OBJECTIVES/GOALS: Despite the large body of evidence concerning the effects of dietary interventions on blood pressure, trials have often reported poor adherence to sodium restriction. We implemented the Sodium Watcher Program-Hypertension (SWPH) program using digital self-monitoring. The purpose of this study was to determine the feasibility of the SWPH program. METHODS/STUDY POPULATION: The SWPH is a pilot two-arm, 2-month randomized controlled trial that enrolls adults with hypertension. The intervention group received personalized feedback on dietary sodium intake and BP and the control group participants received usual care for hypertension. Both groups participated in digital self-monitoring of daily diet and BP over 8 weeks. The primary outcomes were adherence to dietary sodium intake as captured by 24-hour urinary sodium excretion and BP at baseline and at the 2-month follow-up. Feasibility assessment included adherence to dietary sodium intake monitoring and in-home BP monitoring measured by the percentage of days that participant logged their food intake and in-home BP. The preliminary effect on the outcome variables was tested by using a repeated-measures analysis of variance. RESULTS/ANTICIPATED RESULTS: In this feasibility study, we included data from 12 participants (n=9 SWPH, n=3 control) who completed all phases of the study. The patients median age was 56.5 years and 70% were female. The mean baseline BP was 142.7/87.5 mmHg. The mean 24-hour urine sodium of 4853.0 mg (SD=1639.9 mg) with 80% having 24-hour urine sodium >2300 mg at baseline. SWPH group had lower systolic (baseline 142.4 mmHg vs follow-up 124.1 mmHg, p<0.001), diastolic (baseline 87.1 mmHg vs follow-up 77.5 mmHg, p<0.05) BP, and 24-hour urine sodium (baseline 3790.4 mg vs 2609.7 mg, p<0.05) compared with control group. Compared with the control group, the SWPH group had significantly more eligible days of digital food log usage (control 80.0% vs intervention 98.2%, p<0.01) and in-home BP monitor usage (control 77.6% vs intervention 94.6%, p<0.01). DISCUSSION/SIGNIFICANCE: Millions of Americans remain in need of effective interventions to manage their hypertension. Innovative and accessible strategies to sustain a low sodium diet intervention are needed for the majority of individuals not following the low sodium diet. By leveraging digital self-monitoring, a low sodium diet program was feasible and in reducing BP.

OBJECTIVES/GOALS: Dietary approach to stop hypertension (DASH) is a proven intervention to treat hypertension. Despite years of research the immediate physiologic response to its implementation was never characterized.This translational trial describes the biological pathway from nutrition through hormons, urine electrolytes and blood pressure reduction. METHODS/STUDY POPULATION: A single center interventional trial. Stage 1 hypertensive otherwise healthy volunteers were admitted for 14-days, transitioning from American style diet to DASH diet. Nutritional habits were assessed with food frequency questionnaires, and menus designed according to the guidelines of the National Heart Blood and Lung Institute (NHBLI) of the National Institute of Health (NIH). Data were collected daily for vital signs, blood and urine. Participants completed two 24-hour ABPM on days 1,10, and two 24-hour urine collections in parallel. We conducted a follow up visit two weeks after discharge. RESULTS/ANTICIPATED RESULTS: 9 volunteers (78% male, 89% Black individuals) completed the protocol. During an inpatient stay, they consumed a mean daily potassium intake of 5.6 g (±0.7g) and 2.6 g (±0.3g) of Sodium. Serum Aldosterone increased from day 0 (mean 8.3, range 2.8-18.9) to day 5 (mean 17.8, range 10.2-27.2) after intervention, and decreased on day 11 (mean 11.5, range 4.8-18.2) despite continuous exposure (p-value=0.002). The urine electrolyte ratio of ([Na]/[K]) decreased from a mean of 3.5 before intervention to 1.16 on day 4, creating a statistically significant slope (p-value<0.001). Blood pressure by 24-hour ABPM decreased by 3.7 mmHg systolic BP and 2.3 mmHg diastolic BP from day 1 to 10 for the entire period, and for measures taken during sleep or awake time, assessed separately. DISCUSSION/SIGNIFICANCE: Shifting from a high-sodium low-potassium diet to the opposite composition leads to serial physiological changes that are governed by aldosterone and result in blood pressure reduction. Urine electrolyte ratio reflects nutritional changes within 4 days of transition and should guide clinicians in assessing lifestyle modification adherence.

