OBJECTIVES/GOALS: The first aim was to construct a controlled and high resolution FUS water tank characterization system with 1 micron step-sizes. The second aim was to create two unique standardized protocols for mapping the generated acoustic field from FUS transducers; protocol one maps the full 3D field while protocol two rapidly detects changes to the original plot. METHODS/STUDY POPULATION: To accomplish aim one, the focused ultrasound mapping platform was constructed with a water conditioning unit for water degassing and temperature control, a three-axis stage with 1 micron step-size capabilities, and a data plotting software. To measure the outcomes of aim one, the water temperature was monitored, and axis step sizes were measured through ten independent axis translation recordings. To accomplish aim two, FUS acquisitions were executed at different resolutions. For FUS localization at the cellular level, a 1-5 micron step size is required. Once the initial scan was performed, duplicate scans were executed to detect inherent perturbations or errors in the system. Once calculated, the best methods of detecting true changes to FUS signals are proposed. RESULTS/ANTICIPATED RESULTS: The FUS characterization system maintained water temperature and performed 1 micron step-sizes. While pre-existing platforms have demonstrated a resolution of one thousand recordings per cubic millimeter, the proposed system (time and computing power willing) can record one billion recordings per cubic millimeter. In practice, a resolution of 20 micron was sufficient for non-cellular level FUS characterizations. Successive 2D scans were reliably stacked to form a 3D rendering of the generated acoustic field with the average focal point intensity yielding a 1% coefficient of variation between identical scans. This inherent variation can be used as the threshold of significance for true change detection; to rapidly detect changes to the FUS signal, sampling can be performed at regions of high baseline values. DISCUSSION/SIGNIFICANCE: Focused ultrasound medical devices are gaining popularity for treatments including tumor ablation, neuromodulation, and drug delivery; however, the field lacks a standardized method to characterize these FUS transducers. The presented platform and protocols enable a rigorous and high quality translation through verification and validation.

OBJECTIVES/GOALS: The innate immune responses to Multisystem Inflammatory Syndrome in Children (MIS-C) are not fully known. Using samples from MIS-C, we will assess the cellular responses and develop a novel Tri-Specific Killer Engager (TRiKE) that engages innate immune cells to improve those responses. METHODS/STUDY POPULATION: We collected blood samples from 60 pediatric patients from which we isolated plasma and peripheral blood mononuclear cells. We received blood samples from 13 MIS-C, 32 severe acute COVID, 5 COVID-19 asymptomatic, and 15 COVID-19 negative patients. Using plasma, we then performed ELISAs to determine IgG antibody levels against SARS-CoV-2 and plaque reduction neutralization tests to determine neutralizing antibody functions. We isolated DNA to look at Fc receptor genetics. We also utilized utilize flow cytometry assays determine the phagocytosis and killing abilities of the innate cells from these patients. This data will be correlated with clinical outcomes. Additionally, we have developed a novel SARS-CoV-2 TRiKE which directs natural killer (NK) cell killing specifically to of COVID-19 infected cells. RESULTS/ANTICIPATED RESULTS: MIS-C patients had higher IgG antibody titers against SARS-CoV-2 compared to children with symptomatic or asymptomatic COVID. MIS-C patients also neutralized SARS-CoV-2 more effectively than children with acute symptomatic or asymptomatic COVID-19. We found natural killer cells and monocytes are dysfunctional in MIS-C patients and do not kill SARS-CoV-2 infected cells as well. Specifically, NK cells do not kill COVID-19 infected cells as well. To combat this, we have successfully generated and are now testing a Tri-Specific Killer engager (TRiKE) which binds one ends to NK cells, one end to the Spike protein on COVID-19 infected cells and contains IL-15 to improve NK cell function. We anticipate that we can improve NK cell killing of COVID-19 infected cells with this TRiKE. DISCUSSION/SIGNIFICANCE: We found that MIS-C patients have antibodies that can neutralize SARS-CoV-2 but that that innate immune cells that engage antibodies are dysfunctional. We are have successfully developed and are targeting this response with a TRiKE to improve innate immune cell functional; this may serve as an adjunctive therapeutic if proven successful.

