OBJECTIVES/GOALS: Our team has developed a high-throughput 3D patient-derived muscle platform to study signaling pathways associated with skeletal muscle disease. This platform will be used to study pathologies of human muscle that arise from genetic mutations and processes of aging along with pharmacologic interventions to improve mass, function, and performance. METHODS/STUDY POPULATION: In the current study, 3D skeletal muscle is formed from young healthy male samples. Samples are treated with urocortin II (UCNII) or vehicle for ten days and evaluated for tissue performance. Functional assessments include real-time contraction magnitudes using digital image correlation (DIC) analysis of video collected during electrical pulse stimulation and end-point measures of initial and repeated tetanic force production. Functional measures provide indices of patient muscle synchronicity, strength, and endurance related to drug efficacy and toxicity which we will correlate to pro-growth protein signaling via Luminex. A subset of these samples will also be analyzed by histology and microscopy to assess muscle fiber density, type, and size, as well as myotube fusion index and sarcomere uniformity. RESULTS/ANTICIPATED RESULTS: We anticipate that healthy muscle treated with UCNII will have increased synchronicity and contraction magnitudes in DIC analysis throughout their seven-day electrical pulse stimulation protocol. We also expect to see sustained contraction magnitudes in DIC analysis at the end of electrical pulse stimulation indicating fatigue resistance in the drug treated group compared to no-drug control. Like our real-time DIC data, we anticipate increases to initial and sustained maximal force production in the drug treated group. We expect that drug treated muscle will present with an increased fiber density, fiber diameter, and fusion index with uniform sarcomeres. Finally, we expect heightened pro-growth signaling pathways in treated vs. controls. DISCUSSION/SIGNIFICANCE: The current study will serve as an initial investigation of the endogenous ligand UCNII for enhancing skeletal muscle mass and performance in human muscle laying the framework for future drug efficacy and toxicity studies. This platform will ultimately enhance the study of muscle diseases and translation of therapeutics to clinical settings.

OBJECTIVES/GOALS: The proposed study aims to improve physical and mental health outcomes among Hispanic older adults who live alone in a low-income San Juan, Puerto Rico community through weekly PA groups. Specific outcomes include measures of loneliness, social isolation, depression, physical mobility, metabolic indicators, and other health indicators. METHODS/STUDY POPULATION: Data will be collected at three time points: Pre (Week 1), Mid (Week 6), and Post (Week 12) intervention. Currently, the community has 50 residents over 65 years old who live under the poverty index and receive multiple social benefits. Various tools will be implemented to measure loneliness (University of California Los Angeles – Loneliness Scale-10 items), social isolation (Lubben Social Network Scale-6 items), depression (Geriatric Depression Scale-10 items), physical mobility (Time Up and Go Test), metabolic health indicators (hemoglobin A1c and glucose) and other health indicators (i.e., blood pressure, cholesterol, as well as body mass index (BMI)). These measurements will determine if participation in PA groups is associated with improvement of the variables measured. RESULTS/ANTICIPATED RESULTS: It is expected that the baseline scores of older Hispanic adults in terms of loneliness, social isolation, depression, physical mobility, metabolic indicators (i.e., cholesterol level and hemoglobin A1c), and other health indicators (blood pressure or BP and BMI) will be lower compared to those after participation in the Physical Activity Program. More frequent participation will be associated with more significant improvement in measured variables. DISCUSSION/SIGNIFICANCE: Results from this study will determine the effectiveness of community-based PA interventions in addressing loneliness, social isolation, depression, physical mobility, and metabolic factors (hemoglobin A1c and glucose) in elderly minority Hispanic populations as a means of improving their health outcomes and quality of life.

