OBJECTIVES/GOALS: The overall goals of this project are: (1) to demonstrate the utility of staff with cross-disciplinary skills in biostatistics and epidemiology as program managers for CTSA BERD Cores, and (2) examine streamlined processes in project triaging, consultations, and data extractions for CTSA service requests when individuals are in this role. METHODS/STUDY POPULATION: Biostatistics and epidemiology are partnered disciplines incorporated in a variety of research areas, especially in human health sciences and health care. For interdisciplinary teams, including individuals trained in both specialties results in efficient research collaborations. When these individuals are program managers for research navigation, processes become more effective and expedited for project timelines and workflows across the CTSA. Examples of this integration are described in vignettes from project triaging, statistical consultations, and data extractions from CTSA service requests. Process comparisons of previous and current workflows are presented to show the advantages of utilizing this type of program manager within these areas of BERD. RESULTS/ANTICIPATED RESULTS: This project defines three areas of integration for a program manager with cross-disciplinary training in biostatistics and epidemiology: (1) project triaging to statistical teams, (2) study design and analysis consultations, and (3) data extractions. Each of these areas demonstrates prior processes that once would require multiple steps in CTSA service requests (including time, resources, and personnel) and identified common issues (slow response to requests, data re-extractions, and challenges providing statistical support) that are now avoided with a manager with cross-training in biostatistics and epidemiology methods. The advantages and disadvantages of integrating these individuals are also described. DISCUSSION/SIGNIFICANCE: BERD Cores seeking improvements to research navigation processes can gain efficiency by incorporating program managers with cross-disciplinary training in biostatistics and epidemiology. BERD Cores may also use this as a case study for translational science with innovation to longstanding challenges in CTSA research workflows.

OBJECTIVES/SPECIFIC AIMS: To examine how women with OF in Ghana develop strategies for coping in the absence of access to successful surgical repair. To assess the feasibility, acceptability, and appropriateness of an innovation to support coping among women with OF seeking care in a health facility in Ghana. To examine the perceived facilitators and barriers to implementation among additional OF stakeholders regarding the innovation. METHODS/STUDY POPULATION: This study uses a sequential exploratory mixed methods design. The population of study is women in Ghana living with obstetric fistula, as well as additional fistula stakeholders (programmers, policy makers, community leaders). To get an understanding of usual leakage, women carried out at baseline a pad test, where they wore a sanitary pad for 2 hours and leaked freely. We subtracted the dry pad weight from the wet pad weight to estimate urine leakage in mL. Then women inserted the cup for 2 hours and again wore a pad and urine leakage was estimated. Acceptability among women with vesicovaginal fistula was measured by questionnaire. Acceptability among additional stakeholders was examined by semistructured interview. Appropriateness was assessed among the user, additional stakeholders, and organizational setting. RESULTS/ANTICIPATED RESULTS: We observed a 61% mean reduction in leakage with the cup which was also perceived by cup users as a reduction in wetness. Notably, one participant who had 4 previous surgical attempts, experienced a 78% reduction in leakage. No adverse events attributable to use of the cup were observed, unlike some of the strategies women currently use to manage leakage. Acceptability was high as most women could easily insert, remove, and wear the cup over the 2-hour period and fistula stakeholders indicated the innovation content and complexity were acceptable. In community interviews, women shared various coping and self-care strategies to manage their leaking, other related impairments, and stigma. Women using the cup in the health facility expressed that it was useful. Additional stakeholders found the cup a low-cost, low-tech solution to supplement existing programs. Within the stakeholder interviews we heard that the cultural norms and existing activities of the potential implementation partners align with the innovation approach. Stakeholders revealed various implementation facilitators and barriers. The facilitators to implementation reported in the interviews were related to the intervention and organization characteristics in particular. Stakeholders perceived a relative advantage to self-management. Stakeholders had concerns regarding whether women would find the insertable device acceptable and appropriate—questioning whether potential users would have access to water, soap, and safe space to empty cup. DISCUSSION/SIGNIFICANCE OF IMPACT: The innovation is efficacious, acceptable, adds to current coping strategies, and fits within existing fistula programs. Stakeholders pre-implementation perceptions highlight the importance of partnerships and the need for an evidence base related to effectiveness, acceptability, and cost. Challenges to address include access to resources within these contexts (water, soap, and safe space) and development appropriate counseling message.

