OBJECTIVES/GOALS: Studies to improve uptake of Chronic Obstructive Pulmonary Disease Clinical Practice Guidelines (COPD CPG) have yielded inconsistent results. We hypothesized that using implementation science would facilitate rigorous site ‘diagnosis’, and promote effective contextual tailoring of COPD CPG, while piloting the use of telehealth for this. METHODS/STUDY POPULATION: The study was conducted in two Veterans Affairs primary care clinics located in a small sized city. A detailed formative evaluation was conducted using key informant interviews (with VA staff and veterans with COPD who received care at this location) and quantitative data. Multidisciplinary stakeholder group was engaged and strategies to address the determinants identified through the previous step were identified. Telehealth was strongly encouraged as the primary modality for implementing the COPD CPG and we are collecting pilot data on this. Tele-Facilitation, used as the meta-strategy was employed in conjunction with other strategies such as develop/distribute educational materials, tailor strategies, change record systems and revise professional roles. RESULTS/ANTICIPATED RESULTS: Primary Care at the VA is provided by Patient Aligned Care Teams (PACT-teams), where each team consists of multiple health professionals to provide collaborative care to the patient. Discussions with the multidisciplinary stakeholder team suggested that any implementation effort primarily focused on physician and nursing efforts was unlikely to succeed due to competing demands. A pharmacy-centric model that allowed for the PACT-team clinical pharmacist to address most of the COPD CPG (inhaler technique education/assessment, inhaler choice optimization, COPD-specific patient education, spirometry use, smoking and immunization) was developed and implemented with incorporation of telehealth (video visits and telephone). We will present pilot implementation outcomes using RE-AIM framework elements. DISCUSSION/SIGNIFICANCE: This use of implementation science to implement COPD CPG and novel use of telehealth has enormous potential for impact. Increasing reach/adoption by targeting primary care practices can help permeate quality care to the underserved population. This data will allow us to explore generalizability through wider scale implementation studies.

OBJECTIVES/GOALS: Black women participate in the least amount of physical activity in the U.S., and determining methods to increase motivation and consistent exercise are warranted. Purpose: This study assessed differences in psychological and behavioral outcomes in young Black women in a culturally-tailored and theory-based resistance exercise (RE) study. METHODS/STUDY POPULATION: Women (M= 22.7 ± 3.6y) were randomized to the standard exercise group (SEG; n= 6) or motivational exercise group (MEG; n= 8), and completed 10 weeks of RE with a Black woman trainer and 11 weeks of unsupervised RE. The MEG discussed and received text messages about exercise education, self-regulation, autonomy, and competence. Motivation was measured by the Behavioral Regulation in Exercise Questionnaire-3, the Physical Activity Self-Regulation Scale-12 measured self-regulation, and the Basic Psychological Needs in Exercise Scale measured competence, autonomy, and relatedness. Adherence was calculated as # of completed/total sessions, and retention was the percent of women who completed ≥2 days/week of unsupervised RE. An ANOVA and Bonferroni post hoc analyses were used to determine significant findings. RESULTS/ANTICIPATED RESULTS: Significant time effects were found for intrinsic motivation (Λ = .691, F [11, 2]= 17.494, p< .001), basic psychological needs (Λ = .951, F [11, 2]= 22.691, p< .001), and self-regulation (Λ = .881, F [10, 2]= 40.942, p< .001), but no main interactions. Significant increases occurred from pre-testing to 3-mo follow-up for intrinsic motivation (pre: 1.80 ± .90 vs 3mo: 2.71 ± .63, p= .002), competence (pre: 1.98 ± .93 vs 3mo: 3.55±.81, p< .001), autonomy (pre: 2.14 ± .75 vs 3mo: 3.81 ± .74, p< .001) relatedness (pre: 2.78±1.60 vs 3mo: 4.32 ± .58, p< .001), and self-regulation (pre: 18.4 ± 2.88 vs 3mo: 33.79 ± 3.11, p< .001). Adherence rates for both groups were 93%. Retention rates were 33% for SEG and 25% for MEG. MEG had 38% complete RE 1 day/week opposed to none in SEG. DISCUSSION/SIGNIFICANCE: Ten weeks of culturally-tailored, supervised RE showed efficacy in significantly increasing motivation and behavioral practices to help sustain exercise. Future research should further explore strategies to use during unsupervised training to help increase exercise adherence in young Black women.

