OBJECTIVES/SPECIFIC AIMS: (1) To delineate the function of the heavy-chain receptor binding domain (HCR), a portion of botulinum neurotoxin type A (BoNT/A) and synaptic vesicle 2 (SV2) signaling pathway, which provide a novel multipurpose biologic with potential clinical applications in tumor detection/imaging, inhibition of tumor progression, and reduction of bioactive hormone secretion in metastatic neuroendocrine (NE) cancers. (2) To evaluate the expression pattern of SV2 receptors in NE cancer patient-derived tissues for prediction of patient response for recombinant HCR (rHCR) treatment. (3) To assess the in vivo efficacy and toxicity of rHCR in a NE cancer liver metastasis mouse models and in the NE patient-derived 3D MicroTumor system. (4) To collect preclinical data to design and conduct a clinical trial with NE cancer patients, a major goal toward translating our discoveries into much needed therapies. METHODS/STUDY POPULATION: Recombinant botulinum heavy chain (rHCR) was produced using an IPTG-inducible expression vector in E. coli BL21. The rHCR was His-Tag purified and stored in PBS buffer before usage. Cytotoxicity: H727, TT, and MZ cells were plated at a density of 5000 cells/well in 96-well plates and incubated under standard conditions overnight. The next day, cells were treated with 10, 100, or 500 nmol/L of rHCR and incubated for 72 hours. Following incubation, cell viability was assessed by ATP quantification using the CellTiter-Glo (Promega) assay. Fresh NE tumors were dissociated and injected into polydimethylsiloxane bioreactors in a matrigel and collagen suspension for 3D culture experiments. The viability of 3D cultures incubated with various doses of rHCR was assessed by measuring the uptake of the near-infrared dye IR-783 using an IVIS imaging system. Western blot: H727, TT, and MZ cells were seeded in 6-well plates at a density of 3×105 cells/well for 24 hours followed by treatment with 100 nmol/L for 72 hours. Total cellular proteins were isolated and analyzed to assess the level of SV2A expression and the effect of rHCR on the expression levels of NET marker proteins. Immunohistochemistry: Deparaffinized tissue culture slides were incubated with SV2A primary antibody in 1% BSA and incubated overnight at 4°C. Slides were rinsed twice with TBS containing 0.025% Triton, followed by 0.3% H2O2 for 15 minutes. Slides were then incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Detection of protein-protein interaction: Precleared cell lysate was incubated with glutathione-agarose beads in the presence of 10 μg of GST-tagged rHCR for 2 h at 4°C with end-over end mixing. Samples were then centrifuged at 10,000 g for 2 minutes and the supernatant was analyzed by SDS-PAGE. Preclinical models: To allow rHCR testing on NET patient derived cells in a very novel 3D surrogates, sterile collected fresh NET tissues will be obtained from the UAB Tissue Procurement, dissociated into a single cell suspension and injected into a polydimethylsiloxane bioreactors containing extracellular matrix composed of bovine collagen and matrigel. Such 3D cell culture will be maintained in bioreactors with constant supply of media through the microchannels and treated with rHCR at concentrations ranging from 10 nM to 1 mM. Following histologic confirmation of growth and morphology of NET patient-derived 3D surrogates we will test the anticancer activity of rHCR in this system using the standard cytotoxicity assays as well as we will validate the NET hormone expression using immunohistochemistry assay. To create an animal model of NE cancer progression, we will perform intrasplenic injection of NET cell lines. In approximately 4 weeks, the animals should develop NE liver metastases based upon our previous experience. rHCR-iFPs accumulation in the tumor mass: rHCR-iFPs will be injected to the tumor bearing mice after 4 weeks of cells implantation in 1 week interval for total of 4 treatments at the concentrations of 0.125, 1.25, and 12.5 mg/kg. RESULTS/ANTICIPATED RESULTS: Based on the preliminary data, we expect to detect rHCR-iFPs in NE cancer xenografts and in patient derived 3D explants. Our preliminary data revealed that treating NETcells with rHCR significantly reduced NE peptide expression in 3 days. Thus, we expect to see the decrease of NE tumor markers even if the fluorescent detection method is not sensitive enough to monitor the signal. The reduction of the NET markers can be used as an indicator of the rHCR-iFPs uptake by the tumor mass. If HCR exhibit high binding affinity to SV2 receptors in NET models but moderate anticancer efficacy, we plan to use rHCR peptide to conjugate with the nanocarrier for targeted drug delivery. In this case rHCR peptide can be used as a ligand that specifically binds to NE cancer cells and delivers anticancer drug. In 3D NET patient derived explants we expect significant reduction of NET markers and hormones (serotonin and calcitonin) in NE cancer cells upon long-term rHCR-iFPs treatment. In addition, we will perform multiplex protein quantification assay using Luminex to assess the various hormones, cytokines, and growth factors to be repurposed into a diagnostic and detection reagent, or a drug delivery ligand for targeted therapies. DISCUSSION/SIGNIFICANCE OF IMPACT: NE cancers are highly metastatic: NE cancers such as carcinoid, islet cell tumors, and medullary thyroid cancer frequently metastasize to the liver. They are the second most prevalent GI malignancy. Ninety percent of patients with pancreatic carcinoid tumors and 50% of patients with islet cell tumors develop isolated hepatic metastases. Patients with untreated, isolated NE liver metastases have <30% 5-year survival. Thus, there is a critical need for new therapies for NE cancers. Surgery is the only curative therapy available for patients with NE cancers but most cannot be cured: surgical removal is the most effective treatment for NE cancers; however, a very high percentage of patients present with metastatic disease. While surgical resection can be potentially curative, many patients are not candidates for operative intervention due to widespread metastases or the degree of hepatic involvement by the NE cancers. Moreover, other forms of therapy including chemoembolization, radioembolization, cryoablation, and chemotherapy have had limited efficacy. We hope that our in vitro data on rHCR toxicity and specificity to NET, validated in the pre-clinical models will allow the first in-human application of this technology in clinical studies.

