OBJECTIVES/GOALS: Aim 1: We will determine whether temporal changes in the fecal microbiome signature correlate with a clinical multiple system atrophy (MSA) phenotype. Aim 2: We will evaluate whether secretomes cultured from fecal samples from MSA patients enhance intracellular and extracellular α-synuclein (αSyn) aggregation using in vitro functional assays. METHODS/STUDY POPULATION: Aim 1: Gut microbiome profiling will be performed by 16S rRNA gene sequencing, tandem mass spectrometry for expression proteomics, and targeted metabolomics in fecal samples from 30 MSA cases matched to 30 healthy controls, a Parkinson’s disease comparison group, and household controls. Aim 2: Microbial species will be isolated using dilution-to-extinction on MSA fecal samples and then will be cultured to obtain secretomes. To assess the effect of MSA fecal secretomes on αSyn aggregation, culture media from microbial isolates will be used in fluorescence resonance energy transfer (FRET) assays and luciferase reporter assays, both modified to measure αSyn aggregation. Positive tests will undergo expanded metagenomic characterization of the microbes and secretome to identify potential causative agent(s). RESULTS/ANTICIPATED RESULTS: Based on cross-sectional metagenomic studies on MSA, MSA cases are expected to have genus reductions in Blautia and Dorea (acetate production); Paraprevotella (succinic and acetic acid production); and Ruminococcus, Coprococcus, and Faecalibacterium (butyrate production). Increases in genus Bacteroides (clinical pathogen) and Akkermansia (mucin degradation) and pro-inflammatory families Clostridiaceae and Rikenellaceae are also expected. MSA is predicted to be associated with reduced levels of short chain fatty acids and increased lipopolysaccharide. These microbial proteins and metabolites are anticipated to modulate intracellular and extracellular αSyn aggregation in vitro. Microbe isolation and secretome culturing methods are expected to identify additional drivers of αSyn aggregation. DISCUSSION/SIGNIFICANCE: This study’s novel use of longitudinal sampling, household controls, and secretome culturing aim to develop a more comprehensive understanding of the complex interactions between the gut microbiome and MSA. The success of this work offers the potential for new insights into the impact of the gut microbiome and secretome on MSA and αSyn aggregation.

OBJECTIVES/GOALS: SLC6A1-Related Neurodevelopmental Disorder (SLC6A1-NDD) is a leading genetic cause of epilepsy and autism. Haploinsufficiency of SLC6A1 leads to reduced uptake of GABA from the synaptic cleft, and increased extracellular GABA in mice. It is a candidate for gene transfer therapy, but translational read outs are needed. EEG is a promising biomarker. METHODS/STUDY POPULATION: The SLC6A1-NDD program includes a specialty clinic and prospective cohort study run in parallel with pre-clinical gene therapy development. Characterization and pre-clinical testing of a homozygous knock-out, heterozygous knock-out, and two humanized knock-in models are on-going, including EEG analyses before and after treatment. Patients with a confirmed diagnosis of SLC6A1-NDD are seen annually in a specialty clinic and a subset participate in the cohort study which collects standardized questionnaires, EEGs, and MR Spectroscopy to measure glutamate and GABA. Gene Therapy Program investigators meet weekly to discuss progress on pre-clinical and clinical trial readiness on SLC6A1-NDD and to align efforts on translational read outs, including EEG, in both humans and the pre-clinical models. RESULTS/ANTICIPATED RESULTS: We have enrolled the full cohort of 20 participants in the prospective SLC6A1-NDD cohort study. Preliminary results have shown that all but 1 individual has a history of developmental delay, and 8 of the 24 individuals in our clinical cohort had at least one episode of developmental regression. Over 90% have epilepsy, and 17/20 in the cohort study have intermittent rhythmic delta activity on EEG. The full knock out mice have behavioral and learning deficits and abnormal electrical brain activity on telemetry, including bursts of spike trains, analogous to epileptiform activity seen in humans. Next steps include quantitative analysis of both mouse and human EEG to develop a translational brain-based biomarker. We plan to assess delta power and investigate genotype-phenotype correlations in mice and humans. DISCUSSION/SIGNIFICANCE: With targeted therapies in development for SLC6A1-NDD, translational biomarkers that demonstrate engagement with the brain are critical. With clinical heterogeneity in SLC6A1-NDD, biomarkers can objectively capture change. Collaborative translational projects may improve efficiency in rare disease research to facilitate early phase trials.

