OBJECTIVES/GOALS: METHODS/STUDY POPULATION: This study utilizes the IeDEA W. Africa cohort to evaluate the prevalence and burden of hypertension and diabetes type II among people living with HIV (PLWH) in sub-Saharan Africa (SSA), and to assess how sex and aging impact the development of these NACM. The cohort is a large, international collaboration across eight countries and 19 treatment centers. Established by the National Institute of Allergy and Infectious Diseases in 2006, the W. Africa cohort is now in its fourth renewal (2021-2026). Participating countries include Senegal, Mali, Cote d’Ivoire, Burkina Faso, Ghana, Togo, Benin, and Nigeria. Data are collected from affiliated cohorts in the regional data center every 24 months. The cohort currently includes >65,000 adult PLWH on anti-retroviral therapy. RESULTS/ANTICIPATED RESULTS: The prevalence of several NACM, such as hypertension and cardiovascular disease, have significantly increased among PWH over time, surpassing prevalence observed in the general population. We therefore expect similar patterns in the W. Africa IeDEA cohort and a high prevalence and burden of both hypertension and diabetes type II in this sample. Additionally, evidence suggests that increasing age and female sex serve as independent risk factors for the development of NACM in PLWH. We anticipate that both increased age and female sex, separately and synergistically, are associated with increased prevalence and burden of hypertension and diabetes type II in this cohort. DISCUSSION/SIGNIFICANCE: With more than 37 million people currently living with HIV, the HIV/AIDS epidemic continues to pose a serious threat to global public health. SSA bears a disproportionate burden of the epidemic with greater than two-thirds of global cases. NACM are now driving morbidity and mortality among PLWH, and increased age and female sex may modify this effect.

OBJECTIVES/GOALS: Hypermobile Ehlers-Danlos syndrome (hEDS) is a heritable connective tissue disorder with no known genetic etiology. Its complex phenotypic presentation with multi-system involvement delays proper diagnosis and treatment, especially for females. This study examines the risk for common hEDS comorbidities and medical service utilization. METHODS/STUDY POPULATION: Electronic health records from over 150 million patients across 92 American healthcare servers were queried using the TriNetX database to determine phenotypic presentation of hEDS and risk of receiving comorbid diagnoses. Contingency tables were created with hEDS as the condition and postural orthostatic tachycardia syndrome (POTS) or gastroparesis as the grouping variables. Advanced analytics were conducted to compare outcomes of two cohorts: (1) patients diagnosed with hEDS and both POTS and gastroparesis, and (2) patients with a sole diagnosis of hEDS. After propensity score matching, differences in medical service utilization and mental health diagnoses were assessed between these two cohorts. All analyses restricted age (12-70 years) and employed established exclusion criteria (e.g., diabetes). RESULTS/ANTICIPATED RESULTS: TriNetX national health records (N = 1,968) reveal that hEDS predominantly affects females (87%) and is diagnosed at a mean of 35 years of age. People diagnosed with hEDS have a high prevalence of comorbid nervous system (81%), mental health (76%), and digestive system (69%) disorders. They are 29.7 times more likely to be diagnosed with POTS [95% CI: 27.1, 32.6] and 66.3 times more likely to be diagnosed with gastroparesis [95% CI: 56.5, 77.9], compared to people without hEDS. After propensity score matching for sex, race, and ethnicity, people diagnosed with hEDS and both POTS and gastroparesis have significantly greater service utilization (72.2% vs. 56.7%; z = 2.18, p < .05) but not a greater incidence of mental health disorders (34.4% vs. 24.4%; z = 1.47, p = .14) than people diagnosed with hEDS alone. DISCUSSION/SIGNIFICANCE: This study reveals the phenotypic presentation of hEDS and the elevated risk of co-occurring POTS and/or gastroparesis diagnosis. hEDS and its comorbidities are associated with more frequent medical encounters but not with a greater incidence of mental illness. Findings have implications for both clinical practice guidelines and further research.