Objectives/Goals: To determine the acceptability of a mobile/school-based diabetes and prevention clinic to overcome transportation barriers and improve attendance at endocrinology appointments for youth with type 1 diabetes, type 2 diabetes, MODY diabetes, and obesity living in socially vulnerable communities Methods/Study Population: This study utilized a 3-phase implementation process focused on feasibility, demand, and acceptability. Phase 1: identified high-need areas using patient volume and disease control metrics. Collaborated with school districts to define needs, enrolled them through a memorandum of understanding, and subsequently recruited and consented existing patients attending these schools. Phase 2 piloted the program to refine logistics and workflows. Semi-structured interviews and surveys were conducted with caregivers to assess program acceptability and satisfaction. Phase 3 is ongoing and will track clinical outcomes including glycemic control, appointment attendance, hospital admissions, emergency department visits, and self-management metrics. Results/Anticipated Results: Demand for the program was high with 91% (10/11) of approached districts agreeing to participate. District enrollment was staggered, starting with 1 district in August 2023, expanding to 5 in September, 7 in October, and reaching 8 districts by May 2024. From August 2023 to July 2024, the school-based mobile clinic conducted 355 individual clinical encounters addressing diabetes and obesity. The encounters included 180 for type 1 diabetes, 69 for type 2 diabetes, 8 for MODY diabetes, and 98 for obesity. Interviews and surveys with 36 caregivers are ongoing to evaluate program satisfaction. Discussion/Significance of Impact: The data suggest that this innovative healthcare delivery model is feasible. District-level enrollment demonstrates a demand for the program. Results from the interviews and surveys will further characterize the program’s acceptability among caregivers and lay the groundwork for future efficacy testing.

OBJECTIVES/GOALS: Precision care may engage smokers and providers in treatment but is understudied in the community. We piloted guideline-based care (GBC) alone or with Respiragene, a lung cancer polygenic risk score (PRS, 1-10), or metabolism-informed choice of medication using the nicotine metabolite ratio (NMR). METHODS/STUDY POPULATION: Daily smokers (n = 58) with stored biospecimens in the Southern Community Cohort Study were randomized 1:1:1 to GBC, PRS, or NMR, counseled to quit smoking, and co-selected FDA-approved cessation medication (nicotine replacement, varenicline) with a tobacco counselor. In PRS, precision motivational counseling was guided by PRS (i.e., lung cancer risk 10-40-fold that of never-smokers). In NMR, precision medication recommendations consisted of varenicline for faster metabolizers (NMR≥0.31) and nicotine replacement for slow metabolizers (NMR<0.31). Feasibility was defined as achieving at least 50% provider engagement (med prescription) and at least 50% patient engagement (self-reported med use). RESULTS/ANTICIPATED RESULTS: Participants were median age 59, 72% female, 81% Black, 60% with incomes <$15,000; median cigarettes/day was 15 (IQR 8-20) and 52% reported time-to-first cigarette <5 minutes, illustrating moderate nicotine dependence. Providers confirmed medication prescriptions for 40% of patients (32% GBC, 50% PRS, 37% NMR) and 83% of patients reported using medication (prescribed or unprescribed) during the study (90% GBC, 80% PRS, 79% NMR). At 6-month follow-up, 27% (n = 15) reported cessation (39% GBC, 16% PRS, 26% NMR). Among persistent smokers, 46% reported smoking at least 50% fewer cigarettes/day compared to baseline (45% GBC, 38% PRS, 57% NMR). Small sample size precluded statistical comparisons. DISCUSSION/SIGNIFICANCE OF IMPACT: Precision interventions to quit smoking are feasible for community smokers, who engaged at high rates. However, only 40% of providers supported patients’ quit attempts with medication prescriptions. Future research should test strategies to raise provider engagement in precision smoking treatment. CONFLICT OF INTEREST DESCRIPTION: R.F.T. has consulted for Quinn Emmanual and Apotex on unrelated topics. H.A.T. reported providing input on design for a phase 3 trial of cytisine proposed by Achieve Life Sciences and being a principal investigator of National Institutes of Health–sponsored studies for smoking cessation that include medications donated by the manufacturers. Other authors declare no potential conflicts of interest.