OBJECTIVES/GOALS: Early diagnosis of congenital sensorineural hearing loss (SNHL) is of paramount importance in preventing speech and language impairment.Diffusion tensor imaging (DTI) MRI can identify brain microstructural changes that may potentially contribute towards prognosticating rehabilitation. METHODS/STUDY POPULATION: We retrospectively reviewed pediatric patients with SNHL who obtained DTI MRI between 2011 and 2019, identifying 16 pediatric patients (age <18 years) with at least moderate asymmetric/bilateral SNHL., and gender-matched controlswithout neurological, developmental, or MRI-based brain macrostructural abnormalities. The following brainstem regions and tracts of the auditory pathway were assessed: superior olivary nucleus (SON), inferior colliculus (IC), ipsilateral tracts between the inferior colliculus and superior olivary nucleus (IC-SON). Diffusion values for bilateral regions and tracts were generated, then averaged to calculate a mean value for fractional anisotropy (FA) and mean diffusivity (MD) for each subject. RESULTS/ANTICIPATED RESULTS: Significant differences were identified in FA values of the SON between the SNHL cohort and controls (0.377±0.056 vs 0.422±0.052; p=0.009). No other FA or MD values were significantly different. In children £5 years, MD was significantly decreased in the SNHL cohort compared to controls in the IC (0.918±0.051 vs 1.120±0.142; p<0.001). In children >5 years, there were no significant differences in MD (1.124±0.198 vs 0.997±0.103; p = 0.119). There were no significant differences in MD or FA in the white matter fibers of the IC-SON tract [applewebdata%3A//720AAF0C-C4CF-459C-A42A-6BAA56C4E4CA#_msocom_2]. DISCUSSION/SIGNIFICANCE: This is the first study to assess microstructural changes in brainstem auditory pathway regions among children with SNHL. Longitudinal studies are warranted to assess the predictive value of DTI imaging for long-term outcomes and prognosticating intervention.

OBJECTIVES/GOALS: Our study explores the dose-related effects of THC on cardiovascular measures, self-reported effects, balance, and cognitive function among older adults. We also evaluate the acceptability and feasibility of study procedures, to inform future study designs employing this population. METHODS/STUDY POPULATION: Using a within-subject, double-blind, placebo-controlled design and standard behavioral pharmacology methods, reasonably healthy male and female adults aged 55-70 years undergo an eligibility screening, followed by a mock session and 3 experimental sessions (>7 days apart). During experimental sessions, participants are administered cannabis-infused brownies with varying THC doses. Prior to and at multiple intervals post- consumption, subjects complete assessments including self reports and observer ratings, psychomotor and cognitive performance measures, and vital signs. Follow-up interviews regarding the experience will be conducted one day after each session. RESULTS/ANTICIPATED RESULTS: We anticipate our results to mirror those of previously reported studies conducted in adults under 45 years old in that a dose-response relationship exists for subjective drug effects and vital signs with the caveat that this relationship may be exacerbated in our population. We additionally anticipate findings that indicate THC impairs balance and coordination, potentially increasing the risk of falls and accidents among this population, and cognitive function, affecting attention, memory, and executive functions. Feedback provided during the follow-up interviews will help refine procedures for future studies, ensuring that the methodology is acceptable and feasible for this population. DISCUSSION/SIGNIFICANCE: Prior work demonstrates the safety and efficacy of THC in conditions common among older adults, however, no conclusive data regarding tolerability and safety in this population exists. The presented work is vital groundwork for future research on THC as a potential therapeutic for older adults.

OBJECTIVES/GOALS: Previous research has linked inflammation to changes in brain reward circuitry and subsequent negative symptoms in patients with schizophrenia. This project aims to understand brain-immune interactions using diffusion tensor imaging (DTI) to investigate the impact of inflammatory markers on white matter (WM) tracts. METHODS/STUDY POPULATION: Patients with schizophrenia, ages 18 to 45, were recruited at Grady Hospital in Atlanta, GA. All subjects were stable outpatients and underwent extensive medical screening to rule out medical causes of acute inflammation. DTI data was collected from 39 participants on a 3-Tesla Siemens scanner. Blood was collected between 9-11AM for later assay of serum inflammatory markers. Negative symptoms were assessed using the Brief Negative Symptom Scale (BNSS). A diffusion tensor imaging model will be fitted with the data to generate well-known diffusion tensor measures (fractional anisotropy and mean diffusivity). Linear regression will be used to analyze the relationship between DTI measures and inflammation (C-Reactive Protein, CRP), controlling for possible confounders. RESULTS/ANTICIPATED RESULTS: The hypothesis of this proposal is that decreased microstructural integrity in WM tracts between the nucleus accumbens (NAc) and insula will be associated with increased inflammation, which in turn are associated with increased negative symptoms. Negative symptoms include deficits in motivation/pleasure as well as diminished expressivity, and are strongly associated with poor functional outcomes. Based on previous data from this sample demonstrating relationships between CRP and negative symptoms as well as CRP and fMRI functional connectivity between the NAc and insula, we anticipate results that demonstrate similar relationships with WM microstructural integrity, such as functional anisotropy and mean diffusivity. DISCUSSION/SIGNIFICANCE: Given the lack of treatment options for negative symptoms, this research will provide key data to further our understanding of the potential role of inflammation on neural circuits that underlie these symptoms, including WM integrity. This research also has the potential to inform future anti-inflammatory therapies for patients with schizophrenia.

