OBJECTIVES/GOALS: Neuropsychiatric disorders classified as synaptopathies are marked by a glutamate-associated hypofrontality which impacts decision making and impulsivity. We hypothesized that behavioral efficacy of the psychoplastogen ketamine is mediated in part through lasting promotion of markers of synaptic strength in corticoaccumbens circuit. METHODS/STUDY POPULATION: Male, Sprague-Dawley rats received an intraperitoneal (i.p.) injection of saline, single ketamine (10 mg/kg; 1x/day), or repeated ketamine (10 mg/kg; 1x/day for three days). Twenty-four hrs following the dosing regimen, animals were euthanized, and brains dissected to harvest corticoaccumbens structures including the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). mRNA was extracted and converted to cDNA. Levels of brain derived neurotrophic factor (BDNF) exon II mRNA were quantified using reverse transcriptase polymerase chain reaction (RT-PCR); cyclophilin A (PPIA) was used as a loading control. Gene expression differences in ketamine-treated rats were identified versus saline-treated rats. BDNF protein levels were quantified using capillary-electrophoresis immunoblotting. RESULTS/ANTICIPATED RESULTS: Repeated, but not single, ketamine administration decreased mPFC, but increased NAc, BDNF exon II mRNA levels versus saline (p<0.05). Single and repeated ketamine administration increased NAC BDNF protein (p<0.05), while both dosing paradigms induced a trend towards an increase in mPFC BDNF levels. DISCUSSION/SIGNIFICANCE: We discovered a dosing regimen-dependent and sustained effects of ketamine administration on BDNF levels in the rodent brain. Taken together, ketamine-mediated BDNF levels may sustain synaptic strengthening mechanisms supporting future investigation into the utility of ketamine for diseases characterized by synaptopathies.

OBJECTIVES/GOALS: The dominant complication of Systemic Sclerosis (SSc) is clinically severe and commonly fatal pulmonary fibrosis (PF). We sought to determine the downstream regulatory role of the basic Helix-Loop-Helix protein 40 (bHLHe40), in response to Insulin-like Growth Factor II (IGF-II) on Pro-Lysyl Oxidase cleavage products. METHODS/STUDY POPULATION: We examined the response of primary pulmonary fibroblasts cultured from the lungs of control donors and SSc lung explants to IGF-II as well as human recombinant Lysyl Oxidase Propeptide (LOX-PP). In addition, we utilized an experimentally-induced model of lung fibrosis with intratracheal bleomycin administration. We used qPCR and immunoblotting to quantify mRNA and protein levels, respectively. We used sequence-specific small-interfering RNA to silence targeted genes. Immunoblots were quantified in ImageJ (NIH) and statistical analyses were performed in GraphPad Prism. RESULTS/ANTICIPATED RESULTS: IGF-II regulates levels of Pro-LOX, active LOX, and LOX-PP, as well as isoforms of proteases Bone Morphogenetic Protein 1 (BMP1) and Tolloid-like 1 (TLL1). The transcription factorbHLHe40 localizes to the nucleus in response to IGF-II. bHLHe40 silencing downregulated TLL1, abrogating the enzymatic cleavage of Pro-LOX. SSc lungs have higher baseline levels of the total (N-glycosylated/unglycosylated) LOX-PP than normal lung tissues, and baseline levels of LOX-PP correlated with TLL1 Isoform 2 in SSc lungs. LOX-PP contributes to the development and progression of SSc-PF by mediating changes consistent with the extracellular matrix deregulation implicated in SSc-PF: elevated levels of Collagen 3A1 (COL3A1), Fibronectin-1 (FN1), and Plasminogen Activator Inhibitor-1 (PAI1). DISCUSSION/SIGNIFICANCE: Our findings indicate that bHLHe40, TLL1, and LOX-PP may serve as targets of therapeutic intervention to stop the progression of SSc-PF. Since activation of common fibrotic pathways are involved in different diseases characterized by lung fibrosis such as IPF, our findings may have wider implications for lung fibrosis associated with other diseases.

