OBJECTIVES/GOALS: In the United States, oropharyngeal cancer (OPC) is the leading human papilloma-virus (HPV) related malignancy, and OPC rates are increasing among the US veteran population. The purpose of this study is to identify demographic and regional factors that may be associated with low HPV-vaccination rates among the US veteran population. METHODS/STUDY POPULATION: This study will use Veterans Health Administration (VHA) administrative data to create a national cross-sectional cohort of veterans ages 18-45 with a VHA primary care visit from 2018-2020. HPV-vaccination status of each subject (initiation of vaccination series, completion of vaccination regimen, and age vaccinated) will be determined. Differences in the prevalence of HPV-vaccination by smoking status, geographic location, socioeconomic status, race/ethnicity, and rural-urban context will be examined in the unadjusted analysis. Factors associated with low HPV-vaccination rates in the VHA will be identified using multivariable logistic regression to model no (vs any) HPV-vaccination, no completion (vs completion) of HPV-vaccination recommendations, and non-routine (vs routine) HPV-vaccination: RESULTS/ANTICIPATED RESULTS: In 2019, HPV-vaccination guidelines were expanded to include some adults between the ages of 26-45, making many young veterans in the VHA eligible for vaccination. From this study’s recently generated dataset, more than 1.2 million subjects (n=1,219,896) met the study inclusion criteria. Extrapolating from trends in the civilian population, it is anticipated that HPV-vaccination rates will be lower among African Americans compared to non-Hispanic Caucasian Americans, within the South Central and Southeastern regions of the US, and in rural communities. This study will define a veteran’s geographic location by their associated Veteran Affairs Integrated Service Network (or VISN), which are groups responsible for healthcare planning and resource allocation in particular regions of the US: DISCUSSION/SIGNIFICANCE: Identifying factors associated with low HPV-vaccination rates within the VHA will be the first step to reducing future incidence of HPV-related cancer burden among US veterans. Assessing geographic variability in vaccination rates by VISNs will create the potential to generate targeted interventions within an existing VHA framework.

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.

OBJECTIVES/GOALS: ICD-10 coding inconsistencies hinder timely recognition and treatment of metabolic syndrome (MetS), posing a significant risk for cardiometabolic disease progression. This study employed a digital phenotype for MetS and compared odds for medication and lifestyle intervention compared to those coded for MetS. METHODS/STUDY POPULATION: MetS is a cluster of cardiometabolic risk factors that increase risk for numerous adverse clinical outcomes. Patients with MetS were identified through electronic medical records on TriNetX LLC using the standard ICD-10 code or through a digital phenotype, involving grouping codes for the individual components. Percentage of patients with MetS not captured with the standard code was identified. In addition, disparities in blood pressure, glucose, lipid-lowering medication, and lifestyle intervention between the coding schemas were assessed, shedding light on healthcare inequities and informing targeted interventions. Odds ratios (RR) were presented for all outcomes. RESULTS/ANTICIPATED RESULTS: Patient demographics and lab values were similar between the standard code and digital phenotype cohorts. Of the 4.3 million individuals aged 50 to 80 identified as having MetS using the digital phenotype in the TriNetX research network, only 1.78% of participants shared the standard code. Individuals with the digital phenotype for MetS were at lower odds in receiving glucose lowering medication (OR: 2.11, 95% CI: 1.98–2.13, p <0.001) and exercise or nutrition-based intervention advice (OR: 1.76, 95% CI: 1.55–1.96, p <0.001) after controlling for demographics and lab values for each MetS component. DISCUSSION/SIGNIFICANCE: This project utilized TriNetX to create a digital phenotype for MetS, and suggests most patients are not coded for it using the standard ICD-10 system. This is troublesome given those with the standard code are less likely to receive certain interventions.

