ABSTRACT IMPACT: This work will develop a novel drug delivery system that has improved biocompatibility and controlled release than current systems and allow for customizable loading and drug delivery to unique patient and treatment requirements. OBJECTIVES/GOALS: The goal of my project is a novel hybrid core/shell nanoparticle system for controlling in vivo chemotherapeutic concentration. The current goal is to confirm core and shell polymeric nanoparticle formation via emulsion technique and validate predictive model developed to optimize shell formation efficiency and control shell thickness. METHODS/STUDY POPULATION: Though early results are promising, they are not proof that the desired core/shell structure is being formed via my novel process. I constructed a theoretical model to use to optimize and control the process for precise shell thicknesses. Therefore, the current experimental plan focus is to not only visually confirm the predicted formation of my core/shell design but use these experiments to validate the model.

1. 1. Gel-Suspended SEM: nanoparticles suspended in gel matrix, bisected to reveal inner structure

2. 2. Fluorescent Conjugation Microscopy: visually-distinct dyes used to show polymer distribution and validated against the theoretical model predictions.

3. 3. Modified Hydrophobic Dye Release: different mixtures of polymers with release showing if previous promising results due to core/shell structure RESULTS/ANTICIPATED RESULTS: As stated, the experiments will confirm the core/shell nanoparticle structure, validate the developed theoretical model, or provide direct evidence against any formation. This core/shell structure is key to the current design for controlling payload release rate and thus in vivo drug concentration. For the gel-suspension experiment, the interior core will be labeled with ultrasmall SPIONs and thus any layers within the particles will be distinct. While this result is qualitative, high magnification fluorescent microscope images will be analyzed using image processing software to determine core/shell formation efficiency and compared to estimated efficiencies from the model. Finally, the mixed release will clarify previous experiments’ release mechanism and either support or disprove shell influence. DISCUSSION/SIGNIFICANCE OF FINDINGS: The significance of this work is twofold: core/shell particles have been proven to provide variable control of release on the micron scale but not yet at the nanoscale, allowing for a circulating, targeted system that can finely control release. The process is also novel for producing this type of structure, at highly consistent quality and size.

ABSTRACT IMPACT: This work will help to understand a novel therapeutic approach to a common type of acute myeloid leukemia. OBJECTIVES/GOALS: FMS-like tyrosine kinase 3 (or FLT3) mutations occur in ˜30% of acute myeloid leukemia (AML) cases. FLT3 tyrosine kinase domain (TKD) mutations are particularly important in relapsed/refractory FLT3 mutant AML, which portends poor prognosis. This study describes a therapeutic approach to overcoming resistance conferred by FLT3-TKD mutations. METHODS/STUDY POPULATION: To understand the efficacy of a novel type 1 FLT3 inhibitor (NCGC1481), as a monotherapy and combination therapy, several assays were utilized to interrogate functionality of these therapies. Cell lines and patient samples containing aspartate 835 to tyrosine mutations (D835Y, the most common TKD alteration) and phenylalanine 691 to leucine (F691L) were utilized to examine the effects of NCGC1481 with and without other targeted therapies like MEK inhibitors. Specifically, assays measuring viability, cell death using flow cytometry, in vitro clonogenicity, cellular signaling, and xenograft survival were examined in these FLT3-TKD AML models. Synergy was also measured using well described methods, which also allowed for appropriate dose finding for drug combination experiments. RESULTS/ANTICIPATED RESULTS: Our novel type 1 FLT3 inhibitor (NCGC1481) was particularly effective in the most common FLT3 TKD mutant, D835Y. NCGC1481 reduced viability and cell signaling, while also inducing cell death and prolonging xenograft survival in the FLT3-D835Y model system. In contrast, clinically approved FLT3 inhibitors were less effective at suppressing AML cells expressing FLT3-D835Y. In the case of FLT3-F691L, most of the FLT3 inhibitors tested, including NCGC1481, suppressed canonical FLT3 signaling, but did not significantly reduce viability or leukemic clonogenicity. However, when NCGC1481 was combined with other targeted agents like MEK inhibitors, at synergistic doses, eradication of the FLT3-F691L AML clone was substantially increased. DISCUSSION/SIGNIFICANCE OF FINDINGS: In AML, response to FLT3 inhibitor therapy is often short-lived, with resistance sometimes occurring via FLT3-TKD mutations. Given the dismal prognosis of relapsed FLT3 mutant AML, novel therapies are necessary. This study describes efficacy of a novel FLT3 inhibitor, along with its synergistic activity when combined with other targeted agents.

