OBJECTIVES/GOALS: We aimed to determine if GLP-1 receptor agonists exert beneficial effects on surrogate measures of cardiovascular function independently of weight loss. Our objective was to compare the outcomes between GLP-1 receptor agonist treatment versus a similar drug without cardiovascular benefit versus weight loss through diet alone. METHODS/STUDY POPULATION: We enrolled 88 individuals with obesity (BMI ≥ 30kg/m2) and pre-diabetes and randomized them in a 2:1:1 ratio to 14 weeks of the GLP-1 receptor agonist liraglutide, the dipeptidyl peptidase-4 inhibitor sitagliptin, or hypocaloric diet. Sitagliptin blocks degradation of endogenous GLP-1 but does not cause weight loss or lower adverse cardiovascular outcomes. Treatment was double-blinded and placebo-controlled for drug, and unblinded for diet. Primary endpoints were flow-mediated dilation (FMD) to assess endothelial vasodilatory function, and plasminogen activator inhibitor-1 (PAI-1) to assess endothelial fibrinolytic function. We used a general linear model for each outcome and included gender as a covariate for FMD. Baseline characteristics were similar. Mean age was 50, with 32% men and 13% black. RESULTS/ANTICIPATED RESULTS: At 14 weeks, diet and liraglutide caused weight loss (diet -4.3 ± 3.2 kg, P<0.01; liraglutide -2.7 ± 3.2, P<0.01), while sitagliptin did not (-0.7 ± 2.0, P=0.17). Diet did not improve FMD at 14 weeks compared to baseline (+0.9%, 95% CI [-1.5, 3.3], P=0.46). FMD tended to increase after liraglutide and sitagliptin but was not significant (liraglutide +1.2 [-0.3, 2.8], P=0.12; sitagliptin +1.6 [-0.6, 3.8], P=0.15). Given that liraglutide and sitagliptin work through the same GLP-1 pathway, we combined the liraglutide and sitagliptin groups for overall effect on FMD, which was significantly improved from baseline (+1.4 [0.1, 2.8], P=0.04). Diet and liraglutide improved PAI-1 at 14 weeks (diet -4.4U/mL, [-8.5, -0.2], P=0.04; liraglutide -3.4 [-6.0, -0.7], P=0.01), while sitagliptin did not (-1.4 [-5.1, 2.3], P=0.46). DISCUSSION/SIGNIFICANCE: Activation of the GLP-1 pathway by liraglutide or sitagliptin improves FMD independent of weight loss, while PAI-1 improvement is weight-loss dependent and is only seen after liraglutide or diet. Our study suggests the cardiovascular benefit of liraglutide may be due to combined improvements in endothelial vasodilatory and fibrinolytic function.

OBJECTIVES/GOALS: Reduced cerebral blood flow (CBF) along with vascular risk factors (e.g., dyslipidemia) are prevalent in Alzheimers disease (AD) and related dementias. Statins are one of the most effective pharmacologic treatments for vascular risk reduction, which may contribute to CBF in individuals with an increased risk for AD. METHODS/STUDY POPULATION: Cross-sectional analysis of 212 older adults with a family history of dementia. Heart rate via electrocardiogram, mean arterial pressure (MAP) via brachial sphygmomanometers, end-tidal CO2 via capnograph, and CBF velocity at the middle cerebral artery (MCAv) via transcranial Doppler ultrasound were collected following 20-minutes of supine rest. Mean MCAv (cm/s) was measured within each cardiac cycle and averaged over an 8-minute duration. Cerebrovascular conductance was calculated by dividing mean MCAv by MAP. Pulsatility Index was calculated by subtracting systolic MCAv from diastolic MCAv and then dividing by mean MCAv. RESULTS/ANTICIPATED RESULTS: 125 females (68 ± 6 years; 49 statin) and 87 males (70 ± 6 years; 47 statin) were included in analyses. There were no significant differences between heart rate, MAP, or end-tidal CO2 between statin and non-statin users. After controlling for age, sex, and low-density and high-density lipoprotein, statin use did not significantly contribute to MCAv (p = 0.09). However, statin use did significantly contribute to cerebrovascular conductance (MCAv/MAP; p = 0.03) as well as Pulsatility Index (assessment of cerebral health, p < 0.01). DISCUSSION/SIGNIFICANCE: Our findings suggest statin use significantly and positively contributes to resting cerebral blood flow velocity and cerebrovascular health. Further investigation is warranted into statin interventions with other components of cerebrovascular function, as differences may have implications for brain health and disease pathogenesis.