ABSTRACT IMPACT: This research aims to identify changes in visual network function after TBI as a way to define potential therapeutic targets for neuromodulation or neural tissue substrates. OBJECTIVES/GOALS: The objectives of this study are to compare neural activity in the visual cortex following TBI with cortical activity in the uninjured brain. This study aims to characterize functional changes in single neuron activity, spike-field relationships and oscillatory activity. METHODS/STUDY POPULATION: The effects of TBI will be studied by comparing electrophysiologic recordings from Long-Evans rats with a fluid percussion injury (FPI) to rats with a sham injury. Four days after the injury or sham procedure, a laminar probe with multiple electrode contacts will be chronically implanted in the ipsilesional primary visual cortex (V1). Afterwards, rats will be anesthetized weekly for 3 weeks (up to 4 weeks post-injury) to assess visual processing in response to drifting grating visual stimulation. To assess behavioral correlates, neural activity will also be recorded while rats perform a visual discrimination task in an operant, touchscreen chamber twice weekly. Recordings will be analyzed for visually evoked units, unit entrainment to local field potentials (LFPs) and evoked oscillatory activity. RESULTS/ANTICIPATED RESULTS: Consistent with other studies, our preliminary evidence from V1 recordings in naive rats has shown that individual neurons are responsive to visual stimuli, visual stimuli are associated with evoked oscillations and unit activity is correlated with LFPs. While activity of individual V1 neurons in injured animals is expected to recover to resemble activity in uninjured animals over time, patterns of functional organization in the two groups are expected to diverge over time. We anticipate that TBI-associated axonal damage, neuronal loss and changes in synaptic weights will lead to disruptions in the timing of neural activity in V1. These perturbations of neural communication within the visual system are expected to be associated with behavioral deficits in the awake, visual discrimination task. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study helps define how cortical network disruption after TBI. These changes are potential targets for novel TBI therapeutics, including neuromodulation and neural tissue transplantation. Thus, this work lays the groundwork for future studies aimed at mitigating the effects of TBI with rationally designed experimental therapeutics.

OBJECTIVES/GOALS: The prevalence of combusted cigarette (CC) smoking among older adults is stagnant, with zero declines attributable to e-cigarette (EC) use. Normative beliefs predict quitting and switching to ECs (a behavior likely to yield health benefits for those unable to quit), so this study seeks to characterize the role of age in norms about CC and EC use. METHODS/STUDY POPULATION: Data come from Wave 5 (2018-2019) of the adult Population Assessment of Tobacco Use and Health (PATH) study, a nationally-representative, U.S. longitudinal cohort. Analyses were restricted to people with established CC use (smoked CCs in the past year, currently smoke regularly, and smoked ≥100 lifetime CCs; n=8,590). Cross-sectional weighted estimates of the prevalence of normative beliefs about CCs and ECs were calculated by age using the Balanced Repeated Replication (BRR) method with Fay=0.3. We used chi-square tests to examine the association of age group (18-24, 25-34, 35-44, 45-54, 55-64, or ≥65) with the prevalence of 2 descriptive and 4 personal social norms. RESULTS/ANTICIPATED RESULTS: The prevalence of the normative belief that most people disapprove of CCs (p DISCUSSION/SIGNIFICANCE: Older adults are more likely than younger adults to endorse anti-tobacco norms, which prior work shows is associated with quitting smoking. These beliefs could be leveraged to create targeted communications towards older adults encouraging smoking cessation. More research is needed to assess age-related tobacco beliefs and switching from CC to EC.

OBJECTIVES/GOALS: This proposed qualitative study aims to explore the perspectives about PrEP among heterosexual, cisgender Black women currently enrolled at an HBCU. Specifically, this study will elucidate this populations knowledge, attitudes, and beliefs surrounding PrEP uptake and marketing. METHODS/STUDY POPULATION: The Health Belief Model adapted from Chandler, Hull et al. (2020) will be utilized as the conceptual framework to guide this study. Multiple virtual semi structured focus groups will be conducted with individuals at two HBCUs. Study participants (n=20-25) will meet the following inclusion criteria: (a) self-identifies as Black/African American, (b) was assigned female at birth, (c) is enrolled at Morgan State University or Spelman College, (d) is 18 years or older, (e) is sexually active (anal, oral, vaginal, or experimental), (f) is HIV negative, (g) has multiple sexual partners and/or has inconsistent condom usage. A qualitative data analysis will be conducted utilizing MAXQDA software to perform a thematic analysis by creating a codebook and identifying prominent themes related to perceptions and use of PrEP. RESULTS/ANTICIPATED RESULTS: The research is on-going pending IRB approval. Data collection will include questions focused on topics surrounding PrEP, barriers/facilitators to participation in a proposed PrEP campaign, and sexual/reproductive health. These findings will characterize the awareness of PrEP among Black HBCU women, identify factors that may facilitate or cause barriers to Black HBCU womens knowledge and uptake of PrEP, and identify best practices for successful marketing strategies for PrEP campaigns targeting uptake among HBCU women DISCUSSION/SIGNIFICANCE: Findings from this study can help the public health community tailor PrEP campaigns designed for college-age Black women thereby curtailing HIV transmission among this at-risk group.