OBJECTIVES/GOALS: One in six children in the U.S. has a Neurodevelopmental Disability (NDD). Prechtl’s General Movement Assessment (GMA) is a qualitative predictor of early motor dysfunction. However, no quantitative biomechanical assessment exists to more accurately identify all patients with NDD. METHODS/STUDY POPULATION: With UAMS IRB approval, as part of a larger study, healthy infants were filmed while lying supine on a force plate for 2 minutes. We studied 12 healthy full-term infants (gestational age: 38.9±1.5 weeks, age: 2.1-7.0 months; 7M, 5F; length: 64.0±5.2 cm; weight: 7.2±1.3 kg). Within our data set there were 3 infants transitioning to fidgety period (≤3 months), 4 in the fidgety period, (3-5 months), and 5 that matured beyond fidgety period (>5 months). Center of pressure (COP) path-lengths were gathered from the force plate at 1000 Hz. We grouped our data with K-means clustering and performed statistical analysis with ANOVA. RESULTS/ANTICIPATED RESULTS: We divided our data into 3 distinct clusters. The first group contained infants with moderate variability of movements which included 2 infants between 3 and 5 months and 2 infants slightly outside of this range. The second group, with mild variability in movements, included 4 infants between 2 and 3 months as well as 2 infants just older than 5 months. The third group, with little variability in movements, included 2 infants older than 5 months. A GMA reader (TJ) qualitatively confirmed these findings with video footage. Using a threshold of p<0.05, data sets within the clusters were similar and significantly different from other clusters. DISCUSSION/SIGNIFICANCE OF IMPACT: Fidgety infants have greater variability in COP patterns than their mature counterparts. We anticipate additional COP measurements will correspond with qualitative GMA analyses. Artificial Intelligence-based quantification of the GMA may be useful in earlier detection or prediction of NDD outcomes.

OBJECTIVES/SPECIFIC AIMS: This study aims to understand the utility of 5-ALA and SWIG in detecting areas of neoplasm in a murine model of GBM. Primary outcome is the distribution of the two dyes in comparison to the true tumor extent; the sensitivity, specificity, PPV, and NPV of both dyes will be calculated. The secondary outcome is the suitability of existing cell-lines used for GBM research for studies in fluorescence-guided surgery. METHODS/STUDY POPULATION: Two cell lines are used for this research: U87, derived from human GBM; and GL261, derived from rodent stem cells. U87 are implanted intracranially into 6-week old athymic, nude, female mice, while GL261 are implanted intracranially into 10-week old female C57BL/6 mice. The mice are weighed every 3 days to monitor health and bioluminescence imaging is performed between 7-10 days after implantation to confirm tumor implantation and monitor tumor growth. The mice are sacrificed between 10-21 days after implantation. 5mg/kg of intravenous ICG is administered 24-hours prior to harvest and 250mg/kg of intraperitoneal 5-ALA is administered 3-hours prior to harvest. Once the mice are sacrificed, their brains are quickly harvested and placed in cold formalin. Using a high-resolution Odyssey CLx scanner, near-infrared fluorescence from ICG is captured in coronal cross sections of the brains through the tumor. Similarly, 5-ALA fluorescence is imaged using a 405nm LED excitation source and 610-690nm bandpass filter. Afterwards, slices of the brain are stained with H&E, which serves as the gold-standard of the extent of tumor. Images from ICG, 5-ALA, and H&E can then be compared using ImageJ to compare the extent of tumor to the distribution of the dyes. RESULTS/ANTICIPATED RESULTS: In separate, previous studies in humans, both 5-ALA and SWIG have demonstrated utility in detecting residual neoplasm in HGG resections. In general, 5-ALA is more specific for areas of neoplasm, while SWIG is more sensitive. Thus, I anticipate that in this study, SWIG will show a greater distribution than 5-ALA, with SWIG distributing to areas beyond the tumor and 5-ALA distributing within, but not completely covering, the tumor. SWIG’s sensitivity and NPV for detecting tumor should be >90%, while its specificity and PPV may be closer to 50%. For 5-ALA, specificity and PPV should be close to 80-90%, but its sensitivity and NPV may be <50%. In terms of cell-line, preliminary results suggest that U87 cells are not suitable for research involving 5-ALA. We suspect that this is partly due to the limited infiltrative nature of U87 cells; in fact, the cells form a spherical mass, imitating metastases rather than true HGGs. The U87 masses do not have significant vascularity, which likely limits the amount of 5-ALA that can distribute to inside the tumor. DISCUSSION/SIGNIFICANCE OF IMPACT: 5-ALA is currently the only FDA-approved agent for fluorescence-guided neurosurgery. However, it has multiple limitations, which ultimately results in its low sensitivity and NPV. Our novel technique, which has demonstrated much higher sensitivity at the cost of specificity, offers an alternative that may help surgeons better achieve total resections in the operating room. These two agents have not been compared directly in humans or mice. Thus, this experiment sets up an important precedent, on which a human clinical trial comparing the two agents’ effects on resection rates and patient outcome can be performed. Ultimately, this work will lay the foundation for future research into fluorescence-guided neurosurgery, both in the visible and NIR spectrum.