OBJECTIVES/GOALS: Tumor PDL1 signals to immune cells for tumor immune evasion but has cell-intrinsic signals that promote tumor virulence. We identify novel tumor PDL1 depleting drugs (PDDs) to interrupt tumor-intrinsic PDL1 signals and sensitize tumors to targeted therapy in vitro and in vivo. METHODS/STUDY POPULATION: We screened the Prestwick and LOPAC libraries for FDA-approved drugs reducing B16 melanoma PDL1 > 2.6-fold. ?-lactam antibiotics were used at 80 ?M and Chk1 inhibitor rabusertib as indicated in T24 human bladder cancer, and murine ID8agg ovarian cancer, 4T1 breast cancer and B16. Genetic PDL1 KO was by CRISPR or shRNA and re-expression by lentivirus. Viability was by MTT and protein by immunoblot. We challenged 5 NSG mice/group with 2x106 T24 (SQ) cells and 5 BALB/c mice/group with 5x105 4T1 cells (mammary fat pad) and treated with cefepime (200 mg/kg), rabusertib (2.5 mg/kg), vehicle, or combo daily from day 3. RESULTS/ANTICIPATED RESULTS: Structurally-related ?-lactam antibiotics cefepime and ceftazidime are tumor PDDs. Cefepime or ceftazidime reduced tumor PD-L1 and thus its cell-intrinsic signals to deplete the DNA damage sensing Chk2 protein and promote rabusertib synthetic lethality in vitro and in vivo in a tumor PDL1-dependent manner independent of immunity. Structurally distinct ?-lactam antibiotics did not sensitize tumor cells to rabusertib, suggesting ?-lactam antimicrobial functions did not promote PDL1 depletion or rabusertib treatment effects in vivo. Although rabusertib effects were immune-independent, both PDDs induced immunogenic tumor STING signaling, suggesting they can improve tumor immunotherapy. DISCUSSION/SIGNIFICANCE: We show a rapidly translatable way to deplete detrimental tumor-intrinsic PDL1 signals, and sensitize tumors to rabusertib. We are testing PDD structure activity relationships to improve PDD effects and testing PDD effects on other treatments, e.g., PARP inhibitors, immunotherapy.

OBJECTIVES/GOALS: Myocarditis is an inflammatory cardiomyopathy commonly caused by viral infections. The residual burden of this disease after guideline-based therapies is substantial, as there are no pathway-specific therapies. Our long-term goal is to find and translate treatments that reduce acute myocarditis severity and prevent progression of disease. METHODS/STUDY POPULATION: Of available therapies, extracellular vesicles (EVs) are ideally suited to the task of simultaneous, specific reprogramming of multiple pathogenesis mechanisms necessary in diseases like myocarditis, without similar immunosuppressive risks as their pharmacologic counterparts. We obtained plasma from healthy men and women and isolated nanoparticles, which were analyzed for physiochemical markers of human EVs. After confirming presence of EVs, we injected these plasma-derived extracellular vesicles (PEV) into male BALB/c mice vs. PBS control intraperitoneally on days -1, 0, 1 of viral infection (day 0) in a highly translational, mouse model of myocarditis. Hearts were examined at day 10 at the peak of acute myocarditis, using standard histological and cell composition analyses. RESULTS/ANTICIPATED RESULTS: Mice treated with PEV had significantly less myocardial inflammation both histologically and by gene expression of immune markers in the heart. The immunoregulation by PEV treatment decreased many key components of innate immune response networks that are known to be upregulated during acute myocarditis: TLR4+ mast cells and macrophages and complement. These pathways drive the profibrotic gene and protein changes that lead to remodeling, fibrosis, and disease progression observed in patients. We anticipate that when we analyze later timepoints in this model (day 35 post infection) which are normally associated with development of chronic myocarditis, dilated cardiomyopathy, we will see reduction of this fibrosis and of damage-associated changes. DISCUSSION/SIGNIFICANCE: These data suggest that EVs from plasma may be a novel treatment for viral myocarditis, but translation of EVs is hindered by their heterogeneity. We demonstrate characterization both physiochemically and functionally in a well-defined model. Such practices are necessary as these contemporary products challenge current regulation standards.

OBJECTIVES/GOALS: The goal of this study is to evaluate the role of WNT5A and WNT5a-AS1 in sex-differences of GBM progression. In our preliminary studies, we found that a long non-coding RNA WNT5A-AS1 is overexpressed in male GBM patients. We also found that WNT5A-AS1s expression shows a negative correlation with overall survival within male patients. METHODS/STUDY POPULATION: We will define the mechanism by which WNT5A-AS1 regulates WNT5a-mediated glioma stem cell (GSC) maintenance by assessing the effects of inhibiting WNT5A-AS1 expression on transcriptional activity and stemness in GSCs. We will determine if there are distinct Wnt-signaling patterns in male and female isogenic murine astrocytes by examining the expression of downstream proteins in the Wnt signaling pathway and how inhibition of WNT5A-AS1 alters this expression. We will then examine the impact of WNT5A-AS1 on temozolomide (TMZ) resistance in-vitro and in-vivo. We will assess the cell viability and survival of GBM PDX cells upon treatment with TMZ in vitro. Next, we will assess the capacity of knockdown of WNT5A-AS1 to increase sensitivity to TMZ-induced cell death and prolong survival in vivo in intracranial models. RESULTS/ANTICIPATED RESULTS: We hypothesize that WNT5A-AS1 targets Wnt5a and regulates its expression. We anticipate that knockdown of WNT5A-AS1 will upregulate WNT5A expression. We also expect that inhibiting WNT5A-AS1 will alter GSC stem maintenance and functional effects. We expect to see an increase in downstream Wnt5a signaling proteins in males vs females when treated with exogenous Wnt5a. We hypothesize that knockdown of both, WNT5A-AS1 and WNT5A will alter the expression of downstream proteins. We hypothesize that knockdown of WNT5A-AS1 will decrease tumor growth and therapeutic resistance to TMZ while increasing survival in patient derived xenographs in vivo and in vitro. DISCUSSION/SIGNIFICANCE: This study will provide insight into the mechanisms underlying the difference in GBM onset and progression between male and female patients, which is clinically important. We will also characterize the biological role WNT5A-AS1 which is currently unknown to date and elucidate differential role of GSCs in GBM progression between male and female.