OBJECTIVES/GOALS: Primary Objective: To evaluate the safety of venetoclax plus tazemetostat in patients with relapsed and refractory (R/R) Follicular lymphoma (FL) or Diffuse large B-cell lymphoma (DLBCL) Secondary Objectives: 1. To evaluate the tolerability of the combination of T+V using patient reported outcomes (PROs) 2. To evaluate the efficacy of T+V METHODS/STUDY POPULATION: Study design: A phase I trial in two parts: Part 1: a single-arm, open-label sequential dose escalation (3+3) of venetoclax in combination with tazemetostat, given at its recommended phase II dose (RP2D) of 800mg BID, to determine the maximum tolerated dose (MTD) of venetoclax. Part 2: two expansion cohorts (R/R DLBCL and R/R FL) to further characterize the safety and tolerability of the combination, and to estimate the preliminary efficacy. We will perform additional exploratory studies to determine if there are biologic features that correlate with responses. Eligibility: up to 38 patients aged >/=18 years old with histologically confirmed diagnosis of FL or DLBCL who have received at least 2 prior lines of therapy for lymphoma with evidence of disease progression and meet inclusion criteria RESULTS/ANTICIPATED RESULTS: Primary Endpoints: 1. Incidence and severity of adverse events as per CTCAEv5 2. Dose-limiting toxicity (DLT) of T+V, and to establish the maximum tolerated dose (MTD) of V plus fixed dose T Secondary Endpoints: 1. Incidence and Severity of toxicity and quality of life as per PRO-CTCAE and FACT-Lym 2. Overall response rate (ORR), complete response (CR) rate, partial response (PR) rate, as per Lugano criteria 3. Duration of response (DOR), progression-free survival (PFS), overall survival (OS)Exploratory Endpoints: 1. Characterization of tumor cells pre-treatment (including EZH2 mutations and BCL2 translocations) 2. Phenotypic analysis (including BCL2 expression) and quantification of the tumor microenvironment in pre-treatment samples (using image mass cytometry)Explorato DISCUSSION/SIGNIFICANCE: There is a need for novel therapeutic approaches to improve the prognosis for patients with R/R NHL. Preclinical data suggests synergism between the pair.5 Importantly, this represents a chemotherapy-free, oral regimen. If well tolerated, this could present an alternative therapeutic option for patients ineligible for more intensive therapies.

OBJECTIVES/GOALS: We aim to characterize how Heligmosomoides polygyrus bakeri (H. poly) alleviates murine allergic asthma which shares many characteristics of human asthma. This approach of has already identified helminth-produced human immune cell ligand “mimics” that hold great potential for next-generation clinical biologics METHODS/STUDY POPULATION: We examined the lung tissue of C57BL/6 mice infected with H. poly for changes in the pulmonary microenvironment. At ten days post infection, four infected mice and two co-housed uninfected mice were sacrificed, and their lung tissue harvested for examination of RNA via RT-qPCR. This design allows for the comparison between the lung microenvironments of infected and naīve mice. In future experiments, we intend to characterize what small molecules produced by the helminth drive changes in the lung using germ-free models of H. poly infection. RESULTS/ANTICIPATED RESULTS: We found key differences in lung chemokines between mice infected with H. poly and naīve mice. Using a student t-test with naīve correction for variance, we were able to show significant differences in the expression of E cadherin (p = 0.0355), CXCL10 (p = 0.0025), CX3CL1 (p = 0.0029), CCR2 (p = 0.017), and IDO1 (0.0078). We also found that differences in the expression of CCL5 bordered on significant with a p-value of 0.066. The expression of most of these markers (CXCL10, CCR2, CCL5, and IDO1) was elevated in the lungs of infected mice compared to naīve controls. In contrast, E cadherin and CX3CL1 showed the opposite trend with naīve mice showing greater expression. These clear differences in lung tissue gene expression underscore the connection between the gastrointestinal and pulmonary mucosal immune compartments. DISCUSSION/SIGNIFICANCE: The changes are unexpected for an infection that has been shown to attenuate allergic inflammation in the lung with increases in the IFN-Y responsive genes IDO1 and CXCL10 and inflammatory lung markers, CCL5 and CCR2. In contrast, there were decreases in inflammatory lung marker CX3CL1 and the tight junction protein E cadherin in infected mice.

