OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

OBJECTIVES/SPECIFIC AIMS: Inflammatory Bowel Disease (IBD) is a chronic, life-long condition characterized by inflammation of the intestine that greatly affects an individual’s quality of life. While biologic therapy directed against TNFα (anti-TNFα, Infliximab) and α4β7 integrin (anti-α4β7; Vedolizumab) is used to treat IBD, a substantial number of patients remain non-responsive. Using a comprehensive bioinformatics approach, the aim of this study was to characterize immune cell profiles and altered molecular pathways in IBD patient non-responders to anti-TNFα and anti-α4β7 therapy to determine potential mechanisms and/or indicators of treatment non-response. METHODS/STUDY POPULATION: Publicly available whole transcriptomes from 65 healthy control and IBD endoscopic biopsies were assessed (NCBI GEO GSE73661). Specifically, transcript profiles from responders or non-responders to anti-TNFα and anti-α4β7 therapy were utilized. Differentially expressed transcript profiles were obtained by comparing responders or non-responders prior to receiving therapy versus healthy controls using NCBI’s GEO2R after adjustment with Benjamini and Hochberg testing (p<0.05). Immune profiling of DEgs were analyzed by the core LM22 immune signature for subsets of B-, T-, dendritic-, mast-cells, macrophages, and neutrophils (CIBERSORT, cibersort.stanford.edu) (p<0.05). Networks, functional analysis, and interpretation of transcriptomic data were performed using Ingenuity Pathway Analysis (IPA) (Qiagen) (p<0.05). RESULTS/ANTICIPATED RESULTS: Initially, we determined colonic immune profiles in responders and non-responders to anti-TNFα and anti-α4β7 therapy. Compared to responders, in both anti-TNFα and anti-α4β7 non-responders we found elevated neutrophil levels (p<0.05). Specific to anti-TNFα treatment, non-responders demonstrated substantially reduced Treg cells (p<0.05); whereas, exclusive to anti-α4β7 treatment, non-responders showed elevated dendritic cells, activated CD4 T cells, and reduced M2 macrophages (p<0.05). Next we profiled differentially expressed transcripts to determine molecular pathways associated with therapy non-response. In both anti-TNFα and anti-α4β7 non-responders, we observed alterations in pathways specific to cellular growth and metabolism. Among cell growth pathways we found activated growth hormone, Wnt, ErB, and IGF-1 signaling; whereas, among metabolic regulation we found altered triglyceride, tryptophan, and leptin signaling. Moreover, unique to anti-TNFα non-responders, we found activated sphinogosine-1-phosphate and paxillin pathways. While non-response to anti-α4β7 indicated activation of SAPK/JNK and IL-9 signaling. DISCUSSION/SIGNIFICANCE OF IMPACT: Together these data define specific immune profiles and molecular pathways observed in non-responders to anti-TNFα and anti-α4β7 therapy. Our analysis identified substantial alterations in pathways specific to cellular growth and metabolism, identifying a link between non-response to biologic therapy and specific cell functions. These data suggest particular alterations in immune profiles and molecular pathways could play a role in non-response to biologic therapy, highlighting a future direction for personalized treatment regimens that could lead to more targeted use of existing therapies and more favorable patient health outcomes.