OBJECTIVES/SPECIFIC AIMS: The overall goal is to determine if intestinal commensal bacteria play a role in enteric virus evolution. We will use reovirus, an enteric segmented virus, to investigate specific goals. First, we will determine if specific bacterial species enhance the coinfection frequency of 2 separate strains of reovirus. Second, we will determine if the presence/absence of different bacterial species in the microbiota of mice results in different reovirus reassortment frequencies. Finally, we will discover if reassortant reovirus is present in human populations. METHODS/STUDY POPULATION: My first goal is to determine if specific bacterial species enhance the coinfection frequency of 2 strains of reovirus. In our lab, we have a panel of commensal intestinal bacterial strains, as well as a number of lab adapted bacterial strains. We will use this panel of bacteria to determine if reovirus binds to different species of bacteria using a binding assay involving radiolabeled virus. Additionally, we will determine if specific species of bacteria alter the coinfection frequency through a Flow cytometry based assay. This will involve mixing virus with bacteria, infecting cells in culture, and straining for reovirus proteins for flow cytometry. Our second goal is to determine if specific bacteria promote reassortment of reovirus in a mouse model of infection. To do this, we will use gnotobiotic techniques to create mice harboring different intestinal bacteria populations. Mice will be infected with 2 strains of reovirus, and then feces and organs will be collected. Progeny virus will be subjected to a plaque assay on 2 different types of cells. The first type of cells will be normal cells in culture in which all viable viruses will form plaques. The second will be a cell line that stably expresses siRNAs against specific reovirus segments in which only specific reassortants will form plaques. These 2 plaque assays will be used to quantify the total number of viruses present and the total number of reassortant viruses present. Additionally, SDS-PAGE and RT-PCR will be used to confirm reassortants. Our third goal is to determine if reassortant reovirus is present in infected humans. To do this, I will obtain feces from reovirus-infected children and isolate reovirus. One specific reovirus reassortant is known to propogate in dual-infected mice. I will use the plaque assay technique to determine if this reassortant is also present in humans. To determine if other reassortants are present, I will use RT-PCR and SDS-PAGE. RESULTS/ANTICIPATED RESULTS: Based on previous studies with other enteric viruses, we suspect that specific bacterial species bind reovirus strains with different efficiencies. It is likely that a number of bacterial species will promote coinfection. The bacterial strains that binds both reovirus strains at a high efficiency will likely enhance coinfection by the greatest amount. It is likely that mice harboring different bacterial populations will produce different reovirus reassortment frequencies. We predict that bacteria that enhance reovirus coinfection in vitro should also enhance reovirus reassortment in our mouse model. Therefore, mice specifically lacking bacteria that promote coinfection should have significantly lower amounts of reassortant reovirus. It will be important to control for the overall amount of replication within mice with different microbiotas, as this will affect the basal reassortment frequency. We suspect that reovirus reassortants are present in humans. Work done both in vitro and in mouse models indicates that reassortment happens at high frequencies. Additionally, one specific reassortant commonly propogates in mice due to an enhanced cellular attachment phenotype. Therefore, we predict that this reassortant also commonly emerges after coinfection and reassortment in humans. DISCUSSION/SIGNIFICANCE OF IMPACT: Segmented viruses, such as influenza and rotavirus, are important human pathogens. Viral reassortment poses a unique threat to humans, as it enables new viruses to emerge and cause pandemics or epidemics. However, little is known about what factors promote viral reassortment. This study will provide insight into a novel mechanism of segmented virus evolution.

Objectives/Goals: Opioid use disorder (OUD) at delivery increased between 1999 and 2014. Clinical guidelines include medication for OUD (MOUD) for pregnant women with OUD and is associated with better fetal outcomes. Few large studies have compared prenatal MOUD outcomes to no MOUD. We evaluated the association of documented MOUD prescription during pregnancy with maternal outcomes. Methods/Study Population: We utilized aggregated electronic health records using the TriNetX platform to conduct a retrospective cohort study of females, aged 1249 years with a childbirth CPT code and documented opioid use via ICD-10 codes in the nine months before delivery between 2014 and 2020, comparing patients with MOUD prescription of buprenorphine or methadone during the nine months before delivery to demographically matched patients without any documented MOUD, using hazard ratios and 95% CIs for outcomes occurring one week to one or three years after childbirth. Results/Anticipated Results: MOUD cohort (n = 6,945, 85.33% White; 82.77% Non-Hispanic or Latino) was associated with significantly higher subsequent documented MOUD prescription (HR, 9.26 [95% CI, 7.98–10.76]; 6.21 [95% CI, 5.60–6.88]) and new remission codes (HR, 2.79 [95% CI, 2.15–3.62]; 2.85 [95% CI, 2.38–3.40]) at one and three years, lower ED visits at one year (HR, 0.88 [95% CI, 0.81–0.96]), no significant association of ED visits at three years (0.95 [95% CI, 0.89–1.02]), higher outpatient visits (HR, 1.26 [95% CI, 1.20–1.32]; HR, 1.27 [95% CI, 1.21–1.33], and no significant association of inpatient visits at one and three years (HR, 0.93 [95% CI, 0.813–1.06]; 1.06 [95% CI, 0.96–1.18]) than the never-MOUD cohort (n = 4,708, 76.11% White; 75.68% non-Hispanic or Latino). Discussion/Significance of Impact: A documented prescription for MOUD during pregnancy is associated with newly documented remission of OUD, increased outpatient visits, decreased ED visits, and additional documented MOUD prescriptions suggestive of increased access to continuity care. Efforts to increase MOUD use in pregnancy may improve maternal outcomes.

