OBJECTIVES/SPECIFIC AIMS: This research seeks to create a next generation documentation system that exists independent of but is complimentary to the packaging system in R. The new documentation can be manipulated programmatically as with all R objects. It also implements multiple translators for creating documentation from different sources, including documentation pages written in latex and code comments. METHODS/STUDY POPULATION: This work is based on input from the R Documentation Task Force, which is a working group, supported by the R Consortium and the University of Utah Center for Clinical and Translational Science, consisting of R Core developers, representatives from the R Consortium member companies and community developers with relevant interest in documentation. An abstraction of the documentation currently in use was created and extended. This abstraction was translated to a class system in R so that documentation can be stored and manipulated in R. RESULTS/ANTICIPATED RESULTS: The class system representing the documentation and the tools for creating the translators are currently being implemented in R. A preview of the system is scheduled to be available at the time of the conference. DISCUSSION/SIGNIFICANCE OF IMPACT: Good documentation is critical for researchers to disseminate computational research methods, either internally or externally to their organization. This work will facilitate the creation of documentation by making documentation immediately accessible and promote documentation consumption through multiple outputs which can be implemented by developers.

OBJECTIVES/SPECIFIC AIMS: The overall objective of this proposal is to establish and modulate the inflammatory profile of individuals across the spectrum of multiple sclerosis (MS), with a focus on determining the potential of interleukin 4-induced protein 1 (IL4I1) as a possible marker of progression and modulator of inflammation in human blood samples. METHODS/STUDY POPULATION: The proposed experimental approach involves isolating plasma and peripheral blood mononuclear cells (PBMCs) from individuals across the spectrum of MS phenotypes, and analyzing these samples primarily by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) methods. Specifically, study groups include: (1) actively relapsing-remitting MS (a-RRMS), (2) non-actively relapsing-remitting MS (n-RRMS), (3) non-active secondary-progressive MS (SPMS), (4) other autoimmune diseases (OAD), (5) healthy controls (HC). RESULTS/ANTICIPATED RESULTS: We expect that IL4I1 treatment increases regulatory cytokine (eg, IL10, TGFb) expression while decreasing Th1 and Th17-derived cytokines (IFNg, IL17), as well as increasing relative composition of regulatory cells (Th2, Treg, M2) as compared with Th1, Th17, M1 (aim 1). Preliminary data on healthy control cells support this prediction. Our central hypothesis is that IL4I1 level indicates the body’s ability to repair itself. As such, we anticipate that all MS groups are deficient in IL4I1, to varying degrees, such that HC>n-RRMS>a-RRMS>SPMS. HC have full repair capacity. RRMS>SPMS as remission indicates existent repair capacity, which is lost in SPMS. n-RRMS>a-RRMS since both, as RRMS, capable of repair response, but a-RRMS triggered this response more recently in response to more recent relapse. In all groups, we expect IL4I-treatment to mitigate inflammation (aim 2). Finally, we expect that H2O2 production by IL4I1 is a key player in IL4I1 function, and that H2O2 will preferentially induce oxidative stress to pro-inflammatory subsets of PBCMs (aim 3). DISCUSSION/SIGNIFICANCE OF IMPACT: MS is a chronic inflammatory neurodegenerative disease of the central nervous system that, with an average age of onset of 34, afflicts over 2.3 million individuals worldwide during many of the most productive years of their lives. The pathogenesis of MS, which involves autoimmune destruction of myelin, is poorly understood. Accurate biomarkers, which could predict disease progression, are yet to be identified and would provide valuable information to patients and their treating clinicians. Likewise, effective treatments are few and in high demand. IL4I1 is a promising candidate for both roles.