OBJECTIVES/GOALS: Trigeminal Neuralgia (TN) is a debilitating neuropathic condition characterized by electric-shock-like pain attacks. TN is considered a clinical diagnosis, and few proposed objective markers exist. This work studies the ability of advanced MRI techniques to diagnose and classify TN. METHODS/STUDY POPULATION: Anatomical MRI data from patients undergoing radiosurgery to treat TN was collected. A custom deep-learning UNet algorithm was trained to segment trigeminal nerves from the pons to the anterior wall of Meckels cave using segments drawn by an expert in neuroanatomy. 108 radiomics features related to nerve shape, voxel intensity, and image texture were extracted from the segmented nerves. A 2 layer neural network was trained to distinguish TN affected nerves from the pain-free contralateral nerves. Feature selection was performed within a cross-validation scheme to prevent model overfitting. Mean model performance over the validation sets was used to estimate model generalizability. RESULTS/ANTICIPATED RESULTS: 134 patients and 268 nerves were included. The average number of years with TN was 8. The average validation set accuracy was 78% [range: 75-80%]. The average validation set sensitivity and specificity were 0.82 [range: 0.79-0.84] and 0.76 [range: 0.70-0.79]. 34% of patients had undergone a prior invasive procedure to treat their TN. To evaluate whether the model detected signal changes relating to the previous treatment, those patients were excluded and the model was retrained on the surgically naive patients. Model performance in a reduced cohort of patients was similar to the model trained on all the patients, with accuracy of 77% [range: 73-82%]. DISCUSSION/SIGNIFICANCE: This study suggests that radiomics features calculated from MRIs of trigeminal nerves correlate with anatomical changes in TN affected nerves. This technique will need to be verified in a larger, more heterogeneous cohort of TN patients with a range of MRI acquisition parameters.

OBJECTIVES/GOALS: Osteoarthritis (OA) is a cartilage destroying disease. We are investigating abaloparatide (ABL) activation of parathyroid hormone receptor type 1 (PTH1R), which is expressed by articular chondrocytes in OA. We propose ABL treatment is chondroprotective in murine PTOA via stimulation of matrix production and inhibition of chondrocyte maturation. METHODS/STUDY POPULATION: 16-week-old C57BL/6 male mice received destabilization of the medial meniscus (DMM) surgery to induce knee PTOA. Beginning 2 weeks post-DMM, 40 μg/kg of ABL (or saline) was administered daily via subcutaneous injection and tissues were harvested after 6 weeks of daily injections and 8 weeks after DMM surgery. Harvested joint tissues were used for histological and molecular assessment of OA using three 5 μm thick sagittal sections from each joint, 50 μm apart, cut from the medial compartment of injured knees. Safranin O/Fast Green tissue staining and immunohistochemistry-based detection of type 10 collagen (Col10) and lubricin (Prg4) was performed using standard methods. Histomorphometric quantification of tibial cartilage area and larger hypertrophic-like cells was performed using the Osteomeasure system. RESULTS/ANTICIPATED RESULTS: Safranin O/Fast Green stained sections showed a decreased cartilage loss in DMM joints from ABL-treated versus saline-treated mice. Histomorphometric analysis of total tibial cartilage area revealed preservation of cartilage tissue on the tibial surface. Immunohistochemical analyses showed that upregulation of Col10 in DMM joints was mitigated in the cartilage of ABL-treated mice, and chondrocyte expression of Prg4 was increased in uncalcified cartilage areas in ABL-treated group. The Prg4 finding suggests a matrix anabolic effect that may counter OA cartilage loss. Quantification of chondrocytes in uncalcified and calcified tibial cartilage areas revealed a reduction in the number of larger hypertrophic-like cells in ABL treated mice, suggesting deceleration of hypertrophic differentiation. DISCUSSION/SIGNIFICANCE: Cartilage preservation/regeneration therapies would fill a critical unmet need. We demonstrate that an osteoporosis drug targeting PTH1R decelerates PTOA in mice. ABL treatment was associated with preservation of cartilage, decreased Col10, increased Prg4, and decreased number of large hypertrophic-like chondrocytes in the tibial cartilage.

OBJECTIVES/GOALS: Nutrition plays an imperative role in the management of Duchenne and Becker Muscular Dystrophy, but guidelines for nutrition counseling are absent. This study was designed to gain insight into provider experiences with nutritional services to find solutions for future counseling. METHODS/STUDY POPULATION: In this prospective, observational exploratory study, semi structured interviews were conducted at Certified Duchenne Care Centers (CDCCs) to gain insight on providers experiences with nutritional services and to identify barriers and solutions to nutrition education/care/counseling at CDCCs. Interviews were video recorded and then transcribed for themes. Overarching themes gave insight for a quantitative survey to be sent out, assessing all members on the multidisciplinary team perceptions, confidence, barriers, and solutions to providing nutritional care to Duchenne and Becker patients. RESULTS/ANTICIPATED RESULTS: We anticipate this study will provide novel data and key information from providers regarding nutrition education /care/counseling efforts in the multidisciplinary care of neuromuscular diseases. DISCUSSION/SIGNIFICANCE: Results will demonstrate the need for higher standards and more specific recommendations in nutritional services at CDCCs, while providing a framework for referrals, continuing education opportunities, and increasing providers’ confidence and abilities to provide sound nutritional advice.

