidemia, and central adiposity). The research network database on TriNetX was utilized to elucidate risk ratios for neurocognitive decline, Alzheimer’s disease and related dementias (ADRDs), and cerebrovascular disease between a preclinical sample of older adults with and without MetS. Propensity score matching was used to match cohorts on demographic variables, medication use, and relevant comorbidities. Risk ratios (RR) and confidence intervals (95% CI) were presented for all outcomes. RESULTS/ANTICIPATED RESULTS: The digital phenotype for MetS expanded the sample from 29,830 to 274,703, a 10-fold increase. Sensitivity to the standard MetS ICD-10 code was 95.1%, showing strong agreement between coding schema. Older adults with MetS had higher risk of cognitive decline (RR: 1.30, 95% CI: 1.15–1.48, p <0.001), ADRDs (RR: 1.48, 95% CI: 1.25–1.75, p <0.001), and cerebrovascular issues (RR: 1.62, 95% CI: 1.55–1.70, p <0.001) when controlling for demographics, medication, and comorbidities. MetS individuals with cerebrovascular dysfunction had even greater risks for neurocognitive decline (RR: 1.70, 95% CI: 1.38–2.08, p < 0.001) and ADRDs (RR: 2.09, 95% CI: 1.56–2.80, p < 0.001) than those with only MetS. DISCUSSION/SIGNIFICANCE: Implementing a digital MetS phenotype in EMR effectively increased sample size and power for analyses. Older adults with MetS have higher risk for neurocognitive decline, especially among those with cerebrovascular dysfunction, highlighting a critical intervention window prior to overt cardiometabolic disease.

OBJECTIVES/GOALS: At least 15 stroke thrombectomies per proceduralist per year are required for Thrombectomy-Capable and Comprehensive Stroke Centers. We sought to estimate the potential impact of these minimum volume thresholds on geographic access to stroke thrombectomy (ST). METHODS/STUDY POPULATION: Using the Florida State Inpatient Database, we will identify patients discharged with acute ischemic stroke from 2015-2019 and all non-federal facilities that performed ≥1 ST procedure per year. We will then calculate the proportion of stroke patients who live within 20, 65, 115, 165 and 200 miles (correlating with estimated ground transport times of 1, 2, 3, 4 and 5 hours, respectively) of centers that perform ST using ArcGIS software and evaluate the impact of varying the threshold ST volume required by each facility on this proportion. We will then perform multiple two proportion z-tests to compare proportions of patients within driving distance over time. RESULTS/ANTICIPATED RESULTS: We hypothesize that over time, and particularly after the pivotal trials of ST were published in 2015, that the number of facilities that perform ST have increased over time, which would increase the geographic access to ST. We also hypothesize that since the Joint Commission set the minimum procedural volume for proceduralists at Thrombectomy-Capable and Comprehensive Stroke Center to 15 per year, this would work to increase regionalization and could work to decrease geographic access to ST. However, we hope to elucidate the net impact of the interplay between these two opposing factors on regionalization of care over time which is currently unclear. DISCUSSION/SIGNIFICANCE: Current ST volume thresholds have focused on technical proficiency but may impact regionalization of care and geographic access to ST. Since access to ST is time-sensitive, a data-driven approach and better coordination on a regional level may be necessary to ensure timely access to ST.

Objectives/Goals: We aim to establish a systematic approach to distinguish translational science from translational research. Our goal is to create a simple tool that would enable individuals with different backgrounds and levels of expertise to readily determine whether a study truly features translational science. Methods/Study Population: Participants were recruited from a Clinical and Translational Science Award (CTSA) program hub and randomly divided into 2 groups. One group was asked, with minimal guidance, to categorize whether publications described translational science or translational research. The group met to resolve disagreements and identify key indicators and challenges in determining whether a study involves translational science. They provided input on a set of guiding questions intended to facilitate the identification of translational science. The second group did not participate in discussion or tool development. Both groups reviewed a new set of publications, using the tool to guide their assessments. Results/Anticipated Results: Based on publication assessments, we will assess the percent agreement among reviewers in each group for each publication and across the set. We anticipate that the first group will exhibit higher agreement for its second round of review than its first, owing to the benefit of discussion with colleagues and provision of guiding questions. We anticipate that the tool will also promote higher agreement among the second group in their first round of review. We predict that both groups will exhibit high rates of agreement when reviewing with the support of guiding questions. Discussion/Significance of Impact: This study will help us understand interpretations of translational science, a term that has sparked debate and disagreement within CTSA hubs. If successful, the guiding questions will provide CTSAs a tool to improve training, proposal responsiveness, and review for translational science projects.

