OBJECTIVES/GOALS: The objective of this study is to investigate the neurophysiological mechanism of vision therapy in male and female adolescents with persistent post-concussion convergence insufficiency (PPCS-CI). This study may improve diagnostics and inform more effective personalized point of care treatment strategies to remediate symptoms. METHODS/STUDY POPULATION: Participants (ages 11-25) were diagnosed with PPCS by a physician, CI was diagnosed by an optometrist and OBVAT was performed by certified therapists. Patients with PPCS-CI were randomly assigned to a therapy type (immediate therapy or natural recovery). Hemodynamic measures were examined in patients with PPCS-CI at baseline (1-3 months post-concussion), and post OBVAT to evaluate recovery outcomes. Non-invasive techniques were used to measure middle cerebral artery velocity (MCAv), blood pressure, heart rate, and end-tidal CO2 at rest and during objective symmetrical convergence step eye movements. Functional magnetic resonance imaging (fMRI) was acquired during convergence step eye movement experiments contrasted to sustained fixation and resting state data collection. RESULTS/ANTICIPATED RESULTS: To investigate the neural mechanism of OBVAT, eye movements, fMRI and physiological measures were collected in 8 patients with PPCS-CI (4 men and 4 women). Results show an 10% decrease in the MCA during 4-degree symmetrical convergent eye movement responses in males post-OBVAT and a 19% increase in MCA during convergent eye movement responses in females. Furthermore, there was a group level activation of the frontal eye fields, which improves post-OBVAT. The beta weights in the left frontal eye fields show a trending decrease in male patients post-OBVAT and trending increase in females. Males had a decrease in MCAv post OBVAT (baseline 83.6 ± 7.5 cm/sec & 75.7 ± 12.5 cm/sec post-OBVAT), while females show a significant increase post-OBVAT (baseline 53.77 ± 5.2 cm/sec & post-OBVAT 65.13 cm/sec ± 12.5). DISCUSSION/SIGNIFICANCE: This initial pilot demonstrates there may be different underlying pathophysiological outcomes associated with a concussion dependent on sex. This work may have direct implications on treatment strategies for male and female adolescent patients with PPCS-CI.

OBJECTIVES/GOALS: Barriers to childhood vaccine completion include forgeting vaccine appointments, lack of clinic access (distance and funds), and vaccine hesitancy. We tested the impact of automated and real-time appointment reminders, vaccine hesitancy counseling, and targeted vaccine drives on receiving the third dose of the diphtheria vaccine. METHODS/STUDY POPULATION: An implementation study to determine the feasibility and impact of implementing a mobile health-supported intervention bundle. A digital vaccine registry was developed to manage vaccine uptake data. The intervention bundle was applied sequentially: each registered parent received an automated appointment phone reminder (text and voice). If they delayed for >5 days, they received a real time reminder phone call. If during the real time call vaccine hesitancy was deemed to be a barrier, counseling was provided. If access - lack of funds or long distance - to the clinic was the barrier, vaccination was performed at patient's home on the monthly vaccine drives. We compared vaccine completion (all childhood vaccines before 18 months) during the implementation to the preceding three years. RESULTS/ANTICIPATED RESULTS: We anticipate the implementation will be feasible as >90% of all eligible children will be registered. We expect providers will be accepting and would recommend the intervention to other providers. We anticipate the intervention will result in a >10% increase in childhood vaccine completion compared to the average of the past three years. DISCUSSION/SIGNIFICANCE: We anticipate applying a multifaceted intervention will be acceptable to providers, feasible to implement, and significantly improve childhood vaccine completion rates moving Nigeria closer to achieving the global target of >95% childhood vaccine completion rate.