OBJECTIVES/GOALS: Large-panel genomic tumor testing (GTT) is a new technology that promises to make cancer treatment more precise. However, patients may have unrealistic expectations of its benefits. The goal of this project is to assess expectations for GTT among cancer patients in community oncology practices. METHODS/STUDY POPULATION: A survey assessing expectations of the benefits of GTT was administered to cancer patients participating in a statewide study of GTT implementation, prior to receiving test results. Descriptive and regression analyses were conducted to assess expectations and the factors associated with these expectations. The study sample (N = 1,139) consisted of patients with a range of cancer types (22% gynecologic, 14% lung, 10% colon, 10% breast,and 46% other malignancies). Mean age was 64 years (standard deviation = 11); 668 (59%) were women; 71% had no college degree; 57% came from households with less than $50,000 US dollars household income; and 73% lived in a rural area. RESULTS/ANTICIPATED RESULTS: Generally, patients had high expectations that they would benefit from GTT (M = 2.81 on 0-4 scale) and positive attitudes toward it (M = 2.98 on 0-4 scale). Patients also had relatively poor knowledge about GTT (48% correct answers on an objective test of GTT knowledge). Greater expectations for GTT were associated with lower knowledge (b = –0.46; p < .001), more positive attitudes (b = 0.40; < .001), and lower education (b = –0.53; < .001). DISCUSSION/SIGNIFICANCE: This research suggests patients have high expectations that they will benefit from GTT, which is associated with low knowledge, positive attitudes, and low education. Interventions may be needed to boost understanding and moderate expectations, particularly for disadvantaged patients.

OBJECTIVES/GOALS: Immunomodulatory drugs (IMiDs) are critical to multiple myeloma (MM) disease control. IMiDs act by inducing Cereblon-dependent degradation of IKZF1 and IKZF3, which leads to IRF4 and MYC downregulation (collectively termed the “Ikaros axis”). We therefore hypothesized that IMiD treatment fails to downregulate the Ikaros axis in IMiD resistant MM. METHODS/STUDY POPULATION: To measure IMiD-induced Ikaros axis downregulation, we designed an intracellular flow cytometry assay that measured relative protein levels of IKZF1, IKZF3, IRF4 and MYC in MM cells following ex vivo treatment with the IMiD Pomalidomide (Pom). We established this assay using Pom-sensitive parental and dose-escalated Pom-resistant MM cell lines before assessing Ikaros axis downregulation in CD38+CD138+ MM cells in patient samples (bone marrow aspirates). To assess the Ikaros axis in the context of MM intratumoral heterogeneity, we used a 35-marker mass cytometry panel to simultaneously characterize MM subpopulations in patient samples. Lastly, we determined ex vivo drug sensitivity in patient samples via flow cytometry. RESULTS/ANTICIPATED RESULTS: Our hypothesis was supported in MM cell lines, as resistant lines showed no IMiD-induced decrease in any Ikaros axis proteins. However, when assessed in patient samples, Pom treatment caused a significant decrease in IKZF1, IKZF3 and IRF4 regardless of IMiD sensitivity. Mass cytometry in patient samples revealed that individual Ikaros axis proteins were differentially expressed between subpopulations. When correlating this with ex vivo Pom sensitivity of MM subpopulations, we observed that low IKZF1 and IKZF3 corresponded to Pom resistance. Interestingly, most of these resistant populations still expressed MYC. We therefore assessed whether IMiD resistant MM was MYC dependent by treating with MYCi975. In 88% (7/8) of patient samples tested, IMiD resistant MM cells were sensitive to MYC inhibition. DISCUSSION/SIGNIFICANCE: While our findings did not support our initial hypothesis, our data suggest a mechanism where MYC expression becomes Ikaros axis independent to drive IMiD resistance, and resistant MM is still dependent on MYC. This suggests targeting MYC directly or indirectly via a mechanism to be determined may be an effective strategy to eradicate IMiD resistant MM.