OBJECTIVES/GOALS: The long-term goal of our lab is to develop clinical and intra-operative methods to aid in assessment of compressive and traumatic peripheral neuropathies. The overall objective of this project is toidentify the potential of serum neurofilament light chain as a diagnostic biomarker for nerve injury. METHODS/STUDY POPULATION: The objective of this prospective study is to obtain data on serum NfL levels in patients with cubital tunnel syndrome and traumatic nerve injuries. Serum NfL from patients with cubital tunnel and traumatic nerve injuries will be compared to serum NfL of asymptomatic, sex and aged matched controls. Pre-operative and post-operative serum levels will be measured and compared topatient’s pre-operative physical exam findings, motor and sensory function testing, electrodiagnostic studies, ultrasound, presence of intraneural vascularity, and post-operative patient reported outcome measures for cubital tunnel patients. For patients with traumatic nerve injury, acute phase and a subsequent serum NfL measurement will be used to assess temporal changes in NfL. RESULTS/ANTICIPATED RESULTS: The central hypothesis of this study is that symptomatic compression of the ulnar nerve or traumatic injury to the brachial plexus leading to axonotmesis will result in measurable increases in serum NfL proportional to the degree of nerve injury. This hypothesis has been formulated based on clinical experience and published studies demonstrating increased expression of serum NfL levels with axonal injury secondary to varying forms of peripheral neuropathy. DISCUSSION/SIGNIFICANCE: The significance of this project is that characterization of the relationship of clinical symptoms, non-invasive imaging and expression of NfL will lead to better diagnostic and prognostic algorithms in the treatment of compressive and traumatic peripheral neuropathies.

OBJECTIVES/GOALS: Assess molecular and cellular mechanisms of allograft loss in kidney biopsies using digital spatial profiling and clinical outcomes data. METHODS/STUDY POPULATION: Patients with chronic allograft dysfunction (CGD), enrolled in the Deterioration of Kidney Allograft Function (DeKAF) study, with or without eventual allograft loss, were included. CGD was defined as a >25% increase in creatinine over 3 months relative to a baseline. Kidney biopsy tissue was assessed by Nanostring GeoMX digital spatial profiling (DSP) after staining with anti-pan-cytokeratin, anti-CD45, anti-CD68, Syto-13, to identify specific cell populations, and Nanostring’s Whole Transcriptome Atlas (WTA), to quantify the distribution of transcripts across the biopsy. Up to 14 regions of interest (ROIs) were selected, with or without glomerulus. CIBERSORT was used to perform cell deconvolution. Clinical and outcomes data were from the DeKAF study and United States Renal Data System. RESULTS/ANTICIPATED RESULTS: Macrophage (M1) cell population abundance was significantly different in ROIs with glomerulus between graft loss and no graft loss. Principle component analysis of differentially expressed genes resulted in transcriptomes in ROIs that cluster together by clinical outcome of graft loss or no graft loss. There were 203 DEGs in ROIs with glomerulus that were different by graft loss or no graft loss. By pathway analysis, these 203 DEGS were enriched in the T-cell activation, integrin signaling and inflammation pathways. DISCUSSION/SIGNIFICANCE: DSP of kidney allograft biopsies allows for the identification and quantification of specific cell types, such as macrophages and molecular transcripts as potential drug targets. This data can be used to understand mechanisms of kidney allograft loss and may lead to improved immune suppression in kidney transplant recipients.

OBJECTIVES/GOALS: Untargeted metabolomics platforms are powerful biomarker discovery tools. However, the absence of uniform study design, data analysis, and reporting standards limits translation of this research into the clinical lab. The goal was to critically appraise existing untargeted metabolomics platforms that analyzed inborn errors of metabolism. METHODS/STUDY POPULATION: A search strategy was conducted in MEDLINE via PubMed from January 16, 2013, to January 16, 2023. The search strategy was limited to primary literature articles written in English that evaluated human subjects with inborn errors of metabolism (IEMs). Articles that performed targeted metabolomic analysis or analyzed non-human samples were excluded. Information on patient cohorts analyzed, sample types, and method design were extracted using a template. Categorical data are summarized as frequencies and percentages. RESULTS/ANTICIPATED RESULTS: A total of 96 distinct IEMs were evaluated by the different untargeted metabolomics methods included in this review. However, most IEMs (55/96, 57%) were evaluated by a single platform, in a single study, with a limited cohort size. Only one study validated their results using a separate, validation cohort. There was considerable diversity in the separation techniques and mass spectrometry instrumentation used by the studies to create their untargeted metabolomics methods. Slightly over half (59%) of the studies identified at least some of the metabolites detected in their samples with the highest level of confidence. Importantly, most of the included studies reported adherence to quality metrics, including use of quality control material (65%) and internal standards in their analysis (61%). DISCUSSION/SIGNIFICANCE: Future studies analyzing IEM patient samples with untargeted metabolomics platforms should progress beyond single-subject studies and evaluate the reproducibility of the research using a prospective, or validation cohort as well as confirm metabolite annotations with reference metabolites standards to generate clinically useful data.