OBJECTIVES/GOALS: The objective of this project was to determine the research volunteer recruitment capabilities and methodologies currently utilized by CTSA Hubs in order to disseminate recruitment best practices and create collaborations across institutions. METHODS/STUDY POPULATION: The CTSA Recruitment and Retention working group developed a REDCap survey to collect information about what participant recruitment and retention resources and processes are being used at CTSA institutions to support investigators. It was distributed to CTSAs between May and July 2019. The survey, consisting of over 50 multiple choice and short answer questions, is an updated version of a 2016 survey. Institutions reported on registry use, feasibility assessment use, clinical trial listings, experience recruiting special populations, program operations and evaluation, workforce education, social media use and other recruitment resources. RESULTS/ANTICIPATED RESULTS: 40 of the 64 CTSA institutions completed the survey. Almost all of those responding are providing investigators access to a registry either favoring their institutional registry (45%) or ResearchMatch (34%) with most (85%) leveraging ResearchMatch to some extent. Over 80% of the CTSAs are providing investigators recruitment consultations, feasibility assessments, study listings, and EMR Utilization. 73% of the CTSAs are assisting with study materials with 47% offering social media assistance. Additionally, over half the respondents indicated they were successful recruiting healthy volunteers, patients, seniors, pregnant women, minors, low-income populations and underrepresented minorities; however, most found recruiting non or limited English speaking persons and rural populations challenging. DISCUSSION/SIGNIFICANCE OF IMPACT: A variety of recruitment and retention resources exist across CTSA institutions, and this inventory serves as a way to compile these services and foster collaboration across institutions. Additionally, it allows CTSAs to identify services not currently being offered that could improve outcomes while also creating opportunities for collaboration.

OBJECTIVES/GOALS: Myocardial interstitial fibrosis leads to high hemodynamic load resulting in heart failure (HFrEF). Previous studies show that treatment with a left ventricular assist device (LVAD) does not reduce fibrosis. We hypothesize that human cardiac fibroblasts are highly activated in HFrEF and remain unresponsive to hemodynamic unloading by LVAD. METHODS/STUDY POPULATION: Forty human subjects with HFrEF undergoing LVAD implantation were enrolled to provide a portion of myocardium routinely removed during LVAD placement. In addition, 7 biopsies previously collected from transplanted hearts with extended LVAD treatment were also evaluated (LVEX). RESULTS/ANTICIPATED RESULTS: Quantification of PSR-stained sections reveals a significant increase in collagen content in the HFrEF tissue (CVF = 2.8) compared to control tissues (CVF = 0.9) that remained elevated in LVEX hearts (CVF = 3.1). HCFs from LV biopsies were isolated and grown to confluence. HCFs from HFrEF patients and control HCFs were plated on substrates with stiffnesses reflective of normal myocardium (2kPa) or HFrEF myocardium (8kPa). Cells were collected at 4- and 7-day time points and levels of collagen I and alpha-smooth muscle actin were quantified by western blot analysis. Control HCFs were responsive to changes in substrate stiffness producing more Col I and a-SMA on 8kPa versus 2kPa, HCFs from HFrEF patients were unresponsive to changes in stiffness exhibiting no significant difference in protein production on 2 vs. 8kPa. DISCUSSION/SIGNIFICANCE: Our data suggests that HCFs isolated from the failing myocardium do not respond to changes in mechanical load and might contribute to persistent increases in fibrosis. These findings bring us one step closer to elucidating mechanisms behind fibrosis in HFrEF which could lead to targeted therapies to improve patient outcomes from LVAD support.