OBJECTIVES/SPECIFIC AIMS: To compare the accuracy and precision for estimating the diagnostic accuracies (sensitivities and specificities) between differential verification (DV) and partial verification (PV) methods. Comparisons were made under scenarios with different values of design parameters including disease prevalence, proportion of verification for positive results, proportion of verification for negative result, sensitivity and specificity of the brass standard (BS) test in DV method. Through comparing 2 different verification methods under different scenarios, we give suggestions that which verification method is optimal under different design settings. METHODS/STUDY POPULATION: For both PV and DV methods, simulation studies were performed using statistical package R, version 3.1.3. We were primarily interested in studying how the unbiasedness and precision for estimation of diagnostic accuracies (sensitivity and specificity) of an index test change with the following design parameters: disease prevalence, proportion of verification for positive test results, the proportion of verification for negative test results, and the sensitivity and specificity of a BS test. We chose different values for each of the above parameters. For each estimation, we allowed values in only 1 parameter to change by fixing the other 2 parameters, so that the effect of each design parameter on the unbiasedness and precision of both sensitivity and specificity can be determined. For the DV method, we also developed an analytical method to estimate the sensitivity and specificity of an index test using a quadratic equation with a unique solution of the specificity and sensitivity. RESULTS/ANTICIPATED RESULTS: For rare disease with prevalence less than 1%, the PV method resulted in a less biased and more precise estimate of sensitivities and specificities of the index test. If the disease prevalence was between 1% and 10%, the DV method using a BS test with moderate or high sensitivity and specificity (sensitivity and specificity >90%) resulted in a less biased and more precise estimate of diagnostic accuracies of the index test. When the disease prevalence was greater than 10%, the PV method was superior when the BS test had sensitivity and specificity <80%, and the DV method was superior when the BS test had both sensitivity and specificity >90%. When the proportion of verification of positive test results was <30% or >70%, the DV method yielded smaller bias for the estimated specificity than the PV method. However, the PV method generated a much smaller mean square error (MSE) for specificity than the DV method when the proportion of verification for positive test results was >50%. Although the disease prevalence was >10% and the proportion of verification of positive test results was <30%, the DV method resulted in a smaller MSE for specificity. DISCUSSION/SIGNIFICANCE OF IMPACT: Disease prevalence and proportions of verification for patients with positive and negative test results influence the accuracy of a new diagnostic test. If a new index test for a very rare disease is evaluated, the PV method should be used for assessing the performance of the index test. When a disease prevalence is >1%, the DV method will result in a less biased and more precise estimate of diagnostic accuracy of an index test, if the BS test itself used in the DV method has large specificity and specificity. One concern of using BS test for the DV method is the clinical cost. Depending on the disease type, the BS tests usually are imperfect, but may be less aggressive and/or less expensive than the gold standard test. Moreover, as all clinical examinations require professional personnel to perform, verification of the index test for relative large proportion of a large cohort of patients could become a burden on human resources. Thus, the future research of the optimal design method for a diagnostic accuracy study should be based on the comprehensive cost-effectiveness analysis.

The National Center for Advancing Translational Sciences has called for more comprehensive research with priority populations to reduce disparities and for the development of additional resources to assist researchers in implementing these recommendations. Here we report the development and initial evaluation of five Priority Populations Toolkits, which are resources developed by the University of Illinois Center for Clinical and Translational Science to meet these goals. Three aims guide the content: increasing knowledge, facilitating communication, and improving research design. Materials were curated from scientific literature reviews and Internet searches and revised iteratively. Analytics and user surveys provide information about usage. In 22 months, 387 unique users accessed the toolkits. The top reason for usage was to improve research recruitment. Comprehensive toolkits for working with priority populations show promising potential for increasing knowledge and readiness to work with underrepresented populations. Further toolkit development and evaluation of effectiveness are warranted.