ABSTRACT IMPACT: This work sheds diagnostic insight on patients with idiopathic rare disease and has the potential to further their care and treatment as a result. OBJECTIVES/GOALS: Correct diagnosis is imperative to treating patients with idiopathic, suspected genetic conditions, yet sequencing approaches leave up to 70% of these patients undiagnosed. We sought to improve diagnosis rates for a cohort patients referred for sequencing by characterizing deleterious variants within the 5’UTR. METHODS/STUDY POPULATION: We retrospectively analyzed whole exome sequencing (WES) data from 472 unsolved rare disease patients within the Mayo Clinic Center for Individualized Medicine to identify variants within the 5’UTR that affect the presence of upstream open reading frames (uORFs). uORFs are short regions (typically 30bp - 600bp) that typically influence downstream gene translation by sequestering ribosomes. We specifically searched for variants with the potential to disrupt existing uORFs or introduce new uORFs within the 5’UTR, and developed a pipeline to annotate these variants with information including GnomAD allele frequency and gene loss of function intolerance (pLI) score. To aid in variant interpretation, we applied two deep learning tools to predict variant impacts on transcript ribosome load (TITER and FramePool). RESULTS/ANTICIPATED RESULTS: Our pipeline identified a median of 21 variants per patient that were predicted to have a deleterious impact on the translational efficiency of protein coding transcripts, primarily by introducing new start codons within the 5’UTR or by altering the Kozak consensus of existing start codons. A median of 10 of these variants occur upstream of haploinsufficient genes with an existing disease association. We also identified a subset of variants that are predicted to introduce translationally active N-terminal extensions to protein coding transcripts, with the potential to disrupt protein localization and processing. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work demonstrates that analysis of 5’UTR variants can be incorporated into existing WES pipelines, and identifies a group of variants with potential significance to patient disease. Further experimental evidence is necessary to ascertain the pathogenicity of these variants.

ABSTRACT IMPACT: Preliminary results will inform the formal evaluation of the reliability of point-of-care ultrasound (POCUS) done by the gastroenterologist compared to standard of care methods such as MR-Enterography. OBJECTIVES/GOALS: Evaluation of mucosal healing is standard for pediatric patients with inflammatory bowel disease (IBD). Point-of-care ultrasound is a non-invasive, cost-efficient tool for assessing intestinal inflammation. We aim to evaluate the agreement between POCUS and typical cross-sectional imaging, such as MR-Enterography (MRE). METHODS/STUDY POPULATION: In this cross-sectional study, we recruited consecutive patients newly diagnosed with IBD, presenting to the specialty outpatient clinic or hospitalized in a pediatric tertiary care center between August to November 2020. They underwent POCUS performed by a single gastroenterologist, in addition to MRE. The sonographer was blinded to MRE results. Bowel wall thickness (BWT) was measured across different bowel segments and recorded twice in longitudinal view and twice in axial view. An average segmental BWT of the four measurements of more than 3 mm was considered inflamed. Agreement between sections of the bowel measured as inflamed were compared to inflamed bowel segments seen by MRE, using Cohen’s kappa. RESULTS/ANTICIPATED RESULTS: Eight of 12 patients completed both MRE and POCUS.A total of 40 bowel segments were assessed, namely the terminal ileum, ascending, transverse, descending and sigmoid colon. There were 4 girls with a median age of 15 years (IQR 14.25-16 years), and 6 patients were diagnosed with Crohn’s disease. Median PCDAI was 32.5 (IQR 30.6-40), and median PUCAI was 75 (72.5-77.5). Agreement between MRE and point-of-care ultrasound was substantial to perfect for the terminal ileum *(κ= 0.75, 95%CI 0.31-1), transverse colon (κ= 1, 95%CI 1-1) and sigmoid colon (κ= 1, 95%CI 1-1). The agreement was poor for the ascending (κ= 0, 95%CI 0-0) and moderate for the descending colon. (κ= 0.6, 95%CI -0.07-1) DISCUSSION/SIGNIFICANCE OF FINDINGS: In pediatric patients with IBD, we found a high agreement between POCUS and MRE for imaging of the terminal ileum, transverse and sigmoid colon, areas commonly involved in IBD. This reinforces adult data, outlining the potential of POCUS as an evaluation tool of disease activity in clinical practice.

ABSTRACT IMPACT: The key to advancing precision medicine is to deepen our understanding of drug modes-of-action (MOA). This project aims to develop a novel method for predicting MOA of potential drug compounds, providing an experimental and computational platform for more efficient drug discovery. OBJECTIVES/GOALS: To develop (1) a targeted CRISPR-Cas9 chemical-genetic screen approach, and (2) a computational method to predict drug mode-of-action from chemical-genetic interaction profiles. METHODS/STUDY POPULATION: Screening drugs against a gene deletion library can identify knockouts that modulate drug sensitivity. These chemical-genetic interaction (CGI) screens can be performed in human cell lines using a pooled lentiviral CRISPR-Cas9 approach to assess drug sensitivity/resistance of single-gene knockouts across the human genome. A targeted, rather than genome-wide, library can enable scaling these screens across many drugs.

CGI profiles can be derived from phenotypic screen readouts. These profiles are analogous to genetic interaction (GI) profiles, which represent sensitivity/resistance of gene knockouts to a second gene knockout rather than a drug. To computationally predict a drug’s genetic target, we leverage the property that a drug’s CGI profile will be similar to its target’s GI profile. RESULTS/ANTICIPATED RESULTS: Five proof-of-principle screens will be conducted with compounds that have existing genome-wide profiles and well-characterized MOA. I will generate CGI profiles for these five compounds and identify genes that are drug-sensitizers or drug-suppressors. I will then evaluate whether targeted library screens can recapitulate the CGIs found in genome-wide screens. Finally, I will develop a computational tool to integrate these CGI profiles with GI profiles (derived from another project) to predict gene-level and bioprocess-level drug targets. These predictions (from both targeted and genome-wide profiles) will be benchmarked against a drug-target and drug-bioprocess standard. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work will develop a scalable, targeted chemical-genetic screen approach to discovering how putative therapeutics work. The targeted screen workflow provides a method for higher-throughput drug screening. The computational pipeline provides a powerful tool for exploring the MOA of uncharacterized drugs or repurposing FDA-approved drugs.

