OBJECTIVES/SPECIFIC AIMS: Wake Forest Baptist Health (WFBH) is an Academic Learning Healthcare System (aLHS) serving 24 counties in North Carolina and Virginia. Like many aLHSs, WFBH experiences strained community relationships attributable to a history of medical and research abuses against marginalized populations. This legacy accompanies longstanding community mistrust in the healthcare system and research. To overcome these challenges, community-engaged research (CEnR) approaches have potential to repair community-academic relationships, improve public health, and empower groups that traditionally have been neglected by or overlooked in research. To develop and revise our understanding of how CEnR is harnessed at WFBH, semi-structured interviews were conducted with investigators and study staff experienced in CEnR approaches. In-depth interview guides were designed iteratively to capture socio-contextual and detailed descriptions of perceptions, experiences, and strategies specific to the use of CEnR. METHODS/STUDY POPULATION: A keyword search performed within WFBH study records identified 51 investigators whom had submitted research proposals related to CEnR within the past ten years. Sixteen were confirmed eligible based on a review of proposal abstracts, of which 14 responded to email invitations agreeing to participate. Four additional participants were referred by initial participants. Eighteen investigators (16 faculty and 2 research associates) provided consent and completed Interviews. RESULTS/ANTICIPATED RESULTS: The participant sample was 50% female with a mean age of 55 years, 11% Black and 89% White, with representation across various academic backgrounds (e.g., anthropology, medicine, psychology, and public health) A majority of participants (89%) hold doctoral degrees (i.e., PhD, DrPH, EdD, MD, and MD-PhD). On average, participants had been employed at WFBH for 13.9 years, and represented various departments including dermatology, epidemiology and prevention, family medicine, neurology, social sciences and health policy, and psychiatry. Nearly all participants (89%) indicated they had never received formal education or training in CEnR, though 100% reported “on-the-job” training in CEnR. Interviews were audio-recorded, transcribed, coded, and analyzed following an inductive thematic approach, from which twenty-two themes emerged across six domains related to CEnR (Table 2), including: Conceptualization and Purpose, Value and Investment, Community-Academic Partnerships, Sustainability, Facilitators, and Challenges. Results also provided key characteristics that define CEnR (Table 3), and yielded 11 emerging needs necessary to enhance CEnR within aLHSs (Table 4). DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study provide information critical to understanding how CEnR frameworks and approaches can be harnessed not just in Schools of Public Health, but within aLHSs to build and repair community-academic partnerships, inform research and institutional priorities, and address community health concerns. Despite the small sample size, the number of participant interviews was sufficient to achieve saturation while also providing broad and unique perspectives across various fields and CEnR approaches. Overall, participants conceptualized the purpose and goals of CEnR quite similarly, though there was a great deal of variance in how CEnR was defined and operationalized across interviews, indicating a need to more clearly articulate important features that enhance understanding of what CEnR is and what it is not (Table 3). These discrepancies and inconsistencies indicate a potential need for additional formal training in the understanding and use of CEnR approaches, which is supported by the fact that nearly all participants reported receiving no formal training in CEnR. Across all interviews, participants expressed a need for health care providers and researchers to better understand community contexts, social determinants of health, and historical factors influencing community health and participation in research (Table 4). This work and the data presented here are important for informing CEnR approaches and will be useful for guiding the development of a model incorporating the core tenets of CEnR within the mission, vision, and priorities of aLHSs.

OBJECTIVES/SPECIFIC AIMS: Black patients with hepatocellular carcinoma (HCC) receive fewer curative therapies and have higher mortality than other groups. Reducing this disparity will require an in-depth understanding of patient comorbidities, tumor characteristics, and social determinants of health. Our objectives are to a) perform a multi-center retrospective cohort study of black and white patients diagnosed with HCC in the Indianapolis area. b) prospectively enroll black and white patients with HCC to collect clinical characteristics as well as data on the social determinants of health. METHODS/STUDY POPULATION: A retrospective chart review of patients with a diagnosis of HCC from 2010-2017 from five area Indianapolis hospitals will be performed. Demographics, comorbidities, liver disease severity, and tumor characteristics will be collected using the Indiana Network for Patient Care database and compared between black and white patients. Concomitantly, a prospective cohort of black and white patients will be enrolled and surveyed to collect data on socioeconomic status and income adequacy, literacy, functional status, substance abuse history, social support, activation, and adherence. The primary outcomes are the receipt of curative therapies for HCC including liver transplantation, resection or ablation. The secondary outcome is mortality. Multivariable logistic regression models will be used to explore disparities seen in the primary and secondary outcomes. RESULTS/ANTICIPATED RESULTS: These preliminary results include Indiana University Hospital (IUH) findings; a multicenter analysis is underway. The IUH cohort included 1,032 (86%) white and 164 (14%) black patients. Black and white patients had similar Model for End-Stage Liver Disease and Child-Pugh scores. There was a trend toward larger tumor size (5.3 cm vs. 4.7 cm; P = 0.05) in black patients; however, Barcelona Clinic Liver Cancer staging and Milan criteria were similar. Black patients were less likely to undergo liver transplantation than white patients—a disparity that was not attenuated (odds ratio [OR], 0.43; 95% confidence interval [CI]: 0.21-0.90) on multivariable analysis. Substance abuse was more frequently cited as the reason black patients within Milan criteria failed to undergo transplantation than white patients. Survival was similar between the two groups. DISCUSSION/SIGNIFICANCE OF IMPACT: Racial differences in patient and tumor characteristics were small in our single center analysis and did not explain the disparity in liver transplantation. This analysis however only reflects 25% of patients diagnosed with HCC in the Indianapolis metropolitan, highlighting the need for a multicenter study. Higher rates of substance abuse in black patients within Milan criteria who failed to undergo transplantation suggest social factors contribute to this disparity and highlight the need for a prospective study that can explore these and other social factors.