The last 50 years have seen an increasing dependence on academic institutions to develop and commercialize new biomedical innovations, a responsibility for which many universities are ill-equipped. To address this need, we created LEAP, an asset development and gap fund program at Washington University in St. Louis (WUSTL). Beyond awarding funds to promising projects, this program aimed to promote a culture of academic entrepreneurship, and thus improve WUSTL technology transfer, by providing university inventors with individualized consulting and industry expert feedback. The purpose of this work is to document the structure of the LEAP program and evaluate its impact on the WUSTL entrepreneurial ecosystem. Our analysis utilizes program data, participant surveys, and WUSTL technology transfer office records to demonstrate that LEAP consistently attracted new investigators and that the training provided by the program was both impactful and highly valued by participants. We also show that an increase in annual WUSTL start-up formation during the years after LEAP was established and implicate the program in this increase. Taken together, our results illustrate that programs like LEAP could serve as a model for other institutions that seek to support academic entrepreneurship initiatives.

OBJECTIVES/GOALS: Our primary objective was to determine the demographic and linguistic characteristics of research coordinators (RCs) in a large pediatric emergency medicine research collaborative network. Our secondary objective was to determine if the RCs perceived any impact of those characteristics on their duties. METHODS/STUDY POPULATION: We conducted a 15-question electronic survey of RCs at the member institutions of the Pediatric Emergency Care Applied Research Network (PECARN). A total of 74 potential respondents were identified and received the survey. RESULTS/ANTICIPATED RESULTS: Fifty-three surveys (71.6%) were completed. Most respondents identified as female; white; and not Hispanic or Latino. Fourteen respondents (26.4%) identified as underrepresented minorities in medicine (UIM), which is similar to the percentage of UIM among the general population (30%). Twenty-eight respondents (52%) felt that their race/ethnicity positively impacted recruitment efforts. Twenty-three respondents (43%) felt that their ability to speak a language other than English positively impacted recruitment efforts. Four female respondents felt that their gender hindered their recruitment activities and impacted their sense of belonging within the research team. DISCUSSION/SIGNIFICANCE: RCs felt that their backgrounds and attributes positively impacted subject recruitment. However, some female coordinators felt negatively impacted by their gender. Increasing diversity amongst clinical research professionals and incorporating team cultural humility practices, may help increase diversity among clinical research subjects.

OBJECTIVES/SPECIFIC AIMS: Prostate cancer (PCa) is the most common noncutaneous malignancy in men in the United States. A significant fraction of advanced PCa treated with androgen deprivation therapy experience relentless progression to lethal metastatic castration-resistant prostate cancer (mCRPC). The PCa tumor microenvironment is comprised of a complex mixture of epithelial and stroma cell types engaged in multifaceted heterotypic interactions functioning to maintain tumor growth and immune evasion. We recently uncovered the important role played by myeloid-derived suppressor cells (MDSCs) to mediate tumor immune evasion in aggressive PCa (Wang, Lu et al., Cancer Discovery, 2016). Immune checkpoint blockade (ICB) has elicited durable therapeutic responses across a number of cancer types. However, the impact of ICB on mCRPC has been disappointing, which may signal the need to combine mechanistically-distinct ICB agents and/or override immunosuppression in the tumor microenvironment. Our objective is to determine if robust immunotherapy responses in mCRPC may be elicited by the combined actions of ICB agents together with targeted agents that neutralize MDSCs yet preserve T cell function. METHODS/STUDY POPULATION: We created a novel embryonic stem cell-based chimeric mouse model of mCRPC engineered with signature mutations to study the response to single and combination immunotherapy. The efficacy studies were followed with detailed mechanistic investigation and clinical validation. RESULTS/ANTICIPATED RESULTS: Consonant with early stage clinical trials experience, anti-CTLA4 or anti-PD1 monotherapy failed to impact disease progression. Similarly, modest anti-tumor activity was observed with combination ICB as well as monotherapy with targeted agents including Cabozantinib (tyrosine kinase inhibitor), Dactolisib (PI3K/mTOR inhibitor), and Dasatinib (tyrosine kinase inhibitor). In contrast, mCRPC primary and metastatic disease showed robust responses to dual ICB treatment together with either Cabozantinib or Dactolisib, but not with Dasatinib which impaired T cell infiltration in the tumor. Detailed intratumoral immune profiling with mass cytometry (CyTOF) showed that combined ICB and Cabozantinib or Dactolisib was associated with significant depletion of MDSCs. Cabozantinib and Dactolisib blocked the PI3K signaling activity in MDSCs and reduced their immunosupppresive activity. Mechanistically, the combination efficacy was due to the upregulation of IL-1RA and suppression of MDSC-promoting cytokines secreted by mCRPC cells. DISCUSSION/SIGNIFICANCE OF IMPACT: We demonstrated that an antibody cocktail targeting CTLA4 and PD1 was insufficient to generate effective anti-tumor response, but combination of ICB with targeted therapy that inactivates PI3K signaling displayed superior synergistic efficacy through impairing MDSCs in the tumor microenvironment. These observations illuminate a clinical path hypothesis for combining ICB with MDSC-targeted therapies in the treatment of mCRPC. Importantly, conclusions from the study on PCa may have implications in combination immunotherapy for aggressive breast cancer which is also largely resistant to immune checkpoint blockade and replete with immunosuppressive myeloid cells.