OBJECTIVES/GOALS: The goal of this study is to identify if ocular graft versus host disease (oGVHD) patients treated with a systemic JAK inhibitor have a change in their tear cytokine profile (a possible bio-marker) and oGVHD score. oGHVD is a severe inflammatory dry eye disease and major cause of morbidity after a hematopoietic stem cell transplant. METHODS/STUDY POPULATION: Janus Kinase (JAK) is an upstream regulator of cytokine production. A JAK 1/2 inhibitor, Ruxolitinib, was recently FDA approved for the treatment of chronic GVHD. We propose that JAK inhibition results incytokine changes in tears and improvement of oGVHD. To study this, we will quantify tear cytokines in patients with oGVHD, with and without systemic JAK inhibition treatment. Patients with ‘definite’ oGVHD based on the international chronic oGVHD diagnostic criteria (ICOGVHD) whom we have collected tears will be grouped based on JAK inhibition treatment. Tear cytokines are analyzed using Iso spark Meteor bulk quantitative proteomic analysis. RESULTS/ANTICIPATED RESULTS: Seven patients were identified from our patient cohort who met inclusion criteria (oGVHD; tears collected while on Ruxolitinib), five patients were identified whom we have collected tears with oGVHD who have not taken ruxolitinib. The following 10 cytokines will be analyzed in the tears by the Iso spark Meteor bulk quantitative proteomic analysis: GM-CSF, IFN-g, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-a. The change in cytokine levels will be compared with the ICOGVHD score, corneal fluorescein staining, schirmers test (measurement of tear production), conjunctival injection score, ocular surface disease index score (validated symptomatic score of dry eye disease). DISCUSSION/SIGNIFICANCE: OGVHD is a major cause of morbidity for patients who undergo a hematopoietic stem cell transplant and is the result of a highly complex immune process including dysregulation of pro-inflammatory cytokines. It is critical to understand the effect of cytokine changes on the eyes to potentially identify a biomarker and possible treatment targets.

OBJECTIVES/GOALS: We aim to develop an understanding of how polysubstance use (PSU) relates to the general psychopathology factor (p-factor), as well as to individual components of the Hierarchical Taxonomy of Psychopathology (HiTOP) model (e.g., fear, distress). This insight can help identify treatment targets related to substance use and psychopathology. METHODS/STUDY POPULATION: Psychopathology and substance use data, collected at a Baltimore treatment center over several years, will be analyzed. The center aids about 6000 underserved clients per year, and the population is primarily African American clients of all genders. Structural equation modeling (using Mplus software) will be used to develop the latent models and identify relationships between psychopathology and PSU (i.e., direct and indirect pathways). The current latent HiTOP model was developed from symptom checklists completed upon entry at the treatment center. The PSU latent factor will be developed from a biopsychosocial assessment where clients list their drug of choice. Due to the varying organizations of the datasets, smaller-scale preliminary models will be developed to ensure an accurate large-scale final model. RESULTS/ANTICIPATED RESULTS: Current models being tested are derived from January to September 2023 data (i.e., completed months' data), with an N of 1,564. From symptom checklist data collected at the treatment center, a preliminary HiTOP model was derived with reasonable fit (χ2 = 4532.35 (df = 321, p<.001), CFI = .77, SRMR = .07, RMSEA = .09 (.089, .094)). Data analysis is being conducted to derive the PSU factor before relating PSU to the HiTOP model. Given previous work at a local treatment center (Pavuluri etal., 2022) and with the National Comorbidity Survey-Replication data, we expect all positive direct relationships, negative indirect relationships between internalizing factors (fear and distress) and PSU when accounting for p-factor, and a positive indirect relationship between antagonism and PSU when accounting for p-factor. DISCUSSION/SIGNIFICANCE: Given our previous work to develop such models, we want to establish proof of concept in alarger treatment center population. This confirmation will help provide a path towards conducting therapeutic trials to target psychopathology when treating substance use given the shared relations, some of which are less understood (e.g., fear and PSU).

