OBJECTIVES/GOALS: Sudden Unexpected Death in Epilepsy (SUDEP) is a leading cause of death in epilepsy patients. This study aims to determine whether cardiac mechanisms contribute to SUDEP in epilepsy patients with variants in DEPDC5, a gene encoding a member of the mTOR GATOR complex, to identify SUDEP biomarkers. METHODS/STUDY POPULATION: SUDEP has been reported in 10% of epilepsy patients with DEPDC5 loss-of-function variants. We used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to measure changes in cellular excitability that are known to be substrates for cardiac arrhythmias. CRISPR-derived isogenic DEPDC5 iPSC-CMs and DEPDC5 patient-derived iPSC-CMs were used in this study. Whole-cell patch-clamp was used to measure voltage-gated sodium current (INa) and calcium current (I>Ca) in single iPSC-CMs in voltage-clamp mode; and to measure action potentials (APs) in 3-dimentional iPSC-CM-derived micro-tissues in current-clamp mode. RESULTS/ANTICIPATED RESULTS: CRISPR generated heterozygous deletion of 1 base-pair in the first coding exon of DEPDC5 gene, resulting in a premature stop codon, simulated the variants identified in DEPDC5 epilepsy patients. In CRISPR generated heterozygousDEPDC5 iPSC-CMs, whole-cell voltage-clamp recordings revealed that INa was increased and ICa was reduced compared with isogenic control iPSC-CMs. Whole-cell current-clamp recordings revealed that AP duration at 80% and 90% of repolarization, APD80 and APD90, respectively, were prolonged compared to isogenic control iPSC-CMs. Similar measurements will be performed for iPSC-CMs derived from DEPDC5 patients. DISCUSSION/SIGNIFICANCE OF IMPACT: This study shows that epilepsy patients with non-ion channel gene variants in DEPDC5 have altered CM excitability, which may serve as a substrate for cardiac arrhythmias in DEPDC5 patients. Importantly, this work may allow us to identify biomarkers for SUDEP risk in these patients in the future. CONFLICT OF INTEREST DESCRIPTION: L.L.I. is the recipient of a collaborative research grant from Stoke Therapeutics.

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.

OBJECTIVES/GOALS: To streamline the standards and procedures for operating a research-specific, clinical data warehouse, acheived by defining roles, introducing a common language, and categorizing dataset types to provide transparency regarding data security risks inherent in the use of patient data. METHODS/STUDY POPULATION: We established a Bioethics committee responsible for ensuring clinical data is securely procured, maintained, and extracted in a manner that adheres to all federal, state, and local laws. We created an operational framework in the form of an umbrella IRB protocol and shared it with the bioethics committee for feedback and approval. The protocol was approved first by the bioethics committee and subsequently by the IRB. It was then disseminated across the institution and published online for continuous reference and use by committee members, researchers, and the data warehouse service team. RESULTS/ANTICIPATED RESULTS: The resulting framework defined the roles of researchers, data warehouse service team members, and honest brokers; explains the procedures for accessing and securely delivering data; and lists six categories of datasets according to type and implicit risks: datasets that are preparatory for research/aggregate counts, anonymized datasets, coded datasets, limited datasets, identified datasets for recruitment purposes, and defined identified cohort datasets. The protocol is approved and in use enterprise-wide, has reduced the number of questions from stakeholders, and has given researchers, IRB members, and informatics staff confidence in the use of the clinical research data warehouse. DISCUSSION/SIGNIFICANCE: We offer our framework to CTSAs interested in streamlining their data warehouse operations. We believe the adoption of this framework will establish strong procedures for ensuring compliance with IRB requirements, data privacy, and data security while reducing barriers to clinical research.