ABSTRACT IMPACT: Effective healthcare interventions improve access, quality of care, and health outcomes for underserved, high-disparity populations of Los Angeles county and beyond. OBJECTIVES/GOALS: We will expand our successful, Los Angeles-based public-academic partnership to develop and evaluate health system interventions aimed at improving healthcare for underserved communities, as well as develop workforce skilled in healthcare delivery science. METHODS/STUDY POPULATION: Together with the LA County Department of Health Services, the two LA-based CTSA hubs at USC and UCLA have established critical infrastructure for effective cross-sector translational research: (1) New funding mechanisms to evaluate health system interventions in county hospitals and clinics in areas of mutual interest; (2) Specialized research service cores (Safety-net Health Innovation core, Clinical Research Informatics core, and Healthcare Delivery Science core), and (3) Training and mentorship programs tailored for healthcare delivery scientists. RESULTS/ANTICIPATED RESULTS: Outcomes from the first four years of the partnership include: (1) Significant impact on health outcomes from eight funded projects, e.g., lowered A1c levels by 0.9%; (2) Successful, coordinated service to dozens of research projects, e.g., a teleretinal screening program decreased ophthalmology visit wait times from 158 to 17 days; (3) New virtual coursework in seven domains (healthcare delivery science, dissemination and implementation science, systems engineering, behavioral economics, informatics, team science, and community engagement); (4) A published ‘synergy paper’ w/ CTSA hubs in three other urban cities examining common themes of academic-public partnerships; and (5) Rapid and streamlined COVID-19 research policy setting with county leadership. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our sustainable infrastructure is effectively bridging research-policy-practice gaps in Los Angeles and addressing patients’ and the health system’s priorities.

Objectives/Goals: • To determine the impact of liraglutide on inflammation and organ injury during sepsis. • To investigate the protective effects of liraglutide on microvascular dysfunction in a clinically relevant model of sepsis. • To provide evidence for the potential therapeutic use of GLP-1 receptor agonists in endothelial dysfunction in sepsis. Methods/Study Population: Sepsis was induced in mice (N = 34) by intraperitoneal injection of cecal contents (1.8 mg/g body weight) and 24-hour hyperoxia (FiO2 90–95%). Mice received saline or liraglutide (0.1 mg/kg) at 6 and 18 hours post-injection and fluids and antibiotics at 12 hours. At 24 hours, mice were euthanized for plasma, bronchoalveolar lavage (BAL), and tissue collection. Plasma inflammatory markers, organ injury markers, and BAL components were measured. In vitro, primary human lung microvascular endothelial cells (HLMVECs) were treated with saline or liraglutide for 24 hours before exposure to saline or LPS (100 ng/mL). HLMVEC barrier dysfunction was evaluated using express permeability testing (XPerT) and electric cell-substrate impedance sensing (ECIS) to measure transendothelial electrical resistance (TER). Results/Anticipated Results: In murine sepsis, illness severity scores and lung injury were improved in mice pretreated with liraglutide (N = 10). Plasma blood urea nitrogen (BUN; P = 0.0036), alanine transaminase (ALT; P = 0.0311) and vascular inflammatory markers MCP-1 (P = 0.0172), ICAM-1 (P = 0.0356), and Pecam-1 (P = 0.0493) in plasma were reduced in mice treated with liraglutide. In HLMVECs, liraglutide (1.5 nM) significantly reduced LPS-induced barrier dysfunction measured by XPerT assay (P = 0.0030) and ECIS (P = 0.0075). Discussion/Significance of Impact: Liraglutide reduces illness severity, vascular inflammation, and organ injury in a two-hit model of sepsis. Liraglutide has direct effects in the microvascular endothelium, limiting LPS-mediated barrier dysfunction. These findings support a protective role for GLP-1 receptor agonism in sepsis, mediated through the microvasculature.

OBJECTIVES/GOALS: In 2020, Baylor College of Medicine held a datathon to introduce a data warehouse, identify its capabilities/limitations, foster collaborations, and engage trainees. The event was held again in 2022, and lessons learned (e.g., tools for data self-service or team communication) were applied. METHODS/STUDY POPULATION: Senior faculty reviewed proposals with an emphasis on feasibility, impact, and relevance to quality improvement or population health. Selected teams worked with Information Technology (IT) for 2 months and presented findings at a 1-day event. Surveys were administered to participants before and after the event to evaluate their background, team characteristics, collaborations, knowledge before and after the datathon, perceived value of the datathon, and plans for future work. Descriptive statistics of respondents’ self-reports were tabulated. RESULTS/ANTICIPATED RESULTS: In 2022, 19 of 36 projects were accepted (13/33 in 2020). At both events, most projects studied quality improvement or clinical outcomes. Of 82 participants in 2022, 54 completed surveys. In 2022, 72% had no datathon experience (48% in 2020). Median effort was 10 person-hours; median IT time was 20% (20 and 10%, in 2020). Seven respondents finished and 21 partially finished their projects (1 and 11, in 2020); 92% made new collaborations (91% in 2020). Respondents strongly agreed that: the experience was valuable (n=28), they would participate in future datathons (n=30), and they would use the warehouse for future work (n=25). Twenty-seven have planned abstracts; 25 have planned manuscripts. DISCUSSION/SIGNIFICANCE: The 2022 datathon had more participants with less experience, potentially due to improved promotion and training opportunities. Fewer person-hours and a higher percentage of IT time were required as compared to 2020, and more projects were completed, possibly due to increased IT efficiency.