OBJECTIVES/SPECIFIC AIMS: Engaging patients and consumers in research is a complex process where innovative strategies are needed to effectively translate scientific discoveries into improvements in the public’s health (Wilkins et. al., 2013; Terry et. al., 2013). The Clinical Translational Science Awards (CTSA)—supported by the National Institute of Health (NIH) under the auspices of the National Center for Advancing Translational Sciences (NCATS)—aim to provide resources and support needed to strengthen our nation’s clinical and translational research (CTR) enterprise. In 2008, Stanford University was awarded a CTSA from the NIH, establishing Spectrum (Stanford Center for Clinical and Translational Research and Education) and its Community Engagement (CE) Program aimed at building long-standing community-academic research partnerships for translational research in the local area surrounding Stanford University. To date, the CE Pilot Program has funded 38 pilot projects from the 2009-2017 calendar year. The purpose of this study was to understand, through a unique pilot program, the barriers, challenges, and facilitators to community-engaged research targeting health disparities as well as community-academic partnerships. METHODS/STUDY POPULATION: Investigators conducted a qualitative study of the community engagement pilot program. Previous pilot awardees were recruited via email and phone to participate in a one-hour focus group to discuss their pilot project experience—describing any barriers, challenges, and facilitators to implementing their pilot project. RESULTS/ANTICIPATED RESULTS: The focus group revealed that community engage research through the pilot program was not only appreciated by faculty, but projects were successful, and partnerships developed were sustained after funding. Specifically, the pilot program has seen success in both traditional and capacity building metrics: the initial investment of $652,250.00 to fund 38 projects has led to over $11 million dollars in additional grant funding. In addition, pilot funding has led to peer-reviewed publications, data resources for theses and dissertations, local and national presentations/news articles, programmatic innovation, and community-level impact. Challenges and barriers were mainly related to timing, grant constraints, and university administrative processes. DISCUSSION/SIGNIFICANCE OF IMPACT: The Community Engagement Pilot Program demonstrates an innovative collaborative approach to support community-academic partnerships. This assessment highlights the value and importance of pilot program to increase community engaged research targeting health disparities. Challenges are mainly administrative in nature: pilot awardees mentioned difficulties working on university quarterly timelines, challenges of subcontracting or sharing money with community partners, onerous NIH prior approval process, and limitations to carryover funding. However, pilot grants administered through the program strengthen the capacity to develop larger scale community-based research initiatives.

OBJECTIVES/SPECIFIC AIMS: Qualitative approaches help explore poorly understood phenomenon, and are highly engaging, enabling both sides of an encounter greater connection. Historically, Deaf communities have been marginalized and oppressed, with their linguistic needs unrecognized and ignored. As a result, Deaf participants are rarely involved in clinical research. Like other marginalized communities, the Deaf community experiences health disparity compared with others, especially in low- and middle-income settings. The purpose of this project was to assess the feasibility of conducting qualitative research with Deaf Dominicans. METHODS/STUDY POPULATION: We implemented a partnered research process with 59 Deaf community members in the Dominican Republic, conducting preliminary thematic analysis through reviews of interviews and on-site debriefings. RESULTS/ANTICIPATED RESULTS: Participants were highly engaged with the Deaf-Deaf research encounters, indicating satisfaction with both the process and with the opportunity to communicate their needs and interests. Preliminary findings indicated Deaf Dominicans were highly engaged, confirming their interest, and often stated that they felt they were being listened for the first time. Indeed, some participants claimed that this was the first time they communicated their experiences as Deaf Dominicans and appreciated the opportunity to relate this experience to Deaf interviewers. DISCUSSION/SIGNIFICANCE OF IMPACT: This experience confirms that the Deaf Dominican community can be mobilized and will participate in Deaf-Deaf research.

OBJECTIVES/SPECIFIC AIMS: To develop a social network model of collaborations within and external to the University of Rochester Medical Center (URMC) CTSI using data from the annual Research Performance Progress Report (RPPR) as well as other sources, to provide longitudinal evaluation of the CTSI’s engagement with key stakeholder groups. METHODS/STUDY POPULATION: The annually submitted RPPR follows a specific format with well-defined sections. The Highlights, Milestones and Challenges Report includes areas in which CTSI function leaders provide details about program integration and innovation, including collaborations with other functions or external groups. The Highlights, Milestones and Challenges Report was qualitatively coded to identify function-collaborator dyads. Each entity in the dyad became a node in the network. Nodes were connected by edges named by the dyads. The network included two types of nodes. The first were CTSI internal functions/programs, i.e. the entities that submitted RPPR sections and formed an interconnected sub-network. The second type of nodes were entities external to the CTSI (collaborators, internal or external to the CTSI site). These entities were named by functions submitting RPPR narratives. External nodes with similar meanings were consolidated. Duplicate edges were removed. CTSI-external nodes were grouped into five stakeholder categories: URMC, University of Rochester (UR), community, other CTSA institutions, CTSA consortium. Thus, these nodes were connected to the CTSI internal nodes, but not to each other. A second source of collaboration data was function-reported internal metrics. As part of the internal metric data collection, functions list partners who play a role in improving metric data or who are responsible for providing data. Partners identified in the internal metrics data, but not specified in the RPPR, were added to the network. RESULTS/ANTICIPATED RESULTS: Twenty-three internal CTSI functions submitted an RPPR and represent the CTSI internal nodes. Internal CTSI functions identified 235 collaborations (edges): 125 collaborations with other CTSI internal functions, 57 collaborations with URMC entities, 14 with UR entities, 15 with the external community, 15 with other institutions (CTSA hubs and other universities), and 9 with CTSA consortium entities. Thirty-eight of the collaborations were identified in the internal metrics partners section. In total, the network comprised 104 nodes. Graph density was.022 for full network and.21 for the CTSI internal sub-network. The global clustering coefficient, a measure of connectivity, for the CTSI internal sub-network was.252. DISCUSSION/SIGNIFICANCE OF IMPACT: The RPPR provides an underutilized source of data for annually repeated analyses of internal and external CTSI collaborations and is a way to enhance use of this routinely collected information. Analyses of the network yield metrics for measuring CTSI reach and impact on stakeholder groups over time. For example, measures such as number of nodes representing entities external to CTSI and average vertex degree of the CTSI Internal nodes track aspects of CTSI collaborations. Visualizations using different layouts or highlighting different sub-networks provide a representation of CTSI engagement with the communities of stakeholders as well as insights to relationships between functions, regions of collaboration, and areas of gaps. These data also provide an important new mechanism to engage the CTSI leadership and function leads in understanding how their work contributes to the overall network and synergies they have with each other.

