OBJECTIVES/GOALS: An ex-vivo tissue model has been developed to predict target concentrations of tenofovir diphosphate (TFVdp; active metabolite of tenofovir) but has not been utilized to see how vaginal dysbiosis affects TFVdp/dATP exposure in female genital tract (FGT). My central hypothesis is that presence of specific anaerobic bacteria will increase dATP in FGT. METHODS/STUDY POPULATION: De-identified HIV-negative cervical tissues from women undergoing gynecological surgeries will be procured and a punch biopsy will be used to create explants. TFVdp/dATP concentrations were both tested in both aerobic and anaerobic conditions after a 24-hour incubation in tenofovir (TFV) to determine any changes between conditions. TFVdp/dATP in cervical tissue was be measured using LC-MS. Next, media and explants were collected at baseline to characterize donor microbiome for 6 donors. 16S microbiome sequencing was performed on extracted DNA to obtain the relative abundances of each bacteria species present. To test changes in dATP/TFVdp due to the microbiome, explants will be incubated in TFV for 24 hours with Prevotella and Dialister to specifically see how microbiomes dominated by these taxa affect dATP. RESULTS/ANTICIPATED RESULTS: There was no significant difference in TFVdp formation between aerobic and anaerobic conditions after a 24-hour tenofovir incubation (p = 0.2) for 8 donors. dATP was not quantifiable at 24 hours in explants, so explants are being collected before 24hrs during a TFV incubation to determine how quickly dATP depletes after collection. We were able to characterize the donor microbiome in media and tissue at baseline and 24hrs which had inter variability. We did not see any presence of Prevotella or Dialister in any donors. We are working on characterizing bacteria growth over time to see how the donor microbiome would change during a 24 hour experiment in anaerobic conditions. Finally, we anticipate seeing increases in dATP with a Prevotella or Dialister supplemented donor microbiome compared to baseline donor microbiome. DISCUSSION/SIGNIFICANCE: The addition of vaginal dysbiosis to tissue model will increase accuracy of prediction of 100% protective TFVdp concentrations and is likely to provide a translational model that can be used to improve TFV-based PrEP in women and streamline development of future PrEP candidates, bringing more prevention options to women and ending the HIV epidemic.

The National COVID Cohort Collaborative (N3C) is a public–private–government partnership established during the Coronavirus pandemic to create a centralized data resource called the “N3C data enclave.” This resource contains individual-level health data from participating healthcare sites nationwide to support rapid collaborative analytics. N3C has enabled analytics within a cloud-based enclave of data from electronic health records from over 17 million people (with and without COVID-19) in the USA. To achieve this goal of a shared data resource, N3C implemented a shared governance strategy involving stakeholders in decision-making. The approach leveraged best practices in data stewardship and team science to rapidly enable COVID-19-related research at scale while respecting the privacy of data subjects and participating institutions. N3C balanced equitable access to data, team-based scientific productivity, and individual professional recognition – a key incentive for academic researchers. This governance approach makes N3C research sustainable and effective beyond the initial days of the pandemic. N3C demonstrated that shared governance can overcome traditional barriers to data sharing without compromising data security and trust. The governance innovations described herein are a helpful framework for other privacy-preserving data infrastructure programs and provide a working model for effective team science beyond COVID-19.

Community health needs assessments (CHNAs) are important tools to determine community health needs, however, populations that face inequities may not be represented in existing data. The use of mixed methods becomes essential to ensure the needs of underrepresented populations are included in the assessment. We created an in-school public health course where students acted as citizen scientists to determine health needs in New Brunswick, New Jersey adults. By engaging members of their own community, students reached more representative respondents and health needs of the local community than a CHNA completed by the academic hospital located in the same community as the school which relies on many key health statistics provided at a county level. New Brunswick adults reported significantly more discrimination, fewer healthy behaviors, more food insecurity, and more barriers to accessing healthcare than county-level participants. New Brunswick participants had significantly lower rates of health conditions but also had significantly lower rates of health screenings and higher rates of barriers to care. Hospitals should consider partnering with local schools to engage students to reach populations that face inequities, such as individuals who do not speak English, to obtain more representative CHNA data.