OBJECTIVES/GOALS: Using a cell culture model, we will determine the effects of phosphate on primary lung cell cultures and use our results to delineate a pathway through which these changes are carried out. Using animal models, we will determine the effects of phosphate on inflammatory and fibrotic lung injury, both in the presence and absence of CKD. METHODS/STUDY POPULATION: For our in vitro experiments, human lung fibroblasts were treated with concentrations of 1 to 5 mM sodium phosphate and FGFR inhibitors. Expression levels of interleukin (IL)-1beta, IL-6, and IL-8 were analyzed by qPCR and secretion of these cytokines was measured by ELISA. Phosphorylation of PLCy and ERK was measured by western blot. Using an in vivo approach, we placed C57Bl/6 mice on a high phosphate (3%) diet to elevate serum phosphate levels in the absence of kidney injury and administered bleomycin via oropharyngeal aspiration to generate an acute inflammatory response. Serum FGF23 levels were measured by ELISA and serum analysis for phosphate and renal function were obtained. Furthermore, expression of FGF23 pathway and inflammatory markers were analyzed in murine lung tissue using qPCR and western blotting. RESULTS/ANTICIPATED RESULTS: Augmented phosphate concentrations led to increased cytokine expression and secretion from human lung fibroblasts as well as a concomitant increase in PLCy and ERK phosphorylation. Inhibition of FGFR1 reversed the effects of phosphate on the inflammatory cytokines and PLCy/ERK phosphorylation. Serum FGF23 levels were significantly upregulated in mice on a high phosphate diet and further increased in mice subjected to a high phosphate diet with exposure to bleomycin. Both serum phosphate and creatinine levels were significantly elevated as well. Additionally, high phosphate and bleomycin increased local FGF23 expression in murine lung tissue, when compared to controls or each stimulus alone. DISCUSSION/SIGNIFICANCE: Phosphate has a significant impact on inflammation and fibrosis in the lung, indicating that the existence of pulmo-renal crosstalk exaggerates pulmonary injury and that there are biological pathways that may be targeted therapeutically to mediate these effects. These results could have a substantial impact on the quality of life for CKD patients.

OBJECTIVES/GOALS: Time restricted feeding (TRF) in diet induced obesity (DIO) has several health benefits, including improved metabolic rhythms and inflammation. Our lab has shown that TRF in DIO significantly reduces renal and aortic damage. The main goal of our research is to understand how TRF impacts aortic function, organ damage, and T cell activation in DIO. METHODS/STUDY POPULATION: We will use a 20-week DIO model, where mice will be on 20 weeks of normal fat diet (ND) or high fat diet (HFD). During weeks 18-20, mice will go through TRF intervention where food is restricted to the 12-hour active period or continue ad libitum feeding. At the end of the 2-week TRF intervention or continued ad libitum feeding, aortic stiffness will be measured via pulse wave velocity measurements. We will also collect kidney, aorta, and small intestine at the end of the 20-week protocol for flow cytometric analysis of tissue T cell activation as well as histological assessments. This will allow us to determine the relationship with organ damage, organ function, and the T cell response. We will also analyze tissue and circulating levels of inflammatory T cell-derived cytokines such as interleukin-17A (IL-17A) via ELISA. RESULTS/ANTICIPATED RESULTS: DIO mice showed significantly increased aortic stiffness (measured by pulse wave velocity) compared to mice on ND. Interestingly, TRF intervention in DIO mice reduced aortic stiffness compared to DIO ad libitum. Histological assessments also showed that TRF abolished aortic and kidney fibrosis suggesting a role for the timing of feeding in regulating aortic function and organ damage from chronic HFD. We have several ongoing experiments to determine the T cell response with TRF in DIO mice. We predict that TRF in DIO mice will significantly decrease inflammatory T cells and reduce cytokine abundance in target organs. DISCUSSION/SIGNIFICANCE: Our lab has shown that TRF reduces aortic thickness and aortic and kidney fibrosis, but the driving mechanisms are unknown. We propose that TRF reduces T cell activation in DIO mice leading to reduced organ damage. Our work will provide insight on how TRF in DIO regulates the T cell response and may improve inflammation in the kidney and aorta.