OBJECTIVES/SPECIFIC AIMS: Virtual communities are an untested method to enhance community engagement in biomedical research. Our CTSA Hubs collaborated to assess receptivity to engage in a statewide Facebook (FB) group. METHODS/STUDY POPULATION: Cross-sectional online survey administered via iPads at the MN State Fair in 2018 to adults aged 18+ years residing in MN assessed demographics, social media use, interest in participating in a FB group for biomedical research; and open-ended questions on health topics of interest, and what would keep people engaged in this group. RESULTS/ANTICIPATED RESULTS: Respondents (N=487) were 21% racial minorities and 65% female sex. Most (87%, n=422) had created a personal FB profile. Of these, the proportion who agreed/strongly agreed was: 57% that the FB group sounded interesting, 45% were interested in being part of it, 41% were willing to share it with others, 62% that it would allow the community’s thoughts/ideas to be heard and 59% wanted to learn about opportunities to participate in research on health topics they care about. Using content analysis, the top 3 health topics people wanted to learn about were chronic disease and prevention, wellness, and mental health. Top ways to keep people engaged were providing personable, relevant health information; and interactive bi-directional discussions. DISCUSSION/SIGNIFICANCE OF IMPACT: Findings will inform development of a FB group to engage diverse populations in biomedical research.

OBJECTIVES/SPECIFIC AIMS: The overall objective of this study is to develop a novel, clinically-relevant, image-guided mouse model for radiation-induced cardiotoxicity, which can be used to gain insight into clinically-targetable, pathophysiologic mechanisms of cardiac injury in thoracic radiotherapy patients. METHODS/STUDY POPULATION: Photon or sham radiation will be administered at differential doses to a defined portion of the heart and/or lungs of C57BL/6 female mice using micro-CT visualization of the heart with Xstrahl’s MuriSlice Software applied to the Small Animal Radiation Research Platform (SARRP). Cardiac and lung segments from a subset of mice will be harvested at specific time points for confirmation of radiation targeting, local apoptosis assessment, and evaluation of fibrosis and vascular tissue morphology. Quantitative echocardiography, myocardial 18F-fluorodeoxyglucose positron emission tomography computed tomography (18F-FDG PET/CT), and myocardial perfusion imaging (MPI) with Technicium-99 (Tc-99) sestamibi will be implemented to identify sensitive imaging measures of cardiac injury and asses myocardial mechanics, inflammation, and perfusion deficits, respectively. Concurrently, a multiparametric analysis will be conducted to identify novel, circulating biomarkers of cardiotoxicity. RESULTS/ANTICIPATED RESULTS: We hypothesize that a clinically-relevant mouse model can be generated by the in situ, focal irradiation of a portion of heart and/or lung tissue segments, and can be used to elucidate molecular mechanisms of radiation-induced cardiac damage. We anticipate time-dependent and dose-dependent, focal histopathologic changes in the mouse heart, with cardiac fibrosis development, vascular damage, and cellular apoptosis in irradiated mice. Additionally, we anticipate that our mouse model of focal heart irradiation will reveal radiologic and biochemical changes that can be used to characterize and predict radiation-induced cardiac injury. Specifically, we expect our quantitative echocardiography, FDG-PET, and MPI parameters to identify and characterize cardiac damage that topographically matches histopathological analysis, and expect levels of select biochemical markers to differentially vary with time. DISCUSSION/SIGNIFICANCE OF IMPACT: Our mouse model of radiation-induced cardiotoxicity has the potential to shift current preclinical research paradigms to more closely mimic the radiation plans most commonly administered in clinical practice. The primary technologic innovation to be developed here is the use of the SARRP to deliver image-guided, in situ, focal radiation to a defined portion of the mouse heart. From a conceptual perspective, we propose a novel approach for phenotyping radiation-induced cardiac damage in patients undergoing chest radiation therapy, integrating sensitive radiomic and biochemical markers into a predictive model of cardiotoxicity.

OBJECTIVES/SPECIFIC AIMS: Aim 1: To compare frozen MOM to fresh MOM over time as an agent to inoculate DHM and measure the enrichment of commensal microbes and their beneficial bioactive components similar to MOM. Hypothesis: Frozen or fresh MOM inoculated in DHM will produce similar microbial content to MOM over time allowing for the production of beneficial bacterial compounds that may contribute to host immune response. Aim 2: To determine the effect of MOM storage (fresh vs frozen) on the expansion of bioactive components from live microbiota in DHM. Hypothesis: Both fresh and frozen MOM will produce similar results when inoculated into DHM to restore the microbial content (including their bioactive components) similar to each MOM sample. Aim 3: To compare the microbiome found in a mother’s MOM to the microbiome in her infant’s stool. Hypothesis: The mother/infant pair will share a common microbiome between the mother’s MOM and her infant’s stool. METHODS/STUDY POPULATION: Subjects will include 12 pump-dependent mothers of infants born < 34 weeks gestation admitted to the University of Florida Health Shands Hospital, Neonatal Intensive Care Unit (NICU). Inclusion criteria consists of mothers expressing over 100 ml of MOM per day, producing at least 45 ml of MOM at an expression session, at least 18 years of age, and speak English. Mothers are excluded if they have taken antibiotics within 3 days of sample collection, are HIV+, or delivered an infant who has a chromosomal abnormality or is severely ill. An expressed MOM sample will be collected and divided into two fractions: (A) fresh and (B) frozen at -20C for 24 h. The fresh fraction (A) will be processed immediately while the frozen fraction (B) will be processed after 24 h. Each MOM will be inoculated in DHM at dilutions of 10% and 30% and incubated at different time points: 0 h (T0), 2 h (T2), 4 h (T4) at 37°C. At each time point, total viable cell counts as well as microbiome analysis through 16S ribosomal shotgun sequencing will be performed and compared for differences. Bacteria isolated from each MOM will be saved, identified through 16S ribosomal sequencing and grown in culture for future studies. Fecal samples from each corresponding infant will be collected within 48 h after collection of the MOM sample. Stools will be homogenized and subjected to DNA extraction to perform 16S ribosomal shotgun sequencing. Microbiome analysis will be conducted, compared between fecal samples as well as with the microbiome of the MOM. RESULTS/ANTICIPATED RESULTS: Study is ongoing. We anticipate similar results with fresh or frozen MOM to that of a previous pilot study, where enriched microbiota similar to MOM was found when fresh MOM was inoculated and incubated in DHM. The microbiome analysis of the infant fecal samples may illustrate the influence that the microbiome of the MOM may have on the development of the infants’ gastrointestinal microbiota. DISCUSSION/SIGNIFICANCE OF IMPACT: The purpose of this study is to provide evidence on the ability, timing, and efficacy of inoculating DHM with fresh and frozen MOM. Study results will inform future studies to support the implementation of an inoculation procedural protocol to be used in clinical practice and human milk banking. The description of the MOM microbiome, as well as the gastrointestinal microbiome, will expand scientific knowledge on the role breast milk has on the origins of health and disease.

