OBJECTIVES/SPECIFIC AIMS: The objective of this partnership was to create a global network of clinical and public health researchers and communities conducting technology-assisted research in noncommunicable disease. METHODS/STUDY POPULATION: The University of Rochester’s Clinical and Translational Science Institute (CTSI) has successfully leveraged the informatics core’s capacity into an emerging network of organizations that focus on technology and health in settings outside of the mainland United States. The CTSI coordinated with another NIH-funded infrastructure program [the RCMI Translational Research Network (RTRN)] to identify partner institutions interested in technology and health. RTRN identified the University of Puerto Rico and the University of Hawaii, both of which serve as hubs for common research interests in technology and health throughout the Caribbean and the Pacific. This network was formalized as the CDC’s Coordinating Center for its Global and Territorial Health Research Network (the “Global Network”), with additional US partners (Yale, University of Illinois at Chicago, University of North Caroline Chapel Hill, and the University of South Florida) within a wider scope of the CDC’s Prevention Research Centers (PRC) program. RESULTS/ANTICIPATED RESULTS: Through combining 2 main NIH-funded research infrastructure networks (CTSA and RTRN), with a large CDC-funded PRC, the University of Rochester’s Informatics Core was successful in establishing a new productive global health network throughout Latin America and the Caribbean, and in the Pacific, garnering additional research support from NIH Fogarty and other programs. The resulting network not only supports locally-important research in technology and health on compelling health issues (eg, diabetes, ZIka, participation in research), but also facilitates community engagement through local partnerships and the cores of the involved networks. In addition, much of the information and communications technology (ICT)-related research and learnings from the Global Network activity is immediately applicable to populations in the United States, served by the various collaborative networks. In total, while new, the Global Network supports a wide range of projects and engagements throughout the world that expand local informatics capacity and use of technology in the research process and to address global health problems, further enhancing the CTSI’s informatics core to serve the needs of its own constituency and promote research engagement with technology within this population. Local research collaborative projects reinforce the utility of the network and its resources, evidenced by tools, publications, partnerships, and conference presentations that have arisen. Lessons to date from this Global Network collaboration include: specific global research projects provide opportunities for partnership building and meaningful collaboration, team science is of central importance in distributing the work of the network, synergy is multidirectional with expertise and need flowing in all directions, and project team members in all locales learned and contributed substantially in ways that carried into their other responsibilities. DISCUSSION/SIGNIFICANCE OF IMPACT: The overall partnership has created opportunity for South-South collaboration, for adaptation of projects among locales, and has helped boost reputational value for all partners involved. Implications for other CTSA awardees include: global collaboration can serve core research and technical needs for the CTSA itself and its local partners, CTSA status can be leveraged to access resources to support local research, and collaboration in other federally-funded research networks helps expand the insight, scope, and potential for new research.

OBJECTIVES/GOALS: Clinical trials of SGLT2 inhibitors in patients with heart failure (HF) have confirmed a reduction in hospitalization and death. Adoption of novel therapeutics has been slower in Black and female patients. We investigated utilization of SGLT2 inhibitor in patients with HF and type 2 diabetes and if there were utilization differences by race or gender. METHODS/STUDY POPULATION: We created a retrospective cohort of outpatients with HF at Emory Healthcare from 2015 to 2020. Additional inclusion criteria included presence of heart failure and a diagnosis of T2D. SGLT2 inhibitor use was identified by a presence of SGLT2 inhibitor prescription at the time of the clinic visit. We estimated differences in prescription of SGLT2 inhibitors by race and gender using Chi-square analysis. RESULTS/ANTICIPATED RESULTS: The cohort included 5829 patients, age 69.47 years ± 13.44, 47.67 % female, 54.62% Black. Overall prescription of SGLT2 inhibitors was low but increased over time (1.4% in 2015 to 5.6% in 2020; p<0.0001). On average, SGLT2 inhibitor use increased annually by 44.77%. From 2015 to 2020, fewer female than male patients were on an SGLT2 inhibitor (1.94% vs. 2.73%, p=0.0033). A similar percentage of Black and non-Black patients were on an SGLT2 inhibitor (2.13% vs. 2.64%, p=0.0591). DISCUSSION/SIGNIFICANCE: Prescription rates of SGLT2 inhibitors remain low in patients with T2D and HF, especially for female patients, despite evidence of their benefit on hospitalizations and mortality. Implementing use of SGLT2 inhibitors in this population represent an opportunity to improve cardiovascular outcomes.

