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 takes a transcriptomic approach to parse genes and molecular pathways that underlie the fear memory circuitry and, in doing so, identifies therapeutic targets that can further be developed into treatments for fear disorders, such as post-traumatic stress disorder. OBJECTIVES/GOALS: Normal fear learning produces avoidance behavior that promotes survival, but excessive and persistent fear after trauma can lead to development of phobias and post-traumatic stress disorder (PTSD). Our goal is to understand the mechanism and identify novel genetic targets underlying fear responses. METHODS/STUDY POPULATION: Involvement of the basolateral amygdala (BLA) in fear acquisition is well established and requires activation of N-methyl-D-aspartic acid receptors (NMDARs). At a cellular level, NMDAR activation leads to production of nitric oxide (NO) by a process mediated by interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS). To elucidate mechanisms underlying the role of the PSD95-nNOS-NO pathway in conditioned fear, here we use rodent behavioral paradigms, pharmacological treatment with a small molecule PSD95-nNOS inhibitor, RNA-sequencing, and an AAV-mediated knockdown of the nNOS gene in the BLA. RESULTS/ANTICIPATED RESULTS: We show that treatment with ZL006 attenuates rodent cued-fear consolidation. Additionally, with RNA-sequencing, expression of 516 genes was altered in the BLA following fear expression; of these genes, 83 were restored with systemic ZL006 treatment. Network data and gene ontology enrichment analyses further revealed that cGMP effects, insulin-like growth factor binding, and cognition-related pathways were significantly altered. Finally, we show that a BLA-specific knockdown of nNOS attenuates cued fear consolidation, without adverse effects on other memory and motor behaviors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Via a model of NMDAR-mediated fear consolidation, our results reveal novel pathways and genetic targets that underlie plasticity of fear memory circuitry. Importantly, these results will inform future therapeutic strategies for targeting fear related disorders like PTSD.

OBJECTIVES/SPECIFIC AIMS: The objective of this study is to develop patient-derived “personalized” organotypic models of human distal airways, in which basal stem cells (BCs) isolated from the pre-/terminal conducting airway region are co-cultured with autologous stromal cells from the same region to reproduce patient-specific distal airway epithelial-stromal units and their remodeling in COPD. METHODS/STUDY POPULATION: We established a protocol to isolate and propagate epithelial BCs, fibroblasts, and endothelial cells from the distal airways of normal and COPD lung donors. Heterogeneous cellular and molecular phenotypes in the human distal airways were characterized using immunofluorescence and single-cell RNA sequencing. Patient-specific distal airway epithelial-stromal units were reconstructed by co-culturing BCs and autologous stromal cells using an air-liquid interface-based airway wall model and a bronchosphere-based 3D distal airway organoid assay. RESULTS/ANTICIPATED RESULTS: Histologic analysis of derived epithelial-stromal units revealed heterogeneous patient-specific phenotypes characterized by hypo-/hyper-/metaplastic lesions (hypo-regenerative phenotype, mucous cell hyperplasia, squamous metaplasia, distal-to-proximal repatterning) in the epithelial compartment, accompanied, in some samples, by stromal remodeling. Candidate epithelial-stromal cross-talk mechanisms were identified using quantitative real-time RT-PCR analysis of autologous epithelial and stromal compartments of established patient-specific distal airway unit models. DISCUSSION/SIGNIFICANCE OF IMPACT: Epithelial and stromal cells isolated from distal airways of subjects with and without COPD can be assembled into functional, organ-level tissue which mimics the architecture of human distal airways and, in patients with COPD, reproduces several distal airway remodeling phenotypes. Patient-specific models of distal airway epithelial-stromal cross-talk established in this study can be used to identify candidate pathways that mediate disease-relevant airway remodeling and potentially utilized as pre-clinical platforms for developing personalized therapeutic approaches to suppress the progression of distal airway remodeling in chronic lung diseases, including COPD.

OBJECTIVES/GOALS: The objective of this study is to assess the feasibility and preliminary impact of a physiotherapy protocol for developing an individualized home-based physical activity program to increase physical activity (PA) levels in sedentary older adults with Type II Diabetes Mellitus (T2DM) living in Puerto Rico (PR). METHODS/STUDY POPULATION: This will be a pilot study with two phases. In phase 1, we will design a novel patient-centered home-based PA program protocol for adults ≥65 years with T2DM based on the Information-Motivation-Behavioral Skills model. Its content validity will be assessed through focus groups with 10 experts and 10 older adults and analyzed using a directed content analysis. Phase 2 we will be program implementation using a one-group, repeated measures design with 12 adults ≥65 years with T2DM. PA levels will be assessed by recording active minutes with a Fitbit. Risk of falls, balance, strength, and physical function will be assessed through standardized tests validated for this population. Statistical analysis will include descriptive statistics, comparisons via chi-square/Fisher’s exact test, and non-parametric tests. RESULTS/ANTICIPATED RESULTS: We expect to recruit a minimum of 12 participants and to administer the program for 12 weeks at a frequency of two visits per week. We anticipate that implementing and supervising the home-based PA protocol will be feasible as determined by recruitment and retention rates, patients’ satisfaction, and compliance with the program. We also expect that this protocol will increase physical activity levels, improve general strength, balance, physical function, and reduce the risk of falls in sedentary older adults with T2DM. DISCUSSION/SIGNIFICANCE: As the third cause of death in PR, T2DM represents a public health challenge. An effective home-based PA program may decrease morbidity and mortality rates in older adults by increasing PA and functional health. This study will provide data for planning a randomized controlled trial to assess its effectiveness in the outcomes of interest.