ABSTRACT IMPACT: Characterizing the cellular composition of human adipose tissue may contribute to the prevention and/or treatment of obesity-associated metabolic diseases. OBJECTIVES/GOALS: Our aims in this study were to use single-cell techniques 1. to characterize cell types within the stromavascular fraction of human adipose tissue, 2. to identify subsets of cells within each type (sub-clustering), 3. to identify gene sets and pathways that may provide information on the function and significance of each cell cluster. METHODS/STUDY POPULATION: Abdominal subcutaneous adipose tissue samples from n=6 healthy volunteers (1M, 5F, age 28-38 y, BMI 24.5-63.0 kg/m2) were collected by aspiration or during surgery. In 3 subjects, all females, paired femoral samples were also collected. After collagenase digestion approximately n=10,000 cells/sample were used for single-cell RNA sequencing using the 10X Genomics platform. After QC and downstream analysis, data were analyzed in Seurat v.3.1.5. We identified first different cell types and then subclusters in an unbiased fashion. Gene Set Enrichment Analysis (GSEA) was used for pathway analysis. RESULTS/ANTICIPATED RESULTS: Progenitor markers are related to extracellular matrix, eg DCN (logFC progenitors vs other cells types=3.17, expressed in 99.7% (pct.1) of progenitors, 26.1% (pct.2) of others). Endothelial and pericytes shared markers like RBP7 (logFC=1.67, pct.1 0.88, pct.2 0.39); pericytes also showed unique markers, eg RGS5 (logFC=2.29, pct.1 0.89, pct.2 0.17). Progenitors are further divided into 11 sub-clusters, one of which showed enrichment of CD36 (high proliferation potential), FABP4 (differentiation), and of the novel marker PALMD (logFC=7.13, pct.1 0.94, pct.2 0.48). All p<10E-5. GSEA analysis suggests that inflammatory pathways are downregulated in both adipose progenitors and endothelial/pericyte cells in the femoral compared to the abdominal depot. DISCUSSION/SIGNIFICANCE OF FINDINGS: Single cell RNA sequencing provides unique insights into the molecular profile of cell types and the identification of novel subsets of cells within the human adipose tissue. Such cellular heterogeneity may explain differences in adipose function between individuals and eventually in the risk of obesity-associated metabolic diseases.

OBJECTIVES/GOALS: Preeclampsia (PE) is a hypertensive disorder of pregnancy, affecting 5 - 7% of pregnancies worldwide. A major cause of morbidity and mortality, PE is also associated with subsequent adverse health outcomes, including long-term increased risk of cardiovascular disease. The genetics conferring increased risk for PE are incompletely understood. METHODS/STUDY POPULATION: We performed a cross-ancestry, fixed-effects meta-analysis, incorporating both published and unpublished genome-wide association study (GWAS) summary statistics. In addition to publicly available summary statistics from two prior studies, we generated GWAS data from three electronic health record biobanks (BioVU, eMERGE, and PMBB). In total, we utilized data from 359,378 individuals (4,411 cases and 354,967 controls). Leveraging this large-scale biobank data importantly allows for detection of complex factors contributing to the diverse etiology of PE. Cases across cohorts were defined using PE-specific ICD-9/ICD-10 codes and phecodes. Cohorts included pregnant individuals of self-identified non-Hispanic Black, non-Hispanic White, and East Asian ancestry. RESULTS/ANTICIPATED RESULTS: 2 of 20,204,625 loci achieved genome-wide significance (p < 5 × 10–8) when minor allele frequency was limited to common variants (>0.01). The most significant locus was rs138180605 (p = 1.77 × 10–8), located in an intergenic region between FGFR2 and ATE1, both previously associated with breast cancer. The other significant locus was rs137895377 (p = 2.33 × 10–8), located in an intronic region of PLEKHO1. Another 225 loci achieved suggestive significance (p < 1 × 10–5). 203 loci could be mapped to 109 unique genes, some previously associated with related phenotypes such as hypertension. Next steps will focus on functional analyses, including genetically predicted gene expression incorporating placental tissue, followed by construction of a PE polygenic risk score to demonstrate predictive utility of results. DISCUSSION/SIGNIFICANCE: This work has contributed to the limited body of knowledge surrounding maternal genetic susceptibility to PE by identifying several loci warranting further investigation. Further work will expand on these results to improve understanding of genetic factors and clarify clinical risk of disease.