OBJECTIVES/SPECIFIC AIMS: To demonstrate that olanzapine recapitulates the effect of increased lateral hypothalamic (LH) GABAergic activity in the DRN and the DBB. This will provide a potential neural substrate for the observed increase in consumption of food and weight gain. METHODS/STUDY POPULATION: (1) We will examine electrophysiological activity of the DRN and the DBB in response to optogenetic stimulation of LH fibers to these nuclei. (2) We will identify the behavioral phenotype of stimulating these same projections using optogenetic techniques. (3a) Identify the behavioral phenotype of mice possessing cre-loxp-dependent knockout (KO) of LH GABAergic activity, DRN serotonergic activity, and inhibition of DBB cholinergic activity. (3b) Using these mice, we will establish behavioral response to olanzapine in ad libitum feeding and fast-refeeding condition. (4) Using baseline and post-treatment body mass index (BMI), PANSS, and side effect profile scores from a recently completed prospective cohort study of treatment-naive schizophrenic patients receiving atypical antipsychotics for 1 year, we will sequence multiple single nucleotide polymorphisms and explore the correlation of serotonergic, dopaminergic, and cholinergic receptor mutations with the increase in BMI and changes in PANSS score and side effect scores. RESULTS/ANTICIPATED RESULTS: (1) Our preliminary data indicates that the LH exclusively sends GABAergic input to the DBB, and the large majority of its projections to the DRN are GABAergic. (2) We have identified that stimulating LH–>DBB projections produces intense feeding and drinking behavior, a real-time place preference for laser stimulation, and a conditioned place preference for laser stimulation. Preliminary data shows that the LH->DRN also produces feeding behavior. (3a) Our lab has demonstrated that transgenic mice with LH-specific GABA release KO are smaller, have increased anxiety-like behaviors such a repetitive grooming and open field aversion, and have reduced feeding after fasting conditions. We expect the DRN serotonergic KO mice to have increased body weight and reduced anxiety-like behaviors. (3b) Our pilot study demonstrated that the LH GABA KO mice administered olanzapine have a greater consumption of food over 1 hour than controls (n=7, 5, respectively; p=0.08). DRN serotonergic KO mice and mice with inhibition of choline will have an increased baseline feeding behavior, but will not be affected by olanzapine. (4) We believe that SNPs in serotonergic receptors such as 5HT2C, and those affecting dopaminergic and cholinergic receptors, will be more common in schizophrenic patients with increased BMI than those without. Further, we believe that a reduction in the PANSS items reflecting anxiety and aversiveness will correlate with increased BMI, since we postulate that mimicking LH GABAergic activity will produce its previously demonstrated anxiolytic effects. DISCUSSION/SIGNIFICANCE OF IMPACT: Identifying the important role for a reward-oriented feeding center in the brain in producing antipsychotic weight gain will allow a more comprehensive, ethologically sound approach to behavioral modification therapy in these patients. It will lend mechanistic credence to weight control therapies which have used token economy, opioid antagonism, and other inhibition-promoting therapies. This study will also increase the validity for testing further the use of selective serotonin agonists which prevent weight gain such as lorcaserin.

OBJECTIVES/SPECIFIC AIMS: The long-term goal of this project is to develop a cost effective 3D printed multipurpose intravaginal ring (IVR) to prevent against unintended pregnancies and infectious diseases.Our goal is to develop a female-controlled method for prevention using innovative IVRs. METHODS/STUDY POPULATION: In vitro and in vivo characterization. RESULTS/ANTICIPATED RESULTS: Controlled and fine-tuned release kinetics 100% drug release from 3D printed IVRs compared with 10%–15% with traditional injection molded IVRs cost-effective engineering of multipurpose IVR with CLIP 3D printing technology. DISCUSSION/SIGNIFICANCE OF IMPACT: If successful, this project will revolutionize the engineering of IVRs and will have a global impact on human health. Not only we will help save millions of women around the world but also millions of children that are infected by their HIV-positive mothers through gestation or breast feeding.