OBJECTIVES/SPECIFIC AIMS: This poster presents a detailed characterization of the distribution of semantic concepts used in the text describing eligibility criteria of clinical trials reported to ClincalTrials.gov and patient notes from MIMIC-III. The final goal of this study is to find a minimal set of semantic concepts that can describe clinical trials and patients for efficient computational matching of clinical trial descriptions to potential participants at large scale. METHODS/STUDY POPULATION: We downloaded the free text describing the eligibility criteria of all clinical trials reported to ClinicalTrials.gov as of July 28, 2015, ~195,000 trials and ~2,000,000 clinical notes from MIMIC-III. Using MetaMap 2014 we extracted UMLS concepts (CUIs) from the collected text. We calculated the frequency of presence of the semantic concepts in the texts describing the clinical trials eligibility criteria and patient notes. RESULTS/ANTICIPATED RESULTS: The results show a classical power distribution, Y=210 X(−2.043), R2=0.9599, for clinical trial eligibility criteria and Y=513 X(−2.684), R2=0.9477 for MIMIC patient notes, where Y represents the number of documents in which a concept appears and X is the cardinal order the concept ordered from more to less frequent. From this distribution, it is possible to realize that from the over, 100,000 concepts in UMLS, there are only ~60,000 and 50,000 concepts that appear in less than 10 clinical trial eligibility descriptions and MIMIC-III patient clinical notes, respectively. This indicates that it would be possible to describe clinical trials and patient notes with a relatively small number of concepts, making the search space for matching patients to clinical trials a relatively small sub-space of the overall UMLS search space. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results showing that the concepts used to describe clinical trial eligibility criteria and patient clinical notes follow a power distribution can lead to tractable computational approaches to automatically match patients to clinical trials at large scale by considerably reducing the search space. While automatic patient matching is not the panacea for improving clinical trial recruitment, better low cost computational preselection processes can allow the limited human resources assigned to patient recruitment to be redirected to the most promising targets for recruitment.

ABSTRACT IMPACT: This project aims to investigate the impact of spermine oxidase inhibition on amelioration of neuronal injury. OBJECTIVES/GOALS: Our group has recently described a series of triazole-based reversible inhibitors of spermine oxidase (SMOX) (Holshouser et al. 2019). The purpose of the current project is to optimize our most promising inhibitors by structural modification, and to determine whether they can reduce oxidative damage in models of neuronal injury. METHODS/STUDY POPULATION: A small number of SMOX inhibitors have been described in the literature, however, currently available inhibitors lack selectivity for the enzyme and are associated with dose-limiting toxicity. For this project we used multiple medicinal chemistry techniques to synthesize novel triazole-based analogs of our most potent inhibitors as potential SMOX inhibitors. In addition, we plan to utilize virtual and physical screening methods to identify new potential scaffolds. Compounds with demonstrated activity against SMOX via enzymatic assay will then be evaluated in a cell-based model of neuronal injury. In a preliminary study, we investigated the ability of hydrogen peroxide to induce SMOX expression in an SH-SY5Y neuroblastoma cell line using western blot. RESULTS/ANTICIPATED RESULTS: We found that cellular SMOX protein increases in response to hydrogen peroxide exposure in a dose-dependent manner, indicating that this may be a viable cellular model for testing the efficacy of our experimental compounds. To extend these studies, we have developed a SMOX enzymatic assay that will be used for high-throughput screening of commercial libraries, as well as the South Carolina Compound Collection (SC3), which contains 100,000 proprietary, fully annotated analogs. As hits are identified, they will be synthesized and evaluated for potency and selectivity as SMOX inhibitors. The most potent and selective compounds will then be evaluated in our cellular model of neuronal injury. DISCUSSION/SIGNIFICANCE OF FINDINGS: Studies have linked the overexpression of SMOX and the production of associated toxic byproducts with increased susceptibility to excitotoxic stress and neuronal injury. Our objective is to develop potent and selective inhibitors for this enzyme that can serve as chemical probes for elucidating the role of this enzymatic pathway in neuronal injury.

OBJECTIVES/SPECIFIC AIMS: (i) Digitally quantify pathologically relevant glomerular microcompartmental structures in murine renal tissue histopathology images. (ii) Digitally model disease trajectory in a mouse model of diabetic nephropathy (DN). METHODS/STUDY POPULATION: We have developed a computational pipeline for glomerular structural compartmentalization based on Gabor filtering and multiresolution community detection (MCD). The MCD method employs improved, efficient optimization of a Potts model Hamiltonian, adopted from theoretical physics, modeling interacting electron spins. The method is parameter-free and capable of simultaneously selecting relevant structure at all biologically relevant scales. It can segment glomerular compartments from a large image containing hundreds of glomeruli in seconds for quantification—which is not possible manually. We will analyze the performance of our computational pipeline in healthy and streptozotocin induced DN mice using renal tissue images, and model the structural distributions of automatically quantified glomerular features as a function of DN progression. The performance of this structural-disease model will be compared with existing visual quantification methods used by pathologists in the clinic. RESULTS/ANTICIPATED RESULTS: Computational modeling will reveal digital biomarkers for early proteinuria in DN, able to predict disease trajectory with greater precision and accuracy than manual inspection alone. DISCUSSION/SIGNIFICANCE OF IMPACT: Automated detection of microscopic structural changes in renal tissue will eventually lead to objective, standardized diagnosis, reflecting cost savings for DN through discovery of digital biomarkers hidden within numerical structural distributions. This computational study will pave the path for the creation of new digital tools which provide clinicians invaluable quantitative information about expected patient disease trajectory, enabling earlier clinical predictions and development of early therapeutic interventions for kidney diseases.

