Machine learning (ML) provides the ability to examine massive datasets and uncover patterns within data without relying on a priori assumptions such as specific variable associations, linearity in relationships, or prespecified statistical interactions. However, the application of ML to healthcare data has been met with mixed results, especially when using administrative datasets such as the electronic health record. The black box nature of many ML algorithms contributes to an erroneous assumption that these algorithms can overcome major data issues inherent in large administrative healthcare data. As with other research endeavors, good data and analytic design is crucial to ML-based studies. In this paper, we will provide an overview of common misconceptions for ML, the corresponding truths, and suggestions for incorporating these methods into healthcare research while maintaining a sound study design.

ABSTRACT IMPACT: Brain networks can be explored by delivering brief pulses of electrical current in one area while measuring responses in other areas, and this describes an open-source novel algorithm to carry out this exploration. OBJECTIVES/GOALS: If we focus on a single brain site and observe the average effect of stimulating each of many other brain sites, visually-apparent motifs in the temporal response shape emerge from adjacent stimulation sites. There are no existing approaches to identify and quantify the spatiotemporal structure of these motifs. METHODS/STUDY POPULATION: Individual stimulation trials are correlated with one another, then a correlation-significance matrix quantifying similarity between stimulation sites is decomposed with non-negative matrix factorization, in which the inner dimension is iteratively reduced. The dimensionality reduction identifies stimulation sites that produce a common elicited temporal response, and linear kernel PCA is applied to obtain the robust profile of this response cluster. RESULTS/ANTICIPATED RESULTS: We describe and illustrate a data-driven approach to determine characteristic spatiotemporal structure in these response shapes, summarized by a set of unique ‘basis profile curves’ (BPCs). Each BPC may be mapped back to underlying anatomy in a natural way, quantifying projection strength from each stimulation site using simple metrics. Our technique is demonstrated for an array of implanted brain surface electrodes in a human patient, and our code is shared at https://purl.stanford.edu/rc201dv0636. DISCUSSION/SIGNIFICANCE OF FINDINGS: This framework enables straightforward interpretation of single-pulse brain stimulation data, and can be applied generically to explore the diverse milieu of interactions that comprise the connectome.

ABSTRACT IMPACT: We aim to understand how LTCCs impact cerebellar function. OBJECTIVES/GOALS: L-type calcium channels (LTCCs) are important in activity-dependent neurite outgrowth, which comprises neurite initiation and elongation. We used cerebellar granule neurons (CGNs) to differentiate between LTCC effects on neurite initiation vs elongation. We also tested cerebellar function in mice lacking specific LTCCs with behavioral assays. METHODS/STUDY POPULATION: CGNs were cultured from 129SvEv mouse pups at P4-P6. Potassum chloride (50mM) was used to stimulate neuronal cultures for 24 hours. Isradipine (20nM) was added to culture medium to inhibit all LTCCs for 1 hour. For Cav1.2 deletion, we crossed Cav1.2 conditional knockout mice (Cav1.2-cKO) to Syn-Cre mice (for deletion in most neurons) or Atoh1-Cre mice (for deletion in CGNs). The Cav1.2-cKO line was maintained on a 129SvEv background. For constitutive Cav1.3 deletion, mice were maintained on a C57BL/6NTac. Behavioral tasks included open field, rotarod, and Erasmus Ladder. Data were analyzed with sexes combined and separated to assess for sex as a biological variable. Studies were analyzed by one-way ANOVA, two-way ANOVA, or generalized linear mixed model, where appropriate. RESULTS/ANTICIPATED RESULTS: CGNs exhibited an increase in neurite initiation but not elongation when stimulated with potassium chloride, consistent with previous reports of activity-dependent neurite outgrowth in this cell type. LTCC inhibition with isradipine blunted KCl-induced neurite initiation. We observed no change in the length of either primary or secondary neurites with isradipine treatment with or without KCl stimulation. In our behavioral experiments, we observed no deficits in open field, rotarod, or Erasmus Ladder when Cav1.2 was deleted in most neurons (driven by Syn-Cre expression) or in cerebellar granule neurons (driven by Atoh1-Cre expression). In contrast, loss of Cav1.3 was associated with impaired motor learning in the rotarod task without evidence of ataxia on Erasmus Ladder. DISCUSSION/SIGNIFICANCE OF FINDINGS: We show a specific role for LTCCs in activity-dependent CGN neurite initiation. While loss of Cav1.2 does not affect motor learning, loss of Cav1.3 does impair motor learning. Our results help expand our understanding of LTCC function in cerebellar neurodevelopment and function.