OBJECTIVES/GOALS: Recent research has attempted to identify diagnostic, prognostic, and predictive biomarkers, however, currently, no biomarkers can accurately diagnose GBC and predict patients prognosis. Using machine learning, we can utilize high-throughput RNA sequencing with clinicopathologic data to develop a predictive tool for GBC prognosis. METHODS/STUDY POPULATION: Current predictive models for GBC outcomes often utilize clinical data only. We aim to build a superior algorithm to predict overall survival in GBC patients with advanced disease, using machine learning approaches to prioritize biomarkers for GBC prognosis. We have identified over 80 fresh frozen GBC tissue samples from Rochester, Minnesota, Daegu, Korea, Vilnius, Lithuania, and Calgary, Canada. We will perform next-generation RNA sequencing on these tissue samples. The patients clinical, pathologic and survival data will be abstracted from the medical record. Random forests, support vector machines, and gradient boosting machines will be applied to train the data. Standard 5-fold cross validation will be used to assess performance of each ML algorithm. RESULTS/ANTICIPATED RESULTS: Our preliminary analysis of next generation RNA sequencing from 18 GBC tissue samples identified recurrent mutations in genes enriched in pathways in cytoskeletal signaling, cell organization, cell movement, extracellular matrix interaction, growth, and proliferation. The top three most significantly altered pathways, actin cytoskeleton signaling, hepatic fibrosis/hepatic stellate cell activation, and epithelial adherens junction signaling, emphasized a molecular metastatic and invasive fingerprint in our patient cohort. This molecular fingerprint is consistent with the previous knowledge of the highly metastatic nature of gallbladder tumors and is also manifested physiologically in the patient cohort. DISCUSSION/SIGNIFICANCE: Integrative analysis of molecular and clinical characterization of GBC has not been fully established, and minimal improvement has been made to the survival of these patients. If overall survival can be better predicted, we can gain a greater understanding of key biomarkers driving the tumor phenotype.

OBJECTIVES/GOALS: Rib fractures are common traumatic thoracic injuries and are associated with high rates of morbidity and mortality. In those age ≥ 65, the rate of these complications double. This study sought to identify the extent to which injury-related predictors influence clinical outcomes in geriatric patients with rib fractures. METHODS/STUDY POPULATION: A retrospective 5-year review was performed of a single Level 1 Trauma center registry. Geriatric patients (≥65 years of age) diagnosed with rib fractures from January 1, 2014 to December 31, 2019 were included. The primary outcome of interest was in-hospital mortality. Secondary outcomes included hospital and intensive care unit length of stay (HLOS and ICU LOS, respectively) and discharge disposition, as a surrogate for loss of independence. Further, subgroup analysis based on number of rib fractures (i.e. <4 and ≥4 rib fractures) was performed. RESULTS/ANTICIPATED RESULTS: 2,134 adult trauma patients were admitted with at least one rib fracture. Of these, 1,037 (49%) were ≥ 65 years old. This cohort had a mean age of 78.6 years old, injury severity score (ISS) of 11.4, HLOS of 7.4 days and 29% required ICU care with mean ICU LOS of 1.9 days. Only 36% were discharged home compared to 64% who were discharged to a care facility and thus had a loss of independence. Overall mortality was 6.3%. Compared to survivors, non-survivors had a higher ISS (19.3 vs. 10.8, p = < 0.0001) and longer ICU LOS (7.1 vs. 6.5 days, p = 0.04). Analysis based on number of rib fractures showed that those with ≥4 rib fractures had significantly higher mortality (8% vs. 4%, p = 0.008), longer HLOS (8.7 vs. 6.1 days, p < 0.0001), longer ICU LOS (2.6 vs. 1.3 days, p < 0.0001), and significantly lower discharge to home (32% vs. 39%, p = 0.02). DISCUSSION/SIGNIFICANCE: To our knowledge, this is the largest single-center study of geriatric patients with rib fractures. In this study, the observed mortality in patients ≥65 years of age was 6.3% which represents a lower mortality rate than historically reported. Despite this, only 36% were able to be discharged directly to home.

OBJECTIVES/GOALS: Neonatal endotracheal tubes (ETTs) are usually uncuffed to avoid subglottic stenosis and other complications, but cuffed ETTs allow better ventilation. Our goal was to detect and control pressure in the cuff below the limit of occluding venous flow to minimize the risk of subglottic stenosis. METHODS/STUDY POPULATION: We designed a pressure sensor to fit on a 2.5 ETT for prototype testing in 8 age adult female rabbits. Eight uncuffed age- and sex- matched rabbits served as control. Study duration was 2 hours during which pressure in the cuff was limited by novel sensor (intervention) or auscultation (control). Anesthesia was maintained with sevoflurane. Ventilation was provided mechanically. Subsequently the tracheae were removed, sectioned crosswise, and compared histologically for mucosal damage. RESULTS/ANTICIPATED RESULTS: Preliminary data demonstrated an almost 30% greater amount of intact mucosa in the intervention group. The sensor also provided data on heart rate and respiratory rate, although this signal was not optimal. After filing an invention disclosure and provisional patent, we are refining our device to include multiple compartments for local control of cuff pressure and applying for a STTR Phase I/II application. DISCUSSION/SIGNIFICANCE: Ventilation in neonates with uncuffed ETTs can be suboptimal due to leak around the tube, but cuffed ETTs pose the threat of subglottic stenosis and other complications. We have designed a prototype cuffed ETT with a sensor to maintain low cuff pressure while preventing leaks and largely avoiding damage to the tracheal mucosa.