OBJECTIVES/GOALS: Alzheimer’s Disease (AD) displays numerous pathological features, including amyloid-beta deposition, extensive neuroinflammation, and vascular fibrosis. However, putative therapeutic options for alleviating these features remain limited, emphasizing the need to develop comprehensive treatments for patients with AD. METHODS/STUDY POPULATION: CSF from human AD patients treated with nilotinib (n=12), a tyrosine kinase inhibitor, or placebo (n=11) was collected and sequenced, and significantly altered miRNAs were identified and analyzed for alterations to disease-associated genes via gene ontology analysis. TgAPP mice were injected intraperitoneally with one of two novel tyrosine kinase inhibitors, BK40143 or BK40197, or DMSO (n=12 per group) daily for six weeks, during which memory deficits between groups were measured, before brains were harvested for analysis of amyloid-beta load via ELISA, microglial activation via Sholl analysis, and vascular collagen levels via immunohistochemistry. RESULTS/ANTICIPATED RESULTS: CSF obtained from AD subjects treated with nilotinib revealed significantly increased (p<0.05) levels of miRNAs regulating autophagy, neuroinflammation, and collagen production compared to placebo. These results were validated in vivoin TgAPP mice, who displayed improved recall on the novel object recognition test and Morris water maze following treatment with our drugs, correlating with decreased levels of brain amyloid-beta (30% decrease, p=0.002), decreased microglial reactivity and activation (40% decrease, p=0.01), and decreased vascular fibrosis (50% decrease, p=0.005) along small brain blood vessels compared to controls. DISCUSSION/SIGNIFICANCE: These data identify tyrosine kinase inhibition as a valid therapeutic strategy for alleviating various pathological features associated with AD and warrant further investigation as a treatment option for human patients as a means of slowing cognitive decline.

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.

OBJECTIVES/GOALS: To quantify clinical exam in total knee arthroplasty by answering the following questions: (1) What are the magnitudes of forces applied by surgeons during the varus-valgus exam? (2) Is the choice of tibial insert thickness related to the magnitude of the applied forces? (3) How accurately does a surgeon estimate the gaps in the varus-valgus exam? METHODS/STUDY POPULATION: Three cadaveric knees were implanted with standard TKA trial implants. Four pliable force sensors were wrapped around the foot and ankle of each cadaver to measure the push-pull forces applied during the varus-valgus exam. Six surgeons with varying experience independently conducted a varus-valgus exam in extension and flexion and reported the gaps that they observed. Motion capture was used to measure the gaps between femur and tibia by placing cluster of reflective markers on femur and tibia. Subsequently, each surgeon chose the tibial insert that they thought best fit each knee. The measured peak applied forces were related to the insert thickness and the measured gaps were compared to the observed gaps by surgeons. Since insert thickness was in 1 mm increments, 1 mm gap error was considered a meaningful difference. RESULTS/ANTICIPATED RESULTS: The peak forces varied among surgeons for each cadaver. In cadaver one, the peak forces in varus and valgus in extension were 48±20 and 20±12 N, and in flexion were 27±14 and 8±11 N. Peak forces in cadavers two and three were similar; in varus and valgus in extension, 24±14 and 35±10 N, and in flexion, 23±12 and 20±10 N, respectively. It was observed that the larger the valgus force in extension, the thinner was the inserts choice (β = −0.08 mm/N, p = 0.012). In extension, the difference between estimated gaps and measured gaps was > 1 mm for 36% of all assessments and 91% of gaps were underestimated. Only one measure, however, was underestimated by > 2 mm. In flexion, gap estimates were > 1 mm for 35% of all measurements and 59% of all measurements were overestimated. Four measures were overestimated, and one was underestimated by > 2 mm. DISCUSSION/SIGNIFICANCE OF IMPACT: We found that the applied forces varied among surgeons and a negative association between insert thickness and forces in extension valgus exam. We also found that error in gap estimates among surgeons was > 1 mm a third of the time and that underestimation is more common in full extension, which may lead to using smaller inserts that affect knee stability. CONFLICT OF INTEREST DESCRIPTION: The corresponding author has no COI but my coauthors had the following COI:

1. 1. Royalties from a company or supplier: Zimmer; Stryker; Exactech, Inc; Lima; Mathys Ltd.

2. 2. Speakers bureau/paid presentations for a company or supplier Acelity; Flexion Therapeutics; Smith & Nephew; Exactech, Inc; Mallinckrodt Pharmaceuticals; Stryker.

3. 3B. Paid consultant for a company or supplier Acelity; DePuy Synthes; Exactech, Inc; Flexion Therapeutics; Intellijoint; Smith & Nephew; Zimmer; Stryker

4. 4. Stock or stock options in a company or supplier Imagen; Insight Medical; Intellijoint; Parvizi Surgical Innovation; OrthAlign; Orthobond.

5. 5. Research support from a company or supplier as a Principal Investigator Acelity; Exactech, Inc; Intellijoint; Smith & Nephew; Mallinckrodt Pharmaceuticals; Stryker; Lima.