Objectives/Goals: Many left hemisphere stroke survivors have a reading disorder (alexia), which is experienced as decreasing well-being. Therapies produce inconsistent results, demonstrating a need for treatment response predictors. We identify neural correlates of a computational model of reading, which may provide biomarkers to improve therapeutic outcomes. Methods/Study Population: Left hemisphere stroke survivors (LHSS) (n  =  52) performed an oral reading task and tests of semantic and phonological processing. Artificial neural network (ANN) models, mapping between orthography (visual word form), phonology (auditory word form), and semantics (word meaning), were trained to read single words at an adult reading level. Stroke was simulated by removing percentages (in 10% intervals) of the connections into and out of semantics, phonology, and the combination thereof. The lesioned model producing the smallest average Euclidean distance over word and pseudoword reading accuracy to each LHSS was selected as the matched model. Two voxelwise lesion-symptom mapping (VLSM) analyses identified the neural correlates of the percent of phonological and semantic links removed in the matched models. Results/Anticipated Results: Model reading was correlated with LHSS reading (high-frequency regular words, r(48) = 0.96; high-frequency irregular words, r(48) = 0.94; low-frequency regular words, r(48) = 0.97); low-frequency irregular words, r(48) = 0.85; all p’s Discussion/Significance of Impact: Our results show that ANN models of reading, when closely matched to LHSS reading performance, directly connect cognitive processes to the brain. Using matched models as a precision medicine framework to predict therapy response or to identify targets for neurostimulation provides a valuable route toward improving poststroke language outcomes.

OBJECTIVES/SPECIFIC AIMS: An accurate method to identify bleeding in large populations does not exist. Our goal was to explore bleeding representation in clinical text in order to develop a natural language processing (NLP) approach to automatically identify bleeding events from clinical notes. METHODS/STUDY POPULATION: We used publicly available notes for ICU patients at high risk of bleeding (n=98,586 notes). Two physicians reviewed randomly selected notes and annotated all direct references to bleeding as “bleeding present” (BP) or “bleeding absent” (BA). Annotations were made at the mention level (if 1 specific sentence/phrase indicated BP or BA) and note level (if overall note indicated BP or BA). A third physician adjudicated discordant annotations. RESULTS/ANTICIPATED RESULTS: In 120 randomly selected notes, bleeding was mentioned 406 times with 76 distinct words. Inter-annotator agreement was 89% by the last batch of 30 notes. In total, 10 terms accounted for 65% of all bleeding mentions. We aggregated these results into 16 common stems (eg, “hemorr” for hemorrhagic and hemorrhage), which accounted for 90% of all 406 mentions. Of all 120 notes, 60% were classified as BP. The median number of stems was 5 (IQR 2, 9) in BP Versus 0 (IQR 0, 1) in BA notes. Zero bleeding mentions in a note was associated with BA (OR 28, 95% CI 6.5, 127). With 40 true negatives and 2 false negatives, the negative predictive value (NPV) of zero bleeding mentions was 95%. DISCUSSION/SIGNIFICANCE OF IMPACT: Few bleeding-related terms are used in clinical practice. Absence of these terms has a high NPV for the absence of bleeding. These results suggest that a high throughput, rules-based NLP tool to identify bleeding is feasible.

OBJECTIVES/GOALS: The mission of our new Anti-racism in Data and Analysis seminar series is to provide a forum for researchers to discuss the ways that race and racism show up in data and analysis so that we as scientists may work together to advance racial equity and justice in society. METHODS/STUDY POPULATION: Our CTSA’s Biostatistics, Epidemiology, and Research Design (BERD) Program has initiated a seminar series that focuses on the ways that race and racism show up in all phases of the research process. We have hosted guest speakers who have shared their cutting-edge research as well as their reflections on race in research, and have facilitated breakout discussions to provide a forum for researchers to discuss specific questions. Our seminars are intended for practicing clinicians, clinical and population researchers, basic scientists, students, and others. RESULTS/ANTICIPATED RESULTS: We have sponsored three discussion-based seminars and two guest speaker seminars, and have several seminars of each type in the planning stages. We have collaborated with another CTSA to sponsor a discussion-based seminar at their institution, thus outfitting them with the tools to initiate a seminar series of their own. Our seminars have been extremely well attended, attracting researchers from a broad base of departments at our institution, from students to full professors. Feedback has been very positive; complaints cluster around paucity of similar opportunities, and students have asked why such material is not included as a required part of their respective training programs. DISCUSSION/SIGNIFICANCE: Race and racism permeate research, and failure to recognize and address the issues involved perpetuates racism in our society. Researchers want to learn more, but there are few opportunities for them to do so. Our seminar series provides one such forum to researchers at our institution and beyond.