OBJECTIVES/GOALS: Cerebral palsy (CP) is the most common motor disability in childhood in the US1. Augmented reality (AR) has promise enhancing engagement of rehabilitation2. We developed adaptive games for children with CP using Augment Therapy™. The goal of this study was to obtain user experience in the orthopedic clinic to inform a home-based clinical trial. METHODS/STUDY POPULATION: Participants were recruited at a pediatric center from July 1 to September 20, 2023. Inclusion criteria werediagnosis of CP, ages 5-10 years, English/Spanish speaking. Exclusion criteria were cognitive delay, audio or visual impairment, seizures, or recent surgery. The Augment Therapy™ app was delivered through a clinic iPad. The electronic medical record was used to screen eligible patients. 22 patients were screened; 14 were not eligible based on exclusion criteria. 2 patients refused based on not having enough time to participate. Children played 5 games and their movements were tracked using augmented reality. To evaluate the quality of the app, participants were asked to complete a validated questionnaire, the modified Mobile App Rating Scale. Descriptive statistics were used to analyze responses. RESULTS/ANTICIPATED RESULTS: Six participants were eligible and completed phase I. Eighty-three percent (5/6) of participants reported the character was mostly matching their child’s movements and easy for their child to follow instructions. Thirty three percent (2/6) reported there were technical issues where the app did not track or froze. 100% reported being interested in participating in the home clinical trial. Parent reported their child’s favorite games were flying and obstacle course. The mean and median MARS score was 3.8/5 and 4/5, respectively. Regarding engagement, 66.7% (4/6) reported the app was fun to use. DISCUSSION/SIGNIFICANCE: Families of children with CP reported positive experiences overall with Augment Therapy™ mobile app. The technical issues will need to be addressed. Future studies will need to establish efficacy and dosing time of use of the app for adherence to a program using these modules and engagement with the app to increase habitual physical activity.

OBJECTIVES/SPECIFIC AIMS: Rodent models can be used to study neonatal abstinence syndrome (NAS), but the applicability of findings from the models to NAS in humans is not well understood. The objective of this study was to develop a rat model of norbuprenorphine-induced NAS and validate its translational value by comparing blood concentrations in the norbuprenorphine-treated pregnant rat to those previously reported in pregnant women undergoing buprenorphine treatment. METHODS/STUDY POPULATION: Pregnant Long-Evans rats were implanted with 14-day osmotic minipumps containing vehicle, morphine (positive control), or norbuprenorphine (0.3–3 mg/kg/d) on gestation day 9. Within 12 hours of delivery, pups were tested for spontaneous or precipitated opioid withdrawal by injecting them with saline (10 mL/kg, i.p.) or naltrexone (1 or 10 mg/kg, i.p), respectively, and observing them for well-validated neonatal withdrawal signs. Blood was sampled via indwelling jugular catheters from a subset of norbuprenorphine-treated dams on gestation day 8, 10, 13, 17, and 20. Norbuprenorphine concentrations in whole blood samples were quantified using LC/MS/MS. RESULTS/ANTICIPATED RESULTS: Blood concentrations of norbuprenorphine in rats exposed to 1–3 mg/kg/d of norbuprenorphine were similar to levels previously reported in pregnant women undergoing buprenorphine treatment. Pups born to dams treated with these doses exhibited robust withdrawal signs. Blood concentrations of norbuprenorphine decreased across gestation, which is similar to previous reports in humans. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that dosing dams with 1–3 mg/kg/day norbuprenorphine produces maternal blood concentrations and withdrawal severity similar to those previously reported in humans. This provides evidence that, at these doses, this model is useful for testing hypotheses about norbuprenorphine that are applicable to NAS in humans.