OBJECTIVES/GOALS: Decoding the origins of cell-free DNA (cfDNA) released from dying cells in a liquid biopsy sample (e.g. blood) offers the potential to provide insight into the dynamic, organism-wide changes reflective of health and disease. Thus, making cfDNA an ideal target for serial, minimally invasive monitoring of disease-related changes. METHODS/STUDY POPULATION: We develop a probabilistic method that leverages the co-regulation of neighboring CpG sites on individual methylome-wide sequencing (WGBS) reads to more flexibly model cell-specific methylation compared to prior methods that focus on the methylation rate of a single CpG site. We then extend our cross-sectional model to account for sequential sampling within the same subject. The increased sampling frequency is critical to identifying the evolutionary dynamics of disease progression influencing treatment response and resistance, and disease recurrence. We utilize Bayesian inference techniques to model patient-specific longitudinal profiles of cell-type turnover in simulated serial samples. RESULTS/ANTICIPATED RESULTS: We found our model more effective at capturing a range of methylation patterns on cfDNA fragments with lower Root Mean Square Error across simulations compared to a single CpG model. We apply our model to detect significant (p < 0.05, Friedman’s test) increases in cellular contributions from lung and cardiac tissue in breast cancer patients (n=15) undergoing radiation therapy compared to baseline. We also identify signals of radiation induced toxicity to the liver in right-sided breast cancer patients (n=8) receiving radiation treatment compared to left-sided breast cancer patients (n=7). Finally, we show our extended model results in more efficient estimates of simulated cell-type turnover profiles compared to analyzing serial samples cross-sectionally, ignoring the longitudinal nature of the data. DISCUSSION/SIGNIFICANCE: Here we address an unmet need in developing novel statistical methodologies to decode the origins of methylated cfDNA obtained from liquid biopsy samples. We demonstrate the far-ranging clinical utility of serial liquid biopsy sampling to complement and advance the standards of clinical care in oncology and other pathologies.

OBJECTIVES/GOALS: Study the regulatory role of sodium glucose co-transporter 1 (SGLT1) in cardiomyocytes and the therapeutic potential of sotagliflozin in hypertrophic cardiomyopathy (HCM) by (a) quantifying SGLT1 expression in HCM and (b) examining the impact of sotagliflozin on cardiac mechanics. METHODS/STUDY POPULATION: * Use Western Blot in cardiac tissue from HCM and non-HCM patients and pre-existing RNA seq and proteomics datasets to quantify SGLT1 levels in HCM. Hypothesis: SGLT1 is upregulated in HCM * Determine how SGLT1/2 inhibition by sotagliflozin will affect cardiac mechanics using living myocardial slice (LMS) preparations. A vibratome creates 200um-thick slices from (a) failing HCM heart explants, (b) septal myectomy samples from HCM patients, and (c) nonfailing rejected donor hearts. LMS are mounted on a force transducer and work-loops are stimulated under varying pre- and after-loads. Collecting baseline and post-drug work loops allows each slice to function as its own control. Hypothesis: sotagliflozin will improve diastolic mechanics by reducing stiffness in the end-diastolic pressure-volume relationship RESULTS/ANTICIPATED RESULTS: * Preliminary results from RNA seq data indicate that SLC5A1 mRNA (encoding gene for SGLT1) is significantly decreased in HCM. No proteomics study examined thus far has detected SLC5A1, indicating that overall SGLT1 levels in cardiac tissue are quite low. We will examine SGLT1 levels in our own HCM and non-HCM tissue samples with both mass-spectrometry and Western Blot. * We analyze six slices from each heart and expect 15 donor hearts and 15 HCM hearts/myectomy samples. We visualize the work loop by plotting stress/strain. Stress/strain at mitral valve closure represents exponential end diastolic pressure-volume relationship; Stress/strain at aortic valve closure represents linear end systolic pressure volume relationship. A two-sample paired t-test will compare change in stiffness and elastance. DISCUSSION/SIGNIFICANCE: This project contributes to a growing body of research surrounding the currently unknown cardioprotective mechanism of SGLT 1/2 inhibitors, furthers the technique of using living myocardial slices to study cardiac mechanics, and supports a trial examining sotagliflozin in HCM, for which disease modifying therapy remains a prevailing unmet need.

In recent times, there have been calls from within the developing nations for increased ownership by governmental research bodies and universities of the priority research setting and research that aligns with national health strategies. This is a review paper of the studies that have been published on clinical trials in developing countries, with a focus mainly on Pakistan. The literature review used online databases such as PubMed, Scopus, and Google Scholar, World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP), and ClinicalTrials.gov trial registries to search for clinical trials conducted in Pakistan between January 2000 and December 2022 and analyzed. The results revealed that clinical research in Pakistan is hindered by a number of barriers, including a lack of funding, skilled personnel, and regulatory issues. Lack of funding is a common obstacle, and the majority of funding for clinical trials originates from Western countries or pharmaceutical companies established in the West. In conclusion, clinical studies in developing countries, especially in Pakistan, are hindered by a plethora of barriers, and to improve the current state, increasing funding, streamlining ethical approval procedures, simplifying regulatory systems, addressing cultural and religious concerns, and participating in global efforts to bridge the gap in health-based research are crucial.