OBJECTIVES/GOALS: Parents’ empathy toward their children affects their parenting, which can in turn impact child outcomes. Although parental empathy is theoretically distinct from trait empathy, current literature relies on largely self-report measures of parents’ trait empathy. Thus, the current study evaluated new analog assessments of parental empathy. METHODS/STUDY POPULATION: One parental empathy analog measure (Empathy Measure for Parents Analog Task, Emotion Script; EMPAT-ES) was created based on parents’ responses to open ended prompts describing scenarios that elicit different emotions (e.g., happy, mad, sad, scared) in children. These responses were used to create short scripts. A second analog task (EMPAT, Emotion Audio) was created using 20 sec audio clips of children expressing the different emotions wherein participants respond with how they feel hearing the emotions and separately, how they believe the child feels. After an initial pilot, both versions of the EMPAT-E were administered to 120 families enrolled in a prospective longitudinal study. Parents completed self-report measures of trait empathy and parental empathy, as well as the EMPAT-ES and EMPAT-EA analog tasks. RESULTS/ANTICIPATED RESULTS: Internal consistency of both the EMPAT-ES and EMPAT-EA tasks are expected to be robust, demonstrating the reliability of these novel assessments of parental empathy. Results are also expected to demonstrate the construct and convergent validity of both analog tasks. These new measures of parental empathy are expected to be significantly associated with measures of trait empathy. Specifically, parents’ responses indicating how they believe the child feels in the analog are expected to be strongly related to their reported emotion recognition abilities and responses indicating how analog items made parents feel are expected to be related to parents’ empathic concern. Finally, parents’ responses to the analog tasks are anticipated to be strongly associated with parents’ self-reported parental empathy. DISCUSSION/SIGNIFICANCE OF IMPACT: Valid, novel assessments of parental empathy can impact the parenting literature as well as community intervention and prevention efforts with parents. Such analog tasks can bolster parenting research but they may also be translated to the community setting as a training tool wherein parents are taught new skills that promote more positive parenting.

ABSTRACT IMPACT: To identify potential facilitators and barriers to a successful transition in care. OBJECTIVES/GOALS: Improvements in care for children with sickle cell disease (SCD) have increased survival into adulthood. However, mortality rates are increasing in young adults. One of the challenges is providing appropriate care during transition from pediatric to adult care. The goal is to identify facilitators and barriers to a successful transition in care. METHODS/STUDY POPULATION: The UAB SCD Center serves a large area of Alabama. The pediatric program is in Birmingham and has outreach clinics in three other cities. The adult program only has one clinic located in Birmingham. With IRB approval, we performed a retrospective chart review of individuals with SCD (all genotypes) aged 18-24 (as of 1/31/2019) who were seen at least twice prior to age 18 (in pediatrics) and have confirmed SCD. Charts were reviewed for demographics, genotype, last known insurance, SCD therapy, clinic location, and transition status. Analyses were undertaken to determine predictors of successful transition (defined as coming to an appointment with an adult hematologist) and unsuccessful transition (defined as lost to follow-up (LTFU) without transfer of care). RESULTS/ANTICIPATED RESULTS: There were 544 individuals meeting inclusion criteria. Of this group, 234 were LTFU, 189 transitioned, 36 moved, and 15 died. Seventy patients are still under pediatric care and were excluded. Sixty-eight percent of patients that transitioned had their last pediatric visit in Birmingham, compared to only 32% of those that transitioned from outreach sites (p<.01). Patients were more likely to successfully transition if they had sickle cell anemia (HbSS or HbSß0) (p<.01) and if they were receiving hydroxyurea or chronic transfusion therapy (p<.01). DISCUSSION/SIGNIFICANCE OF FINDINGS: Geography, genotype, and SCD therapy are potential drivers for transition. Genotype in pediatrics likely confers disease severity, suggesting patients with worse SCD may be more likely to successfully transition. Novel strategies are needed to improve transition of care for patients outside of Birmingham and those with less severe disease.