OBJECTIVES/SPECIFIC AIMS: Polyglutamine (polyQ) neurodegenerative diseases, associated with the unstable expansion of polyQ tracts, are devastating diseases for which no treatments exist. Moreover, most drug discovery attempts have been hindered by the lack of understanding on the relevant pathogenic mechanisms. Here, using previously reported 3D protein predicted structures of ataxin-2 and ataxin-3, we analyze the effect of polyQ enlargement on hydrogen bonding and water accessibility patterns as a possible mechanism for pathogenesis thought enhanced protein aggregation. METHODS/STUDY POPULATION: Using the I-TASSER predicted structures of ataxin-2 and ataxin-3 with different numbers of glutamine repeats representing polyQ lengths characteristic of both normal and pathological tracts (Journal of Biomolecular Structure and Dynamics, 2016: 1–16), we identified hydrogen bonds (HBs, UCSF Chimera FindHBond module) and calculated solvent-accessible surface areas (SASA, DSSP program) for the polyQ tracts available in the 3D structures. RESULTS/ANTICIPATED RESULTS: The identified HBs were analyzed as the function of the number of glutamines in the polyQ tracts and characterized as those intra-polyQ and exter-polyQ, respectively. The SASA of the polyQ region was also studied as the function of the polyQ tract length. DISCUSSION/SIGNIFICANCE OF IMPACT: The results obtained here indicate that polyQ regions increasingly prefer self-interactions, which consistently can lead to more compact polyQ structures. The results strongly support the notion that the expansion of the polyQ region can be an intrinsic force leading to self-aggregation of polyglutamine proteins and suggest that the modulation of solvent-polyQ interactions could be a possible therapeutic strategy for polyQ diseases.

OBJECTIVES/SPECIFIC AIMS: The soluble adenylyl cyclase (sAC) is a noncanonical source of cAMP in mammalian cells. sAC is an ATP/bicarbonate ion sensor, whose activity responds to intracellular signals such as pH changes and metabolism. Unlike the more traditionally studied transmembrane adenylyl cyclase, sAC is not tethered to the cell membrane and instead is found in subcellular microdomains like the mitochondria and nucleus. In particular, sAC localization in the mitochondria has been implicated in oxidative phosphorylation and mitochondrial metabolism. Specific changes in sAC microdomain localization have diagnostic utility in a wide variety of cancers, namely melanoma. We have recently found that loss of sAC leads to tumorigenesis and a Warburg/cancer-like metabolic phenotype, consisting of an activated flux through glycolysis, increased lactate production, and dependence on glucose metabolism. In addition, computational analysis of the metabolomics profile of sAC null cells suggests an increased flux through serine synthetic pathways. We hypothesized that specific sAC microdomains are responsible for this cancer-like metabolic state. METHODS/STUDY POPULATION: We have established oncogenic SV40 large T antigen and HPV16-E6 expressing mouse embryonic fibroblasts lacking sAC expression (SV40 KO and E6 KO, respectively). Using these parental lines, we reintroduced sAC by targeting the protein to specific microdomains. sAC was either driven into the mitochondria (mito-sAC) or was driven into all possible microdomains (WT sAC). Single clones were generated and sAC expression was confirmed by Western analysis. Stable cell lines were evaluated for mitochondrial metabolism, glucose sensitivity, and serine sensitivity. RESULTS/ANTICIPATED RESULTS: We found that reintroduction of WT sAC into sAC null cells rescued sensitivity to glycolytic inhibition compared with control cells (p<0.01). The effect was not dependent on the method of immortalization as it was seen in both SV40 and E6 KO cell lines. sAC activity was not directly proportional to expression suggesting that additional regulatory pathways exist. Interestingly, targeted delivery of sAC to the mitochondria was not as effective in rescuing glucose sensitivity as untargeted delivery of sAC into all possible microdomains. Therefore, even though mitochondrial sAC is known to influence metabolism, our data suggests that the nonmitochondrial isoform is most important for cancer cell metabolism. Although metabolomics analysis suggested that serine synthetic pathways are activated in sAC null cells, there is no evidence to suggest that serine is required for sAC null cell growth. Neither inhibition of serine synthesis nor serine starvation differentially affected the growth of sAC null cells compared with WT sAC. DISCUSSION/SIGNIFICANCE OF IMPACT: These data suggest that the Warburg metabolic phenotype in sAC null cells is regulated by specific sAC microdomains. By targeting sAC to specific microdomains, we can further distinguish the role of sAC localization in cellular metabolism. Cancer cells have been shown to exhibit altered metabolic circuitry of pathways like glycolysis, which allow them to adapt to increased metabolic demands of cellular proliferation and waning environmental resources. Beyond helping us improve the use of sAC immunolocalization as a cancer diagnostic, a better understanding of sAC microdomains in transformed cells will help us understand how this signaling pathway is important in cancer. Pharmacologic manipulation of sAC signaling may represent a new cancer therapeutic strategy.