OBJECTIVES/SPECIFIC AIMS: Joint injury is a common cause of premature retirement for many equine athletes. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) offer a particularly attractive cell source for cartilage engineering. Although bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine superior equine MSC source for cartilage engineering via a self-assembling process (SAP). METHODS/STUDY POPULATION: MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis via 3D culture and then used to generate cartilage via SAP. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring biochemical, mechanical, and histological properties. RESULTS/ANTICIPATED RESULTS: We found that while BM-MSCs possessed higher tensile properties and collagen content, CB-MSCs had superior compressive properties and GAG content. Moreover, CB-MSCs had lower alkaline phosphatase activity and higher collagen type II, suggesting a more hyaline cartilage-like phenotype. DISCUSSION/SIGNIFICANCE OF IMPACT: In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage, and is therefore the superior MSC source for purposes of cartilage self-assembly.

OBJECTIVES/SPECIFIC AIMS: Immune-based therapies hold great promise for treatment of refractory tumors. However, development is limited by a lack of identified immune correlates to vaccination. We recently showed that dendritic cells (DCs) prolong progression-free survival (PFS) and overall survival (OS) in patients with glioblastoma, and that DC migration to site draining lymph nodes robustly correlates with both PFS and OS. While this appears to be a reliable immune correlate, the complexity of routine labeling for PET and SPECT prohibits validation in a large clinical study. We therefore seek to develop a safe, translatable reporter that can be imaged with a widely available imaging modality. METHODS/STUDY POPULATION: The cationic liposome 1,2-doleoyl-3-trimethylammonium-propane (DOTAP) was loaded with MRI-imageable iron oxide nanoparticles (IONPs) with or without the neutral molecules PEG and cholesterol. The resulting nanoparticles were loaded with RNA to form RNA-NPs that were characterized with transmission electron microscopy (TEM) and used to transfect DCs in vitro; 4.7 T MRI was then used to image particles or cells in agarose gel phantoms. RESULTS/ANTICIPATED RESULTS: TEM images of RNA-NPs indicate the presence of IONP-loaded liposomes. In vitro transfection experiments demonstrate that iron oxide does not reduce RNA-NP-mediated transfection of DCs. Additionally, small amounts of either PEG or cholesterol within RNA-NPs increased transfection of DC2.4s and enhanced T-cell priming by bone marrow-derived dendritic cells. A series of 4.7 T MRI images of particles in cells, spleens, and LNs demonstrated quantifiable differences in particle density between groups. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that IONP-loaded RNA-NPs can be imaged with MRI and manipulated to augment DC function. Future work will include in vivo imaging in mice and safety studies to facilitate translation into first-in-human studies. Successful completion of this project would provide a powerful clinical tool to improve and track patient responses to immune therapy.

OBJECTIVES/SPECIFIC AIMS: Our long-term goal is to elucidate the molecular mechanisms and virulence factors that control the differential presentation of infection with Salmonella typhimurium and Salmonella typhi. The objectives of this study are to study the mechanisms that enable S. typhi to trigger a decreased inflammatory response in comparison with S. typhimurium and evade detection by the immune system, leading to the development of asymptomatic chronic carriage of S. typhi. METHODS/STUDY POPULATION: A loss of function eptB mutant S. typhimurium strain was generated. Lipopolysaccharide (LPS) was isolated from wild-type and eptB mutant S. typhimurium and wild-type S. typhi. Binding of LPS to recombinant intelectin was tested by dot blot and enzyme-linked immunosorbant assay (ELISA). C57BL/6 mice were infected with wild-type or eptB mutant S. typhimurium by oral gavage and inflammatory cytokines in the spleen, liver, and Peyer’s patches were measured by qPCR. RESULTS/ANTICIPATED RESULTS: LPS isolated from wild-type S. typhimurium is not bound by intelectin, a protein that has been proposed to function in innate immunity and that is known to be able to bind certain moieties within LPS. Conversely, LPS isolated from eptB mutant S. typhimurium and wild-type S. typhi, which lacks a functional eptB, is bound by intelectin. Mice infected with an eptB mutant S. typhimurium exhibit decreased expression of inflammatory cytokines in the spleen compared to mice infected with the wild type S. typhimurium, suggesting that loss of eptB function allows a nontyphoidal Salmonella serovar to mimic the stealth phenotype of typhoidal serovars. Together, these results suggest that loss of eptB function allows intelectin to bind to and detoxify Salmonella LPS, leading to decreased systemic inflammation during infection. DISCUSSION/SIGNIFICANCE OF IMPACT: These results have broad implications for how pathogens such as S. typhimurium induce systemic shock during infection and may also help to explain a mechanism for how S. typhi is able to evade immune detection and enhance dissemination to systemic sites, leading to development of the asymptomatic chronic carrier state. Further investigation of this novel virulence mechanism will mark a decisive step forward in understanding the mechanisms underlying the differential pathogenesis of S. typhimurium-induced gastroenteritis and S. typhi-induced typhoid fever. Additionally, these results contribute to our understanding of the interactions between host and pathogen in affecting disease presentation, which will have wide appeal among researchers interested in microbial pathogenesis and the contribution of host-pathogen interactions to health and disease.