OBJECTIVES/GOALS: In 2018, the United Network for Organ Sharing began prioritizing patients on temporary MCS over those on durable MCS for OHT in an effort to prioritize sicker patients and decrease waitlist mortality. We explored the impact of this change by examining if the type of MCS prior to transplant affects the risk of post-transplant infection. METHODS/STUDY POPULATION: We will conduct a retrospective cohort study of approximately 350 patients that have undergone OHT at Tufts Medical Center between January 2014 and July 2021 who survived at least 72 hours post-transplant and have minimum post-transplant follow-up of one year or time to death if before one year. Chart review will determine the type of MCS in place prior to transplant and the occurrence of infections within one year of transplant. Data will also be collected on patient’s age, sex, medical comorbidities, lab values, and open chest management practices. We will examine differences in the incidence rates of a composite outcome (blood stream infection, invasive fungal infection, skin and soft tissue infection of device sites, and mediastinitis) between patients that were on temporary versus durable MCS. RESULTS/ANTICIPATED RESULTS: We anticipate that this study will show a greater frequency of infections of all types in patients that received temporary as compared with durable mechanical circulatory support prior to transplantation. We will use Cox proportional hazards survival models to model multivariable relationships for predictors of infection. DISCUSSION/SIGNIFICANCE: This study will provide insights into the magnitude and type of infectious complications that patients experience after OHT and the impact that type of MCS and other factors have on their outcomes. The data obtained may have implications for choice of mechanical device prior to undergoing OHT surgery as well as antimicrobial prophylaxis.

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

OBJECTIVES/GOALS: Our objective is to investigate the antibacterial and regenerative properties of a novel AHA dental coating for the prevention and treatment of deep caries (cavities). Further, we aim to investigate and compare these properties through in vivo murine models and assessment on human saliva samples and human pulpal cells collected from clinical samples. METHODS/STUDY POPULATION: In vitro antibacterial studies were performed by collecting and culturing human salivary bacteria with AHA substrates and quantifying survival of cariogenic Sm (S. mutans). In vivo, C57BL/6 mice were treated with AHA composite fillings, infected with Sm clinical isolates, and fed a high sucrose diet with cavity formation assessment after 6 weeks. To evaluate regeneration, mice were similarly given composite with AHA or MTA (standard of care) upon pulpal exposure with regeneration quantified by microCT and histological analysis of dentin bridge formation, ALP production, and odontoblast migration. In vitro, AHA substrates were cultured with MC3T3-E1 pre-osteoblast cells and dental pulp stem cells obtained from clinical samples over 21 days, with mineralization and ALP assessed indicating osteogenesis. RESULTS/ANTICIPATED RESULTS: In vivo studies have shown the reduction of cavity formation in mice treated with AHA as well as dentin regeneration upon pulpal exposure using microCT and histological image analysis. Coinciding with these findings, AHA substrates eradicated cariogenic Sm in human saliva samples and single species cultures in vitro. Further, preliminary results in vitro have shown increased mineralization of MC3T3-E1 cells when cultured with AHA substrates in comparison to uncoated substrates. We anticipate similar increased mineralization as well as increased ALP production of human pulpal stem cells from clinical samples when cultured with AHA substrates, suggesting osteogenesis. Further, we anticipate increased odontoblast migration and ALP production upon additional analysis of in vivo tissue samples. DISCUSSION/SIGNIFICANCE: This work will elucidate the antibacterial and regenerative properties of AHA dental coatings. These results further support the translation of AHA coatings into the clinic as a novel therapeutic for the prevention and treatment of dental decay, which may overcome the limitations associated with the current treatments.