Objectives/Goals: Transgender women who have sex with men (TGWSM) have higher HIV risk. The rectal mucosal (RM) immune environment of TGWSM who choose feminizing hormone therapy (FHT) has been shown to be distinct from the RM of cisgender men who have sex with men (MSM). We studied the impact of FHT on the adaptive immune cellular composition of the RM. Methods/Study Population: We sampled cross-sectional and longitudinal cohorts of TGWSM and cisgender MSM from The Silom Clinic in Bangkok, Thailand from December 2020 to December 2023. We included participants aged >18 years, all cisgender MSM and TGWSM with FHT levels in the therapeutic range for cisgender women. We performed RM biopsies and analyzed the adaptive immune cell characteristics via flow cytometry. We will perform binary linear regression to assess the association between systemic FHT levels and the percentage of CD4+ T cells expressing key biomarkers. Primary outcomes include the percentage of CD4+ T cells that express CCR5, with a secondary outcome of the percentage of CD4+ T cells that express Ki67. Results/Anticipated Results: The cross-sectional cohort included 100 TGWSM on FHT and 50 cisgender MSM. The longitudinal cohort included 25 TGWSM who were initiating FHT. Similar primary and secondary outcomes are to be elucidated in both cohorts. We anticipate the RM environment of TGWSM using FHT in both cohorts compared to the RM environment of cisgender MSM in the cross-sectional cohort will be associated with greater percentages of activation/co-receptor expression of CD4+ T cells that express biomarkers of interest. In the longitudinal cohort, we similarly anticipate increased percentages and activation/co-receptor expression of CD4+ T cells expressing biomarkers of interest in TGWSM after in comparison to before initiating FHT. Discussion/Significance of Impact: This is the largest study of its kind to compare HIV target cells in RM of TGSWM, which challenges prevailing perspectives suggesting to group cisgender MSM with TGWSM. Anticipated results will inform HIV prevention strategies and future vaccine studies in this high-risk population.

OBJECTIVES/GOALS: The objective is to assess the effect of pesticide exposure (individually and pesticide mixtures) on the immune response to COVID-19 in children. The goal is to improve scientific knowledge on factors affecting COVID-19 and identify a potentially modifiable factor to reduce disparities in COVID-19 morbidity. METHODS/STUDY POPULATION: Blood samples will be obtained from 50 children with asthma two time points; baseline and 12 months later. SARS-CoV-2 infection or vaccination will be determined with blood exposome RNA analyses.. Immunological response will be measured using neutralizing, phagocytizing, and NK-activating anti-body responses biomarkers. Pesticide exposure will be measured via urinary pesticide metabolites (UPMs). For individual metabolites multivariable analyses for each pesticide will be conducted using generalized estimating equation (GEE) models with compound symmetry correlation to account for the repeated measures design. To assess the pesticide mixture, weighted quantile sum regression (WQS) will be used. RESULTS/ANTICIPATED RESULTS: The main hypothesis is that increased pesticide exposure results in a reduction in the immunological response to SARS-CoV-2 infection and the COVID-19 vaccine. Therefore, we anticipate that increasing concentrations of individual UPMs as well as the increasing index will result in reductions in markers of the immune response to SARS-CoV-2 infection and the COVID-19 vaccine. DISCUSSION/SIGNIFICANCE: Exposure to pesticides is a modifiable environmental factor. If pesticides are found to alter the immune response to COVID-19 infection and vaccination, these data will provide an evidence base for efforts to reduce pesticide exposure in children.

ABSTRACT IMPACT: Our data demonstrate that VC2 oncolytic virotherapy has significant clinical potential. OBJECTIVES/GOALS: Use our novel oncolytic herpes simplex virus type I (HSV-1), VC2, to understand how oncolytic virotherapy affects the immunosuppressive tumor microenvironment as a mechanism of efficacy. METHODS/STUDY POPULATION: We tested the efficacy of VC2 as an oncolytic virotherapy (OVT) in a syngeneic B16F10-derived mouse model of melanoma. We modified the B16F10 to express nectin-1 (B16F10n-1), the major receptor for HSV-1. Engrafted B16F10n-1 tumors were intratumorally treated with either phosphate-buffered saline (PBS) or 1x10^6 pfu VC2. At indicated time points, treated tumors were excised and processed for immunohistochemistry or flow cytometry analysis. For our experimental metastasis studies, mice were intravenously challenged with B16F10n-1 cells. For our depletion studies, CD4+ and CD8+ T cells were depleted in mice by treatment with mouse anti-CD4 and anti-CD8 monoclonal antibodies respectively, while the control mice were given Rat IgG2b isotype. RESULTS/ANTICIPATED RESULTS: We found that VC2 slowed tumor growth rates and significantly enhanced survival times over control treated mice. VC2-treated mice that survived initial tumor engraftment were able to reject a second tumor challenge and were also resistant to lung colonization (experimental metastasis) of tumor cells. Furthermore, VC2 treatment promoted increased intratumoral T cell infiltration and induced a strong antitumor effect that decreased growth rates of distant, untreated tumors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data demonstrate that VC2 OVT has significant clinical potential. Furthermore, due to the increased survival rates and CD8+ T cells dependence, our model will enable study of the immunological correlates of protection for VC2 OVT and OVT in general, as well as to inform the rational design of future OVs with improved therapeutic potentials.