OBJECTIVES/SPECIFIC AIMS: This poster presents preliminary results of using in silico approaches to predict a priori, based on sequence alone, the pathogenesis of novel influenza A virus. METHODS/STUDY POPULATION: Here we analyzed the structure of the NS1 protein of 11 strains of well characterized influenza A virus with known pathogenesis, reported in the literature as LD50 values, and published sequences. We performed homology comparison of these sequences using the ExPASy SIM alignment tool for protein sequences and then predicted their 3D structures using the I-TASSER method for protein structure prediction. We retained the best 20 I-TASSER models for the NS1 sequences considered here and compared their structures with that of the X-ray crystallographic structure of the NS1 protein in the A/blue-winged teal/MN/993/1980 (H6N6). The average RMS between this experimental structure and the best 20 I-TASSER models was used as a measure of structural similarity between the 3D structures among the proteins. RESULTS/ANTICIPATED RESULTS: The sequence homology shows modest correlation between sequence and pathogenicity. Linear correlations with R values as large as 0.6 where observed for the full sequence homology and the homology of the RBD domains of the proteins. The correlations with the other protein domains were significant lower. We did not found overall correlation between the 3D structures and pathogenesis of all the variants considered here, but the initial results suggest that correlations do exists for different subgroups of viruses. In future work we will use advanced data mining methods to better understand clustering and correlation between structure and pathogenesis. DISCUSSION/SIGNIFICANCE OF IMPACT: The results presented in this poster demonstrate, as proof of concept, the use of in silico approaches to determine pathogenesis of viruses with substantial impact on human health. The ability of computationally predicting pathogenesis of rapidly mutating viruses can be an effective way to accelerate the development prevention strategies because computational methods are relatively inexpensive and much more scalable than in vivo approaches.

OBJECTIVES/SPECIFIC AIMS: This study investigates the process configuration parameters involved in targeted drug delivery to the avian respiratory system. Previously, direct intratracheal aerosol delivery in an avian model using a commercial atomizer was found to result in delivery of a high portion of the total dose into one lung lobe. We hypothesize that controlling process configuration will decrease the asymmetric distribution. METHODS/STUDY POPULATION: A 3D printed model of an avian trachea and mainstream bronchi was constructed to create a representative model for direct instillation of aerosols. Construction of the model respiratory tract included the trachea and the first mainstream bronchi bifurcation to measure left/right (L/R) distribution of aerosol delivered. Both liquid aerosol delivery (LAD) using a commercial atomizer and dry aerosol delivery (DAD) using a custom-built dry powder insufflator device were tested. Two experimental variables were controlled: (1) retraction distance from the carina and (2) centering of device shaft in the lumen of the trachea. Measurement of device efficiency (dose delivered to the 3D model at as fraction of total dose), aerosol delivery efficiency (dose captured at L/R bifurcations as a fraction of total dose), and aerosol lateralization (L/R) was conducted. RESULTS/ANTICIPATED RESULTS: The aerosol delivery efficiency for both LAD and DAD devices [73.9% (95% CI: 68.2–79.2) and 73.4% (95% CI: 55.5–91.3), respectively] did not have an appreciable difference. However, the LAD device had a higher efficiency as compared with the DAD device. The L/R distribution for the DAD device was found to be highly dependent on both retraction distance and shaft centering. Appreciable improvement in the L/R distribution was seen using the DAD device by increasing the retraction distance distal to the carina. DISCUSSION/SIGNIFICANCE OF IMPACT: The use of targeted drug delivery to treat pulmonary pathogens requires a careful design, manufacture, and therapeutic positioning of devices. In particular, clinically relevant animal models and treatment regimes requires a sound understanding of the physical processes controlling aerosol distribution in the respiratory system. By using a simulated respiratory model, many of the physical parameters of drug delivery can be tested before using a live animal model. This is especially important from an animal welfare perspective as well as an animal subject availability aspect.