OBJECTIVES/GOALS: This study examines the impact of COVID restrictions on the process of engagement in the Ohio-HCS site. The goals are to: examine the impact of COVID restrictions on the process of engagement; 2) determine differences in process measures by geographic region (rural, urban). METHODS/STUDY POPULATION: Engagement activities collected as part of the Ohio HCS include an engagement log, coalition meeting type and attendance, tracker of implementation strategies. Study period: January 2020 to October 31, 2021. Measures are defined below. Meeting occurrence, type by month: Data report on whether a meeting occurred, was scheduled and cancelled, or a scheduled off-month. The meeting platform was also recorded (in-person, hybrid, virtual). Coalition meeting attendance by month: Number of community members in attendance. Engagement communications by type, by month: Counts and percent (types: email, calls, zoom, or in person). Evidence-Based Practices(EBP) delivery option: in person, virtual, or hybrid. Counties: 9 Ohio counties, 5 rural, 4 urban. RESULTS/ANTICIPATED RESULTS: Coalition meetings were cancelled in 40% of rural counties; none in the urban counties. Two rural counties switched back to in person or hybrid meetings by late 2020; urban counties remain 100% virtual. Rural counties had a drop-off in attendance in June 2021 with no decrease seen in urban counties. During first two months of 2020 engagement in rural and urban communities occurred in person; by March that shifted to 80% by email, which continued within rural counties. In urban counties that dropped to 50% by August of 2020, with zoom calls accounting for 30%. In-person strategies for naloxone distribution remained high in both county types (90%); urban counties use of in-person only strategies for medication for OUD (rural: 83%, urban 52%) and safer prescribing (rural:74%, urban:10%) were much lower than rural counties. DISCUSSION/SIGNIFICANCE: Results show that rural counties continue to rely on in person engagement strategies, making COVID restrictions more disruptive for rural counties. These results suggest that new supports and strategies may be needed to assure that rural regions are equally equipped to engage in research in a virtual environment.

Objectives/Goals: Manual skin assessment in chronic graft-versus-host disease (cGVHD) can be time consuming and inconsistent (>20% affected area) even for experts. Building on previous work we explore methods to use unmarked photos to train artificial intelligence (AI) models, aiming to improve performance by expanding and diversifying the training data without additional burden on experts. Methods/Study Population: Common to many medical imaging projects, we have a small number of expert-marked patient photos (N = 36, n = 360), and many unmarked photos (N = 337, n = 25,842). Dark skin (Fitzpatrick type 4+) is underrepresented in both sets; 11% of patients in the marked set and 9% in the unmarked set. In addition, a set of 20 expert-marked photos from 20 patients were withheld from training to assess model performance, with 20% dark skin type. Our gold standard markings were manual contours around affected skin by a trained expert. Three AI training methods were tested. Our established baseline uses only the small number of marked photos (supervised method). The semi-supervised method uses a mix of marked and unmarked photos with human feedback. The self-supervised method uses only unmarked photos without any human feedback. Results/Anticipated Results: We evaluated performance by comparing predicted skin areas with expert markings. The error was given by the absolute difference between the percentage areas marked by the AI model and expert, where lower is better. Across all test patients, the median error was 19% (interquartile range 6 – 34) for the supervised method and 10% (5 – 23) for the semi-supervised method, which incorporated unmarked photos from 83 patients. On dark skin types, the median error was 36% (18 – 62) for supervised and 28% (14 – 52) for semi-supervised, compared to a median error on light skin of 18% (5 – 26) for supervised and 7% (4 – 17) for semi-supervised. Self-supervised, using all 337 unmarked patients, is expected to further improve performance and consistency due to increased data diversity. Full results will be presented at the meeting. Discussion/Significance of Impact: By automating skin assessment for cGVHD, AI could improve accuracy and consistency compared to manual methods. If translated to clinical use, this would ease clinical burden and scale to large patient cohorts. Future work will focus on ensuring equitable performance across all skin types, providing fair and accurate assessments for every patient.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to examine bioenergetic phenotype of retinoic acid receptor responder 1 (RARRES1)-depleted epithelial cells and to facilitate the discovery of personalized metabo-therapeutics in the context of cancers characterized with loss of or low expression of RARRES1. METHODS/STUDY POPULATION: Anoikis assay and annexinV labeling were used to assess drug resistance and apoptotic phenotype in RARRES1-depleted epithelial cells. Metabolomics, AMP kinase activity, mito-tracker, and extracellular flux assays were used to examine the bioenergetic profile of RARRES1-depleted epithelial cells. Extracellular flux assays were used to assess the phenotype of RARRES1-depleted epithelial cells treated with or without metformin. RESULTS/ANTICIPATED RESULTS: RARRES1 is a major regulator of mitochondrial function. Its depletion in tumors induces an oxidative phosphorylation dependent phenotype and subsequently increases ATP abundance in the cell, enhances anabolic pathways and increases survival. Treatment with FDA approved mitochondrial respiration inhibitor, metformin, reversed the metabolic phenotype of RARRES1 depleted-epithelial cells. Metformin could be the ideal therapeutics to reduce tumor burden in cancers with loss of or low expression of RARRES1. DISCUSSION/SIGNIFICANCE OF IMPACT: Bioenergetic dynamics are emerging as a basis for understanding the pathology of cancer. The malignancy progresses as its metabolic pattern and mitochondrial respiration become more dysfunctional. The regulatory pathways of bioenergetic dynamics are currently poorly understood, and the characterization of proteins implicated in those processes must be assessed. One understudied protein and tumor suppressor is RARRES1. RARRES1 is induced by retinoic acid (a major metabolic regulator) and functions as a putative carboxypeptidase inhibitor. Understanding the connection between this carboxypeptidase inhibitor and intermediary metabolism will enlighten our understanding of the bioenergetic profile of cells and facilitate the discovery of personalized metabo-therapeutics in the context of cancer.

