Objectives/Goals: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer (BC) with limited treatment options. Mortality rate is especially high in African American (AA) women of reproductive age. High levels of intracellular calcium (Ca2+) have been shown in TNBC cells. This study is to investigate Ca2+ channel blockers (CCBs) as therapy for TNBC. Methods/Study Population: Two human TNBC cell lines obtained from ATCC – HCC1806, and MDA-MB-453 are treated with CCBs, Cilnidipine (Cil), and Mibefradil (Mib), in a concentration- and time-dependent manner. Cell proliferation assays are performed by the MTS cell viability assay. Intracellular Ca2+ levels are measured using the fluorescent dye: Fluro 4-AM. Apoptosis is determined by flow cytometry using Annexin V staining and mitochondrial permeability will be assessed by the Mito JC-1 assay. Expression of Ca2+ signaling genes will be quantitated by real-time polymerase chain reaction (RT-PCR). Potential pathways of CCB efficacy will be identified by ingenuity pathway analysis (IPA). Results/Anticipated Results: Our findings show both CCBs decrease cell proliferation in a concentration- and time-dependent manner to a maximum of 80% vs. control in both TNBC cells. Flow cytometry findings on both TNBC cells treated with both drugs at 20 µM for 24 hours depicts late apoptosis. Interestingly, Mib did not change the intracellular Ca2+ level in HCC1806 cells yet decreased in MDA-MB-453 cells by fivefold, while Cil increased the intracellular Ca2+ level in both cells almost twofold. It is anticipated that Mito JC-1 assay depict decreased mitochondrial potential in both cells. For reverse transcription polymerase chain reaction, it is anticipated that CCB treatment will increase transient receptor potential Ca2+ channels and decrease voltage-gated Ca2+ channels in both cells. IPA analysis is expected to show apoptotic pathways are involved in TNBC via CCB treatment. Discussion/Significance of Impact: TNBC lacks the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Treatment options for TNBC remain severely limited. Our findings that both Cil and Mib can inhibit proliferation of human breast cancer cell lines indicate repurposing CCBs as treatment for TNBC warrants further investigation.

Objectives/Goals: NAD+ synthesis is enhanced in glioblastoma (GBM) allowing GBM to resist chemotherapy. NQO1 is upregulated in GBM and may be targeted by β-lapachone (β-lap) to induce NAD-depletion and cell death. This project investigates NQO1 as a selective target for GBM and the contributions of glucose and uridine to NAD+ synthesis. Methods/Study Population: Survival Studies and NQO1 expression. RNA-seq and survival data from TCGA of glioma patients (n = 667) was obtained using the UCSC Xena platform. Western blots were utilized to determine expression levels of NQO1 and NAMPT in normal human astrocytes, U87 cells, and patient-derived GBM cell lines. Immunocytochemistry: γ-H2AX staining was used to evaluate β-lap induced DNA damage. NQO1-dependence was evaluated with the NQO1-inhibitor dicoumarol. Cytotoxicity measurements. Cells were exposed to β-lap and other inhibitors, and cell survival was determined by trypan-blue exclusion assay. Co-culturing experiments were performed with fluorescently labeled U87 cells and unlabeled astrocytes. NAD+ quantification. Intracellular NAD+ was acid extracted and quantified by an enzyme-cycling reaction. Results/Anticipated Results: NQO1 overexpression is linked to decreased survival in glioma patients. In glioma patients, high NQO1 expression was associated with a decreased overall survival and high-grade tumors. β-lap induces selective NAD+ depletion and cell death in NQO1-expressing GBM cells. Western blots demonstrate NQO1 expression to be elevated in GBM cell lines compared to normal human astrocytes. β-lap induces NQO1-dependent NAD+ depletion and cell death in GBM compared to astrocytes in mono- and co-culture experiments. Glucose and uridine facilitate NAD+ regeneration in GBM. We demonstrate extracellular glucose and uridine facilitate NAD+ regeneration and cell survival in β-lap exposed GBM cells. Utilizing inhibitors, we determined that glucose and uridine facilitate NAD+ regeneration through the NAD+ salvage pathway. Discussion/Significance of Impact: GBM is the most common primary adult CNS tumor with a median survival of 14 months. Despite significant research in therapeutic strategies, treatment has not improved in 2 decades. There is a significant need to discover new targets that may improve GBM treatment. We demonstrate here that targeting NQO1 with β-lap induces selective GBM toxicity.

