OBJECTIVES/SPECIFIC AIMS: Modulation of autophagy has the potential to treat inflammatory bowel disease (IBD). IBD is characterized by dysregulated inflammatory pathways and a defective intestinal epithelial barrier. We sought to better understand how autophagy can be utilized to regulate both inflammation and the intestinal barrier. METHODS/STUDY POPULATION: We examined mice with an autophagy defect in only macrophages in an animal model of IBD. To understand the phenotype, we utilized macrophages to investigate the mechanism behind autophagy and proinflammatory cytokine secretion. In addition, we analyzed the development of colonoids in a co-culture system with macrophages with or without a functional autophagy pathway. Lastly, pharmacological modulation of autophagy to control inflammation was assessed. RESULTS/ANTICIPATED RESULTS: Mice with autophagy-deficient macrophages were highly susceptible to intestinal barrier disruption. Susceptibility was due to enhanced proinflammatory cytokine secretion and intestinal permeability. Furthermore, proinflammatory macrophages (due to an autophagy defect) co-cultured with colonoids, significantly decreased the number of mucus producing goblet cells. Finally, pharmacologically modulation of autophagy reduced the secretion of proinflammatory cytokine by macrophages and reduced intestinal permeability. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results strongly suggest autophagy modulation can dampen inflammation and enhance the intestinal epithelial barrier.

OBJECTIVES/GOALS: Genetic variation in L-type voltage-gated calcium channels, including CaV1.3, is associated with increased risk for psychiatric disorders including bipolar disorder and schizophrenia. Additionally, rare mutations in CaV1.3 have been linked to epilepsy, developmental delay, and autism. Deletion of CaV1.3 in mice is associated with impaired consolidation of contextual fear conditioning. Some studies have also observed affective behavior deficits in CaV1.3-deficient mice, but other studies have not found affective phenotypes, perhaps due to differences in genetic backgrounds, sex ratios, or task protocols. CaV1.3 is important for slow afterhyperpolarization in hippocampal and amygdala neurons, which prevents excessive firing in response to sustained excitatory input, and CaV1.3-deficient amygdala neurons exhibit hyperexcitability and impaired LTP. CaV1.3 is also expressed in the cerebellum, but its functional role there is not well understood. Given its importance in shaping neuronal activity in the hippocampus and amygdala, we hypothesized that loss of CaV1.3 would cause abnormalities in motor learning as well as affective and cognitive behaviors. METHODS/STUDY POPULATION: Wild-type (WT), haploinsufficient (Hap), and knockout (KO) mice were maintained on a congenic C57BL/6NTac genetic background and were subjected to behavioral tasks including open field, rotarod, ErasmusLadder, elevated zero maze, forced swim test, and tail suspension test. Data were analyzed with sexes combined and with sexes separated to assess for sex as a biological variable. Studies were analyzed by one-way ANOVA, two-way ANOVA, or generalized linear mixed model, where appropriate. RESULTS/ANTICIPATED RESULTS: CaV1.3 KO was associated with impaired motor learning in the rotarod task (p < 0.05), as well as impaired associative learning in the ErasmusLadder task (p < 0.01), despite intact locomotor function on both tasks. When examined by sex, the rotarod phenotypes were driven by motor learning impairments in males (both Hap and KO, p < 0.05 and p < 0.01, respectively), whereas the ErasmusLadder associative learning phenotypes were present in both sexes only in the KO condition, consistent with previously reported impairments in CaV1.3-deficient mice in consolidation of contextual fear conditioning. Although KO mice learned the motor aspects of the ErasmusLadder task, they learned more slowly. They also failed to learn start cues, which requires intact associative learning. No differences were observed in overall exploration or locomotor activity in open field or elevated zero maze. Analyses from affective tasks are ongoing. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary studies provide new evidence that CaV1.3 is important for the function of neural circuits involved in motor learning, and concur with previous data showing its involvement in associative learning. Our data differ slightly from previous studies of CaV1.3 in motor learning, which could be attributable to differences in task protocols and/or genetic background. These results highlight the importance of CaV1.3 in a variety of behaviors, which may help explain why variation in CaV1.3 expression and function has pleiotropic effects in humans.