OBJECTIVES/GOALS: Determine if the Conserved Transcriptional Response to Adversity transcriptomic profile established in primary blood mononuclear cells (PBMC) of chronically stress caregivers, is present in individuals with early Alzheimer’s disease. Chronic stress is a risk factor for Alzheimer’s, and may be an untapped biomarker for disease risk and pathology. METHODS/STUDY POPULATION: To collect preliminary data on the Conserved Transcriptional Response to Adversity profile in individuals with Alzheimer’s disease, we were able to utilize primary blood mononuclear cell samples from a small open label pilot study called Senolytic Therapy to Modulate the Progression of Alzheimer’s Disease, designed to clear stressed senescent cells. We hypothesized senolytics may beneficially reverse this stress profile. We developed a NanoString assay (measuring 19 inflammatory, 31 type-1 interferon, and 3 antibody synthesis genes) to compare these transcriptomic changes within 4 individuals measured at baseline, post-treatment with an intermittent 12-week senolytic therapy, and at an optional extended post-treatment follow-up time point > 3 months after their post treatment visit. RESULTS/ANTICIPATED RESULTS: There was relative downregulation of expression in transcription in 7 of 19 measured inflammatory genes (FOS, PTGS2, IL8, FOS, Il1b, JUNB, and JUN) in Alzheimer’s disease participants after receiving senolytic treatment (baseline vs. post-treatment). This is consistent with a decrease in the inflammatory arm of the Conserved Transcriptional Response to Adversity profile. These differences were not significant between baseline and the extended follow-up, indicative of a transient effect of senolytic. There were no changes in type 1 interferon or antibody synthesis genes. This data provides preliminary evidence for larger controlled studies to further establish this profile in Alzheimer’s disease, providing exciting evidence for transcript changes that may be reproducible with senolytic therapy. DISCUSSION/SIGNIFICANCE: Literature relevant to Alzheimer’s disease indicates global increases in inflammation paired with deficits in immune response, capturing some genes associated with the Conserved Transcriptional Response to Adversity. This profile may be a useful biomarker for prediction of disease severity or risk of dementia due to chronic stress.

OBJECTIVES/GOALS: Methionine dependence was described 45 years ago as an increased reliance on an exogenous supply of the essential amino acid methionine in most cancer cells compared to normal cells. Methionine depletion, using either synthetic diets or the enzyme methioninase, potentiates the effects of chemotherapy and radiotherapy in tumor-bearing animal models. Two main obstacles prevent methionine dependence from integrating the clinical treatment of cancer. The first is the weight loss associated with methionine depletion therapy, increasing the risk of cachexia in patients. The second is the stubborn absence of a mechanism to explain the inability of cancer cells to adapt to low methionine levels. METHODS/STUDY POPULATION: To address these two obstacles, we are using an immunocompetent murine model of metastatic melanoma to compare the effects of complete methionine deprivation with a moderate, 75-80% methionine restriction similar to the one used to increase lifespan in animal models. In an effort to identify a mechanism of action, we also performed a proteomic screen of two melanoma cell lines divergent for methionine dependence under methionine stress. RESULTS/ANTICIPATED RESULTS: We recently showed that methionine restriction is sufficient to provide gains in treating local and metastatic lesions in vivo, without weight loss. We observed few differences in pathway activation between the two cell lines in response to methionine stress, despite proliferation being cut by half in the methionine dependent cell line. We expect that subcellular translocation events may provide further information on the molecular bases of methionine dependence. DISCUSSION/SIGNIFICANCE OF IMPACT: A moderate restriction in methionine is sufficient to recapitulate the benefits of methionine depletion in cancer, without weight loss. The mechanism behind this effect remains unknown. This work contributes towards the integration of methionine dependence into clinical practice and the discovery of novel drug targets.