OBJECTIVES/GOALS: The goal of this project is to understand vaccine hesitancy and barriers to COVID-19 vaccine uptake among underserved communities in South Florida, to promote vaccine awareness, and to tailor education and communication based on the findings, in partnership with local community-based organizations. METHODS/STUDY POPULATION: By partnering with local community organizations, leaders, faith groups, and other trusted sources, the FL-CEAL teams will conduct a nationally developed CEAL survey in minority communities in South Florida to understand vaccine hesitancy and barriers, conduct outreach activities such as webinars and virtual townhalls to address hesitancy and promote vaccine uptake, and develop and disseminate educational materials and social media communications based on findings. The communities in South Florida include the Latinx farm working community, Latinx sexual/gender minorities, and Haitian and Black populations in South Florida. RESULTS/ANTICIPATED RESULTS: Early findings have indicated that hesitancy drivers vary among specific communities, while lack of trust is an overall barrier. Over 100 outreach activities have been conducted, including events with subject matter experts and community partners addressing vaccines. A credit-earning Community Health Worker (CHW) curriculum on COVID vaccine hesitancy was created in partnership with the Miami CTSI and the Florida CHW Coalition and has been delivered to over 200 Florida CHWs. The training focuses on evidence-informed best practices for CHWs to increase vaccine acceptance. At least 350 surveys will be conducted to understand beliefs and concerns around COVID-19 vaccines, and tailor education materials and communication strategies. DISCUSSION/SIGNIFICANCE: The FL-CEAL Alliance has successfully leveraged their ongoing state-wide effort at addressing COVID-19 disparities to focus on COVID vaccine hesitancy in minority communities and leveraged data and findings to inform community-specific approaches in addressing some of those barriers.

ABSTRACT IMPACT: This work will provide a rational approach to improve the efficacy of current immunotherapy approaches in patients that have historically responded poorly to immune checkpoint inhibitors. OBJECTIVES/GOALS: Recent evidence of immunogenic cell death as a predictor of response to therapy has increased the interest in monitoring the presence of damage-associated molecular pattern protein (DAMPs). By regulating DAMP expression, our lab is interested in discovering new ways to improve the patient response rate to immune checkpoint inhibition. METHODS/STUDY POPULATION: Using cultured cell, and a limited number of patient tumors and serum (n=4), we measured intracellular and extracellular levels of DAMP molecule, high mobility group box 1 (HMGB1) using enzyme-linked immunosorbent assays and immunoblots. Immunological assayed were compared to the expression of immune checkpoint molecules PD-1/PDL1 on patient tumors as presented in pathology reports. RESULTS/ANTICIPATED RESULTS: HMGB1 release was associated with increased levels of PD-L1 on tumor cells. Targeted inhibition of HMGB1 altered the expression of programmed death-ligand 1 (PD-L1), a target for immune checkpoint inhibition therapy. Patients with higher levels of PD-L1 possessed increased levels of HMGB1 in serum. DISCUSSION/SIGNIFICANCE OF FINDINGS: This implies that regulating the expression of HMGB1 could have an effect on the response of patients to immunotherapy. The main objective of the work is to determine the potential benefit of targeting HMGB1 to improve the efficacy of current therapeutic approaches to treating lung cancer.

OBJECTIVES/GOALS: It is estimated that 357,000 children have experienced a school shooting since 1999, yet due to limitations in the firearm violence field broadly, the sequalae are not well understood. The objective of this work is to examine the mental health impacts of school shootings, providing insight into the lasting effects of firearm violence on our communities. METHODS/STUDY POPULATION: We will first conduct a quasi-experimental study using controlled interrupted time series with repeated cross-sectional data to assess school shootings’ impact on US mental health. School shooting data is from the K-12 School Shooting Database, and mental health data will be collected via the Behavioral Risk Factor Surveillance System. Second, we will conduct focus groups with community organizations, school administrators, and the public. Interview guides will be developed to explore the mental health impacts of school shootings, to guide the quantitative results interpretation, and assess educational materials’ usefulness. Qualitative analysis will occur in NVivo software with codebook refinement through thematic analysis. Results will be triangulated through convergence coding. RESULTS/ANTICIPATED RESULTS: This research is situated within the context of the pervasive mental health challenges in the US, where mental illness poses significant health, social, and economic burdens. Thus, we anticipate finding an association between school shootings and decreased self-reported mental well-being among US adults. Literature suggests that there may be a stronger association among specific subgroups, such as parents with school-aged children or individuals living in close proximity to such incidents. We expect to find heterogeneity in the effect estimate based on school shooting attributes, such as the number of casualties. Through focus groups, we anticipate furthering our comprehension of the broad-ranging effects of school shootings on less quantifiable outcomes and the unique trajectories of recovery. DISCUSSION/SIGNIFICANCE: This project will contribute needed information on the impact of school shootings and mental health and assist in reducing the frequency and impact of school shootings. Furthermore, we aim to extend our findings beyond the scientific community, translating them into educational resources advocating for policy and public health interventions.

