OBJECTIVES/GOALS: We recently identified a CA-MRSA strain in Brooklyn, New York (USA300-BKV) causing an outbreak of severe skin infections in predominantly healthy children. The evolution of USA300-BKV included acquisition of a novel prophage, and our objective is to identify the prophage-encoded gene(s) and mechanism responsible for increased bacterial virulence. METHODS/STUDY POPULATION: We deleted candidate genes from a novel mosaic block of phage-encoded genes in USA300-BKV that have been shown to enhance virulence in a murine skin infection model. Deletion mutants and complemented clones will be evaluated in vivo to identify culprit genes and determine the effect of lineage-specific genetic variation on the phenotype. Complementary studies include a comprehensive characterization of phage and bacterial genes expressed during lysogeny in vitro using RNA sequencing (RNA-Seq), and in vivo using a targeted approach focusing on known bacterial virulence and phage lytic pathways as well as candidate genes identified by in vitro studies. RESULTS/ANTICIPATED RESULTS: Comparison of otherwise isogenic lab strains showed that the mosaic block of phage genes present in USA300-BKV enhance skin abscess size in mice, confirming previous results. As this region of the phage, named mΦ11, does not contain known toxin genes, we hypothesize that mΦ11 modulates expression of bacterial host genes to enhance virulence. Thus, transcriptional profiles of CA-MRSA containing mΦ11 and selected deletion mutants are expected to reveal changes in known or novel virulence factors compared to controls. Candidate regulators specific to the mosaic block include an adenine methyltransferase linked to changes in global gene expression of other bacterial species. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results will broaden scientific understanding of phage-bacterial interactions and determine the mechanisms by which phage impact virulence independent from toxin gene carriage. Identification of phage-encoded gene(s) enhancing CA-MRSA contagion will inform surveillance efforts and identify novel therapeutic targets.

Objectives/Goals: Pharmacogenomic (PGx) testing identifies genetic variations affecting medication response but is not yet in routine clinical whole-genome sequencing (WGS) workflows. We aimed to establish a streamlined bioinformatics pipeline for incorporating PGx reporting into clinical WGS and to determine clinical implications for medication treatment. Methods/Study Population: A PGx profiling pipeline based on existing WGS data was developed, integrating three WGS-based PGx calling tools: Aldy, PyPGx, and Cyrius (CYP2D6 only), to provide genotype calls for 17 key pharmacogenes. The pipeline was validated using WGS data from 70 individuals with diverse backgrounds (36% European, 27% African, 27% Asian, and 10% admixed) from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results were manually reviewed against published data. The validated pipeline was then applied to 144 clinical patients previously screened for neurodevelopmental disorders or suspected hereditary diseases, followed by diplotype-to-phenotype translation and preemptive PGx-guided medication recommendations based on consensus guidelines and FDA labeling for commonly used medications. Results/Anticipated Results: Congruent phenotype call rates for GeT-RM samples were 100% for 13 genes (CFTR, CYP2B6, CYP2C19, CYP2C9, CYP3A4, CYP4F2, DPYD, G6PD, IFNL3, NAT2, NUDT15, TPMT, and VKORC1), 99% for three genes (CYP3A5, SLCO1B1, UGT1A1), and 97% for CYP2D6, indicating strong pipeline performance. Among 144 clinical patients, 99.3% had at least one clinically actionable PGx results relevant to 36 of top 300 medications in the USA across psychotropic, cardiovascular, musculoskeletal, gastrointestinal, and other therapeutic areas. The most prevalent drug–gene interactions involved sertraline and CYP2B6, affecting 49% patients: 41% were intermediate metabolizers who may require slower titration and lower maintenance doses, while 8% poor metabolizers may benefit from a lower starting dose or alternative antidepressants. Discussion/Significance of Impact: Our validated WGS-based PGx profiling pipeline successfully extracted actionable PGx data from clinical WGS. By aligning PGx profiles with guideline-recommended clinical actions, we demonstrated the clinical value of integrating PGx reporting in WGS workflows, improving personalized medication management.