OBJECTIVES/GOALS: This presentation outlines a novel approach to evaluating multiple risk factors for the development of dose-limiting toxicities in adolescents and young adults with cancer. This is the first to evaluate biobehavioral predictors originally identified in animal models in clinical human studies. METHODS/STUDY POPULATION: Adolescents and young adults (AYAs) have seen the slowest improvements in cancer survival and have some of the highest rates of dose-limiting mucositis (mouth sores). AYAs receiving chemotherapy with a significant chance of dose-limiting mucositis were recruited for a prospective study. Baseline perceived psychologic stress levels and inflammatory markers were collected at the time of chemotherapy administration and participants completed a daily assessment of mucositis for 14 days following chemotherapy. Logistic regression will be used to evaluate stress and inflammation as predictors of mucositis and Sobels testing will evaluate the role of inflammation as mediators in this relationship. RESULTS/ANTICIPATED RESULTS: We anticipate that, as seen in animal models, stress and inflammation will predict mucositis development. First, we hypothesize that stress levels and inflammatory markers will have a direct correlation and that the level of inflammation at the time of chemotherapy administration will predict mucositis incidence and severity. Through mediation testing, we hypothesize that inflammatory markers will explain a significant amount of the variance in mucositis also explained by stress, identifying inflammation as a mediator in this relationship. In all, we expect that stress and inflammation both predict mucositis development and will be identified as important modifiable factors that can be altered to reduce the risk of toxicity development during cancer therapy. DISCUSSION/SIGNIFICANCE: This work intends to evaluate predictors of chemotherapy-related toxicity development in AYAs with cancer and identify areas of intervention that will reduce toxicity profiles and close the gap in cancer survival for AYAs. Findings are applicable to biomedical, nursing, and psychosocial professionals and will inform future, large clinical studies

OBJECTIVES/GOALS: Almost 8 million Americans live with disability caused by stroke. However, recent advances in stroke rehabilitation are costly and lack resemblance to activities of daily living. The goal of this study was to develop a rehabilitation platform to increase stroke patients paretic limb use using inexpensive virtual reality and exoskeleton devices. METHODS/STUDY POPULATION: We conducted a feasibility study with 2 hemiplegic stroke participants. They reached for targets in a virtual reality environment using both hands. They completed 162 reaches divided into 3 blocks. Following baseline, we used an exoskeleton to provide 50% arm weight compensation to the paretic limb and used wrist weights to provide 50% arm weight resistance to the non-paretic limb. We removed the exoskeleton and wrist weights during the retention block. We used electromyography to approximate muscle activity in the biceps brachii. Relative contribution (RC) was calculated as the displacement of the paretic arm divided by the sum of displacements for both arms. Muscle contribution (MC) was calculated as the root mean square of paretic arm muscle activity divided by the sum of activity for both arms. RESULTS/ANTICIPATED RESULTS: During baseline, RC of the impaired limb was 44% and 48%, and MC of the impaired bicep was 43% and 35% in two mild to moderately impaired patients (Fugl-Meyer Upper Extremity scores of 43 and 37, respectively). During loading, RC increased by 5.6% and 1.1% and MC decreased by 8.3% and 11.8%. These data suggest hemiplegic stroke participants alter limb coordination when our device normalizes muscular output asymmetries between limbs. Importantly, these results closely match data from our previous work in 12 healthy controls, where we found a 2% increase in RC is significantly predicted by a 11% decrease in MC. By collecting more data on stroke patients, we will quantify this tradeoff between coordination and muscle activity modulation, allowing us to optimize the exoskeleton mechanics to maximize paretic limb use. DISCUSSION/SIGNIFICANCE: We demonstrate our platform is well tolerated by mild to moderately impaired stroke patients; this feasibility study forms the basis for low cost at-home technologies for stroke rehabilitation. With further development, clinicians can use our platform to fine-tune the level of limb constraint based on the individual needs of the patient.