OBJECTIVES/GOALS: Pregnant African American (Black women) have higher rates of adverse pregnancy outcomes compared to other races and routine monitoring of lipid levels is not currently in practice in prenatal care. We hypothesized that lipid profiles would show variation across pregnancy indicative of specific requirements during gestation and fetal development. METHODS/STUDY POPULATION: We used an untargeted lipidome analysis approach to investigate lipid metabolism with the progression of pregnancy. Pregnant Black women were recruited at prenatal clinics in Midwest (Metro Detroit, Michigan and Columbus, Ohio), women under 18 or above 45 years of age were not enrolled due to metabolic changes associated with these age groups. Women signed the consent forms and plasma samples were collected at 8-18 weeks at (T1), 22-29 weeks (T2) and 30-36 weeks (T3) of pregnancy. Samples from sixty-three women (mean age 27.41 ± 5.61 years) who had term birth (completed 37 weeks of pregnancy) were subjected to “shotgun” Orbitrap high resolution/ accurate mass spectrometry. Mixed-effects models were used to quantify systematic changes in relative lipid abundances over time using R lme4 and ggplot2 packages. RESULTS/ANTICIPATED RESULTS: Total lipids and some major lipid classes showed a significant increase with the progression of pregnancy. Phospholipids and glycerolipids exhibited a gradual increase throughout pregnancy, while sphingolipids and total sterol lipids displayed a more pronounced increase at the T3 timepoint. Acylcarnitines, hydroxy acylcarnitines and Lyso phospholipids levels significantly decrease from T1 to T3. One of the interesting finding was that non-esterified fatty acids decreased from T1 to T2 and increased again from T2 to T3, suggesting a possible role for these lipids during the later stages of pregnancy. The fatty acids showing this trend included key fatty acids- Linoleic Acid, Arachidonic Acid, Alpha-linolenic acid, Eicosapentaenoic acid, Docosapentaenoic acid, Docosahexaenoic acid. DISCUSSION/SIGNIFICANCE: Mapping lipid trends during pregnancy could lend support to a precision health approach to reduce perinatal health disparities among pregnant Black women. The findings from this study will be used to identify biomarkers and study associations with social and environmental factors responsible for adverse perinatal outcome in pregnant Black women.

OBJECTIVES/GOALS: Ambulatory methods are useful tools to study physical and mental health in everyday life. While many studies show daily activity improves mood, the effects of daily light exposure on mood remain unknown. This study evaluated the effects of daily natural light exposure and activity on daily mood and evaluate whether depression moderate effects. METHODS/STUDY POPULATION: 82 adults with lifetime major depression disorder (25 current) and 49 healthy controls were recruited from the greater Chicago community (N = 131, 62% female, age M = 30.15, SD = 9.94). At baseline, participants completed the Inventory of Depression and Anxiety Symptoms to measure depression symptoms of anhedonia, or loss of pleasure. Positive and negative affect were then measured 3x daily for 14-days via self-report using smartphones while light exposure and activity were continuously recorded from a wrist-worn actigraphy device. Following prior studies, daily natural light exposure was measured as the total number of white light samples greater than 1000 lux each day. Multilevel models were used to separate within-person (daily level) from between-person (subject level) effects. RESULTS/ANTICIPATED RESULTS: Results revealed daily within-person activity (p < .001) and natural light exposure duration (p = .035) were independently associated with increased positive affect. Effects were significantly moderated by baseline anhedonia symptoms (3-way interaction: p = .004). Natural light exposure duration only increased positive affect on lower activity days for high anhedonia and higher activity days for low anhedonia (ps < .018). Significant results remained controlling for between-person light and activity, time of year, age, sex, negative affect, and baseline general depression symptoms. Compared to one’s own daily averages, daily activity and natural light exposure may be independent pathways to boost positive affect, especially for individuals with high anhedonia symptoms. DISCUSSION/SIGNIFICANCE: Results suggest daily natural light exposure may be an accessible, low-cost alternative to independently increase positive affect in depression on days when activity is low. Translational applications are discussed focusing on transdiagnostic implications for physical and mental health conditions that disrupt mood and limit activity.

OBJECTIVES/GOALS: Estimating kidney function for drug dosing poses safety and efficacy concerns with critical medications. This study aims to develop a pragmatic method for measuring kidney function, ensuring that critical clinical decision points based on kidney function are universally applicable to all patients, leading to improved health outcomes. METHODS/STUDY POPULATION: This is a single-dose pharmacokinetic (PK) study to evaluate the concordance between iopamidol- and iohexol-measured glomerular filtration rate (mGFR), as determined by their respective serum clearances, in a cohort of 24 adults with varying kidney function. Participants with estimated glomerular filtration rates (CKD-EPI eGFRcr) ranging from >30 to 120 mL/min will be recruited from the Michigan Medicine health system. Enrolled participants will be stratified into 3 kidney function groups based on conventual kidney dosing considerations. IV micro doses of iohexol and iopamidol will be administered, followed by blood sampling. PK analysis will be used to compare the clearance of these substances. The agreement between iohexol and iopamidol in measuring GFR will be assessed via bioequivalence analysis. RESULTS/ANTICIPATED RESULTS: We expect no statistically significant difference between iopamidol and iohexol CL due to the high similarity of iopamidol and iohexol molecular and PK properties. We also expect that the ordinary least square regression analysis of iopamidol mGFRand iohexol mGFR will show limited variability across GFR measurements. These expected results will support the use of iopamidol as a marker of mGFR and its interchangeability with the gold standard iohexol. DISCUSSION/SIGNIFICANCE: Addressing eGFR errors is crucial for accurately dosing critical medications. This study aims to develop a novel mGFR methodology that accommodates various kidney function levels. This will enable precision dosing and streamline clinical trials. It also eliminates biological variability, enhancing generalizability and health outcomes.