OBJECTIVES/SPECIFIC AIMS: 1) Determine the mutational landscape, including translocation, mutations and mutational signatures as well as copy number variations of pPCL and identify significant differences to non pPCL MM. 2) Determine whether genetic changes pertinent to pPCL could be explored as therapeutic targets to improve the dismal prognosis of this patient population. METHODS/STUDY POPULATION: Samples from overall 19 pPCL patients that presented to the Myeloma Center, UAMS between 2000-2018 were used for this study. We performed gene expression profiling (GEP; Affymetrix U133 Plus 2.0) of matched circulating peripheral PCs and bone marrow (BM) PCs from 13 patients. Whole exome sequencing (WES) was performed on purified CD138+ PCs from BM aspirates from 19 pPCL patients with a median depth of 61x. CD34+ sorted cells, taken at the time of stem cell harvest from the same 19 patients, were used as controls. Translocations and mutations were called using Manta and Strelka and annotated as previously reported. Copy number was determined by Sequenza. RESULTS/ANTICIPATED RESULTS: 1) GEP from the BM and circulating peripheral PCs showed that the expression patterns of the two samples from each individual clustered together, indicating that circulating PCs and BM PCs in pPCL result from the same clone and are biologically clearly related. 2) The clinical characteristics from the patient cohort used for WES analysis were as follows: median age was 58 years (range 36–77), females accounted for 74% (14/19), an elevated creatinine level was found in 78% (14/18) and an elevated LDH level in 71% (10/14). All patients presented with an ISS stage of III. Median OS of the whole dataset was poor at 22 months, which is consistent with OS from previously reported pPCL cohorts. 3) Primary Immunoglobulin translocations were common and identified in 63% (12/19) of patients, including MAF translocations, which are known to carry high risk in 42% (8/19) of patients [t(14;16), 32% and t(14;20), 10%] followed by t(11;14) (16%) and t(4;14) (10%). Furthermore, 32% (6/19) of patients had at least one MYC translocation, which are known to play a crucial role in disease progression. 4) The mutational burden of pPCL consisted of a median of 98 non-silent mutations per sample, suggesting that the mutational landscape of pPCL is highly complex and harbors more coding mutations than non-pPCL MM. 5) Driver mutations, that previously have been described in non-pPCL MM showed a different prevalence and distribution in pPCL, including KRAS and TP53 with 47% (9/19) and 37% (7/19) affected patients respectively compared to 21% and 5% in non-PCL MM. PIK3CA (5%), PRDM1 (10%), EP300 (10%) and NF1 (10%) were also enriched in the pPCL group compared to previously reported cases in non-pPCL MM. 6) Biallelic inactivation of TP53 – a feature of Double Hit myeloma - was found in 6/19 (32%) samples, indicating a predominance of high risk genomic features compared to non-pPCL MM. Furthermore, analysis of mutational signatures in pPCL showed that aberrant APOBEC activity was highly prevalent only in patients with a MAF translocation, but not in other translocation groups. DISCUSSION/SIGNIFICANCE OF IMPACT: In conclusion we present one of the first WES datasets on pPCL with the largest patient cohort reported to date and show that pPCL is a highly complex disease. The aggressive disease behavior can, at least in part, be explained by a high prevalence of MAF and MYC translocations, TP53 and KRAS mutations as well as bi-allelic inactivation of TP53. It is of interest that only KRAS but not NRAS mutations are highly enriched in pPCL. From all highly prevalent genomic alterations in pPCL, only KRAS mutations offer a potential for already available therapeutically targeting with MEK inhibitors, which should be further explored.