OBJECTIVES/GOALS: This project will provide novel insights into mechanism(s) by which differences in inflammation develop & resolve, or fail to resolve, in metabolically different groups of bariatric surgery patients determined by Type 2 Diabetes status. My work may uncover unique differences between cohorts, encouraging development of personalized medicine. METHODS/STUDY POPULATION: I analyzed human blood samples collected before and 3, 6, & 12 months after bariatric surgery at the University of Kentucky through an established tissue bank. Subjects hadnormal glucose tolerance, pre-diabetes, or Type 2 Diabetes, based on HbA1c%. Iisolated peripheral blood mono nuclear cells & will comparecytokine profiles among cohorts across all time points. I will define & perturbmetabolic differencesin immune cells among cohorts & across time via isotope tracing, fuel source limitation, and metabolite inhibition. This will determine causal relationships between cytokine profiles & immune cell metabolism. RESULTS/ANTICIPATED RESULTS: I anticipatecytokine profiles, a functional output of immune cells, will differ among cohorts pre-surgery, and that this difference will diminish post-surgery. Differencesmay be insignificant by the 12 month time point. I also anticipate differences in fuel usage and metabolite productionin immune cells among cohorts pre-surgery, and that these differences only partially resolve post-surgery to poise immune cells for continued chronic inflammatory action. I hypothesize that T2D status has a lasting impact on immune cell function and fuel usage patterns, and will continue to supportchronic inflammation following short term T2D remission and longer-term weight loss. DISCUSSION/SIGNIFICANCE: There has been an alarming increase in obesity and its comorbidities over recent decades, and inflammation is a known supporter of T2D. The anticipated rewiring of immune cell metabolism post-surgery, if incomplete, may poise subjects forweight regain and T2D recurrence.

OBJECTIVES/GOALS: Our aim is to establish soluble salivary biomarkers indicative of increased risk of oral premalignancy to be used in a point-of-service technology. Our goal is to non-invasively assess risk level for premalignancy by characterizing a molecular signature pattern that can be applied to such a diagnostic tool at routine dental or medical visits. METHODS/STUDY POPULATION: Adult patients 18 years of age and older who are non-smokers and patients of the University of Maryland School of Dentistry Oral Medicine Clinic and have been diagnosed with oral premalignancy (proliferative verrucous leukoplakia) are eligible. Exclusion criteria include history of immunosuppression or immune compromise; use of antifungal, antibiotic, and/or antiviral medications within the past three months; and gross dental disease. Serial unstimulated saliva samples will be collected at baseline or diagnosis of oral premalignancy, 6 months and 12 months. Solubility testing will be completed to determine whether malignant markers such as EGFR/mTOR/PI3K/p53 are soluble in saliva, and patient samples will be analyzed by ELISA and compared to appropriate controls. RESULTS/ANTICIPATED RESULTS: We anticipate demonstrating increased activity of molecular pathways known to be involved in malignant transformation, such as EGFR/mTOR/PI3K/p53, or increased burden of select microbial pathogens to be associated with increased risk of oral premalignancy in the form of proliferative verrucous leukoplakia. Preliminary sensitivity and specificity testing of the identified markers will provide additional insight to the utility of a diagnostic tool with salivary specimen. Therefore, the microbiome and/or molecular profile proposed from these results will serve as a translational application to development of future point-of-service test devices to be used in the prevention and detection of oral premalignant lesions. DISCUSSION/SIGNIFICANCE: Oral cancer is the sixth most common cancer worldwide, and presents challenges in its diagnosis and clinical management. Later diagnosis is associated with poorer patient outcomes—therefore, a molecular and microbiome profile that may be used in a noninvasive diagnostic test technology would prove beneficial to providers and patients.

OBJECTIVES/GOALS: Penetrating brain injury (PBI) differs both physiologically and in clinical outcomes when compared to blunt-force traumatic brain injury (TBI). Despite this, there are few pre-clinical models of PBI described in the literature. To address this gap, we will develop a study protocol for a systematic review. METHODS/STUDY POPULATION: Three electronic databases (PubMed, Embase, Web of Science) will be searched using keywords and controlled vocabulary related to animal models, computational models, simulations, and disease key words including traumatic brain injury and penetrating brain injury. The primary outcome will be the method of PBI modeling. Secondary outcomes will be related to bibliographic information, computational analysis, and histochemical, radiographic, behavioral, and human clinical biomarkers and outcome measures used in PBI models. A panel of independent investigators will review publications resulting from this search strategy to identify relevant studies. The protocol will adhere to PRISMA-P guidelines. RESULTS/ANTICIPATED RESULTS: Eligible studies will include both exploratory and descriptive research, and both quantitative and qualitative data. A summary of selected studies will be presented, and the synthesis will follow a narrative framework. DISCUSSION/SIGNIFICANCE: This protocol provides a framework for comprehensively evaluating pre-clinical PBI models with focus on methodology. PBI is a phenotypically unique disease and is under studied. This protocol will be of great benefit to clinicians and scientists in this emerging field and can help monitor future progress in translational research.