6. 6. Royalties, financial or material support from publishers (The following conflicts were disclosed) Exactech, Inc.

7. 7. Medical/Orthopaedic publications editorial/governing board Bone and Joint Journal 360; Journal of Orthopaedics and Traumatology; Techniques in Orthopaedics.

8. 8. Board member/committee appointments for a society Knee Society; Eastern Orthopedic Association.

OBJECTIVES/GOALS: Research Education Towards Opportunities (RETO) Mentoring Offering Training Opportunities for Research (MOTOR) 1 – 2 and the elective courses (INTD 5998/ MDCL 101) in CTR of Title V Coop were designed to provide the participants from higher education institution (HEI) in Puerto Rico (PR), interdisciplinary – interprofesional knowledge in CTR. METHODS/STUDY POPULATION: Since April 2017, Research Education Towards Opportunities (RETO) and Mentoring Offering Training Opportunities for Research (MOTOR) 1 - 2 trainings were offered as part of the Title V Coop. In addition, since January 2020, as part of the institutionalization of the trainings in CTR, two elective courses (INTD 5998 and MDCL 101) were created-offered. The trainings/courses present the main concepts underlying CTR performance through lectures, workshops and presentations, in hybrid modalities, as well as the services-resources of the Center Research Education and Science Commnunication Opportunities (CRESCO). These programs have given students (undergraduate and graduate) and faculty the opportunity to get started in CTR and to integrate in Clinical and Translational Mentoring Teams (CTMT). RESULTS/ANTICIPATED RESULTS: Eight (8) cycles of RETO-MOTOR 1 and seven (7) cycles of RETO-MOTOR 2; two sessions of INTD 5998 and one session of MDCL 101 were offered. The RETO-MOTOR 1 training was completed by 219 participants and RETO-MOTOR 2 by 130 participants. The INTD 5998 course was completed by 22 students and the MDCL 101 course by 18 students. A total of 389 participants have been initiated in the CTR. Of the trainings, 90% indicated that the knowledge acquired in CTR was invaluable, 85% understand that the most significant achievement, as students, was present at a scientific conference, and 100% indicated interest in continuing to do CTR. Of the courses, 100% indicated that they were a good learning experience, helped them increase their knowledge in CTR, met their expectations and would recommend other students to take the course. DISCUSSION/SIGNIFICANCE: The RETO-MOTOR 1, RETO-MOTOR 2 trainings and CTR courses provide a based of research knowledge and valuable interprofessional experience for those who whish to start in the clinical and translational research. The Title V Cooperative Project provides this opportunity to undergraduate and graduate students such as faculty of HEI in PR.

OBJECTIVES/GOALS: Supported by the State of Alabama, the Alabama Genomic Health Initiative (AGHI) is aimed at preventing and treating common conditions with a genetic basis. This joint UAB Medicine-HudsonAlpha Institute for Biotechnology effort provides genomic testing, interpretation, and counseling free of charge to residents in each of Alabama’s 67 counties. METHODS/STUDY POPULATION: Launched in 2017, as a state-wide population cohort, AGHI (1.0) enrolled 6,331 Alabamians and returned individual risk of disease(s) related to the ACMG SF v2.0 medically actionable genes. In 2021, the cohort was expanded to include a primary care cohort. AGHI (2.0) has enrolled 750 primary care patients, returning individual risk of disease(s) related to the ACMG SF v3.1 gene list and pre-emptive pharmacogenetics (PGx) to guide medication therapy. Genotyping is done on the Illumina Global Diversity Array with Sanger sequencing to confirm likely pathogenic / pathogenic variants in medically actionable genes and CYP2D6 copy number variants using Taqman assays, resulting in a CLIA-grade report. Disease risk results are returned by genetic counselors and Pharmacogenetics results are returned by Pharmacists. RESULTS/ANTICIPATED RESULTS: We have engaged a statewide community (>7000 participants), returning 94 disease risk genetic reports and 500 PGx reports. Disease risk reports include increased predisposition to cancers (n=38), cardiac diseases (n=33), metabolic (n=12), other (n=11). 100% of participants harbor an actionable PGx variant, 70% are on medication with PGx guidance, 48% harbor PGx variants and are taking medications affected. In 10% of participants, pharmacists sent an active alert to the provider to consider/ recommend alternative medication. Most commonly impacted medications included antidepressants, NSAIDS, proton-pump inhibitors and tramadol. To enable the EMR integration of genomic information, we have developed an automated transfer of reports into the EMR with Genetics Reports and PGx reports viewable in Cerner. DISCUSSION/SIGNIFICANCE: We share our experience on pre-emptive implementation of genetic risk and pharmacogenetic actionability at a population and clinic level. Both patients and providers are actively engaged, providing feedback to refine the return of results. Real time alerts with guidance at the time of prescription are needed to ensure future actionability and value.