ABSTRACT IMPACT: This work identifies host immune perturbations in sepsis mortality that suggests targets for a precision medicine paradigm in the host response to infection. OBJECTIVES/GOALS: To compare the early whole blood transcriptome during sepsis between 30-day survivors and non-survivors in the Intensive Care Unit (ICU), and to evaluate for pathway enrichment that might explain sepsis lethality. METHODS/STUDY POPULATION: We enrolled 162 sepsis patients in the first 24 hours of ICU admission, particularly targeting individuals requiring vasopressors. Peripheral whole blood was collected in PAXgene vacutainers. Isolated RNA was analyzed with Affymetrix Human Genome ST 2.0 microarray. Differential gene expression was performed with Bioconductor/R/limma using log2 fold-change +/-0.6 as a threshold for differential expression, and a Benjamini-Hochberg adjusted p-value <0.05 to declare significance. Functional gene enrichment was performed using the Gene Ontology (GO) database with PANTHER overrepresentation test (Fisher’s Exact) on all transcripts with adjusted p-value <0.05. Pathways analysis was performed with the Reactome Project using the raw fold change and significance data to identify dysregulated pathways. RESULTS/ANTICIPATED RESULTS: There were 58 non-survivors (36% mortality). We identified 39 genes as differentially expressed between sepsis non-survivors and survivors; 31 were upregulated in non-survivors and 8 had reduced expression. Several of the most overexpressed transcripts are neutrophil-specific, including LCN, MPO, OLF4M4, DEFA3, and DEFA4. A functional gene overrepresentation test further supports this finding, as the most enriched gene ontologies were neutrophil-mediated killing, neutrophil cytotoxicity, neutrophil extravasation, and respiratory burst, all demonstrating higher than 10-fold enrichment and FDR < 0.02. Pathway analysis of the peripheral blood transcriptome was notable for immune response derangement, specifically downregulation of both innate and adaptive immune pathways (FDR < 0.00001). DISCUSSION/SIGNIFICANCE OF FINDINGS: We identified increased expression of neutrophil-related genes in sepsis non-survivors, replicating candidates previously identified in pediatric sepsis mortality and ARDS. These immune perturbations in sepsis mortality may represent key targets for eventually employing precision medicine strategies in sepsis.

ABSTRACT IMPACT: Genomic variation likely plays a role in differences in rates of adverse reactions and efficacy for African populations, and this research will add to the understudied field of pharmacogenomics in African populations. OBJECTIVES/GOALS: We aim to characterize the frequency of variants in clinically relevant genes in East and West African populations to assess the prevalence of potential drug-gene interactions. METHODS/STUDY POPULATION: Our pilot study will consist of 100 Somali patients enrolled at Mayo Clinic Rochester and 100 Ghanaian patients recruited at Teaching Hospitals in Ghana. Germline DNA will be extracted from pre-existing blood samples. Sequencing will be performed using Admera Health’s PGxOne Plus test, interrogating a panel of 62 genes. Variants will be reported along with the predicted response for a list of drugs. Differences between frequencies of variants in East and West African populations will be analyzed. We will look for correlations with reported adverse reaction rates. We will then compare our findings with allele frequency data from publicly available data bases. Additionally, we will analyze the flanking regions of the panel for novel variants, using machine learning to predict gene-drug interactions. RESULTS/ANTICIPATED RESULTS: African populations are known to have more genetic diversity than any other population. Additionally, only African-Americans, African-Caribbeans from Barbados, Esan and Yoruba Nigerians, Gambians, Kenyans, and Sierra Leoneans are accounted for within the publicly available data bases most often used for variant studies. Thus, it is anticipated that we will find differences in the variant allele frequencies of our populations compared to European allele frequencies, and differences in frequencies between the East and West African populations. In the 200 base pair flanking regions that are sequenced in the assay along with the known variant regions, we may find novel previously unreported variants. DISCUSSION/SIGNIFICANCE OF FINDINGS: The lack of knowledge of pharmacogenomic variation in African populations contributes to ethnic disparities in patient outcomes. This study addresses this gap by adding to our comprehension of variants in clinically relevant genes, giving insight into underlying mechanisms of ethnicity-based drug responses.