OBJECTIVES/GOALS: Minor procedures are anxiety-provoking and/or painful for children. Virtual reality (VR) is an emerging technology in medicine and largely has been used as an adjunct to analgesia and opioids. This study reviews the institutional use of VR in lieu of pharmacology and general anesthesia (GA) to perform minor procedures in a pediatric population. METHODS/STUDY POPULATION: A retrospective chart review was performed on all patients that presented to our institution from 2019 to 2022 for hormone implant placement, exchange, or removal with VR distraction. Demographic and procedure information was recorded. The primary outcome was successful procedure completion without requiring pharmacologic sedation or analgesia. RESULTS/ANTICIPATED RESULTS: A total of 111 patients underwent the following minor procedures with VR only. 14 patients underwent more than one procedure resulting in a total of 126 procedures. The mean age was 11.3 ± 3.6 years. 43 patients were female, 23 were female to male, 6 were non-binary, 7 were male, and 32 were male to female. 58% had private insurance. The most common diagnosis was precocious puberty (54%) followed by gender dysphoria (46%). The most common procedure was implant placement (72%). 69% of procedures were performed in the clinic and 31% in a procedural room. All procedures were completed without requiring sedation or GA. None of the patients required intravenous catheter placement for the procedure. No intra-procedural complications were recorded. DISCUSSION/SIGNIFICANCE: Despite the current trend toward minimizing GA and sedation in children, there is no widely accepted substitute. VR is a feasible option that can spare children from sedation or GA for minor procedures. This can enable procedures to be transitioned into more resource efficient settings and improve pediatric patient experience.

OBJECTIVES/GOALS: Clonal hematopoiesis of indeterminate potential (CHIP) is a common age-related condition that confers an increased risk of blood cancer, cardiovascular disease, and overall mortality. Larger proportions of blood cells with the CHIP mutation (clones) lead to worse outcomes. The goal of this study was to characterize CHIP clonal behavior over time. METHODS/STUDY POPULATION: While DNA biobanks have the ability to identify large cohorts of individuals with CHIP, they typically only contain blood from a single timepoint, limiting the ability to infer how CHIP clones change over time. In this preliminary study, we utilized multi-timepoint blood samples from 101 individuals with CHIP in Vanderbilt’s biobank (BioVU) to characterize clonal behavior over time. Using a CHIP gene-specific sequencing pipeline, we were able to characterize each individual’s CHIP mutation(s) and how the fraction of cells with the CHIP mutation expanded/reduced over time. By Spring 2023, we will also include ~300 additional individuals with CHIP in this study. RESULTS/ANTICIPATED RESULTS: CHIP mutations occurred 48% of the time in DNMT3A and 23% of the time in TET2, consistent with previous studies. 21% of individuals had more than one CHIP mutation. The mean difference in time between the two timepoints was 5.2 years (SD=2.9). Surprisingly, we observed both clonal expansion and clonal reduction across timepoints with 30% of DNMT3A, 0.6% of TET2, and 46% of JAK2 clones shrinking over time. The fastest average expansion was seen in TET2 clones (2% growth/year) and the slowest in DNMT3A clones (0.4% growth/year), but there was a significant amount of variation between individuals. In DNMT3A clones, there were no differences observed between loss of function mutations, missense mutations or DNMT3A R882 hotspot mutations. Clonal competition was observed in individuals with multiple driver mutations. DISCUSSION/SIGNIFICANCE: We used multi-timepoint blood samples to quantify the change in CHIP cell fraction over time on a per individual basis and observed novel clonal behavior and competition. Understanding the factors that influence the rate of CHIP progression can lead to personalized disease risk assessment for individuals with CHIP.