OBJECTIVES/GOALS: Determine how a history of unpredictable foot shock in mice affects brain wide patterns of neural activation to future stressors. Additionally, we aimed to characterize how the paraventricular nucleus of the thalamus (PVT) is involved in the fear sensitization process. METHODS/STUDY POPULATION: We used a mouse model of stress enhanced fear learning, where stressed mice are first subjected to a series of unpredictable foot shocks in a novel context while control mice undergo exposure to the novel context without experiencing foot shock. Mice are then left undisturbed for 28 days, following which they are exposed to a single foot shock in a novel context. Mice are tested in the second context 24 hours after single shock, and the amount of time spent frozen in the context provides a measure of fear sensitization. Whole brain patterns of activation during the second context test will be assessed via whole brain optical clearing with antibody staining of immediate early genes. The role of the PVT in fear sensitization will be characterized using chemogenetic approaches. RESULTS/ANTICIPATED RESULTS: Our preliminary results demonstrate that mice display enhanced fear acquisition long after the initial experience of unpredictable shocks. We anticipate to identify regions previously implicated in fear learning and novel regions not previously described through our brain clearing approach. In addition, we anticipate chemogenetic inhibition of the PVT will reduce freezing to an auditory cue associated with the shock in the second context but not to the context itself. DISCUSSION/SIGNIFICANCE: Our findings will provide a comprehensive view of how a history of unpredictable stress affects whole brain processing of subsequent stressful experiences, and describe the role of the PVT in cued fear sensitization.

OBJECTIVES/GOALS: Within our CTSA hub, greater emphasis is on understanding innovation and human health impact of translational science writ large rather than focus on translational research projects. Our program is restructured to reflect distinguishing ethical features of translational science which are complimentary to traditional research ethics issues. METHODS/STUDY POPULATION: This descriptive analysis depicts the development of our ethics program as an exemplar of how to integrate into the research enterprise of an academic health science center that engages in translational research. Our relational approach is predicated on the embodiment of ethical values by all who are involved in research committed to proactive dialog, team building, and collaboration. This translational research culture is facilitated by a multidisciplinary ethics team who are embedded throughout the translational research enterprise. RESULTS/ANTICIPATED RESULTS: Our program is integrated into a translational science enterprise within a CTSA hub in four areas: relational structure (from leadership team to community engagement), education (from trainees to the research community to the public), support (through ethics consultation with multiple touchpoints in the translational science pathway), and team science (from team on-boarding and communication to D&I research of team science interventions). We have developed a research agenda examining research ethics topics that increase quality, applicability and downstream social impact of research; understanding translational science through historical and science & technologies studies lenses, and ethnographic and mixed-method approaches to understanding team science and the science of team science. DISCUSSION/SIGNIFICANCE: The integration of a translational ethics program provides attention to traditional research ethics issues regarding study conduct and integrity but also transcends those concerns to focus on the translational science enterprise itself through relationships, cultivating trust, team science, DEIA, and social responsibility.

OBJECTIVES/GOALS: Our objective is to characterize excitatory, inhibitory, and neuromodulatory components of the voluntary motor command at the level of the spinal motoneuron in people with multiple sclerosis (MS). This information will provide insight into neural mechanisms of motor dysfunction and their heterogeneity among patients with MS. METHODS/STUDY POPULATION: Due to advances in high-density surface EMG (HDsEMG) decomposition and the recent development of a paradigm for reverse engineering of motor unit population discharge, we can feasibly estimate aspects of excitatory, inhibitory, and neuromodulatory components of the voluntary motor command in humans on a person-specific basis. We tested 11 ambulatory patients with MS and mild-moderate disability. We recorded HDsEMG from tibialis anterior (TA) and soleus (SOL) during isometric plantarflexion and dorsiflexion, performed as slow triangle contractions. EMG was decomposed into motor unit spike trains using blind source separation. We calculated a number of motor unit variables, most notably delta-F, which estimates motoneuron excitability and the balance of neuromodulatory and inhibitory inputs. RESULTS/ANTICIPATED RESULTS: There were consistent differences in MS patients vs. controls. For TA, values were decreased for delta-F (3.9 vs. 5.9 pps), initial firing rate acceleration (5.8 vs. 7.1 pps), firing rate range (9.3 vs. 11.9 pps), and max firing rate (12.3 vs. 15.0 pps). SOL had more modest decreases in delta-F (3.0 vs. 3.8 pps) and firing rate range (4.8 vs. 5.6 pps). Self-sustained firing was longer for MS patients. Within a patient, abnormalities in motor unit variables were not consistent across muscles and legs. Interestingly, there were several abnormalities in the patients with a normal clinical motor exam, indicating that perhaps our measures are sensitive to subclinical changes in processing of voluntary motor commands. DISCUSSION/SIGNIFICANCE: Excitatory, inhibitory, and neuromodulatory components of the voluntary motor command must be appropriately balanced for skilled motor output. This study is the first to characterize how they are disrupted in MS, providing foundational information to inform the development of mechanistically-based rehabilitation interventions.

