OBJECTIVES/SPECIFIC AIMS: Previous studies suggest that genetic variants in the oxytocin receptor (OXTR) may alter oxytocin dose requirement for labor induction and may increase risk for preterm labor and neurodevelopmental disorders. However, the mechanisms of actions of these variants remain unknown. The goal of this study was to functionally characterize common missense and noncoding variants in OXTR. First, we aimed to determine the effects of missense variants on two major aspects of receptor function: calcium signaling and β-arrestin recruitment. Second, we used allelic expression imbalance assays in an effort to identify regulatory single nucleotide polymorphisms (SNPs) in noncoding regions of OXTR that alter OXTR mRNA expression. METHODS/STUDY POPULATION: We used the Exome Aggregation Consortium database to identify the 12 most prevalent missense single nucleotide variants in OXTR. To determine the functional effects of these variants, we transfected human embryonic kidney cells (a common model system used to study receptor function) with wild type OXTR, variant OXTR, or empty vector control. We used the calcium-sensitive dye Fluo4 to quantify intracellular calcium flux in response to oxytocin treatment, and used bioluminescence resonance energy transfer assays to measure recruitment of the signaling partner β-arrestin to the receptor. To investigate potential effects of noncoding SNPs on OXTR mRNA expression, we quantified allele-specific expression of OXTR in human uterine tissue obtained from participants at the time of Cesarean section. We used next-generation sequencing (Illumina MiSeq) to count alleles of a reporter SNP in OXTR exon 3. RESULTS/ANTICIPATED RESULTS: Of the 12 most prevalent missense single nucleotide variants, four were predicted to be deleterious by PolyPhen variant annotation software. We anticipate that these variants will alter receptor signaling through calcium or β-arrestin pathways. We further observed that a reporter SNP in OXTR exon 3 exhibits significant allelic expression imbalance in a subset of our myometrial tissue samples, indicating that OXTR expression may be regulated by a functional SNP. Our current work focuses on discovering the functional SNPs in OXTR responsible for the pattern of allelic expression imbalance seen in mRNA. In the future, we will seek to explore the effects of these variants on uterine function by using genome editing of uterine smooth muscle cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that both missense and noncoding variants may affect OXTR expression and function. Future studies may suggest that OXTR sequencing, genotyping, or expression analysis would be useful to identify individuals likely to respond or fail to respond to safe doses of oxytocin for labor induction. Personalizing approaches for labor induction in this way would increase the safety of oxytocin and potentially reduce maternal morbidity and mortality.

OBJECTIVES/GOALS: Navigating the NIH Single IRB Policy has been challenging for investigators, study teams, and Human Research Protection Programs (HRPP). In response, the Indiana Clinical and Translational Sciences Institute (CTSI) created an innovative Single IRB Project Manager role (sIRB PM), uniquely placed within the Indiana CTSI. METHODS/STUDY POPULATION: The Single IRB Project Manager role was created in 2018 by the Indiana CTSI in response to the NIH Single IRB Policy for Multi-Site Research. The role of the sIRB PM is to serve as a liaison between the Indiana University HRPP, lead site, coordinating center, and participating sites when Indiana University serves as the Single IRB. This model has proven useful to both the IRB and lead site, notably in the following ways:

• At study start-up, the sIRB PM can handle complicated communications among sites and the IRB at the same time the lead site is responsible for many other administrative tasks related to start-up. By absorbing the workload of IRB approval for multiple sites, the sIRB PM provides the lead site more capacity to handle other essential tasks.

• The sIRB PM translates new terminology and facilitates processes that are new for sites.

RESULTS/ANTICIPATED RESULTS: Early assessment of this program is predominantly positive. The sIRB PM currently supports 24 external sites. In an NIA-funded 13 site study, all sites were added within 9 months of initial IRB approval of the protocol. This role fills a gap that benefits:

• IRB staff by allowing them to fulfill their duties of screening and review while leaving some of the reliance organization to the sIRBPM.

• Lead PI by allowing them to focus on conducting the research instead of the many administrative tasks required for single IRB review.

• Participating sites by having a liaison to enter their amendments and reportable events into an otherwise closed IRB software system.

• All parties by having the sIRB PM manage document organization, storage, and distribution study-wide.

DISCUSSION/SIGNIFICANCE OF IMPACT: The CTSI sIRB PM role effectively shifts administrative work caused by the sIRB mandate by merging research coordinator experience with regulatory experience while building upon an existing strong relationship with the HRPP. Future focus is on process education, standardizing pricing structure, and ensuring sufficient budget support in grants.