OBJECTIVES/SPECIFIC AIMS: Diabetes mellitus (DM) is a risk factor for the development of microvascular complications. We sought to determine the association between diabetes mellitus status and microvascular circulatory disease, as measured in the sublingual capillary bed. METHODS/STUDY POPULATION: We prospectively recruited adults with cardiovascular (CV) risk factors or established CV disease, with and without DM, who were referred for invasive coronary angiography at an urban tertiary care medical center. All participants underwent non-invasive sublingual sidestream darkfield microscopy. The primary outcome was the perfused boundary region (PBR), a measure reflecting the extent to which red blood cells (RBC) penetrate the sublingual glycocalyx in vessels between 5 and 25 µm in width. RESULTS/ANTICIPATED RESULTS: 57 participants were enrolled. The mean age was 66.1 ± 11.1 years and a majority of participants (66%) were men. DM was present in 18 (31.6%) participants. Sublingual PBR measurements were not different between participants with and without DM overall (1.93µm vs. 1.96µm, p=0.63) or in vessels with high flow (1.48µm vs. 1.59µm, p=0.08). No differences in capillary RBC filling (72.9% vs 73.0%, p=0.95) or perfused microvascular density (3112 vs. 3236 µm/mm2, p=0.32) by DM status were observed. DISCUSSION/SIGNIFICANCE OF IMPACT: In a population of adults with CV risk factors or disease, DM was not associated with impaired sublingual microvascular glycocalyx. Additional investigation into diabetes-induced microvascular impairment is warranted.

OBJECTIVES/SPECIFIC AIMS: Thoracic endovascular aortic repair (TEVAR) is more effective in remodeling the dissected aorta in acute versus chronic type B aortic dissection (TBAD). It has been hypothesized that this is due to differences in dissection flap biomechanical and structural properties but has not been confirmed in explanted human aortic tissue. We aimed to characterize and compare differences in tissue biomechanics and microstructure between acute and chronic dissection flaps that may underlie these findings. METHODS/STUDY POPULATION: Dissection flaps were obtained at time of operative repair for patients presenting for open aortic replacement to treat acute type A (ACUTE, n=7) or chronic type B (CHRONIC, n=7) aortic dissection. Given that the current treatment modality for acute complicated TBAD is TEVAR, it was not feasible to acquire acute TBAD flaps for analysis. Tissues were cryopreserved and subjected to biaxial tensile testing in the circumferential and longitudinal directions. Stiffness was quantified by the tangent modulus (TM) in the low and high linear regions of the compiled equibiaxial response curves for each cohort. Extensibility was defined as the intersection of the fitted line from the high linear region with the x-axis, and the degree of anisotropy (DA) was defined as the mean absolute percentage error of the strains in both directions. Flap architecture and collagen fiber organization were also compared between groups using two-photon microscopy. RESULTS/ANTICIPATED RESULTS: Average age of dissection flaps were 3.4±3.4 days in ACUTE and 1,868.7±1,354.0 days in CHRONIC (p=0.011). There were no differences in age, co-morbidities, maximum aortic diameter, and aortic wall thickness. ACUTE exhibited an anisotropic stress-strain response with increased extensibility longitudinally than circumferentially (0.18 vs. 0.09, p=0.022, DA=0.67) while CHRONIC demonstrated an isotropic response with similar extensibility in either direction (0.11 vs. 0.12, p=0.606, DA=0.26). CHRONIC and ACUTE had comparable stiffness in the circumferential direction (TMlow 439.92 vs. 541.08, p=0.729, and TMhigh 1585.19 kPa vs. 1869.35 kPa, p=0.817). In the longitudinal direction, CHRONIC was significantly stiffer than ACUTE (TMhigh 8347.61 kPa vs. 1201.34 kPa, p=0.049) (FIGURE). Microscopy corroborated these findings with greater collagen fiber organization circumferentially than longitudinally in ACUTE and increasing fibrosis, collagen predominance, and straightening of collagen fibers in CHRONIC. DISCUSSION/SIGNIFICANCE OF IMPACT: Compared to ACUTE, CHRONIC exhibited loss of anisotropy with increased tissue stiffness in the longitudinal direction. Increased dissection flap fibrosis and decreased compliance may explain the worse outcomes for aortic remodeling after TEVAR in chronic TBAD. This study offers biomechanical support for early TEVAR in the acute phase of uncomplicated TBAD.

OBJECTIVES/SPECIFIC AIMS: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is expressed on Wnt/β-catenin-dependent adult stem cell populations of the colon. Cancer stem cells are hypothesized to be the driving force behind tumor progression and metastasis, making them attractive therapeutic targets. Our aim was to analyze the clinicopathologic and prognostic significance of LGR5 expression in a cohort of colorectal cancer patients with peritoneal metastasis. METHODS/STUDY POPULATION: A total of 49 Formalin-fixed paraffin-embedded (FFPE) tissue blocks of primary or metastatic tumors and their respective normal tissues were collected from the tissue bank for time period 2009-2015. LGR5 expression was assessed at the protein level through immunohistochemical (IHC) staining of tissue microarray (TMA) constructs consisting of pairs of tumor and normal colon tissue. The correlation between LGR5 expression and clinicopathologic parameters and prognosis was assessed by statistical analysis. RESULTS/ANTICIPATED RESULTS: Of the 49 patient sample, 30(61.22%) were female vs. 19 (38.78%) males. Age range at initial diagnosis ranged from 31.7 years to 84.4 years, with a median age of 61.29 years. Duration of follow-up ranged from 1 – 9 years with a median of 5 years.LGR5 expression was higher in colorectal cancer than in normal mucosa. In univariate survival analysis overexpression of LGR5 was significantly associated with improved survival (p=0.002).Of significance, LGR5 positivity was an independent prognostic marker for better prognosis in a multivariate survival analysis adjusting for prognostic variables age, stage, gender, tumor histology and grade (HR 2.67. 95% CI 1.01-7.00, P = 0.046). DISCUSSION/SIGNIFICANCE OF IMPACT: LGR5 was significantly over expressed in colorectal cancer compared to normal tissues. LGR5 was noted to be an independent prognostic variable for an improved survival outcome in colorectal cancer patients with peritoneal metastasis, making LGR5 a potential therapeutic target in colorectal cancer patients with peritoneal metastasis.