ABSTRACT IMPACT: Our data reveal a histone modifying enzyme involved in regulating inflammation that may be a novel target for treating non-healing diabetic wounds. OBJECTIVES/GOALS: We investigate molecular mechanisms that regulate the inflammatory phenotype of macrophages in normal and diabetic wound healing. Our goal is to identify novel pathways that may be used to better treat diabetic patients with non-healing wounds. METHODS/STUDY POPULATION: We utilize normal and transgenic murine models on standard chow or high-diet to identify chromatin modifying enzymes involved in regulating macrophage function during wound healing. We validate our murine studies with human blood monocytes or wound macrophages from diabetic patients undergoing limb amputation surgery. RESULTS/ANTICIPATED RESULTS: We have identified the histone methyltransferase SETDB2 as a regulator inflammation in normal and diabetic wound macrophages. We found that SETDB2 was dependent on IFNβ singaling and that both IFNβ and Setdb2 expression were impaired in diabetic wound macrophages. Further, we show that SETDB2 regulates inflammatory response and immune cell trafficking pathways. We also show that SETDB2 genomic localization is dependent on *NFκΒ deposition of the promoter. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results indicate that SETDB2 is a regulator of macrophage plasticity and that SETDB2 expression is impaired in diabetic wound macrophages leading to hyper-inflammatory response and delayed wound healing. These data provide a novel potential therapeutic pathway for treating non-healing diabetic wounds.

OBJECTIVES/SPECIFIC AIMS: Psoriasis is one of the most common inflammatory diseases of the skin, affecting about 2%–3% of the US population. Despite its high prevalence, its pathogenesis remains poorly understood. The ability of the microbiome to modify host immunity and metabolism suggests that it may contribute to the development of psoriasis and its cardiometabolic comorbidities. This study aims to characterize the psoriatic skin microbiome and understand the functional role that these bacteria may play. METHODS/STUDY POPULATION: 16s rRNA sequencing of site-matched skin swabs from 8 psoriasis patients and 8 healthy controls was used to identify bacteria and determine their relative abundance and microbial community diversity in the sample. PICRUSt was used to infer the functional roles of the bacteria from 16s rRNA amplicon data. RESULTS/ANTICIPATED RESULTS: Lesional psoriasis skin had lower α diversity (p=0.04), less Actinobacteria (p=0.0001), but higher Firmicutes (p=0.009) compared with controls. At the genus level, lesional skin had more Alloiococcus (p=0.01) and Aerococcus (p=0.01) and demonstrated a trend towards lower Propionibacterium (p=0.08) and higher Gallicola (p=0.09) compared to controls. Interestingly, Alloiococcus (p=0.003) and Gallicola (p=0.04) were also higher in nonlesional skin compared with controls. Furthermore, lesional and nonlesional skin shared an increased abundance of Acinetobacter sp., Staphylococcus pettenkoferi, and Streptococcus sp., relative to controls. Lesional and nonlesional psoriasis skin did not differ significantly in microbiome composition. Predictive functional analysis revealed that both the healthy and psoriatic skin microbiome were enriched with bacteria capable of amino acid and carbohydrate metabolism suggest these functions might have a general role in host-microbe interaction. DISCUSSION/SIGNIFICANCE OF IMPACT: These data reveal intriguing differences in the cutaneous microbiome of psoriatic individuals and healthy controls and suggest that bacterial metabolism may play an important role in host-microbe interaction.