OBJECTIVES/GOALS: Social isolation/loneliness is a public health crisis and one that is unlikely to be solved through pharmacology. Nonpharmacological approaches, such as dance, are needed. The objective of this study is to investigate the physiological correlates of dance-induced improvements in social connection. METHODS/STUDY POPULATION: Participants were randomly assigned to participate for 4 weeks (2 times per week, 90-minute sessions) in either 1) improvisational dance training (experimental group; n=7); or a 2) dance movie watching experience (control group; n=7). Before and after the intervention, using mobile brain-body imaging techniques, participants and their instructor had their brain (via electroencephalography) and body physiology (via photoplethysmography) recorded during a series of verbal and nonverbal interactive experiences. Participants were also video recorded via 4 surrounding cameras for later motion capture analysis. Neuropsychological assessments were also conducted before and after the intervention. RESULTS/ANTICIPATED RESULTS: We found that dance significantly increased social skills including empathy, interpersonal skills, emotional regulation, mindfulness, and attention. Additionally, we found that dance significantly increased interbrain synchrony during nonverbal experiences including theta (4-8 Hz), beta (12-35 Hz), and gamma (35-45 Hz) frequencies in the occipital lobe. Increases in interbrain synchrony were also positively correlated with increases in empathy. Additionally, intercardiac synchrony between the participant and instructor showed a significant correlation at post-intervention only. Future investigations will focus on the relationship between interbrain, intercardiac, and movement synchrony. DISCUSSION/SIGNIFICANCE: Our findings support the idea that dance increases interpersonal synchrony at the level of the brain, heart, and behavior. Understanding the neural and somatic mechanisms of social behaviors will help promote understanding and development of interventions for the critical problem of social isolation and loneliness.

OBJECTIVES/GOALS: The objective ofthis study is to use GNR technology to track immune cells infiltrating malignant brain tumors that are delivered as part of a novel immunotherapeutic strategy. We seek to implement this new platform to elucidate the underlying mechanisms of therapeutic benefit from ACT via correlation between biodistribution and efficacy. METHODS/STUDY POPULATION: Utilizing the inherent two-photon luminescent signal of GNRs, we will identify uptake and phenotype of lineage negative hematopoietic stem cells (HSCs) in vitro. HSCs will be isolated from the bone marrow of 6-week-old C57bl/6 female mice. Following isolation, HSCs will be co-cultured with varying concentrations of GNRs in DMEM w/o sodium pyruvate for 24 hours, tested for viability, and images to quantify uptake and identify phenotyping. CT contrast of our novel Iodine-capped PEGylated gold nanorods will be confirmed through microCT and biodistribution of HSCs at time points after injection will be identified via CT visualizationin vivo. RESULTS/ANTICIPATED RESULTS: We expect that increased GNR signaling 24 hours post-transplant in the tumors of glioma-bearing mice will be positively correlated with long term survival following ACT. Published data from our labs have revealed that CCR2+ lineage-negative HSCs significantly accumulate in tumor of glioma-bearing mice12. Importantly, CCR2+ lineage-negative HSCs promote differentiation to dendritic cells in the tumor, increase antitumor T cell responses mediated by cross-priming and cross-presentation, and improve efficacy of immune checkpoint inhibition12. Given that HSCs are important in mediating immunotherapy efficacy, we seek to correlate the accumulation of GNR signaling within the tumor as a marker of treatment response. DISCUSSION/SIGNIFICANCE: Adoptively transferred cells have been imaged using numerous published methods. While promising to the field of immunotherapy, these methods lack significant clinical validation. GNRs have not been used to study hematopoietic stem cells in the context of ACT and brain malignancies. Our research is poised to address this gap.

