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/Goals: We will conduct a 12-week pilot randomized controlled trial (RCT) to test the feasibility, acceptability, and preliminary efficacy of a staged-intensity whole foods intervention on hemoglobin A1c (HbA1c) change in adults, diet quality change (via the 2020 healthy eating index [HEI-2020]) in adults and offspring, and diet adherence and social determinants of health (SDOH) considerations via focus groups. Methods/Study Population: In this two-arm, parallel RCT, 30 adults with prediabetes (25–59 years) and their offspring (6–18 years) will be randomized to receive the 1) 12-week whole foods intervention which includes a 2-week feeding period (all foods/recipies provided), a 6-week customizable feeding period (3 dinners/recipies weekly), and a 4-week maintenance period (no food/recipies). The control group will receive standard of care (i.e., single RD-led diet counseling session). Primary outcomes include feasibility (≥80% retention and completion of study outcome measures) and acceptability (≥75% adult self-reported diet satisfaction). Intervention effects include 1) HbA1c change at 12-weeks in adults and 2) adult/offspring HEI-2020 scores assessed via diet records. Focus groups will assess influences of SDOH on diet adherence. Results/Anticipated Results: We have received Institutional Review Board approval, and recruitment is planned for January 2025. We will enroll 30 families from the greater Nashville, TN area. An intent-to-treat analysis will be conducted to test the preliminary effects of the whole foods diet intervention on the 12-week change in HbA1c (adults only) and 2020-HEI diet quality scores during the intervention period (adults and offspring). Focus groups will be conducted to understand how individual and family needs/preferences and SDOH may be perceived barriers or facilitators of diet adherence. Data generated from this study will be used to guide a fully powered RCT of our whole foods intervention to assess long-term effects on additional diabetes and metabolic outcomes and assessment of SDOH influences to support long-term adherence. Discussion/Significance of Impact: A healthy diet pattern is an effective nonpharmacological solution to prevent T2D, but only if it can be maintained. A family-centered whole foods diet pattern that uses “food as medicine” and considers how individual and family needs/preferences, and SDOHs could be an effective and sustainable multigenerational solution to prevent T2D in families.

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.

ABSTRACT IMPACT: The potential to use vaginal pH as a low cost, non-invasive diagnostic test at the point of CIN2 diagnosis to predict worsening of cervical disease. OBJECTIVES/GOALS: We previously reported that persistence/progression of cervical intraepithelial neoplasia-2 (CIN2) was uncommon in women living with HIV (WLH) from the Women’s Interagency HIV Study (WIHS, now MWCCS). Here we examined additional factors that may influence CIN2 natural history. METHODS/STUDY POPULATION: A total of 337 samples from 94 WLH with a confirmed CIN2 diagnosis were obtained from the MWCCS. 42 cervicovaginal HPV types and 34 cervicovaginal cytokines/chemokines were measured at CIN2 diagnosis (94 samples) and 6-12 months prior to CIN2 diagnosis (79 samples). Covariates, including CD4 count and vaginal pH, were abstracted from core MWCCS visits. Logistic regression models were used to explore CIN2 regression (CIN1, normal) vs. persistence/progression (CIN2, CIN3). Log rank tests, Kaplan Meier method, and Cox regression modeling were used to determine CIN2 regression rates. RESULTS/ANTICIPATED RESULTS: The most prevalent HPV types were HPV54 (21.6%) and 53 (21.3%). 33 women (35.1%) had a subsequent CIN2/CIN3 diagnosis (median 12.5 years follow-up). Each additional hr-HPV type detected at the pre-CIN2 visit associated with increased odds of CIN2 persistence/progression (OR 2.27, 95% CI 1.15, 4.50). Higher vaginal pH (aOR 2.27, 95% CI 1.15, 4.50) and bacterial vaginosis (aOR 5.08, 95% CI 1.30, 19.94) at the CIN2 diagnosis visit associated with higher odds of CIN2 persistence/progression. Vaginal pH >4.5 at CIN2 diagnosis also associated with unadjusted time to CIN2 persistence/progression (log rank p=0.002) and a higher rate of CIN2 persistence/progression (adjusted hazard ratio [aHR] 3.37, 95% CI 1.26, 8.99). Cervicovaginal cytokine/chemokine levels were not associated with CIN2 persistence/progression. DISCUSSION/SIGNIFICANCE OF FINDINGS: We found relatively low prevalence of HPV16/18 in this cohort. Elevated vaginal pH at the time of CIN2 diagnosis may be a useful indicator of CIN2 persistence/progression and the rate of persistence/progression.