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

Objectives/Goals: There has been a significant increase in the engagement of researchers with persons with lived and living experience, driven by a growing recognition of the invaluable insights and expertise these individuals bring to the research process. However, there remains a need for research teams to learn how to engage with patients and community partners. Methods/Study Population: The EMPOWER project is a collaborative of members with lived/living expertise of substance use during pregnancy. EMPOWER meets three times per month in support of their goals of patient-centered research, changing the narrative through conversations, and building the capacity of persons with lived experience to co-lead research. Through over 4 years of active collaboration and co-learning, EMPOWER has identified and created methods that support meaningful patient engagement and capacity building. Results/Anticipated Results: EMPOWER has identified the following methods that support meaningful engagement and capacity building: Mind-Mapping Exercises, Personal and Collective Journey Mapping, 8-month research training curriculum, Digital Storytelling, Learning Labs, and World Cafe Meetings. These methods have led to a collaborative of 20 individuals with lived experience of substance use to identify research gaps, prioritize research questions, design data collection tools, analyze research data, disseminate research results, and consult with healthcare teams in efforts to improve clinical care. To-date EMPOWER have given over 10 invited presentations and is currently writing manuscripts. Discussion/Significance of Impact: Meaningful patient and community engagement is critical to developing and implementing healthcare interventions. Unfortunately, engagement efforts are often lack in building capacity of patients and community, which impedes their ability to fully engage in all phases of research.

OBJECTIVES/GOALS: The goal of this project was to perform an exploratory analysis of the research themes of scientific publications from the Miami Clinical and Translational Science Institute using text mining techniques and using bibliometric characterization and network analysis to further assess research trends. METHODS/STUDY POPULATION: Publications were identified from the Web of Science database using Miami CTSI grant numbers as search criteria for the period 2013-2021 and KL2 scholar publications. Following data pre-processing, topic modeling was performed using the Latent Dirichlet Allocation algorithm and cluster analysis in the R programming language. The resulting themes will be further analyzed by employing a citation-based impact assessment approach to identify trends over time. Network analysis of publications will be performed using the VOSviewer package to visualize publication networks using citation and co-authorship relations within each major theme and their evolution over time. Findings will be evaluated for alignment with institutional research strategy. RESULTS/ANTICIPATED RESULTS: About 400 CTSI publications from 2013-2021 to date were used for analysis. Twenty topics and five major research themes were identified among the Miami CTSI publications – neuroscience, cancer, community and public health, metabolics, and HIV/infectious diseases. Top keywords within each topic were aligned with the most frequent author-assigned keywords for that topic. The CTSI research themes were also well-aligned with the institutional vision for research and focus areas. Trends using citations and networks for each research theme are currently being analyzed and results will be included in the overall findings post analysis. DISCUSSION/SIGNIFICANCE: Text mining was successfully used in identifying topics and research themes for clinical and translational research publications of the Miami CTSI, and in combination with bibliometric characterization, will be helpful in shaping CTSI strategy and alignment with the universitys research priorities.

OBJECTIVES/GOALS: Age-related lobular involution (LI) is a physiological process of breast epithelial regression that occurs primarily during perimenopause (ages 45-55); women in this age range for which the process of LI is delayed, defined as LI stagnation, show significantly increased risk of breast cancer as compared to LI progression patients. METHODS/STUDY POPULATION: The Mayo Clinic Benign Breast Disease (BBD) cohort includes ~1000 women who had multiple sequential benign biopsies. 103 patients were found to have sequential biopsies during the perimenopausal period, of which 10 eventually progressed to breast cancer. These patients were assessed for LI stagnation vs LI progression by quantifying 10 lobules per slide and comparing median acini number and median lobule size between initial and subsequent biopsies from the same patients. RNA was derived from whole tissue sections from the initial biopsies, and profiled using NanoString IO360 and BC360, which were normalized using RUVg methods. Differentially expressed genes associated with LI stagnation were defined as having two-tailed, unpaired p-values less than 0.05. RESULTS/ANTICIPATED RESULTS: Analysis showed subsetting patient sets by time between biopsies improves classification of stagnant vs. progression. Differential gene analysis identified 37 genes associated with LI stagnation and LI progression, and 20 of these genes were found to overlap a set of 128 gnese that were differentially expressed between women who subsequently developed breast cancer vs remained cancer-free. These genes represent potential biomarkers of processes that link LI stagnation and increased breast cancer risk. DISCUSSION/SIGNIFICANCE: In future studies, we intend to study these genes that were shown to be upregulated in LI stagnation for their association with subsequent development of breast cancer in independent cohorts of women with BBD. We will use this knowledge to improve individualized risk assessment, which will help focus surveillance and prevention strategies.