OBJECTIVES/SPECIFIC AIMS: We hypothesized that CXCL12, as a biased dimer variant or secreted at dimer-dominant concentrations, would influence PDAC growth and progression. METHODS/STUDY POPULATION: PDAC cells were genetically manipulated to express dimer-promoting levels of CXCL12. These cells were studied in vitro or orthotopically implanted into the mouse pancreas for in vivo studies. As a second approach, recombinant wild-type or engineered CXCL12 monomer or dimer proteins were applied to cells in culture or administered intra-peritoneal to study the effects on tumor growth. RESULTS/ANTICIPATED RESULTS: Mice engrafted with CXCL12-expressing cells had a better survival rate, delayed tumor growth and smaller tumors. Tumors from these mice had significantly less proliferation, measured by Ki-67 staining. In vitro analysis of CXCL12-expressing cells showed decreased viability and growth rates. Percent of cells in the cell cycle G2 phase was also decreased, suggesting cell cycle progression blockade. Viability of human PDAC cells dose-dependently declined upon wild-type CXCL12 treatment, with the non-motile dimer-dominant dose (1000 nM) exhibiting maximal effect. Treatment in an allogeneic mouse model of PDAC with locked-dimer CXCL12, but not wild-type, reduced tumor burden. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results support the notion that biased CXCL12 signaling may be therapeutically exploited to limit pancreatic cancer progression.

OBJECTIVES/SPECIFIC AIMS: The objective of the study was 2-fold; to identify potentially deleterious alleles in a child with Treacher Collins syndrome, and; to demonstrate the value of the iobio analysis platform for intuitively and rapidly analyzing genomic data. METHODS/STUDY POPULATION: We used the iobio suite of web-based applications to analyze quality metrics for the sequencing data and called variants for the proband and his parents. We then visually interrogated variants in genes potentially associated with the syndrome in real-time, using the intuitive gene.iobio application. We sought high impact variants that demonstrated a predicted impact on the protein function, and were simultaneously at low allele frequency in the general human population. Variants were also compared against the ClinVar database of known mutations to identify variants that have already been associated with this, or related syndromes in the literature or clinical studies. Finally, the gene.iobio tool allows users to interrogate the primary sequencing data to ensure that no variants had been missed by the primary variant calling pipeline. This analysis pipeline was performed using intuitive web-based apps in real time, and consequently represents a system that is available to users that traditionally are excluded from these analyses. RESULTS/ANTICIPATED RESULTS: The iobio suite was used to rapidly assess data quality and interrogate genetic variants for a child with Treacher Collins syndrome. A compound heterozygote consisting of 2 missense alleles in the TCOF1 gene was identified as a compelling pathogenic allele, necessitating further functional investigation. The study helped validate the use of the intuitive iobio tools in such analyses, strengthening the case for greater involvement of medical professionals in data analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: The performed analyses demonstrated that the whole genome sequencing data for the family being studied was of a very high quality, although 1 gene demonstrated a local region of almost zero coverage. This ensured that study conclusions can be presented with confidence. A variant associated with Treacher Collins syndrome 1 in ClinVar was uncovered in the TCOF1 gene, however, given it’s benign rating, this variant was not considered further. The most interesting candidate was a compound heterozygote, consisting of 2 missense mutations, also in the TCOF1 gene. These mutations occurred with allele frequencies of 22% and 8% in the general population, and additional molecular and functional studies are currently being pursued.