The critical processes driving successful research translation remain understudied. We describe a mixed-method case study protocol for analyzing translational research that has led to the successful development and implementation of innovative health interventions. An overarching goal of these case studies is to describe systematically the chain of events between basic, fundamental scientific discoveries and the adoption of evidence-based health applications, including description of varied, long-term impacts. The case study approach isolates many of the key factors that enable the successful translation of research into practice and provides compelling evidence connecting the intervention to measurable changes in health and medical practice, public health outcomes, and other broader societal impacts. The goal of disseminating this protocol is to systematize a rigorous approach, which can enhance reproducibility, promote the development of a large collection of comparable studies, and enable cross-case analyses. This approach, an application of the “science of translational science,” will lead to a better understanding of key research process markers, timelines, and potential points of leverage for intervention that may help facilitate decisions, processes, and policies to speed the sustainable translational process. Case studies are effective communication vehicles to demonstrate both accountability and the impacts of the public’s investment in research.

ABSTRACT IMPACT: This research aims to identify changes in visual network function after TBI as a way to define potential therapeutic targets for neuromodulation or neural tissue substrates. OBJECTIVES/GOALS: The objectives of this study are to compare neural activity in the visual cortex following TBI with cortical activity in the uninjured brain. This study aims to characterize functional changes in single neuron activity, spike-field relationships and oscillatory activity. METHODS/STUDY POPULATION: The effects of TBI will be studied by comparing electrophysiologic recordings from Long-Evans rats with a fluid percussion injury (FPI) to rats with a sham injury. Four days after the injury or sham procedure, a laminar probe with multiple electrode contacts will be chronically implanted in the ipsilesional primary visual cortex (V1). Afterwards, rats will be anesthetized weekly for 3 weeks (up to 4 weeks post-injury) to assess visual processing in response to drifting grating visual stimulation. To assess behavioral correlates, neural activity will also be recorded while rats perform a visual discrimination task in an operant, touchscreen chamber twice weekly. Recordings will be analyzed for visually evoked units, unit entrainment to local field potentials (LFPs) and evoked oscillatory activity. RESULTS/ANTICIPATED RESULTS: Consistent with other studies, our preliminary evidence from V1 recordings in naive rats has shown that individual neurons are responsive to visual stimuli, visual stimuli are associated with evoked oscillations and unit activity is correlated with LFPs. While activity of individual V1 neurons in injured animals is expected to recover to resemble activity in uninjured animals over time, patterns of functional organization in the two groups are expected to diverge over time. We anticipate that TBI-associated axonal damage, neuronal loss and changes in synaptic weights will lead to disruptions in the timing of neural activity in V1. These perturbations of neural communication within the visual system are expected to be associated with behavioral deficits in the awake, visual discrimination task. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study helps define how cortical network disruption after TBI. These changes are potential targets for novel TBI therapeutics, including neuromodulation and neural tissue transplantation. Thus, this work lays the groundwork for future studies aimed at mitigating the effects of TBI with rationally designed experimental therapeutics.