ABSTRACT IMPACT: Here, we describe extensive sex-specific differences in the transcriptomes of pancreatic neuroendocrine tumors (PNETs). Given that the clinical course of PNETs differs by sex (female sex is associated with better survival), achieving a greater understanding of the specific molecular sexual dimorphisms is invaluable for advancing personalized treatment. OBJECTIVES/GOALS: Epidemiologic studies demonstrate that pancreatic neuroendocrine tumors (PNETs) exhibit sexual dimorphisms with regards to prognosis, disease recurrence, and complication rates. We sought to compare the transcription and DNA methylation landscapes of PNETs by sex, to elucidate molecular differences that may underlie this sex disparity. METHODS/STUDY POPULATION: RNAseq data was generated from PNETs surgically resected at our institution (9 Female; 12 Male patients). RNA was extracted with the RNeasy Mini Kit, stranded sequencing libraries were prepared with TruSeq, and paired end sequencing was done on the HiSeq 2500/4000 systems. Transcript-level quantification was performed with salmon, and DESeq2 was used for differential expression analysis. To account for significant variation due to covariates other than sex, surrogate variables were computed with the SVA package and adjusted for. The goseq package was used for gene set over representation analysis. Matched DNA methylation (DNAm) and RNAseq data was downloaded from GEO (16 F; 16 M). Raw DNAm data was processed with minfi. Differential methylation analysis was done with limma and bumphunter. Analysis was done in R. RESULTS/ANTICIPATED RESULTS: We found that 826 autosomal genes were differentially expressed (DE) by sex in PNETs (at FDR ≤0.1). Gene set over representation analysis performed on the DE genes revealed significant enrichment for several processes, including ‘ascorbate & aldarate metabolism’ and ‘positive regulation of ERK1 & ERK2 cascade’ (all FDR ≤0.1). When we compared DNAm profiles between sexes, we found 8 CpGs which were differentially methylated by sex (at FDR ≤0.1), 7 of which were proximal to genes. Methylation of one of the sex-associated CpGs, overlapping the gene TIMM8B, was found to be negatively correlated with gene expression (rho=-0.41; p-value=0.02). Interestingly, TIMM8B deletion has been previously reported in other non-pancreatic neuroendocrine tumors. There were no differentially methylated regions between sexes. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our findings demonstrate that PNETs exhibit extensive sexual dimorphisms with regards to gene expression profiles but have largely congruent methylomes by sex. These molecular differences may contribute to the variability in clinical course between men and women, and their characterization is vital for the advancement of personalized medicine.

ABSTRACT IMPACT: This work assesses clinical implementation of a surface guided imaging system to improve the accuracy radiation delivery for treatment of brain lesions using a patient CT derived head phantom. OBJECTIVES/GOALS: Advancements in radiotherapy design have made clinical demand for efficient and accurate methods to deliver stereotactic radiosurgery (SRS) for treatment of intracranial lesions. This study assesses the potential of using surface guided imaging for setup using a 3D patient specific head phantom. METHODS/STUDY POPULATION: A single isocenter, multiple metastases SRS plan was generated on a CT derived RTsafe Prime phantom made of tissue equivalent materials and a polymer gel insert. Five targets of varying diameters were treated with 8Gy of radiation using two different positioning techniques. The first gel insert was irradiated within the phantom according to internal alignment with standard orthogonal x-ray imaging while the second setup used surface guided imaging, based on external anatomy. 42 hours after irradiation, the phantom was scanned in a head coil using a 1.5T MRI. MR images were fused with the patient CT data and structure set to further evaluate calculated and measured dose distributions. RESULTS/ANTICIPATED RESULTS: Discrepancies in phantom setup according to standard orthogonal x-ray imaging compared to surface guided imaging demonstrated to be <1mm in each translational (vertical, longitudinal, and lateral) and angular (rot, roll, pitch) directions. The 3D gel inserts permitted spatial analysis to compare dose distributions of measured values to those calculated in a treatment planning system (TPS). 3D GI (Gamma Index) analysis showed good alignment in high dose regions and resulted in passing rates >94% (5%/2mm) and >87% (3%/2mm). Finally, 3 of 5 targets showed better 3D GI passing rates and less geometric offset for positioning with the surface guided imaging. DISCUSSION/SIGNIFICANCE OF FINDINGS: 3D spatial analysis of human like phantoms demonstrated that patient positioning according to external anatomy performed comparable to standard methods aligning to the internal anatomy, for a multiple met SRS treatment.

ABSTRACT IMPACT: The implementation of DPYD and UGT1A1 pharmacogenetic testing, a promising tool of precision medicine, translates evidence-based research into clinical oncology practice with personalized dosing to better predict interpatient variability in chemotherapy tolerability. OBJECTIVES/GOALS: Patients with DPYD and UGT1A1 genetic variants are at risk for severe toxicity from fluoropyrimidines and irinotecan, respectively. We propose that providing clinicians with the option to order a pharmacogenetic (PGx) test with relevant dose recommendations will increase test uptake to guide pharmacotherapy decisions and improve safety outcomes. METHODS/STUDY POPULATION: We plan to conduct a non-randomized, pragmatic, open-label study in 600 adult patients with gastrointestinal (GI) cancers initiating a fluoropyrimidine- and/or irinotecan-based regimen at three cancer centers within a health system. Implementation metrics of a new, in-house laboratory developed PGx test will be measured, including feasibility of returning results within one week, fidelity of providers following dose recommendations, and penetrance via test ordering rates. Clinical aims will include assessing severe toxicity during the first six months of chemotherapy. Outcomes will be compared to a historical control of GI cancer patients enrolled in a biobank and treated with standard dose chemotherapy. RESULTS/ANTICIPATED RESULTS: We anticipate that there will be an increase in PGx test uptake given its shorter turnaround time to facilitate clinical decision-making prior to the first dose of chemotherapy. Through integration of test results in the electronic health record (EHR) and clinical decision support tools for patients with actionable genotypes, we also expect that providers will have a high level of agreement to the recommended dose adjustments. We anticipate a decreased incidence of severe (Grade >3) toxicity among prospectively genotyped patients in the first six months of chemotherapy compared to DPYD and UGT1A1 variant carriers in the historical control group. Exploratory clinical utility data on costs of hospitalizations, chemotherapy treatment, PGx test, and medical services will also be reported. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study aims to address barriers identified by key stakeholders to implementing PGx testing to better tailor chemotherapy dosing to the genetic profiles to patients. This may prevent adverse event-related hospitalizations, improve quality of life for patients, and reduce health system resource utilization costs.

