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: MicroRNAs (miRNA) affect transcription of a number of genes involved in the development and progression of diabetic nephropathy (DN), and have become attractive therapeutic targets and biomarkers. Elevated renal gluconeogenesis, fibrosis, and albuminuria are early markers of incipient DN. Recent studies report that renal miRNA-451 may protect against DN and reduce renal gluconeogenesis in rodent models. MiRNA-451 is thought to act by targeting select factors resulting from disrupted insulin and growth factor signaling and the mechanistic-target of rapamycin (mTOR) in early DN. This study aimed to elucidate the role of miRNA-451 in the development and progression of DN. METHODS/STUDY POPULATION: To further elucidate the role of miRNA-451 in DN, we placed male insulin-resistant, TALLYHO/Jng mice on a high-fat diet (60% kCal). The mice were divided into 2 treatment groups and received 8 consecutive weekly intraperitoneal injections of locked nucleic acid (LNA) miR-451-inhibitor or LNA-scrambled compound (2 mg/kg·bw; n=8/treatment). Mice were euthanized after 12 weeks (4 weeks sans injections) and kidneys, liver, pancreas and abdominal adipose tissue were harvested for analysis. RESULTS/ANTICIPATED RESULTS: Renal homogenate expression of miRNA-451 was drastically reduced in inhibitor-treated mice (~6-fold) in comparison with scramble-treated mice. Western blotting of cortex homogenates for indicators of fibrosis and targets of miRNA-451 revealed a significant reduction in collagen IV (marker of epithelial integrity) in inhibitor-treated mice. In addition, metalloproteinase type 9 (MMP9, a known type IV collagenase), YWHAZ (a scaffolding protein and known target of miR-451), mTOR, and fructose bisphosphatase (FBP1, a rate-limiting gene in gluconeogenesis) were significantly increased in this group in comparison to scramble-treated mice. However, no differences were found in protein levels for glucose-6-phosphatase (G-6-Pase) or phosphoenolpyruvate (PEPCK), 2 additional gluconeogenic rate-limiting genes. MiRNA-451 antagonist did not significantly affect final body weight or blood glucose; however, mean blood sodium concentrations were slightly, but significantly higher (2%) in the LNA-inhibitor treated group (when compared with the scramble-treated group). No differences in blood potassium or chloride were found. Anion gap was 90% higher in the LNA-inhibitor treated group when compared with scramble-treated mice. No differences in urinary albumin to creatinine ratio were found between the two treatment groups. However, Masson Trichrome scoring revealed a 59% increase in fibrosis in inhibitor-treated mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Collectively, these findings support a potentially protective role of miRNA-451 in attenuating signaling via mTOR that may alter both renal gluconeogenic potential (contributing to the diabetic phenotype) and activation and progression of renal fibrosis. Therapies to enhance miRNA-451 signaling may be beneficial to reduce renal pathology associated with DN.