OBJECTIVES/SPECIFIC AIMS: The overall goal of this project is to understand the genetic and clinical differences in POAG that specifically increase risk in individuals of African genetic ancestry. We will approach this goal by completing the following objectives: (i) localize a genetic signal that accounts for the significantly increased risk for primary open-angle glaucoma in African Americans and (ii) utilize electronic health records (EHR) data to expand our understanding of risk to incorporate endophenotypes of glaucoma and other clinically recorded variables that may influence disease risk. METHODS/STUDY POPULATION: We will genotype at least 200 available African American samples with glaucoma on the Illumina Infinium® Expanded Multi-Ethnic Genotyping Array (MEGAEX) and perform admixture mapping. We will then access EHR data to expand our analysis beyond glaucoma to encompass other relevant risk modifiers captured in the clinical record. RESULTS/ANTICIPATED RESULTS: We anticipate localizing a genetic signal or signals that may account for the increased POAG risk in African Americans. Our calculations indicate that we have ~81% power to detect association at a LOD score of 2 and a risk ratio of 2. Thus, we are well-powered to detect a true signal at this modest level of association DISCUSSION/SIGNIFICANCE OF IMPACT: This project will not only help to achieve precision medicine by filling in the gaps in knowledge regarding glaucoma in African Americans, but it will also address health disparities and aid in the realization of the full potential of “big data” so that all of these elements can be incorporated into a better understanding of health disparities.

OBJECTIVES/SPECIFIC AIMS: Gliomas are the most lethal and commonprimary tumor type in the central nervous system across all age groups; affectedadults have a life expectancy of just 14 months. As glioma cells invade thesurrounding normal parenchyma they remodel the composition and ultrastructure ofthe surrounding extracellular matrix (ECM), suggesting that the native (i.e.,“normal”) microenvironment is not ideal for their survivaland proliferation. Recent reports describe suppressive and/or lethaleffects of mammalian ECM hydrogels derived from normal (nonneoplastic) sourcesupon various cancer types. ECM-based bioscaffolds placed at sites of neoplastictissue resection in humans have never been reported to facilitate cancerrecurrence. The objective of the present research is to evaluate mammalian ECMas a novel approach to glioma therapy. METHODS/STUDY POPULATION: ECMhydrogels from porcine dermis, small intestine, and urinary bladder wereproduced as described previously. Primary glioma cells were graciously suppliedby Drs. Nduka Amankulor and Johnathan Engh, and U-87 MG were ordered throughATCC. Cells were plated onto tissue culture plastic at~60% confluence and allowed to attach for 24 hours beforetreatment. The saline-soluble fraction (SSF) of ECM was obtained by mixinglyophilized, comminuted ECM with 0.9% saline for 24 hours thenfiltering the resulting mixture through a 10 kDa molecular weight cutoff column.All assays and kits were followed according to the manufacturer’sinstructions. Cell viability was measured via MTT assay(Vybrant® MTT Cell Proliferation Assay, Invitrogen)and by live/dead staining(LIVE/DEAD® Cell Imaging Kit, Invitrogen). Timelapse videos were created by taking images every 20 minutes for 18 hours(phase-contrast) or every 10 minutes for 12 hours (darkfield). NucView reagentwas ordered from Biotium. Temozolomide was ordered through Abmole. All in vivowork was conducted according to protocols approved by the University ofPittsburgh’s IACUC office. RESULTS/ANTICIPATED RESULTS:ECM hydrogels derived from porcine dermis, small intestine, or urinary bladderall decreased the viability of primary glioma cells in vitro, with urinarybladder extracellular matrix (UBM) having the most dramatic effects. The SSF ofUBM (UBM-SSF), devoid of the fibrillar, macromolecular components of ECM, wassufficient to recapitulate this detrimental effect upon neoplastic cells invitro and was used for the remainder of the experiments described herein. In acell viability assay normalized to the media treatment, non-neoplastic CHME5 andN1E-115 cells scored 103% and 114% after 48 hours whentreated with UBM-SSF and 2 primary high-grade glioma cell types scored17% and 30.5% with UBM-SSF (n=2).Phase-contrast time-lapse video showed CHME5 and HFF thriving in the presence ofUBM-SSF for 18 hours while most primary glioma cells shriveled and died withinthis time. Darkfield time-lapse video of wells containing Nucview dye,fluorescent upon cleavage by active caspase-3, confirmed that within 12 hoursmost primary glioma cells underwent apoptosis while CHME5 and HFF did not. Inculture with primary astrocytes, high grade primary glioma cells, and U-87 MGglioma cells for 24 hours, UBM-SSF was found to significantly increase thepopulation of primary astrocytes compared with media(p<0.05) while decreasing the 2 glioma cell types toapproximately one-third as many cells as the media control(p<0.0001). A dose-response of temozolomide from 0to 10,000 μM showed that when treating 2 non-neoplastic cell types(CHME5 and HFF) and 2 types of primary glioma cell there was no difference insurvivability at any concentration. Contrasted to this, a dose-response ofUBM-SSF from 350 to 7000 μg/mL showed that thenon-neoplastic cells survived significantly better than the glioma cells atconcentrations of 875 μg/mL and upward(p<0.05). In preliminary animal experiments, largeprimary glioma tumors in the flanks of athymic nude mice were resected andreplaced with either UBM SSF or Matrigel (an ECM product of neoplastic cellorigin). After 7 days the resection sites with UBM-SSF had little tumor regrowthif any compared with the dramatic recurrence seen in the Matrigel injectionsites (n=2). In a separate survival study comparing PBS to UBM-SSFinjections in the flank-resection model, all animals given PBS had to besacrificed at 9, 11, and 11 days (n=3) whereas animals given UBM-SSFwere sacrificed at 15, 24, and 39 days (n=3), indicating a moderateincrease in survival due to the UBM-SSF. DISCUSSION/SIGNIFICANCE OFIMPACT: Since the introduction of the pan-cytotoxic chemotherapeutic agent TMZin 2005, the standard of care for patients with glioblastoma multiforme has notimproved. These findings indicate that non-neoplastic ECM contains potentbioactive regulators capable of abrogating malignancy. Our in vitro data suggestthese molecules appear to have no deleterious effect on non-neoplastic cellswhile specifically inducing apoptosis in glioma cells. Our in vivo data suggestthat these molecules may be useful in delaying glioma recurrence, thus resultingin extended lifespan. Delivering soluble fractions of ECM to a tumor site mayrepresent a novel approach to glioma therapy, sidestepping traditional cytotoxictherapies in favor of utilizing putative endogenous anti-tumor pathways.