OBJECTIVES/SPECIFIC AIMS: Brown adipose tissue (BAT) increases energy expenditure by dissipating chemical energy as heat. The combustion of glucose and lipids produces beneficial metabolic effects and renders BAT an attractive target to battle obesity and associated diseases. The majority of adults do not display active BAT on positron emission tomography (PET) without prior cold exposure. Interestingly, a fraction of individuals with BAT positive PET scans exhibits excessive BAT (eBAT) activity, indicating a possible underlying genetic contributor. We aim to identify genetic determinants of BAT activity by studying individuals with eBAT activity using next-generation sequencing. A cellular model will be used to validate variants and perform in-depth pathway analysis. METHODS/STUDY POPULATION: We performed a retrospective review of PET scans over a period of 12 months in patients presenting with suspected or diagnosed cancer (n=20,348). The distribution of BAT positive individuals (n=1251) was used to implement a threshold to define eBAT activity. Samples from prospectively recruited individuals with BAT activity above the threshold will undergo whole exome sequencing. Variants associated with eBAT activity will be engineered into an immortalized BAT cell line using CRISPR to validate results and perform in-depth pathway analysis. RESULTS/ANTICIPATED RESULTS: We expect to identify genetic variants associated with eBAT. Studying the effects of these variants on thermogenesis followed by in-depth pathway analysis in genetically engineered cellular and mouse models may enable us to find new regulators of BAT activity. These findings may eventually contribute to the development of new drugs targeting obesity and its sequelae. DISCUSSION/SIGNIFICANCE OF IMPACT: The contribution of genetic factors to individual BAT activity is currently unknown. Identifying individuals with eBAT on PET scans and studying the underlying genetic determinants may provide the foundation for the discovery of new pathways for BAT activation.

ABSTRACT IMPACT: A mouse model of minimally-invasive chronic sleep deprivation is essential for elucidating the impact of sleep deprivation on various health issues, and it would lead to the possibility of sleep as a therapeutic target. OBJECTIVES/GOALS: The lack of sleep has been associated with various health conditions. In mice, sleep deprivation has been achieved mainly by physical disturbances, which raises concern about confounding effects by stresses. Without physical disturbance, targeted neuron ablation can address this methodological flaw. METHODS/STUDY POPULATION: AdultVgat-IRES-cre mice undergo a stereotaxic injection of adeno-associated virus (AAV) vector containing mCherry-dtA to bilateral parafacial zone (PZ) to perform GABAergic neuron-specific cell ablation. Control mice receive an injection of AAV vector containing hSyn-DIO-mCherry. All mice are implanted with electroencephalogram and electromyogram (EEG/EMG) electrodes for sleep-wake analysis. After 7-10 days of the postoperative recovery period, mice are kept individually in a cage for sleep-wake state recording. EEG/EMG and video recording are used to measure total wake time, total sleep time, percent of rapid eye movement (REM) and non-REM sleep, and detailed characterization with spectral analysis. RESULTS/ANTICIPATED RESULTS: We anticipate that the ablation of GABAergic neurons in bilateral PZ decreases the fraction of sleep state in mice, especially non-REM sleep. In the Vgat-IRES-cre mice that received the injection of AAV vector containing mCherry-dtA, total sleep time is expected to be decreased constantly during the 8-week observation period. Sleep-wake staging by video activity recording is anticipated to be closely correlated with the gold standard staging by EEG/EMG. Possible stresses caused by the restriction of physical activity and handling of mice for EEG/EMG recording can be further minimized by the sleep-wake staging performed with the video activity recording. DISCUSSION/SIGNIFICANCE OF FINDINGS: The lack of sleep has been associated with negatively affecting overall health and is implicated in major health conditions including obesity, diabetes, and cardiovascular diseases. This chronic sleep deprivation mouse model can be used to understand the mechanisms of such detrimental effects on health, and would improve many health conditions.

OBJECTIVES/GOALS: Children with medical complexity (CMC) experience frequent transitions of care (e.g., hospital to home) and are at increased risk for medication-related harm. This study aimed to identify transition-related medication safety barriers experienced by family caregivers, as they shoulder most of the caregiving responsibility following discharge. METHODS/STUDY POPULATION: We conducted semi-structured qualitative research interviews of 6 family caregivers and 10 healthcare professionals with roles assisting families during hospital discharge. Interviews focused on identifying key stages of the hospital-to-home transition period as well as medication-related tasks, decisions, and contexts. Transcribed audio interviews and research meeting notes were content analyzed to develop journey maps visually depicting key phases of the families experiences managing medication at home and their decision points and unmet needs. RESULTS/ANTICIPATED RESULTS: Journey mapping identified key decision points, medication management needs, and way points navigated by family caregivers during the hospital-to-home transition. Findings were salient for each phase of the family journey: 1) initial admission/intra-unit transfer; 2) in-patient care; 3) peri-discharge planning; 4) discharge; 5) immediate post discharge period (we termed post-discharge configuration); and 6) period of ongoing tasks and needs. Illustrative examples will be presented and discussed. DISCUSSION/SIGNIFICANCE: Family caregivers of CMC have needs that evolve throughout the medication use journey, suggesting a need for interventions that account for the time variant nature of this work. Findings lay a foundation for the next step of our study which aims to develop a prototype medication safety intervention that will be evaluated with family caregivers.