OBJECTIVES/SPECIFIC AIMS: CFTR is the mutant protein that causes cystic fibrosis (CF), a fatal respiratory diseases affecting 1 in 3500 children. CFTR modulators are small molecules that directly address mutant CFTR function. Improving correction of the F508del CFTR mutation (affecting 90% of CF patients) is one of the most pressing unmet needs in CF. Currently available F508del therapeutics only marginally improve CF, In vitro, we have identified a miRNA that impairs utility of CFTR directed therapies. miR-145 is upregulated by TGF-β (a genetic modifier of CF lung disease) with a direct binding site on the 3’-untranslated region of CFTR mRNA. Binding of miR-145 to CFTR destabilizes mRNA transcript and impedes protein translation. Overexpression of miR-145 abolishes benefit of F508del CFTR correction. Antagonists to miR-145 block TGF-β suppression of CFTR function and augment response to CFTR correction. This project evaluate in vivo impact of TGF-beta and miRNA manipulation on CFTR functional readouts including nasal potential difference (NPD) and short circuit current (Isc) across tracheal explants in addition to standard biochemical measures. METHODS/STUDY POPULATION: Wild-type Sprague-Dawley rats were inoculated with an adenoviral vector containing bioactive TGF-beta or sham at 1×109 pfu/animal placed in the left nares. Seven days post-inoculation, functional, and biochemical measures were conducted. NPD was measured with a microelectrode placed in the left nare and grounded the tail. The nare was sequentially perfused with standard Ringer’s solution, amiloride (to block the ENaC sodium channel), low chloride Ringer’s (to stimulate chloride efflux), forskolin (to open the CFTR channel) and CFTRinh-172 (to block the CFTR channel. Tracheal explants were harvested, microdissected, and placed on modified Ussing chambers. RESULTS/ANTICIPATED RESULTS: We have inoculated WT rats with bioactive TGF-β Versus sham delivered by intranasal inoculation of an adenoviral vector. Functional readout of CFTR function is by Isc across tracheal epithelia and NPD. Lung homogenates are analyzed for TGF-β signaling, miRNA expression, and CFTR transcripts. Both tracheal explants and NPD indicate TGF-β stimulation diminishes CFTR function in vivo. In tracheal explants, TGF-β exposure diminishes CFTR response to forskolin-stimulation by 75%. Loss of current after CFTR inhibition (CFTRinh-172) is halved. By nasal PD, TGF-β inoculation similarly halves the bioelectric response to low chloride and forskolin stimulation. Evaluation by qPCR reveals a strong increase in TGF-β signaling demarcated by PAI-1, prompting a reduction in CFTR mRNA. miR-145 is expressed highly in rat pulmonary tissue, but no change in overall miR-145 levels was detected between TGF-β and sham exposed rats. This finding reflects what we have observed in human lungs, with a localized increased miR-145 expression in CF epithelia, but similarly high levels of miR-145 in both CF and non-CF whole lung homogenates. Although expressed at lower levels than miR-145, we did find increased expression in TGF-β relevant miR-101, miR-494, and miR-144 that have a predicted binding site on rat 3’-UTR in TGF-β exposed Versus sham lungs. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data indicate the relevance of TGF-β stimulation to suppress CFTR synthesis and function in vivo. Future work will evaluate whether these additional miRNA with CFTR binding sites may mediate TGF-β suppression of CFTR in the rat model, and the utility of miRNA manipulation to augment F508del CFTR correction.

OBJECTIVES/SPECIFIC AIMS: Cystic fibrosis (CF) is a life-shortening genetic disease that affects approximately 30,000 patients in the United States. CF patients suffer from chronic pulmonary infections that are associated with hyperinflammation and irreversible damage to the lower airways. As CF patients age, Pseudomonas aeruginosa (P.a.) is the predominant pathogen that infects the respiratory tract. The P.a. strains initially infecting the CF lung have a nonmucoid colony morphology, whereas, once chronic infection is established, these bacteria mutate leading to the emergence of mucoid P.a. variants with heightened resistance to both antibiotics and host immunity. Both nonmucoid and mucoid P.a. variants are often co-isolated on microbiological cultures of sputum collected from CF patients. However, the CF lung is known to exhibit heterogeneity in inflammation and infecting microbes across different lung regions that cannot be studied using routine sputum collection alone. Here, using a standardized bronchoscopic protocol, bronchoalveolar lavage (BAL) fluid was prospectively collected from each lobe of a CF cohort undergoing clinically indicated surgical procedures. We sought to investigate if there is an association between infecting P.a. variants (nonmucoid, mucoid, or mixed populations), the lung lobes in which these variants are found, and regional proinflammatory cytokine production. METHODS/STUDY POPULATION: We performed BAL on 16 CF patients with clinically stable disease. For each patient, we obtained BAL fluid from the right upper lobe, right middle lobe, right lower lobe, left upper lobe, lingula, and left lower lobe. We plated BAL fluid on nonselective and P.a.-selective medium to quantitate bacteria and to identify P.a. colony subtypes (nonmucoid, mucoid, or mixed). We further used a V-PLEX human cytokine array to quantitate inflammatory cytokine concentrations (IL-1β, TNF-α, IL-6, IL-8, and IL-10) within BAL fluid specimens. Our specimen collection was approved by the local IRB with informed consent and assent obtained from patient volunteers. RESULTS/ANTICIPATED RESULTS: Based on microbiological analysis, each lobar BAL specimen was classified as uninfected with P. a. or infected with nonmucoid, mucoid, or mixed (both nonmucoid and mucoid) P.a. variants. There was no observed propensity of mucoid or nonmucoid variants to be confined to certain lung lobes in our cohort. However, infection with mucoid P.a. variants was associated with higher concentrations of IL-1β (p<0.001), TNF-α (p<0.001), IL-8 (p<0.001), and IL-10 (p<0.001) within lobar BAL fluid compared with P.a.-free specimens. Specimens with mucoid variants also had greater concentrations of TNF-α (p<0.01), IL-8 (p<0.001), and IL-10 (p<0.05) compared with specimens with only nonmucoid P.a. variants. Patients infected with mixed mucoid and nonmucoid variants showed higher concentrations of TNF-α and IL-10 (p<0.05) as well as nonsignificant trends for higher concentrations of IL-1β and IL-6 compared to P.a.-free samples. Interestingly, the presence of nonmucoid P.a. variants was inversely correlated with IL-6 (p<0.05). Total bacterial burden (both P.a. and non-P.a. species) within BAL fluids was positively correlated with higher proinflammatory cytokine concentrations. Additionally, independent of bacterial colonization, the upper lobes (right upper lobe and left upper lobe) of the lungs showed trends towards higher proinflammatory cytokine concentrations compared with the lower lobes (right lower lobe and left lower lobe). DISCUSSION/SIGNIFICANCE OF IMPACT: Our results demonstrate that P.a. variants (mucoid or nonmucoid) appear not to be geographically restricted in ability to colonize any lobe of the CF lung. Moreover, infection with mucoid P.a. (either alone or in mixed populations with nonmucoid variants) is associated with higher inflammatory cytokine concentrations in the CF lung. Given that infection with mucoid P.a. predicts deterioration in pulmonary function, this study provides a rationale for further investigation of cytokines as diagnostic/prognostic correlates of infection and lung disease in CF.