OBJECTIVES/GOALS: Preeclampsia, a hypertensive disorder in pregnancy, disrupts immune cell profiles at birth in both mice and humans. In mice, it affects offspring’s memory and behavior. This study aimed to investigate whether preeclampsia induces lasting immune cell changes after birth and its impact on astrocyte and microglia cell counts in offspring. METHODS/STUDY POPULATION: Preeclampsia was induced in C57BL/6 females by infusion of vasopressin (24 ng/hr) or saline throughout gestation via osmotic minipump. Parturition was allowed to occur naturally. Offspring were euthanized at various timepoints post-delivery for experimental measures. Total urine protein was determined via bicinchoninic acid assay. Single cell suspensions were prepared from thymus spleen, and brain tissue and separated via density gradient. Cell suspensions were stained with fluorochrome-conjugated monoclonal antibodies for flow cytometry. Statistical significance was determined using a two-tailed Student t test or one way ANOVA multiple comparisons test. The minimal level of confidence deemed statistically significant was p<0.05. RESULTS/ANTICIPATED RESULTS: Preeclampsia resulted in lower body and heart masses in offspring. Although T cell populations in the thymus were not altered in preeclampsia offspring, total T cells, Thelper, and cytotoxic T cells were elevated. Total B and isotype-switched B cells were increased in offspring of preeclampsia. Total dendritic cell percentages were not changed in offspring of preeclampsia, however, total anti-inflammatory markers on dendritic cells were reduced. Lastly, offspring of preeclampsia had a reduction in microglia and astrocytes within the brain. DISCUSSION/SIGNIFICANCE: Our study could establish including in utero data in predicting future disease risk, addressing gaps in understanding rising rates of cardiovascular and behavioral diseases. It also uncovers the impact of preeclampsia on early immune programming and reduced glial cell populations, potentially affecting cognitive and behavioral development.

OBJECTIVES/SPECIFIC AIMS: Virtual communities are an untested method to enhance community engagement in biomedical research. Our CTSA Hubs collaborated to assess receptivity to engage in a statewide Facebook (FB) group. METHODS/STUDY POPULATION: Cross-sectional online survey administered via iPads at the MN State Fair in 2018 to adults aged 18+ years residing in MN assessed demographics, social media use, interest in participating in a FB group for biomedical research; and open-ended questions on health topics of interest, and what would keep people engaged in this group. RESULTS/ANTICIPATED RESULTS: Respondents (N=487) were 21% racial minorities and 65% female sex. Most (87%, n=422) had created a personal FB profile. Of these, the proportion who agreed/strongly agreed was: 57% that the FB group sounded interesting, 45% were interested in being part of it, 41% were willing to share it with others, 62% that it would allow the community’s thoughts/ideas to be heard and 59% wanted to learn about opportunities to participate in research on health topics they care about. Using content analysis, the top 3 health topics people wanted to learn about were chronic disease and prevention, wellness, and mental health. Top ways to keep people engaged were providing personable, relevant health information; and interactive bi-directional discussions. DISCUSSION/SIGNIFICANCE OF IMPACT: Findings will inform development of a FB group to engage diverse populations in biomedical research.