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to determine if dopamine signaling is altered in a mouse model displaying excessive self-grooming and further elucidate the potential utility of compounds targeting the striatal DA system in modulating repetitive behaviors. METHODS/STUDY POPULATION: Here, we report studies using fast-scan cyclic voltammetry (FSCV) in mice lacking the postsynaptic protein SAP90/PSD95-associated protein (SAPAP3 KO mice) as well as control littermates. Rodent self-grooming provides a behavioral output with which one can monitor repetitive, self-directed, patterned behavior that has great translational value to OCD-like disorders. Total time spent grooming was monitored in SAPAP3KO mice and control littermates. To further examine the role of DA in regulating repetitive grooming behaviors the magnitude and kinetics of DA transients were assessed using FSCV in ex vivo slice preparations as well as in anesthetized mice in vivo. DA transients were elicited in the dorsolateral striatum (DLS), dorsomedial striatum (DMS); and nucelus accumbens core (NAcc). In some experiments mice were crossed with DAT-Cre animals and channelrhodopsin 2 (ChR2) was virally expressed in DA neurons to allow optical stimulation of DA transients. RESULTS/ANTICIPATED RESULTS: As previously reported, SAPAP3 KO mice showed excessive grooming compared to control littermates at the age assessed (4-5 months). DA transients evoked by a single electrical pulse in slices from SAPAP3 KO mice were not significantly different from those observed in slices from control littermates in any of the regions tested including the DLS, DMS and NAcc. However, when four electrical pulses were applied at a frequency of 10Hz to mimic DA neuron bursting, the magnitude of DA transients observed in the DMS and NAcc of SAPAP3 mice were greater than those evoked in control littermates.Interestingly, phasic stimulation produced similar DA transients in the DLS of both genotypes suggesting that phasic DA signaling was not globally altered. To confirm this finding we crossed SAPAP3 KO mice with DAT-Cre mice and injected ChR2 containing virus into the midbrain to selectively express ChR2 in DA neurons. Transients were then optically evoked resulting in selective activation of DA neurons. Optical stimulation produced a pronounced enhancement of DA release in SAPAP3 KO mice specifically in the DMS and only following phasic-like stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: These exciting findings suggest that DA signaling in SAPAP3KO mice is dysregulated in a very precise manner that is sub-region specific as well as dependent on the pattern of stimulation. These results suggest that targeted therapies that can modulate these specific modes of dopaminergic signaling in these distinct striatal subregions could provide improved efficacy in OCD patients that are resistant to SSRI treatment.

ABSTRACT IMPACT: By assessing function of mutant (patient-specific) tp53 in zebrafish embryonal rhabdomyosarcoma will inform clinicians of the severity of mutant tp53 alleles. OBJECTIVES/GOALS: This study aims to define loss- and gain-of-function TP53 mutations by comparing effects in tp53-null and wild-type tumors. In addition, it aims to generate a rapid in vivo analysis platform to assign function to patient specific TP53 mutations in the clinic METHODS/STUDY POPULATION: To define tp53 function in ERMS pathogenesis, we previously generated a new tp53-null mutant (tp53-/-) in zebrafish by deleting the entire tp53 genomic locus using TALEN mutagenesis. tp53-/- zebrafish spontaneously develop a spectrum of tumors including sarcomas, leukemia and germ cell tumors (Ignatius…Baxi et. al., eLife) reminiscent of tumors observed in Trp53-null mice. Using the tp53-/- mutants to generate kRASG12D-induced ERMS, we discovered that tp53 is a potent repressor of metastases but rather surprisingly had no effect on self-renewal (Ignatius…Baxi et. al., eLife). Here, using tp53-/- zebrafish, we assessed effects of wild-type and mutant (patient specific) tp53 on tumor initiation, proliferation and apoptosis. RESULTS/ANTICIPATED RESULTS: ERMS tumor initiation in the tp53-/- background is observed in > 97% of animals whereas only <40% of wild-type animals develop ERMS. Additionally, tp53 is a potent suppressor of ERMS proliferation and its effect on apoptosis is minor. Next, we expressed either WT zebrafish or human TP53 in tp53-/- animals along with kRASG12D and both genes suppressed tumor initiation and growth. We co-expressed TP53C176F (found in two ERMS patients) and TP53P153del (identified in a patient with osteosarcoma in our clinic) in zebrafish ERMS, and find that the TP53C176F allele significantly suppressed tumor initiation with effects predominantly on enhanced apoptosis. However, the TP53P153del allele initiated tumors at similar frequency compared to tp53-/- animals but increased the initiation of tumors in the head musculature. DISCUSSION/SIGNIFICANCE OF FINDINGS: Different TP53 alleles identified in patient tumors have very different effects on tumorigenesis in vivo and can respond differently to potentially therapeutic compounds. Thus, the type of precision modeling demonstrated here promises to help further define patient-specific TP53 biology and improve clinical strategies in the future.