Objectives/Goals: Unsafe sleep practices contribute to sleep-related infant mortality, a leading cause of preventable sudden unexplained infant death (SUID). Infant illness represents a risk for SUID. However, the mechanism behind this increased risk is unknown. The objective of this study was to measure changes to safe sleep practices during infant illness. Methods/Study Population: We performed a prospective cohort survey study of infants aged 0–12 months presenting to the pediatric emergency department. We assessed sleep practices prior to illness, during illness, and 2 weeks and 1 month following illness. We assessed adherence to American Academy of Pediatrics safe sleep recommendations at each point in time. Wilcoxon sign rank test was used to examine changes between time points. Regression models compared caregivers who reported a change to unsafe sleep practices with those who did not. Results/Anticipated Results: Of enrolled participants (n = 142), 110 (77%) completed all three follow-up surveys. For those with complete follow-up, 62.4% were female, 60.3% were Black, non-Hispanic, 25.7% were White, non-Hispanic. The most common chief complaint was respiratory illness (35.7%), followed by fever (22.7%), and 70.3% of patients were discharged home. Across all sleep behaviors surveyed, caregivers reported, on average, a 12% change to unsafe sleep practices during illness. These changes were sustained at the 2-week and 1-month follow-ups. Factors associated with a change to unsafe sleep practice were parental age. Discussion/Significance of Impact: Over 10% of infants experienced a change to unsafe sleep practices during illness, sustained at 2-week and 1-month follow-ups. This may explain the association of infant illness with SUID. Interventions promoting safe sleep adherence during illness are key to decreasing sleep-related infant mortality.

Objectives/Goals: In neurological animal research, statistical misapplication may lead to overoptimism in a therapy’s potential for successful translation. This pilot study investigated whether human clinical trials that fail have higher prevalence of statistical misapplication in preceding animal experiments, compared to human trials that succeed. Methods/Study Population: Phase 2 clinical trials for 3 neurological conditions were identified on ClinicalTrials.gov and classified as successful or failed based on advancement to Phase 3 and/or preplanned efficacy test results. PRISMA guideline methods were used to systematically search for preclinical animal experiments (same indication and intervention) preceding the start of the human trial. Data were gathered from animal articles by collectors blinded to human trial outcome and included items describing reporting transparency, experimental design and sample sizes, and statistical tests applied. Statistical mistakes were coded based on mismatch between test and design. Rates of mistakes were compared between articles preceding successful and non-successful human trials using weighted point estimates and 95% confidence interval. Results/Anticipated Results: The final sample included 24 trials (16 successful) and 70 associated animal studies. Transparency was poor, with infrequent reporting of group allocation method (39%), sample sizes adequate to evaluate attrition (Discussion/Significance of Impact: Statistical misapplication is common in animal research, and this pilot study has demonstrated that preclinical statistical mistakes may indeed occur more frequently prior to failed human trials. Mistakes and lack of transparency may lead to overoptimism in preclinical experimental findings, with consequences for subsequent human translation.

OBJECTIVES/GOALS: Bronchiolitis is a major cause of PICU admission, yet identifying best practices is limited by the lack of existing databases containing the needed demographic and clinical variables. We used probabilistic linkage to join the Virtual Pediatric System (VPS) and Pediatric Health Information Systems (PHIS) databases to overcome this data barrier. METHODS/STUDY POPULATION: We performed a single site study joining VPS and PHIS data. These national databases contain clinical (VPS) and billing and resource use (PHIS) data. Limits on the use of patient identifiers (PI) for multi-center research, makes direct linkage techniques impossible. To demonstrate that probabilistic linkage can accurately link VPS and PHIS records, we obtained our single site VPS and PHIS records and linked them using probabilistic linkage without PI and compared this to a gold standard linkage created with PI. We also compared demographic features of linked and unlinked records to assess the ability of probabilistic linkage to create a representative sample. RESULTS/ANTICIPATED RESULTS: We obtained 920 VPS records of patients with bronchiolitis and linked 91% (839/920) to a PHIS record with 4 (0.5%) false-positive matches. Characteristics of linked and unlinked records are compared in Table 1. Comparison of probabilistically linked and unlinked records showed no difference in median age in years (0.7 [Interquartile range (IQR) 0.3-1.5] v 0.7 [IQR 0.2-1.5], p = 0.76), median number of complex chronic conditions (0 [IQR 0-1] v 0 [IQR 0-1], p = 0.16), median Pediatric Index of Mortality 3 severity of illness scores (-4.6 [IQR -4.7 – 4.4] v -4.6 [IQR -4.7 – 4.4], p = 0.44), median days of PICU stay (4 [IQR 3-6] v 4 [IQR 2-6], p = 0.36), proportion female ( 44% v 46%, p = 0.82), or proportion of patients intubated (28% v 24%, p = 0.41). DISCUSSION/SIGNIFICANCE: Probabilistic linkage creates an accurate combined VPS-PHIS database. Extending our methodology to join data from all 38 hospitals contributing to VPS and PHIS will allow creation of a large database containing the demographic, treatment, and outcome data needed to enable Comparative Effectiveness Research of bronchiolitis care.