OBJECTIVES/GOALS: Clinical indicators predictive of venous thromboembolism (VTE) in trauma patients at multiple time points are not well outlined, particularly at time of discharge. We aimed to describe and predict inpatient and post-discharge risk factors of VTE after trauma using a multi-variate regression model and best of class machine learning (ML) models. METHODS/STUDY POPULATION: In a prospective, case-cohort study, all trauma patients (pts) who arrived as level 1 or 2 trauma activations, from June 2018 to February 2020 were considered for study inclusion. A subset of pts who developed incident, first time, VTE and those who did not develop VTE within 90 days of discharge were identified. VTE were confirmed either by imaging or at autopsy during inpatient stay or post-discharge. Outcomes were defined as the development of symptomatic VTE (DVT and/or PE) within 90 days of discharge.A multi-variate Cox regression model and a best in class of a set of 5 different ML models (support-vector machine, random-forest, naives Bayes, logistic regression, neural network]) were used to predict VTE using models applied a) at 24 hours of injury date or b) on day of patient discharge. RESULTS/ANTICIPATED RESULTS: Among 393 trauma pts (ISS=12.0, hospital LOS=4.0 days, age=48 years, 71% male, 96% with blunt mechanism, mortality 2.8%), 36 developed inpatient VTE and 36 developed VTE after discharge. In a weighted, multivariate Cox model, any type of surgery by day 1, increased age per 10 years, and BMI per 5 points were predictors of overall symptomatic VTE (C-stat 0.738). Prophylactic IVC filter placement (4.40), increased patient age per 10 years, and BMI per 5 points were predictors of post-discharge symptomatic VTE (C-stat= 0.698). A neural network ML model predicted VTE by day 1 with accuracy and AUC of 0.82 and 0.76, with performance exceeding those of a Cox model. A naīve Bayesian ML model predicted VTE at discharge, with accuracy and AUC of 0.81 and 0.77 at time of discharge, with performance exceeding those of a Cox model. DISCUSSION/SIGNIFICANCE: The rate of inpatient and post-discharge VTEs remain high. Limitations: single institution study, limited number of patients, internal validation only, with the use of limited number of ML models. We developed and internally validated a ML based tool.Future work will focus on external validation and expansion of ML techniques.

OBJECTIVES/GOALS: Annually, 1.5 million global patients receive maxillofacial reconstruction. The gold standard, involving bone particulate, lacks reproducibility. To improve this, we have developed a custom 3D-printable, porous cover-core design. This study optimizes the hydrogel core properties and growth factor (GF) release for enhanced bone regeneration. METHODS/STUDY POPULATION: Different ratios of Methacrylated Gelatin (GelMa), Methacrylated Alginate (AlgMa) and tricalcium phosphate (α²-TCP) were combined to optimize cell viability, GF sequestration and mechanical stability. Material characterization was performed using a rheometer to determine the viscoelastic properties of the blends. Release from disks loaded with FGF-containing PLGA microparticles was quantified with an ELISA kit. Furthermore, scanning electron microscopy (SEM) was conducted to quantify hydrogel porosity. In vitro studies were performed using NIH 3T3 murine fibroblasts in Corning Transwells while immunofluorescent, metabolic and osteogenic studies were performed in 96 well plates to investigate cell infiltration, cell adhesion, viability and differentiation, respectively. RESULTS/ANTICIPATED RESULTS: By adjusting the AlgGelMa ratio, we manipulated matrix properties. GelMa possesses excellent durability and cell adhesion due to intrinsic RGD-binding motifs. AlgMa enhanced swelling by 30%, growth factor sequestration by 50% in 24hrs, and matrix storage modulus without increasing the loss modulus which could cause cell migration away from the hydrogel. Varying the AlgGelMa ratio lowered pH, promoted cell infiltration, and reduced fibronectin accumulation. The addition of β-TCP is anticipated to improve cell differentiation towards an osteogenic lineage due to improved elastic modulus, calcium and phosphate ion concentration improving mineral deposition. DISCUSSION/SIGNIFICANCE: These findings suggest through the use of this composite, early cell infiltration can be increased and promoted due to FGF release, leading to increased osteointegration. Our porous cover-core design ensures efficient clot integration and early cell infiltration, enhancing osteointegration through FGF release.

