OBJECTIVES/GOALS: The overall goal of this study was to determine the effect of early life stress (ELS) on the intestinal CD4+ T cell immune compartment, at homeostasis and after induction of experimental Inflammatory Bowel Disease (IBD). METHODS/STUDY POPULATION: We used a mouse model of ELS, maternal separation with early weaning (MSEW). We used IL-10 reporter mice to enable analysis of IL-10-producing cells. Mice were examined on postnatal day 28 to determine the impact of ELS on gut regulatory T cells. Plasma levels of corticosterone (rodent stress response hormone) was determined by ELISA. Colitis was induced in MSEW and normal rear (NR) mice via intraperitoneal injection of α-IL-10R every 5 days until day 15. Mice were euthanized on days 20 and 30. Colonic tissue sections were stained for histological analysis. Remaining tissue was further processed for flow cytometric analysis of CD4+ T cells and innate lymphoid cells. RESULTS/ANTICIPATED RESULTS: Plasma corticosterone was elevated in MSEW mice compared to their NR counterparts at 4 weeks of age. We observed that the MSEW stress protocol does not affect the baseline colonic CD4+ T cell or innate lymphoid cell populations. There was a reduction in the intestinal CD4+ T cells and regulatory T cells on day 20 in α-IL-10R MSEW mice compared to NR counterparts. This difference disappeared by day 30. Histological scoring showed no difference in disease severity between α-IL-10R treated MSEW and NR mice on day 20. However, on day 30, when α-IL-10R NR mice are recovering from colitis, MSEW mice showed persistent histological inflammation, mainly attributable to sustained epithelial damage. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that ELS prolongs intestinal inflammation and impairs epithelial repair. Future studies will focus on elucidating the mechanisms responsible for ELS-dependent impairment of mucosal repair in experimental colitis.

OBJECTIVES/GOALS: High aortic stiffness is associated with increased cardiovascular morbidity and mortality. The purpose of this work is to demonstrate the potential of our compliant stent-graft design to therapeutically increase aortic compliance over a standard aortic stent-graft. METHODS/STUDY POPULATION: The aorta from a human cadaver will be excised and placed into a pulse duplicator circuit. The stiffness of the system will be estimated using the pulse wave velocity (PWV), which will be calculated using the time delay between pressure measurements at proximal and distal locations in the system. Baseline measurements with the unstented aorta will be compared to two cases: (1) with a standard stent-graft placed, and (2) with our compliant stent-graft prototype in the descending thoracic aorta. PWV is calculated as the distance between the pressure sensors divided by the time delay. Faster PWV is associated with a stiffer vessel, or lower aortic compliance. RESULTS/ANTICIPATED RESULTS: Prior work in vitro showed that the compliant stent-graft reduced peak and pulse pressures compared a standard, rigid stent-graft. We also expect the compliant device to exhibit lower PWV compared to a rigid stent-graft. Depending on the aortic tissue stiffness, the compliant stent-graft could raise or lower PWV compared to no stent. Mean pressure in the compliant case is likely to be slightly higher than the other two cases because the compliant stent-graft’s narrower lumen increases flow resistance. Although mean pressure will be higher, peak pressure should be lower than in the standard stent-graft because the added compliance decreases overall pressure swing between systole and diastole. DISCUSSION/SIGNIFICANCE OF IMPACT: Lower PWV in the compliant stent-graft over the standard stent-graft will indicate its potential to therapeutically lower aortic stiffness in patients needing aortic stenting. Positive outcomes from this study will be a step toward the eventual translation of a compliant stent-graft to clinical use.