OBJECTIVES/GOALS: 1. Establish the prevalence of amenorrhea among women Ebola survivors of reproductive age. 2. Determine whether amenorrhea in Ebola survivors is associated with hyperthyroidism, primary ovarian failure, and/or low weight METHODS/STUDY POPULATION: This study will enroll a cohort of 150 women Ebola survivors and 150 uninfected controls from Kenema, Sierra Leone. Participants will complete women’s health questionnaires with detailed menstruation history and provide blood samples. Serum levels of thyroid stimulating hormone, follicle stimulating hormone, and albumin will be measured in order to explore the role of thyroid dysregulation, ovarian failure, and low weight in amenorrhea associated with prior Ebola infection. RESULTS/ANTICIPATED RESULTS: Enrollment for the study is still in progress. As of November 14, 2022, 68 female Ebola survivors of reproductive age and 124 uninfected controls have been enrolled. Among this preliminary group, there is a high baseline level of menstrual irregularities in both survivors and controls. Prior to the Ebola outbreak, 97% of all participants reported less than 6 periods per year and 59% reported periods lasting 3 days or less. The prevalence of missed periods increased after the Ebola outbreak in both groups. Four Ebola survivors (5.9%) reported missing periods before infection, compared to 16 (23.5%) survivors after infection. Two uninfected controls (1.6%) reported missing periods before the Ebola outbreak, compared to 12 (9.7%) after the outbreak. DISCUSSION/SIGNIFICANCE: The next steps of this project are to complete enrollment, conduct data analysis, and perform laboratory studies. An enhanced understanding of amenorrhea is needed to develop novel medical interventions, to inform healthcare guidelines and policies, and to develop personalized treatment strategies to better care for women who have survived Ebola.

OBJECTIVES/SPECIFIC AIMS: We have reported that radiofrequency renal denervation (RF-RDN) in SHR at 20-weeks of age, decreased blood pressure (BP) and fibrosis in kidney cortex and medulla when rats were sacrificed at 6 months. However, whether RF-RDN can have similar benefits in older rats remains unknown. This study examined whether performing RF-RDN in older rats also has a beneficial effect on BP and renal fibrosis. METHODS/STUDY POPULATION: Baseline systolic and diastolic BP (SBP/DPB) was measured (telemetry) in nine-month-old SHR and Wistar Kyoto rats (WKY). Groups of rats then received bilateral RF-RDN or Sham-RDN (SHR-RDN, n=9; SHR-Sham, n=10; WKY-RDN, n=5; WKY-Sham, n=8). Rats were then sacrificed at 12-months of age. Kidneys were harvested, sectioned, and assessed for fibrosis by Masson’s trichrome stain. A pathologist, who was blinded to treatment groups, evaluated each kidney section for fibrosis. RESULTS/ANTICIPATED RESULTS: Compared to SHR with Sham-RDN, RF-RDN prevented a further increase in systolic and diastolic BP from baseline (9-month) in SHR as they aged to 12-months (SHR-Sham mmHg: 9-month 193±4/127±4; 12-month 207±3/142±5; SHR-RDN mmHg: 9-month 197±3/132±2; 12-month 197±4/132±3). RF-RDN did not alter SBP or DBP in aged WKY. One-year-old SHR with prior Sham-RDN showed extensive renal fibrosis in kidney cortex and medulla. In contrast, RF-RDN significantly decreased renal fibrosis in the medulla, but not cortex. There was no fibrosis in kidneys of age matched WKY. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that RF-RDN may be a potential therapy for halting progression of hypertension and decreasing medullary fibrosis in the aged population.