OBJECTIVES/GOALS: Biliary atresia (BA) is a progressive congenital disease that is characterized by periductular inflammation and fibrosis that leads to bile duct destruction and cholestasis in neonates. Galectin-3 (Gal3) plays a key role in inflammation and fibrosis. The aim of this study was to evaluate plasma Gal3 levels in early and late BA. METHODS/STUDY POPULATION: Samples from our institutional Pediatric Liver Biobank were used for this study. Patients were categorized as early BA (at diagnosis), late BA (at liver transplant), early other cholestatic liver disease (CLD), late other CLD, or controls without cholestasis or structural liver disease. Plasma Gal3 levels were measured by standard ELISA. Inflammatory cytokines were measured in a subset of samples using MSD Proinflammatory Panel 1 multiplex ELISA. Liver fibrosis was categorized as none (Ishak or METAVIR 0), mild (Ishak 1-2 or METAVIR 1), moderate (Ishak 3-4 or METAVIR 2-3), and severe (Ishak 5-6 or METAVIR 4) based on histology. Data are presented as median (IQR) and compared using Kruskal-Wallis test. Spearmans correlation was used to assess the relationship between Gal3 and clinical and inflammatory markers. RESULTS/ANTICIPATED RESULTS: Samples from 10 controls, 26 early BA, 24 late BA, 13 early other CLD, and 8 late other CLD patients were used for this study. Gal3 levels in late BA (20.8 [12.4-30.5] ng/mL) and late other CLD (21.8 [16.9 – 27.2] ng/mL) were significantly higher than in controls (10.2 [7.6 – 14.5] ng/mL, p < 0.02) and early BA (11.3 [8.7 – 16.8] ng/mL, p < 0.01), but not significantly different from early other CLD (15.7 [11.9 – 21.4] ng/mL, p > 0.05). Gal3 positively correlated with fibrosis score (rho 0.3, p = 0.01), total bilirubin (rho 0.3, p = 0.002), ALT (rho 0.3, p = 0.01), AST (rho 0.3, p = 0.005), and APRI score (rho 0.3, p = 0.009), and negatively correlated with albumin (rho -0.3, p = 0.01). Out of the 10 cytokine proinflammatory panel, Gal3 was significantly correlated with IL-6 (rho 0.3, p = 0.006). DISCUSSION/SIGNIFICANCE: Gal3 is elevated in late BA and other CLD at time of transplant and correlated with degree of fibrosis, suggesting it may play a role in disease progression to cirrhosis. If targeted in the early disease stage, blocking Gal3 in pediatric cholestatic liver diseases may help delay the progression to cirrhosis and need for transplant.

OBJECTIVES/GOALS: We have shown that small extracellular vesicles (exosomes) isolated from patients with a history of ACS disrupt the endothelium in vitro. Sequencing of miRNA contents of these vesicles suggested that miR let-7c was differentially expressed. The current study was designed to determine the relationship between miR let-7c levels and ACS. METHODS/STUDY POPULATION: We identified 16 subjects from the SCD Lungomics biobank at the University of Chicago Comer and La Rabida Childrens Hospitals who had samples obtained at baseline. Among them, 9 had a history of ACS (ACS(+)) and 7 did not (ACS(-)). For all subjects, we reviewed clinical data relevant to their SCD and laboratory data (including hemoglobin, absolute reticulocyte count, white blood cell count) obtained at the same time as the baseline samples. RNA was isolated from the plasma and miR let7c was quantified using quantitative RT-PCR. RESULTS/ANTICIPATED RESULTS: Subjects were similar clinically, except that those with a history of ACS were more likely to be on hydroxyurea (p<0.05) and to have obstructive sleep apnea (p<0.05). Hematologic laboratory values were similar irrespective of ACS history. The mean miR let-7c level was 2-fold less in subjects with a history of ACS than in those who had never had ACS (p<0.05). A plasma miR let-7c level < 1 (normalized to the control subjects) had a positive predictive value of 0.78 for history of ACS and a sensitivity of 78% and specificity of 71%. Among subjects with a history of ACS, the let7c levels did not correlate with time since the last ACS event. However, among subjects who developed ACS following the baseline samples, higher miR let-7c levels correlated with increased length of time to next ACS event (R=0.8). DISCUSSION/SIGNIFICANCE: Our results in a group of subjects with SCD show that plasma miR let-7c levels are decreased in subjects with a history of ACS. They suggest that miR let-7c may be protective against development of ACS and that measurement of its levels could be a useful biomarker to assess or predict risk for this complication of SCD.