OBJECTIVES/SPECIFIC AIMS: Histones are nuclear proteins that associate with DNA to facilitate packaging into condensed chromatin. Histones are dynamically decorated with post-translational modifications (PTMs), which regulate such processes as DNA repair, gene expression, mitosis, and meiosis. Histone 3 Family 3 (H3F3) histones (H3.3), encoded by H3F3A and H3F3B, mark active genes, maintain epigenetic memory, and maintain heterochromatin and telomeric integrity. Specific somatic mutations in H3F3A and H3F3B have been strongly associated with pediatric glia and other tumors, but no germline mutations have been reported. The goal of our study was to further understand the functional effects of germline mutations of H3F3A and H3F3B. METHODS/STUDY POPULATION: We analyzed 32 patients bearing de novo germline missense mutations in H3F3A or H3F3B with core phenotypes of progressive neurologic dysfunction and congenital anomalies, but no malignancies. Patient histones were analyzed by quantitative mass spectrometry (qMS). RESULTS/ANTICIPATED RESULTS: qMS results revealed that the mutant histone proteins are present at a concentration similar to that of wild-type H3.3. qMS analysis showed strikingly aberrant PTM patterns that suggested local dysregulation. These patterns are distinct from the dominant negative somatic mutations, which cause more global PTM dysregulation. Patient cells also demonstrated upregulation of the expression of genes related to mitosis and cell division, and had a greater proliferative capacity. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggests that the pathogenic mechanism of germline histone mutations is distinct from that of the published cancer-associated somatic histone mutations, but may converge on control of cell proliferation. Further clarification of the pathophysiology in these patients can elucidate the roles of histones and histone PTMs in human development and non-syndromic neurodegeneration. In addition, it provides a framework for targeted therapy development for this and related progressive neurologic disorders.

OBJECTIVES/SPECIFIC AIMS: Alzheimer’s disease (AD) is the leading cause of dementia, and a rapidly growing public health crisis as life expectancy increases. Two hallmark symptoms of the disease are memory impairment and the pathological accumulation of amyloid beta protein. The hippocampus is a brain region critical for the consolidation of new memories, and one of the first regions in which amyloid accumulation is observed. Our lab and others have observed a disruption to hippocampal network activity that is critical for memory consolidation in amyloid-accumulating mice. However, the mechanisms and neuronal micro-circuitry underlying this disruption are under-explored, a critical gap that warrants exploration if we are to understand memory disruption in the disease. In this study we have investigated the hypothesis that a preferential disruption to inhibitory PV neurons and the extracellular matrix that surrounds this cell type underlies downstream network alterations. METHODS/STUDY POPULATION: We have employed the 5xFAD mouse model of familial Alzheimer’s disease crossed with transgenic lines that selectively fluoresce in different neuronal sub-types. In a multi-modal approach, we have investigated behavioral, electrophysiological, and biochemical alterations between 3-month-old amyloid-accumulating 5xFAD mice and littermate controls. RESULTS/ANTICIPATED RESULTS: We observe a 35% increase in the incidence of synchronous hippocampal oscillations known as sharp wave ripples (SWRs) in amyloid-accumulating mice versus littermate controls (n = 28, p = 0.01), as well as a 95% increase in the power of slow gamma oscillations (p = 0.002). This hyperexcitability of the hippocampal network is correlated with an impairment in hippocampal-dependent memory, assayed with the Barnes Maze, a behavioral test of spatial memory (172% increase in latency to find escape hole, n = 8, p = 0.01). To elucidate the micro-circuitry that underlies this network disruption, we have investigated the integrity of peri-neuronal nets (PNNs), part of the extracellular matrix of proteins that preferentially ensheathe inhibitory PV neurons and support their function. We observe a 60% decrease in intensity of PNNs (n = 5, p = 0.005), suggesting PNN integrity is impaired in amyloid-accumulating mice. Ongoing experiments into the activity and synaptic input to both inhibitory PV and excitatory pyramidal neurons seek to determine the effects of this PNN disruption on downstream micro-circuitry. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that a preferential impairment to PNNs and inhibitory PV cells underlie hippocampal hyperexcitability in a mouse model of AD. As hippocampal network activity is critical for memory consolidation, these effects contribute to our understanding of memory disruption during early disease progression, which has been poorly understood to date. These findings provide a foundation for future in vivo studies employing optogenetic stimulation to this neuronal sub-type, to determine if restoring physiological network balance can ameliorate memory decline.

OBJECTIVES/SPECIFIC AIMS: (1) Assess if the total duration of EEG suppression during a protocolized exposure to general anesthesia predicts cognitive performance in multiple cognitive domains immediately following emergence from anesthesia. (2) Assess if the total duration of EEG suppression in the same individuals predicts the rate of cognitive recovery in a three-hour period following emergence from anesthesia. METHODS/STUDY POPULATION: This was a non-specified substudy of NCT01911195, a multicenter investigation taking place at the University of Michigan, University of Pennsylvania, and Washington University in St. Louis. 30 healthy volunteers aged 20-40 years were recruited to receive general anesthesia. Participants in the anesthesia arm were anesthetized for three hours at isoflurane levels compatible with surgery (1.3 MAC). Multichannel sensor nets were used for EEG acquisition during the anesthetic exposure. EEG suppression was detected through automated voltage-thresholded classification of 2-second signal epochs, with concordance assessed across sensors. Following return of responsiveness to verbal commands, participants completed up to three hours of serial cognitive tests assessing executive function, reaction time, cognitive throughput, and working memory. Non-linear mixed effects models will be used to estimate the initial cognitive deficit and the rate of cognitive recovery following anesthetic exposure; these measures of cognitive function will be assessed in relation to total duration of suppression during anesthesia. RESULTS/ANTICIPATED RESULTS: Participants displayed wide variability in the total amount of suppression during anesthesia, with a median of 31.2 minutes and range from 0 minutes to 115.2 minutes. Initial analyses suggest that greater duration of burst suppression had a weak relationship with participants’ initial cognitive deficits upon return of responsiveness from anesthesia. Model generation of rate of recovery following anesthetic exposure is pending, but we anticipate this will also have a weak relationship with burst suppression. DISCUSSION/SIGNIFICANCE OF IMPACT: In healthy adults receiving a standardized exposure to anesthesia without surgery, burst suppression appears to be a poor predictor of post-anesthesia cognitive task performance. This suggests that burst suppression may have limited utility as a predictive marker of post-operative cognitive functioning, particularly in young adults without significant illness.