OBJECTIVES/GOALS: Weaimed to compare subjective and objective sleep in individuals with chronic post-concussive symptoms. We hypothesized an association between self-reported sleep quality and objective sleep parameters, which is different for concussed and control cohorts. METHODS/STUDY POPULATION: 28 individuals with chronic post-concussive symptoms and 13 age-matched controls (no concussion history) completed the ISI, PSQI, PROMIS Depression, Anxiety, Stress and Cognitive questionnaires at enrollment. Objective sleep parameters were obtained for a minimum of 7 days and up to 30 days with a validated sleep monitoring device placed under the subject’s bed (Emfit). For each night, raw activity data per minute were analyzed to determinein-bed, sleep, wake, andout-of-bedtimes. These measures were used to calculate total sleep time (TST), sleep onset latency (SOL), and wake after sleep onset (WASO) for each night. RESULTS/ANTICIPATED RESULTS: Concussed individuals reported worse sleep with PSQI and ISI scores significantly higher than controls. They also showedsignificant associations between PSQI and Dep ression, ISI and Depression, and ISI and Anxiety scores. There was no difference between objective sleep parameters in the concussed and control cohorts (in-bed/sleep/wake/out-of-bed times, TST, SOL, and WASO). Instead, higher PSQI, ISI, Depression, Anxiety, and Stress scores (greater symptom burden) were all associated with later sleep times, where as higher Cognitive scores (greater cognitive function) were associated with earlier sleep times, regardless of group status. DISCUSSION/SIGNIFICANCE: Concussed individuals report worse subjective sleep but no differences to controls when objectively assessing sleep. Depression/anxiety, and not concussion status, determine objective sleep parameters. Psychiatric comorbidities should inform the treatment of post-concussive sleep disturbances.