OBJECTIVES/GOALS: The goal of this project was to conduct a preliminary assessment of in vivo feasibility early on in the drug-discovery process in an effort to expedite the translation of novel drug scaffolds to potential clinical candidates. The data gathered in this study will be used to direct analog synthesis of our current lead compounds through rational drug design. METHODS/STUDY POPULATION: Based on virtual and physical high-throughput screening efforts and subsequent similarity searching, we identified a set of potent and selective spermine oxidase (SMOX) inhibitors adhering to a common structural scaffold. In order to address potential barriers to in vivo use, we then conducted a robust optimization analysis in an effort to identify analogs with improved drug-like characteristics. Docking simulations to predict binding were performed and visualized using molecular modeling software (MOE and PyMol). ADMET properties were calculated using a variety of software resources including SwissADME and CDD Vault. RESULTS/ANTICIPATED RESULTS: Through these optimization efforts, we were able to successfully identify analogs with improved drug-like characteristics, including increases in predicted CNS penetration, isosteric replacement of metabolically labile functional groups, increased lipophilicity, and elimination of structural attributes suggestive of off-target activity. Analogs were ranked according to predicted binding and properties of in vivo feasibility. Compounds achieving the highest scores were then selected as scaffolds to guide analog synthesis. DISCUSSION/SIGNIFICANCE: Despite evidence implicating induction of SMOX as a mechanism contributing to neuronal pathology, the lack of potent and selective inhibitors with profiles conducive for in vivo use has significantly impeded clinical investigation of this target. In this presentation, rational drug design focusing on translational optimization will be discussed.

OBJECTIVES/GOALS: Age-specific N-glycosylation occurs on follicle- stimulating hormone (FSH) in pituitaries of post-menopausal women and results in a higher ratio of fully glycosylated to hypoglycosylated FSH. Our goal is to identify in vivo the N-glycosylation pathway enzymes and the regulatory mechanisms in gonadotropes of young and old female mice. METHODS/STUDY POPULATION: Pituitaries were isolated from female mice (at 4m and 8m; n=5 per group) carrying an Fshb-Cre transgene on a Rosa mT/mG genetic background and the GFP-tagged gonadotropes were purified by FACS. RNA-Seq analysis, and subsequent qPCR assays were performed on GFP+ cells from pituitaries of female mice at 4m (reproductively young), 8m (reproductively mid age) and ≥ 12m (reproductively old) of age. To identify the role of progesterone signaling in age-dependent N-glycosylation in gonadotropes, a gonadotrope-specific knockout of Pgr was achieved. Gonadotropes from these mutant mice at 4-, 8-, and ≥12 months of age (n=5 per group) were isolated for qPCR analysis of N-glycosylation enzyme gene expression. Predicted progesterone receptor (PR) promoter binding sequences was performed using JASPAR. RESULTS/ANTICIPATED RESULTS: RNA-seq identified 28 differentially expressed N-glycosylation enzyme-encoding mRNAs in gonadotropes of female mice at 4- and 8-months. Three genes showed significant differences between ages (Man2a1, Man1c1, and B4galt5.), and further qPCR analyses revealed six out of eight genes analyzed showed age-dependent expression, including Man2a1, Man1c1, and B4galt5. The promoters of all N-glycosylation enzyme genes showed strong predicted binding sequences for PR. Further qPCR analysis showed age-and genotype-dependent differences in N-glycosylation enzyme expression in Pgr cKO females, with the most striking differences observed at 13 months, where B4galt5, Man1a2, Mgat5, and Man2a1 were downregulated in Pgr cKO gonadotropes compared to controls. DISCUSSION/SIGNIFICANCE: We identified changes in the N-glycosylation machinery in female mouse gonadotropes and confirmed the age- and Pr-dependent regulation of the corresponding mRNAs. Our results provide insights into the mechanisms at the level of the pituitary by which old age-specific FSH glycoform regulates osteoporosis and weight gain in post-menopausal women.

OBJECTIVES/GOALS: In triple negative breast cancer (TNBC), obesity is associated with poor outcomes. Adipose stem cells (ASCs) from obese patients (obASCs) secrete higher levels of adipokines compared to ASCs from lean individuals. Leptin, one of these adipokines, has been implicated in many cancers. This study seeks to examine the role of leptin signaling in TNBC. METHODS/STUDY POPULATION: Previous work in conjunction with a collaborating lab has shown that leptin signaling promotes metastasis and increased expression of epithelial-mesenchymal transition (EMT) markers in triple negative breast cancer cell lines. This project expands upon this work through using both patient-derived cell lines and and patient-derived xenografts (PDX), and examines the role of leptin signaling both in vitro and in vivo. To determine the effects of obesity upon a PDX model of TNBC, a high fat diet was used to induce obesity in vivo. A pharmacological inhibitor of the leptin receptor was used to test the requirement for leptin signaling both in vivo and in vitro. RESULTS/ANTICIPATED RESULTS: Exposure to conditioned media harvested from obASCs increased the percentage of TNBC cells that expressed cancer stem cell markers, whereas exposure to an inhibitor of the leptin receptor decreased the percentage of cells with cancer stem cell markers. PDX tumors implanted into mice with diet-induced obesity had an increased volume compared to tumors implanted into lean controls. Further analysis will be conducted on metastasis, circulating tumor cells, and survival in both lean and obese mice. DISCUSSION/SIGNIFICANCE: Understanding the complex signaling events in the obese tumor microenvironment is essential, as these molecular differences may contribute to different outcomes for obese and lean individuals with triple negative breast cancer. Therefore, study of the crosstalk between obASCs and TNBC cells is critical.