OBJECTIVES/GOALS: Congenital anomalies (CAs) affect 3% of live births, yet the cause of 80% of CAs is unknown and for the 20% with an identified cause, variability in penetrance suggests additional risk drivers exist. Our method for identifying and categorizing CAs in electronic health record (EHR) linked biobank databases can expand and improve CA etiologic research. METHODS/STUDY POPULATION: We identified individuals with CAs in three groups: 1. Those with at least one CA 2. Those with multiple CAs (MCA), those with two or more ‘major’ CAs, and 3. Those with CAs in a specific organ system. We also created a novel quantitative approach, using phenome-wide association studies (pheWAS), for determining CA-associated genetic disease billing codes in order to separate individuals that have a known genetic cause for their CAs from those with idiopathic CAs. We updated CA phecodes, aggregates of clinical billing codes, which we used to identify CA cases in Vanderbilt’s EHR-linked biobank database, BioVU. We create a new phecode, ‘All CAs’, for researchers to quickly identify all individuals with at least one CA. We evaluate the definition of MCA using pheWAS analyses to compare ‘minor’ vs ‘major’ CA. RESULTS/ANTICIPATED RESULTS: The new CA phecode nomenclature includes 5.8 times more codes for CAs compared with the previous version (365 vs 56), improving granularity. 85 (19.7%) CA-associated genetic disease billing codes were identified through literature review. PheWAS analyses revealed an additional 16 (3.7%) genetic disease billing codes with one or more significant (p< 2.75 x10-5) association with CA-related phecodes. Identifying CA-associated genetic disease billing codes allows researchers to differentiate between idiopathic CAs and those that have a known genetic cause. PheWAS analyses of individuals with previously considered “minor” CAs showed many associated severe health problems, revealing that the differentiation between “minor” vs “major” CAs when identifying individuals with MCA in the EHR is arbitrary. DISCUSSION/SIGNIFICANCE: Our CA identification method is scalable for the growing number of EHR-linked biobanks. Differentiating between idiopathic CAs from those with known causes will increase power in studies discovering additional genetic drivers of CAs. Our novel method allows for expansion and acceleration of CA epidemiological research in EHR-linked biobank data.

OBJECTIVES/GOALS: Platform trials gain efficiency by sharing placebo controls among different study arms. However, the varying routes of administration make it unclear whether participants exposed to different placebos have similar outcomes. As such, we seek to compare outcomes between participants receiving tablet and inhaler placebos in the ACTIV-6 trial. METHODS/STUDY POPULATION: ACTIV-6 is a large, decentralized platform trial exploring repurposed drugs for the treatment of adults with mild to moderate COVID-19. Enrolled participants were randomly assigned to a study arm vs. placebo and then mailed the study drug. They were monitored until symptom resolution or Day 28. Here, we compare outcomes for control participants contributing to the fluticasone furoate study arm, in which 251 were assigned to a tablet placebo and 370 an inhaler placebo. Time to sustained recovery and time to resolution of individual symptoms are compared between groups using Kaplan-Meier curves and unadjusted log-rank tests. A step-down procedure is applied to control the false discovery rate. RESULTS/ANTICIPATED RESULTS: Control participants assigned to tablet placebos had shorter time to sustained recovery (adjusted hazard ratio (HR) 1.34 (95% CI 1.11, 1.62)). When examining each of the eleven individually reported symptoms on study Day 14, nasal symptoms (adjusted odds ratio (OR) 0.44 (0.27, 0.72), p<0.01), dyspnea (OR 0.44 (0.22, 0.87), p = 0.02), and cough (OR 0.54 (0.35, 0.83), p<0.01) were identified as symptoms in which the tablet-placebo group performed notably better than those who received inhaler-placebos. In the follow-up, longitudinal analysis, we anticipate similar results. DISCUSSION/SIGNIFICANCE: Among ACTIV-6 control participants, those receiving a tablet placebo had a significantly shorter time to sustained recovery than those receiving an inhaler placebo. Platform trials using shared controls should consider efficiency in the context of the additional variability when sharing controls with a different route of administration.