OBJECTIVES/GOALS: Septal cholinergic innervation to the hippocampus is critical for normal learning and memory and is severely degenerated in Alzheimer’s disease. To understand the molecular events underlying this loss, we optimized a primary septal-hippocampal co-culture system that facilitates study of central cholinergic synapses. METHODS/STUDY POPULATION: We developed an optimized in vitro septal-hippocampal co-culture system modified from previous published protocols. Briefly, hippocampal and septal tissue were harvested from embryonic day 19 (E19) Sprague-Dawley rats, digested with 0.1% trypsin, and an equal number of cells from each region plated onto coverslips coated with poly-D-lysine and laminin at a final density of 300 cells/mm2. We use immunostaining with validated primary antibodies and a fluorescent binding assay, together with confocal microscopy, to determine the structure of cholinergic synapses that are 1) native, 2) mammalian, 3) CNS derived, 4) comprised of physiological synaptic partners, and 5) developmentally mature. RESULTS/ANTICIPATED RESULTS: After DIV21, co-cultures maintained a healthy morphology. A subpopulation of neurons strongly expressed the cholinergic markers vesicular ACh transporter (vAChT), choline acetyltransferase (ChAT), and the high-affinity choline transporter (ChT1), whereas most neurons lacked vAChT expression and were presumably glutamatergic or GABAergic. The percentage of cholinergic neurons in the co-culture attained up to ~5-7%, depending on conditions such as embryo age at dissection or ratio of septal to hippocampal cells. We also report on cholinergic synapse structure by examining postsynaptic markers (excitatory and inhibitory) and staining for nicotinic acetylcholine receptor subunits. DISCUSSION/SIGNIFICANCE OF IMPACT: Primary septal-hippocampal co-cultured neurons have not been exploited extensively in the field, perhaps due to the difficulty in maintaining such cultures for extended periods. Here, we optimized an in vitro septal-hippocampal co-culture system, a powerful tool to comprehensively analyze central cholinergic synapse formation and dysfunction.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to evaluate a potential strategy to overcome tamoxifen (tam) resistance by using tam in combination with high-intensity focused ultrasound (HIFU). Tam is the most commonly used anti-cancer therapeutic agent in estrogen receptor positive (ER+) breast cancer (BC) which accounts for ~70% of BC cases. Tam treatment decreases a woman’s risk of recurrence by 50%; however, BC that is initially responsive to tam often develops resistance. METHODS/STUDY POPULATION: HIFU deposits acoustic energy locally to a cancerous region, which induces strong vibrations of molecules inside and outside of the cells. The resulting absorption causes rapid heating and mechanical disruption. This clinically relevant, noninvasive, and nonionizing physical force modality, has been shown to synergistically enhance chemical anticancer therapies. RESULTS/ANTICIPATED RESULTS: In this study we found that treatment of MCF7 cells with HIFU and tam has additive antiproliferative effects and mediates increased cell death. Additionally, we used tam resistant (TR) MCF7 cells that had been exposed to low-dose tam over time until they acquired resistance. When MCF7 TR are treated with tam there is no change in viability; however, treatment with HIFU in combination with tam decreased viability of both MCF7 and MCF7 TR to 19% and the viability of the cell lines was indistinguishable. We next evaluated the effect on MCF7 Y537S mutant ESR1, where ER is mutated to be constitutively active. Treatment of MCF7 Y537S had no significant decrease in viability of combination therapy compared with viability after HIFU alone. Analysis of ERalpha gene expression showed that HIFU treatment increased ERalpha expression in MCF7 TR cells, thus resensitizing these cells to tam and allowing these therapies to work synergistically. Our team developed a system to evaluate the potential of this combination of therapies in a patient-derived xenografts (PDX) model. PDX have emerged as a novel translational tool for cancer research with the potential to more accurately recapitulate the molecular and behavioral aspects of cancer. The WHIM20 PDX is a tamoxifen resistant tumor where the patient developed the Y537S mutation in ESR1. Ex vivo experiments on PDX tumor pieces demonstrated that combination therapy of HIFU and tam work synergistically to increase cell death of these tumors. Further, cryogenic-scanning electron microscopy was utilized to directly demonstrate the physical disruption to both cellular and tumor microenvironment post exposure to combination treatment. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies present a novel translational strategy to overcome tamoxifen resistance in ER+BC.

OBJECTIVES/SPECIFIC AIMS: Angiotensin type 1 receptor blockers (ARBs) and angiotensin-converting enzyme inhibitors (ACEIs) are frequently prescribed for hypertension and associated cardiovascular and renal complications. In animal models, these drugs also reduce anxiety and depression. OBJECTIVE—to determine if Veteran Affairs (VA) clinical pharmacy data indicate a protective effect of ARBs and/or ACEIs for major depression. METHODS/STUDY POPULATION: Pharmacy records from nationwide VA electronic medical records (EMR) were extracted for patients prescribed ARBs, ACEIs, α-blockers, β-blockers, calcium channel blockers, or diuretics (n=4,081,359). Patients were excluded if: they had not received medications for 6 months with >70% coverage; were diagnosed with substance/alcohol abuse, dementia, psychosis, schizophrenia, or prescribed insulin. The study population was categorized as “ARB/ACEI” (A/A) or “Never ARB/ACEI” (NA/A). Using the Greedy Matching Algorithm, subjects were matched on a 1:1 ratio for sex and race over a 5 year age range resulting in 2 equal groups of n=1,350,236 each. Subjects were older (M=71.6, SD=12) and mostly men (97%). RESULTS/ANTICIPATED RESULTS: In the A/A Versus NA/A, respectively, the incidence of anti-depressant use was greater during (9.9% vs. 8.9%) but was lower after (11.8% vs. 12.2%) the study period. PHQ-2 scores (Mean±SD) were statistically lower, albeit similar, during (0.79±1.56 vs. 0.85±1.63) and after (1.00±1.73 vs. 1.07±1.79) the study period. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary data suggest that inhibiting angiotensin II action does not provide a protective effect on major depression when compared with other classes of antihypertensive drugs. This study illustrates how “Big Data” may inform the design, or obviate the need, for large-scale randomized-controlled trials.