ABSTRACT IMPACT: The implementation of nasal nitric oxide (nNO) as a diagnostic tool to understand the phenotypic/genotypic profiles of Primary Ciliary Dyskinesia (PCD) in Puerto Rico (PR) will be translated in early disease diagnosis, avoidance of comorbidities, and increase survival in our population. OBJECTIVES/GOALS: This study aims to evaluate the role of nNO levels in PCD diagnosis in the Puerto Rican population. Also, we aim to describe the clinical, genetic, and physiological characteristics of PCD in Puerto Ricans to develop a better understanding of the disease. METHODS/STUDY POPULATION: We plan to conduct a cross-sectional study on participants recruited from patients of the Pediatric Rare Lung and Asthma Institute in PR. We will compare nNO levels among genetically confirmed PCD patients, suspected PCD patients with variant of unknown significance (VUS) mutations, suspected PCD patients without genetic mutations, and age-matched healthy subjects. We plan to analyze clinical data and genetic variants to understand the natural history of the disease. The nNO measurements will be completed following previous published protocols. We will also assess the accuracy of the nNO measurements by repeating the measurements two weeks after the initial measurement. RESULTS/ANTICIPATED RESULTS: We hypothesize that many of the VUS present in our population may represent potential new founder mutations not previously reported in the literature. Our expectation is to identify new atypical PCD phenotypes contemplating the heterogenous genetic Puerto Rican pool. We anticipate that nNO levels will help to screen, identify, and confirm diagnosis of patients with clinical PCD in PR. Our findings will be translated in avoidance of further comorbidities and mortality due to earlier disease PCD diagnosis and will expand our genetic understanding about PCD in PR and other diverse populations with heterogenous genetic admixture. DISCUSSION/SIGNIFICANCE OF FINDINGS: We present a significant and novel research proposal that plan to impact the quality of life of patients living with PCD in PR. The implementation of state-of-the-art diagnostic tools like nNO measurement will positively impact and expand our current capabilities to diagnose rare lung diseases like PCD on the island.

ABSTRACT IMPACT: This study deepens knowledge with respect to the associations between depression, cardiometabolic conditions, and accelerated aging with a clinically accessible marker in a population with disproportionate risk for comorbidity. OBJECTIVES/GOALS: The aim of this secondary analysis is to examine associations between DNA methylation age acceleration (DNAm AA) and depressive symptoms in African American women (AAW) considering the presence of cardiometabolic conditions (CMCs) including hypertension, diabetes, obesity. METHODS/STUDY POPULATION: Genomic and longitudinal clinical data (collected 2015-2020) from the Intergenerational Impact of Genetic and Psychosocial Factors on Blood Pressure Study (InterGEN) cohort (n=227) were utilized for this analysis. DNA methylation age (estimated by the Horvath method) incorporates DNA methylation status at 353 CpG sites. DNAm AA is the residual of DNA methylation age regressed on chronological age in a linear model. Spearman’s correlations and linear regression examine the relationship between DNAm AA and depressive symptoms (Beck Depression Inventory) and cardiometabolic status. The potential association and impact of SES, trauma, substance use, and stress were also considered. RESULTS/ANTICIPATED RESULTS: Contrary to our hypothesis, DNAm AA did not associate with the severity of depressive symptoms. Correlation between DNAm AA and affective symptom subscore (BDI) approached significance (p = 0.06). We observed significant correlations between DNAm AA and specific depressive symptoms including participants’ reported disappointment, disgust, or hatred toward themselves (p < 0.05), difficulty with making decisions (p < 0.05), and worry about their physical health (p < 0.05). DNAm AA was also significantly correlated with BMI (p > 0.001). Significant relationships were not evident in the subsequent regression analysis examining potential relationships between DNAm AA and depression. To our knowledge, this is the first study to examine associations between DNAm AA and depressive symptoms in AAW. DISCUSSION/SIGNIFICANCE OF FINDINGS: Depression limits life quality and quantity and is highly comorbid in CMCs. AAW have a high risk of comorbidity. This study deepens knowledge with respect to the associations between depression, CMCs, and aging with a clinically accessible marker in a population with disproportionate risk.