A clinical and translational scientist (CTS) often seeks to increase their knowledge of statistical topics to effectively conduct biomedical research studies. A common method for obtaining this knowledge is through existing online educational materials that are suggested by a biostatistical collaborator or identified by the CTS. However, the volume of available educational materials on diverse statistical topics makes the task of identifying high-quality educational resources at an appropriate level challenging and time consuming for CTSs and collaborative biostatisticians. In response to these challenges, the Biostats4You website was created, where existing online educational materials for a variety of statistical topics are vetted to identify those most appropriate for CTSs. In this manuscript, we describe the resource review process, provide information about statistical topics and resources currently available, and make recommendations for how CTSs and collaborative biostatisticians can utilize the Biostats4You website to improve training, mentoring, and collaborative research practices.

Persistent barriers exist to engage rural providers in research and training. Provider shortages exacerbate these challenges, leading to a scarcity of time and limiting motivation to participate in research. We present application of an innovative engagement model to increase rural primary care provider participation in research. Using our Community Engagement and Research Core, we demonstrate that fundamental principles of training and expertise, attention to efficiency and multitasking, and commitment to community are important for addressing provider recruitment barriers. We encourage other Clinical and Translational Science Centers to provide similar services to their local investigators to enhance provider engagement in research.

This exploratory study investigated perceptions of competent vs. contentious communication in the workplace as experienced by Clinical Research Professionals (CRPs) managing or coordinating clinical research. Qualitative data collected from a 90-min focus group interview were thematically analyzed using open and axial coding and constant comparison. Findings suggest CRPs associate contentious communication with uncertainty, stress, and emotional labor. Further, although many participants regularly utilize effective conflict and emotion management strategies, they lack confidence in both knowledge and efficacy of competent communication, stress management, and emotion management skills. Conclusions support revising “Wheel of Competencies” figure representing the Joint Task Force for Clinical Trial Competency framework. Study limitations and suggestions for future research and educational training are discussed.

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.

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/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: 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.

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: 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: 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: 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.