OBJECTIVES/SPECIFIC AIMS: Objective: To assess the impact of corticosteroid therapy on in-hospital mortality in IPF patients admitted with acute respiratory failure. METHODS/STUDY POPULATION: Methods: Patients with IPF were retrospectively identified in the University of California San Francisco medical center’s electronic health records from January 1, 2010 to June 1, 2018. Cases with IPF were defined as age 50 years or older, having at least two codes one month apart for idiopathic fibrosing alveolitis or post-inflammatory fibrosis (ICD-9 516.3, 516.31 or 515.0 or ICD-10 codes J84.9, J84.10, J84.111 or J84.112), and a subsequent hospitalization for acute respiratory failure or acute respiratory symptoms. The prevalence of pre-selected co-morbidities, clinical events (ICU admission, mechanical ventilation, lung transplantation) and clinical outcomes were assessed. A propensity score model for corticosteroid use was constructed using a multivariable logistic regression with inclusion of corticosteroid-associated demographic and baseline variables (univariate p-value < 0.25). A marginal structural model (MSM) was used to address time-dependent confounding and mediating effects of ICU admission and mechanical ventilation by applying inverse probability weighting for receipt of corticosteroid treatment. Secondary outcome analysis was performed on patients who survived hospital admission. RESULTS/ANTICIPATED RESULTS: Results: A total of 132 patients with IPF and an acute respiratory admission were identified. 48 patients (36%) received corticosteroids during their admission. Applying inverse weighting to time-dependent co-variates (ICU admission and invasive mechanical ventilation) in a MSM, corticosteroid therapy was not associated with risk of in-hospital mortality (odds ratio 1.82; 95% CI, 0.47-6.99; p = 0.39). After adjusting for corticosteroid therapy using a propensity score, corticosteroid therapy remained unassociated with in-hospital mortality (odds ratio 1.53, 95% confidence interval [CI] 0.37, 6.29; p = 0.55). There was no difference in discharge disposition or time to hospital readmission by corticosteroid treatment. There was a possible increase in time to death following discharge in patients receiving corticosteroids (Figure). DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusions: This study suggests that treatment of acute exacerbations of interstitial lung disease with corticosteroids does not improve short-term outcomes, including in-hospital mortality, all-cause non-elective re-hospitalization or death within 6 months of discharge. Further research in larger cohorts is needed to more definitively assess this relationship.

OBJECTIVES/GOALS: To determine in vitro mechanisms by which fruits and vegetables (FV) contribute to colon barrier development in Latin American infants. We hypothesize that simulated colonic fermentation of FVs will stimulatein vitro cell barrier function by activating the hypoxia-inducible factor (HIF) pathway in colonocytes. METHODS/STUDY POPULATION: FVs consumed by US-based Latin American infants 6-12 months old (identified from NHANES-What We Eat in America Surveys) will be combined with human breast-milk samples from women self-identified as Hispanic or non-Hispanic, and then subjected to in vitro digestion and anaerobic colonic fermentation using human feces. FV fermenta will be incubated with Caco2 monolayers to measure in vitro cell permeability and protein levels of cellular tight junction, metabolic, and HIF signaling enzymes. To examine their effects in vivo, FVs identified to modulate in vitro barrier function, will be fed (5% freeze dried powder) to wild-type mice and the above parameters will be examined. If in vivo effects are found, intestinal specific HIF knockout mice will be used to examine the role of HIF signaling in mediating these effects. RESULTS/ANTICIPATED RESULTS: We expect that fermenta derived from human milk and FVs will reduce in vitro gut permeability in Caco2 monolayers by increasing gene and protein expression of the HIF signaling complex relative to fermenta of human milk alone. This will be reflected with higher cellular trans-epithelial resistance and greater expression levels of tight junction proteins. We expect FV powder consumption will similarly increase in vivo gut permeability and expression of related genes in mice as compared to mice fed diets without FVs. As we expect an increase in HIF signaling in the colon, we expect that FV powder consumption will not enhance in vivo gut permeability in mice colons with an intestinal specific knockout of HIF. DISCUSSION/SIGNIFICANCE: Data from this study will provide mechanistic evidence to help clinicians promote relevant FVs recommendations for Latin American infants and families. Due to the link between gut permeability and obesity, our next step will be to conduct a dietary intervention in this population.