OBJECTIVES/SPECIFIC AIMS: The objective of this project is the application of complex fusion models, which combine observed and modeled data, to areas with sparse monitoring networks with multiple chemical components is under-developed. Such models could provide improved accuracy and coverage for air quality measurement predictions, an area greatly limited by the amount of missing data. METHODS/STUDY POPULATION: This project focuses on the development of methods for improved estimation of pollutant concentrations when only sparse monitor networks are found. Sparse monitoring networks are defined as areas where fewer than three criteria air pollutants (based on EPA standards) are monitored. Particularly, a multivariate air pollutant statistical model to predict spatio-temporally resolved concentration fields for multiple pollutants simultaneously is developed and evaluated. The multivariate predictions allow monitored pollutants to inform the prediction of nonmonitored pollutants in sparse networks. RESULTS/ANTICIPATED RESULTS: Daily, ZIP code level pollutant concentration estimates will be provided for 8 pollutants across South Carolina, and goodness of fit metrics for model variants and previously established methods will be compared. DISCUSSION/SIGNIFICANCE OF IMPACT: These methods utilize only widely available data resources, meaning that the improved predictive accuracy of sparsely monitored pollutant concentrations can benefit future studies in any US area by improving estimation of health effects and saving resources needed for supplemental air pollutant monitoring campaigns. Our method for estimation attempts to improve predictive accuracy and data availability for sparsely monitored pollutants and areas.