OBJECTIVES/GOALS: We designed the Biocascade Exhaled Breath Sampler (BEBS) to characterize viral aerosol shedding among individuals with influenza and other respiratory virus infections. We first aimed to test the BEBS on volunteer COVID-19 cases and report the aerodynamic size distribution of exhaled breath aerosol particles carrying SARS-CoV-2 RNA. METHODS/STUDY POPULATION: From June 15 through December 15, 2022, we recruited 27 PCR-confirmed COVID-19 cases from a college campus and the surrounding community to provide 30-minute breath samples into a well-validated Gesundheit-II (G-II) exhaled breath aerosol sampler. Among these individuals, 17 provided an additional exhaled breath sample into the newly designed BEBS. We quantified samples for viral RNA using reverse transcription digital polymerase chain reaction (RT-dPCR) and determined the viral RNA copies collected within two aerosol size fractions (≤5 µm and >5 µm in diameter) from the G-II, and four aerosol size fractions (<1.15 µm, 1.15–3.2 µm, 3.3–8.2 µm, and >8.2 µm) from the BEBS. RESULTS/ANTICIPATED RESULTS: Individuals with a SARS-CoV-2 Omicron BA.4 or BA.5 infection shed virus in aerosols at an average rate of 7.5x103 RNA copies per 30-minute G-II sample, with 78% of the total RNA in aerosols ≤5 µm in diameter. Among the BEBS samples, 10% of the total viral RNA was detected in aerosols <1.15 µm, 43% in 1.15–3.2 µm, 37% in 3.3–8.2 µm, and 10% in the >8.2 µm size fraction. Based on viral RNA loads, our results indicate that exhaled aerosols ≤3.2 µm contribute the majority of SARS-CoV-2 inhalation exposure. DISCUSSION/SIGNIFICANCE: Our data provide additional evidence that respirable aerosols contribute to the spread of SARS-CoV-2. Thus, our data suggest that mitigation measures designed to reduce infectious aerosol inhalation, such as ventilation and the use of air cleaners and respirators, are needed to control the spread.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this study was to evaluate the performance of a bone fracture targeted systemically administrable bone anabolic as a potential therapeutic for bone fracture repair. Currently all bone fracture repair therapeutic require local administration during surgery. However, the population that need the most assistance in repair bone fractures are not eligible for surgery. So, it was our goal to design an inject-able therapeutic to assist in bone fracture repair to reduce the invasiveness. The injectable nature of it allows for repair administration of the bone anabolic and for therapeutic effect throughout the entire bone fracture healing process. Targeting it to the bone fracture site reduces the toxicity and increases the efficacy. METHODS/STUDY POPULATION: METHODS To achieve the above objective, a bone mineral-(hydroxyapatite-) targeting oligopeptide was conjugated to the non-signaling end of an engineered parathyroid hormone related protein fragment 1-46 with substitutions at Glu22,25, Leu23,28,31, Aib29, Lys26,30 (ePTHrP). The negatively charged oligopeptide has been shown to target raw hydroxyapatite with remarkable specificity, while the attached PTHrP has been demonstrated to induce sustained and accelerated bone growth under control of endogenous morphogenic regulatory factors. The conjugate’s specificity arises from the fact that raw hydroxyapatite is only exposed whenever a bone is fractured, surgically cut, grafted, or induced to undergo accelerated remodeling. The hydroxyapatite-targeted conjugate can therefore be administered systemically (i.e. without invasive surgery or localized injection) and still accumulate on the exposed hydroxyapatite at the fracture site where it accelerates the healing process Murine in vivo experiments were conducted on female Swiss Webster mice (10 per group). Femoral fractures were induced with a 3-point bending device and stabilized. Mice were dosed with 3 nmol/kg/d of targeted-ePTHrP, non-conjugated (free) ePTHrP, or saline. Following a 4-week study, fracture callus densities were measured using microCT. Canine in vivo experiments were conducted on 1-year-old male beagles. Beagles underwent a 10 mm bilateral ulnar ostectomy. Two dogs in the treatment group and Three dogs in the control group were dosed daily with either targeted-ePTHrP 0.5nmol/kg/d or saline respectively. Dogs were x-rayed weekly for the first 6 weeks and then every other week thereafter. One tailed ANOVA followed by Dunnett’s post-hoc test was used to establish significance. All animal experiments were conducted as described in approved IACUC protocols. P<0.05 was considered significant. RESULTS/ANTICIPATED RESULTS: RESULTS SECTION: In the murine studies we observed a marked increase in fracture callus size and a 2-fold increase in bone deposition was observed in the targeted-ePTHrP group over the saline group (P<0.01). A significant doubling in bone density was also observed. Targeted-ePTHrP group fractured femurs were able to achieve their pre-fracture strength as early as 3 weeks compared to 9 weeks in the saline mice representing a 66% reduction in healing time. In the canine studies, we observe a significantly higher closure of the ostectomy gap than saline controls (P<0.05). In addition, no significant differences in weight are observed in the treatment vs. saline controls. No significant difference between the control group and treatment groups was found in a histological investigation of the organs. DISCUSSION/SIGNIFICANCE OF IMPACT: DISCUSSION: Although attempts have been made in developing a systemically administered fracture therapeutic for fracture repair, i.e. teriparatide, to date, no such anabolics have been approved for this use. In these studies there is evidence that anabolic activity was occurring at the fracture site, but at a level that did not meet FDA required end-points.2 It is plausible that if sufficient drug were to be delivered to a fracture site then improved fracture repair would be possible. In previous studies, we demonstrated fracture specific accumulation bone anabolics can be achieved by modifying the drug with acidic oligopeptides.3 Here, by modifying a safe, clinically proven, parathyroid hormone receptor agonist with an acidic oligopeptide we observe improved bone deposition and strength in mice. Furthermore, when administered to canine critical sized defect ostectomies, a more relevant and difficult model, we observe improved ostectomy closure. CLINICAL RELEVANCE:: The ability to accelerate bone fracture repair is a fundamental need that has not been addressed by conventional methods. By targeting bone anabolic agents to bone fractures, we can deliver sufficient concentrations of anabolic agent to the fracture site to accelerate healing, thus avoiding surgery and any ectopic bone growth associated with locally-applied bone anabolic agents.