OBJECTIVES/GOALS: The goal of this study is to highlight the unique characteristics of Miami CTSIs Master of Science in Clinical and Translational Investigation (MSCTI) as part of CTSIs overall translational workforce development program and to describe program outcomes. METHODS/STUDY POPULATION: The MSCTI at the University of Miami offers a structured, individualized, and mentored educational program that trains students in the principles and practice of translational science and clinical research. Based on student feedback and needs assessment, three courses were specifically created for the program to include additional scientific cognates and promote essential skills for the translational research workforce – team science and entrepreneurship, research ethics, and writing for clinical and translational science. A fourth course on applied statistics is currently under development that provides training in practical statistical knowledge required for clinician-researchers. Career trajectory of graduates and academic output were tracked through surveys and secondary data collection. RESULTS/ANTICIPATED RESULTS: The MSCTI Program has enrolled 76 students over its lifetime, including KL2 scholars, NIH K scholars and T32 trainees; with 25% of students holding foreign degrees. In addition to traditional outcomes such as funding and publications, qualitative case studies will highlight scientific impact and career progress of students. The program has performed exceptionally well in recruiting underrepresented persons in research, with 41% of the students identifying as Hispanic, and 52% of the students as female. Of the graduates, 84% have gone on to achieve leadership positions in NIH and pursue research careers in academic institutions, while 16% have moved to industry, government, or non-profit careers. Overall, 92% of graduates were engaged in research after two years of graduating from the program. DISCUSSION/SIGNIFICANCE: Miami CTSIs MSCTI Program has trained students with a wide range of incoming experience to establish successful careers in clinical and translational research and is a critical component of CTSIs translational workforce development program.

OBJECTIVES/GOALS: The aim of this study was to quantify balance impairments in stance in individuals with degenerative cervical myelopathy (IwDCM) in response to external perturbations. IwDCM have damage to their spinal cord due to degeneration of the cervical vertebral column, but little is known about balance. METHODS/STUDY POPULATION: Recovery time following a perturbation may be an important measure of balance. Changes in recovery time were measured in 7 IwDCM (2m, 58.59±15.00y) and 6 controls without DCM (2m, 56.91±11.04y) as they stood on an instrumented treadmill and received cued (predictable) and uncued (unpredictable) lateral pulls to the waist at 12% (high) and 6% (low) pull magnitudes. Individuals stood with feet together, shoulder width, and wide. Recovery time was defined as the time following pull onset when the absolute value of the center of pressure velocity returned to <1x baseline standard deviation. Repeated measures ANOVA was performed on recovery time. RESULTS/ANTICIPATED RESULTS: We anticipate that feet together standing, unpredictable, higher magnitude perturbations will be most challenging evidenced by longer recovery times. For waist pull recovery time, there was a trend for a Group x Predictability x Magnitude x Stance Width interaction (p = 0.1) which we anticipate being greater with additional participants. There were significant Group x Predictability x Stance Width (p = 0.01) and Group x Magnitude x Predictability (p = 0.01) interactions. IwDCM had exaggerated recovery times in narrow and wide stances with unpredictable pulls. IwDCM recovered more slowly in response to unpredictable higher magnitude pulls. DISCUSSION/SIGNIFICANCE OF IMPACT: Balance responses in IwDCM are most impaired in narrow stances and when perturbations are unpredictable. Rehabilitation should focus on shortening latency of response timing and increasing power utilization during balance response to promote quicker recovery.

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.

OBJECTIVES/GOALS: A growing number of older adults in the United States have multiple social determinants of health (SDoH) that are barriers to effective medical care. We used generalizable machine learning methods to identify and visualize subtypes based on participant-reported SDoH profiles, and their association with delayed medical care (self-reported yes/no). METHODS/STUDY POPULATION: Data. All participants aged >=65 in All of Us with complete data on 18 SDoH self-reported variables, selected through consensus by 2 experienced health services researchers, and guided by Andersen’s behavioral model. Covariates included demographics, and the outcome was delayed medical care . Cases (n=4090) consisted of participants with at least one of the 18 SDoH variables, and controls (n=7414) consisted of participants with none of them. Method. (1) Used bipartite network analysis and modularity maximization to identify participant-SDoH biclusters, and visualize them through ExplodeLayout. (2) Used multivariable logistic regression (adjusted for demographics and corrected through Bonferroni) to measure the odds ratio (OR) of each participant bicluster to the outcome, compared with the controls. RESULTS/ANTICIPATED RESULTS: The analysis identified 7 SDoH subtypes (https://postimg.cc/Vd7Pg4xZ) with statistically significant modularity compared with 100 random permutations of the data (All of Us=.51, Random Mean=.38, z=20, P DISCUSSION/SIGNIFICANCE: The results identified 7 distinct subtypes based on SDoH profiles and their risk for delayed medical care, highlighting the importance of addressing specific combinations of barriers, with affordability having the highest risk. Furthermore, the analytical methods used are generalizable and have been made publicly available on CRAN and All of Us.

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.