OBJECTIVES/SPECIFIC AIMS: Parkinson’s disease (PD) is a condition that affects over a million Americans, and despite current medical therapies, the progression of the disease results in impaired generation of internally timed or guided (IG) movements. To address this loss of motor function, previous rehabilitation therapies have focused on remediating the affected striatal-thalamic-cortical circuits (STC), primarily thought to be responsible in generating timed motor patterns. However, given the disease leads to the cell death of dopaminergic cells that are essential for proper STC function, we propose a motor therapy aimed at utilizing a compensatory parallel cerebellar-thalamic-cortical (CTC) pathway, recruited to perform externally guided (EG) movements, in which gait initiation is driven from sensory input. Our previous study has shown efficacy in our novel argentine tango therapy and improves behavioral measures above the relevant MCID threshold, but it has not been established that the CTC are in the causal pathway that are responsible for these changes. Using neural measures from task fMRI, we have begun to characterize networks that have changed and quantify any associations with behavioral metrics. METHODS/STUDY POPULATION: Patients were randomly assigned to an IG (n=18), EG (n=18), or education contact control (n=14). Participants were assessed preintervention and postintervention for behavioral motor and cognitive measures and neurophysiologically with task based fMRI. In the task, participants performed a foot tapping task under both IG (tap their foot in previously learned rhythm) or EG (tap immediately after receiving a tactile cue on their hand) conditions. The fMRI data were preprocessed using AFNI and registered to MNI standard space. The brainnetome atlas was applied and the average time series of each region of interest (ROI) was used to increase the signal to noise ratio. The activation of these ROI with respect to the stimulus was modeled using GLM, and we estimated the area under the curve during the task blocks. A 1-way ANOVA analysis on these betas were performed between the pre and the post intervention time points and the ROIs that were above a significance of 0.95 were identified and corrected for multiple comparisons. The change in beta in all ROIs for each individual were calculated and then correlated with the changes in the behavioral data, to see which changes in ROI areas matched the best with the behavioral changes. RESULTS/ANTICIPATED RESULTS: The EG group showed significant changes only in the EG task in 2 areas—inferior frontal gyrus and inferior temporal sulcus. Correlating to the cognitive behavioral measures show reduced error from the Inferior frontal gyrus (corr>0.5) best reflect changes in observed. There were no changes to either the STC or the CTC pathways. The IG group showed no changes behaviorally and showed no changes neurally as well. The control group showed no changes behaviorally, but neuronally certain DMN nodes, such as the precuneus and inferior temporal regions showed a significant change for both tasks. DISCUSSION/SIGNIFICANCE OF IMPACT: Addressing the damaged STC pathway directly through IG therapy may not be effective. The EG therapy may not be able to enhance the STC pathway. However, the therapy appears to utilize new areas in the frontal regions and correlates with positively with changes in spatial memory and balance tasks. Contrary to our hypothesis the CTC circuit was not upregulated for performance of the IG or EG task, but therapy may have enhanced recruitment of other cognitively engaged areas. The educational control group interestingly showed changes in the DMN network, which has been shown to be linked to attention during tasks blocks.

OBJECTIVES/SPECIFIC AIMS: Autism spectrum disorder (ASD) affects 1 in 68 people and includes restricted, repetitive behavior, and social communication deficits. Aspects of face processing (i.e., identity, emotion perception) are impaired in some with ASD. Neuroimaging studies have shown aberrant patterns of brain activation and connectivity of face processing regions. However, small sample sizes and inconsistent results have hindered clinical utility of these findings. The study aims to establish consistent patterns of brain responses to faces in ASD and provide directions for future research. METHODS/STUDY POPULATION: Neuroimaging studies were identified through a multi-database search according to PRISMA guidelines. In total, 23 studies were retained for a sample size of 383 healthy controls and 345 ASD. Peak coordinates were extracted for activation likelihood estimation (ALE) in GingerALE v2.3.6. Follow-up ALE analyses investigated directed Versus undirected gaze, static Versus dynamic, emotional Versus neutral, and familiar Versus unfamiliar faces. RESULTS/ANTICIPATED RESULTS: Faces produced bilateral activation of the fusiform gyrus (FG) in healthy controls (−42 −52 −20; 22 −74 −12, p<0.05, FDR) and left FG activation in ASD (−42 −54 −16, p<0.05, FDR). Activation in both groups was lateral to the mid-fusiform sulcus. Follow-up results pending. DISCUSSION/SIGNIFICANCE OF IMPACT: Reduced right FG activation to faces may inform biomarker or response to intervention studies. Mid-fusiform sulcus proved a reliable predictor of functional divides should be investigated on a subject-specific level.