OBJECTIVES/GOALS: HIV-specific CD8+ T-cells play a critical role in partially controlling viral replication in infected-individuals, but ultimately fail to eliminate infection. Enhancing these T-cell responses through lymphocyte engineering approaches has the potential as a novel therapy capable of achieving durable control or eradication of infection. METHODS/STUDY POPULATION: IL-15 Superagonist (IL-15SA) potently supports the in vivo persistence and antiviral activity of adoptively transferred CD8+ T-cells. The Deep-PrimingTM technology platform, developed by Torque, allows for loading of immunomodulators onto the surface of T-cells via electrostatic ‘nanogels’, which slowly release to deliver sustained autocrine immune stimulation without the harmful effects of systemic exposure. Here, we investigate the impact of IL-15SA Deep-Priming on HIV-specific CD8+ T-cells in a humanized mouse model of HIV infection. Humanized mice were generated by engrafting NOD-scid-IL2Rgnull mice with memory CD4+ T-cells isolated from an ARV-suppressed HIV+ donor. An autologous HIV-specific Cytotoxic T-Lymphocyte (CTL) clone was isolated, and killing potential confirmed. Four weeks post humanization, mice were infected with HIV and received an infusion of unmodified HIV-Specific CTLs, or IL-15SA Deep-Primed HIV-specific CTLs (CTL-DP). T-cell numbers and plasma viral loads were quantified weekly by flow cytometry and qRT-PCR. RESULTS/ANTICIPATED RESULTS: Mice receiving unmodified CTLs trended toward reduced viral loads compared to the No Treatment condition, while mice receiving CTL-DP saw significant, 2-Log10 reductions in VL (p < 0.01). At 41 days post-infection 100% (5/5) of the No Treatment, 66.7% (4/6) of the CTL treatment, and 16.7% (1/6) of CTL-DP treatment mice had detectable viremia. IL-15SA Deep-Priming increased CTL expansion and persistence in peripheral blood which correlated with improved CD4+T-cell preservation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we demonstrate the first in vivo analysis of IL-15SA Deep-Priming of HIV-Specific CTLs. These data suggest that Deep-Priming of patient T-cells can enhance in vivo function and persistence, leading to improved viral suppression; a significant advancement in the field of HIV cure research. CONFLICT OF INTEREST DESCRIPTION: Austin Boesch, Thomas Andresen, and Douglas Jones are employees of Torque. Darrell Irvine is a co-founder of Torque and Chairman of Torque’s Scientific Advisory Board.

Expanded access (EA) provides a pathway for the clinical use of investigational products (drugs, biologics, and medical devices) for patients who are without satisfactory therapeutic options and for whom a clinical trial is not available. Academic medical centers (AMCs) are likely to encounter EA requests, but it is unknown what support is available at these institutions for physicians seeking EA for patients. Methods: A landscape assessment was conducted at AMCs, focused on those within the Clinical and Translational Science Awards (CTSA) consortium. Results: Forty-seven responses were evaluated including 42 CTSA hubs. The large majority (43 of 47 respondents) reported using single-patient EA, while 37 reported multi-patient industry sponsored EA and 37 reported multi-patient investigator-initiated EA. Only half reported central tracking of EA requests. Support was available at 89% of sites for single-patient EA but less often for multi-patient EA. Types of support varied and were focused largely on the initial submission to the FDA. Conclusion: Use of and support for EA is widespread at AMCs, with support focused on single-patient requests. Gaps in support are common for activities after initial submission, such as FDA reporting and data collection.

We analyzed the publication productivity supported by the Puerto Rico Consortium for Clinical and Translational Research (PRCTRC) using the structured process of scientometrics. The objective of this study was to evaluate the impact of the research and collaborations as presented in publications. Manuscripts published from 2010 to 2018 and that had the PRCTRC award number and a PMCID number were retrieved from the Science Citation Index database. Scientometric indicators included h-index (HI), average citation (AC), collaboration coefficient (CC), collaboration index (CI), and degree of collaboration (DC) analysis, and relative citation ratio (RCR) was done with Web of Science Platform, iCite, and Stata software. Joinpoint Trend Analysis Software was used to calculate the annual percent change (APC). From 2010 to 2018, 341 publications were identified with an average of 38 publications per year and a total of 3569 citations excluding self-citations. A significant growth (APC: 17.76%, P < 0.05) of scientific production was observed. The overall HI was 31, and the AC per item was 11.04. The overall CC was 0.82, the CI was 8.59, and the DC was 99.1%. This study demonstrates a statistically significant increase in the PRCTRC scientific production. Results allow for the assessment of the progress resulting from the provided support and to plan further strategies accordingly.