OBJECTIVES/GOALS: Frontiers CTSI developed applications to ensure its science teams have technological tools to advance their community engagement and trial management. The Trial Finder app is a mobile application that allows users to navigate available trials. The Accrual app will help study teams monitor their recruitment performances in real time. METHODS/STUDY POPULATION: The Data Science team at the University of Kansas Medical Center (KUMC) had previously developed similar applications for The University of Kansas Cancer Center. Both retrieve information from KUMC’s clinical trial management system and ClinicalTrials.gov. This was replicated to include KUMC Pulmonary Critical Care (PCC) and KUMC Neuromuscular (NM) trials. Frontiers CTSI is working with both groups for piloting and feedback. Recruiting and marketing strategies for investigators to add their trials to both apps will be done through existing communication channels and be highlighted on Frontiers trial resource website. Recruiting and marketing strategies of the Frontiers Trial Finder app to the external community will have a focus on, but not limited to, paid social media advertising. RESULTS/ANTICIPATED RESULTS: The Trial Finder app can help providers search for trials their patient may be eligible for during clinic visits and to engage with the community by allowing anyone to download and browse on their Android/iOS device. Built in REDCap forms are used to capture contact information. The Accrual app is a web-based application that helps study teams monitor their recruitment performances in real time and provide an opportunity to adjust strategies. It uses an in-house algorithm to predict if trials will meet timeline goals. This data is conveniently laid out on a single web page so that science teams can overview all their trials’ recruitment performances simultaneously. The next phase of developing these applications is to market their use within Frontiers CTSI and its community catchment area. DISCUSSION/SIGNIFICANCE: Through collaboration, Frontiers CTSI is developing resources to support community engagement and trial management. New innovative applications like these ensure all the main stakeholders involved with clinical trial execution are always engaged and have access to iterative contemporary technologies that support their research.

OBJECTIVES/GOALS: 1) Assess the patient-reported, perceived change in olfactory function after bimodal visual-olfactory training (OT) 2) Assess change in olfactory function after bimodal visual-olfactory training with a smell identification test 3) Assess which scents are most important to people with olfactory dysfunction (OD) METHODS/STUDY POPULATION: The participants are adults with subjective or clinically diagnosed OD with post-surgical or traumatic etiologies within the last 5 years. At the first of two study visits, participants complete the University of Pennsylvania Smell Identification Test (UPSIT) and complete general health (SF-36) and olfactory-related quality-of-life questionnaires. From a list of 34 scents, participants chose the 4 scents most important to them and smelled the scents twice daily for 3 months. Olfactory testing and the quality-of-life questionnaires were repeated at the final visit. RESULTS/ANTICIPATED RESULTS: 10 participants have enrolled in the study. There was one screen fail and one withdrawal. Six participants are currently undergoing OT and two have completed the study. Seven participants have post-surgical etiology and three have post-traumatic etiology of their OD. Of the two participants who have completed the study, one had an UPSIT score improvement from 25 to 33 out of the 40 questions correct. The minimally clinically important difference on the UPSIT is 4. She reports improvement subjectively. The second participant had a UPSIT score change from 25 to 24 and reports ability to smell is neither better nor worse. DISCUSSION/SIGNIFICANCE OF IMPACT: Traumatic and post-surgical, particularly post-transphenoidal hypophysectomy, are common etiologies of OD and no effective treatments exist. The results from our pilot study will help better inform the best way to undergo OT, how effective it is, and the planning of future studies.

OBJECTIVES/GOALS: This study evaluates the impact of an updated and expanded training for social and behavioral health researchers. Participants’ experience with training modules focused on community engagement is a focus of this evaluation as is the application of this training by participants in teams. METHODS/STUDY POPULATION: The Social and Behavioral Research training series for health researchers and team members was first created by faculty and staff of the Michigan Institute for Clinical and Health Research in 2018. This training was updated and expanded in 2021 with support from the National Institutes of Health to include new material regarding community-engaged health research as well as updates concerning technology and new federal regulations. Past participants of the training were invited to retake the training, as were clinical and translational researchers at University of Michigan who were new to the training. Surveys were sent to all participants after completing the training and a focus group of research staff was conducted to identify how they utilized the training in support of their teams. RESULTS/ANTICIPATED RESULTS: It is anticipated that at least 100 individuals will participate in the evaluation of the Social and Behavioral Research training between October 2022 and February 2023. Data extracted from U-M’s learning management platform will demonstrate how well participants performed on key knowledge checks embedded in the modules and how quickly they progressed through the sections of the training. These results will be compared to benchmarks derived from evaluations of the prior course which were conducted in 2018. A focus group of at least 10 individuals will demonstrate how health research staff utilized the training and associated resources to advance the scientific work of their study teams. DISCUSSION/SIGNIFICANCE: Comprehensive training programs for research best practices in social and behavioral health need to have tailored and up-to-date information for this group of researchers and staff. The results of this evaluation will demonstrate how this program contributed to the professional development of the health research workforce.