OBJECTIVES/SPECIFIC AIMS: Background: Children with autism spectrum disorder (ASD) show a broad range of unusual responses to sensory stimuli and experiences. It has been hypothesized that early differences in sensory responsiveness arise from atypical neural function and produce “cascading effects” on development across a number of domains, impacting social and communication skill, as well as broader development in children affected by ASD. A primary challenge to confirming these hypotheses is that ASD cannot be definitely diagnosed in the earliest stages of development (i.e., infancy). A potential solution is to prospectively follow infants at heightened risk for ASD based on their status as infant siblings of children who are diagnosed. We examined the developmental sequelae and possible neurophysiological substrates of three different patterns of sensory responsiveness—hyporesponsiveness (reduced or absent responding to sensory stimuli) and hyperresponsiveness (exaggerated responding to sensory stimuli), as well as sensory seeking (craving of or fascination with certain sensory experiences). Infants at high risk (HR) for ASD were compared with a control group of infants at relatively lower risk for ASD (LR; siblings of children with typical developmental histories). Objectives: Research questions included: (a) Do HR infants differ from LR infants in early sensory responsiveness?, (b) Does sensory responsiveness predict future ASD and related symptomatology? and (c) Is sensory responsiveness predicted by resting brain states? METHODS/STUDY POPULATION: Methods: To answer these questions, we carried out a longitudinal correlational investigation in which 20 HR infants and 20 LR controls matched on sex and chronological age were followed over 18 months. At entry to the study, when infants were 18 months old, sensory responsiveness was measured using the Sensory Processing Assessment and the Sensory Experiences Questionnaire, and a number of putative neural signatures of early sensory differences were measured via resting state EEG. When infants were 24 and 36 months of age, ASD and related symptomatology was evaluated in a comprehensive diagnostic evaluation. RESULTS/ANTICIPATED RESULTS: Results: HR infants trended towards increased hyporesponsiveness and hyperresponsiveness and showed significantly elevated levels of sensory seeking relative to LR controls at 18 months of age. Both groups, furthermore, displayed a high degree of heterogeneity in sensory responsiveness. Atypical sensory responsiveness (increased hyperresponsiveness and/or hyporesponsiveness, as well as sensory seeking behavior) predicted several aspects of ASD and related symptomatology, including social, communication, and play skill, and was associated with differences in resting brain state, including metrics of oscillatory power, complexity, and connectivity, as well as hemispheric asymmetry. Moderation analyses revealed that several relations varied according to risk group, such that associations were stronger in magnitude in the HR Versus LR group. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion: Findings provide empirical support for the notion that early sensory responsiveness may produce cascading effects on development in infants at heightened risk for ASD. Differences in resting brain states may underlie atypical behavioral patterns of sensory responsiveness. From a clinical standpoint, results suggest that early sensory differences may be useful for predicting developmental trajectories, and be potentially important targets for early preventive intervention, in infants at risk for autism.

OBJECTIVES/SPECIFIC AIMS: Approximately 1.6 million Americans suffer from inflammatory bowel diseases (IBD), ulcerative colitis, and Crohn’s disease. It is a challenge for both physicians and patients alike to manage the disease, primarily due to lack of disease specific biomarkers. Endoscopy remains the gold standard to diagnose and evaluate IBD activity. Current biomarkers or their combinations cannot adequately predict IBD progression or relapse, and response to therapy. METHODS/STUDY POPULATION: In total, 97 IBD patients recruited at University of Kentucky undergoing endoscopy. Patients medical information was collected from electronic database including C-reactive protein (CRP), fecal calprotectin (FC), endoscopy/pathology report. RESULTS/ANTICIPATED RESULTS: The mean CRP and FC levels were 1.3 (normal <1) and 679 (normal <162), respectively. FC (sensitivity 74%) was more reliable to predict mucosal inflammation compare to CRP (sensitivity 36%). However, 52% of patients did not have FC performed (vs. CRP only 4%), and 45% of these patients failed to submit stool sample for analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data suggests FC is the most promising noninvasive marker for disease monitoring in IBD. It correlates well with endoscopic activity and mucosal inflammation. However, further analysis must be done to evaluate barriers to testing and issues with compliance from patients. We feel strongly that a blood biomarker for disease activity is vital for disease monitoring and response to therapy in IBD.

OBJECTIVES/SPECIFIC AIMS: To understand the role of PGF2a and to characterize a novel cyclooxyrgenase (COX)-independent prostaglandin synthesis pathway in epithelial ovarian cancer. METHODS/STUDY POPULATION: We used high grade epithelial ovarian cancer cell line (OVCAR3) as a model to study our pathway. Our main mode of PGF2a detection is through mass spectrometry. RESULTS/ANTICIPATED RESULTS: Our current results suggest the OVCAR3 cells may synthesize PGF2a independently of COX enzymes. We anticipate this novel pathway may be dependent on the TGFb pathway. DISCUSSION/SIGNIFICANCE OF IMPACT: Understanding the role and synthesis pathway of PGF2a may allow us to uncover a novel therapeutic pathway for high grade ovarian cancer.