OBJECTIVES/GOALS: A UCLA Clinical and Translational Science Institute (CTSI) science team partnered with the second largest US school district, with over 500,000 K-12 students, to design and implement a statistical process control dashboard to guide COVID-19 response, including mitigation and vaccination outreach. METHODS/STUDY POPULATION: District data for students, teachers, and staff are updated daily and include COVID-19 test results, counts of quarantine after positive tests, and COVID-19 vaccination rates. Displays used a new hybrid Shewhart control chart to detect changes in test positivity rates and distinguish meaningful signals from noise (random day-to-day variation). The dashboard uses the Shiny and plotly packages in R to display interactive graphs of each data stream (cases, tests, and vaccinations) charted at multiple levels (districtwide, subdistricts, schools). Displays of variation over time show policy impacts and inequities. Selected displays use municipal COVID-19 data to complement district data. RESULTS/ANTICIPATED RESULTS: The district has used the displays to assess the impact of their COVID-19 response and to identify variation in close to real-time to suggest areas with need for additional resources for mitigation or vaccination. The CTSI team has continued to edit and add displays in response to the district’s changing operational needs and questions. DISCUSSION/SIGNIFICANCE: The UCLA CTSI team developed and implemented a robust data visualization dashboard to monitor COVID-19 case rates and plan vaccination outreach efforts. Control charts enabled the district to distinguish noise from signal, thereby rapidly identifying when specific parts of the district needed targeted support to achieve equity goals.

OBJECTIVES/GOALS: The goal of this study is to leverage a national database to see if autologous reconstruction rates differ in patient and clinical characteristics, readmission rates, and overall survival (OS) compared to other forms of reconstruction. Autologous reconstruction has not been looked at in this way before. METHODS/STUDY POPULATION: • Aim 1: Use the National Cancer Data Base to construct three patient cohorts for women under 70 and above 18 treated surgically for breast cancer with A) mastectomy only, B) implant-based reconstruction, and C) autologous breast reconstruction. • Aim 2: Examine receipt rates of surgical intervention in Cohorts A vs. B vs. C based on clinical and patient demographic/socioeconomic characteristics. • Aim 3: Compare readmission and overall survival (OS) rates for Cohorts A vs. B vs. C while controlling for age and other key variables. RESULTS/ANTICIPATED RESULTS: Based on the literature, we expect rates of autologous reconstruction (Cohort C) to be lower for patients of minority backgrounds compared to white individuals. In addition, we do not expect overall survival to differ between implant-based (Cohort B) and Cohort C reconstruction. Still, we expect mastectomy-only (Cohort A) survival to vary from the two cohorts even when adjusting for different clinical factors, as similar but smaller studies have shown. Finally, we expect readmission rates to be higher for Cohort C, compared to Cohorts A & B, as it is a more complicated procedure typically done in academic institutions with skilled surgeons. DISCUSSION/SIGNIFICANCE: Autologous reconstruction is now considered the gold standard due to its ability to restore the breast shape with higher patient satisfaction and superior long-term outcomes. Multiple studies have documented ongoing racial disparities in post-mastectomy breast reconstruction and autologous reconstruction, with lower rates and referrals.

OBJECTIVES/SPECIFIC AIMS: To identify cardiac structural and function parameters, obtained on usual stroke-care TTE evaluation, associated with cardioembolic stroke (CE) in patients without AF. Hypothesis—left atrial (LA) size and valve dysfunction will be strongly associated with incident CE. METHODS/STUDY POPULATION: Inclusion criteria: July 1, 2013 to July 1, 2015 admission with imaging-confirmed ischemic stroke, no AF, TTE within 1st 7 days. TTE structure/function parameters were recorded. Stroke subtype (CE vs. other) defined using TOAST criteria, blinded to TTE. New AF definition: AF on ECG, telemetry or event monitor. CE/New AF outcome of interest in separate multivariable logistic regression models testing associations with TTE parameters (adjusting for demographics/vascular risk factors). RESULTS/ANTICIPATED RESULTS: Participants (n=332) were ~60 years hypertensive black males with moderate NIHSS and normal ejection fraction. In adjusted models, odds of CE increased with increasing LA systolic diameter (per 0.1 cm), mitral E point velocity(cm/s), mitral valve dysfunction, wall motion abnormality. New AF also associated with increasing LA systolic diameter. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings may suggest cardiac structural changes independent of AF that are on the CE causal pathway. Understanding the relationship between such TTE parameters and stroke subtype would impact clinical practice, as such TTE data is underutilized when considering stroke mechanism and management.