ABSTRACT IMPACT: This study has uncovered a novel surprising mechanism involving the epithelial adherens junctions and transposon regulation that can deepen our understanding of tumorigenesis. OBJECTIVES/GOALS: Recent studies show that genomic instability in 50% of tumors can be attributed to increased transposon activity, but the the reasons for this activity are unknown. We have evidence of a novel mechanism linking adherens junctions with transposon regulation. We hypothesize that adherens junctions suppress transposons to maintain genomic integrity. METHODS/STUDY POPULATION: We observed co-localization of PIWIL2 with adherens junction components of well differentiated epithelial breast, kidney and colon cell lines MCF10A, MDCK and CACO2, respectively, through immunofluorescence staining, confocal microscopy, and co-immunoprecipitation studies. Breast cancer cell lines MCF7 and MDA-231 were also observed using immunofluorescence to determine the localization of PIWIL2 in cancer cell lines. shRNA knockdown of PIWIL2 in MCF10A cells, followed by western blot, immunofluorescence, and qRT-PCR was performed to confirm the knockdown, observe if transposons were upregulated, and determine the extent of DNA damage to the genome by the marker gamma-H2AX. RNA-seq will be performed to determine piRNA sequences and possible targets of PIWIL2. RESULTS/ANTICIPATED RESULTS: Our data have revealed an interaction of E-cadherin and p120 catenin, core components of adherens junctions, with PIWIL2, a member of the Argonaute family of proteins and a key component of the piRNA processing pathway that is responsible for transposon silencing. piRNAs (PIWI-interacting RNAs) are a distinct class of small RNAs that bind to PIWI proteins, and aid in transposon degradation. We found co-localization of PIWIL2 with E-cadherin and p120 catenin at adherens junctions of well-differentiated epithelial cells, whereas this association was lost in cancer cells. Furthermore, our data show that E-cadherin depletion results in mis-localization of PIWIL2 and TDRD1, another member of the PIWI complex. E-cadherin depletion also results in upregulation of transposons and ?-H2AX, an indicator of DNA damage. DISCUSSION/SIGNIFICANCE OF FINDINGS: Since both loss of junctional integrity and increased transposon activity are universal events in cancer, this study has the potential to further our understanding of the causes of tumorigenesis. Understanding the mechanisms of transposon regulation has the potential to lead to a therapeutic target in the future.

OBJECTIVES/GOALS: The objective of this project was to recruit racially and ethnically diverse participants with an accelerated timeline into a COVID-19 vaccine trial. METHODS/STUDY POPULATION: Recruitment of diverse populations was a priority. Culturally appropriate strategies were implemented to increase enrollment of African Americans and Hispanics from the DC Metropolitan area. These strategies included the use of the Community Advisory Board for consultation, faith-based community events, town halls, radio advertisements which targeted the African American community, and the NIAID COVID-19 registry. Meaningful patient engagement by the study recruiter/coordinator throughout the study was key to recruitment and retention of participants (i.e. listening attentively, sharing personal experiences with vaccination, providing participants with enough information to make an informed decision). Education on the importance of research and vaccines was a component throughout the process. RESULTS/ANTICIPATED RESULTS: The Howard University site screened 188 participants. Of those, 150 were recruited into the vaccine trial from diverse racial and ethnic backgrounds (30% AA, 21% Hispanic, 5% Asian, 1% mixed race), a total of 57% over seven weeks. Of the 150 participants 64% were male and 36% females. The study involved a 2:1 randomization with vaccine vs. placebo. All of the 150participants received the first and second COVID-19 vaccine/placebo doses administered 21 days apart. DISCUSSION/SIGNIFICANCE: Culturally-relevant recruitment strategies allowed for meaningful community and patient interactions and were important determinants for effectively recruiting a racially and ethnically diverse group of participants within a short amount of time. Strategies employed could be applied effectively in other trials.

OBJECTIVES/GOALS: Test the effects of a community health worker supported model to deliver home-based COVID-19 testing in the Yakima Valley (Washington) and Flathead Reservation (Montana) METHODS/STUDY POPULATION: A pragmatic, randomized controlled clinical trial evaluating the effects of a community health worker supported model to deliver home-based COVID-19 testing in the Yakima Valley (Washington) and Flathead Reservation (Montana) vs. a modified direct-to-consumer. 400 participants will be enrolled, 200 from each community. Outcomes include comparing the number of completed testing kits as well as the number of testing kits with successful (detected vs not-detected) results. RESULTS/ANTICIPATED RESULTS: The poster presents preliminary results from 191 participants, blinded to study assignment. To date, 53% of enrolled participants returned a sample for testing and 39% received a usable (detected or not-detected) result. Our populations experienced a high-rate (16%) of sample errors, required 28 replacement kits and had 20 participants randomized to the control arm receive the intervention to ensure participants received testing during the pandemic. DISCUSSION/SIGNIFICANCE: Home-based testing models are build for those who are proficient in verbal and written English, have high tech. literacy and continuous access to internet. For home-based testing to have similar success rates as white Americans, cultural and demographic differences and disparities will need to be accounted for in development and implementation.