OBJECTIVES/GOALS: To examine the individual and combined association between preoperative sleep disturbance (SD) and depression and 12-month disability, back pain, and leg pain after lumbar spine surgery (LSS). METHODS/STUDY POPULATION: We analyzed prospectively collected multi-center registry data from 700 patients undergoing LSS (mean age=60.9 years, 37% female, 89% white). Preoperative SD and depression were assessed with PROMIS measures. Established thresholds defined patients with moderate/severe symptoms. Disability (Oswestry Disability Index) and back and leg pain (Numeric Rating Scales) were assessed preoperatively and at 12 months. We conducted separate regressions to examine the influence of SD and depression on each outcome. Regressions examined each factor with and without accounting for the other and in combination as a 4-level variable. Covariates included age, sex, race, education, insurance, body mass index, smoking status, preoperative opioid use, fusion status, revision status, and preoperative outcome score. RESULTS/ANTICIPATED RESULTS: One hundred thirteen (17%) patients reported moderate/severe SD alone, 70 (10%) reported moderate/severe depression alone, and 57 (8%) reported both moderate/severe SD and depression. In independent models, preoperative SD and depression were significantly associated with 12-month outcomes (all p’s<0.05). After accounting for depression, preoperative SD was only associated with disability, while preoperative depression adjusting for SD remained associated with all outcomes (all p’s<0.05). Patients reporting both moderate/severe SD and moderate/severe depression had 12.6 points higher disability (95%CI=7.4 to 17.8) and 1.5 points higher back (95%CI=0.8 to 2.3) and leg pain (95%CI=0.7 to 2.3) compared to patients with no/mild SD and no/mild depression. DISCUSSION/SIGNIFICANCE: Preoperative SD and depression are independent predictors of 12-month disability and pain when considered in isolation. The combination of SD and depression impacts postoperative outcomes considerably. The high-risk group of patients with moderate/severe SD and depression could benefit from targeted treatment strategies.

OBJECTIVES/GOALS: In this study, we aim to report the role of porins and blaCTX-M β-lactamases among Escherichia coli and Klebsiella pneumoniae, focusing on emerging carbapenem resistant Enterobacterales (CRE) subtypes, including non-carbapenemase producing Enterobacterales (NCPE) and ertapenem-resistant but meropenem-susceptible (ErMs) strains. METHODS/STUDY POPULATION: Whole genome sequencing was conducted on 76 carbapenem-resistant isolates across 5 hospitals in San Antonio, U.S. Among these, NCP isolates accounted for the majority of CRE (41/76). Identification and antimicrobial susceptibility testing (AST) results were collected from the clinical charts. Repeat speciation was determined through whole genome sequencing (WGS) analysis and repeat AST, performed with microdilution or ETEST®. Minimum inhibitory concentrations (MIC) were consistent with Clinical and Laboratory Standards Institute (CLSI M100, ED33). WGS and qPCR were used to characterize the resistome of all clinical CRE subtypes, while western blotting and liquid chromatography with tandem mass spectrometry (LC-MS-MS) were used to determine porin expression and carbapenem hydrolysis, respectively. RESULTS/ANTICIPATED RESULTS: blaCTX-Mwas found to be most prevalent among NCP isolates (p = 0.02). LC-MS/MS analysis of carbapenem hydrolysis revealed that blaCTX-M-mediated carbapenem hydrolysis, indicating the need to reappraise the term, “non-carbapenemase (NCP)®” for quantitatively uncharacterized CRE strains harboring blaCTX-M. Susceptibility results showed that 56% of all NCPE isolates had an ErMs phenotype (NCPE vs. CPE, p < 0.001), with E. coli driving the phenotype (E. coli vs. K. pneumoniae, p < 0.001). ErMs strains carrying blaCTX-M, had 4-fold more copies of blaCTX-M than ceftriaxone-resistant but ertapenem-susceptible isolates (3.7 v. 0.9, p < 0.001). Immunoblot analysis demonstrated the absence of OmpC expression in NCP-ErMs E. coli, with 92% of strains lacking full contig coverage ofompC. DISCUSSION/SIGNIFICANCE: Overall, this work provides evidence of a collaborative effort between blaCTX-M and OmpC in NCP strains that confer resistance to ertapenem but not meropenem. Clinically, CRE subtypes are not readily appreciated, potentially leading to mismanagement of CRE infected patients. A greater focus on optimal treatments for CRE subtypes is needed.