OBJECTIVES/GOALS: Susceptible mucocutaneous membranes of the eye and nasal cavity are easily infected by viruses leading to pink eye or respiratory infections whose direct cost has been estimated as $16 billion annually in the United States. We have developed a novel and effective barrier that will be agnostic to variants enveloped viruses like coronaviruses. METHODS/STUDY POPULATION: We evaluated the efficacy of hydroxypropyl cyclodextrin barrier in preventing respiratory coronavirus infections using 25 humanized angiotensin converting enzyme-2 receptor (hACE-2) mice under a BSL3 laboratory setting. We have shown the barrier is safe and efficacious in preventing coronavirus infections in in vitro respiratory cell lines. We instilled 10 uL aliquot of the barrier into the nostril of the mouse 30 minutes before exposing them to a 10uL titer containing 10,000 plaque forming units of the SARS-CoV-2 delta variant. The control mice received the SARS-CoV-2 infection but not the barrier. The mice were observed for 5 days after which they were sacrificed. We analyzed the lungs and nasal palates for viral load using reverse transcription-polymerase chain reaction. RESULTS/ANTICIPATED RESULTS: We observed our barrier to be effective in preventing SARS-CoV-2 delta variant infection in hACE2 mice models. The lungs and nasal secretions of treated mice were less infectious with lower viral load than the control mice. The lungs of treated mice showed decrease in IFN gene expression and many cytokines and chemokines that regulate virally induced inflammatory responses such as IL-1b, IL-8, CXCL9, CXCL10, and the CCLs. We observed the plasma Angiotensin I and Angiotensin II decreased with barrier treatment, correlating with the viral load observed in the lungs. These peptides may be useful biomarkers for monitoring viral load within the lungs of virally infected individuals. DISCUSSION/SIGNIFICANCE: This supports the barrier’s efficacy to reduce transmission and prevent infections of SARS-CoV-2. This easy to use barrier can augment the mucocutaneous layers of the eye and nasal cavity. Our agnostic barrier will reduce the economic and public health burden of seasonal respiratory and eye viral infections and their related deaths amongst the public.

OBJECTIVES/GOALS: Congenital cytomegalovirus (cCMV) remains to be the leading infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells during cCMV remains largely unknown. The objective of this study is to define the transcriptomes of fetal NK cells exposed to human cytomegalovirus (HCMV) infection during gestation. METHODS/STUDY POPULATION: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV seropositive (HCMV+) and two HCMV seronegative (HCMV-) fetuses that did not experience any complications during gestation. These samples were provided by the Medical College of Wisconsin Tissue Bank and were processed within 24 hours following live birth. CD7+ CD3e-CD14-CD19-CD20- fetal NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. RESULTS/ANTICIPATED RESULTS: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV+ and two HCMV- fetuses. We were able to successfully sort and capture fetal NK cells and perform scRNA-seq on these samples. Following unbiased clustering, we observed and characterized five distinct fetal NK cell subsets in the umbilical cord blood and four fetal NK cell subsets in the corresponding umbilical cord tissue. Our findings revealed that HCMV+ fetal NK cells primarily consisted of mature NK cell subsets, while HCMV- fetal NK cells constituted the majority of the immature subsets. Importantly, we identified a unique subset of NKG2CHi fetal NK cells that were significantly elevated in the HCMV+ fetuses. Finally, we defined a group of transcription factors involved in the formation of antiviral fetal NK. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of distinct NK cell subsets and drive their unique transcriptional profiles. These findings have the potential to guide the development of an innovative NK cell immunotherapy that could help prevent fetuses from developing symptomatic cCMV.