OBJECTIVES/GOALS: This study examines psychological resilience in adults living with Sickle Cell Disease (SCD) in the U.S. aiming to explore how psychological resilience is defined by this community. METHODS/STUDY POPULATION: Participants were recruited between 2014 – 2018, from across the U.S. as part of an ongoing cross-sectional study: Insights into Microbiome and Environmental Contributions to Sickle Cell Disease and Leg Ulcers Study (INSIGHTS). Inclusion criteria included age of 18 or older, with a clinical history of SCD, and were interviewed if they completed the Brief Resilience Scale (BRS) as part of INSIGHTS. 150 study participants were separated by their BRS scores into “High” and “Low” scoring quartiles. 30 participants were randomly selected, 15 from the lowest quartile and 15 from the highest. All participants completed the Connor Davidson Resilience (CD) measure at the end of their qualitative interview. All identified as Black with an average age of 42.5 (13 F, 17 M). RESULTS/ANTICIPATED RESULTS: Three main concepts emerged within both groups in response to the question “How do you define resilience?” (a) not giving up (b) how one deals with challenges and (c) moving forward. DISCUSSION/SIGNIFICANCE: Our analysis shows that the BRS may not be a precise or accurate indicator of the resilience of adults living with SCD. Therefore, it remains to be seen if these measures are descriptive of these individuals true psychological or physiological state as they have not been used in this community until now.

OBJECTIVES/GOALS: Neonatal hypoxic-ischemic encephalopathy (HIE) is an acute neurologic syndrome where decreased blood flow and oxygen to the brain causes acute and chronic brain dysfunction. The only proven neuroprotective intervention for HIE is hypothermia treatment started within 6 hours of birth and 50% of survivors have long-term deficits. METHODS/STUDY POPULATION: Pre-clinical adult stroke studies demonstrated that vagus nerve stimulation (VNS) has anti-inflammatory effects and attenuates brain damage. Transcutaneous auricular VNS (taVNS) is safe and feasible in infants and may improve the motor skill of bottle feeding. We hypothesize that a combined hypothermia-taVNS treatment shortly after HIE birth will have neuroprotective effects, improve motor function, attenuate infarct volume inflammation compared to hypothermia alone. The HIE model includes ligation of the right common carotid artery in postnatal day 7 (P7) rats followed by 90min hypoxia (8% oxygen) and 2hr hypothermia. taVNS or sham taVNS was administered using a bipolar electrode placed on the auricular concha region for 30min, [30sec trains, 0.5msec duration, 20Hz frequency, followed by 4.5min breaks] RESULTS/ANTICIPATED RESULTS: Experimental groups include +HIE/+taVNS, +HIE/-taVNS, and -HIE/-taVNS. To assess motor function, grasping reflex and forelimb grip strength tasks were assessed prior to surgery through P10. Infarct volume was assessed at 72h after injury by staining coronal sections with cresyl-violet. Thirty-four rat pups underwent surgery with an 8.82% mortality rate. taVNS was well tolerated by the P7 rats when delivered below perceptual threshold (0.4-1.1mA). There was no difference in elementary motor function or infarct volume between any group. DISCUSSION/SIGNIFICANCE: Future studies will include 2.5hr hypoxia for a more severe brain injury and a -HIE/+taVNS control group. These initial pre-clinical studies in neonates are important in determining whether taVNS may translate as a treatment to improve outcomes after neonatal HIE.

OBJECTIVES/GOALS: While anti-seizure medications are effective, nearly one third of patients have seizures that go untreated. Prior studies using evoked seizure models have shown that activation of the Superior Colliculus (SC) display anti-seizure effects. Here we monitored and modulated the DLSC to suppress spontaneous seizures in a genetic model of epilepsy. METHODS/STUDY POPULATION: WAG/Rij rats (4 months old) were employed as study subjects. Animals were surgically prepared for virus injection (ChR2 excitatory opsin, or control vector), fiber optic implantation and cortical EEG for optogenetic studies. For In vivo electrophysiology, animals were implanted with a 16 wire multi-electrode array into the DLSC. In optogenetic experiments, we compared the efficacy of continuous neuromodulation to that of on-demand neuromodulation (real time detection of seizures) paradigms on a within-subject basis. We compared three stimulation frequencies on a within-subject basis (5, 20, 100 Hz). We quantified the number and duration of each spike wave discharge (SWD) during each two-hour-long trial. Electrode array single units were sorted and analyzed for activity before, during and after seizures. RESULTS/ANTICIPATED RESULTS: In vivo electrophysiology found there to be a significant decrease in single unit activity leading up to the start of SWDs. Interestingly, on-demand neuromodulation was effective in both females and males – where the greatest reduction in seizure duration was under 100 Hz light delivery. As expected, male and female animals injected with a control vector did not show a reduction in seizures in response to light delivery. In the open-loop (continuous) stimulation paradigm, optogenetic activation of the DLSC was without effect on the number or duration of SWDs at any of the frequencies examined. DISCUSSION/SIGNIFICANCE: SC activity is significantly decreased prior to the start of seizures. Furthermore, activation of the SC displays anti-seizure effects in a model of spontaneous seizures. A striking difference between open and closed-loop neuromodulation approaches underscores the importance of stimulation paradigm in determining therapeutic effect.