OBJECTIVES/SPECIFIC AIMS: We recently reported that PRMT5 epigenetically activates androgen receptor (AR) in prostate cancer cells. Because targeting AR signaling through androgen deprivation therapy is clinically used as a radiosensitization approach to treat high-risk prostate cancer, our finding raised an exciting possibility that targeting PRMT5 may improve RT for prostate cancer patients. Contrary to our expectation, targeting PRMT5 sensitized both AR expressing and AR negative (AR−) prostate cancer cell lines to radiation. The goal of our study was therefore to determine the role of PRMT5 in repair of IR-induced DSBs and to translate these findings to improving radiation therapy for cancer patients in general (not just prostate cancer patients). METHODS/STUDY POPULATION: The majority of experiments were basic science experiments analyzing PRMT5’s role in the DNA damage response in normal and cancer cell lines. For example, to extend our findings and determine if PRMT5’s role in DSB repair is conserved across multiple cell types, we performed similar experiments in AR− prostate cancer cells, luminal breast cancer cells, glioblastoma cells, and human embryonic kidney cells. To determine the clinical significance of our finding, we also analyzed mRNA expression of PRMT5, AR, and both PRMT5 and AR target genes involved in DSB repair across 43 clinical cancer data sets. RESULTS/ANTICIPATED RESULTS: (1) Targeting PRMT5 sensitizes prostate cancer cells to IR in an AR-independent manner, (2) PRMT5 regulates the repair of IR-induced DSBs in an AR-independent manner, (3) RNA-seq analysis reveals that PRMT5 likely regulates genes involved in the DNA damage response, (4) PRMT5 activates expression of several genes in the DDR including those involved in DSB repair, (5) PRMT5 functions as an epigenetic activator of genes involved in DDR, (6) PRMT5 is required for NHEJ, HR, and G2-Arrest upon IR treatment, (7) Upregulation of PRMT5 correlates with formation and repair of IR-induced DSBs, (8) PRMT5’s role in repair of IR-induced DSBs is conserved in several normal and cancer cell types, and (9) PRMT5 expression correlates with expression of DSB repair proteins in clinical cancer samples. DISCUSSION/SIGNIFICANCE OF IMPACT: In summary, we provide evidence that PRMT5 is a master epigenetic regulator of IR-induced DSB repair through epigenetic activation of multiple target genes involved both HR and NHEJ as well as G2 arrest. Interestingly, the majority of genes regulated by PRMT5 are well-characterized, “core repair proteins” involved in HR (RAD51, BRCA1, BRCA2, RAD51D, and RAD51AP1), NHEJ (NHEJ1, Ku80, XRCC4, and DNAPKcs), and G2 arrest (Cdk1, CDC25C, CCNB2, and WEE1), which may explain why PRMT5 is essential to repair IR-induced DSBs in several cell lines. Although AR may also regulate DSB repair via both HR and NHEJ, several pieces of evidence in our study suggest that PRMT5 also regulates DSB repair independent of AR. First, PRMT5 targeting sensitizes both AR+ and AR− prostate cancer cells to IR. Second, exogenous expression of AR only partially rescues the impairment of IR-induced DSB repair by PRMT5 knockdown. Third, PRMT5 knockdown increases IR-induced DSB in AR− DU145 cells and several other cancer cell lines and normal cells. Fourth, PRMT5 expression correlates positively with the expression of its target genes in multiple human cancer tissues. During preparation of this project, Braun et al. reported that PRMT5 post-translationally regulates the splicing out of detained-introns (DI)s of genes to modulate gene expression. However, analysis of their data showed that the majority of DEGs we identified either do not contain DIs or DI splicing was not affected by targeting PRMT5. In addition, Clarke et al. reported that PRMT5 participates in the DSB repair choice process and promotes HR through methylation of RUVBL1. It is therefore likely that PRMT5 regulates repair of IR-induced DSB via multiple mechanisms. As PRMT5 is overexpressed in many human cancers and its overexpression correlates with poor prognosis, our findings suggest that increased DSB repair by PRMT5 overexpression in these human cancers may confer survival advantages particularly following DNA damaging treatment. Because targeting DSB repair has been proven to be a valid therapeutic approach for cancer treatment, our findings here also suggest that PRMT5 targeting may be explored as a monotherapy or in combination therapy with RT or chemotherapy for cancer treatment.