OBJECTIVES/GOALS: Effective translation of data to inform real-time patient care is lacking in addiction inpatient settings. The current study presents the optimization of a Comprehensive Rehabilitation Assessment summary report that is used by clinicians to individualize treatment. METHODS/STUDY POPULATION: A multi-aim approach was taken that utilized aspects of various implementation science frameworks. Participants were clinical staff (N = 7; female = 71%). A quantitative survey was used for aims 1 and 2 to assess motives and context around the report as well as evaluate the design of it. Aim 3 focused on optimization via semi-structured interviews. Descriptive and modified content analyses were utilized appropriately for each aim. RESULTS/ANTICIPATED RESULTS: Five versions of the assessment report were created between February 2021 and August 2022, the most recent of was adapted into patients’electronic medical records based on study results. Each report version, participants’results/feedback, and researchers’perceived barriers to this translational process will be discussed. DISCUSSION/SIGNIFICANCE: The current study highlights a replicable approach for optimizing the translation of assessment data into treatment for patients with disorders of addiction.

OBJECTIVES/GOALS: Despite the importance of addressing maternal weight to promote healthy pregnancies, previous gestational weight management programs have overlooked rural women. Accordingly, this study used an optimization framework to develop and refine a prototype for an online gestational weight management intervention targeting rural women. METHODS/STUDY POPULATION: Methods were guided by the Obesity-Related Behavioral Intervention Trials (ORBIT) model, an optimization framework focused on intervention development and refinement. In line with this framework, three major steps were followed. First, qualitative interviews were conducted with 16 rural women who were currently or had recently been pregnant to identify barriers, facilitators, and desired resources for gestational weight management. A template analysis approach was applied to the resulting interview transcripts to identify pertinent themes. Second, themes derived from the initial interviews were used to inform the development of an online intervention prototype. Third, feedback on this prototype was sought from an additional sample of 15 rural women who were currently or had recently been pregnant. RESULTS/ANTICIPATED RESULTS: Themes from the initial interviews highlighted numerous barriers, facilitators, and desired resources for rural gestational weight management that aligned with common social determinants of health (e.g., neighborhood and built environment, social and community context) and pregnancy-specific factors. Women also described wanting an online gestational weight management program that included a user-friendly interface, psychoeducation, tailored health recommendations, accountability, and simple behavior-logging tools. Using this feedback, an online intervention prototype was developed. Results from the feedback interviews are currently being qualitatively analyzed for themes and will be used to further refine the prototype prior to feasibility testing. DISCUSSION/SIGNIFICANCE: This study used an optimization framework to develop an online intervention aimed at supporting healthy maternal weight outcomes in rural communities. Because rural women experience notable weight disparities compared to their urban peers, this intervention has the potential to promote more equitable maternal health outcomes in rural areas.

OBJECTIVES/GOALS: This project aimed to examine the impacts of biological sex and alexithymia on frontal lobe connectivity in executive functioning (EF)-related neural networks during successful inhibition as a means to index vulnerability for future cognitive decline. METHODS/STUDY POPULATION: Healthy, cognitively intact older adults (n=43, 33 female, Mage=79) completed the 20-item Toronto Alexithymia Scale (TAS-20) and the stop-signal task in this study. We used electroencephalography (EEG) source estimation to investigate EF-related frontal connectivity during successful inhibition in stop-signal task trials. Connectivity was measured in bilateral frontal ROIs relevant to inhibition using time series correlations over the N200 (186-350ms) and P300 (340-616ms) time windows, associated with the inhibitory subprocesses of conflict processing and performance evaluation, respectively. RESULTS/ANTICIPATED RESULTS: Those with higher alexithymia, as measured by the difficulty identifying feelings (DIF) facet of the TAS-20, had lower right anterior cingulate cortex (ACC)-left superior frontal gyrus (SFG) connectivity in the P300 window, suggesting impaired performance evaluation. Further, in females specifically, those with higher DIF had greater right inferior frontal gyrus (rIFG)-bilateral ACC connectivity in the N200 window than those with lower DIF, suggesting greater resources were allocated for conflict processing and inhibition. Right ACC-rIFG connectivity also correlated with better stop accuracy and faster stop-signal reaction time, supporting this network’s role in successful inhibition. DISCUSSION/SIGNIFICANCE: Overall, during successful inhibition, higher DIF was associated with reduced performance monitoring efficiency as well as greater resource allocation for conflict processing during motor stopping in women only. Thus, alexithymia (via DIF) may exacerbate age-related EF dysfunction and risk for future cognitive decline, especially for females.