OBJECTIVES/GOALS: We are launching a multi-center prospective registry for patients with metastatic invasive lobular carcinoma (ILC), the second most common type of breast cancer, to better understand patterns of progression, imaging features of metastatic sites, and if serial cell free DNA measurements can serve as a surrogate marker of disease progression. METHODS/STUDY POPULATION: Patients with biopsy proven metastatic ILC of any receptor subtype will be included in the registry. We will exclude patients with ductal histology only or those with multiple primary malignancies. Patients will be enrolled at four large academic medical centers across the country. Cell free DNA measurements using a tumor informed assay will be obtained every 3 months concurrent with regular clinical imaging. Disease status will be determined by the patient’s medical oncologist by taking into account imaging, tumor markers, symptoms, and cell free DNA measurement. At each time point, patients will be surveyed on their quality of life and their medical oncologists will be asked to rate the clinical utility of the cell free DNA value. Patients will be followed indefinitely. RESULTS/ANTICIPATED RESULTS: We will explore whether the use of serial cell free DNA or a combination of blood-based biomarkers and clinical endpoints can reliably identify treatment response and disease progression in patients with metastatic ILC. Many patients with metastatic ILC have unmeasurable disease on imaging and are thereby excluded from clinical trials. The end goal of this registry is to determine if blood-based biomarkers can be used as a proxy for measurable disease in ILC patients and therefore increase clinical trial enrollment for this subgroup of patients. DISCUSSION/SIGNIFICANCE: The creation of this prospective registry will open the door for future studies of blood-based markers that reflect disease stability and progression, which is an unmet need specifically in ILC. Identification of such markers could lead to a novel treatment response endpoint, changing the way patients are enrolled in trials and managed clinically.

OBJECTIVES/GOALS: During the pandemic, alcohol related deaths increased by 25%. To help understand how we might mitigate this negative outcome, we sought to examine the association of new diagnosis of alcohol use disorder (AUD) with SARS-CoV2 through two years of the pandemic. METHODS/STUDY POPULATION: Using a non-date-shifted TriNetX database, we conducted a retrospective cohort analysis of electronic health records of patients age ≥12 years who had been diagnosed either with COVID-19 (n=1,359,817) or other respiratory infections with no record of COVID-19 (n=2,013,031). Patients were then matched for propensity score risk for AUD, and results were analyzed by three-month intervals from January 2020 through January 2022, in blocks numbered 1-8. Results were expressed as hazard ratios (HR) and 95% confidence intervals (CI) for diagnosis of AUD from two weeks to six months following COVID-19 diagnosis. RESULTS/ANTICIPATED RESULTS: There was significant excess risk compared to control cohorts of AUD following COVID-19 diagnoses made during the first three months of the pandemic (HR (CI)): block 1: 2.41(1.89,3.08); no excess risk was seen for the remainder of 2020 (blocks 2-4) (HR1.01-1.14, NS). The excess risk increased again in 2021 as the delta and omicron variants emerged (HR and 95% CI): block 5 were: 1.26(1.11, 1.43)); block 6: 1.88(1.62-2.18)); block 7: 1.24(1.10,1.41); block 8: 1.12(1.0-1.25). COVID-19 diagnosis was associated with clinically-evident AUD under some pandemic circumstances. DISCUSSION/SIGNIFICANCE: COVID-19 early in the pandemic (block 1) was associated with substantial excess risk for new diagnosis of AUD, with smaller excess risk after COVID-19 during 2021 (blocks 5-7), and no excess risk otherwise. Diagnosis of COVID-19 and pandemic contextual factors are associated with increased risk for AUD.