OBJECTIVES/GOALS: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030. Though many other cancers have seen improvements in patient survival rates, patients diagnosed with PDAC have a 5-year survival rate of only ~9%. A major contributor to decreased survival is late-stage diagnosis of the disease. New methods of early detection are urgently needed. Extracellular vesicles (EVs) are secreted from cells of all tissue types into the circulation. EVs play important roles in a variety of diseases. They have shown to promote cancer progression and they are being studied as potential biomarkers for disease diagnosis. The purpose of this study was to perform qualitative and quantitative characterization of small-molecule profiles of EVs derived from various pancreatic cancer (PC) and normal pancreas cell lines, to provide proof-of-concept for evaluating the efficacy of leveraging EVs as potential biomarkers of PDAC. METHODS/STUDY POPULATION: EVs were isolated from the conditioned media of six PC and two normal pancreas cell lines using differential ultracentrifugation with filtration. EV enrichment was validated using quantitative ELISA, immunoblot and transmission electron microscopy. Targeted liquid chromatography coupled to mass spectrometry (LC-MS/MS) and untargeted (UPLC-QTOF-MS) metabolomics were used to analyze the biochemical composition of EVs. RESULTS/ANTICIPATED RESULTS: The biochemical profile of PC EVs was found to be significantly different from the profiles of normal cell EVs. Interestingly, amino acids were downregulated in PC EVs as compared to normal cell EVs. However, PC EVs were enriched in lactate and malate. PC EVs also had significant upregulation in other small molecules such as xanthosine, guanosine diphosphate and nicotinamide. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results indicate that the biochemical characterization of EVs using metabolomics has the potential to yield biomarkers which can delineate cancer cell-derived EVs from normal cell-derived EVs. Further work will test the clinical significance of these findings by similar analyses of plasma of PDAC patients. Furthermore, these profiles may be detectable before progression of the disease to late-stage PDAC, leading to the development of assays for earlier diagnosis in patients.

OBJECTIVES/GOALS: The glucocorticoid receptor (GR) is a ubiquitous steroid hormone receptor that is emerging as a mediator of breast cancer metastasis. We aim to better understand the biology associated with phospho-GR species in TNBC and their contribution to tumor progression. METHODS/STUDY POPULATION: To better understand how p-S134 GR may impact TNBC cell biology, we probed GR regulation by soluble factors that are rich within the tumor microenvironment (TME), such as TGFβ. TNBC cells harboring endogenous wild-type or S134A-GR species were created by CRISPR/Cas knock-in and subjected to in vitro assays of advanced cancer behavior. RNA-Seq was employed to identify pS134-GR target genes that are uniquely regulated by TGFβ in the absence of exogenously added GR ligands. Direct regulation of selected TGFβ-induced pS134-GR target genes was validated accordingly. Bioinformatics tools were used to probe publicly available TNBC patient data sets for expression of a pS134-GR 24-gene signature. RESULTS/ANTICIPATED RESULTS: In the absence of GR ligands, GR is transcriptionally activated via p38-MAPK-dependent phosphorylation of Ser134 upon exposure of TNBC cells to TME-derived agents (TGFβ, HGF). The ligand-independent pS134-GR transcriptome primarily encompasses gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TGFβ-induced TNBC cell migration, anchorage-independent growth in soft-agar, and tumorsphere formation, an in vitro readout of breast cancer stemness properties. Finally, a 24-gene pSer134-GR-dependent signature induced by TGFβ1 predicts shortened survival in breast cancer. We expect to find similar results using an in-house tissue microarray. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospho-S134-GR is a critical downstream mediator of p38 MAPK signaling and TNBC migration, survival, and stemness properties. Our studies define GR as a required effector of TGFβ1 signaling and nominate pS134-GR as a biomarker of elevated risk of breast cancer dissemination.

OBJECTIVES/GOALS: In this study, we established a high-throughput chemical screening platform to identify small molecules that facilitates efficient differentiation of stem cells derived β (SC-β) cells. Using this platform, we identified several compounds that potentially increase the differentiation efficiency. METHODS/STUDY POPULATION: Differentiation of human embryonic stem cells (HUES8) into SC-β was carried out using previously published protocols in a 3D cell suspension. Single cells were replated in Matrigel-coated well plates at the start of different stages depending on experiments. Differentiation medium supplemented with small molecules at a final concentration of 2 M and 0.2 M was used throughout the stage. All the cells were then fixed and permeabilized. Immunocytochemical staining was performed. Images of each well were taken and analyzed. Numbers of the total cell, insulin-positive cell, NKX6.1-positive cell, and co-positive cell were recorded. Candidate compounds were validated using flow cytometry or ICC. RESULTS/ANTICIPATED RESULTS: We identified several hit compounds that significantly increase the NKX6.1 positive cell percentage compared to the DMSO-treated controls when treated at the PP1 cell stage. Follow up assays demonstrated that at least one of these putative hits reproducibly increased NKX6.1 expression. In addition, we identified other compounds that significantly increase the insulin and NKX6.1 copositive SC-β cell population when treated at the later PP2 cell stage during the differentiation. We expect a dosage-dependent response when the candidate hits are validated using more accurate assays. DISCUSSION/SIGNIFICANCE OF IMPACT: We established a high-throughput screening platform to identify small molecules that increase the efficiency of SC-β direct differentiation. Successful generation of SC-β allows cell replacement therapy in diabetes patients, and a better understanding of pancreatic biology and development.