OBJECTIVES/GOALS: This project will design, develop and pilot-test a Spanish-mobile application for breast cancer patients. It is intended that patients will improve treatment adherence, have a personal information storage system for their follow-up healthcare and can have greater control in the processes of diagnosis, treatment, education, and recovery. METHODS/STUDY POPULATION: In the first phase, a team of undergraduate engineering students was recruited to work on the areas of: Front End, Database Development, Security Protocols, and Integration. Team Venus will provide the cancer-related content. In the second phase, the engineering team will identify bugs, usability, satisfaction, and workability of the application among a pilot convenience sample of 20 participants from four cancer clinics and the community. The inclusion criteria are being > 21 years old and having a cell phone. The exclusion criterion is not knowing how/being able to use a mobile application. Participants will complete a questionnaire on sociodemographic data, mobile phone and application use, efficacy, usability, and security. Univariate and bivariate statistical analysis will be performed using SPSS. RESULTS/ANTICIPATED RESULTS: The researchers are defining specific visual and content features they would like the application to have. They are also searching and collecting reliable information about cancer from primary sources to incorporate into the tools available to the application’s users. In addition, they have also begun to identify potential cancer treatment facilities and medical personnel to assist in the recruitment of patients for pilot-testing the application. The researchers have divided them into geographical areas in Puerto Rico and each Team Venus member will approach and orient patients and medical personnel about the project. DISCUSSION/SIGNIFICANCE: It is intended that patients will improve treatment adherence, have a storage system for their follow-up healthcare and greater control in their processes of diagnosis, treatment, education, and recovery. Future goals include use of the database feature by researchers and expanding the testing of the application with a larger patient-base.

OBJECTIVES/GOALS: Novel EEG source localization and functional connectivity will assess frontal and cerebellar activity as predictors of subsequent memory and executive functioning (EF) decline in healthy, asymptomatic older adults who carry the Apolipoprotein-E ε4 allele (ε4+), which conveys up to 12x increased risk for Alzheimers disease (AD). METHODS/STUDY POPULATION: Healthy, cognitively intact ε4+ older adults (n = 23; ages 65-89) completed neuropsychological testing (focus on memory and EF) and EEG at Time 1, returning an average of one year later for neuropsychological retesting. EEG data during successful stop-signal inhibitory control trials will be used for advanced source localization and functional connectivity, with a focus on frontal and cerebellar regions of interest (ROIs). Source analyses will focus on the N200 time window (~200-350ms) to assess conflict processing and P300 (~300-550ms) for performance evaluation. Connectivity analyses are frequency-based, and will focus on theta band connectivity to assess conflict processing and delta to assess performance evaluation. RESULTS/ANTICIPATED RESULTS: Using hierarchical linear regression models, the magnitude of source activation within ROIs and connectivity metrics between ROIs will be used to predict residualized change in memory and executive function metrics between Time 1 and Time 2. We anticipate that 1) greater, compensatory activation in frontal ROIs during the N200 window, and 2) less cerebellar activation during P300, will predict memory and executive decline over the retest interval. Decline will also be predicted by 3) greater inter-hemispheric frontal connectivity in the theta band (conflict processing) and 4) less frontal-cerebellar delta connectivity (performance evaluation). DISCUSSION/SIGNIFICANCE: At most, ~50% of ε4 carriers will develop AD. Thus, identifying which carriers will decline is crucial to enabling successful, early intervention. Cerebellar dysfunction and impaired connectivity may be among the earliest indicators of incipient AD. Cutting-edge cerebellar EEG may enable an accessible option for early discernment of AD risk.

OBJECTIVES/GOALS: #NAME? METHODS/STUDY POPULATION: Cell culture & protein identification: human T cells were purified from healthy blood, then activated & cultured for 5d. CAR-T cells were collected from infusion bags of cancer patients undergoing CAR-T. Silver staining of naive & activated healthy T-cell lysates was compared; B-II spectrin was upregulated and confirmed by Western blot. Migration assays: naive & activated T-cells were imaged during migration on ICAM-1 and ICAM-1 + CXCL12 coated plates. T-cells were transfected with BII-spectrin cDNA & the chemokine dependence of migration was compared with controls. In-vivo studies: in a melanoma mouse model, BII-spectrin transfected or control T-cells were injected; tumors were followed with serial imaging. Human patient records were examined to correlate endogenous BII-spectrin levels and CAR-T response. RESULTS/ANTICIPATED RESULTS: Activated T-cells downregulate the cytoskeletal protein B-II spectrin compared to naive cells, leading to chemokine-independent migration in in vitro assays and off-target trafficking when CAR-T cells are given in vivo. Restoration of B-II spectrin levels via transfection restores chemokine-dependence of activated T-cells. In a mouse melanoma model, control mice injected with standard activated T-cells showed fewer cells in the tumor site and more cells in the off-target organs (spleen, lungs) when compared to mice injected with B-II spectrin transfected cells. Furthermore, among 3 human patients undergoing CAR-T therapy, those with higher endogenous B-II spectrin levels experienced fewer side-effects, measured by the neurotoxicity and cytokine release syndrome grades. DISCUSSION/SIGNIFICANCE: A major hurdle to widespread CAR-T therapy for cancer is significant, often fatal side-effects. Our work shows that the protein B-II spectrin is downregulated during CAR-T production, and that restoring B-II spectrin levels decreases side-effects while increasing tumor clearance--hopefully translating to better CAR-T regimens for the future.