OBJECTIVES/GOALS: Using biomarkers to identify vulnerabilities from cocaine use disorder (CUD) is a focus of recent investigations. Current clinical efforts focus on psychiatric recovery in CUD, however other body systems are missed. Applying blood-based transcriptomics to investigate how clinical conditions relate to CUD can alter current treatment approaches. METHODS/STUDY POPULATION: We conducted a comprehensive longitudinal study of 12 individuals (mean 53 yrs.; M/F ratio 9: 3) with CUD abstinent from cocaine. 44 blood samples collected repeatedly every 3 months for 9 months were bulk RNA sequenced. We began with phenotype harmonization grouping individuals with the following metrics; cocaine withdrawal, cue craving, generalized craving, perceived stress, and days of abstinence. We ran differential gene and transcript expression with time across grouping of metrics using the dream software and used the multivariate test to examine their associations. We used the association of gene-transcripts to determine genetic predispositions with clinical traits using the Multi-marker Analysis of GenoMic Annotation assessing their overlap to a reference GWAS database. RESULTS/ANTICIPATED RESULTS: Individuals were grouped in 2 clusters based on scores of cue craving, generalized craving, cocaine withdrawal, and 3 clusters based on days of abstinence from cocaine use. Gene-transcript(s) associationsrevealed genetic predisposition towards certain clinical conditions and substance use traits. Cannabis use disorder showed significant enrichment between the greater vs. lesser abstinent days, and lesser vs. least abstinent days at 9 months. The “drinks per week” trait showed significant gene enrichment between greater vs. lesser abstinent days at 9 months. Coronary artery disease was also enriched with greater vs. least abstinent days at 3 months. Lastly, significant baseline differences in predisposition to small vessel ischemic stroke were seen in responders with high vs low perceived stress. DISCUSSION/SIGNIFICANCE: These results from a robust and feasible pipeline suggest genetic predisposition in CUD for other substances and cardio-neurovascular diseases. This pilot study may lead to larger studies into whole genome-based blood biomarker approaches for monitoring abstinence and other clinical co-morbidities to be addressed in cocaine addiction recovery.

OBJECTIVES/GOALS: Inexpensive, accurate home monitoring is the standard-of-care in many diseases like hypertension or diabetes; however, it has yet to be widely used in neurodegenerative diseases. We used wearable activity monitors and computer-vision evaluated assessments to estimate Parkinson’s disease (PD)-related disease burden. METHODS/STUDY POPULATION: We recruited 22 people from the University of Iowa Movement Disorders Clinic. Each person completed a standardized set of 3 fine motor tasks using their hands. We recorded a video of this activity, which was evaluated using MediaPipe - an open-source pose classification program from Alphabet - as well as had an nurse-practitioner evaluate the performance on a validated scale (UPDRS). Participants wore a Fitbit Inspire 3 activity tracker at home for the next two weeks. We quantified disease burden using the Parkinson’s Disease Questionnaire 39 - a validated 39-item survey about the intensity of PD-related impairment. Using data from the videos and activity trackers, we estimated 1) the standardized UPDRS assessment of motor impairment and 2) the total PDQ-39 score. RESULTS/ANTICIPATED RESULTS: We found observationally recorded fastest sustained (at least 5 minutes) walking speed was a strong predictor of PDQ-39, explaining over one third of the variability in the measure. Range of motion in the videos was a significant predictor of UPDRS scores; however, was only weakly related to the overall PDQ-39 score. Further processing of the signals from the video, including wavelets and frequency domain analysis, may provide better predictive capabilities. PDQ-39 subscores (e.g., cognition, social support, mobility) will be the subject of further analysis. DISCUSSION/SIGNIFICANCE: Home monitoring has become the standard in other fields because of the better generalizability of home measurements. Improving the detection and evaluation of PD using home monitoring will lead to more timely and accurately changes in medication and less need for clinic visits - especially off levodopa.

OBJECTIVES/GOALS: This study was performed to explore the capabilities of simultaneous [89 Zr]trastuzumab-PET/MRI acquisition in a cohort of metastatic HER2+ breast cancer. The insights derived provide additional noninvasive characterization and precise intratumoral analysis tools for healthcare providers. METHODS/STUDY POPULATION: A total of 13 patients, aged between 40 and 70, diagnosed with HER2-positive breast cancer, were selected to participate in this study. Whole-body [89 Zr]trastuzumab-PET/MR imaging was performed 5 ± 1 days post-injection of the radiopharmaceutical during ongoing HER2-directed therapy. Concurrently acquired T1-weighted MRI facilitated the identification of normal organ and tumor regions of interest, which were further analyzed for mean ADC and mean standardized uptake value. Multiparametric intratumoral habitat analysis was performed. Utilizing the median metric values, tumors were evaluated for heterogeneity, specifically assessing high and low HER2 expression through an image processing framework in conjunction with ADC metrics. Long-term treatment response evaluation is ongoing. RESULTS/ANTICIPATED RESULTS: Initial analysis indicate all tumors exhibited higher overall uptake of [89 Zr]trastuzumab across various sites including the bone (p=0.019), brain (p=0.014), and breast (p=0.069), when compared to corresponding normal organs. Additionally, increased ADCmean values were observed in all regions besides brain tumors (bone: p=0.002, brain: p=0.5, breast: p=0.03, juxtapulmonary: p=0.037), indicating distinct patterns of cellularity. Notably, one of five patients with a breast lesion, who exhibited a complete response to HER2-targeted therapy, exhibited the highest breast lesion SUVmean. Brain and lymph node lesions demonstrated intratumoral heterogeneity of HER2 expression. Qualification of multi parametric maps is anticipated to inform on intratumoral heterogeneity DISCUSSION/SIGNIFICANCE: Despite limitation in clinical applications of quantitative approaches due to lack of standardization of processing, initial investigations, in combining molecular imaging of HER2 and quantitative MRI demonstrate potential in characterizing metastatic HER2+ breast cancer for intratumoral classification and therapeutic stratification.