OBJECTIVES/GOALS: During and after cancer treatment, cancer-related fatigue (CRF) is a debilitating symptom reported by up to 80% of cancer patients Our understanding of the pathology underlying CRF is limited. Preliminary RNA sequencing data suggest that increased levels of KIR3DL1, the natural killer cell (NK) immunoglobulin-like receptor 3DL1a, may be associated with CRF. METHODS/STUDY POPULATION: Fatigue was measured using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACT-F). Functional validation of the NK cell finding was performed from whole blood obtained from fatigued and non-fatigued subjects. NK cells were isolated from freshly collected whole blood using a human NK cell isolation kit based on CD56 microbead positive selection. NK cell function was assessed using the NK cell direct cytotoxicity assay. Briefly, isolated NK cells were co-cultured in a 2:1 ratio with calcein AM-labelled K562 cells, which are NK cell-sensitive due to the very low MHCI expression. NK cell-mediated cytotoxicity was assessed with Cytation 1 Cell Imaging Multi-Mode reader. Flow cytometric protocols were used to examine NK subset differences between the fatigued and non-fatigued groups. RESULTS/ANTICIPATED RESULTS: NK cells isolated from the fatigued group exhibited decreased cytotoxicity at 12.28% compared to NK cells isolated from non-fatigued controls at a mean of 40.6% cytotoxicity. Flow cytometry analysis revealed a decrease in the CD56dim CD16bright population in the fatigued group (87.1% of CD56+CD4- cells) compared to the control (91.4% of CD56+CD4- cells). Furthermore, there was a decrease in NKG2A expression in mature NK cells (CD56dim CD16bright) isolated from the fatigued group compared to the non-fatigued group. DISCUSSION/SIGNIFICANCE OF IMPACT: Results from the pilot study suggest that there was a decrease in NK cell cytotoxicity in the fatigued group. In addition, there may be a shift in NK cell subpopulations associated with fatigue. Findings from this pilot study suggest that impaired NK cell function may be associated with CRF pathogenesis.

OBJECTIVES/GOALS: Fatigue is a distressing side effect of cancer and its treatment. It is a subjective symptom that can include mental, physical, emotional, and motivational components. We sought to determine whether preventing inflammation affects fatigue-like behavior in a mouse model of radiation therapy. METHODS/STUDY POPULATION: C57BL/6 mice received three consecutive 8 gray doses of daily peripheral irradiation. We used voluntary wheel running activity to measure fatigue-like behavior before and after this period. Minocycline, an antibiotic with anti-inflammatory effects, was administered beginning a week before irradiation and continued throughout the experiment. We also tested mice lacking the toll-like receptor adaptor protein, MyD88. Cognitive abilities were tested using spontaneous alternation in a Y-maze. RESULTS/ANTICIPATED RESULTS: We found that minocycline reduces fatigue-like behavior exhibited after irradiation, but had no effect on pre-irradiation activity levels. Similarly, fatigue-like behavior after radiation was partially reversed by genetic loss of MyD88. Y-maze spontaneous alternation performance remained similar in all groups. DISCUSSION/SIGNIFICANCE OF IMPACT: Both pharmacological and genetic anti-inflammatory manipulations increased voluntary activity levels after irradiation. Our results suggest that inflammation is an important factor in the development of fatigue-like behavior. Modulators of inflammatory processes hold potential for alleviating fatigue.