OBJECTIVES/GOALS: Numerous diseases, including AL amyloidosis, are due to expression of aberrant antibodies. Significant effort has gone into plasma cell toxic therapies with varying degrees of success, but no therapies preventing antibody synthesis have been developed. The goal of this study is to assess BiP targeting to prevent antibody secretion in plasma cells. METHODS/STUDY POPULATION: Using 4 multiple myeloma cell lines (KMS11, RPMI8226, ANBL-6, U266), we knocked down BiP expression with RnaseH dependent siRNA or subA toxin, a bacterial toxin that specifically cleaves BiP, and measured changes in unfolded protein and intracellular light chains by flow cytometry during drug induced ER stress created by the intracellular calcium depleting agent thapsigargin. BiP is the master regulator of the unfolded protein response (UPR), an ER stress pathway important for protein folding. BiP is also an ER resident protein folding chaperone important for proper antibody folding. We hypothesized that BiP downregulation will lead to decreased folded antibody in the cell, increased unfolded antibody and constitutive activation of the UPR. RESULTS/ANTICIPATED RESULTS: 1 to 4 hours after treatment with thapsigargin plus siRNA against BiP, levels of BiP are significantly decreased. The levels of intracellular light chains decrease, and the level of unfolded protein within the cells increases dramatically. Interestingly, in alignment with the UPR literature, 24 hours post treatment, these levels have normalized again in surviving cells. SubA treatment increased BiP expression by 4 hours, contrary to our hypothesis, and minimally increased unfolded proteins and minimally decreased intracellular light chains. We expect that further functional testing of antibody secretion by ELIspot assays will show decreased secretion of antibody with BiP siRNA treatment. Combination therapies with other UPR stressing agents may act synergistically to affect antibody production. DISCUSSION/SIGNIFICANCE: BiP knockdown reduces antibodies and boosts unfolded proteins. SubA toxin ineffectiveness likely stems from increased BiP due to feedback loops. Combining anti-BiP treatments with UPR stressing drugs like bortezomib may halt antibody synthesis and induce cell death. These findings support BiP as a viable drug target for antibody-related diseases.

OBJECTIVES/GOALS: Childhood psychopathology is a worsening public health crisis leading to negative life outcomes, including self-harm and suicide. Difficulty in self-control as early as 3 years old predicts psychopathology, but the mediating mechanisms of brain function are unknown. Here, we tested one mechanism: functional connectivity (FC) integration. METHODS/STUDY POPULATION: We studied a sample of 204 children [53 F/149 M/2 NC; mean age (SD)=11 years (1.7)] with diverse self-control difficulties (e.g., attention deficit disorder [n=80]; autism spectrum disorders [n=91]). We extracted a general factor of psychopathology (“p-factor”) from the parent-reported Child Behavior Checklist. For participants with high quality fMRI data on 3 self-control tasks (n=79), testing flexibility, working memory, and inhibition, we calculated FC connectomes reflecting a general self-control state, and applied connectome predictive modeling (CPM) to reveal connections predicting overall task impairment. We then measured individual variance in cross-network integration of regions with the most predictive connections and tested for association with p-factor in a multiple linear regression. RESULTS/ANTICIPATED RESULTS: We repeated CPM 1,000 times with 10-fold cross validation to generate a distribution of accuracies for predicted vs. observed task impairment scores (mean r=0.25, permutation p=0.02). Connections selected a maximum of 10,000 times (10 folds * 1,000 repetitions) were strongly predictive of task impairment (r=-0.5, p<0.001), highlighting connectivity of canonical executive networks as well as the default mode network. Regions (n=22) with the top 5% most selected connections were in lateral parietal and frontal cortices and implicated motor control. Between-network integration, operationalized with the graph theory metric participation coefficient, of one of these regions in left posterior superior frontal gyrus significantly predicted p-factor (R2=0.26, F(22,56) = 0.87; B =-0.49, p<0.05). DISCUSSION/SIGNIFICANCE: A portion of dorsolateral prefrontal cortex, associated with executive control, explained individual variance in p-factor. We plan to test alternative predictive models. Identification of such a neuro behavioral mechanism underlying psychopathology may lead to novel intervention targets.