OBJECTIVES/GOALS: A disease-agnostic translational science framework for data mining is proposed for use across disciplines to: Answer clinical questions, justify future clinical research recruitment, and explore under-represented populations. As a case example, male puberty demonstrates utility of the framework. METHODS/STUDY POPULATION: As a case example using the generalizable framework, the following interdisciplinary question was asked: Does early pubertal timing increase the risk of developing type II diabetes (T2d) in boys? A digital phenotype of males < 18 years old was created in the TriNetX Diamond Network utilizing Boolean operator data queries. TriNetX contains patient electronic health record information (ICD-10 diagnoses, anthropometric measures). A case control analysis leveraging patient counts from various digital phenotypes allowed for outcome (T2d) comparison of boys diagnosed with precocious puberty (E30.1, ICD code for early pubertal timing) to those without, while controlling for body mass index (BMI). RESULTS/ANTICIPATED RESULTS: Subjects (N=12,996,132) displayed the following digital phenotype: Male, < 18 years old, without ever having a BMI documented >85th percentile. Boys diagnosed with precocious puberty (E30.1) were 6.89 times more likely to develop T2d when aged 14-18 years old than those without (OR 6.89, 95%CI: 5.17-9.19, p. DISCUSSION/SIGNIFICANCE: Boys are under-represented in the early pubertal timing literature, justifying future human subjects research on male puberty. This case example demonstrates a broader disease-agnostic framework which can be adapted across disciplines. Opportunities may include public health digital phenotyping.

Objectives/Goals: We aim to understand how estradiol (E2) modulates proliferation and migration in distinct breast cancer subtypes within a collagen-based 3D platform that serves to better recapitulate human tissue architecture. This work seeks to bridge in vitro hormone biology with translational modeling of hormone-driven tumor behaviors. Methods/Study Population: MDA-MB-231 (triple-negative) and MCF-7 (hormone receptor-positive) breast cancer cells, tagged with red fluorescent protein, were exposed to varying E2 concentrations (0 to 10-4 M) in both 2D and 3D culture. In 2D, 2,000 cells/well were seeded in 96-well plates. Wells were imaged on Day 1 using confocal fluorescent microscopy, treated with E2, and reimaged on Day 5 to assess post-treatment counts. Normalized proliferation was calculated as Day 5/Day 1 cell count. In 3D, per well, 60,000 cells were embedded in a 12 µL base layer of 0.3% type I collagen, overlaid with 40 µL plain collagen, and topped with 150 µL E2-containing media. On Day 5, microscopy and a machine learning algorithm assessed cell counts and migration, defining migratory cells as those >1 standard deviation from the mean z-position of the tumor body. Results/Anticipated Results: In 2D, MCF-7 cells demonstrated significant dose-dependent proliferation to E2. Normalized cell counts increased progressively from 0 M to 10-10 M, at which point proliferation peaked, followed by a significant decline at higher concentrations. In contrast, MDA-MB-231 cells showed stable proliferation across the 0 to 10-6 M range, with a marked reduction in count at 10-4 M. In 3D, MDA-MB-231 cell proliferation remained stable across low and intermediate E2 doses, with a marked reduction at 10-4 M. MCF-7 cells, however, showed no significant differences in normalized proliferation across any E2 concentration in 3D. Quantitative migration analysis revealed that the percentage of migratory cells remained statistically unchanged across all E2 concentrations for both MCF-7 and MDA-MB-231 cells in the 3D matrix. Discussion/Significance of Impact: Dimensionality influences hormone responsiveness in breast cancer, highlighting limitations of 2D assays in modeling clinical behaviors and predicting outcomes. Advancing biomimetic 3D platforms like these may ultimately enable patient-specific screening of endocrine response and guide personalized therapy for hormone-sensitive cancers.