Objectives/Goals: The aging population faces unique health issues, many of which are exacerbated by aging-associated immune function decline. However, the driving mechanisms behind this decline are poorly understood. We use a mouse model to study the relationship between extracellular matrix (ECM) stiffness and cell mobility within the lymph node (LN) as one potential driving mechanism. Methods/Study Population: We will collect LN from young (6 weeks old) mice and acquire LN from aged (11 month old) mice from a collaborator. We will section LN for ex vivo analysis, including quantification and localization of collagen I using immunofluorescent staining, analysis of microrheological properties using multiparticle tracking (MPT) with PEGylated fluorescent nanoparticles, and migration assays to track the movement of B and T cells. Results/Anticipated Results: We hypothesize a positive correlation between collagen deposits and stiffness within murine LNs due to known mechanisms underlying age-related fibrosis. We also hypothesize that areas of increased stiffness (as revealed by MPT) will exhibit decreased cell migration due to physical hindrance to B and T cell mobilization. Furthermore, we hypothesize that aged murine LN will exhibit a significant increase in stiffness and resultant decreased cell mobility when compared to young murine LN, particularly in areas with increased collagen localization. Discussion/Significance of Impact: These studies will elucidate structure–function relationships driving age-associated LN fibrosis and stiffness, and the resultant impedance to cell migration, thus clarifying some of the potential driving mechanisms behind immune aging and providing data capable of informing the development of relevant models and interventions.

Recruitment of diverse populations into research studies continues to be a challenge. There remains a gap in knowledge and practice on how to best engage with and recruit diverse populations, specifically among Latinos who account for 11% of research participants nationally. Our study focused on Latinas with pre-cervical and cervical cancer in the Pacific Northwest. Our research team took a multilevel approach to diversify recruitment and enrollment processes, focused on methods within healthcare, community-based, and paid media advertisements. This article shares strategies and lessons learned that helped increase participant diversity, meet enrollment goals, and expand relationships with community-based organizations.

Quality clinical research is essential for health care progress and is the mission of academic health centers. Yet ensuring quality depends on an institution’s ability to measure, control, and respond to metrics of trial performance. Uninformed clinical research provides little benefit to health care, drains institutional resources, and may waste participants' time and commitment. Opportunities for ensuring high-quality research are multifactorial, including training, evaluation, and retention of research workforces; operational efficiencies; and standardizing policies and procedures. Duke University School of Medicine has committed to improving the quality and informativeness of our clinical research enterprise through investments in infrastructure with significant focus on optimizing research management system integration as a foundational element for quality management. To address prior technology limitations, Duke has optimized Advarra’s OnCore for this purpose by seamlessly integrating with the IRB system, electronic health record, and general ledger. Our goal was to create a standardized clinical research experience to manage research from inception to closeout. Key drivers of implementation include transparency of research process data and generating metrics aligned with institutional goals. Since implementation, Duke has leveraged OnCore data to measure, track, and report metrics resulting in improvements in clinical research conduct and quality.

ABSTRACT IMPACT: Interdisciplinary networks represent critical components of translational science and learning system development. Our work impacts translational research by presenting an evidence-based approach to developing interdisciplinary networks in response to the COVID-19 pandemic; the approach presented may have broad applications within other academic institutions and medical centers. OBJECTIVES/GOALS: As a local response to the COVID-19 pandemic, we established the University of Alabama at Birmingham COVID-19 Collaborative Outcomes Research Enterprise (CORE) as an interdisciplinary learning health system (LHS) to achieve an integrated health services and outcomes research response amid the pandemic. METHODS/STUDY POPULATION: We adapted a learning system framework, based upon a scoping review of the literature and the Knowledge to Action Framework for implementation science. Leveraging this framework, we developed an institutional-level collaborative network of extant expertise and resources to rapidly develop an interdisciplinary response to COVID-19. The network was designed to quickly collect newly published or clinical information related to COVID-19, to evaluate potential usefulness of this information, and to disseminate the new knowledge throughout the interdisciplinary network; we strove to engage a wide variety of expertise and skills in the network. Thus, we subsequently used social network analysis to examine the emergence of informal work patterns and diversified network capabilities based on the LHS framework. RESULTS/ANTICIPATED RESULTS: We identified three principal characteristics of institutional LHS development including: 1.) identifying network components; 2.) building the institutional collaborative network; and 3.) diversifying network capabilities. Seven critical components of LHS were identified including: 1.) collaborative and executive leadership, 2.) research coordinating committee, 3.) oversight and ethics committee, 4.) thematic scientific working groups, 5.) programmatic working groups, 6.) informatics capabilities, and 7.) patient advisory groups. Evolving from the topical interests of the initial CORE participants, three scientific working groups (health disparities, neurocognition, and critical care) were developed to support the learning network. DISCUSSION/SIGNIFICANCE OF FINDINGS: Interdisciplinary collaborative networks are critical to the development of LHS. The COVID-19 CORE LHS framework served as a foundational resource that may support further institutional-level efforts to develop responsive learning networks. The LHS approach presented may have broad applications within other academic institutions and centers.