OBJECTIVES/SPECIFIC AIMS: Early life stress is known to greatly impact neurodevelopment during critical periods, conferring risk for various psychopathologies, including the onset and exacerbation of schizophrenia and anxiety disorders. The endocannabinoid system is highly integrated into the stress response and may be one means by which early life stress produces such deleterious effects. Using a naturalistic, ecologically valid animal model, this study explored interactions between the stress response and endocannabinoid systems within the cerebellum, a region dense with the CB1 endocannabinoid receptors and shown to be susceptible to stress. METHODS/STUDY POPULATION: This study explored behavioral and neural impacts of early life stress in Long-Evans rats reared with or without limited access to bedding material during postnatal day (PND) 2-9. Corticosterone (CORT) levels were measured at PND8 and 70. During PND50-70, rats were assessed on Novel Object Recognition to test memory, Rotarod to evaluate cerebellar integrity, Elevated Plus Maze to assay anxiety, Social Preference, and Eyeblink Conditioning, a cerebellar-dependent and endocannabinoid-mediated task. Lipid analysis was performed on PND70 tissue samples of cerebellar interpositus (IP) nucleus via high-performance liquid chromatography and tandem mass spectrometry. RESULTS/ANTICIPATED RESULTS: Both male and female rats experiencing early life stress exhibited significantly impaired recognition memory (N = 16-20/group). Female rats having undergone stress exhibited decreased social preference compared to normally reared females (N = 11/group). Stressed males showed facilitated eyblink conditioning compared to normally reared males (N = 7-9/group). There were no group differences in rotarod or elevated plus maze performance or CORT levels at PND8 or 70 across rearing groups. At PND70, male rats experiencing early life stress exhibited a significant decrease in 2-arachidonoyl glycerol (2-AG) and arachidonic acid levels in the IP nucleus compared to normally reared males (N = 8-9/group). Compared to normally reared females, those experiencing early life stress exhibited a significant increase in prostaglandin E2 levels in the IP nucleus (N = 6-7/group). DISCUSSION/SIGNIFICANCE OF IMPACT: Early life stress, induced by limited bedding, resulted in sex-specific behavioral and lipid impairments. Results suggest that stress causes long-term alterations in endocannabinoid dynamics in males in the cerebellar IP nucleus and sex-related lipids in female cerebellum. These changes may contribute to observed long-term behavioral aberrations. Moreover, findings suggest these behavioral changes may be the result of negative-feedback dysfunction (as evidenced by decreased endocannabinoids in males) or increased neural inflammation or proliferation (as evidenced by increased prostaglandins in females). Future analysis will quantify mRNA and protein for cannabinoid receptors to better characterize aberrations to this system. Moreover, other neural regions dense with cannabinoid receptors (i.e., PFC, hippocampus) will be investigated. This work provides a basis for understanding stress impacts on the development of cognitive deficits observed in psychotic and anxiety disorders. Specifically, facilitation of eyblink conditioning complements research in humans with anxiety disorders. Broadly, understanding stress-related endocannabinoid dysregulation may provide insights into risks for, and the development of, psychopathology and uncover novel therapeutic targets with high translational power.