OBJECTIVES/GOALS: Intra-aortic balloon pumps are commonly used as circulatory support in patients with critically reduced cardiac function. The goal of this study is to estimate the incidence of mesenteric ischemia as an understudied vascular complication and to describe the clinical and radiographic characteristics of patients experiencing this complication. METHODS/STUDY POPULATION: We will be conducting a retrospective analysis of the electronic medical records of all patients who underwent intra-aortic balloon pump (IABP) placement between October 2020 and April 2023 at our academic medical center to identify the incidence of mesenteric ischemia. We will describe the clinical course of these patients and characterize them based on demographic features and risk factors for vascular complications including medical comorbidities. Finally, we will assess available chest x-ray and thoracoabdominal CT imaging for adequacy of balloon tip positioning, concordance between balloon size and aortic dimensions, and compromise of any visceral arteries in patients who experienced mesenteric ischemia vs. those who did not. RESULTS/ANTICIPATED RESULTS: We anticipate approximately 150 patients to have recieved IABPs over this period with at least 4 known cases of mesenteric ischemia. We will describe the clinical presentation of these cases and their often fatal outcomes. We expect several known risk factors will be present in these patients, including history of peripheral vascular disease, diabetes, or smoking history. On chest x-ray, we predict balloon tip positioning to be suboptimal (defined as >5cm below the aortic arch) in many patients, both those with mesenteric ischemia and those without, but a greater discordance on CT imaging between balloon size and aortic dimensions with greater evidence of visceral compromise in patients with mesenteric ischemia compared to those without. DISCUSSION/SIGNIFICANCE: Mesenteric ischemia is a serious and poorly studied complication of intra-aortic balloon pumps. Understanding the hospital course, clinical characteristics, and radiographic features present in these patients may guide clinicians in the early recognition and management of this potentially fatal complication.

OBJECTIVES/GOALS: Falls are very common among persons with multiple sclerosis (PwMS) due to the disabling symptoms associated with the disease. The relationship between pain and falls is underexplored. This study investigated the relationship between the facets of pain (intensity and interference) and falls in the context of co-occurring symptoms of MS. METHODS/STUDY POPULATION: This is a survey-based study that included 915 adults with MS. Participants provided data on demographics, clinical data, concerns about falling, symptom severity, and occurrence of falls in the past 6 months. Participants also completed the Patient Reported Outcome Measurement Information System (PROMIS) pain interference and pain intensity short forms. Pain interference and pain intensity were separately entered into univariate and multivariable logistic regression models developed to examine the associations between falls incidence and pain. Multivariable models were adjusted for age, sex, years since diagnosis, MS type, Patient Determined Disease Steps, MS status, concerns about falling, fatigue severity, PROMIS depression short form, and PROMIS physical function short form. RESULTS/ANTICIPATED RESULTS: Univariate regression analyses indicated that pain interference (OR = 1.05; 95% CI 1.03 to 1.06; p < 0.01) and pain intensity (OR = 1.03; 95% CI 1.02 to 1.04; p < 0.01) were both associated with falls. Only pain interference remained significantly associated with falls in multivariable regression analysis (OR = 1.02; 95% CI 1.00 to 1.05; p = 0.03). The model explained 25% of the variance in falls. Pain intensity was not associated with falls (OR = 0.98; 95% CI 0.95 to 1.01; p > 0.05) in multivariable regression analysis. DISCUSSION/SIGNIFICANCE: The findings suggest that pain is associated with falls among PwMS. Interventions designed to reduce falls incidence among PwMS may consider the inclusion of pain management as an integral component of those programs.

OBJECTIVES/GOALS: Familial Hypercholesterolemia (FH) is a common disorder that is vastly underdiagnosed and causes an increased risk for sudden cardiac death. Cardiology providers (CHCPs) are in an ideal position to care for patients with FH. This research aimed to assess the knowledge of CHCPs in the screening, diagnosis, and management of FH. METHODS/STUDY POPULATION: Adaptation of an existing knowledge tool guided survey development. FH knowledge domains included description of FH, prognosis, prevalence, inheritance, diagnostic criteria, and management options. CHCPs were asked to select their provider type (MD, PA, NP, RN) and years in clinical practice (less than 1-5 years, 6-10 years, 11-20 years, and greater than 20 years).Convenience and snowball sampling recruited CHCPs in the Division of Cardiology at Columbia University Irving Medical Center (CUIMC). Descriptive statistical analysis was performed on quantitative survey data using R. Frequency counts of provider type and years in clinical practice were calculated. Comparisons of scores between provider types and years in clinical practice were made using ANOVA. RESULTS/ANTICIPATED RESULTS: 70 surveys were analyzed (30.2% response rate). 50% of CHCPs identified as MDs, 24.2% as RNs, 12.9% as NPs, and 12.9% as PAs. With regards to clinical experience, 21.4% of CHCPs had 1-5 years, 25.7% had 6-10 years, 24.3% had 11-20 years, and 28.6% had greater than 20 years. The average overall score across all CHCPs was 55.4%, with the highest on the description knowledge domain (81.4% correct), followed by management (61.8%), diagnostic criteria (60.6%), inheritance (58.6%), prevalence (44.3%), and prognosis (25.2%). Physicians had the highest average score of 66.0%, followed by NPs (50.3%), PAs (49.7%), and RNs (39.3%). There was no significant difference in scores across experience levels, provider types, and knowledge domains based on experience levels. DISCUSSION/SIGNIFICANCE: CHCPs across all provider types and years of experience had limited FH knowledge. There exists an opportunity to improve CHCPs’ knowledge of FH through education (didactic knowledge) or practice (experiential knowledge). Future interventions should aim to increase didactic and experiential knowledge of CHCPs through a variety of methods.