ABSTRACT IMPACT: The pathophysiologic features of a metabolomic endotype that predicts patient outcomes due to sepsis have the potential to direct new therapies that target immune dysregulation and bioenergetic insufficiency. OBJECTIVES/GOALS: Acute respiratory failure (ARF) requiring mechanical ventilation is a frequent complication of sepsis and other disorders. It is associated with high morbidity and mortality. Despite its severity and prevalence, little is known about metabolic and bioenergetic changes that accompanying ARF. METHODS/STUDY POPULATION: In this study, semiquantitative and quantitative ultrahigh performance liquid chromatography mass spectrometry (UHPLC MS) analysis was performed on patient serum collected from the Trial with Acute Respiratory failure patients: evaluation of Global Exercise Therapies (TARGET). Serum from survivors (n=15) and nonsurvivors (n=15) was collected at day 1 and day 3 after admission to the medical intensive care unit as well as at discharge in survivors. Pathway analysis of the biochemical changes was performed to determine whether the disruption in specific metabolic pathways can identify the bioenergetic and metabolomic profile of these patients. RESULTS/ANTICIPATED RESULTS: Significant metabolomic differences were related to biosynthetic intermediates of redox cofactors nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP), increased acyl-carnitines, and decreased acyl-glycerophosphocholines in nonsurvivors compared to survivors. The metabolites associated with poor outcomes are substrates of enzymatic processes dependent on NAD(P), while the abundance of NAD cofactors rely on the bioavailability of dietary vitamins B1, B2 and B6. Changes in the efficiency of the nicotinamide-derived cofactors’ biosynthetic pathways also associate with an alteration of the glutathione-dependent drug metabolism as characterized by the substantial differences observed in the acetaminophen metabolome. DISCUSSION/SIGNIFICANCE OF FINDINGS: This metabolomic endotype represents a previously unappreciated association between severity of outcomes and micronutrient deficiency, thus pointing to new pharmacologic targets and highlighting the need for nutritional remediation upon hospitalization to improve patient outcomes due to ARF.

ABSTRACT IMPACT: As newborn screening is now available for X-linked adrenoleukodystrophy, there is a need to establish meaningful disease markers to detect the onset of the severe demyelinating cerebral form of this disease at the earliest possible stage, and to quantify early disease progression to evaluate the relative efficacy of therapies. OBJECTIVES/GOALS: Longitudinal testing of neurocognitive and motor function using smartphone and tablet-based applications holds promise for early detection and quantification of brain white matter changes in patients with adrenoleukodystrophy (ALD) and other rare demyelinating diseases, but this methodology requires validation in pediatric populations. METHODS/STUDY POPULATION: We developed an iPad application with a game-like interface to assess interhemispheric transfer across the corpus callosum, the brain structure where cerebral demyelinating disease typically begins in patients with ALD. Feasibility data from remote test administrations with healthy children were collected to analyze and speed and timing of finger tapping movements requiring bimanual coordination on a touchscreen. RESULTS/ANTICIPATED RESULTS: Among our pilot sample of healthy school-aged children, age-related improvements in finger tapping speed were observed in both single-hand and alternating-hand conditions. Results indicate that remote testing using iPad applications is a viable way to collect psychometric testing data rapidly in pediatric populations and is feasible during a pandemic. Next steps in this research project will be: (1) evaluating the stability of repeated test administrations (test-retest reliability), (2) assessing agreement between performance on our iPad application and validated measures of interhemispheric transfer and fine motor function, and (3) comparing performance of children with known corpus callosum white matter abnormality to performance of healthy children. DISCUSSION/SIGNIFICANCE OF FINDINGS: Brief neurocognitive tests that can be frequently administered may have the ability to capture subtle brain changes in developing children. Approaches enabling remote (virtual) testing will facilitate research during the covid-19 pandemic and are especially well-suited for data collection in rare disease populations.

ABSTRACT IMPACT: Alterations in mitochondrial metabolism affect any tissue, especially those with the highest demand for energy. As the symptoms and clinical manifestations are heterogenous, disease diagnosis is challenging. The implementation of genetic-first approach in the diagnosis of mitochondrial diseases will expedite confirmation, treatment, management, and counseling of affected Puerto Rican individuals. OBJECTIVES/GOALS: Mitochondrial diseases are rare, and diagnosis is complex due to the heterogeneity of clinical manifestations. We aim to develop and implement a testing algorithm using a genetics-first approach, facilitating the identification of variants that contribute to mitochondrial disease’s etiology and influence onset and progression in Puerto Ricans. METHODS/STUDY POPULATION: This is a cross-sectional study for characterizing clinical laboratory results from profiles used to evaluate metabolic diseases in individuals with suspected mitochondrial disorders from 2018 to 2021. A subset of 25 individuals from biochemical profile will be recruited to analyze their medical and family history, metabolic biomarkers in blood and urine, hearing test, imaging and chromosomal microarray. The implementation of a genetic testing algorithm using whole exome and mitochondrial DNA sequencing will be performed in a subset of 11 randomized individuals. Descriptive analysis will be reported, including a catalog of all variants. Multivariate analysis will be performed to estimate the statistical association between variants and phenotypes reported and adjusting for potential confounders. RESULTS/ANTICIPATED RESULTS: The biochemical profile of pediatric Puerto Rican individuals suspected of having mitochondrial diseases will be altered and can be used to differentiate among other metabolic causes. We expect to find altered levels of lactate, pyruvate and carnitines in serum, as well as altered organic acids in urine. The implementation of a testing algorithm using both, mitochondrial DNA and whole exome sequencing as first approach will be enabling the identification of disease-causing variants, thus enhancing and confirming the diagnosis of mitochondrial disease in Puerto Ricans. We will be able to identify rare/novel variants specific to our Hispanic population, for both nuclear and mitochondrial DNA. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study will help to characterize the metabolic profile of pediatric Puerto Ricans. No previous study has been reported that describes testing algorithms for genetic diagnosis of mitochondrial disease in our population. Variants found will contribute to a deep understanding of the genetic contribution to phenotypes and disease susceptibility.