OBJECTIVES/SPECIFIC AIMS: The purpose of this study is to characterize children with nonalcoholic fatty liver disease (NAFLD) living in the Southeastern United States. METHODS/STUDY POPULATION: This retrospective electronic medical record chart review was conducted on a random sample of 206 children identified with NAFLD. Patients were included if they met the following criteria: confirmed NAFLD through either an ultrasound or liver biopsy or clinical suspicion of fatty liver disease alongside elevated alanine aminotransferase (ALT) in the absence of other etiologies causing elevated transaminases. Patients were excluded if they had hepatitis or other documented liver disease. Data collected at initial presentation included age, gender, ethnicity, height, weight, body mass index (BMI), BMI percentile, blood pressure, HbA1c, aspartate aminotransferase (AST), ALT, γ-glutamyl transferase (GGT), total cholesterol, total triglycerides, low-density lipoprotein, and high-density lipoprotein. Statistical analysis: for descriptive statistics, frequency counts and percentages alongside means, standard deviation, range, min/max values for the continuous variables were calculated. RESULTS/ANTICIPATED RESULTS: This study included 206 children diagnosed with NAFLD. Subjects were primarily male (n=136, 66%) and Caucasian (n=133, 66%), followed by Hispanic (n=42, 21%), Black (n=25, 12%), and Asian (n=2, 1%). Mean age at diagnosis was 12.3±3.5 years. Mean weight (lbs), height (in), and BMI (kg/m2) of subjects at diagnosis were 192±77 lbs, 61.7±6.6 in, 34.6±9.7 kg/m2, respectively. Patients had an average systolic blood pressure of 124±15.4 mmHg and diastolic blood pressure of 69.6±10.6 mmHg. Mean ALT was 91.8±67.2 U/L, AST was 61±38.8 U/L, and GGT was 55.1±64.6 U/L. Mean HbA1c was 5.8±1.4%, cholesterol was 176±36.3 mg/dL, triglycerides were 200±134 mg/dL, low-density lipoprotein was 107.6±32.1 mg/dL, and high-density lipoprotein was 39.9±8.4 mg/dL. DISCUSSION/SIGNIFICANCE OF IMPACT: In addition to having significantly elevated liver enzymes, children with NAFLD had several derangements in their metabolic profile, most notably high triglyceride levels and HbA1c values in the prediabetic range. Although lifestyle modification is the gold standard treatment for NAFLD, pharmacotherapy may need to be included to address metabolic syndrome.

ABSTRACT IMPACT: The car telemetry system may be an ideal method to accurately and reliably evaluate and compare at-risk driving errors between older drivers with and without glaucoma. OBJECTIVES/GOALS: Our project aims to determine whether an in-car telemetry system used during an on-road driving evaluation can accurately and reliably evaluate driving errors in lane maintenance and visual scanning and objectively quantify the frequency and severity of these errors in glaucoma patients. METHODS/STUDY POPULATION: This is a single center, cross-sectional study of 180 participants (125 with glaucoma and 55 controls), ages 55 or older, who underwent a comprehensive clinical assessment, including vision, cognition, motor function, followed by an on-road evaluation by a trained occupational therapist. Driving errors were recorded through a dual method including: 1. An in-car trained occupational therapist 2. In-car telemetry system. The frequency and severity of errors in lane maintenance and visual scanning from the in-car telemetry will be assessed and compared between participants with varying severity of glaucoma and normal controls. In addition, we will compare the frequency and severity of errors in lane maintenance and visual scanning to those recorded by the in-car driving evaluator. RESULTS/ANTICIPATED RESULTS: We anticipate (or predict) that the in- car telemetry system will be able to detect frequency and severity of driving errors in lane maintenance and visual scanning in glaucoma participants. We also predict that participants with worsening glaucoma severity will commit more driving errors. In addition, the in-car telemetry will detect a similar frequency and severity of driving errors as the in-car driving evaluator. DISCUSSION/SIGNIFICANCE OF FINDINGS: The type and frequency of vision-related driving errors that place individuals at risk for a car accident is not well known. Without this critical information, it is extremely challenging to help older adults with glaucoma to be safe drivers.