OBJECTIVES/GOALS: Early age at menopause has been linked to increased risk of cardiovascular disease; however, there is limited evidence for a relationship between early menopause and heart failure (HF). We examined whether early menopause is associated with incident HF among women in the southeastern United States. METHODS/STUDY POPULATION: The Southern Community Cohort Study enrolled ~86,000 low-income black and white adults from 2002 to 2009. Participants for this analysis were 11,948 women who were postmenopausal at enrollment, had no history of HF, and were on Medicaid or Medicare. HF events were ascertained using ICD-9 codes 428.x via linkage of the cohort with CMS Research Identifiable Files through December 31, 2010. Early menopause was defined as self-reported age at menopause less than 45 years. Hazard ratios (HRs) and 95% confidence intervals (CIs) were computed from multivariable Cox regression models, overall and by race, adjusting for demographic, lifestyle, and reproductive factors, including reason for menopause. RESULTS/ANTICIPATED RESULTS: At baseline, mean age was 58±9 years, and 65% of participants were black. Among women with early menopause, 76% (n = 4,836) had menopause due to hysterectomy or oophorectomy. In women with later menopause, 74% (n = 4,102) reported natural menopause. During a median follow-up of 5.0 years (range 3.1-6.7), 2,157 incident HF events occurred. Compared with women with later onset of menopause, those with early menopause had increased HF risk (HR: 1.27, 95% CI: 1.10–1.47). Risk of HF associated with early menopause was similar in white and black women (p-value for interaction: 0.13). DISCUSSION/SIGNIFICANCE OF IMPACT: In this largely low-income population, early menopause was associated with an increased risk of developing HF. Women with early menopause represent a potential target population for future interventions to decrease risk of HF and cardiovascular risk factors.

OBJECTIVES/GOALS: Uterine fibroids (UF), non-cancerous myometrial neoplasms, affect 11 million women in the US. In qualitative research, experiences of Black women (BW) and Latinas (HL) with UF are understudied. UF etiology is poorly understood, and most symptoms are focused on decreasing UF symptoms. This study will showcase a translational approach in studying UF. METHODS/STUDY POPULATION: A community-engaged research process will be used throughout the study. We have a partnership with the Fibroid Foundation (FF), a UF advocacy organization. For our first aim, we have held community-engagement (CE) studios to provide insight for best practices in creating and implementing culturally appropriate studies with ethnoracially diverse women regarding topics of UF. Our CE studios set the foundation for the second aim, which will be in-depth interviews assessing the sociocultural impacts among pre-menopausal BW and HL with UF. The women will be recruited through the FFs social media platform. To address lack of treatment options in UF, we will be focusing on JAK 2/3 pathway. We will be performing immunofluorescence on tissue microarrays on over 100 patient samples with extensive clinical and survey data. RESULTS/ANTICIPATED RESULTS: We held two CE studios, one with BW with UF and another with HL without UF. These participants were chosen as patient stakeholders. One theme from the CE studio was that both groups mentioned a cultural impact around menstrual health and UF discussions. The participant stakeholders provided insight to other influences in their journey that will be used to develop the interview guide questions for Aim 2. We expect the individual interviews to show similar experiences as our CE studio experiences. We anticipate more in-depth conversations about the sociocultural and ethnoracial factors impacting UF treatment. For Aim 3, we hypothesize that there will be increased JAK 2/3 expression in UF of patients with higher sympton burden and larger fibroid volume. DISCUSSION/SIGNIFICANCE: Implementing a community-engaged research framework provides a foundation for qualitative and translational gynecologic research in BW and HL. This project will contribute insight to their UF experience. There is a lack of innovative treatments, and we have access to valuable patient tissue and data that can be used for potential molecular targets.

OBJECTIVES/GOALS: The objective of this presentation is to describe different recruitment tools implemented by the Miami Clinical and Translational Science Institute (CTSI) to facilitate participant recruitment into research studies. METHODS/STUDY POPULATION: Participant recruitment is critical to the success of all research studies. In the effort of advancing clinical and translational science and to help investigators recruit volunteers for research studies, the University of Miami has two recruitment tools: 1) Consent to Contact (CTC), an opt-in research registry where University of Miami Health System patients are asked for permission to be contacted about studies matching their demographic and/or health profiles; and 2) UMiamiHealthResearch.org (UMHR), implemented with the Michigan CTSA, a community-based registry for volunteers to sign up and be contacted about studies. Study investigators can use these tools once they have obtained IRB approval for their research. RESULTS/ANTICIPATED RESULTS: The CTC was launched in 2016; to date, over 130,000 patients have enrolled in CTC; 69 studies have been approved with over 75,000 patients’contact information released to study teams. UMHR was launched in 2020. To date, the site lists 237 studies. A total of 2,727 portal visitors have expressed interest in participating in specific studies. Study team members were successful in engaging interested participants, and enrolling participants into studies. Overall, teams reported a positive impact on recruitment. Data collection on utilization and satisfaction of these recruitments tools is ongoing. In addition, focus groups of study team members are being conducted to identify best practices for using these tools, and findings will be presented. DISCUSSION/SIGNIFICANCE: The CTC and UMHR recruitment tools have demonstrated positive impact in helping study teams identify potentially eligible research volunteers. The continued promotion of these tools at the University of Miami Health System and in the community will be crucial to the recruitment process and execution of research studies.