ABSTRACT IMPACT: Novel adipokines like tetranectin help explain why some people progress from obesity to diseases like diabetes, atherosclerosis, and dislipidemia OBJECTIVES/GOALS: Obesity has an established association with diabetes, dyslipidemia, and atherosclerosis. Preventing progression from obesity to insulin resistance requires understanding of the regulatory mechanisms involved in the loss of insulin sensitivity. Adipose tissue is well known to function as an endocrine organ that produces many kinds of adipokines. METHODS/STUDY POPULATION: Blood sample analysis from human patients and mice was used to determine associations between tetranectin and obesity. Samples were tested with a monoclonal anti-tetranectin antibody for detection with western blot. A tetranectin mutant knock out mouse line was compared to wild type littermates on high fat diet for 4 months. Insulin tolerance tests and glucose tolerance were used to determine progression to insulin resistance and glucose intolerance. Histological analysis of metabolic tissue was used to demonstrate adipocyte hypertrophy and liver steatosis. RESULTS/ANTICIPATED RESULTS: In the current study, we report the identification and initial characterization of a novel adipokine tetranectin. Tetranectin, which is coded by the C-type lectin domain family 3 member B (CLEC3B) gene, is ubiquitously expressed in various mouse tissues, whereas it is highly enriched in white adipose tissue. We found that the serum level of tetranectin was much higher in both obese and diabetic patients. Knocking out the tetranectin gene in mice protected against glucose intolerance in males but reduced insulin and glucose tolerance in females, without effects on food intake and body weight for either sex. Mechanistically, tetranectin targets liver tissues and its deficiency increases lipid accumulation in hepatocytes in females. DISCUSSION/SIGNIFICANCE OF FINDINGS: We have identified a novel adipokine which mediates a different metabolic crosstalk among tissues to maintain systemic glucose and lipid metabolism in different genders. Further investigation of tetranectin’s function could yield a new target for precise therapeutic treatment for obesity and its associated metabolic diseases in different genders

OBJECTIVES/SPECIFIC AIMS: These studies seek to evaluate hormonal regulation of luminal breast cancer lipid metabolism and to identify targetable progesterone-mediated changes in lipid biology that contribute to therapeutic resistance in breast cancer. METHODS/STUDY POPULATION: Established and patient-derived luminal breast cancer cell lines, which express ER and PR, were used for this study. RNA transcript and protein expression levels were evaluated by qRT-PCR and immunoblot, respectively. Broad scale lipidomics of progesterone-treated cells was conducted via ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS) through the UCD Skaggs School of Pharmacy Mass Spectrometry Core. RESULTS/ANTICIPATED RESULTS: Data mining of previously published microarray data of CK5+ and CK5− syngeneic cancer sublines revealed that CK5+ cells have increased expression of lipid processing genes, including LPL and PPARG. As progestin treatment induces a subpopulation of cells to turn on CK5 expression in luminal breast cancers, UHPLC-MS-based lipidomics analysis will expose whether modulation of the lipid landscape occurs in all cells with progesterone treatment, or whether this phenomenon is heightened specifically in CK5+ cells. I also expect that ER+ breast cancers with progestin induced-altered lipid content, such as lipid droplet formation, will evade therapy-induced death. DISCUSSION/SIGNIFICANCE OF IMPACT: There are numerous approved and developmental therapeutics targeting lipid biology. By determining if progestins alter lipid metabolic genes specifically in CK5+ CSCs, which are endocrine resistant, strategies may be devised to target these resistant cells using combination therapy in conjunction with existing therapies to prevent tumor recurrence.

OBJECTIVES/GOALS: Social anhedonia is considered a key feature of schizophrenia that leads to social withdrawal. Research in general anhedonia suggests those with schizophrenia exhibit deficits in anticipated (predicted), but intact consummatory (in-the-moment), pleasure. This study will determine if these temporal differences apply to daily social anhedonia. METHODS/STUDY POPULATION: This project will use experience sampling methods (ESM) to measure real-world social pleasure in people with schizophrenia and healthy controls. Using electronic surveys, participants will predict social activities they plan to do throughout their day and report anticipated pleasure for each. Then, in subsequent surveys, participants will report their consummatory social pleasure shortly after engaging in each of these activities. We will use a time-lagged approach to match anticipated/consummatory pleasure ratings for specific social events and compute a difference score to determine discrepancy. Multi-level modeling will be used to determine if clinical status (schizophrenia/control) predicts anticipated pleasure, consummatory pleasure, and/or their discrepancy. RESULTS/ANTICIPATED RESULTS: Data collection for this project was recently completed. The schizophrenia and control groups (n = 30 per group) were demographically matched for age, sex, race, and ethnicity. Data processing and analyses are currently underway. We hypothesize that, in line with laboratory research in general anhedonia, those with schizophrenia will exhibit deficits in anticipated, but not consummatory, social pleasure throughout their daily lives. Moreover, when anticipated and consummatory pleasure ratings for the same social activities are matched, we expect those with schizophrenia to exhibit larger discrepancies between predicted and in-the-moment pleasure. DISCUSSION/SIGNIFICANCE: This project serves as a critical intermediate step to bridge the gap between laboratory research and patient treatment. ESM bypasses limitations of laboratory studies and increases ecological validity. Results will provide a nuanced understanding of social anhedonia and help identify precise targets to treat social withdrawal in schizophrenia.