ABSTRACT IMPACT: Our improved understanding of the changes in chromatin accessibility that occur in persistent pain states may identify regulatory genomic elements that play essential roles in modulating gene expression in the DRG. OBJECTIVES/GOALS: Efforts to understand genetic variability involved in an individual’s susceptibility to persistent pain support a role for upstream regulation by epigenetic mechanisms. Our objective was to examine the transcriptomic and epigenetic basis of persistent pain following nerve injury. METHODS/STUDY POPULATION: We used a multiomic approach to identify novel molecular pathways associated with nerve injury-induced pain hypersensitivity. Adult Sprague Dawley rats were randomized to Chronic Constriction Injury (CCI) to the sciatic nerve or no treatment (naive). The ipsilateral L4-L6 dorsal root ganglia (DRG)s were removed on Day 14 and used for ChIP-seq for H3K4me1, ATAC-seq, and RNA-seq. We assessed for differential chromatin accessibility, transcription factor motifs, and enrichment for biological processes in chromatin accessible regions associated with cis-regulatory regions identified by ATAC-seq and H3K4me1 enrichment. Luciferase assays determined the functional significance of these sequences. RESULTS/ANTICIPATED RESULTS: We identified 58,446 genomic regions where H3K4me1 enrichment overlapped with chromatin accessibility. Differential analysis identified 2145 of these 58,446 regions that had changes in accessibility after CCI. The majority of these regions were located in introns or intergenic regions. Functional annotation of the differentially accessible regions identified disparate molecular functions enriched following nerve injury which suggests that altered chromatin structure plays a role in the development of mechanical hypersensitivity. Motif analysis identified specific transcription factor families whose binding sequences were enriched in regions of increased or decreased accessibility. Luciferase assays showed significant enhancement or repression of gene transcription. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data provides a comprehensive map of chromatin accessibility changes in the DRG after CCI and emphasizes the importance of chromatin structure in the development and maintenance of chronic pain.

OBJECTIVES/GOALS: Individuals with a family history of alcoholism (FH+) are more likely to develop an alcohol use disorder than those with no such history. Early life adversity has a high coincidence with FH+ making pathogenic studies difficult in clinical studies. Here, we developed a mouse model to study pathogenic mechanisms underlying these risk factors. METHODS/STUDY POPULATION: Male and female C57BL6/J mice were exposed to increasing concentrations of ethanol (3-21%) or water for 15 days prior to breeding. Ethanol was not present during gestation. Offspring were either removed from the home cage and isolated for 3 hours or left undisturbed from postnatal days 1-21. Beginning on PND 56 offspring mice were assessed for clinically relevant behavioral disruptions in social behavior, cognitive working memory, locomotor activity, anxiety-like phenotypes, ethanol preference and binge drinking behavior. In a separate experiment, brains of Cx3cr2+/GFPxCcr2+/RFP mice from ELA or control conditions were collected every 7 days after birth for assessment of neuroinflammation and central immune cell morphology and density. RESULTS/ANTICIPATED RESULTS: Mice with a family history of ethanol exposure and ELA are predicted to exhibit behavioral changes (impaired working memory, reduced social behavior, increased anxiety-like behaviors, increased ethanol consumption) to a greater extent than mice with a family history of ethanol exposure or ELA alone. We expect markers of neuroinflammation (cytokine expression, immune cell activation) to predict the behavioral changes in these mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Alcohol consumption and stressful life events are known environmental precipitants to neuroinflammation, which in turns may predispose individuals to anti-social and risky behavior. A mouse model of these early postnatal conditions will allow basic scientists to unravel the biological underpinnings of the behaviors driven by these factors.