OBJECTIVES/GOALS: Rare mutations in CHCHD10 gene are found in 1% of patients with familial Amyotrophic lateral sclerosis (ALS). The overall goal of this study is to utilize induced pluripotent stem cells (iPSCs) as an in vitro model organism for rare ALS variants to evaluate the mechanism of transcription adaptation of CHCHD10/2 as a potential therapeutic. METHODS/STUDY POPULATION: Point mutations on normal iPSCs was performed via Donorguide CRISPR/Cas9. The single stranded RNA/DNA donors contain genetic alterations of CHCHD10: Pro12Ser, Arg15Leu, Pro23Leu, Pro34Ser, Ser59Leu, Gly66Val, Pro80Leu, Tyr92Cys and Gln102His. Ribonucleoprotein electroporation was used to transfect iPSCs and DNA sequencing was used to validate gene editing. To validate transcriptional adaption, changes in levels of protein and gene expression were measured via immunoblot and quantification of CHCHD10 and CHCHCD2 was performed from whole cells lysates of the edited iPSCs. RESULTS/ANTICIPATED RESULTS: We anticipate that CHCHD2 transcriptional adaptation can functionally compensate for the locus loss of function of CHCHD10. This mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of patient phenotypes. DISCUSSION/SIGNIFICANCE: This study supplies further evidence for genetic modification as a treatment option for diseases with point mutation causal or enabling mechanisms, including some variants of ALS. Future work will explore the gene-correction from an ALS patient with a known CHCHD10-R15L variant.

OBJECTIVES/GOALS: The objective of my study is to test my overall hypothesis that CDK4/6 inhibitor-induced cellular senescence has long-lasting consequences on the tumor microenvironment that affect tumor growth and disease progression. METHODS/STUDY POPULATION: There is a lack of mechanistic understanding of the crosstalk of drug effects on different cellular pathways. One less-studied consequence of CDK4/6 inhibitors (CDKI) is their ability to induce cellular senescence in the non-malignant host cells (“stroma”), which causes secretion of a plethora of proteins that have lasting effects on the tumor microenvironment. Stromal cells are known to be able to enter senescence and promote a state of chronic inflammation that contributes to disease progression. I propose to study the stromal contribution to the tumor microenvironment and how this affects tumor growth and disease progression by using genetically engineered mouse models of breast cancer. RESULTS/ANTICIPATED RESULTS: We have found that palbociclib, a CDKI, inhibited growth and induced senescence of breast cancer cell lines xenografted into mice, and that while most on-target effects of palbociclib were reversed after treatment cessation, there was long-lasting down-regulation of interferon-γ and inflammatory response signaling pathways in the mouse host stroma that persisted after cessation of palbociclib treatment. In syngeneic models, we have found that palbociclib treatment influenced migration of T cells into CDKI-resistant tumors. DISCUSSION/SIGNIFICANCE: These studies will shed light on how senescence-inducing anticancer therapies, such as CDKI, affect tumor growth and disease progression. My studies will help elucidate new therapeutic avenues in combination with CDKI to combat therapeutic resistance in advanced breast cancer.

OBJECTIVES/GOALS: A functional precision medicine platform to identify therapeutic targets for a glioblastoma patient with Li Fraumeni syndrome was performed. Comparative transcriptomics identified druggable targets and patient derived organoids and a 3D-PREDICT drug screening assay was used to validate the pipeline and identify further therapeutic targets. METHODS/STUDY POPULATION: A comparative transcriptomics pipeline was used to identify druggable genes that are uniquely overexpressed in our patient of interest relative to a cancer compendium of 12,747 tumor RNA sequencing datasets including 200 GBMs. Mini-ring patient derived organoid-based drug viability assays were performed to validate the comparative transcriptomics data. Additionally, a spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. RESULTS/ANTICIPATED RESULTS: Using comparative transcriptomics STAT1 and STAT2 were found to be significantly overexpressed in our patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitor, as a potential therapy. Druggable pathways predicted using comparative transcriptomics corresponded with ruxolitinib sensitivity in a panel of patient derived organoids screened with this compound. Cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. Additionally, 3D-PREDICT screening identified the mTOR inhibitor everolimus as a potential candidate. These two targeted therapies were selected for our patient and resulted in radiographic disease stability. DISCUSSION/SIGNIFICANCE: This research illustrates the use of comparative transcriptomics to identify druggable pathways irrespective of actionable DNA mutations present. Our results are promising and serve to highlight the importance of functional precision medicine in tailoring treatment regimes to specific patients.