ABSTRACT IMPACT: Through its interdisciplinary, tailored approach, the FLOW program could change the way that we approach promoting healthy lifestyle changes in the primary care field. OBJECTIVES/GOALS: The goal of this project is to assess patient outcomes associated with the implementation of the Fitness, Lifestyle, and Optimal Wellness (FLOW) Program. The ultimate aim of this program is two-fold: increasing patient-reported wellness and improving objective health measurements. METHODS/STUDY POPULATION: The FLOW program consists of a multidisciplinary team of sports medicine physicians, nutritionists, fitness trainers, and clinical psychologists. Patients who choose to participate in the program undergo a comprehensive physician-guided assessment, including lifestyle and metabolic evaluation, biomarker profile, and body composition analysis. Based on the patient’s goals and results of evaluation, he/she is then connected with other members of the FLOW team to develop a comprehensive plan and offer resources for potential improvements in physical activity, nutrition, and/or behavior. The patient will undergo follow-up assessments and questionnaires at three and six months to track their objective measurements and reported progress. RESULTS/ANTICIPATED RESULTS: The anticipated results of the FLOW program are an overall improvement in patient health and wellbeing. More specifically, we anticipate seeing increased levels of exercise from initial reported levels, as well as better nutrition habits. We expect to see improvements in follow-up body composition assessments, with gains in fat-free mass and decreased body fat, in addition to patient-reported improvements in behavioral health as measured by PHQ-9, GAD-2, and the Perceived Stress Scale. We will also assess reported sleep health with the hopes to see improvement in follow-up assessments. DISCUSSION/SIGNIFICANCE OF FINDINGS: The FLOW program is designed to address health inequities that disproportionately affect the Deep South. Through this program, we propose a new role of the primary care team in promoting healthy lifestyle habits and disease prevention through exercise, nutrition, and behavioral health services.

Regulatory Science

ABSTRACT IMPACT: It provides insight in the relationship between pediatrics and clinical research and how pediatric participation in CT translates to clinical significance in form of drug labels, which inform clinicians on how to prescribe pediatric medications. OBJECTIVES/GOALS: Assessing the extent that the Best Pharmaceuticals for Children Act (BPCA) advances pediatric inclusion in clinical trials (CTs) and the availability of pediatric-specific drug information METHODS/STUDY POPULATION: The BPCA provides the U.S. Food and Drug Administration (FDA) authority to solicit sponsors whose drugs may benefit pediatric populations. Participation is voluntary and provides additional market exclusivity and pediatric information. CTs that received marketing exclusivity from 2016-2018 under BPCA were reviewed using Clinicaltrials.gov to access the legislation’s impact. CTs were categorized according to eligibility: (1) pediatric and adult groups, (2) pediatrics, and (3) pediatric sub-groups. Studies were excluded for ambiguous age data. Studies open to both groups were evaluated for pediatric participation. Each drug was searched in DailyMed.com for published pediatric indications. RESULTS/ANTICIPATED RESULTS: Between 2016 - 2018, 22 drugs received marketing exclusivity under BPCA. Of the 196 CTs conducted for these drugs, 135 were available to adults and pediatrics, 10 were available to the entire pediatric population, and 51 were available to specific pediatric sub-populations. Exclusion criteria permitted only 118 of the CTs for assessment where eligibility included both pediatric and adult populations, of which 65 of these had less than 1% pediatric representation. Of the 22 drugs, 20 have pediatric indications. Over this three-year period, the number of CTs where adults and pediatrics were eligible were greater than CTs for pediatric only or pediatric subpopulations. DISCUSSION/SIGNIFICANCE OF FINDINGS: It is prevalent for BPCA compliant CTs to include both; 65% of drugs (13/20) with pediatric indications had more studies involving both groups than only pediatrics. Adequate pediatric CT representation is necessary for developing pediatric drug labeling with meaningful data for clinical indications.

ABSTRACT IMPACT: Lack of regulatory knowledge and education is a key barrier to the translation of medical devices and we describe the design and results for a university graduate-level course providing training on medical device regulatory submissions for approval that can help fill this unmet need and improve and accelerate translational success. OBJECTIVES/GOALS: Within the Indiana CTSI, the Medical Technology Advance Program (MTAP) in the Purdue University Weldon School of Biomedical Engineering (BME) offers three courses in regulatory science and regulatory affairs for medical devices. One course is focused on regulatory submissions for approval, and this report details the course design and evaluation. METHODS/STUDY POPULATION: For Fall 2020, the Regulatory Submissions for Approval course was enhanced to increase participation from regulatory professionals in US FDA and industry, with the core content, curriculum and course design led by BME faculty. The course was taught two days per week and included both in-person and remote (synchronous or asynchronous) attendance options. During the first class session each week a topic was covered in standard lecture format by BME faculty with industry regulatory experience. During the second class session, guests from both industry and FDA were invited to provide in-depth discussion on the topic, share perspectives and viewpoints, present real-world examples, experiences, and case studies, and answer student questions. An end of semester survey evaluated the effectiveness of the course design. RESULTS/ANTICIPATED RESULTS: Medical Device regulatory submissions and related activities were taught including product classification, presubmissions and meetings, 510(k), de novo, EUA, PMA, HDE, and advisory panels. FDA history, regulatory careers, regulatory science, and EU, China, and Japan regulations were also discussed. Overall, 29 speakers from FDA and industry participated live via video calls. A survey completed by 21/23 studentsrevealed overall satisfaction: all reported increased regulatory understanding and 20/21 learned ‘a lot’ or ‘an incredible amount’. The weekly lecture was the top factor contributing to learning, and guest speakers were the next most important factors. Nearly all students indicated FDA and industry speakers were ‘very’ or ‘extremely’ valuable/helpful. Additional results will be presented. DISCUSSION/SIGNIFICANCE OF FINDINGS: The three courses are designed to improve medical device translation by training students to better understand regulatory processes and pathways. Survey results and feedback indicated this course was successful. Continued participation from FDA and industry is critical to the learning. Additional case studies will also help enhance learning.