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

OBJECTIVES/GOALS: Virtual surgical planning and 3D printing enable streamlined surgeries and increased complexity. These technologies, however, require CT scans and radiation exposure. This project’s goal is to optimize and demonstrate the accuracy of Black Bone MRI for surgical planning in reconstructive surgery. METHODS/STUDY POPULATION: Four common craniofacial surgeries were planned and performed on cadaver specimens (maxillary advancement, orbital floor reconstruction with patient-specific implants, cranial vault reconstruction, and fibular free flap reconstruction of the mandible). For each surgical procedure, ten cadaver heads were used. Five of each surgery were planned and 3D printed guides were created utilizing Black Bone MRI versus five with CT scans. Following mock surgeries, all specimens underwent a post-operative CT scan. 3d reconstruction was performed and surgical accuracy compared to the plan was assessed using GeoMagic Wrap, assessing average post-operative deviation from plan. RESULTS/ANTICIPATED RESULTS: In all surgeries, guides created from Black Bone MRI demonstrated high accuracy to surgical plan. Average osteotomy (cut) deviation from plan was not statistically significantly different when Black Bone MRI was used compared to CT scans for planning and guide creation in the wide variety of craniofacial surgeries performed. The average deviation of post-operative anatomy from pre-operative plan was also not statistically significant when Black Bone MRI versus CT scans were utilized in the surgeries. These results then enabled the translational application of this technology clinically, and we demonstrate a clinical reconstructive craniofacial case planned utilizing Black Bone MRI. DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates that virtual surgical planning and 3d surgical guide creation can be performed using Black Bone MRI with comparable accuracy to CT scans in a wide variety of craniofacial procedures. This could dramatically reduce radiation exposure for patients. The successful segmentation, virtual planning, and 3d printing of accurate guides from Black Bone MRI demonstrate potential to change the pre-operative planning standard of care. This project, overall, also demonstrates the development of new solutions to advance clinical care, thus serving as an example of moving translational science from a concept to the operating room.

Objectives/Goals: Pregnancy increases vulnerability to stress and mental health symptoms, particularly among Hispanic women in Puerto Rico (PR), a population with unique socioenvironmental adversities, such as poverty and natural disasters. This study examined the relationships between life adversities and psychological distress in this at-risk population. Methods/Study Population: Participants (n = 50) in this cross-sectional study were recruited from an obstetrician’s office in Southern PR. All participants provided written consent and completed the Adverse Life Experiences Scale (ALES, Cronbach’s alpha = 0.71) to identify lifelong adversities faced and the overall duration (chronicity). Measures of psychological distress included the Perinatal Anxiety Screening Scale (PASS, Cronbach’s alpha = 0.96), the Edinburg Postnatal Depression Scale (EDPS, Cronbach’s alpha = 0.87), the Perceived Stress Scale (PSS, Cronbach’s alpha = 0.79), and the Brief Resilience Scale (BRS, Cronbach’s alpha = 0.86). Descriptive and Spearman’s rho correlation analyses were conducted. Results/Anticipated Results: The mean age of the participants was 27.90 years (SD = 6.05), with most in the first trimester (66.0%). On average, participants reported 4.32 (SD = 3.1) out of 23 lifetime adversities. The most common adversities were natural disasters (60.0%), loss of a beloved (58.0%), and financial difficulties (38.0%). Nearly half (44.0%) experienced five or more adversities. A significant number of participants met the clinical threshold for anxiety (38.0%, PASS), depression risk (22.0%, EPDS), moderate-to-severe perceived stress (52.0%, PSS), and low resilience (24.0%, BRS). The overall duration of adversities was significantly associated with anxiety (rs = 0.50, p = 0.001) and stress (rs = 0.50, p = 0.007). Discussion/Significance of Impact: Hispanic pregnant women in PR face high levels of adversity and distress, which can negatively affect both maternal health and fetal development, influencing long-term child outcomes. Early identification and targeted interventions addressing adversities, can improve maternal mental health and child health-development outcomes.