Objectives/Goals: The never in mitosis kinase (NEK) family regulates vital processes, namely cell cycle progression, but their potential as therapeutic targets in TNBC has not been fully explored. Our studies aim to develop a toolkit to investigate the functional roles of NEKs in pathologies including carcinogenesis. Methods/Study Population: To assess differential NEK expression in normal and tumor tissues and correlation of gene expression with patient survival, we used Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier Plotter (KMPlot) pan-cancer analysis, respectively. Basal NEK protein levels were determined by immunoblot across a panel of cell lines, including breast cancer, osteosarcoma, hepatocellular carcinoma, and non-cancerous cells, to identify appropriate systems for evaluation of NEK function. Doxycycline-inducible cell lines were generated by transduction with lentiviral stocks of NEK shRNA and overexpression constructs and antibiotic selection. Expression was analyzed by qPCR and immunoblot. Results/Anticipated Results: Expression of NEK2, 4, 5, 6, 8, and 11 was higher in breast tumors compared to normal tissue by GEPIA analysis. Further examination using KMPlot showed a correlation between elevated NEK6 expression and decreased overall survival in patients with aggressive cancers. As an initial proof-of-concept study, we analyzed NEK6 protein expression in breast cancer cells. Levels of NEK6 were elevated in TNBC cells (MDA-MB-231) compared to hormone receptor positive (HR+) breast cancer cells (MCF7). Using complementary approaches to investigate the functional role of NEK6 in breast cancer, we depleted NEK6 expression using shRNAs in TNBC cells and expressed NEK6 in HR+ cells Discussion/Significance of Impact: Because kinase dysregulation promotes oncogenesis and metastasis, targeting kinases is a key strategy in therapeutic development. A NEK-specific molecular toolkit allows researchers to elucidate NEK functions and contributions to carcinogenesis, promoting advancement of novel therapies.

OBJECTIVES/GOALS: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks effective targeted treatment options. TNBC's greater degree of immunogenicity than other breast tumors makes immunotherapy a viable strategy. Strategies to improve the immunotherapy response includes targeting the cGAS-STING innate immune pathway with STING agonists. METHODS/STUDY POPULATION: We have previously shown in vitro that eribulin, a microtubule destabilizer currently used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm. Separately, eribulin also significantly enhances type I interferon expression induced by STING agonists measured by qRT-PCR through a second TBK1-dependent mechanism downstream of STING activation through detecting higher amounts of phosphorylated IRF-3 by western blot protein analysis. Mechanisms of eribulin-mediated interferon expression occur in immune and TNBC cells and are shared with other microtubule destabilizers but not with the microtubule stabilizing agent paclitaxel. RESULTS/ANTICIPATED RESULTS: We determine that the enhancement of type I interferon expression by eribulin is pharmacologically synergistic with multiple STING agonists. The significant enhancement by eribulin led us to evaluate the antitumor efficacy of eribulin in combination the STING agonist ADU-S100 in a challenging spontaneous mammary tumor model MMTV-PyVT. We show that the combination treatment significantly decreased tumor growth which allowed for longer survival compared to other groups. This is particularly interesting because of our previous studies showing that eribulin alone, but not paclitaxel, promotes the activation of CD4+ T-cells in the spleen and draining lymph nodes of BALB/c mice with 4T1 tumors through flow cytometric analysis. DISCUSSION/SIGNIFICANCE: These data contribute to accumulating evidence that there are important mechanistic differences between the microtubule targeted chemotherapeutics currently used in the treatment of TNBC and suggest that eribulin can act as an immune adjuvant in addition to its anti-mitotic effect.