ABSTRACT IMPACT: We are developing the 3D perfusion system for use with patient-derived bacteria to further characterize the mechanism behind bacterial-induced inflammation and cancer. OBJECTIVES/GOALS: We previously reported the adherent invasive E. coli NC101 promote colorectal cancer (CRC) in mice. FimH, a mannose-specific adhesin on type 1 fimbriae, is involved in bacterial surface adhesion. Herein, we investigated the role of FimH in E. coli NC101-induced adherence and carcinogenesis in a novel 3D perfusion culture imaging plate. METHODS/STUDY POPULATION: E. coli NC101 gene fimH was deleted byï ŲRed Recombinase System. Biofilm formation was assessed by crystal violet and congo red staining. 5 dpf (wild-type strain) zebrafish embryos were infected in 6x107 cfu/ml wild type (WT) or fimH-deleted (ï ,,fimH) E. coli NC101 for 24hr and gut dissected for bacterial culture. A 2D/3D infection culture system for IEC-6 and HT-29 cells was infected for 4 hr and imaged and then DNA damage examined by comet assay, cell cycle andÎ3H2AX accumulation. Germ-free (GF) Il10-/- (colitis) mice were orally gavaged with 108 cfu WT orï ,,fimH E. coli NC101 for 16 weeks. E. coli colonization were quantified by plate culture and qPCR. Lipocalin2 was quantified by ELISA. PCNA and β-catenin were evaluated by immunohistochemistry (IHC). RESULTS/ANTICIPATED RESULTS: Biofilm formation was reduced by more than 40% (p<0.05) in E. coli NC101ï ,,fimH compared to WT strain. Zebrafish larvae showed a 41% decrease in intestinal colonization ofï ,,fimH compared to WT (p<0.05). E. coli NC101-induced DNA damage was reduced by 67% (p<0.0001) in HT-29 cells infected withï ,,fimH compared to WT strain. Using the 3D infection system, a 46% decrease in yH2AX (p<0.05) and 42% decrease in G2 cell cycle arrest (p<0.05) was observed inï ,,fimH infected IEC-6 cells compared to WT strain. Furthermore, ï ,,fimH infected Il10-/- mice showed decreased colonization (p<0.01), decreased intestinal inflammation (p<0.05), decreased stool lipocalin2 level (p<0.01), and reduction of PCNA positive cells in the intestine (p<0.05) compared to mice infected with WT strain. DISCUSSION/SIGNIFICANCE OF FINDINGS: Adhesin protein FimH is required by E. coli NC101 to colonize and promote colitis and carcinogenesis both in a 3D perfusion culture and in mice and may serve as potential therapeutic target.

ABSTRACT IMPACT: In a global pandemic where data development and dissemination are integral to combating the disease, the Clinical Research Support Center at the University of Minnesota provides a model of comprehensive virtual support, helping to attain and disseminate novel research on COVID-19, its individual and community impact, and treatment initiatives/outcomes. OBJECTIVES/GOALS: The pandemic created massive disruption to the conduct of clinical research with an unprecedented reorientation towards COVID-19. In this fast-paced environment, the Clinical Research Support Center (CRSC) rapidly developed innovative means of supporting diverse research initiatives. METHODS/STUDY POPULATION: The CRSC rapidly transitioned into a virtual environment and developed tools for the clinical research community to enhance remote clinical trial start up. This includes supporting remote consent, eBinders, COVID-19 research training for clinical staff, and easier identification of potential participants for COVID-19 studies; all through virtual support. Support provided research teams guidance on study protocols, regulatory requirements, informatics, biostatistics, financial management, recruitment strategies to support critical, urgent COVID-19 research. We outline proactive examples of how the CRSC now provides support to research teams through the pandemic. RESULTS/ANTICIPATED RESULTS: From March-November 2020, 116 COVID-19 projects received virtual support from the CRSC for COVID-19 research: disease understanding (n=27), treatment (n=23), pandemic impact (n=20), clinical care innovation (n=18), disease control and surveillance (n=10), prevention (n=9), detection (n=5), and impact on minorities (n=4). The diversity of these studies demonstrates the demand for and benefit from multidisciplinary expertise supporting study design and implementation. Through successful articulation and acceleration of research activities, the CRSC met the need for speed and rapidly adapted to new challenges created by the pandemic. DISCUSSION/SIGNIFICANCE OF FINDINGS: In a global pandemic where rapidly changing barriers to research is ongoing, through multidisciplinary efforts, the CRSC continues to provide comprehensive, virtual support to attain and disseminate novel research on COVID-19, its individual and community impact, and treatment initiatives/outcomes.