OBJECTIVES/GOALS: A collaboration among Indiana CTSI community health partnerships (CHeP), bioethics, and regulatory programs identified and reviewed human research protection training programs targeting community engaged research, and pilot tested CIRTification with community partners working across a range of contexts. METHODS/STUDY POPULATION: We searched community human research protection training programs from across the county, identified three, examined each based upon criteria identified by community partners (time, relevance, online delivery) and our Human Research Protection Program (HRPP), and selected CIRTification (CIRT) to pilot. Ten community research partners volunteered to complete CIRT and a debriefing interview. Four completed CITI training previously. Participants included local and state-wide organizations, a resident, a state agency, and a hospital, and came from rural and urban communities. Interviews covered practical issues (ease of use, language, time), relevance, and comparison to CITI. Results were shared with HRPP for approval. RESULTS/ANTICIPATED RESULTS: Most felt CIRT was easy to navigate and engaging, and those who had done CITI felt CIRT was more relevant and engaging. The sections on historical background and recruitment were cited as most valuable. Suggestions were made to increase the diversity of examples beyond health care research. Community members mentioned several applications for CIRT including: (1) helping their own community work; (2) empowering them to be an advocate; (3) referring others to CIRT; (4) influencing approaches to recruitment and community engagement; and (5) applying ethics principles to their other community work. The Human Research Protection Program approved CIRT in place of CITI for community researchers. DISCUSSION/SIGNIFICANCE: Our process represents collaboration across the Indiana CTSI, HRPP and community partners, and use of best practices. Exemplifying “nothing about us without us”, actions were based on direct input from community partners, with the goal of decreasing barriers to engaging communities in research.

Academic discovery in biomedicine is a growing enterprise with tens of billions of dollars in research funding available to universities and hospitals. Protecting and optimizing the resultant intellectual property is required in order for the discoveries to have an impact on society. To achieve that, institutions must create a multidisciplinary, collaborative system of review and support, and utilize connections to industry partners. In this study, we outline the efforts of Case Western Reserve University, coordinated through its Clinical and Translational Science Collaborative (CTSC), to promote entrepreneurial culture, and achieve goals of product development and startup formation for biomedical and population health discoveries arising from the academic ecosystem in Cleveland. The CTSC Office of Translation and Innovation, with the university’s Technology Transfer Office (TTO), helps identify and derisk promising IP while building interdisciplinary project teams to optimize the assets through key preclinical derisking steps. The benefits of coordinating funding across multiple programs, assuring dedicated project management to oversee optimizing the IP, and ensuring training to help improve proposals and encourage an entrepreneurial culture, are discussed in the context of a case study of therapeutic assets, the Council to Advance Human Health. This case study highlights best practices in academic innovation.