OBJECTIVES/GOALS: Idiopathic Pulmonary Fibrosis (IPF) is a fatal disease of lung scarring. Aberrant vascular remodeling is a contributor to IPF progression. We have identified CCN3 as an endothelial gene that is upregulated in resolving but not in persistent lung fibrosis in mice. Here we tested the role of CCN3 in lung microvascular endothelial function. METHODS/STUDY POPULATION: RNAi loss of function experiments were used to evaluate the role of CCN3 in lung endothelial biology. Human lung microvascular endothelial cells (HLMECs) were transfected with human CCN3 siRNA and analyzed via qPCR to assess expression of endothelial transcripts, via wound healing assay for assessment of migratory function, and via 2D tube formation assay for assessment of angiogenic function. To ascertain the effect of HLMECs on Normal Human Lung Fibroblasts (NHLFs), conditioned media (CM) from endothelial cells with control and CCN3 siRNA was applied to TGFβ primed fibroblasts and qPCR was used to measure expression levels of pro-fibrotic transcripts. Recombinant human CCN3 protein was subsequently used to confirm the gain of function role of CCN3 in lung endothelial biology using a subset of these assays. RESULTS/ANTICIPATED RESULTS: CCN3 is a secreted matricellular protein thought to be involved in angiogenesis, cell adhesion, cell migration, and inflammatory responses in endothelial cells. In other organs, CCN3 suppresses expression of fellow matricellular protein, CCN2 (CTGF); importantly, CCN2 is a known pro-fibrotic mediator of aberrant tissue remodeling in the fibrotic lung. Upon CCN3 knockdown in HLMECs, we observed reduced transcripts for inflammatory and pro-fibrotic genes, along with impaired endothelial function in wound healing and angiogenesis assays. CM from CCN3 knockdown endothelial cells enhanced the pro-fibrotic effects of TGFβ in NHLFs. Addition of recombinant CCN3 to HLMECs generated, conditioned media that reduced fibroblast pro-fibrotic activation. DISCUSSION/SIGNIFICANCE: We have shown that matricellular protein- CCN3 plays a fundamental role in endothelial identity and function and could be a promising therapeutic target in IPF. A future goal is to restore levels of genes such as CCN3 in the aged vasculature in the setting of lung fibrosis to test their capacity to promote vascular repair and fibrosis resolution.

OBJECTIVES/GOALS: The goal of this study was to understand the impact of a high sodium diet on gene networks in the kidney that correlate with blood pressure in female primates, and translating findings to women. METHODS/STUDY POPULATION: Sodium-naïve female baboons (n=7) were fed a low-sodium (LS) diet for 6 weeks followed by a high sodium (HS) diet for 6 weeks. Sodium intake, serum 17 beta-estradiol, and ultrasound-guided kidney biopsies for RNA-Seq were collected at the end of each diet. Blood pressure was continuously measured for 64-hour periods throughout the study by implantable telemetry devices. Weighted gene coexpression network analysis was performed on RNA-Seq data to identify transcripts correlated with blood pressure on each diet. Network analysis was performed on transcripts highly correlated with BP, and in silico findings were validated by immunohistochemistry of kidney tissues. RESULTS/ANTICIPATED RESULTS: On the LS diet, Na+ intake and serum 17 beta-estradiol concentration correlated with BP. Cell type composition of renal biopsies was consistent among all animals for both diets. Kidney transcriptomes differed by diet; analysis by unbiased weighted gene co-expression network analysis revealed modules of genes correlated with BP on the HS diet. Network analysis of module genes showed causal networks linking hormone receptors, proliferation and differentiation, methylation, hypoxia, insulin and lipid regulation, and inflammation as regulators underlying variation in BP on the HS diet. Our results show variation in BP correlated with novel kidney gene networks with master regulators PPARG and MYC in female baboons on a HS diet. DISCUSSION/SIGNIFICANCE: Previous studies in primates to identify molecular networks dysregulated by HS diet focused on males. Current clinical guidelines do not offer sex-specific treatment plans for sodium sensitive hypertension. This study leveraged variation in BP as a first step to identify correlated kidney regulatory gene networks in female primates after a HS diet.