OBJECTIVES/SPECIFIC AIMS: The morbidity and mortality in adults with single ventricular hearts who have undergone Fontan palliation is poorly defined. These patients have a high burden of arrhythmia, heart failure, and re-operation. We hypothesized that age and type of Fontan predict occurrence of arrhythmia. METHODS/STUDY POPULATION: In total, 205 patients aged 18 years who had undergone a Fontan procedure were identified. Those with incomplete data were excluded. Demographic, anatomic, pharmacologic, imaging, hemodynamic, and electrophysiologic data were collected. The χ2 and Mann-Whitney U tests were used to test significance defined as p<0.05. RESULTS/ANTICIPATED RESULTS: Of the 205 patients identified, 59 had been lost to follow-up. Of the 146 patients (77, 53% female) actively followed 18 (12%) had died at a median (IQR) age of 27 (21–34.3); in patients alive as of 10/2016 the median age was 26 years (22–34). Fontan types were lateral tunnel (LT) (n=79, 54.1%), extracardiac (EC) (n=32, 22%), right atrial to pulmonary artery (RV-PA) (n=28, 19%), and Fontan with Bjork modification (n=4, 2.7%). Systemic left ventricle (n=96, 66%) was more common than systemic right ventricle (n=43, 30%). Of the 146 patients, 101 (69%) had significant morbidity or mortality: 86 (59%) were diagnosed with arrhythmia, 18 (12%) died, and 11 (8%) underwent heart transplants. Frequent procedures included: Fontan revisions/cryoablation in 28 (19%), electrophysiology studies with ablation in 73 (50%), and pacemakers in 53 (36%). Of the arrhythmia diagnoses, 57 (64%) were atrial tachyarrhythmias. RV-PA Fontan procedures were associated with significantly more atrial arrhythmia than all other Fontan types (70% vs. 30%; p<0.01). There was no statistical difference in occurrence of atrial arrhythmia in adults with LT Versus EC Fontans (p=0.3). While patients who had undergone RV-PA and Bjork Fontans were older with median age 34 years, there was no significant difference in age between LT and EC (median 24.0 and 24.5). DISCUSSION/SIGNIFICANCE OF IMPACT: Adult survivors of the Fontan procedure suffer from significant morbidity and mortality. The single most prevalent morbidity is atrial arrhythmia. We conclude that RV-PA Fontans, now obsolete, have the highest prevalence of arrhythmia and that there is no difference in arrhythmia burden between LT and EC Fontans. Given the high prevalence of morbidity and mortality in this population, it is imperative that they be followed by cardiologists with expertise in congenital heart disease.

OBJECTIVES/GOALS: Parental SES may influence the trajectory of students applying to orthopaedic surgery residency, perpetuating opportunistic disparities. Thus, we sought to examine the relationship between parental occupation/education and applicant match rate, education financing, and medical school background. METHODS/STUDY POPULATION: Data from the Association of American Medical Colleges (AAMC) documented parental occupation and education levels of 10,697 orthopedics applicants from 2011 to 2021. Parental occupations were categorized into physician vs non-physician, healthcare vs non-healthcare, working class vs non-working class, and STEMM (Science, Technology, Engineering, Mathematics, Medicine) vs non-STEMM. Parental education levels spanned from no college degree to doctorate degrees and were used as a proxy for SES. Outcomes analyzed included match success, premedical and medical school debt, total educational debt, scholarships, and representation from top 40 research medical schools as determined by NIH funding. RESULTS/ANTICIPATED RESULTS: Physician parent applicants (20.1%) had better match rates (75.5% vs. 73.5%), lower debts, lesser scholarships, and higher top 40 school representation. Healthcare parent applicants (37.0%) had similar match rates, less debt and scholarships. Working class parent applicants (6.0%) had more debt and scholarships. STEMM parent applicants (48.6%) had higher match rates, lesser debts and scholarships, and higher top 40 representation. Applicants with parents without college degrees had lower match rates (68.6% vs 74.5%), more debt and scholarships. Doctorate parent applicants had better match success (75.9% vs 72.9%), lesser debts, and higher top 40 school representation (34.9% vs 29.6%). DISCUSSION/SIGNIFICANCE: Parental SES was associated with substantial variation in applicant financial burden and educational pedigree. Notably, applicants with parents lacking degrees had lower match rates, underscoring the need for supportive strategies to ensure equitable opportunities for aspiring orthopaedic surgeons.