Clinical research professionals (CRPs) are integral to the academic medical center workforce, research operations, and daily clinical research tasks; however, due to inconsistent training, there is a shortage of qualified CRPs. The Joint Task Force for Clinical Trial Competency created a competency framework for CRPs, which has demonstrated positive results from various institutions, but training programs have been limited in standardization, replicability, and dissemination. To improve this, we designed the University of Texas Southwestern (UTSW) Medical Center Clinical Research Foundations (CRF) training program, which is a competency-based online self-paced CRP training curriculum hosted via the Collaborative Institutional Training Initiative (CITI) portal. We examined feasibility, acceptability, and uptake of the UTSW CRF training on an institutional scale and were pleased to find this curriculum is not only feasible but has high levels of acceptability. Furthermore, faculty, clinicians, and trainees voluntarily completed this training program indicating utility across diverse groups. The UTSW CRF combines the existing CITI training modules with UTSW-created material, providing an optimal balance between generalized clinical research education and institutionally tailored content. We believe the UTSW CRF curriculum could serve as a plug-and-play foundational model for other research centers to tailor according to their audience and institutional needs.

Little has been published on the demographic composition of the clinical and translational science research workforce within the Clinical and Translational Science Awards (CTSA) Program despite the well-documented need for greater diversity in the biomedical research workforce. Analyses of workforce demographic reveal that women and members of underrepresented groups remain persistently underrepresented in the CTSA hub and training components principal investigators. In contrast, in the CTSA Program career development and training programs, females have greater representation as participants, and non-Whites were better represented in training programs.

Objectives/Goals: Objective: To examine whether familial longevity modifies the relationship between chronic kidney disease (CKD) and cardiovascular disease (CVD) in older adults. Goals include assessing the impact of familial longevity on 1) CVD prevalence, 2) CVD incidence, and 3) mortality among individuals with CKD. Methods/Study Population: An observational, longitudinal study. We examined 1,236 Ashkenazi Jewish adults (ages 65–94) from the LonGenity cohort. Estimated glomerular filtration rate (eGFR) was the independent variable, calculated using the CKD-EPI equation 2021. CVD prevalence, incidence, and mortality were our outcomes. CVD was defined as a composite of MI, PCI, CABG, or stroke. Exceptional longevity was defined as living past 95 years, grouping participants into OPEL (offspring of parents with exceptional longevity, n = 576) and OPUS (offspring of parents with usual survival, n = 604). Stratified logistic regression and Cox proportional hazards models assessed eGFR’s association with CVD, testing effect modification through eGFR × OPEL interaction terms. Median follow-up was 5.5 years. Results/Anticipated Results: A significant association was observed between eGFR and CVD prevalence in OPUS, but not in OPEL. No significant link was found between eGFR and CVD incidence or mortality in either group. Familial longevity did not modify the association between eGFR and CVD prevalence, nor the composite of CVD incidence and mortality, as the interaction term was nonsignificant. The LonGenity cohort’s health status may have influenced these results due to selection and survivor biases, limiting generalizability. Further research is needed to clarify familial longevity’s role in kidney function and cardiovascular outcomes. Discussion/Significance of Impact: Our findings suggest that kidney function, as measured by eGFR, may be associated differently with cardiovascular risk in those with and without familial longevity. The study highlights potential limits of familial longevity in modifying CVD risk associated with CKD, underscoring the need for tailored CVD prevention strategies.