ABSTRACT IMPACT: Approximately 15% of US adults have chronic kidney disease with over 700,000 of those in the end stages where treatment options are severely limited to dialysis or kidney transplant; the research presented here will help identify novel strategies that address oxidative imbalance and preserve renal function. OBJECTIVES/GOALS: Chronic Kidney Disease patients often develop secondary cardiovascular disease - Cardiorenal Syndrome Type 4. RNA sequencing data show increased apelin receptor expression in 5/6 nephrectomy rats. The Apelinergic (APJ) system is deemed beneficial in normal physiological systems. Here we explore links between stress and the APJ system in CRS4. METHODS/STUDY POPULATION: In preliminary studies performed in NRK cells, inflammatory cytokines, IL-1β and IL-6, caused increases in apelin receptor transcripts and decreased apelin transcripts, respectfully. The literature describes inflammatory processes that contribute to degradation of many organs (kidneys, heart, and liver) suggesting an oxidative imbalance. To investigate this imbalance within CRS4, three rat cell types’’ H9c2 cardiomyocytes, HII4E hepatocytes, and NRK renal epithelial cells” will be used to assess the role of exogenous apelin on pro- and anti-oxidant levels. Cells will be pretreated with apelin or vitamin E 48 hours prior to the addition of toxins or cytokines (uremic: uric acid and d-galactose or hydrogen peroxide; cytokines: IL-1β and IL-6), to assess pro- and anti-oxidant protein levels via Western Blot. RESULTS/ANTICIPATED RESULTS: We anticipate with toxin or cytokine addition (either uremic, hydrogen peroxide, IL-1β or IL-6) in all cell types, an increase in protein levels for GPX’‘ a known measure of oxidative stress’‘ should be greater than the increases in antioxidants’‘ SOD1 and Catalase. After pre-treatment of vitamin E, GPX protein levels should decrease compared to toxin/cytokine control, while SOD1 and catalase protein levels increase; this coincides with vitamin E inducing antioxidant activity in animals and humans. The anticipated results for this study after exogenous apelin addition should reveal in all three cells types reduced levels of GPX and increased levels of SOD1 and Catalase comparable to the vitamin E treatment. DISCUSSION/SIGNIFICANCE OF FINDINGS: If addition of apelin causes an antioxidant response in all three cells types, this can build on evaluation of the APJ system as a therapeutic option for those with CKD and CRS4 to minimize both inflammatory and oxidative stress. With the data gathered here, we expect to recreate the results in a CKD rat model that highlights these same manifestations.

ABSTRACT IMPACT: Cholecystokinin-B Receptor -Mediates Growth of Hepatocellular Carcinoma with the use proglumide. Proglumide is a non-selective antagonistic drug therefore, strategies that block signaling at the CCK-BR may provide to be a novel therapeutic option for Hepatocellular Carcinoma treatment OBJECTIVES/GOALS: Cholecystokinin (CCK)and gastrin mediate the growth of Hepatocellular Carcinoma (HCC) through CCK-R and interruption of this signaling pathway could decrease HCC. CCK-Receptors are overexpressed in HCC and proliferation may be mediated through CCK-B. Blockade of the CCK-BR with proglumide decreased both growth in vitro and tumor growth in vivo. METHODS/STUDY POPULATION: RNA was extracted from murine Hepa1-6, RIL-175 and human HepG2 cells and was evaluated by qRT-PCR for expression of CCK-AR, CCK-BR and gastrin. CCK-R protein expression was analyzed by flow cytometry. HCC cells were treated in vitro with CCK peptide, the CCK-AR antagonist or the CCK-BR antagonist. Proliferation of selective CCK-R KO cells was compared to that of wild-type cells. To determine the effect of a CCK-R antagonist on tumor growth in vivo two cohorts of mice bearing subcutaneous Hepa1-6 or RIL-175 HCC tumors were treated with an oral bioavailable CCK-R antagonist proglumide or untreated water for 3-4 weeks. The mice bearing Hepa1-6 tumors were placed on a high-fat diet to raise blood CCK levels. Mice bearing RIL-175 tumors were fed standard chow to determine if proglumide could block autocrine growth by gastrin. RESULTS/ANTICIPATED RESULTS: The mRNA expression of CCK-AR, CCK-BR and gastrin were increased 80-90-fold in all HCC cell lines compared to that of normal liver. CCK-BRs were detected on >85% of the cells by flow cytometry. CCK peptide (1nM) stimulated HCC growth in vitro in both wild-type cells and in CCK-AR KO cells but not in CCK-BR KO cells. CCK-BR antagonist blocked CCK-stimulated growth in vitro but the CCK-AR antagonist did not, suggesting that the CCK-BR was responsible for mediating proliferation. In vivo tumor growth was significantly reduced with proglumide treatment by 70% (p<0.05) in Hepa1-6 and by 73% (p<0.001) in RIL-75 tumors, respectively. DISCUSSION/SIGNIFICANCE OF FINDINGS: CCK-Rs are overexpressed in HCC and proliferation appears to be mediated through the CCK-BR. Downregulation with CRISPR Cas9 or blockade of the CCK-BR with an antagonist decreases growth in vitro and proglumide therapy decreases tumor growth in vivo. Strategies that block signaling at the CCK-BR maybe a novel therapeutic option for HCC treatment.