OBJECTIVES/GOALS: There is a need to better understand how governments develop strategies to adopt, evaluate, and implement novel health technologies in a public healthcare system. The goal of this project is to understand this strategy development process for the translation of pharmacogenomic (PGx) testing in Ontario, Canada. METHODS/STUDY POPULATION: This observational case study of the Ontario Health PGx Working Group focused on developing recommendations for a PGx testing implementation strategy in the province. The group included 9 individuals affiliated with Ontario Health and 13 healthcare experts from multiple clinical fields. Ontario Health is the government agency that oversees provincial healthcare planning and service delivery. Guided by the Translational Thinking Framework and qualitative research methods, we observed the working group’s activities for eight months. We collected meeting recordings, slideshow decks, emails, and group characteristics. We used descriptive statistics and a nine-step inductive approach to analyze the data to create process maps, a case report, and key decision summaries. RESULTS/ANTICIPATED RESULTS: There were 19 meetings conducted remotely with videoconferencing technology. Throughout the working group’s activities, we identified 15 key decisions related to either administrative processes or PGx scientific content. We further stratified these two categories into four main themes relating to decisions about 1) membership involvement, 2) logistical management, 3) discussion and recommendation scope, and 4) information dissemination. These four decision themes represent tools by which Ontario Health guided the expert group activities and achieved their goal of generating a strategic roadmap for PGx testing implementation in Ontario. DISCUSSION/SIGNIFICANCE: The Ontario government makes decisions about how expert groups function by monitoring and controlling the group’s activities to ensure efficiency, standardization, and practicality. Describing expert group decision-making increases transparency and highlights the critical role they play in the translational pathway of health technologies.

OBJECTIVES/GOALS: This study aimed to characterize urinary exosomal miRNA content in African American adults with diabetic kidney disease. METHODS/STUDY POPULATION: Male and female participants between the ages of 18 and 65 were recruited from the Diabetes Treatment Center and the Nephrology Clinic at the Howard University Hospital. Exosomes were isolated from cleared urine of healthy controls (n=3), type 2 diabetics (n=3), and participants with chronic kidney disease (n=3). The purity and size of isolated microparticles was evaluated using NanoSight technology (30nm to 120nm size range) and western blot analysis for exosome-specific markers (TSG101 and CD81) RESULTS/ANTICIPATED RESULTS: Expression of 5 selected microRNAs, miR-4534, miR-320c, miR-451, miR-362-3p and miR-877-3p were evaluated by qRT-PCR. miR-4534 and miR-451 was increased between healthy controls and the type diabetic group. MiR-320c was increased in the CKD group, in comparison to healthy controls. Conversely, there was no difference in miR-877-5p between the three groups. DISCUSSION/SIGNIFICANCE: These findings will provide insight into the use of circulating miRNAs as early markers of DKD, ultimately creating more effective treatments and preventive measures.