OBJECTIVES/SPECIFIC AIMS: Issues with recruiting the targeted number of participants in a timely manner often results in underpowered studies, with more than 60% of clinical studies failing to complete or requiring extensions due to enrollment issues. The objective of this study is to develop and implement a scalable, organization wide platform to enhance accrual into clinical research studies. METHODS/STUDY POPULATION: We are developing and evaluating an informatics platform called Utah Utility for Research Recruitment (U2R2). U2R2 consists of 2 components: (i) Semantic Matcher: an automated trial criterion to patient matching component that also reports uncertainty associated with the match, and (ii) Match Delivery: mechanisms to deliver the list of matched patients for different research and clinical settings. As a first step, we limited the Semantic Matcher to utilize only structured data elements from the patient record and trial criteria. We are now including distributional semantic methods to match complete patient records and trial criteria as documents. We evaluated the first phase of U2R2 based on a randomized trial with a target enrollment of 220 participants that compares 2 treatment strategies for managing back pain (physical therapy and usual care) for individuals consulting a nonsurgical provider and symptomatic <90 days. RESULTS/ANTICIPATED RESULTS: U2R2 identified 9370 patients from the University of Utah Hospitals and Clinics as potential matches. Of these 9370, 1145 responded to the Back Pain study research team’s email or phone communications, and were further screened by phone. In total, 250 participants completed a screening visit, resulting in the current study enrollment of 130 participants. Forty-three of 1145 patients refused to participate, and 50 participants no-showed their screening visit. DISCUSSION/SIGNIFICANCE OF IMPACT: A recruitment platform can enhance potential participant identification, but requires attention to multiple issues involved with clinical research studies. Clinical eligibility criteria are usually unstructured and require human mediation and abstraction into discrete data elements for matching against patient records. In addition, key eligibility data are often embedded within text in the patient record. Distributional semantic approaches, by leveraging this content, can identify potential participants for screening with more specificity. The delivery of the list of matched patient results should consider characteristics of the research study, population, and targeted enrollment (eg, back pain being a common disorder and the possibility of the patient visiting different types of clinics), as well as organizational and socio-technical issues surrounding clinical practice and research. Embedding the delivery of match results into the clinical workflow by utilizing user-centered design approaches and involving the clinician, the clinic, and the patient in the recruitment process, could yield higher accrual indices.

Objectives/Goals: Synthetic data holds potential for inclusion in medical product development pipelines. Therefore, we explored the current regulatory and practice landscape to identify best practices used to ensure, and communicate to key stakeholders, synthetic data quality, relevance, and reliability in regulatory settings. Methods/Study Population: We identified areas in which synthetic data created using generative AI holds the most value for health researchers. We reviewed regulatory documents, published literature, and expert insights to examine how regulators currently use, define, apply, and govern synthetic data created using generative AI. Next, we identified data management tools, best practices, ethical considerations, and regulatory developments that are necessary for generating fit-for-use synthetic datasets. Using this information, we developed a risk-based credibility assessment framework that aligns with current governmental standards that can be useful for users of synthetic data derived from generative AI applications in regulatory settings. Results/Anticipated Results: Synthetic data created using generative AI holds value for health researchers across four key areas: Acting as a 1) privacy-enhancing technology, 2) data science ’sandbox’ for training and exploration, 3) mechanism to navigate legalities around data sharing and/or use, and 4) method to augment underrepresented subgroups in datasets. However, synthetic data raise ethical and legal concerns, particularly regarding privacy, consent, stakeholder engagement, and ownership. Regulators and health technology assessment bodies, including FDA, EMA, MHRA, and Canada’s Drug Agency, are exploring synthetic data to supplement medical datasets, validate external control arms, enhance model performance, and inform regulatory decision-making. Discussion/Significance of Impact: Our work underscores the need to continue cultivating transparent, ethical, and fit-for-use approaches to synthetic data generation using generative AI. Moving forward, effective synthetic data use and development requires a culture of learning and transparency among regulators, end users, and those involved in data generation and exchange.