OBJECTIVES/GOALS: Haptoglobin (Hp) phenotypes may affect inflammatory response after neurologic injury. We are investigating the relationship between patient Hp phenotypes and inflammatory cytokine concentrations in plasma and CSF after aneurysmal subarachnoid hemorrhage (aSAH), a severe form of hemorrhagic stroke. METHODS/STUDY POPULATION: Following IRB approval, all patients with angiographically-proven aSAH and who underwent extraventricular drain (EVD) placement were included. Patients were excluded if they were not expected to survive hospitalization or were on pre-existing anti-inflammatory agents. For all enrolled patients, plasma and CSF samples were taken on post-bleed days 3, 5, 7, and 10, processed and then frozen for later analysis. In this interim analysis, Hp phenotype was assessed through ELISA analysis of plasma samples and cytokine concentrations were determined using multiplex ELISA kits for both plasma and CSF samples. Hp phenotypes were dichotomized to either HP1 (Hp 1-1 or 1-2) or HP2 (Hp 2-2). RESULTS/ANTICIPATED RESULTS: To date, 23 aSAH patients have been enrolled in this IRB-approved study. An interim analysis of the first 13 patients has revealed eight Hp1 patients (Hp 1-1 n = 1, Hp 1-2 n = 7) and five Hp2 patients. CSF levels of IL-6 and TNF-a were greater than plasma levels in all patients at all time points. CSF levels of IL-6 appear to peak on PBD 5 (1890 ± 767 pcg/mL) and 7 (1612 ± 899 pcg/mL). The CSF IL-6 concentrations in the Hp2 group were lower (1792 ± 806 pcg/mL vs. 1952 ± 791 pcg/mL) on PBD5 but were higher (1635 ± 930 pcg/mL vs 1598 ± 943 pcg/mL) at PBD7; however, these differences did not reach statistical significance. DISCUSSION/SIGNIFICANCE OF IMPACT: This interim analysis demonstrated no statistically significant differences in plasma or CSF cytokine concentrations between patients with different Hp phenotypes. This may be due to the low number of samples or the potential confounding effect of disease-specific secondary neurological injuries.

OBJECTIVES/GOALS: Intermuscular adipose tissue (IMAT) has been associated with insulin resistance and type 2 diabetes, yet mechanistic studies addressing the functional role of IMAT are lacking. The aim of this work was to identify novel mechanisms by which IMAT may directly impact skeletal muscle metabolism. METHODS/STUDY POPULATION: We quantified the secretome of IMAT, subcutaneous adipose tissue (SAT), and visceral adipose tissue (VAT) to determine if there are differences between depots in the secretion of cytokines, eicosanoids, FFAs and proteins that influence metabolic function. SAT and VAT biopsies from patients undergoing laparoscopic bariatric surgery and IMAT extracted from vastus lateralis biopsies of individuals with Obesity were cultured for 48 hours in DMEM, and the conditioned media was analyzed using nanoflow HPLC-MS, multiplex ELISAs and LC/MS/MS for proteins, cytokines and eicosanoids/FFA, respectively. RESULTS/ANTICIPATED RESULTS: IMAT secretion of various extracellular matrix proteins (fibrinogen-β, collagenV1a3, fibronectin) was significantly different than VAT and SAT. Pro-inflammatory cytokine secretion of IFNg, TNFa, IL-8 and IL-13 from IMAT was higher than VAT and significantly higher than SAT (p < 0.05). IMAT secretes significantly more pro-inflammatory eicosanoids TXB2 and PGE2 than VAT (p = 0.02, 0.05) and SAT (p = 0.01, 0.04). IMAT and VAT have significantly greater basal lipolysis assessed by FFA release rates compared to SAT (p = 0.01, 0.04). DISCUSSION/SIGNIFICANCE OF IMPACT: These data begin to characterize the disparate secretory properties of SAT, VAT and IMAT and suggest a metabolically adverse secretome of IMAT, that due to its proximity to skeletal muscle may play an important functional role in the pathogenesis of insulin resistance and type 2 diabetes.

OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.