OBJECTIVES/GOALS: Estrogen monotherapy in postmenopausal women can reduce kidney function, while dual therapy combining estrogen with a progestin improves renal health. Using the female Long Evans rat as a novel animal model of postmenopausal cardiovascular disease, we found similar results where estrogen worsens renal health while co-administration of medroxyprogesterone acetate (MPA) was protective. MPA cross-activates glucocorticoid receptors (GR), which are targeted clinically for their anti-inflammatory actions. Therefore, our goal was to determine if estrogen monotherapy induces renal damage by increasing inflammation, while dual therapy with MPA opposes inflammation by cross-activating GR. METHODS/STUDY POPULATION: Female Long Evans rats underwent OVX at 11 months of age and received a subcutaneous implant containing E2, E2+MPA or vehicle for 40 days. RESULTS/ANTICIPATED RESULTS: Coadministration of MPA prevented the E2-induced increase in proteinuria (Veh: 0.27 ± 0.07; E2: 3.53 ± 1.16; E2 + MPA: 1.20 ± 0.58 mg/mg creatinine; P = 0.03) and decline in glomerular filtration rate (Veh: 0.51 ± 0.02; E2: 0.24 ± 0.05; E2+MPA: 0.39 ± 0.05 ml/min; P < 0.01). Co-administration of MPA significantly increased renal GR transcript levels compared with E2 alone (Veh: 0.96 ± 0.02; E2: 0.94 ± 0.10; E2+MPA: 1.24 ± 0.04 fold change; P < 0.01). Inflammatory marker COX 2 renal transcript levels were significantly reduced by a similar degree in both mono and dual therapies compared with vehicle (Veh: 1.07 ± 0.06; E2: 0.81 ± 0.04; E2+MPA: 0.81 ± 0.04 fold change; P < 0.01). Neither TNF-alpha and IL-6 mRNA nor urinary beta-microglobulin levels (Veh: 1.71 ± 0.31; E2: 2.88 ± 0.78; E2+MPA: 3.07 ± 1.15 mg/day; ns) were altered. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results show that the effect of E2 on renal pro-inflammatory markers was not altered by the addition of MPA despite the significant increase in renal GR levels. Therefore, the renoprotective effects of MPA in midlife hormone therapy may be independent of renal GR-mediated changes in the immune profile.

OBJECTIVES/GOALS: Through the Community Mini-Grant program, the University of Kentucky Center for Clinical and Translational Science Community Engagement and Research Core (CERC) provides a unique funding mechanism designed to empower community response by supporting local solutions to complex health issues facing central Appalachian Kentucky communities. METHODS/STUDY POPULATION: Four $2500 grants are awarded annually to Appalachian organizations to implement evidence-based programs responsive to community-identified priority health needs. The CERC also supports program implementation and evaluation by facilitating collaborations between the organizations, community practitioners, and academic researchers. RESULTS/ANTICIPATED RESULTS: Since inception, grants have been awarded to 20 community organizations in 14 Appalachian counties. Health issues addressed have ranged from Alzheimers disease, cancer treatment and prevention, obesity, healthy lifestyle, and chronic disease management and prevention. Evidence-based programs have focused on improving health outcomes among older adults, caregivers, youth, children, women and infants, and families. Program outcomes have included immediate health benefits and long-term benefits resulting from community adoption of and ongoing financial support for programs. As example, results of an evidence-based educational program to improve diabetic foot assessment among clinicians in a large Appalachian healthcare network resulted in establishment of a traveling podiatrist program. DISCUSSION/SIGNIFICANCE: Community mini-grant recipients have successfully implemented projects that address the most significant health disparities in the region. Also of benefit are expanded partnerships that are foundational to the creation of new academic-community collaborations to address the challenging health issues of Appalachian populations in Kentucky.

OBJECTIVES/GOALS: DR-TB care in South Africa includes decentralized treatment with shorter, all-oral regimens. Treatment guidelines direct regular clinical and laboratory evaluation to assess patient improvement. We therefore measured sputum collection frequency and follow-up time to assess fidelity to these guidelines in Gauteng Province, South Africa. METHODS/STUDY POPULATION: We included Rifampicin-resistant (RR) sputum specimens from the South African National Health Laboratory Service, which provides pathology services to 80% of the population, submitted between August 2022-September 2023. Patient data were obtained from a DR-TB registry and additional sputum specimen data were collected from follow-up laboratory worksheets. Follow-up spanned from first sputum collection date (baseline) to patient outcome date (e.g., completion, lost) or study closure date (if still on treatment). Monthly sputum submission rate was measured for those with ≥1 additional sputum submitted. We compared patient data by treatment site: at the specialized hospital vs. any other site, using Wilcoxon ranksum and χ2 tests. RESULTS/ANTICIPATED RESULTS: Baseline RR-TB specimens were available for 142 patients, of whom 28 (20%) had specimens submitted from the specialized hospital. Patients at the specialized hospital were older (median age 41 vs. 35.5 years, p=0.03), had higher baseline fluoroquinolone resistance (10% vs. 1%, p=0.01), and longer follow-up (median 5.2 vs. 3.5 months, p=0.01) compared to patients elsewhere. Further, 43 (30%) patients had ≥1 additional sputum submitted during follow-up. Among these, monthly sputum collection rates did not differ by site (0.3 vs. 0.3 sputum per month, p=0.89). We anticipate that increased sputum frequency will be associated with successful TB treatment outcomes based on preliminary findings. DISCUSSION/SIGNIFICANCE: These findings highlight ongoing challenges with routine laboratory follow-up according to DR-TB guidelines across treatment sites in South Africa. Future research is needed to determine reasons for low sputum collection rates, such as low patient adherence, variation in practice of healthcare workers, loss to follow-up, and clinical challenges.