OBJECTIVES/GOALS: Although the approval of immune checkpoint inhibitors (ICIs) revolutionized the treatment of metastatic breast cancer (BC), yet about 30% remain unresponsive. Since the potency of ICIs depends on the efficient presentation of tumor-specific antigens by cancer cells, compounds which increase such presentation could increase efficacy of ICIs. METHODS/STUDY POPULATION: A library of the ester and urethane derivatives of polyether ionophore antibiotic, monensin (MON) has been synthesized. MTT cell viability assays were performed on the panel of human and mouse BC cell lines, and non-cancerous breast epithelial cells to determine IC50 values of MON and its derivatives. Selectivity Indexes were calculated to identify the most selective compounds towards cancer versus non-cancer cells. Major Histocompatibility Complex (MHC) class I and II presentation and Programmed death-ligand 1 (PD-L1) expression have been determined using flow cytometry. Proteins involved in apoptosis, autophagy and immunogenic cell death were identified through immunoblotting. At least three biological replicates have been performed for each experiment. RESULTS/ANTICIPATED RESULTS: MON and several of its derivatives shown activity in nanomolar range against MDA-MB-231 human BC cell line. MON and its most potent derivatives significantly increased MHC class I and II presentation and downregulated the expression of PD-L1 in BC cell lines. DISCUSSION/SIGNIFICANCE: Present findings will lead to the development of new therapeutic approaches that can serve as single agents or be used in combination with existing ICIs for the treatment of metastatic BC. By pushing the boundaries of our understanding and developing new therapies, this research can make an impact in improving outcomes for patients with metastatic BC.

OBJECTIVES/GOALS: Distinguishing indolent from aggressive prostate cancer and early identification of men at risk of developing aggressive, metastatic disease is of great importance. We aim to explore the relationship between N-glycan and collagen composition in prostate tumor tissue and the long-term outcome of the disease. METHODS/STUDY POPULATION: Matrix assisted laser desorption/ionization mass spectrometry can be utilized to characterize N-glycan profiles in formalin fixed paraffin embedded tissues. Collagen may also be characterized using ECM-targeted collagenase MALDI imaging. These approaches were used to analyze prostatectomy samples with different clinical outcomes. Tissue microarrays containing tissues from 75 non-progressors (no evidence of disease; NED) and 50 metastatic cases (MET) were examined. From a combined list of 90 N-glycans and 500 collagenase peptides, the average AUC intensity value for each glycan and collagen peptide was extracted and assessed as a predictor of metastatic progression. Ancestral informative markers were analyzed and polygenic hazard risk scores were generated for samples as well. RESULTS/ANTICIPATED RESULTS: Three N-glycans and three collagen peptides were found to discriminate between NED and MET cases with statistical significance. The best performing N-glycan was Hex6HexNAc6Fuc1 with an AUC of 0.77 (p<0.001). While the best performing collagen peptide was COL1A2 with an AUC of C 0.77 (p<0.001). DISCUSSION/SIGNIFICANCE: Both a collagen peptide and N-glycan were discovered as promising biomarkers to predict metastasis. Future validation studies are needed to confirm biomarker potential and to determine if the addition of these biomarkers can strengthen current genomic classifier’s ability to predict metastatic prostate cancer.

OBJECTIVES/GOALS: RETgene fusions in sarcoma are rare and their impact on pathogenicity is unknown. Malignant peripheral nerve sheath tumors (MPNST) are a deadly, genomically heterogenous soft tissue sarcoma rarely harboring targetable aberrations. We present a case of a CCDC6-RET fusion MPNST sensitive to RET-inhibitor therapy in a xenograft model. METHODS/STUDY POPULATION: Lung tumor tissue was obtained per an approved collection protocol from a 21yo male patient with a spontaneous MPNST harboring an inactivating mutation in NF-1 and a CCDC6-RET gene fusion detected by a commercially available sequencing panel (Signatera). To confirm pathogenicity of the RET fusion, fresh tumor tissue was engrafted into immunocompromised NSG mice in the anterior and posterior flanks, harvested at ~10 weeks, and re-transplanted into bilateral flanks. When tumor diameters reached 0.5-1cm (~4 weeks), mice were randomized into 3 groups (n=6/group) and treated with either vehicle (V) (PBS), the RET-specific inhibitor selpercatinib (S) (20mg/kg twice daily), or the multi-kinase inhibitor cabozantinib (C) (30mg/kg daily) by oral gavage. Mice were monitored weekly for weight and tumor size. RESULTS/ANTICIPATED RESULTS: 92% (33/36) of implanted tumors were evaluable for treatment response. Pre-treatment tumor volumes (mm3) across all three groups were similar (mean/Std Dev – V: 230/111, S: 271/132, C: 230/123). At day 7, tumor growth was significantly inhibited by S and C versus V (ANOVA p < 0.001, post-hoc Tukey’s V vs S p= 0.0178, V vs C p< 0.0001, S vs C p= 0.0005). V-treated tumors increased in volume by 60% while S reduced tumor volume by ~80% and C reduced tumor volume by ~20%. S and C treatments were tolerated well. and S improved survival with 100% of mice alive at day 63 vs 0% in V and C groups. 6 of the 12 implanted tumors treated with S, 50% increased in size after ~6-weeks following a >90% initial tumor reduction in tumor volume. Follow-on molecular studies in S-resistant tumors are ongoing. DISCUSSION/SIGNIFICANCE: Targetable genomic changes in MPNST, especially in RET, are infrequent and often considered stochastic. Our findings suggest that precision medicine approaches pairing genomic sequencing and in vivotesting of target gene pathogenicity may guide treatment planning and novel discovery for rare, difficult to treat sarcomas.