OBJECTIVES/GOALS: We have shown that glioblastoma upregulates blood-brain barrier drug efflux transporters via a mechanism that likely involves TNFα and PI3K/Akt. Our goal is to repurpose FDA-approved PI3K/Akt inhibitors to increase anticancer drug brain concentrations, which holds the potential for translation into the neuro-oncology clinic. METHODS/STUDY POPULATION: GL261 Red-FLuc and MBR525-1 Red-FLuc cells (2μl; 2.5K cells/μl; 1μl/min) were injected into the right hemisphere of 8-week old female J:NU mice (coordinates relative to bregma: AP -2 mm, ML -2 mm, DV -3 mm). Tumor burden was assessed weekly with IVIS® Spectrum in vivo imaging; tumor volume and invasiveness were measured by MRI and histopathology, respectively. On day 14 post-injection, mice received 5-ALA (200 mg/kg ip), and tumors were resected with a 2 mm punch biopsy tool and surgical fluorescence microscope (ex/em: 405/635nm). Drug efflux transporter expression and activity in isolated brain capillaries were determined by Western blot and substrate fluorescence assays, respectively. Cytotoxicity was assessed after 48-hour drug incubation using CyQuant MTT Cell Proliferation Assay kits. RESULTS/ANTICIPATED RESULTS: IC50 values of temozolomide, lapatinib, alpelisib, and miltefosine were N/A, 32, 20, and 190 μM for GL261 Red-FLuc cells and N/A, 49, 36, and 148 μM for MBR525-1 Red-FLuc cells, respectively. Median survival of GL261 Red-FLuc mice was 26.5d and significantly increased to 34d with resection (p=0.116). In GBM mice, drug efflux transporter expression and activity levels in brain capillaries isolated from the contralateral hemisphere were significantly upregulated compared to sham controls. Furthermore, treatment with FDA-approved PI3K/Akt inhibitors, alpelisib and miltefosine, significantly reduced drug efflux transporter expression and activity to control levels. In PK and survival studies, we expect that PI3K/Akt inhibition will increase brain uptake of anticancer drugs and prolong GBM mouse survival. DISCUSSION/SIGNIFICANCE: We have previously shown that PI3K/Akt inhibition reduces P-gp/BCRP levels in brain capillaries. Here, we vertically extend this strategy by repurposing the FDA-approved PI3K/Akt inhibitors alpelisib/miltefosine to improve brain uptake of anticancer drugs in GBM resection models.

OBJECTIVES/GOALS: Studies have shown that SARS-CoV-2 specific memory B cells can be maintained at least a year after exposure. However, reports show an altered B cell response during infection in severe COVID-19 cases. This study aims to describe the B cell response during COVID-19 convalescence with a focus on signatures that contribute to durable and robust immunity. METHODS/STUDY POPULATION: Our study cohort consisted of individuals who had recovered from non-severe (hospitalized) or severe (hospitalized and requiring invasive mechanical ventilation) COVID-19. In our comparative analysis, samples from both groups were carefully matched to fall within 4-5 weeks post-symptom onset. We also performed a longitudinal analysis of non-severe patients with sampling ending 5 months post-symptom onset. Using high parameter flow cytometry, we characterized the phenotype of memory B cells using 19 distinct cell markers and fluorescently labeled probes to identify B cells reactive with SARS-CoV-2 spike and receptor-binding domain protein. Additionally, serum collected from individuals was used to quantify antibody titers. RESULTS/ANTICIPATED RESULTS: The frequency of spike-specific B cells and serum antibody titers were similar between severe and non-severe groups. However, we observed that individuals recovered from severe COVID-19 have a significantly reduced frequency of spike specific IgG+ memory B cells expressing Tbet and FcRL5 (markers associated with long lived immunity). In the non-severe patients, we observed IgG+Tbet+ B cells targeting the spike protein peak at 2-3 weeks post-symptom onset, decrease by almost fifty percent 4-5 weeks post-symptom onset, and return to baseline 5 months post-symptom onset. Our study also validated previous findings of a short-lived primary response of IgM+ B cells targeting the spike protein. DISCUSSION/SIGNIFICANCE: Our findings highlight potential implications for long-term immunity against re-infection or severity of the resulting disease in patients with severe COVID-19. Further investigation will be necessary to determine whether the maintenance of immunological protection is hindered in patients who overcame severe COVID-19.