OBJECTIVES/SPECIFIC AIMS: A major limitation of cardiac stem cell transplantation following myocardial infarction (MI) is poor retention of cells in the ischemic microenvironment. Our study aims to better understand and promote the survival and differentiation of human cardiosphere-derived cells (hCDCs) in anoxia, a feature of infarcted myocardium. METHODS/STUDY POPULATION: We previously demonstrated that TGFβ1 and heparin-containing hydrogels (TH-hydrogel) can promote murine CDC survival. In this study, hCDCs were incubated in either normoxia or anoxia for 8 hours with and without TH-hydrogel. In addition, hCDCs without TH-hydrogel were assessed in 16 hours of anoxia. Following incubation, hCDCs were assayed for viability using calcein dye and immunostained for CD31, a marker of endothelial differentiation. RESULTS/ANTICIPATED RESULTS: hCDCs incubated for 8 hours in anoxia in both models equally demonstrated increased survival up to 30% when compared with cells incubated in normoxia. However, in contrast to hCDCs alone, hCDCs with TH-hydrogel additionally demonstrated increased differentiation into endothelial cells in both anoxia and normoxia. We found that hCDCs alone were able to upregulate CD31 only when subjected to 16 hours of anoxia. DISCUSSION/SIGNIFICANCE OF IMPACT: We demonstrate a new, previously unknown response of hCDCs to anoxia. This induces increased viability and differentiation of hCDCs into endothelial cells. The differentiation in anoxia was time dependent and could be expedited with use of TH-hydrogel. Anoxic preconditioning of hCDCs together with the TH-hydrogel system may improve the therapeutic potential of stem cell transplantation following MI.

OBJECTIVES/SPECIFIC AIMS: Allergic asthma is a chronic lung disease driven by inappropriate inflammatory responses against inhaled allergens. Neuropilin-2 (NRP2) is a pleiotropic transmembrane receptor expressed in the lung, but its role in allergic airway inflammation is unknown. Here, we characterized NRP2 expression in lung immune cells and investigated the effects of NRP2 deficiency on airway inflammation. METHODS/STUDY POPULATION: NRP2 expression by lung immune cells from NRP2 reporter mice was determined by flow cytometry. NRP2 expression by human alveolar macrophages (AM) from healthy individuals was determined by mRNA analysis and flow cytometry. Airway inflammation in NRP2-deficient mice was assessed by bronchoalveolar lavage (BAL) cytology and inflammatory gene expression in lung tissue. RESULTS/ANTICIPATED RESULTS: NRP2 expression in lung immune cells was negligible under steady-state conditions. In contrast, inhalational exposure to lipopolysaccharide (LPS) adjuvant dramatically induced NRP2 expression in AM, as 63.3% of AM from LPS-treated mice were NRP2+ compared with 1.5% of AM from control mice. Ex vivo treatment of human AM with LPS resulted in a 1.5-fold and 2.6-fold increase in NRP2 mRNA and surface protein expression, respectively. Compared to littermate controls, NRP2-deficient mice had greater numbers of BAL leukocytes and increased lung expression of the T helper type 2 cytokines IL-4 and IL-5. Furthermore, NRP2 deficiency resulted in stochastic development of allergic airway inflammation, as spontaneous airway eosinophilia was detected in 25% (2/8) of NRP2-deficient mice compared with 0% (0/8) of littermate controls. DISCUSSION/SIGNIFICANCE OF IMPACT: NRP2 is expressed by activated human and murine AM and suppresses the spontaneous development of allergic airway inflammation. These findings suggest that NRP2 may play a key role in allergic asthma pathogenesis, and could prove to be an important therapeutic target in patients with asthma and other allergic diseases.