OBJECTIVES/SPECIFIC AIMS: To learn the edit distance costs of a symbolic univariate time series representation through a stochastic finite-state transducer to predict patient outcomes in intensive care units. METHODS/STUDY POPULATION: High frequency data of patients in intensive care units were used as a data set. The nearest neighbor method with edit distance costs (learned by the FST) were used to classify the patient status within an hour after 10 hours of data. Several experiments were developed to estimate the parameters that better fit the model regarding the prediction metrics. RESULTS/ANTICIPATED RESULTS: Different metrics were obtained for the several parameters. These metrics were metrics (ie, accuracy, precision, and F-measure). DISCUSSION/SIGNIFICANCE OF IMPACT: Our best results are compared with published works, where most of the metrics (ie, accuracy, precision, and F-measure) were improved.

OBJECTIVES/GOALS: To describe the impact of Trial-CARE (Coordination, Acceleration, and Recruitment Enhancement), a centralized service core at Washington University (WU), aimed at enhancing the capability of investigator-initiated multi-site clinical trials at WU and partner CTSA institutions. METHODS/STUDY POPULATION: Through review of our marketing materials and service tracking system we defined the following Trial-CARE offerings: Infrastructure: Trial-CARE is a centralized service core at WU. Whereby, a team member is sourced to an investigator-initiated multi-site clinical trial (II-MCT) to ensure the achievement of study milestones across any phase of the clinical trial life cycle. Team: Our team consists of research professionals with expertise in II-MCT project management, data management, and administrative/regulatory management. Services: * One free 60-minute case consultation * Tiers of Service * Academic Research Organization (ARO) support * Clinical Coordinating Center * Data Coordinating Center * II-MCT project management support * Time-limited targeted support RESULTS/ANTICIPATED RESULTS: Trial-CARE has completed 94 consultations in support of WU and partner CTSA institutions enabling the streamlining of study start-up; guidance for study recruitment, implementation, and operations; and offering resources to foster career development. Consultations can be completed at any phase in the clinical trial lifecycle, with Trial-CARE completing: * 12% in the idea phase * 45% in the grant development and submission stage * 28% after funding has been awarded Top reasons researchers connect with Trial-CARE about II-MCTs: * regulatory guidance (40%) * general information about Trial-CARE Services and II-MCTs (35%) * protocol development (21%) * data management (20%) * study budgeting (20%) DISCUSSION/SIGNIFICANCE: In response to a WU wide survey, Trial-CARE plans to generate informational webinars and is creating a clinical trial tracking dashboard, to pro-actively offer support services to researchers prior to grant funding. The goal is also to increase the percentage of consultations completed in the idea phase and the grant development and submission stage.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to assess how quality of life scores change in menopausal women before and after implementation of this aid. In addition, we are also interested in 2 process evaluation objectives: (1) determine if MyChart, the patient portal, is an effective way for this patient population to provide insight their quality of life to their providers and (2) to evaluate providers use of and reactions to the decision support tool. METHODS/STUDY POPULATION: This project is a collaboration between University of Rochester Medical Center and S.U.N.Y. Upstate Medical University. Participants were recruited through Upstate’s Family Medicine and OB/GYN practices via a MyChart invitation sent by the practices. Participating patients will be asked to complete a survey, through MyChart, every 3 months for 18 months. Participating health providers will be trained to use the decision support tool and participate in 3 interviews with the researchers to gain insight into the usefulness and effectiveness of the tool. RESULTS/ANTICIPATED RESULTS: Of the 465 eligible women, 117 women responded to our MyChart invitation to join our study. Of these, 105 agreed to participate and 98 met eligibility criteria. Only half of the women currently enrolled in our study had spoken to a provider about menopause related symptoms (56.1%) prior to study enrollment. DISCUSSION/SIGNIFICANCE OF IMPACT: The goal of this study is to improve menopause related symptoms in women, thus increasing their quality of life, but it will also provide important process evaluation for using EPIC and MyChart for future research studies.