OBJECTIVES/SPECIFIC AIMS: We aim to examine the epidemiological characteristics of prosopagnosia by querying and analyzing a large deidentified clinical data set from 12 New York City-based hospitals and Federally Qualified Health Centers (FQHCs). The PCORI-funded New York City Clinical Data Research Network (NYC-CDRN) contains ~4.5 million deidentified ICD-coded electronic health records (EHRs) with comprehensive longitudinal information on demographics, patient visits, clinical conditions/diagnoses, laboratory and radiology results, medications, and clinical procedures. The NYC-CDRN will be expanded to include other data sources, including insurance claims, social determinant of health, patient reported outcomes, and patient generated data. The central hypothesis was that systematic mining of this database would reveal new epidemiological information about prosopagnosia. We developed a computable phenotype for prosopagnosia, using the International Classification of Diseases version 9 (ICD-9). The computable phenotype consisted of the diagnostic code for the condition under study, prosopagnosia (ICD-9 code 368.16), as well as the codes for known surrogate diagnoses. We expected to identify cases of acquired prosopagnosia, where the condition occurs only after brain damage, due to stroke, trauma, or meningitis for example, and cases of developmental prosopagnosia, where the condition is present from an early age, with no history of brain damage. The goals of this project were to provide new information about the condition’s prevalence rate in the New York City area, which could be furthermore translated into wider geographical areas and to yield novel details about its antecedents and comorbid conditions. METHODS/STUDY POPULATION: To determine the presence of the diagnosis of interest, prosopagnosia, and common co-occurring conditions among a New York City-based study population, we investigated a large database in collaboration with the NYC-CDRN. At the time the large database was mined it contained ~4 million ICD-9 coded EHRs. We first created a search paradigm; applicable for screening the database that consists of ICD-9 coded EHRs. We generated a list of ICD-9 codes indicative for the patients’ difficulties with the perception of faces (368.16), which indicates the presence of the condition as part of the psychophysical visual disturbances complex, and this code identified 871 patients. Furthermore, we collected codes that indicate the presence of conditions that are known to be surrogate diagnoses of prosopagnosia. ICD-9 codes for surrogate diagnoses included for example, 854.* (coding for personal history of traumatic brain injury, n=1,409), 434.01, 434.11, and 434.91 (coding for cerebral thrombosis, embolus and artery occlusion unspecified with cerebral infarction, n=19,409), and 191.2 (coding for malignant neoplasm of the temporal lobe, n=566). In October 2015, coding was changed to the new ICD-10 coding system. No additional patients were revealed from the data set when the cohort was searched for the presence of corresponding ICD-10 codes, as institutions are currently in transition from ICD-9 to ICD-10. Using this search query with the large database, we extracted novel information about the epidemiological and demographical distribution of prosopagnosia and furthermore, gained new knowledge about commonly associated diseases. The fact that it must be presumed that the majority of diagnoses of prosopagnosia have been made on the basis of patients’ self-reports and clinicians’ judgments represents a limiting factor in this study. We are currently exploring machine-learning strategies to identify potential false-negative cases among the patients with surrogate diagnoses. RESULTS/ANTICIPATED RESULTS: Investigations and application of our search query revealed a total number of n=129,549 patients carrying either the diagnosis code for prosopagnosia or the codes for the known surrogate diagnoses. There were 871 patients who carried the ICD-9 code 368.16, indicating the potential presence of prosopagnosia among other visual disturbances. Remaining patients (n=128,678) carried codes for known surrogate diagnoses, contained in the search query. Statistical analyses revealed elevated odds ratios for men (OR=1.55, 95% CI: 1.36, 1.77, p<0.0001), and for Black/African Americans Versus White individuals (OR=2.09, 95% CI: 1.74, 2.51, p<0.0001). DISCUSSION/SIGNIFICANCE OF IMPACT: Currently, the prevalence of prosopagnosia remains unknown. Face blind individuals are struggling to recognize their social contacts by their face only in every day life and are therefore prone to experience reduced quality of life. We searched the large NYC-based clinical database, containing more than 4.5 million deidentified ICD-coded health records, for cases of prosopagnosia to shed light into its prevalence and epidemiological characteristics. We furthermore, mined the database for cases carrying known surrogate diagnoses to explore the magnitude and characteristics of individuals potentially under increased risk. Our efforts address a great healthcare need, as they revealed new epidemiological knowledge of a vulnerable and understudied population. The results of this project reveal new insights into the epidemiological characteristics of prosopagnosia and its surrogate diagnoses, and demonstrate the feasibility of mining large clinical databases to identify rare clinical populations. Our results suggest the need for a more targeted diagnostic assessment of face perception abilities in populations under increased risk.