OBJECTIVES/SPECIFIC AIMS: Incomplete spinal cord injury typically results in life-long disability, often in the form of profound loss of locomotion capability. Individuals who have experienced incomplete spinal cord injury exhibit persistent eccentric motor deficits, which are particularly prevalent in the weight acceptance phase of gait and emphasized in sagittal plane knee motion and frontal plane hip motion. METHODS/STUDY POPULATION: Motion analysis can capture the kinematic and joint-level deficits of these individuals, but it is impossible to directly calculate the contributions of individual muscles to weight acceptance due to the complexity of the musculoskeletal system. Instead, those muscle contributions must be simulated in order to approximate muscle power during locomotion. RESULTS/ANTICIPATED RESULTS: The traditional method for driving these simulations with electromyography readings is unavailable for individuals who have neuromuscular deficits (e.g., spasticity or paralysis), due to the need to generate reliable maximum voluntary isometric contractions for baseline purposes. Instead, this research develops a novel method for using resting electromyography data to drive musculoskeletal simulations using a muscle activation threshold paradigm. DISCUSSION/SIGNIFICANCE OF IMPACT: The simulation results of this method more closely resemble experimental results, but further simulation refinement is needed to fully capture the true muscle activity.

OBJECTIVES/SPECIFIC AIMS: More than half a million adult patients nationally undergo cardiac surgery each year. Reintubation following cardiac surgery is common and associated with higher short- and long-term mortality, increased cost, and longer lengths of stay. The reintubation incidence is estimated at 5-10%. Patients undergoing cardiac surgery are increasing in age and comorbidity burden, and receive increasingly complex cardiac surgical procedures, complicating decision making around when to extubate postoperative patients. Compounding this complexity are financial pressures to maintain high throughput and maximize ICU bed availability. Providers are often compelled to extubate high-risk patients earlier, despite the potential for an increased risk of reintubation. Understanding the risk factors for reintubation after cardiac surgery and identifying effective interventions to reduce these reintubations is of critical importance to optimize patient outcomes. High-flow nasal cannula (HFNC) provides up to 60 liters per minute of 100% oxygen, dead space washout, and humidification to improve secretion clearance, and has shown some benefits in improving hypoxia and reducing reintubation in select populations. However, its benefit in high-risk patients undergoing cardiac surgical procedures is not known and therefore clinicians may still be reluctant to extubate these patients early and introduce HFNC, despite the known risks of prolonged intubation. To address this important issue, we aim to develop and validate a model to predict postoperative reintubation after cardiac surgery using data readily available from the electronic health record (EHR) and use this data to complete a pilot randomized controlled trial (RCT) of post-extubation HFNC to prevent reintubation in cardiac surgery patients identified as at high risk for reintubation. METHODS/STUDY POPULATION: Based on retrospective data demonstrating a 4.7% reintubation incidence within 48 hours in our CVICU, we estimate that there will be 340 reintubations available for analysis of the risk factors for reintubation to develop our predictive model from November 2, 2017 (our EHR go-live). We require 15 events per predictive variable to avoid overfitting the model, giving us at least 22 variables for analysis and inclusion in the model. Model validation and calibration will be performed using a bootstrapped validation cohort. Next, we will prospectively study 120 patients with a greater than 10% predicted risk of reintubation (double the baseline risk of the overall population) and randomly assign them to either HFNC or usual care, to test the hypothesis that HFNC decreases the rate of reintubation in high-risk patients. RESULTS/ANTICIPATED RESULTS: In addition to developing a predictive model, refining it, and validating its ability to predict the primary outcome of reintubation within 48 hours, I will further assess whether HFNC reduces total duration of mechanical ventilation, hospital length of stay, and ICU length of stay in this high-risk population. I will use these data to establish the feasibility of EHR-integrated predictive modeling and randomization, as well as to guide a future multicenter clinical trial that will pragmatically leverage the EHR for patient selection, enrollment, randomization, and data collection. DISCUSSION/SIGNIFICANCE OF IMPACT: Assuming HFNC decreases reintubation rates by 50%, at a 1:1 ratio of cases to controls, we will require 435 patients in each group (970 total), to have an 80% power and alpha of 0.05 to detect a difference. As this will require a multicenter study, we will instead focus on using data from this pilot study to: 1) refine our sample size estimates. 2) demonstrate the feasibility of our novel EHR-integrated pragmatic trial design. 3) identify and screen collaborators at other institutions, including obtaining important regulatory and legal approval. 4) establish a data safety monitoring board for the trial. 5) refine the data collection infrastructure, leveraging commercially available resources in one of the largest enterprise EHR systems (Epic) and associated resource-sharing products, such as Epic’s App Orchard.