OBJECTIVES/GOALS: To identify novel panel of plasma protein biomarkers to improve prediction and diagnosis of Alzheimer’s disease (AD) for African Americans (AA), who are at greater risk of developing AD compared to non-Hispanic White individuals but are underrepresented in AD research. METHODS/STUDY POPULATION: Pre-existing plasma samples from 460 AA individuals with clinical diagnoses of AD, cognitively unimpaired (CU), mild cognitive impairment (MCI), or dementia with Lewy bodies (DLB) will undergo untargeted proteomics using the SomaScan assay, where modified single stranded DNA aptamers bind to protein targets which are quantified by DNA microarray. Protein expression levels will be compared between diagnostic groups to identify differentially expressed proteins. Additional clinical, genetic, and lifestyle factors will be compared with protein expression when available. Proteins of interest, identified by differential protein expression analysis results, will be included in receiver operating characteristic analyses to identify the optimal set of proteins for diagnostic classification. RESULTS/ANTICIPATED RESULTS: A pilot experiment utilizing plasma from 40 individuals identified multiple differentially expressed proteins (DEPs) between AD and non-AD groups. Eight proteins were nominated from the differential protein analysis into a receiver operating characteristic (ROC) analysis based on pvalue and previous implication in AD genome wide association studies. Proteins involved in microglial activation, neuronal adhesion, cell proliferation, and innate immunity were nominated. The ROC model achieved 100% classification accuracy of AD and CU groups using age, sex, and the eight nominated proteins. It is expected that there will be more significant associations when utilizing the full cohort of 460 AA and that DEPs between AD, CU, MCI, and DLB will be identified. DISCUSSION/SIGNIFICANCE: The nomination of a novel panel of plasma biomarkers developed from an AA cohort will directly serve the AA community by improving access to an early and accurate diagnosis of AD. Access to improved prediction and diagnosis will likely improve disease management, thus improving patient outcomes and decreasing burden on families and caregivers.

OBJECTIVES/SPECIFIC AIMS: The overall objective of this study is to develop a novel, clinically-relevant, image-guided mouse model for radiation-induced cardiotoxicity, which can be used to gain insight into clinically-targetable, pathophysiologic mechanisms of cardiac injury in thoracic radiotherapy patients. METHODS/STUDY POPULATION: Photon or sham radiation will be administered at differential doses to a defined portion of the heart and/or lungs of C57BL/6 female mice using micro-CT visualization of the heart with Xstrahl’s MuriSlice Software applied to the Small Animal Radiation Research Platform (SARRP). Cardiac and lung segments from a subset of mice will be harvested at specific time points for confirmation of radiation targeting, local apoptosis assessment, and evaluation of fibrosis and vascular tissue morphology. Quantitative echocardiography, myocardial 18F-fluorodeoxyglucose positron emission tomography computed tomography (18F-FDG PET/CT), and myocardial perfusion imaging (MPI) with Technicium-99 (Tc-99) sestamibi will be implemented to identify sensitive imaging measures of cardiac injury and asses myocardial mechanics, inflammation, and perfusion deficits, respectively. Concurrently, a multiparametric analysis will be conducted to identify novel, circulating biomarkers of cardiotoxicity. RESULTS/ANTICIPATED RESULTS: We hypothesize that a clinically-relevant mouse model can be generated by the in situ, focal irradiation of a portion of heart and/or lung tissue segments, and can be used to elucidate molecular mechanisms of radiation-induced cardiac damage. We anticipate time-dependent and dose-dependent, focal histopathologic changes in the mouse heart, with cardiac fibrosis development, vascular damage, and cellular apoptosis in irradiated mice. Additionally, we anticipate that our mouse model of focal heart irradiation will reveal radiologic and biochemical changes that can be used to characterize and predict radiation-induced cardiac injury. Specifically, we expect our quantitative echocardiography, FDG-PET, and MPI parameters to identify and characterize cardiac damage that topographically matches histopathological analysis, and expect levels of select biochemical markers to differentially vary with time. DISCUSSION/SIGNIFICANCE OF IMPACT: Our mouse model of radiation-induced cardiotoxicity has the potential to shift current preclinical research paradigms to more closely mimic the radiation plans most commonly administered in clinical practice. The primary technologic innovation to be developed here is the use of the SARRP to deliver image-guided, in situ, focal radiation to a defined portion of the mouse heart. From a conceptual perspective, we propose a novel approach for phenotyping radiation-induced cardiac damage in patients undergoing chest radiation therapy, integrating sensitive radiomic and biochemical markers into a predictive model of cardiotoxicity.