OBJECTIVES/GOALS: Childhood Sjögren’s disease (cSD) is a rare autoimmune disease. Despite the profound impact on children and their families, pediatric-specific clinical trials to inform therapeutic strategies in cSD are lacking. In 2022 we participated in the Trial Innovation Network (TIN) Design Lab with the purpose of designing a series of N-of-1 trials for cSD. METHODS/STUDY POPULATION: New medications have the potential to be safe/effective treatments for cSD but must be evaluated in randomized trials. To overcome limitations of traditional parallel-group designs given the rarity of cSD, we developed an N-of-1 trial approach. Our proposal was selected by the Tufts TIN Design Lab. The Design Lab multi-stakeholder process involved parents of and patients with cSD, pediatric and adult rheumatologists, and experts in clinical trial design and outcomes. We engaged all stakeholders in protocol development to maximize the impact of the proposed approach on clinical care, ensure a successful recruitment plan, and inform the choice of endpoints as there are no widely accepted cSD outcome measures to determine treatment efficacy. RESULTS/ANTICIPATED RESULTS: Using the Design Lab methodology, we clarified the N-of-1 study goals and engaged in an iterative process to develop a “briefing book” that ensured a sound premise for our study. We reviewed and accumulated published literature to support our focus on mucosal/glandular manifestations, identified potential interventions to be used in the N-of-1 trials, and enumerated possible outcomes, including outcomes important to patient/parents. This work culminated in a full-day Design Lab event that included multiple stakeholders who provided expertise from different perspectives on the full drug development pathway. Study design feedback focused on three specific areas. 1) Inclusion and exclusion criteria; 2) Identification of outcome measures; 3) Treatment and washout periods. DISCUSSION/SIGNIFICANCE: To address the critical need and move treatment of cSD forward, we are designing a prototype N-of-1 trial in children with rheumatic disease. We will continue to engage stakeholders by using a series of Delphi surveys and an in-person meeting to create composite outcome measures to test cSD therapies in personalized trials.

OBJECTIVES/GOALS: modonko1@jhmi.edu METHODS/STUDY POPULATION: Michelle Odonkor RESULTS/ANTICIPATED RESULTS: I accept DISCUSSION/SIGNIFICANCE: No, I do not want my poster published JCTS.

OBJECTIVES/GOALS: There is a high burden of lung cancer in persons living with HIV (PLWH). The role that HIV status, by levels of immune function and viral load, has on survival from lung cancer is not fully understood. The study’s objectives were to assess 1) the association of HIV with survival in non-small cell lung cancer (NSCLC) and 2) prognostic factors in PLWH with NSCLC. METHODS/STUDY POPULATION: Participants were from a cohort of lung cancer patients diagnosed between 2004-2017 in the Bronx, NY, with vital status ascertainment at least annually. We compared survival from NSCLC diagnosis between HIV-negative patients (HIV-, N = 2881) and PLWH (N = 88), using Cox regression, accounting for clinical and sociodemographic factors including smoking status. In three separate comparisons to HIV-, PLWH were dichotomized by CD4 count (<200 vs. ≥200 cells/μL), CD4/CD8 ratio (median, <0.43 vs. ≥0.43) and HIV viral load (VL) suppression (<75 vs. ≥75 copies/mL). In PLWH only, we assessed the relationships of CD4 count, CD4/CD8 ratio, and VL at diagnosis with survival adjusting for age, sex, and cancer stage. CD4 count and CD4/CD8 ratio were also examined as time-varying variables using a counting process approach. RESULTS/ANTICIPATED RESULTS: PLWH were younger (median 56 years, IQR 51-52 vs. 68, IQR 60-76) and more likely to be current smokers (58% vs. 37%) at diagnosis than HIV- patients. Median survival was lower in PLWH [1.1 years, 95% confidence interval (95%CI): 0.6-1.3] than in HIV- [1.6 (1.5-1.7)]. Survival comparing PLWH with higher CD4/CD8 to HIV- was similar [hazard ratio (HR), 95%CI: 0.63 (0.37-1.07)], but those with lower CD4/CD8 experienced worse survival (HR = 1.74, 95%CI: 1.07-3.89). Among PLWH, having a CD4 count < 200 cells/μL was associated with over twice the risk of death compared to those with CD4 ≥ 200 cells/μL (HR = 2.37, 95%CI: 1.14-4.92). VL and CD4/CD8 ratio were not associated with survival. Lower time-updated CD4 count was also associated with worse survival (HR = 2.19 for CD4 <200 vs. >200 cells/μL, 95%CI: 1.16-4.13). DISCUSSION/SIGNIFICANCE OF IMPACT: Among persons with NSCLC, CD4/CD8 ratio nearest diagnosis was shown to distinguish mortality risk in PLWH compared with HIV- patients. In addition, PLWH with low CD4 had worse prognosis than PLWH who had higher CD4 counts. These results suggest HIV immune status to be an essential component influencing survival in lung cancer.