OBJECTIVES/GOALS: Research objectives include prototyping a mobile phone application that allows physicians to monitor CD19-directed CAR T-cell therapy patients remotely after discharge. This app will also enable standardized data collection across different centers that provide CAR-T cell therapy and allow for the harmonization of follow-up protocols. METHODS/STUDY POPULATION: Literature review and semi-structured interviews with patients, clinical coordinators, and other experts in the field will be used to determine what parameters must be included in the mobile application prototype to effectively monitor the side effects of CD19-directed CAR T-cell therapy. The mobile phone application will be designed using process mapping to integrate data from self-reporting and wearable technologies, including the Garmin smart watch. Figma will then be used to develop new screens based on an existing patient monitoring app for Allogeneic Stem Cell Transplant follow-up. Finally, a preliminary feasibility study will be conducted to collect feedback on the app prototype from CAR T-cell therapy patients, providers, and stakeholders. RESULTS/ANTICIPATED RESULTS: The anticipated results of this study include an app prototype that will include the functionalities required to monitor patients for adverse effects of CD19-directed CAR T-cell therapy. This will include the parameters that will be recorded or measured using a combination of self-reporting, a reliable body temperature sensor, and the Garmin watch which monitors basic vitals, activity, and sleep. Additional parameters may be added during the stakeholder co-design process. The app prototype will include a physician interface where doctors can monitor their patients and will be alerted if they require further physician assessment. It is expected that the app will provide standardized monitoring of patients when they are discharged from the hospital after receiving CAR T-cell therapy. DISCUSSION/SIGNIFICANCE: This app will allow physicians to monitor patients for general follow-up and adverse effects, including cytokine release syndrome and neurotoxicity. Future studies may utilize this app to develop best practices for harmonizing CAR-T follow-up protocols across Canada.

OBJECTIVES/GOALS: Preeclampsia increases cardiovascular (CV) risk, likely via persistent endothelial dysfunction and angiotensin II type 1 receptor autoantibodies (AT1R-Ab). We aim to assess coronary endothelial function (CEF) and AT1R-Ab levels in postpartum preeclampsia with a hypothesis this mediates CV risk. METHODS/STUDY POPULATION: We prospectively enrolled age and CV risk factor matched postpartum women. Coronary MRI was performed at rest and with isometric handgrip stress, an endothelial dependent stressor. CEF was quantified as % stress-induced change in coronary cross-sectional area (%CSA) and in coronary blood flow (%CBF). AT1R-Ab was measured using a novel antigen capture enzyme-linked immunosorbent assay. RESULTS/ANTICIPATED RESULTS: Women with and without preeclampsia were similar in age (mean 32.7+5.0 years), BMI (mean 28.0+6.3 kg/m2) and race/ethnicity (58% White, 35% Black and 4% Hispanic). %CSA was lower with (-2.1+13.6) vs without preeclampsia (8.8+17.1), p=0.023. %CBF was also lower with (11.3 [-11.8, 25.2]) vs without preeclampsia (25.7 [-0.7, 62.9]), p=0.039. AT1R-Ab was higher among women with preeclampsia (p=0.029) and was inversely associated with %CBF (beta coefficient -4.6 [-8.9, -0.3], p=0.037) but not with %CSA. DISCUSSION/SIGNIFICANCE: Women with preeclampsia have elevated AT1R-Ab and impaired CEF demonstrated by insufficient coronary reserve with exercise. Coronary endothelial dysfunction and dysregulation of the renin-angiotensin pathway likely contribute to long-term CV risk and should be considered for targeted risk reduction.