OBJECTIVES/GOALS: Research in Non Alcoholic Fatty Liver Disease Genetics is ongoing. In this project, we aim to: 1.Identify and describe a histological NAFLD Cohort in the MVP Biobank by extracting liver biopsy proven NAFLD patients using natural language processing (NLP) 2.Confirm previously defined NALFD genetic loci (via look up-GWAS) in the histological NLP cohort METHODS/STUDY POPULATION: We will utilize the Million Veteran Program Biobank where a total of 10,959 subjects have been identified using liver biopsy reports in the EMR via CPT codes. Cases will be based on i) steatosis, steatohepatitis, inflammation or fibrosis in liver biopsy reports with ii) exclusion of other causes of liver disease. Controls will be comprised of the general VA population. In collaboration with the Applied NLP and Precision Medicine groups at the VA Informatics and Computing Infrastructure(VINCI); we will attempt to create and validate a histological cohort of NAFLD in the MVP database by: 1)Annotation of biopsy reports for the NLP algorithm 2)Automation/training of the NLP algorithm We will then perform a multi-ancestry genetic lookup of previously established genetic loci among the cases identified. RESULTS/ANTICIPATED RESULTS: Recently published data: the MVP NAFLD research led by Dr. Chang and Dr. Vujkovic had first validated a proxy NAFLD phenotype based on chronic alanine aminotransferase elevation (cALT). A GWAS was then performed using this phenotype which revealed 77 loci of genome-wide significance including 10 established NAFLD- and 52 ALT-associated SNPs were identified. Replication in external Liver Biopsy and Imaging Cohorts validated 17 SNPs of which 9 were novel. Preliminary data on Liver Biopsy reports: Using CPT codes for Liver Biopsy we estimated a total of 10,959 unique patients and 18,812 notes in the MVP biobank. We anticipate 2,000-3,000 NAFLD cases (based on the prevalence of NAFLD in the general population). Initial review reveals 90% concordance between analysts for the purposes of developing an NLP. DISCUSSION/SIGNIFICANCE: NAFLD is a major cause for morbidity and mortality in liver disease. Gold standard for diagnosis is based on biopsy. GWAS studies with biopsy proven phenotypes are limited. The study will aim to isolate a histologically defined NAFLD cohort in MVP to conduct further GWAS that can provide new clinically relevant knowledge for future research.

Contemporary Research Challenges

OBJECTIVES/SPECIFIC AIMS: Modulation of autophagy has the potential to treat inflammatory bowel disease (IBD). IBD is characterized by dysregulated inflammatory pathways and a defective intestinal epithelial barrier. We sought to better understand how autophagy can be utilized to regulate both inflammation and the intestinal barrier. METHODS/STUDY POPULATION: We examined mice with an autophagy defect in only macrophages in an animal model of IBD. To understand the phenotype, we utilized macrophages to investigate the mechanism behind autophagy and proinflammatory cytokine secretion. In addition, we analyzed the development of colonoids in a co-culture system with macrophages with or without a functional autophagy pathway. Lastly, pharmacological modulation of autophagy to control inflammation was assessed. RESULTS/ANTICIPATED RESULTS: Mice with autophagy-deficient macrophages were highly susceptible to intestinal barrier disruption. Susceptibility was due to enhanced proinflammatory cytokine secretion and intestinal permeability. Furthermore, proinflammatory macrophages (due to an autophagy defect) co-cultured with colonoids, significantly decreased the number of mucus producing goblet cells. Finally, pharmacologically modulation of autophagy reduced the secretion of proinflammatory cytokine by macrophages and reduced intestinal permeability. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results strongly suggest autophagy modulation can dampen inflammation and enhance the intestinal epithelial barrier.