OBJECTIVES/SPECIFIC AIMS: Diabetic retinopathy is an increasingly prevalent disease, difficult to screen for across the globe. We have developed and began optimizing an innovative technique to visualize and quantify retinal blood flow, to elucidate the role of the choroid in retinal pathologies such as diabetic retinopathy or choroidopathy. METHODS/STUDY POPULATION: Preliminary retinal was obtained from a surgical retina video library (Truvision, Goleta, CA, USA). Videos of different organs were recorded while vessels were occluded via a blood pressure cuff, using consumer-grade digital video cameras (NEX-5T, a7sii; Sony, New York, NY, USA). All other retinal videos were taken using a fundus camera (50×; Topcon, Oxland, NJ, USA) modified to support the above digital video cameras. All videos were processed using experimental software (MATLAB, Mathworks, Natick, MA, USA). RESULTS/ANTICIPATED RESULTS: Video imaging of the retina was optimized for lighting conditions and software requirements. Parameters were defined for the software imaging pipeline, such as frequency range of interest, sampling rate, and noise minimization. Software was developed to stabilize frames, accounting for eye saccades. Use of a biosensor enabled accurate measurement of pulse waveform, increasing signal-to-noise ratio. The optimal light requirements were determined such that adequate exposure of the retina is reproducible yet still comfortable for use in human subjects. DISCUSSION/SIGNIFICANCE OF IMPACT: This novel technique allows for an inexpensive, noninvasive, and reproducible ocular blood flow imaging platform. By optimizing this technique, we can proceed with our future plans for a pilot study to compare our imaging technique with the current standard, paving the way for future clinical studies.

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/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/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: 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/SPECIFIC AIMS: We aimed to develop an assay to measure new protein synthesis after Antisense Oligonucleotide treatment, which we hypothesized to be the earliest biochemical identification of RNA-targeting therapy efficacy. METHODS/STUDY POPULATION: We treated 2 transgenic animal models expressing proteins implicated in neurodegenerative disease: human tau protein (hTau) and human superoxide dismutase 1 (hSOD1), with ASO against these mRNA transcripts. Animals received isotope-labeled 13C6-Leucine via drinking water to label newly synthesized proteins. We assayed target protein synthesis and concentration after ASO treatment to determine the earliest identification of ASO target engagement. RESULTS/ANTICIPATED RESULTS: hTau ASO treatment in transgenic mice lowered hTau protein concentration 23 days post-treatment in cortex (95% CI: 0.05%–64.0% reduction). In the same tissue, we observed lowering of hTau protein synthesis as early as 13 days (95% CI: 29.4%–123%). In hSOD1 transgenic rats, we observed lowering of 13C6-leucine-labeled hSOD1 in the cerebrospinal fluid 30 days after ASO treatment compared with inactive ASO control (95% CI: 12.0%–48.4%). DISCUSSION/SIGNIFICANCE OF IMPACT: In progressive neurodegenerative diseases, it is crucial to develop measurements that identify treatment efficacy early to improve patient outcomes. These data support the use of stable isotope labeling of amino acids to measure new protein synthesis as an early pharmacodynamics measurement for therapies that target RNA and inhibit the translation of proteins.