OBJECTIVES/GOALS: Many economic evaluations rely on clinical trial data that may not represent real world populations and intervention effectiveness. We compare risk and cost-effectiveness for the Diabetes Prevention Program (DPP) clinical trial cohort and a real world population eligible for the national DPP to assess the impact of using real world data. METHODS/STUDY POPULATION: To produce real world (US population) representative results, we identified National Health and Nutrition Examination Survey (NHANES) subjects eligible for the national DPP and adjusted projections using survey weights. We used clinical predictive models to estimate individual diabetes risk, and microsimulation to estimate lifetime costs, benefits, and net monetary benefits (NMB) for lifestyle intervention and metformin. We compared results across the DPP clinical trial and NHANES populations. RESULTS/ANTICIPATED RESULTS: Three-year risk of diabetes onset for the DPP trial population (mean of 19.7%, median of 10.3%) exceeded corresponding risk for the NHANES population (mean of 14.6%, median of 4.8%). The proportion of individuals with a three-year diabetes risk < 10% for the DPP trial population (49%) was less than the corresponding proportion for NHANES (67%). Mean NMB for metformin for the DPP trial population ($9,749) exceeded the corresponding value for NHANES ($5,391). The proportion of subjects with negative NMB was 49% for the DPP trial population and 67% for NHANES. Lifestyle intervention had a mean NMB of $34,889 for the DPP trial population and $28,652 for NHANES. Only 20% of the NHANES population eligible for national DPP met inclusion/exclusion criteria for the DPP trial. DISCUSSION/SIGNIFICANCE: Real world populations eligible for the national DPP include a greater proportion of low-risk individuals, and for these people, prevention programs may confer smaller benefits. Technology assessments based on clinical trial data should be revised using real world population and treatment effect data.

OBJECTIVES/GOALS: Glioblastoma (GBM) is the most malignant brain tumor in adults and remains incurable with an average survival of 15 months after diagnosis. There is great need for treatment options without side effects that are devastating to the quality of life for patients. GBM tumors can circumvent cellular damage by upregulating antioxidant production. METHODS/STUDY POPULATION: Highly aggressive tumors tend to exhibit increased oxidative metabolism, and thus rely on a mechanism to eliminate reactive oxygen species (ROS) in order for cells to evade autophagy and cell death. We propose that recurrent GBM cells achieve this is by promoting methionine metabolism, upregulating glutathione production and preventing ROS accumulation. We investigated the expression of AHCY and MAT2A, two key enzymes in the methionine pathway, at the gene and protein level in both GBM and non-GBM tissues. We probed for markers of cell death following pharmacological inhibition and siRNA knockdown, and performed metabolite-mediated rescue experiments. Finally, we evaluated changes in cellular respiration using the Seahorse XFe96 real-time mitochondrial stress test following inhibitor treatment. RESULTS/ANTICIPATED RESULTS: The selective AHCY inhibitor markedly reduced cell viability in different cancer cell types, but significantly reduced cell viability in recurrent GBM cells compared to newly diagnosed GBM (p=.009; MD: -0.828, 95% CI -1.350 to -0.306) and normal astrocytes (p=.073; MD: -0.609, 95% CI -1.305 to 0.085). AHCY and MAT2a protein expression appeared to be higher in GBM cells compared to normal astrocytes, medulloblastoma cells and other cancer cell lines. Genetic knockdown of AHCY and MAT2A demonstrated reduced cell viability, increased Caspase, SOD2, LC3-II and Transferrin receptor expression. Acute treatment with the AHCY inhibitor induced cell death, markedly reduced oxygen consumption rate and ablated spare respiratory capacity in recurrent GBM cells compared to newly diagnosed GBM cells. DISCUSSION/SIGNIFICANCE: Oxidative damage was induced following interference with key methionine pathway enzymes by pharmacological inhibition, while a similar concentration of drug largely preserved normal astrocyte viability. These results point to a novel targetable mechanism of disease progression and expand the realm of treatment options for recurrent GBM.

OBJECTIVES/GOALS: Transcriptional adaptation is a phenomenon in which a mutation in one gene leads to the genetic compensation of another homogenous gene. Understanding the mechanism of transcriptional adaptation may contribute to an explanation for variation in clinical manifestations of rare Amyotrophic lateral sclerosis patient phenotypes. METHODS/STUDY POPULATION: The presence of a premature termination codon triggers transcriptional activation. Therefore, we utilized CRISPR-Cas9 tool to generate a premature termination codon in CHCHD10 gene in multiple types of cells, including induced pluripotent stem cells derived from patient samples with known CHCHD10 mutations causative for Amyotrophic lateral sclerosis. CRISPR-Cas9 tool was delivered via ribonucleoprotein electroporation and transfect cell’s DNA was sequenced to validate gene editing. To confirm transcriptional adaption, changes in levels of protein and gene expression will be measured via immunoblot and quantification of CHCHD10 and CHCHCD2 from whole cells lysates of the edited cells. 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: Our approach would advance discovery science towards by exploring transcriptional adaptation mechanism in humans, which can lead to novel therapies for rare Amyotrophic lateral sclerosis, such as CHCHD10.