OBJECTIVES/GOALS: The cytoskeletal protein α²II spectrin interacts with actin and ankyrin-2 in cardiomyocytes which is essential to orchestrate ion channels and membrane proteins in the cardiac dyad. Our goal is to understand molecular mechanism causing severe ventricular arrhythmias due to spectrin dysfunction and explore novel therapies to treat such conditions. METHODS/STUDY POPULATION: We previously published a case of a 36-year-old woman with an ankyrin-2 p.R990Q (ANK2) variant, presented with severe ventricular arrhythmias and sudden cardiac arrest, caused by a novel mutation in the ankyrin-B gene (c.2969G>A) that disrupts the interaction of ankyrin-B/βII spectrin. To model the condition, we will use human induced pluripotent stem cell (DF 19-9-7T, WiCell)-derived ventricular cardiomyocytes (iPSC-CMs) having ANK2 variant, engineered using CRISPR/Cas9 method (Synthego Corp.). We will validate the differentiation of iPSCs into ventricular lineage and characterize the ANK2 ventricular phenotype. Next, we will express light-gated cation channel Channelrhodopsin (ChR2) in the ANK2 iPSC-CMs and investigate the potential role of optogenetics in treating such severe arrhythmias. RESULTS/ANTICIPATED RESULTS: Immunostaining shows 87.339% of iPSC-CMs, treated with All-trans retinoic acid (RA) (1 uM) on days 7 and 12 [RA 7,12], and 23.84% of those, treated on days 3 and 5 [RA 3,5], expressed MLC-2V (p<0.001). Calcium reuptake (τ) is 0.5914 s in RA 7, 12 while 0.2247s in RA 3, 5 (p<0.001). APD90 and APD50 of RA 7, 12 are 2- and 5-fold higher than RA 3, 5, showing distinct ventricular and atrial phenotypes. Protein expression of βII-spectrin and ankyrin-2 and their co-localizations were reduced in the ANK2 phenotype compared to the healthy phenotype. We found prolongation of Ca2+ waves and τ with blue light on iPSC-CMs, expressing ChR2. We anticipate that such prolongation of calcium transients would prevent aberrant calcium spikes, rescue Ca2+/calpain-induced βII-spectrin loss and provide electrical stability. DISCUSSION/SIGNIFICANCE: Animal models cannot accurately recapitulate human cardiac electrophysiology. The proposed human iPSC-CM-based ANK2 model would provide better mechanistic insights of severe ventricular arrhythmias. Also, the proposed optogenetic cardioversion has the potential to provide safe, targeted and painless cardioversion to manage arrhythmias.

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values <0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P<0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.

OBJECTIVES/GOALS: The objective of this research was to determine the associations of candidate genetic variants withdrug-induced long QT syndrome (diLQTS) risk, an adverse effect of over 150 FDA-approved drugsthat can lead to cardiac arrhythmias and sudden cardiac death. METHODS/STUDY POPULATION: This was a retrospective observational study of the genomic biobank at the University of Michigan Health System. Patients treated with a high-risk QT-prolonging drug and ECG measurements were included. The primary outcome was exaggerated prolongation of the QTc interval (i.e., >60 ms change from baseline and/or >500 ms absolute value) corrected using Bazett. We analyzed 3 genetic variants: KCNE1-D85N (rs1805128), SCN5A-G615E (rs12720452) and KCNE2-I57T (rs7415448) in the dominant genetic model. A Bonferroni-corrected p-value of 0.017 was considered statistically significant using logistic regression adjusted for clinical covariates. RESULTS/ANTICIPATED RESULTS: In total 6,083 self-reported white patients were included (12% event rate). The adjusted odd ratio for KCNE1-D85N was 2.24 (95%CI: 1.35-3.57; p=0.0011). The adjusted odds ratio forKCNE2-I57T was 1.40 (95%CI: 0.26-5.78, p=0.662). Only 4 total patients carried the SCN5A-G615E variant, and none of the carriers had prolonged QTc. DISCUSSION/SIGNIFICANCE: This is the largest study of candidate genetic variants in cardiac ion channels associated with the diLQTS risk. KCNE1-D85N was associated with diLQTS risk, while KCNE2-I57T was suggestive of a potential association. KCNE1-D85N should be considered in clinical guidelines as a risk factor of diLQTS.