OBJECTIVES/GOALS: This study was conducted in order to identify novel chemical compounds that exhibit anti-leishmanial activity and to further characterize their efficacy and toxicity in in vitro and in vivo systems in the hopes of future chemotherapeutic developments. METHODS/STUDY POPULATION: We developed a novel, target-free fluorometric high-throughput screen (HTS) to identify small molecules with anti-leishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVERset-EXP library cassette #5 yielded 210 compounds that killed 80% of parasites. One hundred nine (109) molecular scaffolds were represented within the hit compounds, including the 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP). A total of 27 novel 1,4-DAPP compounds were synthesized and anti-leishmanial efficacy and host cell toxicity was determined using L. donovani mCherry expressing amastigotes and THP-1 macrophages. Additional pharmacokinetic analyses of a potent 1,4-DAPP compound were conducted. RESULTS/ANTICIPATED RESULTS: Four experimental compounds had IC50 values less than 5μM, providing similar anti-leishmanial activity to miltefosine. Compound 9279817 had a clearance almost twice the rate of normal hepatic blood flow and had a relatively high volume of distribution, indicating this compound is rapidly cleared and distributes into tissues. in vitro rat liver microsome assays suggest a rapid metabolism of 9279817, and MS/MS results suggest this metabolite is most likely formed via oxidation of the sulfur on the lower aromatic ring. DISCUSSION/SIGNIFICANCE OF IMPACT: This study revealed a novel structural class of compounds that have anti-leishmanial activity. in vitro experiments show compounds with similar efficacy as miltefosine while having significantly less toxicity, suggesting that this class could be further developed as a potential chemotherapeutic.

OBJECTIVES/GOALS: We have recently shown that mice exposed to six days of 60% caloric restriction acutely display reduced hypoglycemia-induced glucagon release following refeeding, and that this effect is concurrent with low leptin levels. The current study was conducted to ascertain if leptin treatment during caloric restriction would reverse this effect. METHODS/STUDY POPULATION: Three groups of mice were used, an ad libitum (Ad-lib) fed group and two caloric restriction (CR) groups, one of which received twice daily leptin injection (0.5-1μg/g; IP) and the other vehicle (saline) during their caloric restriction. CR mice were placed on 60% caloric restriction for 6 consecutive days. Ad lib mice were housed in an identical manner but fed ad libitum during this same period. Following 6 days of restriction, CR mice were given ad lib access to food for 16 h. After the 16 h period of refeeding, both CR and ad lib mice began a 6 h fast which was immediately followed by a hypoglycemic insulin tolerance test (ITT). ITTs consisted of a variable dose of insulin intended to achieve a blood glucose of ~45 mg/dL within 60 minutes, at which time blood was collected for glucagon and corticosterone assays. RESULTS/ANTICIPATED RESULTS: The mean blood glucose levels during the ITT at 45 and 60 minutes post injection across all three groups were 46.8 + 3.1 and 37.0 + 2.4, respectively. There were no significant differences in glucose levels between the three groups at these two time points. As expected, saline treated CR mice displayed significantly reduced serum glucagon levels in response to the ITT relative to Ad-lib mice (23.5 + 10.9 vs. 91.7 + 20.8 pg/mL, p = 0.009). In contrast, leptin-treated CR mice maintained their hypoglycemia-induced glucagon response to the ITT (78.0 + 16.8 pg/mL, p>0.99 vs. Ad-lib group). In addition, although corticosterone levels in saline treated CR mice were numerically lower than in Ad-lib mice, this difference was not statistically significance (3928 + 277 vs. 4571 + 178 pg/mL, p = 0.179). DISCUSSION/SIGNIFICANCE OF IMPACT: Diabetes patients on insulin therapy often develop impaired hypoglycemic counter-regulation which can lead to life-threatening hypoglycemic complications. Our results suggest that leptin may hold promise as a therapeutic intervention for the prevention of impaired hypoglycemic counter-regulation in persons with diabetes.

OBJECTIVES/GOALS: The history of immune suppression, especially CD4 nadir, has been shown to be a strong predictor of HIV-associated neurocognitive disorders (HAND). However, the potential mechanism of this association is not well understood. This study examined the relationship between CD4 nadir and brain atrophy. METHODS/STUDY POPULATION: Fifty-nine people with HIV participated in the cross-sectional study (mean age, 56.5 ± 5.8; age range, 41-69; 15 females; 46 African-Americans). High resolution structural MRI images were obtained using a 3T Siemens scanner. From a comprehensive 7-domain neuropsychological test battery, a global deficit score (GDS) and HAND diagnoses were determined for each participant. The correlation between CD4 nadir (the lowest ever lymphocyte CD4 count) and cortical thickness was investigated using a vertex-wise non-parametric approach with a conservative statistical threshold of p < 0.05 (FWE-corrected). RESULTS/ANTICIPATED RESULTS: Out of the 59 participants, 12 met standard Frascati criteria for asymptomatic neurocognitive impairment (ANI) and two met the criteria for mild neurocognitive disorder (MND). Across all participants, low CD4 nadir was associated with widespread cortical thinning, especially in the frontal and temporal regions. Higher GDS (indicating worse global neurocognitive function) was associated with bilateral frontal cortical thinning, and the association largely persisted in the subset of participants who did not meet HAND criteria. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that the low CD4 nadir may be associated with widespread neural injury in the brain, especially in the frontal and temporal regions. This spatial profile might contribute to the prevalence/phenotypes of HAND in the cART era, such as the frequently observed deficits in the executive domain.