ABSTRACT IMPACT: Insights from this project will provide clinical guidance in treatment of immunotherapy in triple negative breast cancer and identify early imaging biomarkers of treatment response. OBJECTIVES/GOALS: Significant research that addresses monitoring and predicting patient response of triple negative breast cancer (TNBC) to immunotherapy is needed. Using positron emission tomography (PET) imaging to probe the tumor microenvironment (hypoxia, T-cell activation), we aim to predict early response to immunotherapy for in mouse models of TNBC tumors. METHODS/STUDY POPULATION: Female Balb/c mice with 4T1-luciferase mammary carcinoma cell tumors were administered paclitaxel (PTX; 10 mg/kg), anti-PD1 (200 µg), both, or vehicle (saline) intraperitoneally. Treatment was given on days 0, 2, and 5 for cohort 1 (n=16) who underwent granzyme B specific (GZP) PET imaging (T-cell activation) and days 0, 2, 5, and 8 for cohort 2 (n=12) who underwent [18F]-fluoromisonidazole (FMISO)-PET imaging (hypoxia). Bioluminescence (BLI) imaging and caliper measurements were performed to track tumor size changes at multiple timepoints and tumors were collected for histological validation on day 20. Mean standard uptake value (SUVmean) was calculated as percent of day 0, and statistical analyses were performed with unpaired t-tests and Wilcoxon-rank sum tests. RESULTS/ANTICIPATED RESULTS: Non-responders to treatment had a significantly higher tumor volume compared to responders starting on day 6 (p<0.05). Although no significant differences in BLI between control and single-agent therapies were found, BLI data revealed that treatment with combination PTX and anti-PD1 significantly decreased viability signal between days 3 and 6 (p=0.04). SUVmean from GZP-PET was over 250% higher in responders compared to non-responders by day 6 (p=0.03). SUVmean from FMISO-PET was 80% less in responders compared to nonresponders, indicating less tumor hypoxia (p=0.04). DISCUSSION/SIGNIFICANCE OF FINDINGS: Non-invasive PET imaging of the tumor microenvironment can provide data on T cell activation and hypoxic response predicting response to combination immunotherapy and chemotherapy. Utilizing advanced imaging to understand biologically distinct features of the TNBC tumor microenvironment can aid in personalizing anti-cancer therapies.

OBJECTIVES/GOALS: Natural killer (NK) cells are a potential cancer therapeutic but expanding NK cells efficiently in vitro is difficult. Natural killer cell deficiency (NKD), a primary immune deficiency affecting only NK cells, is caused by defects in several DNA replication proteins. By studying NKD we will achieve better NK cell in vitro differentiation. METHODS/STUDY POPULATION: One patient with NKD has a compound heterozygous mutation in the essential DNA replication protein MCM10. We hypothesize that in individuals with NKD, dramatic telomere erosion from abnormal DNA replication leads to premature senescence and the loss of NK cells. To test our hypothesis, we will knockout one allele of MCM10 or over express MCM10 in NK cells isolated from blood. We will then monitor telomere length, expansion and cytotoxic activity of these NK cells. To understand the role of MCM10 in early stages of NK cell development we will deplete MCM10 in induced pluripotent stem cells and differentiate these cells into NK cells. During this differentiation we will monitor progression through NK cell developmental stages as well as telomere length and senescence markers. RESULTS/ANTICIPATED RESULTS: Telomeres insulate chromosomes and induce permanent growth arrest (senescence) when they are critically short. We have demonstrated that depletion of a DNA replication protein causes telomere erosion and increases senescence markers. NK cells have shorter telomeres and lower telomerase expression than other immune cells. We predict, this relatively poor telomere maintenance sensitizes NK cells to telomere loss upon depletion of replication proteins. During in vitro differentiation, we expect NK cell precursors to undergo premature senescence secondary to telomere shortening. Furthermore, we expect supplementation of DNA replication proteins will enhance NK cell expansion and maturation. DISCUSSION/SIGNIFICANCE OF IMPACT: NKD patients have provided the scientific community with clues as to what proteins NK cells rely on for their development. This project aims not only to understand why these proteins are critical, but to harness that information for cellular anti-cancer therapeutics.

OBJECTIVES/GOALS: Researchers include community health workers and promotoras (CHW/Ps) on research teams to increase community engagement; however, no formal training on research best practices exists for this group. Study objectives were to examine perceived relevance of a new culturally and linguistically appropriate CHW/P training and optimal delivery modes. METHODS/STUDY POPULATION: We conducted six focus groups (FGs), three each in English and Spanish, at three study sites, University of Florida, University of Michigan, and University of California Davis from February to August 2021. The CHWs/Ps were purposively selected to include diverse age, race/ethnicity, educational level, and work experience. Separate FGs were conducted for CHWs/Ps in English and Spanish as appropriate. All FGs were audio recorded, translated to English from Spanish, transcribed and analyzed using RADaR (Rigorous and Accelerated Data Reduction) technique. RESULTS/ANTICIPATED RESULTS: Forty CHWs/Ps (95% women, mean age 45) participated, with the majority (58%) identifying as Hispanic/Latino. Of the sample, most identified as White (50%) or Black (25%). The proposed training was mentioned as relevant and would help them to be confident, comfortable, knowledgeable and effective in the community. Online training, though advantageous due to its flexibility also reportedly had barriers such as internet access, computer availability and technological know-how of CHWs/Ps. A hybrid training approach, online plus peer-led, was recommended due to the importance of personal guidance by an experienced CHW/P’ especially for a newly recruited CHW/P. DISCUSSION/SIGNIFICANCE: Findings indicated that a culturally and linguistically appropriate CHW/P training that is flexible and easily accessible in its mode of delivery is relevant and useful. In-person guidance to a new CHW/P was reported as an important training component. Poster will include the detailed quotes on relevance, usefulness, and mode of delivery of training.