OBJECTIVES/SPECIFIC AIMS: Hematopoietic stem and progenitor cells (HSPCs) function to maintain steady state production of new blood cells and to rapidly respond to blood cell loss. Little is known regarding how HSPCs develop the ability to sense and respond to blood cell loss during embryogenesis. Gaining insight into the robust ability of HSPCs to regenerate blood during early development may allow us to develop therapies to rejuvenate this capacity at any stage. METHODS/STUDY POPULATION: We generated a new hematopoietic-specific and inducible cell ablation zebrafish model to uncover the origins of regenerative capacity in HSPCs during development. These transgenic zebrafish express a cyan fluorescent protein (CFP)-nitroreductase (NTR) fusion construct under the control of the draculin (drl) promoter (drl:CFP-NTR), which restricts NTR expression to blood cells. Co-expression analyses of drl:CFP-NTR with known markers of other blood types including HSPCs (runx1+23:mCherry), erythroid cells (gata1:dsRed), and lymphoid cells (rag2:RFP), revealed drl:CFP-NTR was restricted to HSPCs and erythrocytes. To delineate the regeneration potential of embryonic HSPCs, we exposed drl:CFP-NTR transgenic zebrafish embryos to Metronidazole (MTZ), which results in selective ablation of only NTR-expressing blood cells. Embryos were treated from 2 to 3 days postfertilization and recovery of drl+ and gata1+ cells was evaluated over a 7-day recovery period. RESULTS/ANTICIPATED RESULTS: Following MTZ exposure, the nadir of drl+ cell ablation occurs at 2 days post MTZ (dpM) treatment. During the renewal phase of blood regeneration, we first observe recovery of drl+ cells by about 4 dpM, while more mature blood cells like gata1+ erythrocytes show a delayed recovery at about 6 dpM. Our results suggest that HSPCs can respond to injury as early as 2 days of life and that the HSPC-driven regeneration of embryonic blood cells occurs in a hierarchical fashion, similar to regeneration of the adult blood system. DISCUSSION/SIGNIFICANCE OF IMPACT: We have established a quantitative method for in vivo real-time monitoring of embryonic and larval blood regeneration. A significant advantage of our system is that we can use these insights to guide an in-vivo drug screen for factors that accelerate HSPC-driven blood regeneration in a complex biological environment.

OBJECTIVES/GOALS: For shoulder osteoarthritis (OA), the understanding of the patient-specific factors that determine success of both non-operative and operative treatment options is limited. This study aims to identify key factors associated with the response and the heterogeneity of outcomes for both types of treatment. METHODS/STUDY POPULATION: Patients diagnosed with shoulder OA and treated with either reverse/anatomic total shoulder arthroplasty (rTSA/TSA) or non-operative management at the University of California, San Francisco were enrolled in this study. They were followed for a year to ascertain phenotypic traits and patient-reported outcomes (PROs). Magnetic Resonance Imaging (MRI) was used to calculate the Shoulder Osteoarthritis Severity (SOAS) score, a semi-quantitative global assessment of shoulder OA, and to measure fat fractions of rotator cuff muscles. A Microsoft Kinect camera was used to determine the Reachable Workspace (RWS). Linear regression models were used to assess the associations between baseline demographic and radiographic factors on outcomes related to shoulder function. RESULTS/ANTICIPATED RESULTS: It is anticipated that the pre-operative MRI-based SOAS score will be inversely correlated with the magnitude of improvement in PROs 1 year after rTSA/TSA and non-operative management of shoulder OA for the surgical replacement and non-operative cohorts, respectively. Additionally, the non-operative patients who convert to rTSA/TSA within 1 year of observation will have higher SOAS scores compared to patients who continue with non-operative management. The surgical replacement patients with an infraspinatus fat fraction of more than 5% will have worse shoulder function, as measured by RWS, compared to patients with an infraspinatus fraction less than 5%. DISCUSSION/SIGNIFICANCE: MRI may be a novel technique to better predict prognosis of shoulder OA management. This will allow for the development of appropriate algorithms in the prescription of treatments and may be used to counsel patients regarding their expected outcomes or to recommend alternative treatments.