ABSTRACT IMPACT: This study will provide the essential characterization of intrinsic neural activity in human brain organoids, both at the single cell and network levels, to harness for translational purposes. OBJECTIVES/GOALS: Brain organoids are 3D, stem cell-derived neural tissues that recapitulate neurodevelopment. However, to levy their full translational potential, a deeper understanding of their intrinsic neural activity is essential. Here, we present our preliminary analysis of maturing neural activity in human forebrain organoids. METHODS/STUDY POPULATION: Forebrain organoids were generated from human iPSC lines derived from healthy volunteers. Linear microelectrode probes were employed to record spontaneous electrical activity from day 77, 100, and 130 organoids. Single unit recordings were collected during hour-long recordings, involving baseline recordings followed by glutamatergic blockade. Subsequently, tetrodotoxin, was used to abolish action potential firing. Single units were identified via spike sorting, and the spatiotemporal evolution of baseline neural properties and network dynamics was characterized. RESULTS/ANTICIPATED RESULTS: Nine organoids were recorded successfully (n=3 per timepoint). A significant difference in number of units was seen across age groups (F (2,6) = 6.4178, p = 0.0323). Post hoc comparisons by the Tukey HSD test showed significantly more units in day 130 (51.67 ±14.15) than day 77 (16.33 ±14.98) organoids. Mean firing rates were significantly different in organoids based on age, with drug condition also trending toward significance (F (6,12) = 9.97; p = 0.0028 and p = 0.08 respectively). Post hoc comparisons showed a higher baseline firing rate in day 130 (0.99Hz ±0.30) organoids than their day 77 counterparts at baseline (0.31Hz ±0.066) and glutamate blockade (0.31Hz ±0.045). Preliminary network analysis showed no modularity or small-world features; however, these features are expected to emerge as organoids mature. DISCUSSION/SIGNIFICANCE OF FINDINGS: Initial analysis of brain organoid activity demonstrates changes in single unit properties as they mature. Additional work in this area, as well as further network analyses, will confer better sense of how to rationally utilize brain organoids for translational purposes.

OBJECTIVES/GOALS: In spite of significant growth in undergraduate research, there is little accessible RCR training material targeting this group. The goal of this project was to develop level appropriate RCR materials and test them for effectiveness of teaching basic knowledge of RCR. It was developed cognizant of the type of learning preferred by this group. METHODS/STUDY POPULATION: Todays undergraduate students are digital natives accustomed to using on-line resources for learning and value the ability to work independently. An asynchronous, online series of eight RCR modules were developed using PowerPoint. Audio instruction was overlaid over the presentations and uploaded to YouTube. Each module includes quiz at the end that must be completed with a 90% score in order to continue and students can retake each quiz until correct. Case studies, a plagiarism game, and additional resources such as infographics created by the Office of Research Integrity were included. 275 first year undergraduates completed the pilot and the on-line retrospective pre-post assessment that included 28 Likert scale items and two open-ended questions that were analyzed with RapidMiner. RESULTS/ANTICIPATED RESULTS: Participants rated their ability prior to and after the course to recognize the roles/responsibilities of a mentor, maintain and organize data records, recognize what constitutes research misconduct and multiple types of plagiarism, define ethics of authorship and the types of peer review, recognize responsible handling of human and animal subjects and to define collaborative research. Responses were analyzed using t-Tests and all items showed a significant increase. Students reported between a 28% and 50% increase in knowledge in each topic and gains in understanding where to report RCR violations. Text analysis using RapidMiner provided developers with suggestions on how to enhance the project. Students reported finding the course concise and easy to understand and valued the included case studies. DISCUSSION/SIGNIFICANCE: This study demonstrated that RCR materials developed specifically for todays undergraduate students, presented an easily accessible format, effectively conveys the basics of RCR. The on-line, asynchronous format made it accessible to those for whom in person course is not possible. This model fills the gap in RCR training for early researchers.

OBJECTIVES/GOALS: Current clinical practice recommends occlusive dressings (e.g., film and hydrocolloid) for wounds with variable regenerative capacities. However, clinical evidence suggests that occlusion may hinder regeneration. Our objective was to test the impact of occlusion on regeneration using animal models. METHODS/STUDY POPULATION: The murine wound-induced hair neogenesis (WIHN) is a well-established model of regeneration characterized by de novo hair follicle (HF) formation in the center of large full-thickness wounds. The quantity of neogenic HFs depends on the robustness of Wnt signaling. Optimal tissue mechanics is also required for WIHN. Utilizing the murine WIHN model, we tested the hypothesis that wound occlusion impedes regeneration. We determined how (i) the timing and duration of wound occlusion impacts WIHN, (ii) Wnt signaling influences the occlusion-induced effects on regeneration and (iii) occlusion alters the tissue mechanics of the wound, which establishes the morphogenetic field needed for WIHN. RESULTS/ANTICIPATED RESULTS: Occlusion completely eliminated WIHN. Only a brief period of occlusion between post-wound days 0-3 or 4-7 was sufficient to abrogate WIHN. Microarray and qPCR of open and occluded wounds demonstrated that occlusion promotes fibrosis by upregulating TGF-β2 and mechanotransduction, a mechanosensitive profibrotic pathway. Recruitment of these potent profibrotic pathways generated a symmetrically rigid wound incapable of de novo HF regeneration. Using transgenic animal models with enhanced Wnt signaling, we determined that the ligand-dependent Wnt signaling protected against the occlusion-induced inhibition of WIHN, as well as the occlusion-induced upregulation of both profibrotic pathways. DISCUSSION/SIGNIFICANCE: In animal models, occlusion promoted fibrosis at the expense of regeneration during acute wound healing. Augmented Wnt signaling protected against this effect. Occluding wounds may reduce regeneration. Further studies are warranted to validate these findings.