The emphasis on team science in clinical and translational research increases the importance of collaborative biostatisticians (CBs) in healthcare. Adequate training and development of CBs ensure appropriate conduct of robust and meaningful research and, therefore, should be considered as a high-priority focus for biostatistics groups. Comprehensive training enhances clinical and translational research by facilitating more productive and efficient collaborations. While many graduate programs in Biostatistics and Epidemiology include training in research collaboration, it is often limited in scope and duration. Therefore, additional training is often required once a CB is hired into a full-time position. This article presents a comprehensive CB training strategy that can be adapted to any collaborative biostatistics group. This strategy follows a roadmap of the biostatistics collaboration process, which is also presented. A TIE approach (Teach the necessary skills, monitor the Implementation of these skills, and Evaluate the proficiency of these skills) was developed to support the adoption of key principles. The training strategy also incorporates a “train the trainer” approach to enable CBs who have successfully completed training to train new staff or faculty.

ABSTRACT IMPACT: This work may lead to new treatments for crystalline nephropathies. OBJECTIVES/GOALS: This study investigated obeticholic acid (OCALIVA ®) as a potential treatment for 2,8-dihydroxyadenine (2,8-DHA) nephropathy using a mouse model. The treatment was investigated in both sexes at two timepoints. METHODS/STUDY POPULATION: Male and female C57BL/6J mice (12 weeks of age) were fed chow (Research Diets D19120401i) or chow admixed with adenine (0.2% w/w) ad lib for either 3.5 or 7 weeks. Mice were treated with either vehicle (corn oil) or obeticholic acid (10 mg/kg BW) by gavage 5 days per week. Each of the 16 combinations of sex/diet/timepoint/treatment groups had an n = 6 (96 mice in total). Food and body weights were measured twice per week, and 24-hour urines were collected prior to euthanasia. Serum and organs were collected and processed for biochemical and histopathological analyses. RESULTS/ANTICIPATED RESULTS: At both the 3.5-week and 7-week timepoints, dietary adenine robustly increased BUN and serum creatinine compared to control diet in vehicle-treated male and female mice (P < .01, all comparisons). At the 3.5-week timepoint, obeticholic acid reduced BUN in male (P < .05) but not female adenine mice. Obeticholic acid did not affect serum creatinine at this timepoint. At the 7-week timepoint, obeticholic acid reduced BUN in female (P < .05) but not male adenine mice. At the 7-week timepoint, obeticholic acid reduced serum creatinine in both male (P < .05) and female (P < .01) mice. Biochemical and histopathological analyses are ongoing, and we anticipate that the results will agree with the serum chemistries. DISCUSSION/SIGNIFICANCE OF FINDINGS: Obeticholic acid is FDA-approved for primary biliary cholangitis, and it is in clinical trials for several other hepatobiliary diseases. Although currently untested in humans, it is nephroprotective in many preclinical models of kidney disease. This study is the first to investigate obeticholic acid in a model of crystalline nephropathy.