OBJECTIVES/GOALS: Strip craniectomy with orthotic helmet therapy (SCOT) and open cranial vault reconstruction (OCVR) are mainstay surgical treatments for sagittal craniosynostosis. The purpose of this study is to compare long-term morphologic outcomes of the skull, subjective appearance, and perioperative outcomes between patients who underwent SCOT and OCVR. METHODS/STUDY POPULATION: Patients who underwent SCOT or OCVR for isolated, non-syndromic sagittal craniosynostosis with preoperative computerized tomography (CT) imaging were identified at UCSF between 2000 and 2020. Perioperative outcomes were extracted from the medical chart. Anthropometric measurements will be used to assess baseline severity using preoperative CT imaging and postoperative skull morphology using 3D camera imaging. Patient satisfaction surveys will be administered to parents at the time of postoperative 3D imaging. Subjective appearance will be rated among adolescents and craniofacial surgeons using patients’ postoperative 3D imaging. Descriptive statistics will be calculated using Student’s t test and Mann-Whitney U. Chi-square tests and Fisher exact tests will be used to compare categorical outcomes. RESULTS/ANTICIPATED RESULTS: 47 patients were included in the study (18 SCOT and 29 OCVR) with similar follow-up between groups (SCOT 2.3 +/- 1.5, OCVR 2.6 +/- 2.0). There were no significant differences in sex or race. OCVR had longer operative times (p-value 0.0003), higher estimated blood loss (p DISCUSSION/SIGNIFICANCE: SCOT was associated with superior perioperative outcomes compared with OCVR due to its minimally invasive technique. Our results for long-term healing outcomes and subjective appearance may further guide craniofacial surgeons in selecting the most optimal operative technique.

OBJECTIVES/GOALS: Target: Computationally identify the markers of ulcer severity and risk of amputation from datasets that include demographics data, clinical, laboratory data, and medical history over 6000 patients. METHODS/STUDY POPULATION: In this study we will use tables of demographics such as age, gender, and ethnicity/race. Inspired by previous research we’ll include wound age (duration in days), wound size, number of concurrent wounds of any etiology, evidence of bioburden/infection, Wagner grade, being non ambulatory, renal dialysis, renal transplant, peripheral vascular disease, and patient hospitalization. Another table will include laboratory vital signs to include physiological variables such as height, weight, body mass index, pulse rate, blood pressure, respiratory rate, and temperature. We’ll include also social data like smoking status, socio-economic status, housing condition. RESULTS/ANTICIPATED RESULTS: Our project aligns with previous efforts to identify high risk Diabetic Foot Ulcer individuals but also takes a different perspective by collecting and marking clinical data from a subset of patients (e.g., severity, Hispanic versus non-Hispanic) and computationally process these data to provide a tool that can identify DFU severity and high-risk patients. We will obtain samples from Hispanics and non-Hispanics because these two groups are likely to have significant differences in the progression of ulcer severity. The rationale is that by comparing these two groups, we will assess and study the factors that are differentially present. It is our expectation that the proposed project will provide an easy-to-use tool for DFU progression and risk of amputation and contribute to identify high-risk individuals. DISCUSSION/SIGNIFICANCE: Diabetes prevalence estimates in Bexar County, TX exceeds national estimates (15.5% vs. 11.3%) and diagnosed cases are higher among Hispanic adults (13.4%) compared to their non-Hispanic white counterparts (9.5%). Late identification of severe foot ulcers minimizes the likelihood of reducing amputation risk.

OBJECTIVES/GOALS: Cardiovascular disease (CVD) is the most common cause of death in systemic lupus erythematosus (SLE). Genome-wide association studies have identified single nucleotide polymorphisms linked with CVD risk, but the association with SLE is not well established. We aimed to determine associations using machine learning in a multi-ethnic lupus cohort. METHODS/STUDY POPULATION: We will use data from the established SLE cohort study named Genetic Profile Predicting the Phenotype (PROFILE). PROFILE was constituted in 1998 by combining existing cohorts at multiple sites which are also of defined ethnicity (Hispanics of Mexican ancestry and Puerto Rico, African American, and Caucasian). The cohort consists of 3,118 individuals and the database contains socioeconomic–demographic, clinical, laboratory, and genetic variables. Genetic data consist of 196,524 single nucleotide polymorphisms. To detect risk genes and predict an individual’s SLE risk will design a random forest classifier using SNP genotype data. Logistic regression models will be performed with CVD as the outcome, adjusted for age, sex, ethnicity, disease duration, and traditional and nontraditional risk factors for CVD. RESULTS/ANTICIPATED RESULTS: We expect to find several established and new susceptibility genes associated with CVD. DISCUSSION/SIGNIFICANCE: This approach offers an opportunity to characterize distinct genetic risk factors and the relationship of CVD with SLE. These data may be important in the identification of patients at high risk for such events and may allow the design of preventive strategies which may beneficially have an impact on the morbidity and mortality of SLE patients.