OBJECTIVES/GOALS: The primary objective is to measure the independent association of hospital-level and sociodemographic variables on the rate of hospital-onset bacteremia among infants admitted to the neonatal intensive care unit in a United States of America retrospective cohort. The secondary outcome will be relative blood culture collection rate. METHODS/STUDY POPULATION: The study is an analysis of a retrospective cohort comprised of infants admitted to 322 neonatal intensive care units (NICUs) in the United States of America between 2016-2021. The primary outcome will be hospital-onset bacteremia (HOB), defined as a positive blood culture with a bacteria or fungi after day 3 of admission. Independent risk factors will include birthweight, postnatal age, central venous catheter presence, sociodemographic variables (race, ethnicity, insurance status and ZIP code-level demographic data from the US Census American Community Survey (ACS), and hospital-level variables. Infants will be stratified by sociodemographic groups and a Poisson model will be utilized to measure the adjusted association between risk of HOB and clinical and hospital-level variables. RESULTS/ANTICIPATED RESULTS: I anticipate that infants in sociodemographic groups with a history of socioeconomic marginalization will have a higher unadjusted rate of HOB; however, sociodemographic variables will not be independently associated with HOB risk after adjusting for markers of hospital quality and acuity, such as quartiles of the following: mean admissions per year, percentage of infants born <1500g, annual blood culture contamination rate, and percentage infants born at another facility. DISCUSSION/SIGNIFICANCE: Neonatal bacteremia has high morbidity and mortality; however, its contribution to known infant mortality inequities is unknown. This study will estimate the burden of infant HOB stratified by sociodemographic groups and measure the independent association of sociodemographic and hospital-level variables on the adjusted rate of HOB.

ABSTRACT IMPACT: Understanding the biology underpinning head and neck cancer invasion and metastasis could lead to novel targeted therapies that are both effective and tolerable for patients with this debilitating disease. OBJECTIVES/GOALS: The bromodomain and extra-terminal (BET) family of epigenetic regulators has been implicated in the tumorigenesis of various cancers. In head and neck squamous cell carcinoma (HNSCC), the majority of morbidity is due to invasion and metastasis, so there is special interest in understanding the development of these phenotypes in HNSCC cells. METHODS/STUDY POPULATION: Stable SCC9 knockdown cell lines were generated by infecting cells with a lentivirus encoding Cas9-KRAB and a lentivirus encoding gRNA targeting each of the candidate BET proteins: BRD2, BRD3, BRD4, and BRDT. Knockdown was confirmed by qRT-PCR. Next, standard assays for proliferation (CellTiter-glo), invasion (Matrigel), and migration (scratch-wound healing assay) were performed for all candidate knockdowns and compared to a non-target control. RESULTS/ANTICIPATED RESULTS: Proliferation assay results revealed that BRD4 knockdown had a significant negative effect on the proliferative capacity of SCC9 cells in vitro. Similarly, BRD4 knockdown SCC9 cell lines were less invasive and less migratory. Interestingly, knockdown of BRD2, BRD3, and BRDT had no effect on proliferation, invasiveness, or migration. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have identified BRD4 as a key driver of the HNSCC tumorigenic phenotype. In the future, we plan to investigate the role of JQ1, a pan-BET inhibitor, on HNSCC cell phenotypes. Additionally, we will identify the downstream targets of BRD4, which may serve as potential therapeutic targets for both HNSCC as well as other cancers more broadly.

OBJECTIVES/SPECIFIC AIMS: Drug repositioning has the potential to accelerate translation of novel cancer chemotherapeutics from bench to bedside. The goal of this study was to determine the effects of ciclopirox olamine (CPX) on esophageal tumor cells. METHODS/STUDY POPULATION: We tested the effect of CPX on four esophageal cancer cell lines, assessing cell proliferation and viability by hexosaminidase and clonogenicity assay, respectively. We analyzed the effects of CPX on three-dimensional (3D) esophageal tumor cell spheroids. We also analyzed effects on cell cycle by flow cytometry. For mechanism, we performed western blots for proteins involved in cell cycle regulation, apoptosis and the Wnt/β-catenin pathway. For in vivo effects, we performed a murine xenograft model with intraperitoneal administration of CPX (100 mg/Kg body weight daily). RESULTS/ANTICIPATED RESULTS: CPX inhibited growth of all cell lines in a time and concentration-dependent manner. CPX also inhibited growth of esophageal spheroids. Cell cycle analysis demonstrated G0/G1 arrest in cells treated with CPX. Western blot analyses demonstrated decreased expression of cyclinD1, CDK4, CDK6, and transcriptionally active β-catenin, supporting the role of CPX in cell cycle inhibition and decreased β-catenin activity. Finally, treatment of nude mice with CPX significantly decreased tumor xenograft volume. DISCUSSION/SIGNIFICANCE OF IMPACT: CPX demonstrates anti-tumor properties in esophageal cancer cell lines. The current results justify further research into the mechanism of this inhibition. Additionally, given its established safety in humans, CPX is a potential candidate for repositioning as an adjunct treatment for esophageal cancer.