ABSTRACT IMPACT: Reducing methionine levels has repeatedly been shown to reduce cancer growth in vivo, while at the same time increasing lifespan in healthy animals. However, the mechanisms behind the beneficial effects of methionine restriction are currently unknown. OBJECTIVES/GOALS: We hypothesized that comparing the response of a cancer cell line to depletion of the amino acids methionine and cysteine would give us insight into the critical role of these two closely related amino acids in cancer, and help advance methionine restriction on the translational science spectrum. METHODS/STUDY POPULATION: We used the human melanoma cell line A101D to analyze the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion in vitro. We measured proliferation/viability, gene expression patterns, and glutathione levels. We also assessed ferroptotic versus apoptotic cell death. We then used a normal human fibroblast cell line to compare responses. RESULTS/ANTICIPATED RESULTS: The transcription factors ATF4 and NRF2 were activated by all three tested conditions in melanoma cells. Glutathione levels were decreased by ˜40% in cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Inhibiting ferroptotic cell death partially rescued proliferation in cysteine-depleted but not in methionine-depleted cells. Almost 70% of cells grown in methionine-depleted media stained positive for Annexin V, an indicator of apoptosis, compared to only 20% of cells grown in cysteine-depleted media. In normal cells, ferrostatin recued proliferation/viability to 86% of control levels in cysteine-depleted cells. Ferrostatin did not affect methionine-depleted normal cells. DISCUSSION/SIGNIFICANCE OF FINDINGS: These results indicate that methionine depletion leads to apoptosis, while cysteine depletion leads to ferroptosis. We found overlapping pathways activated by methionine and cysteine depletion at the gene expression levels, but divergences in cell death pathways ultimately activated.

ABSTRACT IMPACT: Small molecule readthrough compounds are a promising therapeutic with the potential to overcome nonsense mutations thereby enabling the production of functional ATM protein in patients with Ataxia Telangiectasia OBJECTIVES/GOALS: To generate a novel mouse model of Ataxia-Telangiectasia for testing small molecule readthrough compounds that both expresses a clinically relevant nonsense mutation and recapitulates the major symptoms of the disease, including a progressive loss of motor coordination not previously observed in prior A-T animal models. METHODS/STUDY POPULATION: Using a double-hit strategy to increase genotoxic stress, we generated a novel A-T mouse model that expresses a clinically relevant (c.103C>T) mutation in the Atm gene and a knockout of the functionally related Aptx gene. We then characterized the mouse across multiple domains related to the various symptoms related to the disease. This includes examination of survivability, immunologic function, cancer prevalence, and motor behavior and its associated cerebellar dysfunction and atrophy. Lastly, we tested the ability of small molecule readthrough compounds to enable production of ATM from tissue explants extracted from these ATM deficient mice. RESULTS/ANTICIPATED RESULTS: The double mutant mice display reduced survivability compared to control mice (53% vs. 97%; p<0.0001), dying at a clinically relevant rate of about 30% from thymomas. At postnatal day 400 (P400), only AtmR35X/R35X; Aptx-/- mice, and none of the controls expressing at least one wildtype Atm or Aptx gene develop a motor behavioral deficits that are associate with reduced Purkinje neuron diameter (8.0 ±0.4 µm vs. 9.92 ±0.5; p<0.01) and density (4.3 ±0.2 vs. 6.0 ±0.3 per 100 µm; p<0.05) as well as cerebellar atrophy (cerebellum/forebrain area 0.26 ±0.01 vs. 0.31 ±0.01; p<0.001). ATM deficient mice also display disrupted thymocyte development and metabolic function. When exposed to small molecular readthrough compounds, greater than 50% of the ATM protein is restored. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have created a novel, clinically relevant A-T mouse model that develops a severe ataxia associated with changes in cerebellar function and atrophy as well as demonstrate the potential of SMRT compounds as an A-T therapeutic.