OBJECTIVES/GOALS: Vascularized composite allotransplantation (VCA) restores devastating soft tissue injuries. However, graft viability may be compromised during ischemia time, thus preservation techniques continue to evolve. Here we summarize our preliminary findings from preservation techniques utilizing hyperosmolar extracellular solution (HES) and electric stimulation in a 6 hour ex-vivo perfusion model. METHODS/STUDY POPULATION: A published, MGH rodent hindlimb ex-vivo perfusion model was utilized for this project. Three baseline control elements were taken to compare our results including: a baseline muscle biopsy, harvested hindlimb preserved on ice(4°C) in static cold storage (SCS), and 6 hour perfusion (SNMP) were used to compare results of our four aims. The four aims are shown in Table 1. HES was composed of muscle media and the addition of mannitol until 3 concentrations were made: 300, 500, and 800 mOsm concentrations. In aim 4, the perfusate composition was changed to test a hyper-oncotic purfusate. After 6 hours a muscle biopsy was taken to analyze energy cofactors via liquid chromatography-mass spectrometry, referred to as energy charge. Weight gain (edema), lactate levels, oxygen consumption and energy charge (EC) were used as markers for muscle tissue viability. RESULTS/ANTICIPATED RESULTS: In Aim 1, the higher osmolarity of HES indirectly reduced weight gain but consequentially reduced the EC below 5% when compared to SCS control group. We next incorporate HES into the perfusion model, Aim 2, and noticed a diminution in weight gain. The 500 mOsm group had substantial improvement in EC, lactate production and improved oxygen exchange when compared to a controls: fresh muscle biopsy and SNMP. In Aim 3, after a 6 hour perfusion with the addition of electric stimulation, graft edema improved by 10%, EC improved by 23% and O2 dissociation was highest of all 4 aims. Consequentially, due to muscular contraction the lactate levels were highest. In Aim 4, using a hyper-oncotic perfusate, edema reduced the most during the 6 hour perfusion but revealed lower EC and similar lactate/O2 results. However when left on for 24 hours, edema was significantly higher with lactate build up, EC improved with time as well as O2 dissociation. DISCUSSION/SIGNIFICANCE OF IMPACT: Here we have shown our preliminary results comparing our known ex-vivo perfusion model to multiple hypothesis to improve VCA graft viability. These preservation techniques demonstrate promising results but further studies are ongoing to confirm this encouraging outcome.

OBJECTIVES/GOALS: There is an excess risk of upper respiratory infection (URI) among Black and Mexican-American children in the US. Factors that underpin these disparities are largely unknown. We evaluated the extent to which socioeconomic status (SES), serum cotinine, obesity, and household size explained the association between race/ethnicity and URI. METHODS/STUDY POPULATION: We studied children, 6-17 years of age, who identified as Black, Mexican-American, or White in the National Health and Nutritional Examination Survey (2007-2012). URI was defined as a self-reported cough, cold, phlegm, runny nose, or other respiratory illness (excluding hay fever and allergies) in the past 7 days. The proportion of the association between race/ethnicity and URI explained by SES, serum cotinine, obesity, and household size was estimated as the average causal mediation effect (i.e., the indirect effect of race/ethnicity via the mediator) divided by the total effect of race/ethnicity. The average causal mediation effect was derived from survey weighted logistic regression models adjusted for age and sex. RESULTS/ANTICIPATED RESULTS: Black children were nearly 40% and Mexican American children were ~60% more likely to report a URI than those who identified as White (OR, 1.37; 95% CI, 1.06-1.77 and OR, 1.61; 95% CI, 1.21-2.13, respectively). Lower SES explained ~25% of the association between Black and Mexican American identity and URI (percent mediated 24.7; 95% CI, 23.0-26.6 and 26.1; 95% CI, 24.2-28.2, respectively). Obesity explained ~7% of the association between Black and Mexican-American identity and URI (percent mediated, 7.6; 95% CI, 7.3-8.0 and percent mediated, 6.7; 95% CI, 6.4-6.9, respectively). Nicotine exposure explained 8% of the association between Black identity and URI (percent mediated, 7.9; 95% CI, 5.6-10.1). DISCUSSION/SIGNIFICANCE: Lower SES explained a quarter of the association between race/ethnicity and URI. Low SES is a broad concept that may work through different mechanisms to lead to disparities in URI by race/ethnicity. Future research is needed to better understand these mechanisms and to identify modifiable aspects that can serve as targets for intervention.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this effort is to develop and distribute an easy to use i2b2 component that is capable of evaluating diverse complex relationships for a wide variety of exposures and outcomes over time. In this manner we are able to leverage the unique design of the i2b2 database to support health services research, comparative effectiveness, and quality improvement using a single tool. Furthermore, our novel database redesign has the potential to provide user-friendly access to individual and group CHC data for CER. METHODS/STUDY POPULATION: For this project we used software experts, clinical informatics specialists, and the existing i2b2 open-source software to convert our legacy HOME Cell into a web-client version. The tool will be used to study health outcomes within a network of Boston based Community Health Centers and the largest safety-net hospital in New England, Boston Medical Center. RESULTS/ANTICIPATED RESULTS: The new web-client HOME Cell will allow i2b2 users to model virtually any exposure (including therapeutic interventions such as medications or tests) in i2b2 against any outcome accounting for complex temporal relationships and other factors. In addition we plan to use our new Community Health Center views to enhance our community engagement activities by allowing direct access to their data for our partners. DISCUSSION/SIGNIFICANCE OF IMPACT: Our project addresses multiple national priorities related to data sharing, clinical research informatics, and comparative effectiveness. The web-client version of the HOME Cell substantially improves our community’s access to HOME Cell functionality and is a novel, sharable resource for use within the CTSA/NCATS community. Our approach provides a new way to perform large-scale collaborative research without the need to actually move patient-level data and has demonstrated that CER, health services research, and quality measurement can share a common framework. In addition, and as demonstrated in our earlier pilot work, the HOME Cell also has the potential to support large-scale multivariate analyses in a distributed manner that does not require sharing of patient-level data. We believe our approach has great promise for supporting the reuse of clinical data for rapid, transparent, health outcome assessments on a national scale. Our efforts support multiple strategic goals including: (1) support for building national clinical and translational research capacity by enhancing a broadly adopted informatics tool (i2b2); (2) enhanced consortium-wide collaborations by offering a tool that can be easily shared within the CTSA network to support multi-institutional collaboration; and (3) improving the health of our communities by offering a tool that has the potential to provide new insights into health care processes and outcomes that could drive innovation and improvement activities.