OBJECTIVES/SPECIFIC AIMS: This study aims to determine if a bronchiolitis specific microbiome exists and how it evolves through disease course. Objective 1. Determine the microbiome profile of the airway in virus induced bronchiolitis-associated respiratory failure. Objective 2. Identify changes in the airway microbiome profile through the course of virus induced bronchiolitis associated respiratory failure, and the relationship between microbiome composition and clinical respiratory status. Objective 3. Determine the impact of rhinovirus infection on the lung and stool microbiome in a murine asthma model. METHODS/STUDY POPULATION: Objectives 1 &2: We are conducting a single-center prospective case-control study of patients admitted to the Komansky - Weill Cornell Pediatric Critical Care Unit. Infants less than two years of age with a diagnosis of bronchiolitis requiring intubation and mechanical ventilation are enrolled as subjects. Infants less than two years of age intubated and requiring mechanical ventilation without primary lung pathology are enrolled as controls. To evaluate our primary objective, tracheal aspirates will be collected from both subjects and controls on the day of intubation. We will perform 16s RNA sequencing on the tracheal aspirate samples and compare the resulting microbiomes. To evaluate secondary objective, we will collect tracheal aspirates of our study population on a daily basis and map the microbiome in parallel with objective measures of respiratory status including oxygen index and successful extubation. Both subjects and controls are being enrolled as a convenience sample. Objective 3: Mice, heterozygous for the sptlc2 gene (Spltc2 +/−) demonstrate reduced de-novo sphingolipids and increased airway hyperresponsiveness with methacholine challenge. Airway hyper-responsiveness is a cardinal feature of asthma. This airway hyperresponsiveness is exacerbated in the setting of rhinovirus (Figure 1). Using 16s sequencing, we will examine the lung microbiome of Sptlc2 +/− ad Sptlc2 +/+ at 1- and 7-days following rhinovirus infection. RESULTS/ANTICIPATED RESULTS: This clinical study is currently IRB approved and enrollment is ongoing. We have enrolled 12 subjects and 5 controls. Sample analysis will begin following the 2018-2019 respiratory season, with an anticipated cohort of 20 subjects and 20 controls. With regards to the murine studies, we have demonstrated that the lung microbiome of Sptlc2 +/− and Sptlc2 +/+ mice is similar at baseline (Figure 2) and remains similar following 1-day infection with rhinovirus. We do however, see a distinct change in the microbiome profile of the stool of Sptlc2 +/− mice following rhinovirus infection (Figure 3). Lung analysis at day 7 post infection is pending. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will lay the groundwork for detailing the functional role of the airway microbiome in bronchiolitis, with the objective of developing new modalities for disease treatment and prevention. In addition our murine studies allow us to view the microbiome in the context of sphingolipid deficiency, providing a potential mechanistic link to rhinovirus and ORMDL3 associated asthma.