OBJECTIVES/GOALS: Clinical and translational science needs to address roadblocks to translational processes. We conducted a survey at two institutions, a private medical school and a large public university, to understand the frequency and distribution of barriers and roadblocks to research. METHODS/STUDY POPULATION: We reviewed the literature to compile a pool of barriers and roadblocks and convened a panel of relevant stakeholders to develop a 20-item questionnaire. Survey respondents were asked to select and prioritize the five leading clinical and translational roadblocks, provide information regarding their academic degrees and rank/position, complete open-ended items regarding their areas of research, and optionally add additional remarks in a comment box. The survey was disseminated in August 2022 via REDCap to faculty and staff with active research protocols at Baylor College of Medicine and the University of Houston. RESULTS/ANTICIPATED RESULTS: In total, 227 respondents completed the survey. Their disciplines were basic science (29.5%), translational research (52.9%), clinical research (55.5%), community-engaged research (9.7%), and educational research (9.7%). Respondents identified 1) lack of access to trained research coordinators, 2) lack of understanding about different resources that facilitate research, 3) complex regulatory environment and delays, 4) fragmented infrastructure for administrative and fiscal processes, and 5) inadequate funding for pilot projects to foster new research. Other roadblocks included lack of established community stakeholder partnerships, inadequate access to medical record data, and limited biostatistical support. In the comments, several respondents noted that all items included were important. DISCUSSION/SIGNIFICANCE: Research workforce recruitment/training was the highest priority followed by lack of access to information and administrative bottlenecks. We are building an online portal to increase awareness and simplify access to competency-based training and research services. Initiatives are underway to address other roadblocks.

OBJECTIVES/SPECIFIC AIMS: The primary objective of this research is to determine whether being hand-reared, and deprived of early maternal interaction, will affect telomere length in orphaned kittens. The secondary goal is to examine how early maternal separation impacts the health, growth and behavior of orphaned kittens. METHODS/STUDY POPULATION: Kittens were fostered through local rescue groups and shelters. We collected blood samples from 42 orphaned kittens during the first week of their lives. Due to high mortality of this population, we obtained a second blood sample at eight weeks of age from only 30 of these kittens. We collected blood samples from 12 control kittens raised with mothers at during the first and eighth weeks of life. Blood samples are currently being processed with real time quantitative PCR (qPCR) by the Real-time PCR Research and Diagnostics Core Facility at the UC Davis School of Veterinary Medicine (SVM). This includes RNA extraction, cDNA synthesis, Reference Gene Validation, and qPCR analysis. Relative telomere length (RTL) will be calculated by comparing the average telomere abundance across three samples cells with that of a reference gene (single copy number) for each sample. The resulting T/S ratio (telomere to single copy) is proportional to the average telomere length. If T/S = 1, then telomere length in the sample and the reference are the same. RESULTS/ANTICIPATED RESULTS: Because telomeres show the fastest rate of shortening early in life, we predict that maternal separation will increase the rate of telomere shortening in kittens. We also predict that the telomeres of orphaned kittens will be shorter at both one week and eight weeks of age, compared to controls. DISCUSSION/SIGNIFICANCE OF IMPACT: This study will increase our understanding of early life adversity, a finding that can translate to other mammals. It will inform the practice of fostering neonatal kittens, and illuminate whether these kittens might be at higher risk than mother-reared kittens for health problems (which could be investigated in future studies). If significant telomere shortening occurs between collection periods, then future studies can take more frequent blood samples to determine what stages of early development are potentially most sensitive. If differences between groups are found, this will establish a protocol for several future research projects, such as testing whether these detrimental effects can be mitigated by environmental enrichment via activation of telomerase. Telomerase is an enzyme that appears to counteract some shortening of telomeres, and is activated by several external factors, including exercise. Thus, a logical follow up study would be developing and testing age-specific and appropriate enrichments that may activate telomerase and reduce telomere loss. Physical contact, whether human, mother, or siblings, is another possible source of telomerase activation in young kittens. Future studies also could quantify the effects of different sources of physical contact on telomere shortening. Finally, a positive finding would establish a need for longitudinal studies of the effects of early weaning on feline health and behavior and whether differences in early-life telomere lengths predict health and longevity of cats.