OBJECTIVES/GOALS: The current opioid epidemic has placed post-operative pain management under scrutiny. Limiting post-operative pain can decrease overall opioid usage in the recovery period, especially after orthognathic surgery. Several studies have illustrated the efficacy of pregabalin in decreasing postoperative pain and opioid usage in adults undergoing orthognathic surgery. We aim to study the effects of a single dose of preoperative pregabalin on postoperative pain and total opioid consumption after orthognathic surgery in individuals with cleft lip and palate. METHODS/STUDY POPULATION: This was a retrospective cohort study of consecutive patients who received Le Fort I midface advancement between June 2012 and July 2019 by one of two surgeons at a single institution. We took advantage of our institution’s implementation, beginning in 2016, of a one-time dose of preoperative pregabalin for LeFort I midface advancement. All patients had diagnosed cleft lip and palate. The treatment group received a one-time preoperative dose of pregabalin. The control group did not receive pregabalin. Total morphine milligram equivalents (MME) consumption was calculated by adding intraoperative opioid administration and postoperative opioid consumption during admission. Postoperative pain control during admission consisted of oral oxycodone and intravenous (IV) hydromorphone or morphine. Duration of hospitalization and pain intensity assessed with the numeric pain rating scale (0-10) were also recorded. The mean postoperative pain assessment scores during admission was calculated for each patient. The median of these individual mean pain assessment scores for each group was subsequently computed. RESULTS/ANTICIPATED RESULTS: Twenty-three patients (14 males, 9 females) were included in this study; 12 patients received pregabalin (median dose: 150mg, range: 100-200mg). Mean age (years) at operation of the pregabalin (18.3 ± 1.9) and control groups (17.8 ± 1.9) were also equivalent (p = 0.571). Median hospital stay for both groups was 1.0 day. The pregabalin group had significantly lower consumption of total opioids during admission (total MME 70.95 MME; IQR: 24.65-150.17) compared to the control group (138.00 MME; IQR: 105.00-232.48) (MU = 31.00, p = 0.031). Although pain scores in the treatment group (3.21 ± 2.03) were lower than in the control group (3.71 ± 2.95), the difference was not statistically significant (p = 0.651, 95% Cl [−1.75, 2.75]). DISCUSSION/SIGNIFICANCE OF IMPACT: Based on the results, a one-time preoperative oral dose of pregabalin before orthognathic surgery in patients with cleft lip and palate reduced total opioid consumption during admission. However, there was no difference in length of stay or pain scores within the two groups. A single preemptive oral dose of pregabalin should be considered an effective adjunct to pain management protocols in patients undergoing orthognathic surgery.

OBJECTIVES/GOALS: The inclusion of underrepresented racial and ethnic groups (URGs) in clinical research is critical for ethical and scientific reasons. This initiative aimed to assess the perspectives, barriers, needs, and recommendations encountered by research teams when enrolling and retaining URGs in clinical research. METHODS/STUDY POPULATION: An anonymous, web-based survey comprised of quantitative and qualitative questions was administered to individuals involved in clinical research at an academic medical center. The survey assessed three main domains: 1. Research teams' perceptions and experiences with enrolling URGs in clinical research, 2. Factors that discourage URGs from participating in clinical research, and 3. Research teams’ overall willingness to support URG enrollment. Demographics were also collected. The survey was reviewed by experts in clinical research, research ethics, and diversity, equity, inclusion, and accessibility (DEIA). The assessment was piloted among research professionals and edits were made accordingly prior to official dissemination. Data were analyzed using descriptive statistics. RESULTS/ANTICIPATED RESULTS: There was a total of 63 responses. A majority of respondents have more success enrolling patients whose primary language is the same as their own and that time arranging for an interpreter has negatively impacted enrollment efforts. Approximately half of the respondents believe that the race and/or ethnicity of the potential study participant influences enrollment success. Factors discouraging URGs from participating in clinical research include unavailability for follow-up visits due to transportation issues, distrust in doctors and/or researchers, fear of unknown side effects, and unavailability of medical interpreters. Respondents report that they are not discouraged from enrolling URGs and would utilize resources related to encouraging the inclusion of URGs DISCUSSION/SIGNIFICANCE: Language appears more influential than ethnicity or race when it comes to enrolling and retaining URGs. Additionally, it appears that enrolling is a bigger challenge than retaining. Major themes that emerge with respect to retaining enrolled participants include the inability to attend follow-up visits and the lack of incentives/compensation.

OBJECTIVES/GOALS: The use of Human Centered Design (HCD) to improve the quality of team science is a recent application, and HCDs benefits and challenges have not been rigorously evaluated. We conducted a qualitative study with health sciences researchers trained in HCD methods to determine how they applied HCD methods and perceived its benefits and challenges. METHODS/STUDY POPULATION: The University of Pittsburgh offered HCD training to three cohorts of research scientists (staff as well as faculty) over a three-year period. The training was provided by the LUMA Institute, a premier HCD design firm with a highly regarded training program. We then evaluated this training by conducting 1-hour, semi-structured interviews with trainees from three training cohorts. Interviews focused on perceptions of the training, subsequent uses of HCD, barriers and facilitators, and perceptions of the utility of HCD to science teams. Data analysis was conducted using Braun and Clarkes process for thematic analysis. RESULTS/ANTICIPATED RESULTS: We interviewed 18 researchers (nine faculty and nine staff) trained in HCD methods and identified distinct themes regarding HCD use and its perceived benefits and challenges. Trainees found HCD relevant to research teams for stakeholder engagement, research design, project planning, meeting facilitation, and team management. They also described benefits of HCD in five distinct areas: creativity, egalitarianism, structure, efficiency, and visibility. We also identified challenges, including tensions between HCD approaches and academic culture. DISCUSSION/SIGNIFICANCE: Our data suggest that HCD has the potential to help researchers work more inclusively and collaboratively on interdisciplinary teams and generate more innovative and impactful science. The application of HCD methods is not without challenges; however, we believe these challenges can be overcome with institutional investment.