OBJECTIVES/SPECIFIC AIMS: Hepatitis C viral (HCV) infections are rising significantly both in young adults and as newly diagnosed cases in “baby boomers.” New HCV therapeutics cure over 95% of cases, and a call has been made for elimination of the epidemic by 2030; yet major HCV cascade of care (CoC) barriers exist. We secured CTSA pilot funding to obtain preliminary data for an innovative clinical trial utilizing big data modeling toward HCV elimination. METHODS/STUDY POPULATION: Our pilot work has developed a coordinated, real-time clinical data management process across 3 major CTSA affiliated hospital systems (MedStar Health, Emory-Grady, and UT-Southwestern), and additional data will be obtained from a pragmatic clinical trial. Electronic medical records data will be mapped to the OHDSI model, securely transmitted to Oak Ridge National Laboratory, Knoxville, TN and exposed to integrated data, analytics, modeling and simulation (IDAMS). RESULTS/ANTICIPATED RESULTS: Our U01 CTSA application proposes that HCV-IDAMS will model modifications to the established HCV CoC at community and population levels and thus simulate future outcomes. As data volume increases, system knowledge will expand and recursive applications of IDAMS will increase the accuracy of our models. This will reveal real-world reactions contingent upon population dynamics and composition, geographies, and local applications of the HCV CoC. DISCUSSION/SIGNIFICANCE OF IMPACT: Only an innovative, integrated approach harnessing pragmatic clinical data, big data and supercomputing power can create a realistic model toward HCV elimination.

OBJECTIVES/SPECIFIC AIMS: Objective: To summarize the diagnosis and management of two acute rejection (AR) episodes in the first penis transplant patient in the U.S. Background: Vascularized composite allotransplantation (VCA) has been utilized for state-of-the-art reconstruction of devastating craniofacial defects, limb loss, and recently, severe genitourinary defects. To date, more than 200 VCA’s have been performed, of which four successful penis transplants have been achieved worldwide (Two in the U.S.). However, despite the technical success of VCAs in general, acute rejection episodes remain a significant postoperative management problem, with 80-85% experiencing at least one episode in the first-year post-transplantation. The incorporation of skin in VCAs, which is highly immunogenic, allows early visible recognition of rejection but requires prompt management to prevent allograft failure as well as the progression of chronic rejection, which has been associated with the frequency of acute rejection episodes in preclinical models. We present the first report of acute rejection in a penile allograft. AR episodes in VCA typically manifest with erythema of the allograft skin and/or maculopapular lesions that can be either patchy, focal or diffuse. Histopathologic assessment is essential for diagnosis and management. The Banff classification of histopathologic criteria for degree of AR is most commonly utilized to direct clinical management. METHODS/STUDY POPULATION: We reviewed the clinical course of the first American patient who underwent penis transplantation at Massachusetts General Hospital in 2016. Postoperatively, routine clinical and chemical assessment (immunosuppression levels, routine blood work) was performed, with increased frequency during AR episodes. Skin punch biopsies were obtained during (suspected) AR episodes, analyzed and graded according to the Banff 2007 classification of rejection of skin-containing composite allografts. Histopathologic tissue assessment included CD3, C4d, CD4/8, CD20 FOXP3 and cellular infiltration (hyper keratinization, lymphocytic infiltrate, dermal erosion, macrophage, eosinophilia, T-cell infiltration) and epidermal