OBJECTIVES/SPECIFIC AIMS: The objective of the present study is to determine if decreased adiponectin and increased leptin levels are associated with the development of MetS and identifiable endothelial dysfunction in a cohort of Hispanic pre-pubertal children. To do so we propose the following aims: (1) To measure expression of adiponectin and leptin levels in a Hispanic pre-pubertal cohort and determine their correlation with features of the MetS. (2) To perform proteomic analysis in a Hispanic pre-pubertal cohort. (3) Evaluate early onset of endothelial dysfunction and its correlation with expression of adiponectin and leptin levels in a Hispanic pre-pubertal cohort. METHODS/STUDY POPULATION: A cross-sectional pilot study will obtain a random representative sampling of children aged 6–12 years from all geographical areas of Puerto Rico. Children will be assessed regarding pre-pubertal status through Tanner staging and later divided into pre-MetS Versus MetS groups as well as controls. MetS will include children meeting 3 or more of the current International Diabetes Federation (IDF) criteria. Pre-MetS will include children with at least 1 criterion for MetS. Anthropometric data, blood pressure readings, ultrasound-based noninvasive testing for endothelial dysfunction, and laboratory assays will be performed to the study population and data analyzed for correlation. Total adiponectin and leptin levels will be measured using a commercially available quantitative sandwich enzyme-linked immunoassay test. The study will be submitted to the University of Puerto Rico Medical Sciences Campus’ Institutional Review Board (IRB) for approval. Written consent and assent will be obtained from parents and children respectively to ensure patient anonymity. RESULTS/ANTICIPATED RESULTS: We hypothesize that low levels of adiponectin and high levels of leptin will correlate with features of the MetS as defined by the IDF consensus statement, as well as with clinical features of MetS in undiagnosed Hispanic pre-pubertal youth. We also hypothesize that non-invasive testing of endothelial function will correlate both with clinical features of the MetS and with low levels of adinopectin and high levels of leptin. DISCUSSION/SIGNIFICANCE OF IMPACT: The correlation of findings suggestive of endothelial dysfunction and biomarker expression (mainly adiponectin and leptin levels) in a pre-pubertal cohort has yet to be established and could also provide information regarding early atherogenesis in otherwise unidentified youth at risk. Therefore, by using a proteomic approach, this study aims to measure associations between clinical features of the MetS and expression of proteins associated with an adverse cardiometabolic profile in a Hispanic pre-pubertal population. We will concurrently measure the degree of endothelial dysfunction and evaluate whether a correlation exists between previously mentioned protein expression and early onset of dysfunction.

OBJECTIVES/SPECIFIC AIMS: The aim of this study is to determine whether quantitative measures of knee structures including effusion, bone marrow lesions, cartilage, and meniscal damage can improve upon an existing model of demographic and clinical characteristics to classify accelerated knee osteoarthritis (AKOA). METHODS/STUDY POPULATION: We conducted a case-control study using data from baseline and four annual follow-up visits from the osteoarthritis initiative. Participants had no radiographic knee osteoarthritis (KOA) at baseline. AKOA is defined as progressing from no KOA to advance-stage KOA in at least 1 knee within 48 months. AKOA knees were matched 1:1 based on sex to (1) participants who did not develop KOA within 48 months and (2) participants who developed KOA but not AKOA. Analyses were person based. Classification and regression tree analysis was used to determine the important variables and percent of variance explained. RESULTS/ANTICIPATED RESULTS: A previous classification and regression tree analysis found that age, BMI, serum glucose, and femorotibial angle explained 31% of the variability between those who did and did not develop AKOA. Including structural measurements as candidate variables yielded a model that included effusion, BMI, serum glucose, cruciate ligament degeneration and coronal slope and explained 39% of the variability. DISCUSSION/SIGNIFICANCE OF IMPACT: Knee structural measurements improve classification of participants who developed AKOA Versus those who did not. Further research is needed to better classify patients at risk for AKOA.

OBJECTIVES/SPECIFIC AIMS: The goal of this study was to investigate whether RF-RDN attenuates renal fibrosis and inflammation in SHR with established hypertension. METHODS/STUDY POPULATION: Twenty-two-week-old SHR received bilateral RF-RDN or Sham-RDN (Biosense Webster Stockert 70 generator and RF-probe). Four weeks later, SHR were sacrificed and paraffin sections of kidneys were stained for fibrosis by Masson’s trichrome staining. Kidney tissue were homogenized for measurement of cytokines levels by ELISA. RESULTS/ANTICIPATED RESULTS: The results showed that Sham-RDN treated SHR had extensive fibrosis as demonstrated by moderate thickening of Bowman’s capsule, collagen deposition in glomerulus, extensive tubulointerstitial fibrosis, and segmental glomerulosclerosis. In contrast, RF-RDN significantly reduced each of these pathological components of fibrosis in kidney cortex and medulla as compared with Sham-RDN treated kidneys. In other studies, RF-RDN decreased B cells, CD4+ T cells, and CD8+ T cells in the kidney of SHR as measured by flow cytometry. Meanwhile, kidney tissue levels of IL-17, INF-γ, MIP-3a, TNF-α, and TGF-β were decreased as compared with respective levels in Sham-RDN. DISCUSSION/SIGNIFICANCE OF IMPACT: Together, these findings demonstrate that removal of the influence of heightened renal sympathetic activity by RF-RDN decreases kidney inflammatory markers and attenuates renal fibrosis in hypertensive SHR.