Despite their documented benefits, the widespread adoption of community-engaged and participatory approaches among health researchers remains limited. Institutional practices and policies influence the uptake of community engagement and participatory approaches. We examine the role of financial arrangements between university researchers and community partners, by exploring efforts to bridge the gap between research administration and researchers at two research-intensive institutions. The type of financial arrangement a researcher has with a community partner plays an important role in setting the stage for the structure of the partnership as it relates to shared decision-making and ownership of the research. Continued efforts to clarify and streamline subcontracting processes are needed as is infrastructure to support community partners and researchers as they navigate financial arrangements if progress is to be made.

OBJECTIVES/GOALS: The goal of this proposal is to develop a technology that combines calcium imaging via confocal microscopy, and force measurement via monolayer stress microscopy to perform simultaneous quantitative measurements of agonist-induced Ca2+ and mechanical signals in HASMCs. METHODS/STUDY POPULATION: The methods by which second messenger signals and changes in mechanical forces determine specific physiological responses are complex. Recent studies point to the importance of temporal and spatial encoding in determining signal specificity. Hence, approaches that probe both chemical and mechanical signals are needed. We combine hyperspectral imaging for second messenger signal measurements, monolayer stress microscopy for mechanical force measurements, and S8 analysis software for quantifying localized signals. Imaging was performed using an excitation-scanning hyperspectral microscope. Hyperspectral images were unmixed to identify signals from fluorescent labels and microparticles. Images were analyzed to quantify localized force dynamics through monolayer stress microscopy. RESULTS/ANTICIPATED RESULTS: Results indicate that localized and transient cellular signals can be quantified and mapped within cell populations. Importantly, these results establish a method for simultaneous interrogation of cellular signals and mechanical forces that may play synergistic roles in regulating downstream cellular physiology in confluent monolayers. DISCUSSION/SIGNIFICANCE: We will measure the distribution of chemical and mechanical signals within cells, providing insight into the dynamics of cell signaling. Studies will have implication in the understanding of infections, drug delivery in which non-uniform distributions of drugs are a certainty, and in understanding coordinated responses in cellular systems.

OBJECTIVES/GOALS: Aim 1: Define the genetic events that cooperate with Trp53 mutations to mediate the transformation of MDS to sAML at the stem cell level. Aim 2: Use Bayesian networks to model the signaling pathway activation states identify aberrant regulators of self-renewal in LSCs. METHODS/STUDY POPULATION: To model the transformation of MDS, I will utilize a mouse model of MDS and sAML. The Trp53 mutated mouse mimics the genetics of human MDS and develops MDS. To discover how additional mutations contribute to disease progression, I use the Sleeping Beauty transposon system which induces random mutations. I propose to use this mouse model to define the molecular mechanisms of transformation of MDS to sAML. I will also measure levels of activated signaling intermediates in sAML using CyTOF. CyTOF is a method of flow cytometry that measures up to 40 proteins simultaneously at single-cell resolution. I will also use Bayesian network to model the signaling architecture of the MDS/sAML stem cells. RESULTS/ANTICIPATED RESULTS: I will identify putative mediators of self-renewal in gene expression data and the corresponding Bayesian networks model. I will prioritize genes and signaling intermediates that act in pathways that rank highly in both methods and those that have previously published roles in self-renewal. Based on prior work, I will focus on EZH2, NFï «B, and mTOR pathways in addition to candidate pathways revealed by these studies. Small molecule inhibitors that target candidate signaling intermediates will be used for experimental validation. I will test the impact of these small molecule inhibitors on LSC self-renewal using serial colony forming assays as well as in vivo mouse reconstitution assays. DISCUSSION/SIGNIFICANCE: We focus on the role of stem cells in the transformation of MDS to sAML. Understanding the mutations and signaling relationships that mediate self-renewal in Trp53 mutant stem cells will allow for precise therapeutic target of this disease and improve outcomes for these patients.