or perivascular fibrosis. RESULTS/ANTICIPATED RESULTS: The patient is a 65-year-old male with history of penile carcinoma requiring subtotal penectomy in 2012. He is currently 30-months post penile transplantation (as of 11/15/2018). First Rejection Episode: At 28 days post-transplantation, the patient noted induration, swelling and erythema of the allograft, which was diagnosed as AR clinically (Image 1A). Biopsy showed a Banff Grade III AR, with focal keratinocyte apoptosis with lymphocytic infiltration in epidermis and arteriolar endothelialitis with perivascular inflammation. Initially this episode was treated for 2 days with 2 pulse doses of methylprednisolone (500mg/d IV) with clinical improvement. However, recurrent allograft erythema was observed on postoperative day 32 and an acute rejection grade III according the Banff classification was confirmed by a second biopsy that demonstrated epidermal perivascular lymphocytic infiltrates, spongiosis and dyskeratosis, deep dermis focal lymphocytic infiltrates and focal infiltrates in arterioles as well as endothelialitis in venules. Donor specific antibodies and C4d were negative. CD3+ T cells were present in the epidermis and perivascular space. This was treated with anti-thymocyte globulin (thymoglobulin) course for 4 days (1.5mg/kg/day IV) and 3 more pulse doses of methylprednisolone (500mg/d IV.), followed by a prednisone (250mg/d) taper to baseline. This resulted in complete resolution of AR. Second Rejection Episode: At 10.8 months post VCA the patient presented with penile erythema and scrotal swelling suggestive of AR and received three doses of methylprednisolone (day 1: 500mg/d IV, day 2: 1000mg/d IV and day 3: 500mg/d IV respectively) followed by increased baseline prednisone (10mg PO daily; increased dose compared to previous AR episode). A skin biopsy confirmed Banff Grade III AR. Compared to the previous biopsy, this biopsy demonstrated an increased density of lymphocytic inflammation of the dermis with endarteritis. Prominent involvement of epidermis and adnexal structures corresponding to acute T-cell mediated rejection was also observed (Figure 1). Donor specific antibodies and C4d were again negative. Three doses of ATG (1.5mg/kg/day IV) were administered. In addition, tacrolimus was increased and local tacrolimus (1% ointment) treatment was begun. Clinical signs of rejection improved and repeat biopsy showed dramatic histopathological improvement. Current maintenance immunosuppressive regimen consists of tacrolimus, sirolimus, prednisone, mycophenolic mofetil acid (MMF), rapamycin, and tacrolimus ointment, with no new clinical or histopathological signs of rejection (Image 1B). DISCUSSION/SIGNIFICANCE OF IMPACT: We report the first described case of acute T-cell mediated rejection in penile transplantation. These rejection episodes demonstrated that, even on stringent immunosuppressive regimens, severe acute rejection episodes in VCA may still occur. Edema and acute induration preceded the development of erythema in our cases, representing a harbinger for the more severe grade of rejection that eventually developed. Our experience was consistent with other VCAs in that donor specific antibodies did not develop, despite a severe Banff Grade. Consistent use of topical calcineurin inhibitor based immunosuppression on the allograft skin may be helpful in warding off future episodes, as our patient has been rejection free now for 18 months. To date, no histologic signs of chronic rejection were present on 2-year protocol surveillance biopsy. We have added rapamycin to the current drug regimen, with concurrent reduction of tacrolimus dosing for renal protection, which has been demonstrated in cardiac transplantation to deter the intimal hyperplasia/vasculopathy associated with chronic rejection.

OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the amygdala in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecular PSD95-nNOS inhibitor, co-immunoprecipitation, Western blotting, and RNA-sequencing. RESULTS/ANTICIPATED RESULTS: We show that fear conditioning enhances the PSD95-nNOS interaction and that the small-molecule ZL006 inhibits this interaction. Treatment with ZL006 also attenuates rodent cued-fear consolidation and prevents fear-mediated shifts in glutamatergic receptor and current densities in the basolateral amygdala (BLA). With RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored by systemic ZL006 treatment. Network data and gene ontology enrichment analysis with Ingenuity Pathway Analysis and DAVID software found that cell-cell interaction, cognition-related pathways, and insulin-like growth factor binding were significantly altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results reveal novel genetic targets that underlie plasticity of fear-memory circuitry via their contribution of NMDAR-mediated fear consolidation and can inform future strategies for targeting fear related disorders like PTSD. CONFLICT OF INTEREST DESCRIPTION: Anantha Shekhar and Yvonne Lai are co-founders of Anagin, Inc., which is developing some of the related molecules for the treatment of PTSD.

ABSTRACT IMPACT: Gaining a better understanding on the role of opioids in opioid use disorder (OUD) can help us find better diagnostics, treatments, and procedures to treat the disorder. OBJECTIVES/GOALS: While we are familiar with brain areas and pathways that are implicated in opioid use disorder (OUD), we do not have a full understanding of the neural circuits activated upon drug exposure. METHODS/STUDY POPULATION: In order to identify areas of the brain most activated by opioids, we ran a pilot study using transgenic cFos-GFP mice that were injected with saline or heroin and examined the brain-wide activity patterns using a quantitative high-resolution mapping method. We observed many brain regions highly activated upon drug exposure. To examine cFos based brain activation in rats, we also ran a pilot study using a tissue clearing and 3D immunolabeling method combined with light sheet microscopy. RESULTS/ANTICIPATED RESULTS: We would expect to see higher cFos activation for brain areas in the reward pathway [including the Nucleus Accumbens (NAc), Ventral Tegmental Area (VTA), Prefrontal Cortex (PFC)] in heroin animals compared to saline animals. We can also expect higher activation in more novel areas like the lateral hypothalamus. DISCUSSION/SIGNIFICANCE OF FINDINGS: If we are able to track OUD effects through imaging in mice and rats, this can help us find better diagnostics, therapeutics, and procedures to treat the disorder. We can also eventually have a human brain atlas that outlines these affected areas as well in order to gain a better understanding on OUD particularly in the human population.

OBJECTIVES/GOALS: To determine the signs, symptoms, and diagnoses that are significantly upregulated in cases of long COVID while identifying risk factors and demographics that increase one’s likelihood of developing long COVID. METHODS/STUDY POPULATION: This is a retrospective, big data science study. Data from Veterans Affairs (VA) medical centers across the United States between the start of 2020 and the end of 2022 were utilized. Our cohort consists of 316,782 individuals with positive COVID-19 tests recorded in the VA EHR with a history of ICD10-CM diagnosis codes in the record for case-control comparison. We looked at all new diagnoses that were not present in the six months before COVID diagnosis but were present in the time period from one month after COVID through seven months after. We determined which were significantly enriched and calculated odds ratios for each, organized by long COVID subtypes by medical specialty / affected organ system. Demographic analyses were also performed for long COVID patients and patients without any new long COVID ICD10-CM codes. RESULTS/ANTICIPATED RESULTS: This profile shows disorders that are highly upregulated in the post-COVID population and provides strong evidence for a broad definition of long COVID. By breaking this into subtypes by medical specialty, we define cardiac long COVID, neurological long COVID, pulmonary long COVID, and eight others. The long COVID cohort was older with more comorbidities than their non-long COVID counterparts. We also noted any differences regarding sex, race, ethnicity, severity of acute COVID-19 symptoms, vaccination status, as well as some analysis regarding medications taken. DISCUSSION/SIGNIFICANCE: This profile can be utilized to decisively define long COVID as a clinical diagnosis and will lead to consistence in future research. Elucidating an actionable model for long COVID will help clinicians identify those in their care that may be experiencing long COVID, allowing them to be admitted into more intensive monitoring and treatment programs.

Objectives/Goals: Methicillin-resistant Staphylococcus aureus is a nefarious bacterial pathogen and is classified as a serious threat. MRSA is resistant to most B-lactam antibiotics (penicillins, cephalosporins, etc.). The goal of this study is to identify an antibiotic adjuvant capable of resurrecting B-lactam antibiotics for treatment of MRSA infections. Methods/Study Population: A fluorescence-reporter assay was used to screen a compound library. Minimum-inhibitory concentrations were assessed for the compounds against various MSSA and MRSA strains. A common resistance mechanism to B-lactams by MRSA is by the function of the bla operon. One gene in this operon encodes for a B-lactam sensor/signal transducer protein BlaR, the primary target of this study. Inhibition of BlaR by compound 1 (best potentiator of oxacillin) was studied by nano-differential scanning fluorimetry (nanoDSF), surface plasmon resonance (SPR), scanning electron microscopy (SEM), and in vivo assays. Results/Anticipated Results: We identified 80 compound hits from a 1,974-compound NCI library. Twenty-four compounds showed potentiating ability (2- to 4,096-fold decrease in MIC for oxacillin). Seven compounds exhibited melting temperature shifts by nanoDSF of BlaR, indicating binding. SPR determined compound 1 has a binding affinity of 31 mM to BlaR-SD. SEM images showed disruption in the S. aureuscell wall on exposure to compound 1and oxacillin. S. aureusN315 showed 3-log reduction in bacterial count treated with a mixture of compound 1 and oxacillin. Discussion/Significance of Impact: Compound 1 targets BlaR-SD, which restores S. aureussusceptibility to treatment by oxacillin. There are currently few antibiotics available in the clinic capable of treating MRSA infections. The combination hold promise of a treatment option for MRSA.