OBJECTIVES/GOALS: Win ratio (WR) is an increasingly popular composite endpoint in clinical trials. A typical set up in cardiovascular trials is to use death as the first and hospitalization as the second layer. However, the power of WR may be reduced by its strict hierarchical structure. Our study aims to release the oracular hierarchical structure of the standard WR. METHODS/STUDY POPULATION: Addressing the power reduction of WR when treatment effects lie in the subsequent layers, we propose an improved method, Shrinking Coarsened Win Ratio (SCWR), that releases the oracular hierarchical structure of the standard WR approach by adding layers with coarsened thresholds shrinking to zero. A weighted adaptive approach is developed to determine the thresholds in SCWR. We conducted simulations to compare the performance of our improved method and the standard Win Ratio (WR) under different scenarios of follow-up time, association between events, and treatment effect levels. We also illustrate our method by re-analyzing real-world cardiovascular trials. RESULTS/ANTICIPATED RESULTS: First, the developed Shrinking Coarsened Win Ratio (SCWR) method preserves the good statistical properties of the standard WR and has a greater capacity to detect treatment effects on subsequent layer outcomes. Second, the SCWR method outperforms the standard approach under the scenarios in our simulations in terms of gaining higher power. In practice, we expect that SCWR can better detect the treatment effects. Finally, we will offer convenient software tools and clear tutorials for implementing the SCWR method in future studies, which include both unstratified and stratified designs. DISCUSSION/SIGNIFICANCE: The developed SCWR provides a more flexible way of combining the top layer and subsequent layers (e.g., the fatal and non-fatal endpoints) under the hierarchical structure and achieves a higher power in simulation. This nonparametric approach can accommodate different types of outcomes, including time-to-event, continuous, and categorical ones.

OBJECTIVES/SPECIFIC AIMS: While Hb-induced toxicity has been assumed to cause PT dysfunction, the development of tubular proteinuria from this dysfunction in SCD patients is not well understood. We previously found that free Hb, at concentrations predicted to be present chronically and during hemolytic crisis in the tubular filtrate of SCD patients, impairs uptake of albumin by PT cells via direct competition for binding to megalin and cubilin receptors. The purpose of this study is to further evaluate the consequences of increased filtered Hb concentrations on vitamin D reabsorption and activation. METHODS/STUDY POPULATION: We have developed a PT cell culture model that closely mimics in vivo PT cell structure, morphology, and endocytic capacity. Using this model, we treated cells with physiologic levels of cell-free Hb estimated in SCD and measured protein endocytosis and toxicity/oxidative stress. Endocytosis of fluorescently-tagged DBP and RBP were evaluated and quantified by confocal imaging and spectrofluorometry. Cellular toxicity and oxidative stress were assessed by measuring aconitase activity and accumulation of mitochondrial reactive oxygen species. RESULTS/ANTICIPATED RESULTS: PT cell uptake of DBP was significantly inhibited by both concentrations of Hb estimated to be filtered into the tubule lumen under chronic conditions (0.6μM Hb; 39% inhibition) and hemolytic crisis (≤20μM Hb; up to 92% inhibition) in SCD patients. ****p<0.0001 by one-way ANOVA, Dunnett’s multiple comparisons test. PT cell uptake of RBP was minimally affected by the same concentrations of Hb that profoundly inhibited internalization of DBP. RBP uptake was not significantly inhibited by all concentrations of Hb tested except the estimated hemolytic crisis maximum concentration (20μM; 27% inhibition). RBP uptake inhibition at 20μM Hb treatment was dramatically less than DBP uptake inhibition under the same treatment condition (27% RBP inhibition vs 92% DBP inhibition). *p<0.05 by one-way ANOVA, Dunnett’s multiple comparisons test. Mitochondrial oxidative stress, measured as a decrease in aconitase activity, was significantly increased in cells exposed to Hb (~43% aconitase activity reduction after 72h 20μM Hb treatment, and ~11% aconitase activity reduction after 72h 1μM Hb treatment). *p<0.05, **p<0.01 by one-sample t-test of the differences between treatment and untreated control conditions. We are currently assessing changes in PT cell vitamin D hydroxylase expression levels and vitamin D metabolism after exposure to chronic and hemolytic concentrations of Hb. Because oxidative stress has been previously reported to affect hydroxylase expression and activity, we expect to find a decrease in vitamin D hydroxylase expression and/or activity, resulting in decreased vitamin D activation. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that competition for megalin/cubilin binding between Hb and normally-filtered proteins, including DBP, may be the primary cause of tubular proteinuria in SCD patients. This inhibition appears to be selective for proteins that are largely α-helical in structure, such as albumin and DBP. Understanding the structural basis for Hb competition with filtered proteins for PT uptake could identify biomarkers to detect tubular proteinuria in SCD patients prior to the onset of kidney disease. This may also help develop therapeutic compounds that would selectively inhibit Hb binding to megalin/cubilin receptors. We have developed a highly sensitive fluorescence-based assay to test for such compounds. Inhibition of DBP uptake and vitamin D metabolism in the PT could lead or contribute to vitamin D deficiency. To our knowledge, our study is the first to suggest a mechanism for vitamin D deficiency commonly observed in SCD patients. Ongoing studies focus on measuring vitamin D metabolism in both cell and mouse models of SCD.