OBJECTIVES/GOALS: Adoption of the Observational Medical Outcomes Partnership (OMOP) common data model promises to transform large-scale observational health research. However, there are diverse challenges for operationalizing OMOP in terms of interoperability and technical skills among coordinating centers throughout the US. METHODS/STUDY POPULATION: A team from the Critical Path Institute (C-Path) collaborated with the informatics team members at Johns Hopkins to provide technical support to participating sites as part of the Extract, Transform, and Load (ETL) process linking existing concepts to OMOP concepts. Health systems met regularly via teleconference to review challenges and progress in ETL process. Sites were responsible for performing the local ETL process with assistance and securely provisioning de-identified data as part of the CURE ID program. RESULTS/ANTICIPATED RESULTS: More than twenty health systems participated in the CURE ID effort.Laboratory measures, basic demographics, disease diagnoses and problem list were more easily mapped to OMOP concepts by CURE ID partner institutions. Outcomes, social determinants of health, medical devices, and specific treatments were less easily characterized as part of the project. Concepts within the medical record presented very different technical challenges in terms of representation. There is a lack of standardization in OMOP implementation even among centers using the same electronic medical health record. Readiness to adopt OMOP varied across the institutions who participated. Health systems achieved variable level of coverage using OMOP medical concepts as part of the initiative. DISCUSSION/SIGNIFICANCE: Adoption of OMOP involves local stakeholder knowledge and implementation. Variable complexity of health concepts contributed to variable coverage. Documentation and support require extensive time and effort. Open-source software can be technically challenging. Interoperability of secure data systems presents unique problems.

OBJECTIVES/SPECIFIC AIMS: MicroRNAs (miRNA) affect transcription of a number of genes involved in the development and progression of diabetic nephropathy (DN), and have become attractive therapeutic targets and biomarkers. Elevated renal gluconeogenesis, fibrosis, and albuminuria are early markers of incipient DN. Recent studies report that renal miRNA-451 may protect against DN and reduce renal gluconeogenesis in rodent models. MiRNA-451 is thought to act by targeting select factors resulting from disrupted insulin and growth factor signaling and the mechanistic-target of rapamycin (mTOR) in early DN. This study aimed to elucidate the role of miRNA-451 in the development and progression of DN. METHODS/STUDY POPULATION: To further elucidate the role of miRNA-451 in DN, we placed male insulin-resistant, TALLYHO/Jng mice on a high-fat diet (60% kCal). The mice were divided into 2 treatment groups and received 8 consecutive weekly intraperitoneal injections of locked nucleic acid (LNA) miR-451-inhibitor or LNA-scrambled compound (2 mg/kg·bw; n=8/treatment). Mice were euthanized after 12 weeks (4 weeks sans injections) and kidneys, liver, pancreas and abdominal adipose tissue were harvested for analysis. RESULTS/ANTICIPATED RESULTS: Renal homogenate expression of miRNA-451 was drastically reduced in inhibitor-treated mice (~6-fold) in comparison with scramble-treated mice. Western blotting of cortex homogenates for indicators of fibrosis and targets of miRNA-451 revealed a significant reduction in collagen IV (marker of epithelial integrity) in inhibitor-treated mice. In addition, metalloproteinase type 9 (MMP9, a known type IV collagenase), YWHAZ (a scaffolding protein and known target of miR-451), mTOR, and fructose bisphosphatase (FBP1, a rate-limiting gene in gluconeogenesis) were significantly increased in this group in comparison to scramble-treated mice. However, no differences were found in protein levels for glucose-6-phosphatase (G-6-Pase) or phosphoenolpyruvate (PEPCK), 2 additional gluconeogenic rate-limiting genes. MiRNA-451 antagonist did not significantly affect final body weight or blood glucose; however, mean blood sodium concentrations were slightly, but significantly higher (2%) in the LNA-inhibitor treated group (when compared with the scramble-treated group). No differences in blood potassium or chloride were found. Anion gap was 90% higher in the LNA-inhibitor treated group when compared with scramble-treated mice. No differences in urinary albumin to creatinine ratio were found between the two treatment groups. However, Masson Trichrome scoring revealed a 59% increase in fibrosis in inhibitor-treated mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these findings support a potentially protective role of miRNA-451 in attenuating signaling via mTOR that may alter both renal gluconeogenic potential (contributing to the diabetic phenotype) and activation and progression of renal fibrosis. Therapies to enhance miRNA-451 signaling may be beneficial to reduce renal pathology associated with DN.