Objectives/Goals: Our lab’s novel adoptive cellular therapy (ACT) significantly improves survival in brain tumor models. However, there is a lack of biomarkers to assess immunotherapy responses. Our objective is to use gold nanorods to track hematopoietic stem cell migration, a critical arm of ACT, and validate it as a prognostic biomarker. Methods/Study Population: Hematopoietic stem cells (HSCs) were isolated from the bone marrow of 6-week-old C57BL/6J mice and co-cultured with varying gold nanorod (GNR) concentrations and time points. GNR uptake in HSCs was evaluated with inductive coupled plasma mass spectrometry, two-photon luminescence, and tissue histology. After GNR co-culture, HSC viability and differentiation were quantified with flow cytometry and colony forming unit assays. To evaluate the impact of GNRs on HSC reconstitution, mice received myeloablative total body irradiation and intravenously received GNR-labeled HSCs. Computed tomography (CT) contrast of GNRs will be confirmed through microCT. Lastly, mice will intracranially receive KR158b glioma and GNR-labeled HSC bio-distribution will be measured after ACT and correlated with survival outcomes. Results/Anticipated Results: We have demonstrated that GNRs are readily taken up by HSCs within 30 minutes, and retained within intracellular compartments, via TPL. Incubation of GNRs with HSCs did not significantly alter cell viability or differentiation, supporting the GNR’s favorable biosafety profile. Colony-forming unit assays revealed that GNR incubation did not significantly disrupt the total number of colonies formed and qualitatively, colonies did not demonstrate significant lineage differences. GNR-labeled HSCs demonstrated significant reconstitution after myeloablative total body irradiation in mice. We expect that GNR-labeled HSCs will distribute to the glioma microenvironment and draining lymph nodes, positively correlating with long-term survival after ACT. Discussion/Significance of Impact: GNRs harbored high biosafety and feasibility for tracking HSC migration after ACT. We seek to translate this theranostic tool into the current first-in-human clinical trials at our institution for patients diagnosed with neuroblastoma and diffuse intrinsic pontine glioma to improve immunotherapies against brain malignancies.

OBJECTIVES/GOALS: A major barrier to older adult (65+) over-the-counter (OTC) safety is the lack of actionable research on factors affecting older adult decisions during OTC selection. This goal of this study is to develop and validate a survey instrument based on the Protection Motivation Theory to measure older adults perceived risk of OTCs. METHODS/STUDY POPULATION: The 24-item OTC Protection Motivation survey underwent revision using a consult from the UW Survey Center as well as rigorous cognitive interviewing and pilot testing with 8 older adults. It was administered to 103 community-dwelling older adults. OTC misuse data was gathered from 15 of the participants. OTC misuse evaluated by three pharmacy experts on the basis of OTC appropriateness given participants age, existing conditions, concurrent medication use, and intention to adhere to label guidelines. Multivariate linear regression was used to examine the relationship between OTC misuse and protection motivation. Exploratory factor analysis (EFA) was used to determine a model of best fit to describe the factor structure of the OTC Protection Motivation survey. RESULTS/ANTICIPATED RESULTS: The EFA yielded a 5-factor model of protection motivation, which included the components of deliberative risk perception, a combination of experiential and affective risk perception, threat severity, protective behavior intent, and perceived efficacy in protective behavior engagement. The EFA-based item reduction resulted in a final 18-item OTC Protection Motivation survey. DISCUSSION/SIGNIFICANCE: Older adults, who have the lowest health literacy levels, are responsible for 30% of OTC medication use and 61.5% of emergency department visits due to adverse drug events. This study is the first to characterize the relationship of OTC protection motivation and OTC misuse to inform patient-centered interventions for older adult OTC safety.