OBJECTIVES/GOALS: Maternal health and exposures during pregnancy play a major role in shaping the neurodevelopment of our offspring—one influence is maternal immune activation (MIA). Here we explore the association of MIA during pregnancy and the developing human connectome through analysis of 46 markers of activation. METHODS/STUDY POPULATION: 74 healthy women with singleton pregnancies underwent blood draws between 34-37 weeks gestation. 46 markers of maternal immune activation, both adaptive (e.g., IgG) and innate (e.g., cytokines and acute phase reactants), were collected. In addition, for preliminary analyses of MIA in relation to the newborn brain, we utilized 30 participants with MRIs between the ages of 0-6 months. RESULTS/ANTICIPATED RESULTS: Principal component analysis (PCA) identified the first 5 PCs explains ~68% of the variance and the first 10 explains ~83% (top PC is 42.1%). Using the top PC each edge in the connectome was correlated with the immune profiles. Several regions trended towards significance–one survived correction and included 359 edges, showing. The highest number of edges was observed in the inferior parietal lobe of the left hemisphere–a region associated with functions from basic attention toa social cognition, suggesting that deviations in fetal exposure to MIA can longitudinally impact offspring behavior in areas essential for human interaction. DISCUSSION/SIGNIFICANCE: This is the first study in understanding how interruptions (i.e., MIA) influence later development. Identification of alterations, and long-term outcomes could lead to the development of mechanism-based healthcare, facilitate timely referral for appropriate interventions and provide family support.

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: 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: Accurate classification of disorders of consciousness (DoC) is key in developing rehabilitation plans following brain injury. The Coma Recovery Scale-Revised (CRS-R) is a sensitive measure of consciousness. We explore feasibility, safety and impact of CRS-R guided rehab in hemorrhagic stroke patients with DoC and evaluate predictors of recovery. METHODS/STUDY POPULATION: Consecutive patients with non-traumatic hemorrhagic stroke, defined as subarachnoid hemorrhage (SAH) or intracerebral hemorrhage (ICH), receiving serial CRS-R assessments during their ICU stay at University of Maryland Medical Center from 2017-2021 were retrospectively identified. Outcomes of interest included the association with CRS-R and discharge disposition, therapy-based function and mobility and occurrence of safety events during CRS assessment. We also examined the association between CRS-R and physiological and anatomical injury pattern on electroencephalography (EEG) and magnetic resonance imaging (MRI), respectively. CRS-R RESULTS/ANTICIPATED RESULTS: 76 patients with≥2 CRS-R assessments were identified (22 SAH, 54 ICH, median age = 59, 50% female). Median CRS-R completed was 3 with no SAEs identified during sessions. We identified 4 patterns: persistent VS/UWS (49%), persistent MCS or better (13%), emergence from VS/UWS to MCS or better (27%) and regression from MCS or better to VS/UWS (11%). Persistent low CRS-R correlated with older age in SAH (p=0.01), female gender in ICH (p=0.04), and history of diabetes (p=0.01). 2% of patients with final CRS-R DISCUSSION/SIGNIFICANCE: Early neurorehabilitation guided by CRS-R appears to be feasible and safe acutely following hemorrhagic stroke complicated by prolonged DoC and may enhance access to inpatient rehabilitation with a lasting benefit on recovery. Further characterization of DoC patterns and their correlation to clinical markers, including EEG and MRI is needed.

ABSTRACT IMPACT: Chronic kidney disease (CKD) affects ˜15% of the US population and the majority of patients are diagnosed too late to benefit from early intervention. We are developing a new diagnostic imaging tool (RadioCF-PET) for the kidney to enable early detection of diseases and to monitor overall kidney health. OBJECTIVES/GOALS: Nephron mass, or the number of functioning nephrons, is a measure of the functional capacity of the kidney. RadioCF-PET may enable early detection of nephron loss in patients with or at risk of CKD before changes are clinically detectable, facilitating early interventions to improve outcomes in these patients. METHODS/STUDY POPULATION: RadioCF-PET, labeled with Cu-64, has the advantage of using sub pharmacological doses for imaging, carrying low risk and can be used with the FDA’s exploratory IND (eIND) mechanism for early in human testing. We are developing the technology to be used in pre-eIND toxicology and pharmacology studies. We are also developing other aspects of translational science to propel this technology toward translation, including: market analysis, critical path to market, customer discovery, and commercialization strategy. RESULTS/ANTICIPATED RESULTS: Milestone 1: Apply technology in mouse model study and in human kidneys rejected for transplant. Anticipated Result 1: PET signal from RadioCF-PET correlates with glomerular density in healthy and diseased model male mice (R2 = 0.98). RadioCF-PET signal correlates with glomerular number in a donated human kidney (R2= 0.78). Milestone 2: Application to federal funding (STTR) and gap funding mechanisms to enable pre-eIND studies. Anticipated Result 1: Application for funding will aid to clarify and validate our market analysis and commercialization strategy. Milestone 3: Continued research and development with the technology in new studies. Other Anticipated Results: Future work with RadioCF-PET will enhance technology performance in preparation for pre-eIND studies. DISCUSSION/SIGNIFICANCE OF FINDINGS: We foresee a large clinical and commercial potential for RadioCF-PET to provide precise, early monitoring in patients at risk for or with CKD. The two biggest hurdles for clinical translation are validating safety and proving efficacy. This work targets both issues to facilitate RadioCF-PET toward clinical translation.