OBJECTIVES/GOALS: Super refractory status epilepticus (SRSE) is associated with high mortality, often due to withdrawal of life sustaining therapy (WLST) based on perceived poor neurological prognosis. Factors influencing decision making are underreported and poorly understood. We surveyed clinicians who treat SRSE to identify factors that influence WLST. METHODS/STUDY POPULATION: Health care providers (HCP), including physicians, pharmacists, and advanced practice providers, who treat SRSE answered a 51-question survey on respondent demographics, institutional characteristics and SRSE management that was distributed though professional societies. Respondents described approaches to prognostication and rated the importance of clinical factors in the management of two hypothetical clinical cases followed by their prediction of recovery potential for the same two cases. Neurointensivists and other HCP responses were compared using descriptive statistics to differentiate group characteristics; a p-value <0.05 was considered significant. Logistical regression models were employed to identify associations between clinician specific factors and prognostication. RESULTS/ANTICIPATED RESULTS: One-hundred and sixty-four respondents were included in the analysis. Compared to other HCPs (neurologists, epileptologists, neurosurgeons, other intensivists; n=122, 74%), neurointensivists (n=42, 26%) [Odds ratio (OR) 0.3, 95% confidence interval (CI) 0.14-0.68), p=.004)] were less likely to use prognostic severity scores and were less likely to prognosticate likelihood of good functional recovery (OR: 0.28 (95% CI: 0.13-0.62), p=.002) compared to non-neurointensivist HCPs, controlling for potential confounders including professional degree, years of experience, country of practice, and annual volume of SRSE cases. There was, however, significant overlap in factors deemed necessary for determining futility in care escalation. DISCUSSION/SIGNIFICANCE: Neurointensivists value similar clinical factors to other HCPs when evaluating medical futility in SRSE but are less likely to predict definitive outcomes. Pending final survey results, future studies aimed at understanding why neurointensivists may be less likely to decisively prognosticate (i.e. avoiding nihilism) in SRSE may be warranted.

The proposal of improving reproducibility by lowering the significance threshold to 0.005 has been discussed, but the impact on conducting clinical trials has yet to be examined from a study design perspective. The impact on sample size and study duration was investigated using design setups from 125 phase II studies published between 2015 and 2022. The impact was assessed using percent increase in sample size and additional years of accrual with the medians being 110.97% higher and 2.65 years longer respectively. The results indicated that this proposal causes additional financial burdens that reduce the efficiency of conducting clinical trials.

OBJECTIVES/GOALS: Xylazine is a strong sedative and fentanyl contaminant which has been increasingly detected in drug overdose deaths in Maryland. The goal of this project is to analyze the demographic characteristics and time trends of xylazine-related overdose deaths (XROD) in Maryland from 2020-2022. METHODS/STUDY POPULATION: This cross-sectional study utilizes the Maryland medical examiner's autopsy reports from 2020-2022. These reports include every death in the state that was investigated by the medical examiner, with demographic and toxicological data showing the presence of various substances at the time of death. An XROD was defined as someone who died from drug overdose and had a positive serum xylazine test at time of death. Demographic characteristics and time trends for XROD were analyzed. Multivariable logistic regression modeled associations between demographic variables and the presence of other substances with XROD. RESULTS/ANTICIPATED RESULTS: A total of 1,509 people died from XROD, of which the mean age was 44.4 years and 73.3% were male. The majority were White (57.6%), 39.2% were Black, and 3.2% identified as another race. Over 99.9% of individuals who died from XROD tested positive for fentanyl. XROD peaked in January 2021 and has been trending downwards since then. Adjusted multivariable logistic regression revealed that White individuals had greater odds of XROD relative to Black individuals (OR=1.22, 95% CI=1.07-1.37), and adults aged 30-45 years had higher odds of XROD relative to adults over age 60 (OR=1.26, 95%CI=1.04-1.54). Individuals who used fentanyl had higher odds of XROD relative to those who did not use fentanyl (OR=327.4, 95%CI=46.0-2331.3). DISCUSSION/SIGNIFICANCE: This study demonstrates that middle age, White race, and fentanyl use are associated with xylazine-related overdose deaths in Maryland. Efforts to reduce xylazine-related mortality in the state should address the unique social and geographic factors that influence substance use in this population.