OBJECTIVES/SPECIFIC AIMS: Our primary objective is to determine whether the bacteria exerts its effect intra- or extra-cellularly. We have genomic and microscopy preliminary evidence indicating that the bacteria is capable of invading endometrial cells. Our secondary objective is to identify what type of impact the bacteria have on the host cells and whether they are capable of transforming the host cells from a benign into a malignant phenotype. We are currently testing a putative mechanism by which the bacteria may cause the overexpression of the hypoxia inducible factor (HIF), a hallmark of endometrial cancer. METHODS/STUDY POPULATION: We are utilizing our custom built optofluidics platform (microfluidics platform incorporated into an advanced microscope with optical laser tweezers) to isolate single cells from the endometrial tissues of 150 patients with and without endometrial cancer. We are utilizing single cell whole genome amplification followed by qPCR to identify if the bacteria is present intracellularly. We are coupling this procedure with standard microbiological invasion assays with endometrial cell line cultures and P.somerae. We are also utilizing our optofluidics platform to perform single cell transcriptomic amplification, followed by sequencing of cells invaded or in the presence of the bacteria to determine the impact in the transcriptome of the host cell. We are coupling this with western blots of factors hypothesized to be impacted by the bacteria in the overexpression of HIF. RESULTS/ANTICIPATED RESULTS: Based on our preliminary data we anticipate to find evidence that P.somerae is invading the host cells, in particular the cells in tumor tissues. We also expect to find that the intracellular presence of the bacteria is causing the overexpression of the HIF pathway, hence resulting in a cancerous phenotype. DISCUSSION/SIGNIFICANCE OF IMPACT: Our long-term goal is to develop primary prevention strategies that will reduce endometrial cancer incidence rates. A confirmation of our hypothesis could suggest that it is sufficient for endometrial cancer prevention efforts to eliminate P.somerae, in line with gastric and cervical cancer efforts. It could also mean that targeting P.somerae in cancer treatment is necessary to contain the disease and prevent recurrence.

OBJECTIVES/SPECIFIC AIMS: Our objective was to examine serum E2 levels in dcSSc males in relation to disease characteristics (i.e autoanitbody profile and internal organ involvement) and its impact on survival. METHODS/STUDY POPULATION: We measured serum E2 levels in 83 dcSSc men >50 years old from the University of Pittsburgh Scleroderma Center and healthy controls of similar age. Using statistical modeling, we examined the associations between circulating E2 levels, internal organ involvement, autoantibody profiles, and survival. RESULTS/ANTICIPATED RESULTS: Male dcSSc patients had significantly higher serum E2 levels compared to healthy male controls and compared to dcSSc post-menopausal women of similar age. Male dcSSc patients with high serum E2 levels had significantly more heart involvement and worse survival. Using Cox regression modeling for risk of death, increasing serum E2 levels in anti-Scl-70 antibody positive dcSSc males were associated an increased risk of death. DISCUSSION/SIGNIFICANCE OF IMPACT: DcSSc male patients have higher levels of E2 compared to healthy controls and dcSSc postmenopausal women. Elevated serum E2 levels in dcSSc males >50 are associated with heart involvement and, if anti-Scl-70 antibody positive, worse survival. Our current study expands on our previous work, not only that that E2 exerts pro-fibrotic effects on skin, but also internal organ involvement, overall survival. These data suggest an important role of estrogen imbalance in SSc.