OBJECTIVES/SPECIFIC AIMS: Voiding postponement is common cause of LUT dysfunction in which children void infrequently with large volumes. This condition is modeled in mice that are subjected to social stress who show decreased voiding frequency and increased voided volumes along with increases in corticotropin-releasing hormone (CRH) expression in Barrington’s nucleus (BN) (i.e., the pontine micturition center). Optogenetics is a technique to selectively stimulate cells or neurons of interest via light activated channel receptors [i.e., channel-2 rhodopsin (ChR2)]. Here we examined the effects of optogenetic manipulation of CRH BN neurons on the in vivo voiding phenotype and urodynamics in awake mice. We hypothesized that stimulating these neurons at higher frequencies (10–50 Hz) would lead to CRH-dependent alterations in voiding phenotype (i.e., larger voided volumes and longer intermicturition intervals. METHODS/STUDY POPULATION: Double transgenic mice expressing ChR2 in CRH cells were generated using the Cre-lox recombinase system and had fiberoptic probes implanted into BN at 8 weeks of age. The mice also underwent simultaneous catheter placement into the bladder for in vivo cystometry. In vivo cystometry before and during optogenetic stimulation at various frequencies was performed 5–7 days postoperatively. Saline was perfused at 10 µL/minute and baseline stable voiding cycles were established. Bladder capacity, threshold pressure, voiding pressure, and voided volume were recorded at baseline and at each optogenetic setting. In some mice, the protocol was repeated in the presence of CRH antagonist (NBI 30775). RESULTS/ANTICIPATED RESULTS: Fiberoptic stimulation (470 nm at 25 and 50 Hz) produced a significant rise in the intermicturition interval, bladder capacities and increased void volumes. This effect was especially pronounced in females in whom bladder capacity and intermicturition interval more than doubled at 50 Hz stimulation. Fluoroscopic images confirmed complete bladder emptying with each void. The increased bladder capacity at higher frequencies (25 and 50 Hz) was CRH-dependent as injection of a CRH antagonist (NBI 30775) blocked the optogenetic effect. Control non-double mice showed no effects from optogenetic stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that optogenetic stimulation of CRH-expressing neurons in BN at high frequency (25 and 50 Hz) inhibits micturition and recapitulates the voiding phenotype seen in socially stressed mice (large, infrequent voids). Lower frequencies of optogenetic stimulation (2 and 10 Hz) had no effects on cystometry parameters or voiding phenotype. In addition, females had a greater response to optogenetic stimulation compared with males with larger bladder capacities and longer intermicturition intervals. The changes in voiding phenotype seen were CRH dependent as blockage of the CRH receptor prevented changes in cystometry parameters with optogenetic stimulation. Further elucidation of these and other neural subpopulations in BN are warranted to understand micturition and how it may be manipulated in disease states such as voiding postponement and acute urinary retention.

OBJECTIVES/GOALS: Trichomonas vaginalis (TV) has a prevalence of 26% in Baltimore and is associated with preterm delivery (PTD). Yet screening and treatment of TV is not advised due to conflicting data on harms. Our goal is to investigate the association between asymptomatic TV treatment and PTD. METHODS/STUDY POPULATION: This is a retrospective cohort study of women who delivered a child at The Johns Hopkins Hospital between 7/1/16 – 11/19/19. Exclusion criteria included multiple gestation, stillborn, miscarriage, diabetes, hypertension/ preeclampsia, HIV, and history of PTD. Chart review and ICD-10 diagnosis codes were used to collect data on demographics, STI test results, lab encounter diagnoses, STI treatment during pregnancy, and labor encounter diagnoses. Preliminary analysis for crude incidence of PTD in asymptomatic and symptomatic women treated for TV was performed using TriNetx, a global research network compiling all de-identified data within the Hopkins system. RESULTS/ANTICIPATED RESULTS: Three hundred and eighty women were tested for TV, 240 (63%) were asymptomatic and 140 (37%) women were symptomatic. Mean ages were 26 (SD:5) and 26 (SD:5) years, respectively. Black women comprised 87% of the asymptomatic cohort and 93% of the symptomatic cohort. Women of Hispanic ethnicity were represented by 4% of the asymptomatic cohort and 7% of the symptomatic cohort. Crude incidence of PTD was 4.1% among asymptomatic women and 7.1% among symptomatic women. Incidence ratio comparing asymptomatic PTD incidence to symptomatic PTD incidence was 0.58 with 95% CI (0.22, 1.56). DISCUSSION/SIGNIFICANCE OF IMPACT: Preliminary data from our study suggests there is no difference in PTD between asymptomatic and symptomatic women treated for TV. Future steps include multiple linear regression using a larger dataset. These preliminary data suggest TV should be considered for screening during pregnancy.

OBJECTIVES/SPECIFIC AIMS: The purpose of this study was to characterize the pharmacokinetics of phosphatidylethanol (PEth) 16:0/20:4 homolog in uncoagulated, human blood samples taken from 18 participants in a clinical laboratory setting after consumption of 2 doses of ethanol. METHODS/STUDY POPULATION: Male and female participants received either 0.4 or 0.8 g/kg oral doses of ethanol during a 15-minute period. Blood samples were collected before and throughout 6 hours immediately after alcohol administration, then after 2, 4, 7, 11, and 14 days of administration day. PEth 16:0/20:4 levels were quantified by liquid mass spectrometry. Breath ethanol concentrations were measure concurrently with each blood collection during the administration day, as well as transdermal ethanol concentrations monitored constantly before, during and after ethanol administration day. RESULTS/ANTICIPATED RESULTS: (1) Single doses of 0.4 and 0.8 g ethanol/kg produced proportional increases in BrAC and PEth 16:0/20:4 levels; (2) the increase of Peth 16:0/20:4 from base line to Cmax was less than either PEth 16:0/18:1 or PEth 16:0/18:2 during the 6-hour period after ethanol administration; (3) the mean rate of formation of PEth 16:0/20:4 was lower than those of the other 2 homologs; (4) the mean half-life of PEth 16:0/20:4 was 2.18 days, which was shorter than that of either PEth 16:0/18:1 and PEth 16:0/18:2, which were 6.80 and 6.62, respectively. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study further confirm that PEth homologs are a sensitive biomarker for ethanol consumption. The measurement of three PEth homologs appears to provide additional information about the level and time frame of drinking.