ABSTRACT IMPACT: Quantifying the types of dental procedures patients with healthcare needs receive can help understand and improve modalities of dental care to improve healthcare outcomes. OBJECTIVES/GOALS: 1. Quantify how medical complexity based on special needs diagnosis impacts dental treatment rendered

2. Comprehend how medical diagnosis of a special need can affect rate of treatment and type of treatment METHODS/STUDY POPULATION: This study consists of a chart review of all active patients in a dental school setting who have one of the following diagnoses of a special need: autism, developmental disorder, epilepsy, cerebral palsy, neuromuscular disorder, and hydrocephaly. Medical diagnoses were used to extract records and quantify the types of dental treatment rendered for these patients (preventative, restorative, and surgical), as well as the rates of appointments for this patient population. RESULTS/ANTICIPATED RESULTS: The medical complexity of patients of this population, as defined by the number medical diagnoses, impacts the type of treatment rendered as well as how frequently these patients are seen for dental care (rate of appointments). DISCUSSION/SIGNIFICANCE OF FINDINGS: Over- or under-utilization of dental treatment modalities can impact the oral health status and outcomes for patients with special needs. The differences in types and frequency/ rate of dental treatment rendered for patients of different medical complexity can further impede their oral health and systemic health status and health outcomes.

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: Introduction: Between 2014 and 2019 the National Institute of Health (NIH) through the National Center for the Advancement of Translational Science (NCATS) has awarded about $2.7 billion to U.S. Academic Medical Centers to build a national network of clinical and translational science program hubs that serve to meet their key goals and initiatives. Today there are about 60 Clinical and Translational Science Award (CTSA) program hubs. Each CTSA program hub has a corresponding website highlighting its clinical and translational science centered programs and activities. These websites are a critical communication gateway to promote NCATS goals and initiatives. Objective: The objective of this research is to evaluate the NIH funded Clinical and Translational Science Award (CTSA) program hub websites for NCATS goals and initiative content alignment, navigability, and interactivity. METHODS/STUDY POPULATION: Methods: Each CTSA program hub website was systematically evaluated for information or tools that align with the five NCATS / CTSA Goals and eight CTSA nationally identified program initiatives. Each NCATS goal and CTSA initiative was subsequently ranked by information diversity level (text, tool, interactivity) and navigation level (click distance from the home page). RESULTS/ANTICIPATED RESULTS: Results: Four of the five NCATS goals are thoroughly and consistently represented among the CTSA Consortium with workforce development, patient and community engagement, and quality and efficiency of research being the top three. Informatics is thoroughly and consistently represented, but not always clearly identified on the home page. The most underrepresented goal is integration of special and underserved populations which was identified on only 60% of CTSA program hub websites. The most common focus of the eight CTSA program initiatives is the Trial Innovation Network in CTSA program hub websites. The Smart IRB comes in a distant second. The remaining six initiatives are severely underrepresented. DISCUSSION/SIGNIFICANCE OF IMPACT: Discussion: The identification of these gaps among the CTSA program hubs presents an understanding of content management and website functionality among the consortium from 3 principal approaches. First it creates an understanding of CTSA program hub content alignment with its funding source goals and initiatives. Such an understanding presents an opportunity to promote ways to create a better aligned consortium with improved collaboration pathways by the funding source through program hub website content standards. Second, it creates an opportunity for program hubs to understand and respond to the messaging their websites are presenting as it relates to the funding source. Third, it provides an opportunity to identify specific program initiatives and goals the CTSA institutions independently chose to highlight which can open a dialog to the better understanding the value of the program initiatives as they relate to the needs of CTSA program hubs. Ultimately, CTSA websites through content alignment should lead to an improved user experience.