ABSTRACT IMPACT: My work focuses on understanding the role of an immunomodulatory pathway in Alzheimer’s disease as a potential therapeutic target. OBJECTIVES/GOALS: My long-term goal is to develop effective drugs to halt the progression of Alzheimer’s disease (AD). The overall objective of this translational work is to study the deregulation of the Lanthionine Synthetase C-like 2 (LANCL2) immunomodulatory pathway in AD. I hypothesize that stimulation of the LANCL2 pathway will inhibit progression of AD. METHODS/STUDY POPULATION: These studies use a transgenic rat model of AD, and a LANCL2-binding drug, BT-11, that crosses the BBB. Tg-AD Fisher 344 rats express human APP with the Swedish mutation and human presenilin-1 with the ? exon 9 mutation. Tg-AD rats develop age-dependent AD-like pathology. Rodent chow containing BT-11 (10mg/Kg bw) was administered orally from 5 months of age (pre-pathology) to 11 months of age. Eight groups of rats (8 rats/group) were included: WT and Tg-AD x 2 sexes x 2 conditions (BT-11 treated and untreated). Rats are analyzed for: (1) Spatial learning and memory with an active place avoidance test. (2) AD pathology including amyloid plaques and activated microglia, with hippocampal immunohistochemistry. (3) LANCL2-signaling and other AD relevant pathways, with hippocampaI western blot and RNAseq analyses. RESULTS/ANTICIPATED RESULTS: I predict that BT-11 will prevent and/or mitigate some aspects of AD pathology displayed by the AD transgenic rats (Tg-AD). Tg-AD rats treated with BT-11 should perform better in the spatial learning and memory tasks, than the untreated Tg-AD rats. I also expect that the immunohistochemical analysis will reveal reduced pathological hallmarks, including amyloid plaques and reactive microglia, in hippocampal tissue of BT-11 treated versus untreated Tg-AD rats. Through western blot and RNAseq analyses, I will establish how BT-11 treatment affects the LANCL2 signaling cascade as well as other AD relevant pathways, in treated and untreated rats from both sexes and both genotypes. DISCUSSION/SIGNIFICANCE OF FINDINGS: My proposed translational research will address the potential of targeting the LANCL2 pathway to improve AD treatment. Discovering the effects of stimulating the LANCL2 pathway on AD pathology is a novel approach in AD drug development. I expect that my studies with BT-11 will positively influence therapeutic outcomes of this devastating condition.

The availability of large healthcare datasets offers the opportunity for researchers to navigate the traditional clinical and translational science research stages in a nonlinear manner. In particular, data scientists can harness the power of large healthcare datasets to bridge from preclinical discoveries (T0) directly to assessing population-level health impact (T4). A successful bridge from T0 to T4 does not bypass the other stages entirely; rather, effective team science makes a direct progression from T0 to T4 impactful by incorporating the perspectives of researchers from every stage of the clinical and translational science research spectrum. In this exemplar, we demonstrate how effective team science overcame challenges and, ultimately, ensured success when a diverse team of researchers worked together, using healthcare big data to test population-level substance use disorder (SUD) hypotheses generated from preclinical rodent studies. This project, called Advancing Substance use disorder Knowledge using Big Data (ASK Big Data), highlights the critical roles that data science expertise and effective team science play in quickly translating preclinical research into public health impact.

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.

For the past 4 years, as part of the National Institutes of Health (NIH) Clinical and Translational Science Award (CTSA) grant award number UL1TR001436, the Clinical Translational Science Institute of Southeast Wisconsin (CTSI) has used process engineering approaches to identify and understand barriers that local researchers and other stakeholders face when engaging in clinical and translational science. We describe these approaches and present preliminary results. We identified barriers from published and unpublished work at other CTSA hubs, supplemented by surveys and semi-structured interviews of CTSI faculty. We then used a multifaceted approach to organize, visualize, and analyze the barriers. We have identified 27 barriers to date. We ranked their priority for CTSI to address based on the barrier’s impact, the feasibility of intervention, and whether addressing the barrier aligned with CTSI’s institutional role. This approach provides a systematic framework to scope and address the “barriers to research problem” at CTSI institutions.