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to determine if dopamine signaling is altered in a mouse model displaying excessive self-grooming and further elucidate the potential utility of compounds targeting the striatal DA system in modulating repetitive behaviors. METHODS/STUDY POPULATION: Here, we report studies using fast-scan cyclic voltammetry (FSCV) in mice lacking the postsynaptic protein SAP90/PSD95-associated protein (SAPAP3 KO mice) as well as control littermates. Rodent self-grooming provides a behavioral output with which one can monitor repetitive, self-directed, patterned behavior that has great translational value to OCD-like disorders. Total time spent grooming was monitored in SAPAP3KO mice and control littermates. To further examine the role of DA in regulating repetitive grooming behaviors the magnitude and kinetics of DA transients were assessed using FSCV in ex vivo slice preparations as well as in anesthetized mice in vivo. DA transients were elicited in the dorsolateral striatum (DLS), dorsomedial striatum (DMS); and nucelus accumbens core (NAcc). In some experiments mice were crossed with DAT-Cre animals and channelrhodopsin 2 (ChR2) was virally expressed in DA neurons to allow optical stimulation of DA transients. RESULTS/ANTICIPATED RESULTS: As previously reported, SAPAP3 KO mice showed excessive grooming compared to control littermates at the age assessed (4-5 months). DA transients evoked by a single electrical pulse in slices from SAPAP3 KO mice were not significantly different from those observed in slices from control littermates in any of the regions tested including the DLS, DMS and NAcc. However, when four electrical pulses were applied at a frequency of 10Hz to mimic DA neuron bursting, the magnitude of DA transients observed in the DMS and NAcc of SAPAP3 mice were greater than those evoked in control littermates.Interestingly, phasic stimulation produced similar DA transients in the DLS of both genotypes suggesting that phasic DA signaling was not globally altered. To confirm this finding we crossed SAPAP3 KO mice with DAT-Cre mice and injected ChR2 containing virus into the midbrain to selectively express ChR2 in DA neurons. Transients were then optically evoked resulting in selective activation of DA neurons. Optical stimulation produced a pronounced enhancement of DA release in SAPAP3 KO mice specifically in the DMS and only following phasic-like stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: These exciting findings suggest that DA signaling in SAPAP3KO mice is dysregulated in a very precise manner that is sub-region specific as well as dependent on the pattern of stimulation. These results suggest that targeted therapies that can modulate these specific modes of dopaminergic signaling in these distinct striatal subregions could provide improved efficacy in OCD patients that are resistant to SSRI treatment.

ABSTRACT IMPACT: By assessing function of mutant (patient-specific) tp53 in zebrafish embryonal rhabdomyosarcoma will inform clinicians of the severity of mutant tp53 alleles. OBJECTIVES/GOALS: This study aims to define loss- and gain-of-function TP53 mutations by comparing effects in tp53-null and wild-type tumors. In addition, it aims to generate a rapid in vivo analysis platform to assign function to patient specific TP53 mutations in the clinic METHODS/STUDY POPULATION: To define tp53 function in ERMS pathogenesis, we previously generated a new tp53-null mutant (tp53-/-) in zebrafish by deleting the entire tp53 genomic locus using TALEN mutagenesis. tp53-/- zebrafish spontaneously develop a spectrum of tumors including sarcomas, leukemia and germ cell tumors (Ignatius…Baxi et. al., eLife) reminiscent of tumors observed in Trp53-null mice. Using the tp53-/- mutants to generate kRASG12D-induced ERMS, we discovered that tp53 is a potent repressor of metastases but rather surprisingly had no effect on self-renewal (Ignatius…Baxi et. al., eLife). Here, using tp53-/- zebrafish, we assessed effects of wild-type and mutant (patient specific) tp53 on tumor initiation, proliferation and apoptosis. RESULTS/ANTICIPATED RESULTS: ERMS tumor initiation in the tp53-/- background is observed in > 97% of animals whereas only <40% of wild-type animals develop ERMS. Additionally, tp53 is a potent suppressor of ERMS proliferation and its effect on apoptosis is minor. Next, we expressed either WT zebrafish or human TP53 in tp53-/- animals along with kRASG12D and both genes suppressed tumor initiation and growth. We co-expressed TP53C176F (found in two ERMS patients) and TP53P153del (identified in a patient with osteosarcoma in our clinic) in zebrafish ERMS, and find that the TP53C176F allele significantly suppressed tumor initiation with effects predominantly on enhanced apoptosis. However, the TP53P153del allele initiated tumors at similar frequency compared to tp53-/- animals but increased the initiation of tumors in the head musculature. DISCUSSION/SIGNIFICANCE OF FINDINGS: Different TP53 alleles identified in patient tumors have very different effects on tumorigenesis in vivo and can respond differently to potentially therapeutic compounds. Thus, the type of precision modeling demonstrated here promises to help further define patient-specific TP53 biology and improve clinical strategies in the future.