OBJECTIVES/SPECIFIC AIMS: The objective of this study is to use machine Learning techniques to generate maps of epithelium and lumen density in MRI space. METHODS/STUDY POPULATION: Methods: We prospectively recruited 39 patients undergoing prostatectomy for this institutional review board (IRB) approved study. Patients underwent MP-MRI before prostatectomy on a 3T field strength MRI scanner (General Electric, Waukesha, WI, USA) using an endorectal coil. MP-MRI included field-of-view optimized and constrained undistorted single shot (FOCUS) diffusion weighted imaging with 10 b-values (b=0, 10, 25, 50, 80, 100, 200, 500, 1000, and 2000), dynamic contrast enhanced imaging, and T2-weighted imaging. T2 weighted images were intensity normalized and apparent diffusion coefficient maps were calculated. The dynamic contrast enhanced data was used to calculate the percent change in signal intensity before and after contrast injection. All images were aligned to the T2 weighted image. Robotic prostatectomy was performed 2 weeks after image acquisition. Prostate samples were sliced using a 3D printed slicing jig matching the slice profile of the T2 weighted image. Whole mount samples at 10 μm thickness were taken, hematoxylin and eosin stained, digitized, and annotated by a board certified pathologist. A total of 210 slides were included in this study. Lumen and epithelium were automatically segmented using a custom algorithm written in MATLAB. The algorithm was validated by comparing manual to automatic segmentation on 18 samples. Slides were aligned with the T2 weighted image using a nonlinear control point warping technique. Lumen and epithelium density and the expert annotation were subsequently transformed into MRI space. Co-registration was validated by applying a known warp to tumor masks noted by the pathologist and control point warping the whole mount slide to match the transform. Overlap was measured using a DICE coefficient. A learning curve was generated to determine the optimal number of patients to train the algorithm on. A PLS algorithm was trained on 150 random permutations of patients incrementing from 1 to 29 patients. Slides were stratified such that all slides from a single patient were in the same cohort. Three cohorts were generated, with tumor burden balanced across all cohort. A PLS algorithm was trained on 2 independent training sets (cohorts 1 and 2) and applied to cohort 3. The input vector consisted of MRI values and the target variable was lumen and epithelium density. The algorithm was trained lesion-wise. Trained PiCT models were applied to the test cohort voxel-wise to generate 2 new image contrasts. Mean lesion values were compared between high grade, low grade, and healthy tissue using an ANOVA. An ROC analysis was performed lesion-wise on the test set. RESULTS/ANTICIPATED RESULTS: Results: The segmentation accuracy validation revealed R=0.99 and R=0.72 (p<0.001) for lumen and epithelium, respectively. The co-registration accuracy revealed a 94.5% overlap. The learning curve stabilized at 10 patients with a root mean square error of 0.14, thus the size of the 2 independent training cohorts was set to 10, leaving 19 for the test cohort. DISCUSSION/SIGNIFICANCE OF IMPACT: We present a technique for combining radiology and pathology with machine learning for generating predictive cytological topography (PiCT) maps of cellularity and lumen density prostate. The voxel-wise approach to mapping cellular features generates 2 new interpretable image contrasts, which can potentially increase confidence in diagnosis or guide biopsy and radiation treatment.