OBJECTIVES/GOALS: Our goal is to develop a non-invasive stimulation technique using magneto-electric nanoparticles (MENs) for inducing and enhancing neuronal activity with high spatial and temporal resolutions and minimal toxicity, which can potentially be used as a more effective approach to brain stimulation. METHODS/STUDY POPULATION: MENs compose of core-shell structures that are attracted to strong external magnetic field (~5000 Gauss) but produces electric currents with weaker magnetic field (~450 Gauss). MENs were IV treated into mice and drawn to the brain cortex with a strong magnetic field. We then stimulate MENs with a weaker magnetic field via electro magnet. With two photon calcium imaging, we investigated both the temporal and spatial effects of MENs on neuronal activity both in vivo and in vitro. We performed mesoscopic whole brain calcium imaging on awake animal to assess the MENs effects. Furthermore, we investigated the temporal profile of MENs in the vasculatures post-treatment and its toxicities to CNS. RESULTS/ANTICIPATED RESULTS: MENs were successfully localized to target cortical regions within 30 minutes of magnetic application. After wirelessly applying ~450 G magnetic field between 10-20 Hz, we observed a dramatic increase of calcium signals (i.e. neuronal excitability) both in vitro cultured neurons and in vivo treated animals. Whole brain imaging of awake mice showed a focal increase in calcium signals at the area where MENs localized and the signals spread to regions further away. We also found MENs stimulatory effects lasted up to 24 hours post treatment. MEN stimulation increases c-Fos expression but resulted in no inflammatory changes, up to one week, by assessing microglial or astrocytes activations. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study shows, through controlling the applied magnetic field, MENs can be focally delivered to specific cortical regions with high efficacy and wirelessly activated neurons with high spatial and temporal resolution. This method shows promising potential to be a new non-invasive brain modulation approach disease studies and treatments.

OBJECTIVES/GOALS: We recently identified a CA-MRSA strain in Brooklyn, New York (USA300-BKV) causing an outbreak of severe skin infections in predominantly healthy children. The evolution of USA300-BKV included acquisition of a novel prophage, and our objective is to identify the prophage-encoded gene(s) and mechanism responsible for increased bacterial virulence. METHODS/STUDY POPULATION: We deleted candidate genes from a novel mosaic block of phage-encoded genes in USA300-BKV that have been shown to enhance virulence in a murine skin infection model. Deletion mutants and complemented clones will be evaluated in vivo to identify culprit genes and determine the effect of lineage-specific genetic variation on the phenotype. Complementary studies include a comprehensive characterization of phage and bacterial genes expressed during lysogeny in vitro using RNA sequencing (RNA-Seq), and in vivo using a targeted approach focusing on known bacterial virulence and phage lytic pathways as well as candidate genes identified by in vitro studies. RESULTS/ANTICIPATED RESULTS: Comparison of otherwise isogenic lab strains showed that the mosaic block of phage genes present in USA300-BKV enhance skin abscess size in mice, confirming previous results. As this region of the phage, named mΦ11, does not contain known toxin genes, we hypothesize that mΦ11 modulates expression of bacterial host genes to enhance virulence. Thus, transcriptional profiles of CA-MRSA containing mΦ11 and selected deletion mutants are expected to reveal changes in known or novel virulence factors compared to controls. Candidate regulators specific to the mosaic block include an adenine methyltransferase linked to changes in global gene expression of other bacterial species. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results will broaden scientific understanding of phage-bacterial interactions and determine the mechanisms by which phage impact virulence independent from toxin gene carriage. Identification of phage-encoded gene(s) enhancing CA-MRSA contagion will inform surveillance efforts and identify novel therapeutic targets.