OBJECTIVES/SPECIFIC AIMS: While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received considerably less attention. Mac-1 is a heterodimeric beta-2 integrin primarily expressed by myeloid-lineage cells. In its activated state, Mac-1 mediates cell-cell interactions by engaging a variety of surface molecules, including the endothelium-expressed glycoprotein ICAM-1. Here, our goals were (1) to determine the extent to which APS neutrophils adhere to healthy, resting endothelial cells under physiologic flow conditions, and (2) to identify potential therapeutic targets by elucidating the molecules required for that adhesion. METHODS/STUDY POPULATION: Primary APS patients (meeting Sydney criteria) and non-autoimmune controls were matched for age and gender. Freshly isolated human umbilical vein endothelial cells (HUVECs) were utilized within five passages. Samples were introduced into a flow channel via a programmable syringe pump, and perfused across a resting HUVEC monolayer. After 15 minutes of perfusion, the chamber was flushed, and the remaining adherent cells were quantified. Flow cytometry was used to identify differentially-expressed molecules on the surface of APS neutrophils. Neutrophil extracellular trap (NET) release was assessed in static neutrophil-HUVEC cultures. RESULTS/ANTICIPATED RESULTS: Pre-treating control neutrophils with APS plasma resulted in increased adhesion as compared with control plasma (>2.5-fold for n = 12 plasma samples; p < 0.05). This was true under both venous conditions (low shear) and conditions representative of the microvasculature (pulsatile flow and higher shear). Control neutrophils treated with APS plasma demonstrated upregulation of CD64, CEACAM-1, beta-2 glycoprotein I, and activated Mac-1 on the neutrophil surface, as well as shedding of L-selectin. Upregulation of activated Mac-1 and shedding of L-selectin were also triggered by IgG purified from APS plasma. For these changes to be meaningful clinically, we reasoned that they should be present on neutrophils in the peripheral blood of APS patients. Indeed, perfusion of anticoagulated blood through the flow chamber resulted in increased adhesion of patient neutrophils as compared with controls (>5-fold for n = 18 patients; p < 0.05). Similarly, patient neutrophils demonstrated upregulation of CD64, CEACAM-1, beta-2 glycoprotein I, and activated Mac-1 on the neutrophil surface. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils to HUVECs in the flow-chamber assay (>2-fold reduction for n = 5 patients; p < 0.05). Importantly, the same monoclonal antibody reduced NET release in neutrophil-HUVEC co-cultures. DISCUSSION/SIGNIFICANCE OF IMPACT: APS neutrophils have an increased adhesive potential, which is dependent upon the activated form of Mac-1. This may lower the threshold for both neutrophil-endothelium engagement and NET release in patients, and thereby have implications for events such as venous thrombosis. Studies are underway to determine the extent to which Mac-1 is a viable therapeutic target in preclinical models of APS.

ABSTRACT IMPACT: We hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases. OBJECTIVES/GOALS: The role of IFNLR1 receptor dynamics and plasticity in regulating the type-III IFN response is largely unknown. As a specific, powerful component of innate immunity, understanding how the type-III IFN system is regulated could lead to the development of novel therapeutic targets and strategies to face a multitude of viral illnesses. METHODS/STUDY POPULATION: To facilitate our investigation, we will generate doxycycline-inducible FLAG-tagged IFNLR1-expression plasmids representing all known transcriptional variants. These plasmids will allow us to: 1) Evaluate the effect of IFNLR1 surface abundance on the type-III IFN transcriptional profile and 2) Assess the extent of IFNLR1-FLAG co-localization with several notable intracellular structures using immunofluorescence, before and after stimulation with IFNL3. RESULTS/ANTICIPATED RESULTS: We have successfully generated three IFNLR1-FLAG transcriptional variants and confirmed inducible-expression and function in vitro. We are currently assessing the role of surface abundance, internalization, differential isoform expression, and trafficking. DISCUSSION/SIGNIFICANCE OF FINDINGS: By completing this study, we hope to provide a more nuanced understanding of the type-III IFN system, thereby exploring its therapeutic potential in the realm of infectious diseases.