CONTEXTperts (Context and Topic Experts) bring real-world experience in the context of the study topic and setting to enhance research team expertise, capacity, and creativity. They complement but do not replace authentic patient and community engagement, professional consultants, or formal advisory boards. Individual CONTEXTpert consultations or group meetings can help improve research questions, study designs, implementation plans, dissemination, and application of findings. They have added value to a variety of projects with varied research methods and study designs, including research with and without patient or community involvement. CONTEXTperts can bring vision, challenge, and reality checks to a variety of research teams with a practical, affordable model.

As the COVID-19 pandemic took hold in the USA in early 2020, it became clear that knowledge of the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among asymptomatic individuals could inform public health policy decisions and provide insight into the impact of the infection on vulnerable populations. Two Clinical and Translational Science Award (CTSA) Hubs and the National Institutes of Health (NIH) set forth to conduct a national seroprevalence survey to assess the infection’s rate of spread. This partnership was able to quickly design and launch the project by leveraging established research capacities, prior experiences in large-scale, multisite studies and a highly skilled workforce of CTSA hubs and unique experimental capabilities at the NIH to conduct a diverse prospective, longitudinal observational cohort of 11,382 participants who provided biospecimens and participant-reported health and behavior data. The study was completed in 16 months and benefitted from transdisciplinary teamwork, information technology innovations, multimodal communication strategies, and scientific partnership for rigor in design and analytic methods. The lessons learned by the rapid implementation and dissemination of this national study is valuable in guiding future multisite projects as well as preparation for other public health emergencies and pandemics.

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/SPECIFIC AIMS: We aim to show that amyloid accumulation in an animal model of Alzheimer’s disease leads to a preferential disruption of inhibitory parvalbumin-expressing interneurons, and the peri-neuronal nets that surround them, resulting in downstream network alterations to potentially explain early mechanisms of memory impairment in the disease. METHODS/STUDY POPULATION: We employ the 5xFAD mouse model of familial Alzheimer’s disease crossed with transgenic mouse lines which fluoresce red or green in specific neuronal populations. We conducted immunostaining and immunoblotting in amyloid accumulating animals compared with healthy littermate control. Future experiments will be performed in human postmortem tissue to translate these results from mouse model to the human population. Electrophysiological recordings from acute mouse brain slices were conducted as a functional assay. RESULTS/ANTICIPATED RESULTS: Preliminary results indicate that PNNs are disrupted and that activity-associated levels of PV are reduced. Both inhibitory PV and excitatory pyramidal cell populations exhibit altered spiking and synaptic activity during sharp wave ripple events. DISCUSSION/SIGNIFICANCE OF IMPACT: By elucidating the specific neuronal sub-type that is responsible for hippocampal network disruption, future studies could attempt a targeted optogenetic or pharmacological intervention to restore network activity important for memory consolidation.

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: Current techniques for immunosuppressant therapeutic drug monitoring (TDM) are not patient-centric which is prohibitive for those in remote areas. We conducted stakeholder engagement meetings to identify context, attitudes, design requirements, and potential unintended consequences for developing an at-home test for immunosuppressant TDM. Methods/Study Population: We conducted two virtual focus group discussions with 10 organ transplant recipients, some of whom were located in rural and remote locations, including members of the community engagement committee of the Center for Innovations in Cancer & Transplant and the Transplant Recipient Advisory Council. We also conducted a virtual focus group discussion with transplant providers and had individual conversations with one provider and one patient who were unable to attend the focus groups. Results/Anticipated Results: Patients had strong interest in at-home testing to reduce financial and time burdens. Providers wanted faster test turnaround times than available with current centralized labs, especially for managing patients in remote settings. Both patients and providers agreed that at-home tests could enhance patient engagement and empowerment in healthcare, and they preferred a device that would provide highly quantitative and accurate results available on the same day. Finally, both patients and providers also expressed concerns about potential increased work created for caregivers and healthcare workers. Discussion/Significance of Impact: We will incorporate these findings into our design engineering and continue iterative stakeholder engagement to co-design a device that can transform TDM and transplant care.

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.