OBJECTIVES/SPECIFIC AIMS: This study aims to understand the utility of 5-ALA and SWIG in detecting areas of neoplasm in a murine model of GBM. Primary outcome is the distribution of the two dyes in comparison to the true tumor extent; the sensitivity, specificity, PPV, and NPV of both dyes will be calculated. The secondary outcome is the suitability of existing cell-lines used for GBM research for studies in fluorescence-guided surgery. METHODS/STUDY POPULATION: Two cell lines are used for this research: U87, derived from human GBM; and GL261, derived from rodent stem cells. U87 are implanted intracranially into 6-week old athymic, nude, female mice, while GL261 are implanted intracranially into 10-week old female C57BL/6 mice. The mice are weighed every 3 days to monitor health and bioluminescence imaging is performed between 7-10 days after implantation to confirm tumor implantation and monitor tumor growth. The mice are sacrificed between 10-21 days after implantation. 5mg/kg of intravenous ICG is administered 24-hours prior to harvest and 250mg/kg of intraperitoneal 5-ALA is administered 3-hours prior to harvest. Once the mice are sacrificed, their brains are quickly harvested and placed in cold formalin. Using a high-resolution Odyssey CLx scanner, near-infrared fluorescence from ICG is captured in coronal cross sections of the brains through the tumor. Similarly, 5-ALA fluorescence is imaged using a 405nm LED excitation source and 610-690nm bandpass filter. Afterwards, slices of the brain are stained with H&E, which serves as the gold-standard of the extent of tumor. Images from ICG, 5-ALA, and H&E can then be compared using ImageJ to compare the extent of tumor to the distribution of the dyes. RESULTS/ANTICIPATED RESULTS: In separate, previous studies in humans, both 5-ALA and SWIG have demonstrated utility in detecting residual neoplasm in HGG resections. In general, 5-ALA is more specific for areas of neoplasm, while SWIG is more sensitive. Thus, I anticipate that in this study, SWIG will show a greater distribution than 5-ALA, with SWIG distributing to areas beyond the tumor and 5-ALA distributing within, but not completely covering, the tumor. SWIG’s sensitivity and NPV for detecting tumor should be >90%, while its specificity and PPV may be closer to 50%. For 5-ALA, specificity and PPV should be close to 80-90%, but its sensitivity and NPV may be <50%. In terms of cell-line, preliminary results suggest that U87 cells are not suitable for research involving 5-ALA. We suspect that this is partly due to the limited infiltrative nature of U87 cells; in fact, the cells form a spherical mass, imitating metastases rather than true HGGs. The U87 masses do not have significant vascularity, which likely limits the amount of 5-ALA that can distribute to inside the tumor. DISCUSSION/SIGNIFICANCE OF IMPACT: 5-ALA is currently the only FDA-approved agent for fluorescence-guided neurosurgery. However, it has multiple limitations, which ultimately results in its low sensitivity and NPV. Our novel technique, which has demonstrated much higher sensitivity at the cost of specificity, offers an alternative that may help surgeons better achieve total resections in the operating room. These two agents have not been compared directly in humans or mice. Thus, this experiment sets up an important precedent, on which a human clinical trial comparing the two agents’ effects on resection rates and patient outcome can be performed. Ultimately, this work will lay the foundation for future research into fluorescence-guided neurosurgery, both in the visible and NIR spectrum.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this study was to evaluate the performance of a bone fracture targeted systemically administrable bone anabolic as a potential therapeutic for bone fracture repair. Currently all bone fracture repair therapeutic require local administration during surgery. However, the population that need the most assistance in repair bone fractures are not eligible for surgery. So, it was our goal to design an inject-able therapeutic to assist in bone fracture repair to reduce the invasiveness. The injectable nature of it allows for repair administration of the bone anabolic and for therapeutic effect throughout the entire bone fracture healing process. Targeting it to the bone fracture site reduces the toxicity and increases the efficacy. METHODS/STUDY POPULATION: METHODS To achieve the above objective, a bone mineral-(hydroxyapatite-) targeting oligopeptide was conjugated to the non-signaling end of an engineered parathyroid hormone related protein fragment 1-46 with substitutions at Glu22,25, Leu23,28,31, Aib29, Lys26,30 (ePTHrP). The negatively charged oligopeptide has been shown to target raw hydroxyapatite with remarkable specificity, while the attached PTHrP has been demonstrated to induce sustained and accelerated bone growth under control of endogenous morphogenic regulatory factors. The conjugate’s specificity arises from the fact that raw hydroxyapatite is only exposed whenever a bone is fractured, surgically cut, grafted, or induced to undergo accelerated remodeling. The hydroxyapatite-targeted conjugate can therefore be administered systemically (i.e. without invasive surgery or localized injection) and still accumulate on the exposed hydroxyapatite at the fracture site where it accelerates the healing process Murine in vivo experiments were conducted on female Swiss Webster mice (10 per group). Femoral fractures were induced with a 3-point bending device and stabilized. Mice were dosed with 3 nmol/kg/d of targeted-ePTHrP, non-conjugated (free) ePTHrP, or saline. Following a 4-week study, fracture callus densities were measured using microCT. Canine in vivo experiments were conducted on 1-year-old male beagles. Beagles underwent a 10 mm bilateral ulnar ostectomy. Two dogs in the treatment group and Three dogs in the control group were dosed daily with either targeted-ePTHrP 0.5nmol/kg/d or saline respectively. Dogs were x-rayed weekly for the first 6 weeks and then every other week thereafter. One tailed ANOVA followed by Dunnett’s post-hoc test was used to establish significance. All animal experiments were conducted as described in approved IACUC protocols. P<0.05 was considered significant. RESULTS/ANTICIPATED RESULTS: RESULTS SECTION: In the murine studies we observed a marked increase in fracture callus size and a 2-fold increase in bone deposition was observed in the targeted-ePTHrP group over the saline group (P<0.01). A significant doubling in bone density was also observed. Targeted-ePTHrP group fractured femurs were able to achieve their pre-fracture strength as early as 3 weeks compared to 9 weeks in the saline mice representing a 66% reduction in healing time. In the canine studies, we observe a significantly higher closure of the ostectomy gap than saline controls (P<0.05). In addition, no significant differences in weight are observed in the treatment vs. saline controls. No significant difference between the control group and treatment groups was found in a histological investigation of the organs. DISCUSSION/SIGNIFICANCE OF IMPACT: DISCUSSION: Although attempts have been made in developing a systemically administered fracture therapeutic for fracture repair, i.e. teriparatide, to date, no such anabolics have been approved for this use. In these studies there is evidence that anabolic activity was occurring at the fracture site, but at a level that did not meet FDA required end-points.2 It is plausible that if sufficient drug were to be delivered to a fracture site then improved fracture repair would be possible. In previous studies, we demonstrated fracture specific accumulation bone anabolics can be achieved by modifying the drug with acidic oligopeptides.3 Here, by modifying a safe, clinically proven, parathyroid hormone receptor agonist with an acidic oligopeptide we observe improved bone deposition and strength in mice. Furthermore, when administered to canine critical sized defect ostectomies, a more relevant and difficult model, we observe improved ostectomy closure. CLINICAL RELEVANCE:: The ability to accelerate bone fracture repair is a fundamental need that has not been addressed by conventional methods. By targeting bone anabolic agents to bone fractures, we can deliver sufficient concentrations of anabolic agent to the fracture site to accelerate healing, thus avoiding surgery and any ectopic bone growth associated with locally-applied bone anabolic agents.