OBJECTIVES/GOALS: Genetic variation in L-type voltage-gated calcium channels, including CaV1.3, is associated with increased risk for psychiatric disorders including bipolar disorder and schizophrenia. Additionally, rare mutations in CaV1.3 have been linked to epilepsy, developmental delay, and autism. Deletion of CaV1.3 in mice is associated with impaired consolidation of contextual fear conditioning. Some studies have also observed affective behavior deficits in CaV1.3-deficient mice, but other studies have not found affective phenotypes, perhaps due to differences in genetic backgrounds, sex ratios, or task protocols. CaV1.3 is important for slow afterhyperpolarization in hippocampal and amygdala neurons, which prevents excessive firing in response to sustained excitatory input, and CaV1.3-deficient amygdala neurons exhibit hyperexcitability and impaired LTP. CaV1.3 is also expressed in the cerebellum, but its functional role there is not well understood. Given its importance in shaping neuronal activity in the hippocampus and amygdala, we hypothesized that loss of CaV1.3 would cause abnormalities in motor learning as well as affective and cognitive behaviors. METHODS/STUDY POPULATION: Wild-type (WT), haploinsufficient (Hap), and knockout (KO) mice were maintained on a congenic C57BL/6NTac genetic background and were subjected to behavioral tasks including open field, rotarod, ErasmusLadder, elevated zero maze, forced swim test, and tail suspension test. Data were analyzed with sexes combined and with sexes 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: CaV1.3 KO was associated with impaired motor learning in the rotarod task (p < 0.05), as well as impaired associative learning in the ErasmusLadder task (p < 0.01), despite intact locomotor function on both tasks. When examined by sex, the rotarod phenotypes were driven by motor learning impairments in males (both Hap and KO, p < 0.05 and p < 0.01, respectively), whereas the ErasmusLadder associative learning phenotypes were present in both sexes only in the KO condition, consistent with previously reported impairments in CaV1.3-deficient mice in consolidation of contextual fear conditioning. Although KO mice learned the motor aspects of the ErasmusLadder task, they learned more slowly. They also failed to learn start cues, which requires intact associative learning. No differences were observed in overall exploration or locomotor activity in open field or elevated zero maze. Analyses from affective tasks are ongoing. DISCUSSION/SIGNIFICANCE OF IMPACT: These preliminary studies provide new evidence that CaV1.3 is important for the function of neural circuits involved in motor learning, and concur with previous data showing its involvement in associative learning. Our data differ slightly from previous studies of CaV1.3 in motor learning, which could be attributable to differences in task protocols and/or genetic background. These results highlight the importance of CaV1.3 in a variety of behaviors, which may help explain why variation in CaV1.3 expression and function has pleiotropic effects in humans.

OBJECTIVES/GOALS: A collaboration among Indiana CTSI community health partnerships (CHeP), bioethics, and regulatory programs identified and reviewed human research protection training programs targeting community engaged research, and pilot tested CIRTification with community partners working across a range of contexts. METHODS/STUDY POPULATION: We searched community human research protection training programs from across the county, identified three, examined each based upon criteria identified by community partners (time, relevance, online delivery) and our Human Research Protection Program (HRPP), and selected CIRTification (CIRT) to pilot. Ten community research partners volunteered to complete CIRT and a debriefing interview. Four completed CITI training previously. Participants included local and state-wide organizations, a resident, a state agency, and a hospital, and came from rural and urban communities. Interviews covered practical issues (ease of use, language, time), relevance, and comparison to CITI. Results were shared with HRPP for approval. RESULTS/ANTICIPATED RESULTS: Most felt CIRT was easy to navigate and engaging, and those who had done CITI felt CIRT was more relevant and engaging. The sections on historical background and recruitment were cited as most valuable. Suggestions were made to increase the diversity of examples beyond health care research. Community members mentioned several applications for CIRT including: (1) helping their own community work; (2) empowering them to be an advocate; (3) referring others to CIRT; (4) influencing approaches to recruitment and community engagement; and (5) applying ethics principles to their other community work. The Human Research Protection Program approved CIRT in place of CITI for community researchers. DISCUSSION/SIGNIFICANCE: Our process represents collaboration across the Indiana CTSI, HRPP and community partners, and use of best practices. Exemplifying “nothing about us without us”, actions were based on direct input from community partners, with the goal of decreasing barriers to engaging communities in research.

Academic discovery in biomedicine is a growing enterprise with tens of billions of dollars in research funding available to universities and hospitals. Protecting and optimizing the resultant intellectual property is required in order for the discoveries to have an impact on society. To achieve that, institutions must create a multidisciplinary, collaborative system of review and support, and utilize connections to industry partners. In this study, we outline the efforts of Case Western Reserve University, coordinated through its Clinical and Translational Science Collaborative (CTSC), to promote entrepreneurial culture, and achieve goals of product development and startup formation for biomedical and population health discoveries arising from the academic ecosystem in Cleveland. The CTSC Office of Translation and Innovation, with the university’s Technology Transfer Office (TTO), helps identify and derisk promising IP while building interdisciplinary project teams to optimize the assets through key preclinical derisking steps. The benefits of coordinating funding across multiple programs, assuring dedicated project management to oversee optimizing the IP, and ensuring training to help improve proposals and encourage an entrepreneurial culture, are discussed in the context of a case study of therapeutic assets, the Council to Advance Human Health. This case study highlights best practices in academic innovation.