OBJECTIVES/SPECIFIC AIMS: To investigate the performance of a metric for passive localization of central sulcus. METHODS/STUDY POPULATION: We studied 7 patients with intractable epilepsy undergoing intra-cranial EEG (icEEG) monitoring at Yale, in whom central sulcus (CS) localization was obtained by standard methods. Our method takes advantage of inherent properties of the primary motor cortex (MC), which exhibits enhanced icEEG band-power and coherence across the CS. For each contact x we calculated the z-score of a composite power and synchrony value log10(px)x;cx, where px is sum of the root mean square of the icEEG in the high gamma band (80–115 Hz) for contact x over the 6–10 minutes of NREM sleep studied, and cx is the mean magnitude squared coherence in the same band using a 500-ms Hamming window between contact x and all other contacts. z-score values lower than threshold (th) were set to 0. Finally, we calculated a metric m=z/d, where d is the mean Euclidian distance of each contact from contacts with z scores greater than 0. The last step was implemented to emphasize local network activity. RESULTS/ANTICIPATED RESULTS: We report the results of a pilot study to test the performance of a new operator independent method for passive identification of CS with intractable epilepsy undergoing icEEG monitoring at Yale, in whom CS localization was obtained by standard methods. The sensori-motor (SM) cortex exhibited higher EEG-gamma power compared with non-SM cortex (p<0.0002). There was no significant difference between the motor/premotor and sensory cortex (p<0.47). CS was successfully localized in all patients with thresholds between 0.4 and 0.6. In 2 patients, knowledge of anatomy was needed to distinguish the MC from adjacent epileptic foci. The primary hand and leg motor areas exhibited the highest metric values consistently followed by the tongue motor area. Higher threshold values were very specific (94%) for the anterior bank of the CS but not sensitive. Intermediate threshold values achieved a reasonable trade-off (0.4: 89% specific and 70% sensitive). DISCUSSION/SIGNIFICANCE OF IMPACT: We present and successfully implement a rapid procedure for task-free and stimulation free localization of the central sulcus during sleep based on intrinsic electrophysiological properties of the primary motor strip which exhibits increased power and enhanced local connectivity. We successfully localized the central sulcus in all patients. When implementing appropriate thresholds, our proposed metric M is very specific for the anterior lip of the central sulcus which may make it ideal to identify this important anatomical landmark. Our method is sensitive for epileptogenic regions as well, therefore basic knowledge about central sulcus anatomy may be needed in cases where there is an epileptogenic lesion in the vicinity of the central suclus. Our method makes a few a priori assumptions: The regions around the central sulcus are adequately sampled and the occipital or parieto-occipital regions are not included in the analysis. In order for the method to function properly, nonsensori-MC should be sampled adequately as well. In the future, normative data could be generated for the composite product of connectivity×power which may replace within-patient z-scoring. Our method is rapid and can be implemented on short segments of ECoG data. The proposed method may be potentially used for identification of seeds in the motor cortex for subsequent network analysis and further studies may delineate its potential use in the operating room.

OBJECTIVES/SPECIFIC AIMS: Determine PTL’s mechanism(s) of action in a panel of canine hematopoietic cell lines; this will enable us to 1) verify that PTL is working as expected and 2) rationally select combination therapeutics. Characterize the in vitro sensitivity of canine hematopoietic cell lines to PTL in combination with other chemotherapeutic agents. Determine immunohistochemical NFκB expression in tissue microarrays of spontaneous canine neoplasms and correlate with outcome-linked data. Characterize the in vivo sensitivity of canine hematopoietic cell lines to PTL using a murine xenograft model. METHODS/STUDY POPULATION: Growth inhibition assays were performed using a panel of canine mast cell, histiocytic sarcoma, lymphoma, and leukemia cell lines, with PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence and immunoblotting were used to assess NFκB localization and phosphorylation of NFκB p65 (transcriptional activation), respectively. Intracellular glutathione with and without PTL and combination chemotherapeutics will be assessed spectrophotometrically. Archived spontaneous canine tumors will be evaluated immunohistochemically (IHC) for increased NFκB pathway activation relative to normal control tissues. Nude mice will receive intravenous, intraperitoneal, or subcutaneous injections of canine HS cells and will be treated with PTL or with PTL in combination with standard-of-care chemotherapeutics. RESULTS/ANTICIPATED RESULTS: Results: All immortalized canine cell lines evaluated are sensitive to PTL therapy and undergo dose-dependent apoptosis following exposure to drug. PTL exposure leads to inhibition of NFκB, as evidenced by immunofluorescent nuclear exclusion and decreased p65 phosphorylation. Some chemotherapeutics appear to synergize with PTL in vitro. Anticipated results: We expect to find increased IHC NFκB pathway activation in malignantly transformed tissues relative to controls. We expect standard-of-care therapeutics to synergize with PTL in vivo based on preliminary in vitro data. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will determine whether PTL therapy may be beneficial in dogs with a variety of hematopoietic neoplasms, either alone or in combination with other therapeutics that are currently in clinical use. Dogs with mast cell or histiocytic neoplasia are an excellent model for rare and deadly human diseases, which may also benefit from PTL therapy.