OBJECTIVES/GOALS: The research objectives of this project are to elucidate the effects of Bruton’s tyrosine kinase inhibitors (BTKi) of varying target specificity on platelet function with regard to platelet aggregation, adhesion, spreading, and intracellular signaling as measured by kinase phosphorylation. METHODS/STUDY POPULATION: Blood from healthy volunteers was obtained and processed to obtain both washed platelets and platelet-rich plasma. The samples were then treated with one of the BTKi drugs or with vehicle (DMSO) at concentrations matching patient blood concentrations derived from clinical trials and pharmacokinetic studies. The incubated samples were then analyzed in an aggregometer using one of several agonists. Aggregation was stopped after five minutes with a perchloric acid-based lysis buffer. The samples were then analyzed by SDS-PAGE and immunoblotting to quantify BTK protein and BTK phosphorylation. Adhesion was assessed by incubating washed platelets treated with BTKi on microtiter wells coated with fibrinogen or collagen and quantifying adherent platelets by their endogenous acid phosphatase activity. RESULTS/ANTICIPATED RESULTS: We found that ibrutinib, zanubrutinib, and pirtobrutinib all completely inhibited collagen-induced platelet aggregation, whereas they did not inhibit aggregation induced by thrombin, ristocetin, arachidonic acid, or the PAR1 activator peptide SFLLRN (T6). Acalabrutinib inhibited collagen-induced platelet aggregation only at high concentrations (1-2 micromolar). At the lower concentration of 200 nanomolar, comparable to the concentration required for the other BTK inhibitors to completely inhibit platelet aggregation, acalabrutinib failed to inhibit aggregation but did inhibit auto-phosphorylation, indicating an impact on signaling. None of the BTKi drugs inhibited adhesion of platelets to collagen-coated surfaces. DISCUSSION/SIGNIFICANCE: Our data show the inhibitory effect of BTKi on collagen-induced platelet aggregation and signaling. However, it remains unclear whether the inhibition is due to an effect on BTK itself or other related kinases. Better insight into the mechanisms of platelet inhibition by BTKi may help guide the development of BTKi with a lower risk of hemorrhage.

OBJECTIVES/GOALS: Chronic kidney disease (CKD) impacts 15% of US adults and African American (AA) persons are disproportionately affected with more than 3 times higher risk of kidney failure when compared to Caucasian persons. This study evaluated the physiological and metabolomic effects of increased fruits and vegetables (F&V) on cardio-renal risk factors. METHODS/STUDY POPULATION: This pilot trial used a prospective, 2-group, randomized study design to evaluate a F&V intervention (N=46), where participants received a prescribed amount of fresh, base-producing F&V compared to a wait-list control (WL) condition (N=45). All participants were African American adults (≥18 years), had self-reported hypertension, and had CKD (Stage 1-3) on screening spot-urine microalbumin test. Participants were measured at baseline and 6 weeks post-intervention. Clinical data (i.e., systolic and diastolic blood pressure, lipid panel, hemoglobin A1C, BMI [body mass index], and albumin to creatinine ratio) were collected. Targeted metabolomic quantitative analysis was performed followed by LC-MS/MS and FIA-MS/MS. Linear mixed models evaluated analyte expression and clinical data. RESULTS/ANTICIPATED RESULTS: AA participants (N=91) were aged 58 ± 10.2 years, 66% female, and 54% had incomes ≤$50,000. T-tests compared change scores (baseline to 6-weeks) between groups. The F&V group demonstrated a significant reduction in BMI of -4.7 ± 10.5 kg/m² compared to a 1.9 ± 8.3 kg/m² increase in the WL group, p<.01. Further, the F&V group demonstrated a reduction in total cholesterol of -15.4 ± 58.8 mg/dL compared to a 17.7 ± 68.8 mg/dL increase in the WL group, p<.05. Non-significant reductions in hemoglobin A1c were found in the F&V versus the WL group. Metabolomic analysis indicated significant variation with an increase of suggestive key biomarkers for worse CKD in the WL versus F&V groups at 6-weeks. DISCUSSION/SIGNIFICANCE: Consumption of only 2 cups of F&V via a community-based intervention reduced CVD risk factors in AA adults with CKD and HTN and resulted in molecular/biochemical changes which may improve long-term kidney health. Further investigation may lead to development of cost-effective dietary intervention models to improve CKD outcomes in AA persons.

OBJECTIVES/GOALS: The major obstacle to an effective cure or remission for HIV infection is the integration of HIV into the genome of long-lived resting cells which constitute the so-called viral reservoir. With this study we want to elucidate the changes of the gut-associated HIV reservoir at different stages of viral suppression METHODS/STUDY POPULATION: Recent studies have shown that after long-term (>7 years) clinical suppression of peripheral HIV RNA, the circulating viral reservoir does not seem to decline further and, in fact may expand. The gastrointestinal associated lymphoid tissue (GALT) harbors by far the largest fraction of the latently infected cells, however not much is known about its changes over time.We thus quantified the HIV viral reservoir in the GALT by identifying HIV viral transcripts via 10X single-cell RNA sequencing at two GALT-sites in five PWH and compared the amount of HIV RNA found in the group of PWH with early (< 7years) vs late (> 7years) peripheral virological suppression (plasma HIV RNA <20copies/mL). RESULTS/ANTICIPATED RESULTS: Study participants had been diagnosed with HIV infection for a median (IQR) of 31 (32-34) years and had consistently undetectable peripheral blood HIV RNA for the previous 8 (4-15) years. In PWH with consistent viral suppression < 7yrs, 4 (2-6) HIV transcripts were identified in the ileum and 25 (13 – 38) in the colon. In PWH with consistent viral suppression > 7yrs, 0 (0-4) HIV transcripts were identified in the ileum and 7 (14-11) in the colon. Based on these preliminary results we plan to expand our cohort and confirm these results using Proviral DNA quantification. We anticipate that the viral decay in the GALT will follow a slower dynamic than what has been reported for the peripheral blood achieving a steady state after more than 7 years of peripheral viral suppression. DISCUSSION/SIGNIFICANCE: Despite the remarkable progress the survival and quality of life of PWH, after forty years from its first discovery, HIV infection remains uncurable. Considering its critical role, efforts are needed to better understand the dynamics of the GALT-associated HIV reservoir.