OBJECTIVES/GOALS: The goal of this study is to determine the role of the tRNA modifications in the translation of Hfq. Hfq is an RNA chaperone that acts as a co-factor for the action of the largest class of small RNAs in E. coli. RNA modifications have been known to play critical roles in the translational fidelity of many cellular proteins in bacteria. METHODS/STUDY POPULATION: In this study, we used an hfq-lacZ translation fusion to screen several RNA modification mutant genes to uncover additional RNA modifications that may play a role in Hfq translation. We measured hfq-lacZ activity in genetic backgrounds mutated for several additional RNA modification enzymes previously untested for Hfq effects. RESULTS/ANTICIPATED RESULTS: We identified 5 RNA modification genes that were defective for hfq-lacZ fusion activity, and we subsequently performed western blot analysis on the Hfq protein in the absence of these modification mutant genes to determine the effect of these mutants more directly on Hfq protein levels. We identified 2 out of these 5 RNA modification mutants that also affect Hfq protein levels. DISCUSSION/SIGNIFICANCE: Since Hfq is critically important for small RNA function is a wide range of bacteria, it is possible tRNA modifications regulate Hfq expression in other bacteria. These processes, when further investigated, could provide us with the basic information to develop new antibiotics needed to address emerging antibiotic resistance.

OBJECTIVES/GOALS: Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer and does not have targeted therapy. GPR30, a 7-transmembrane estrogen receptor, may play a role in regulating cell growth and proliferation of cancerous cells. Here, we evaluated changes in gene expression while inhibiting GPR30 to determine putative targets to treat IBC. METHODS/STUDY POPULATION: IBC cell lines (SUM149PT) were cultured in medium with serum stripped from growth factor and hormones for 48 hours. Cells were then exposed to either G15 (GPR30 inhibitor) at a concentration of 1µM or ETOH (vehicle negative control) 3 hours in triplicates. After exposure, total RNA was extracted using the Qiagen RNAeasy Mini kit and RNA was sequenced using the Illumina NextSeq (2 X 75bp). The higher-quality reads were aligned, annotated, and quantified to the human genome (HG38) using STAR and RSEM softwares. Gene expression analysis was performed in R statistical software (packages tximport and DESeq2). Functional and enrichment analyses were performed using Metascape and STRING database, respectively. RESULTS/ANTICIPATED RESULTS: There were 656 significantly expressed genes (p < 0.05) between groups (G15 vs. ETOH). The top 5 significant genes include: SMIM7, FANCG, ARID1A, MAML2, and ATF3. Significantly impacted biological processes and pathways include: electron transport chain, mitotic cell cycle process, microtubule cytoskeleton organization, cellular component morphogenesis and DNA-dependent DNA replication (adj p < 0.05). Additionally, physical and functional interaction networks showed 3 major clusters (≥ 12 genes), which contained several gene hubs including BRCA1, BRCA2, FOS (proto-oncogene), PLK1 and PAK1 (both serine/threonine-protein kinases), among others. Interestingly, the network analysis showed the previously known interaction between FANCG and BRCA2, which were both dysregulated by GPR30 inhibition. DISCUSSION/SIGNIFICANCE: Through gene expression, functional and enrichment analyses we found several targets genes that could be associated with the pathogenesis of IBC. Validation of candidates genes (qRT-PCR and Western blot), and functional assays (cell proliferation, motility, and invasion) will be performed to understand the potential of these genes in treating IBC.

OBJECTIVES/GOALS: This research project aims to isolate and identify bioprospects capable of metabolizing estrogen using culture-dependent, culture independent methods and the identification of the gene/genes responsible for the metabolization of estrogen by the bioprospects. METHODS/STUDY POPULATION: For the culture dependent technique, samples were collected from the water treatment plant in Mayagüez, cultivated on TSA medium and selected specific and diverse colonies were patched on M-9NC (no carbon sources), M-9-glucose (M9G) and M-9-hormone mixture (M9H: 17-beta-Estradiol and 17-alpha-Ethynylestradiol). After the 48hrs incubation at 25 and 37 Celsius, growth was scored on the different media, to choose those potential bioprospects that use the hormones as the sole carbon source. For the culture independent approach, metagenomic clones from libraries generated from the Guajatacas water reservoir and Cabo Rojos Saltern microbial mats were cultivated on the appropriate M9NC, M-9G and M-9H, supplemented with Leucine and Thiamine for 48 hours at 37 Celsius. RESULTS/ANTICIPATED RESULTS: The culture-dependent approach showed a total of 6 potential bioprospects capable of growing on hormones as a sole carbon source on M9H at 25 and 37 Celsius and no-growth on the M-9 media used. Currently, we are in the process of determining the identities of the cultivable isolates genetically, any preference on hormone metabolization, monitoring a larger number of metagenomic clones and optimizing the selection conditions. DISCUSSION/SIGNIFICANCE: The identification of bioprospects capable of metabolizing hormones could potentially lead to the generation of new water treatments that could help remove unwanted chemicals, such as high concentrations of estrogens.