OBJECTIVES/SPECIFIC AIMS: In the first aim, we will evaluate the proportion of highly HIV-susceptible memory CD4+ T cells present in the rectal mucosa, based on the proliferation status and expression of the HIV susceptibility markers, CCR5 and α4β7, between HIV-negative adolescent MSM and adult MSM engaging in RAI. The second aim will assess differences between the two study groups in the ratio of Th17 cells (CD4+ IL17+) to Treg cells (CD4+ FoxP3+ CD25+) in the rectal mucosa as a determinant of mucosal inflammation. Finally, in the third aim, we will utilize ex vivo rectal biopsy explant challenge experiments to examine whether HIV target cell availability and the Th17/Treg ratio influence rectal mucosal HIV susceptibility. METHODS/STUDY POPULATION: Rectal biopsy specimens are being collected from healthy, HIV-negative men that comprise the two study groups: 40 adolescent MSM 18-21 years of age who have engaged in RAI at least once previously in their lifetime and 40 adult MSM ≥35 years of age who have engaged in RAI for the previous 5 consecutive years with a minimum of 12 episodes annually. To identify CD4+ subsets of interest for aims 1 and 2, rectal mucosal mononuclear cells are isolated and phenotyped with CD45, CD3, CD4, CD45RA, CCR7, CD69, CCR5, α4β7, Ki67, FOXP3, and CD25 antibodies. To identify the Th17 cell subtype, the cells are stimulated with PMA/Ionamycin and stained with an antibody specific to IL-17A. Using cross-sectional analyses, we will compare the frequencies of mucosal CD4+ T cells that express certain phenotypic characteristics and evaluate differences in the Th17/Treg ratio between adolescent and adult MSM. For aim 3, rectal biopsy specimens are inoculated with HIV virus and the culture supernatant is assayed for p24 concentration on days 3, 7, 10 14, and 18. Longitudinal analyses will be performed to detect differences in p24 concentration at each time point and assess associations with mucosal target cell availability and with the Th17/Treg ratio. RESULTS/ANTICIPATED RESULTS: We hypothesize that younger age will be associated with enhanced memory CD4+ T cell proliferation and increased expression of HIV susceptibility markers (CCR5 and/or α4β7). In addition, we expect that the rectal mucosa of adolescent MSM will demonstrate a higher Th17/Treg ratio as compared to adult MSM, which could facilitate HIV transmission. It is also anticipated that rectal mucosal immune phenotypes characterized by increased HIV target cell availability and high Th17/Treg ratios will be associated with enhanced mucosal HIV susceptibility in the explant challenge model. DISCUSSION/SIGNIFICANCE OF IMPACT: There is a paucity of information regarding the mechanisms of rectal HIV transmission, and no studies to date investigate the immunologic effects of aging on transmission in the rectal mucosa. The results from this study will provide important information regarding age-related differences in the immune cell composition of the rectal mucosa as a critical step in better understanding immunologic factors that influence rectal HIV transmission.

Patient and public involvement (PPI) must be more frequently embedded within clinical research to ensure translational outcomes are patient-led and meet patient needs. Active partnerships with patients and public groups are an important opportunity to hear patient voices, understand patient needs, and inform future research avenues. A hereditary renal cancer (HRC) PPI group was developed with the efforts of patient participants (n = 9), pooled from recruits within the early detection for HRC pilot study, working in collaboration with researchers and healthcare professionals (n = 8). Patient participants had HRC conditions including Von Hippel–Lindau (n = 3) and Hereditary Leiomyomatosis and Renal Cell Carcinoma (n = 5), and public participants included two patient Trustees (n = 2) from VHL UK & Ireland Charity. Discussions among the enthusiastic participants guided the development of a novel patient information sheet for HRC patients. This communication tool was designed to aid patients when informing family members about their diagnoses and the wider implications for relatives, a gap identified by participants within group discussions. While this partnership was tailored for a specific HRC patient and public group, the process implemented can be employed for other hereditary cancer groups and could be transferable within other healthcare settings.

Retrospective case studies are one approach to help identify processes underlying the translation of successful health interventions. This case study investigates the development of RealRisks and Breast Cancer Risk Navigation (BNAV), decision support tools for breast cancer risk assessment, and risk-stratified prevention. Following a recently developed protocol for retrospective translational science case studies, we examined the career trajectory of Dr Katherine Crew as she expanded from basic science to interdisciplinary, patient-oriented research in oncology and began collaboration with Dr Rita Kukafka, a public health informatician focused on communicating risk. Data collection methods included key informant interviews and examination of peer-reviewed publications, funded grants, and news articles associated with the research. Data were analyzed to identify key milestones in the development of RealRisks and BNAV and to elucidate facilitators and barriers to the translational process. Facilitators to translation included funding and infrastructure provided by a Clinical and Translational Science Award (CTSA), the creation of an interdisciplinary team, and broad support from stakeholders including patient advocacy groups. Barriers to translation included limited mid-career support, ongoing costs for technology, and the time required to establish interdisciplinary, team science efforts. The findings reported here can be used to inform ongoing efforts to develop a more robust science of translation.