OBJECTIVES/GOALS: Maternal mental health, such as post-traumatic stress disorder (PTSD), is closely linked to child mental health. PTSD in mothers is associated with their children’s emotional responses. We examined associations between maternal PTSD and child brain function during emotion regulation. METHODS/STUDY POPULATION: Eight children ages 10-12 years, whose mothers had trauma histories, performed the Emotional N-Back task during functional MRI scanning. Mothers and children each reported on their trauma exposure and PTSD symptom severity. BOLD response to fearful faces during the Emotional N-Back was extracted from two specific brain regions of interest, amygdala and anterior cingulate cortex. These regions are involved in emotional response and attentional control, which are processes intrinsic to emotion regulation. An independent samples t-test was conducted on children’s BOLD response to fearful faces, with maternal PTSD symptom severity (high, low) as the independent variable. A parallel analysis was conducted with child PTSD symptom severity (high, low) as the independent variable. RESULTS/ANTICIPATED RESULTS: We found a main effect of maternal PTSD within brain regions of relevance to implicit emotion regulation. Compared to children whose mothers reported low PTSD symptom severity (n=4), children whose mothers reported high PTSD symptom severity (n=4) showed greater responsiveness to fearful faces in anterior cingulate cortex (t=2.04, p=.09,d=1.44) and amygdala (t=2.44, p=.05, d=1.72) at trending significance. A parallel analysis with child PTSD symptom severity showed no differences in brain function by this factor (ps=.55-.61). DISCUSSION/SIGNIFICANCE: Our pilot study is the first, to our knowledge, to examine associations between maternal PTSD and brain function during emotion regulation in their children. This study lays a foundation for future work; our goal is to explore dysfunction in emotion regulation neurocircuitry as one mechanism linking maternal PTSD to their children’s mental health.

OBJECTIVES/GOALS: The goal of this project is to characterize the efficacy of FOXA2 as a potential biomarker for patients with metastatic castrate-resistant prostate adenocarcinoma (CRPC) before transitioning to neuroendocrine prostate cancer (NEPC). NEPC currently has no therapeutic options and poor mechanistic understand of its origins. METHODS/STUDY POPULATION: In our study, we have utilized a multi-omics approach to characterize the potential efficacy of FOXA2 as a prognostic biomarker in numerous patient-derived castrate-resistant prostate cancer (CRPC) models. We have performed ATAC-, ChIP-, RNA-seq and proteomics to fully characterize where FOXA2 is binding genome-wide, how FOXA2 alters chromatin accessibility dynamics, identify regulatory gene targets of FOXA2, and identify FOXA2 protein-protein interactors. We have supported these findings using publicly available data from independent CRPC and NEPC patient cohorts and prostate cancer cell models. RESULTS/ANTICIPATED RESULTS: Our findings show that FOXA2 overexpression suppressed androgen signaling and promoted progression to a NEPC phenotype under short- and long-term androgen deprivation conditions, respectively. Further, FOXA2 redirected the chromatin accessibility landscape to be consistent with an NEPC gene expression program, including increased chromatin accessibility for key NEPC transcription factors. FOXA2 ChIP-seq showed FOXA2 to be bound at known NEPC driver genes and epigenetic modifiers across multiple stages of prostate cancer progression. Lastly, we discovered that FOXA2 physically interacts with key NEPC TFs and epigenetic regulators, suggesting that these FOXA2 physical interactions are required for NEPC progression. DISCUSSION/SIGNIFICANCE: This project will determine the efficacy of FOXA2 as a biomarker in advanced prostatecancer samples, which will translate as a potentially useful tool for clinicians to use for treatment of advanced prostate cancer patients.