ABSTRACT IMPACT: Approximately 15% of US adults have chronic kidney disease with over 700,000 of those in the end stages where treatment options are severely limited to dialysis or kidney transplant; the research presented here will help identify novel strategies that address oxidative imbalance and preserve renal function. OBJECTIVES/GOALS: Chronic Kidney Disease patients often develop secondary cardiovascular disease - Cardiorenal Syndrome Type 4. RNA sequencing data show increased apelin receptor expression in 5/6 nephrectomy rats. The Apelinergic (APJ) system is deemed beneficial in normal physiological systems. Here we explore links between stress and the APJ system in CRS4. METHODS/STUDY POPULATION: In preliminary studies performed in NRK cells, inflammatory cytokines, IL-1β and IL-6, caused increases in apelin receptor transcripts and decreased apelin transcripts, respectfully. The literature describes inflammatory processes that contribute to degradation of many organs (kidneys, heart, and liver) suggesting an oxidative imbalance. To investigate this imbalance within CRS4, three rat cell types’’ H9c2 cardiomyocytes, HII4E hepatocytes, and NRK renal epithelial cells” will be used to assess the role of exogenous apelin on pro- and anti-oxidant levels. Cells will be pretreated with apelin or vitamin E 48 hours prior to the addition of toxins or cytokines (uremic: uric acid and d-galactose or hydrogen peroxide; cytokines: IL-1β and IL-6), to assess pro- and anti-oxidant protein levels via Western Blot. RESULTS/ANTICIPATED RESULTS: We anticipate with toxin or cytokine addition (either uremic, hydrogen peroxide, IL-1β or IL-6) in all cell types, an increase in protein levels for GPX’‘ a known measure of oxidative stress’‘ should be greater than the increases in antioxidants’‘ SOD1 and Catalase. After pre-treatment of vitamin E, GPX protein levels should decrease compared to toxin/cytokine control, while SOD1 and catalase protein levels increase; this coincides with vitamin E inducing antioxidant activity in animals and humans. The anticipated results for this study after exogenous apelin addition should reveal in all three cells types reduced levels of GPX and increased levels of SOD1 and Catalase comparable to the vitamin E treatment. DISCUSSION/SIGNIFICANCE OF FINDINGS: If addition of apelin causes an antioxidant response in all three cells types, this can build on evaluation of the APJ system as a therapeutic option for those with CKD and CRS4 to minimize both inflammatory and oxidative stress. With the data gathered here, we expect to recreate the results in a CKD rat model that highlights these same manifestations.

OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.

OBJECTIVES/SPECIFIC AIMS: The goals of this study are to develop a human-based screening assay for testing individual drug reactions and investigate the mechanism underlying susceptibility to develop diLQT. METHODS/STUDY POPULATION: We derived iPSC-CMs from 10 subjects with a high sensitivity to Sotalol (high-S group) and 10 subjects with no changes in QT interval after administration of the same drug (low-S group). Multielectrode array (MEA) was used to measure field potential duration, a surrogate to the QT interval in the electrocardiogram, in iPSC-CMs under basal conditions and in response to increasing concentrations of Sotalol. Transcriptomic profiling of iPSC-CMs from high-S Versus low-S groups was performed using RNA-sequencing. A parameter sensitivity analysis was performed on the Paci et al. iPSC-CM mathematical model to further support the lead hits identified via RNA-sequencing. RESULTS/ANTICIPATED RESULTS: Cardiac differentiation resulted in the generation of iPSC-CMs with appropriate cardiac channel expression and response to a hERG blocker E4031. MEA recordings showed a significantly higher response to Sotalol in iPSC-CMs from high-S compared with low-S subjects. Transcriptomic profiling identified upregulation or downregulation of genes (DLG2, KCNE4, PTRF, HTR2C, CAMKV) involved in downstream regulation of cardiac repolarization and calcium handling machinery as underlying high sensitivity to Sotalol. In silico parameter sensitivity analysis corroborated transcriptomic profiling of select genes; upregulated KCNE4 and downregulated CAMKV were predicted to positively and negatively correlate with iPSC-CM action potential duration when exposed to Sotalol, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest subject-specific iPSCs can be used to model functional abnormalities observed in diLQTS and offer novel insights into iPSC-based screening assays for toxic drug reactions. Success of this study may help identify key components underlying diLQT susceptibility to ultimately develop novel therapeutic agents.

Racially and ethnically minoritized populations have been historically excluded and underrepresented in research. This paper will describe best practices in multicultural and multilingual awareness-raising strategies used by the Recruitment Innovation Center to increase minoritized enrollment into clinical trials. The Passive Immunity Trial for Our Nation will be used as a primary example to highlight real-world application of these methods to raise awareness, engage community partners, and recruit diverse study participants.