ABSTRACT IMPACT: Genomic data can be used by policy and decision makers to guide, and assess the impact of, public health responses to the COVID-19 pandemic. OBJECTIVES/GOALS: Our objective is to investigate the transmission and population dynamics of SARS-CoV-2 in New Mexico and other Mountain West states using whole genome sequencing. Understanding how the virus is spreading within and between communities is vital for the design of rational, evidence-based control measures. METHODS/STUDY POPULATION: We obtained an aliquot of 500ul - 1 ml of inactivated viral transport media (VTM) from positive SARS-CoV-2 nasopharyngeal swabs as determined by qPCR from the New Mexico Department of Health, TriCore Reference Laboratory, Idaho Bureau of Laboratories, and Wyoming Public Health Laboratory. We extracted viral RNA from the VTM, and sequenced the genomes using the methodology as described by the widely adopted ARTIC amplicon tiling protocol for SARS-CoV-2. Viral genomes were then sequenced on either an Illumina MiSeq or an Oxford Nanopore Technologies (ONT) GridION. We placed these samples within the context of globally representative sequences made available via the GISAID database. Consensus sequences were aligned and added into this global dataset using the Nextstrain augur pipeline. RESULTS/ANTICIPATED RESULTS: We sequenced over 1,000 SARS-CoV-2 genomes thus far from New Mexico (n=861), Wyoming (n=213) and Idaho (n=44). We used this sequence data to infer the transmission dynamics and spread of the virus, both within states and in context of regional and international spread. We inferred at least 128 separate introductions of the virus into New Mexico and at least 29 introductions into Wyoming. The origins of these introductions are diverse, spread across multiple regions in the US and abroad. We also sequenced samples from an individual who had multiple positive tests over time. Our results suggest that this individual was re-infected with a different strain than that of the initial infection. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data show that New Mexico and other Mountain West states have continually experienced many introductions of the virus that then seed local outbreaks. By understanding the number of introductions over time, we can assess the impact of travel restrictions on transmission. Our data also supports that some individuals can be re-infected.

OBJECTIVES/SPECIFIC AIMS: We previously demonstrated that products released by cultured B cells from patients with Multiple Sclerosis (MS) are cytotoxic to neurons and oligodendrocytes, while minimal toxicity was observed in response to B cell secretory products from age- and sex-matched normal controls. The goal of this proposal is to identify the range of brain cells susceptible to MS B cell-mediated cytotoxicity, to define the cytotoxic factor(s) released by MS B cells, and to determine whether particular subset(s) of MS B cells harbor the greatest pathogenic potential. METHODS/STUDY POPULATION: The toxicity of B cell products will be demonstrated by incubating primary rat cultures of neurons, oligodendrocytes, and oligodendrocyte progenitor cells (OPCs) with B cell supernatants. B cells will be isolated from the peripheral circulation of untreated relapse-remitting MS (RRMS) patients and age- and sex-matched normal controls. The identification of specific toxic factor(s) in MS B cell supernatants will be achieved through a combination of exosome-depletion/enrichment of conditioned media, proteomics, next generation sequencing, and lipidomics. Determining pathogenic B cell subsets will be achieved by cell sorting into memory and naïve B cell subsets prior to collection of supernatants. RESULTS/ANTICIPATED RESULTS: We hypothesize that the toxicity of MS B cell products is mediated, at least in part, by extracellular vesicles, such as exosomes. We expect depletion of these exosomes from the B cell conditioned media or inhibition of their biogenesis will mitigate the observed toxicity. Furthermore, differences in B cell-derived exosomal content, such as proteins, (mi)RNAs, or lipids, likely explain the differences in observed toxicity. Lastly, we hypothesize that memory B cells, which are enriched in the CNS of MS patients and demonstrate a more pro-inflammatory profile than naïve B cells, are responsible for the toxicity observed in supernatants of total B cells. DISCUSSION/SIGNIFICANCE OF IMPACT: MS is the most prevalent chronic inflammatory disease of the CNS, affecting more than 2 million people worldwide. Although over a dozen disease-modifying therapies are approved for the treatment of RRMS, none are meaningfully effective at limiting disease progression. This proposal will provide new insight into immune-CNS interactions in progressive MS and provide much-needed novel targets for therapeutic intervention, either via blocking identified toxic molecule(s) or by selectively depleting pathogenic B cell subsets.