OBJECTIVES/SPECIFIC AIMS: Despite significant advances in screening and treatment, colorectal cancer is the second leading cancer killer in the United States today. Some of the most promising recent developments in cancer therapy have come from immune-based therapy. Immune-based therapy, however, has shown limited utility in patients with colorectal cancer. Studies have previously shown that certain chemotherapy regimens may be more effective in combination with immune-based therapy due to induction of inflammation in the tumor microenvironment. In this study, we sought to determine how standard chemotherapy (FOLFOX) affects the generation of antigen-specific anti-tumor immunity in colorectal cancer. METHODS/STUDY POPULATION: To determine the how antigen-specific immunity and T cell responses are affected by FOLFOX, we utilized a model antigen expressing murine colon cancer cell line syngeneic to C57BL/6 (MC38-CEA). Treatment was initiated when tumor size reached 50 mm2. Mice were treated with either vehicle (PBS), 5-Fluorouracil (5-FU), Oxaliplatin, or combination (FOLFOX). Antigen-specific cytotoxic T cell (tet+Tc) were detected using Db-CEA-tetramer obtained from the NIH-tetramer core facility. Flow cytometry was performed for phenotypic analysis and tetramer positivity. Tumor growth was measured using standard caliper measurements. Statistical analysis was performed using t-test for continuous variables and ANOVA was used when comparing multiple groups. Statistical analysis was performed using SPSS. All arms were completed with n=3–7. RESULTS/ANTICIPATED RESULTS: To determine how systemic treatment with chemotherapy affects cytotoxic T cell development (Tc), we established that we could detect antigen-specific Tc (tet+Tc) in the spleen, tumor, and draining lymph nodes of tumor-bearing mice. After establishing that the system worked appropriately, tumor-bearing mice were treated with different chemotherapy regimens and tumor growth was monitored. As expected, the combination of FOLFOX was significantly better than either drug individually (2-way ANOVA, p<0.01). FOLFOX therapy also showed a significant (p<0.05) increase in the number of tumor-associated tet+Tc, and tet+Tc expressing phenotypic markers of effector (Te) and resident memory (Trm) subsets. Tumor-associated tet+Tc highly expressed PD-1 (>50%); however, this was not significantly different between treatment or vehicle arms. Since 5-FU, one component of FOLFOX has previously shown a selective reduction of myeloid-derived suppressor cells, we also investigated the myeloid compartment. There were no significant differences in conventional or plasmacytoid dendritic cells, myeloid-derived suppressor cells, or tumor-associated macrophages. DISCUSSION/SIGNIFICANCE OF IMPACT: The future of cancer care involves multi-modality care tailored to patients. To more effectively combine therapy it is critical that we understand how currently utilized therapy works. In this study, we show that the primary chemotherapy regimen utilized in colorectal cancer increases tumor-associated antigen-specific cytotoxic T cells and the majority of these cells are PD-1 positive. This suggests that FOLFOX may work in concert with immune-based therapy when selected appropriately. Further study is warranted to determine optimal combination therapy and ways to maximize anti-tumor immunity in order to improve the treatment of patients with this deadly disease.

The consent process for research studies can be burdensome for potential participants due to complex information and lengthy consent forms. This pragmatic study aimed to improve the consent experience and evaluate its impact on participant decision making, study knowledge, and satisfaction with the In Our DNA SC program, a population-based genomic screening initiative. We compared two consent procedures: standard consent (SC) involving a PDF document and enhanced consent (EC) incorporating a pictograph and true or false questions. Decision-making control, study knowledge, satisfaction, and time to consent were assessed. We analyzed data for 109 individuals who completed the SC and 96 who completed the EC. Results indicated strong decision-making control and high levels of knowledge and satisfaction in both groups. While no significant differences were found between the two groups, the EC experience took longer for participants to complete. Future modifications include incorporating video modules and launching a Spanish version of the consent experience. Overall, this study contributes to the growing literature on consent improvements and highlights the need to assess salient components and explore participant preferences for receiving consent information.