Objectives/Goals: Diabetic kidney disease (DKD) affects 40% of diabetic patients, leading to renal failure, yet the molecular drivers remain elusive. MicroRNAs, noncoding regulators of gene expression, may hold the key. This study aims to identify key miRNAs in DKD, providing crucial insights for early intervention. Methods/Study Population: miRNA sequencing was conducted on kidneys from 8-week old male BTBR wild type and BTBR ob/ob mice. BTBR ob/ob mice lack the hormone leptin and spontaneously develop type 2 diabetes, with morphological renal lesions characteristic of human DKD. Total RNA was extracted from whole kidney sections and processed using the QIAseq miRNA library kit. Sequencing was performed on an Illumina NextSeq 550 platform. GeneGlobe analysis was used to identify differentially expressed miRNA functional pathways, while ingenuity pathway analysis (IPA) was employed to predict master regulators and causal networks involved in DKD. Results/Anticipated Results: miRNA sequencing identified significantly differentially expressed miRNAs (p < 0.05) between 8-week-old BTBR WT and BTBR ob/ob male mice, including miR-34a (-6.86 fold), miR-122 (-5.01 fold), miR-129 (-2.23 fold), miR-142a (+2.78 fold), miR-346 (+4.66 fold), miR-547 (-2.49 fold), miR-592 (+11.81 fold), miR-802 (-6.95 fold), and miR-6539 (-7.93 fold). Qiagen GeneGlobe analysis revealed biological processes potentially targeted by these miRNAs, including endocytosis, phagocytosis, hyperglycemia (p = 7.59e-3), and insulin-dependent diabetes (p = 4.32e-4). IPA predicted activation of RRAS, a small GTPase regulating cell growth and signaling (Z-score +2), with miR-34a and miR-122 targeting MYC, PI3K, and TGF-β in DKD progression in BTBR ob/ob mice. Discussion/Significance of Impact: We identified kidney miRNA expression in BTBR ob/ob mice at a pivotal disease stage. miR-34a, miR-122, and RRAS emerged as key drivers in DKD progression, showing remarkable early biomarker potential. These findings lay the groundwork for early detection and innovative therapies to halt DKD and improve patient outcomes.

ABSTRACT IMPACT: Racial differences in the prevalence of hypertension and endothelial (dys)function are well established, yet research investigating the mechanism(s) underlying this disparity is still lacking. OBJECTIVES/GOALS: Investigate the influence of race and the effect of serum collected from hypertensive donors on Protein Phosphatase 2A (PP2A) and endothelial nitric oxide synthase (eNOS) expression and activity in human umbilical vein endothelial cells (HUVECs) from Caucasian (CA) and African American (AA) donors. METHODS/STUDY POPULATION: HUVECs from 3 CA & 3 AA donors were cultured in parallel. Experiments were conducted between passages 5-7. At ?90% confluency, cells were serum starved ˜12hrs prior to incubating for 24 or 48 hours in one of the following conditions: 1) Control (Fetal Bovine Serum), 2) serum from normotensives (NT; 5 CA & 5 AA donors), or 3) serum from hypertensives (HT; 5 CA & 5 AA donors). NT and HT serum was pooled from donors with the following characteristics: Male, 30-50 years, nonsmokers, no comorbidities, and non-obese (BMI < 30 kg/m2). Western blotting was used to measure protein expression of total eNOS, p-eNOSS1177, total PP2A, and p-PP2AY307. For activity p-eNOSS1177/total eNOS and p-PP2AY307/ total PP2A ratio was used. A two-way ANOVA was used for statistical analysis. RESULTS/ANTICIPATED RESULTS: Irrespective of the donors’ race, there was no influence of serum treatment or interaction effect in any of the measured proteins of interest. Moreover, compared to CA, HUVECs from AA had lower expression of eNOS irrespective of condition (race p=0.01). Compared to CA, HUVECs from AA tended to have lower expression of p-eNOSS1177 irrespective of condition (race p=0.07). However, there was no racial differences in eNOS activity (p=0.68). There was no racial difference in the expression of PP2A (p=0.35), p-PP2AY307 (p=0.30), or PP2A activity (p=0.97) in all conditions. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our preliminary results suggest no influence serum constituents from hypertensive donors or race on PP2A or eNOS expression and activity in HUVECS. Future research should consider conducting proteomics profiling to compare NT and HT serum.