OBJECTIVES/SPECIFIC AIMS: 1. To identify novel genetic modifiers that result in a mild bleeding phenotype in patients with FVIII <1%. 2. To examine the feasibility of a practice model that incorporates the principles and methods for both obtaining consent for NGS and returning individual research results from the sources described above. METHODS/STUDY POPULATION: 1. We plan a 3-step approach for identifying novel genetic modifiers of hemophilia: a. Obtain samples from individuals with an extremely mild bleeding phenotype: The study will be narrowed to patients with confirmed FVIII <1%, a null mutation in the gene for FVIII, and a mild bleeding phenotype according to a detailed bleeding history. b. Identify variants that modify phenotype: Whole exome sequencing will be performed, followed by a focused analysis of genes known or suspected to be involved in thrombosis and hemostasis and prediction of variant impact using algorithms that account for conservation and deleteriousness of all variants. c. Verify the impact of novel variants in independent samples: In silico (analyze genetic databases for suspected variants), in vivo (assess bleeding in animal models of hemophilia after introducing presumed modifier variants). 2. We will employ a model for obtaining informed consent and communicating individual genetic research results and results with potential clinical impact to research participants: a. The informed consent process will be performed after potential participants read a pamphlet entitled “Genetic Research at The Rockefeller University Hospital and Center for Clinical and Translational Science.” The pamphlet includes 16 questions that the potential participants are urged to ask the investigator, including, “What will you look for in my genetic information?”, “Will I receive results from this study?”. Potential participants will also be informed of the meaning of clinically actionable variants, either pathogenic variants related to phenotype or secondary (“incidental”) findings (i.e. variants unrelated to phenotype, the knowledge of which could lead to actions that may improve health). Participants who do not want to receive information about potentially actionable variants will be excluded from the study to avoid a situation where the investigator has clinically important information that cannot be shared with the participant. b. Genetic testing will be performed in a CLIA-certified lab to allow investigators to share the results with the study participants. c. Results will be reported to study participants according to a standard operating procedure (SOP) that classifies the report of variants according to the relation to phenotype and the pathogenic potential. d. Participant satisfaction with the informed consent process and the return of results will be assessed by a questionnaire for obtaining participants’ perceptions of their research experience, based on a standard set of validated research participation experiences measures (Kost RG et al, J Clin Transl Sci. 2018;2:31). RESULTS/ANTICIPATED RESULTS: Samples from individuals with severe null mutation hemophilia and a mild bleeding phenotype will be enriched in genetic modifier variants. After completing participation, participants will express satisfaction with the informed consent process and the results of the return of genetic information. DISCUSSION/SIGNIFICANCE OF IMPACT: Genetic risk assessment to predict bleeding risk has the potential to provide hemophilia patients with tailored therapy, allowing for very early initiation of treatment (prophylactic thrice weekly IV administration of FVIII) in patients with a high bleeding risk and deferring this costly and burdensome treatment in patients who are expected to be mild bleeders. Genetic modifier variants of hemophilia may be found to predict thrombosis in non-hemophiliac patients and profoundly impact the treatment of venous thrombosis. A structured process for obtaining consent for NGS and return of genetic results to study participants can protect them from uncertain genetic information. Moreover, this process will prevent a situation in which investigators have knowledge about clinically actionable variants but they are not allowed to report them to the participants or do not have a process for doing so. Sharing individual research results and results with clinical significance with participants of studies that involve whole exome sequencing can promote transparency and engagement of participants throughout the research enterprise.

OBJECTIVES/SPECIFIC AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in the United States and increases risk for cirrhosis and liver cancer. Identifying modifiable risk factors for NAFLD could allow better targeting of prevention programs. Insulin resistance (IR) plays a significant role in the development and progression of NAFLD. IR is also an important precursor to the development of type 2 diabetes (T2DM). However, the development and duration of IR during young adulthood and its association with NAFLD and T2DM in midlife is unclear. To test whether trajectories of IR using homeostatic model assessment (HOMA-IR) change throughout early adulthood are associated with risk of prevalent NAFLD and T2DM among persons with NAFLD in midlife independent of current or baseline HOMA-IR. METHODS/STUDY POPULATION: Participants from the CARDIA study, a prospective multicenter population-based biracial cohort of adults (baseline age 18–30 years), underwent HOMA-IR measurement (≥8 h fasting and not pregnant) at baseline (1985–1986) and follow-up exam years 7, 10, 15, 20, and 25. At Year 25 (Y25, 2010–2011), liver fat was assessed by noncontrast computed tomography (CT). NAFLD was defined as CT liver attenuation <51 Hounsfield Units after exclusion of other causes of liver fat (alcohol/hepatitis/medications). Latent mixture modeling was used to identify 25-year trajectories in HOMA-IR over time. Multivariable logistic regression models were used to assess associations between HOMA-IR trajectory groups and prevalent NAFLD with adjustment for baseline or Y25 HOMA-IR. RESULTS/ANTICIPATED RESULTS: Among 3060 participants, we identified 3 distinct trajectory groups for HOMA-IR for individuals free from diabetes in middle adulthood: qualitatively low-stable (46.7% of the cohort), moderate-increasing (42.0%), and high-increasing (11.3%) with a NAFLD prevalence at Y25 of: 8.3%, 33.4%, and 63.5%, respectively (p-trend<0.0001). After adjustment for confounders (baseline smoking status, alcohol use, body mass index, physical activity score, systolic blood pressure, antihypertensive medication use, and total/HDL cholesterol ratio) and baseline HOMA-IR, increasing HOMA-IR trajectories were associated with greater NAFLD prevalence compared with the low-stable trajectory group [odds ratio (95% CI): 5.8 (4.3–7.9) and 22.3 (14.2–34.9) for moderate and high, respectively]. These associations were attenuated, but remained significant, even after controlling for current Y25 HOMA-IR [OR=3.6 (2.6–5.0) for moderate and 5.9 (3.4–10.3) for high (referent: low)]. Among participants with NAFLD (n=511), high-increasing HOMA-IR trajectory was associated with greater prevalent [OR=6.5 (1.6–25.7)] and incident [OR=8.7 (2.2–34.4)] T2DM at Y25 independent of confounders and Y25 HOMA-IR (referent: low-stable). DISCUSSION/SIGNIFICANCE OF IMPACT: In this community-based sample of individuals free from diabetes at baseline, an increasing HOMA-IR trajectory through young adulthood was associated with greater NAFLD prevalence in midlife. Knowledge of changes in IR throughout adulthood provides new information on the risk of T2DM among persons with NAFLD in midlife independent of current level of IR. These findings highlight early identification of increasing IR as a potential target for primary prevention of T2DM in the setting of NAFLD.