ABSTRACT IMPACT: Younger patients receiving biologics for rheumatoid arthritis have higher medical expenditure. OBJECTIVES/GOALS: TNF inhibiting biologic disease modifying antirheumatic drugs are among the most highly regarded treatment options for rheumatoid arthritis (RA). We aimed at evaluating the medical and prescription costs associated with monoclonal antibody use vs. other RA treatment options in subjects diagnosed with RA. METHODS/STUDY POPULATION: Records from the Medical Panels Expenditure Survey (MEPS) database made available by the Agency for Healthcare Research and Quality were used to identify all RA subjects (n=_____). Demographics and MEPS-provided flags for RA were abstracted from the medical condition files for all the subjects surveyed (2008-2018). Prescribed biologics were identified based on generic and brand names following a manual review to detect any misspellings. Total medical expenses and prescription expenses were abstracted for all identified RA subjects. Subject were surveyed for two consecutive years, thus expenses were assessed for each of the two surveyed years. Costs were adjusted for inflation and expressed in 2018 dollars. The relationship between biologics use, cost and age or gender was evaluated by Fisher’s exact test. RESULTS/ANTICIPATED RESULTS: Most RA subjects did not use biologics. RA was more prevalent in women than in men with no significant correlation between sex and the use of biologics in year 1, year 2, or the combined years (p=.44, p=.63, and p=.65, respectively). Biologics users were found to be significantly younger (p<.001), with a mean of 52.8 years compared to 59 years in those who did not use biologics. The 95% confidence interval was 3.7 to 8.6 years younger than non-users. Total medical and prescription costs were higher for biologics users (p<.001) in all analyses. The mean prescription cost difference was $24,038 more per year for biologics users, and $26,296 more total medical expenses, CI $20,502-$27,230 and CI $21,947-$30,646, respectively. There was a trend for biologics users to have higher non-prescription medical expenses (p=.05). DISCUSSION/SIGNIFICANCE OF FINDINGS: Interestingly, biologics non-users had some extreme outliers with expenses far higher than any biologics users, possibly due to poorly controlled RA due to age and/or comorbidities. Yet, our most interesting findings are the higher use of biologics among younger RA subjects and the elevated costs of care being driven mainly by prescriptions cost.

OBJECTIVES/GOALS: In this study we sought to determine the role of glucagon-like peptide-1 (GLP-1), one of the main gut hormones in regulating glucose metabolism, after protein ingestion in patients with a history of Roux-en-Y gastric bypass (GB) and sleeve gastrectomy (SG). METHODS/STUDY POPULATION: We examined the glucose and islet-cell secretory responses to 50 g protein ingestion with and without a potent GLP-1 receptor antagonist, exendin-(9-39) [Ex-9], in 10 GB-treated subjects, 9 SG-treated, and 7 non-operated controls (CN). The groups were matched for age, BMI, fat-free mass, fasting glucose and insulin, and HbA1c. The surgical groups also were matched for weight loss and time post-surgery. No subjects had diabetes. RESULTS/ANTICIPATED RESULTS: Protein ingestion resulted in an early rise in glycemia (AUCGlucose1hr) in GB and SG, whereas CN had minimal change in glucose (p<0.05). Protein ingestion enhanced C-peptide responses in all groups, but to a larger extent in GB and SG when compared to CN (p<0.05). Early glucagon response to protein ingestion (AUCGlucagon1hr) tended to be larger in GB and SG subjects when compared to CN (p=0.07). Ex-9 increased premeal and prandial glycemia in all groups (p<0.05), but increase in early glycemia (AUCGlucose1hr) was most notable in GB (p=0.1, interaction). This glycemic effect of Ex-9 was associated with a ~25% reduction in prandial C-peptide secretion in GB and SG and ~8% increase in CN (p<0.05, interaction). Early prandial glucagon responses were larger during studies with Ex-9 compared to those without (p<0.05). DISCUSSION/SIGNIFICANCE: Our findings indicate that glucose metabolism after protein ingestion is altered after GB and SG. To our knowledge, this is the first report to demonstrate that endogenous GLP-1 contributes to glucose and islet-cell secretory response to protein ingestion, and that GB and SG exaggerate GLP-1 contribution to insulin secretion after protein ingestion.