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p<0.001), as well as higher levels of IFN-γ (all p<0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p<0.01) and CXCL10 (p<0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p<0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

OBJECTIVES/GOALS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a major public health concern due to its increasing prevalence and association with type 2 diabetes mellitus. Non-invasive magnetic resonance-based biomarkers can aid in the monitoring of disease progression and identification of patients at risk for chronic liver disease. METHODS/STUDY POPULATION: Over 600 subjects will be recruited from the San Antonio Mexican American Family Study and from a second study, which consists of (i) T2DM patients diagnosed with either MASLD or metabolic dysfunction-associated steatohepatitis (MASH) or (ii) metabolically healthy controls. Hydrogen-1 MRS and diffusion-weighted MRI (DW-MRI) will be used to measure liver fat fraction and liver stiffness biomarkers, respectively. Several potential biomarkers of liver stiffness will be evaluated in vivo using the intravoxel incoherent motion (IVIM) model for DW-MRI. To further improve the diagnostic accuracy of patients with liver fibrosis, we will integrate MRI/MRS data with relevant clinical indicators of hepatic metabolism. Results will be compared to biopsy samples to evaluate the model’s diagnostic accuracy. RESULTS/ANTICIPATED RESULTS: Based on preliminary data, we predict that IVIM will be able to accurately diagnose hepatic fibrosis in patients with MASLD, allowing it to be implemented in clinics with high-field MRI units easily. Previous studies have shown correlations between IVIM estimates and fibrosis stages, however, none included additional clinical indicators of liver disease in their models. We have already found significant differences in metabolic measurements such as fasting plasma glucose and HbA1c levels. Additionally, the use of machine learning in developing these models has shown improvements in the ability to extract features from the data. The aim is to achieve high accuracy and robustness in the staging of liver fibrosis. DISCUSSION/SIGNIFICANCE: Over 100 million people in the US are affected by MASLD. Without treatment, it progresses from hepatic steatosis to MASH, fibrosis (liver stiffening), and ultimately to hepatic cirrhosis and hepatocellular carcinoma (HCC). Continued research efforts and clinical implementation of MRI and MRS are vital in combating the growing burden of MASLD.

OBJECTIVES/GOALS: To identify empiric neuropsychiatric symptom (NPS) clusters in behavioral variant frontotemporal dementia and to determine the role of early anxiety/depression on functional progression. METHODS/STUDY POPULATION: Analyses were conducted using data from the ARTFL-LEFFTDS Longitudinal Frontotemporal Lobar Degeneration (ALLFTD) study, an established consortium with an ongoing cohort study of FTD patients across 18 clinical sites which includes comprehensive cognitive, neuropsychiatric, and structural neuroimaging data. A polychoric cluster analysis was performed on subjects from the ALLFTD cohort [applewebdata%3A//044E463E-34DA-4677-9EDC-B8309D14C337#_msocom_1] with early-stage disease (N=145, male 61%, median age 62 years) in order to identify empiric NPS clusters. Cox proportional hazard regression was then used to examine the association between early affective symptoms in bvFTD and subsequent functional disabilities adjusted for age, sex, level of education, and FTLD CDR global score. RESULTS/ANTICIPATED RESULTS: We identified a four-factor model as the best fit for the data: (1) an affective cluster with prominent depression, anxiety, agitation, and irritability, (2) a disinhibited symptom cluster with prominent elation and disinhibition, (3) an obsessive symptom cluster with prominent obsessive/ritualistic behavior and hyperorality, and (4) a psychotic symptom cluster with prominent delusions and hallucinations. The hazard of developing impairments in transactions, language, self-care, meal preparation, and incontinence was significantly elevated in those with early affective symptoms (depression/anxiety). DISCUSSION/SIGNIFICANCE: In this study we show that, NPS cluster into four discrete groups: (1) affective symptoms, (2) disinhibited symptoms, (3) obsessive symptoms, and (4) psychotic symptoms. Anxiety and depression are prominent within the affective symptom cluster and are associated with accelerated functional decline in a number of domains.