OBJECTIVES/SPECIFIC AIMS: The current study is the first investigation of frontal alpha asymmetry in distressed violent (DV) and distressed nonviolent (DNV) partners during a placebo-controlled alcohol administration and emotion-regulation study. Because this is the first study of the pharmacological effects of alcohol on FAA, the first portion of the study was conducted to characterize alcohol effects in DV and DNV partners during the baseline condition. The subsequent portions of the study were conducted to characterize the effects of alcohol and evocative stimuli on FAA in DV and DNV partners. We hypothesized that DV partners would demonstrated greater left frontal alpha asymmetry when intoxicated and viewing evocative partner stimuli than DNV partners. Lastly, we attempted to replicate previous research that has found associations between baseline measures of FAA and the State-Trait Anger Expression Inventory – 2 (Spielberger, 1999) subscales of Trait Anger, Anger Expression-Out, Anger Expression-In, Anger Control-Out, Anger Control-In (Hewig, Hagemann, Seifert, Naumann, & Bartussek, 2004). METHODS/STUDY POPULATION: Partners in the present study were drawn from a larger study investigating over-arousal as a mechanism between alcohol use and intimate partner violence (AA022367). Couples were recruited from the community via radio, television and newspaper advertisements, and eligibility screening occurred at the couple level. Participants included in the present analysis were 23 DV partners (12 female, 11 male), and 15 DNV partners (7 female, 9 male). The mean age of the sample was 32 (SD 4.8 years, range 23-40 years). Data from two DV partners were not included in the analyses of the FAA in the emotion-regulation tasks due to movement artifacts during the alcohol condition leaving too little data for analysis. RESULTS/ANTICIPATED RESULTS: The expected beverage by couple type interaction did not reach significance [F (1, 36) = 3.93, p = .055], but the between-subjects effects of couple type revealed a significant difference [F (1, 36) = 4.425, p = .042]. Contrary to our hypothesis, however, these results suggest that under conditions of alcohol, DV partners evidenced significantly greater relative right frontal alpha power asymmetry whereas DNV partners evidenced greater relative left frontal alpha power asymmetry. Although there was no significant between-subjects effect, there was a nearly significant interaction between beverage type and emotion regulation condition [F = (1, 36) = 4.032, p = .052] and a significant main effect of emotion regulation condition [F (1, 36) = 7.579, p = .009]. It appears that asking the participants to “not react” to their partners’ evocative stimuli caused significantly greater right frontal alpha asymmetry. Because intimate partner violence is best understood in the context of conflict between two partners, we also examined partner-reported experiences of anger as predictors of DV participant’s FAA. The model as a whole predicted 67.4% of the variance in DV partner FAA, R squared change =.674, F Change (5, 15) = 6.21, p = .003. Three anger experience scales were statistically significant. The partner Anger Control-Out (B = -1.23, p =.001) scale recorded a higher standardized beta value and accounted for 40% of the variance in this model. Anger Control-In (B = .63, p = .022) accounted for 14% of the variance in the model, and Anger Expression-Out scale (B = .57, p = .024) accounted for 13.7% of the variance in the model. DISCUSSION/SIGNIFICANCE OF IMPACT: The current study is the first pharmacological study of the effects of alcohol on frontal alpha asymmetry in distressed violent and distressed nonviolent partners. Contrary to our hypothesis, under acute alcohol intoxication during the baseline condition, DV partners exhibited significantly greater relative right FAA compared to DNV partners who exhibited significantly greater relative left FAA. Because intimate partner violence is best understood in the context of couple conflict, we examined the ability of partners’ anger experiences to predict DV and DNV partners’ FAA, and a very interesting pattern emerged among our DV participants and their partners. The anger experiences of our DV participants’ partners accounted for 67% of the variance in the FAA of our DV participants when they were intoxicated and viewing evocative stimuli.

OBJECTIVES/SPECIFIC AIMS: In the present study, we examined the functional consequences of 3α-PVA on OATP1B1-mediated PVA transport. To elucidate this, we determined the effect of SLCO1B1 genotype on PVA transport and the role of 3αPVA as a competitive inhibitor of OATP1B1, which could serve as another covariate that disrupts the systemic and hepatic exposure of pravastatin in children and adults. METHODS/STUDY POPULATION: Site directed mutagenesis was performed to generate SLCO1B1 genotypes of interest (*1a, *1b, *5, *15). Human embryonic kidney (HEK293) cells were grown and plated at 200 000 cells per well in 24-well plates. Twenty-four hours later the cells were transfected with the aforementioned plasmids. Forty-eight hours later cell-based transport was performed with radiolabelled [3H]-pravastatin sodium salt. Non-radioactive pravastatin sodium salt and 3’α-iso-pravastatin sodium salt was used for PVA transport and 3αPVA studies, respectively. Cells were washed 3 times with warm uptake buffer, incubated for 1 minute with uptake solutions containing PVA and 3αPVA at varying concentrations. Transport was terminated by four 1-ml washes with ice-cold uptake buffer. Cells were lysed with 300 µl 1% Triton X-100 in PBS at room temperature for 30 minutes prior to analysis. Radioactivity was measured in a MicroBeta2 liquid scintillation counter. The remaining cell lysates were transferred to 96-well plates to determine total protein concentration using the bicinchonic acid protein assay. All transport measurements were corrected by the total protein concentration. All experiments were performed 3 to 4 times independently with 2-3 determinations. Data were analyzed for significant differences amongst genotype groups using ANOVA followed by Tukey’s multiple comparisons test. IC50 and kinetic parameters were calculated using non-linear regression analysis. RESULTS/ANTICIPATED RESULTS: Pravastatin transport in SLCO1B1 variants (*5, *15) was significantly decreased compared to the reference genotype *1a and *1b (Km [µM]: *1a 18.2 ± 0.9; *1b 17.9 ± 3.3; *5 34.2 ± 9.7; *15 34.1 ± 6.1; p≤0.05; Vmax [pmol/mg/min]: *1a 104.9 ± 13.1; *1b 93.7 ± 16.7; *5 44.8 ± 15.9; *15 62.3 ± 22.5; p≤0.05). *1a and *1b were not significantly different with respect to pravastatin transport. Intrinsic clearance was diminished nearly 4 to 5-fold in SLCO1B1 variants compared to reference genotypes (Vmax/Km [µl/min/mg]: *1a 5.8 ± 0.8; *1b 5.7 ± 1.9; *5 1.3 ± 0.2; *15 1.8 ± 0.3; p≤0.01). Pravastatin transport was inhibited by 3αPVA for all genotypes. However, there was more pronounced inhibition in the SLCO1B1 variant genotypes compared to reference genotypes (IC50 [µM]: *1a 15.9 ± 1.9; *1b 18.6 ± 5.7; *5 3.9 ± 2.0; *15 4.4 ± 0.8; p≤0.01; Ki: *1a 15.0 ± 1.8; *1b 17.5 ± 5.4; *5 3.8 ± 2.9; *15 4.3 ± 0.8; p≤0.01). DISCUSSION/SIGNIFICANCE OF IMPACT: In vitro PVA transport is altered according to SLCO1B1 genotype, consistent with previous in vitro and human experience. Our data suggest that the significantly different maximal transport velocity (Vmax) in variant versus non-variant genotypes is consistent with decreased membrane expression of OATP1B1 with the variant c.521T>C allele. However, in contrast to data involving typical model substrates (e.g. estrone-3-sulfate), the PVA binding affinity (Km) was significantly different between variant and non-variant genotypes, consistent with altered binding of the substrate to OATP1B1. Collectively, we conclude that decreased OATP1B1 expression and function in variant genotypes influence altered transport for PVA. Finally, the functional consequences of 3αPVA formation on PVA transport was confirmed in our study. Mechanistically, we confirmed our observation in humans that 3αPVA inhibits OATP1B1 transport. However, this effect is more pronounced in variant genotypes as shown by lower IC50 values compared to the reference genotypes. This highlights another source of variation that must be taken into consideration when trying to optimize the pravastatin dose-exposure relationship in humans.