OBJECTIVES/SPECIFIC AIMS: Retinitis pigmentosa (RP) is a hereditary retinal degeneration disease that affects ~1 in 4000 individuals globally, and there are currently no effective treatment options available. In order to identify potential drug treatments, we optimized our existing a behavioral assay around a transgenic zebrafish carrying a truncated human rhodopsin transgene [Tg(rho:Hsa.RH1_Q344X)]. This line was also crossed with the Tg(-3.7rho:EGFP) reporter for rod visualization. The Q344X larvae experiences significant rod photoreceptor death by 7 days postfertilization (dpf) (Nakao et al., 2012). METHODS/STUDY POPULATION: To assess the vision of the Q344X zebrafish, the VMR assay was run under a dim-light condition based on recorded rod b-waves in larval fish (Moyano et al., 2013) and the minimum cone activation threshold in mice (Cachafeiro et al., 2010). Specifically, Q344X and control larvae at 7 dpf were placed into a 96-well plate and acclimated to a dim-light source (1.802e-05 μ W/cm2 at 500 nm) for 1 hour. The VMR was tracked and quantified during light offset. The total distance traveled was averaged and analyzed at 1 second poststimulus. Retinas were dissected from Q344X and control larvae and whole-mounted to validate the rod degeneration in the Q344X model. RESULTS/ANTICIPATED RESULTS: We found that the Q344X larvae displayed an attenuated VMR (0.121±0.041 cm) to the dim-light offset as compared with the control larvae (0.2751±0.038 cm) (two-sample t-test; p-value=4.619e-14, n=19). Analysis of whole-mounted retinae indicated significant rod degeneration at 7 dpf compared with controls (control: 87 rods/retina, Q344X: 9.3 rods/retina, Welch two-sample t-test, p-value=1.4e4). It is unlikely that the cones of the zebrafish contributed to this VMR since the light intensity of the assay was below the cone detection threshold of mice. As the only apparent difference between the 2 groups of larvae is significant rod degeneration, it can be concluded that the behavioral phenotype was a result of the degeneration. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that the attenuated Q344X VMR is a result of the rod photoreceptor death. This behavioral phenotype can be taken advantage of to develop a drug screening assay. Future steps will screen chemical libraries to identify compounds that ameliorate the rod degeneration. Compounds that prevent degeneration are expected to result in a significant increase in locomotion in response to the dim visual stimulus.

OBJECTIVES/SPECIFIC AIMS: This project aims to synthesize an angiogenic decorin mimic (VEGFp-DS-SILY) with varying densities of QK and characterize its angiogenic potential and synergism with VEGF by evaluating (1) endothelial cell (EC) migration and proliferation, (2) EC VEGF receptor activation, (3) EC tubule formation in collagen scaffolds, and (4) angiogenesis from a chick chorioallantoic membrane (CAM assay) growing into the scaffold, reflecting the ability of the collagen scaffold to integrate into existing vasculature. The next main goal is to develop and characterize an MMP-degradable nanoparticle system for controlled release of VEGF. Future work will evaluate in vivo effects of VEGFp-DS-SILY bound to a 3D collagen scaffold on ischemic wound repair in a combined excisional wound/bipedical dorsal skin flap rat model. METHODS/STUDY POPULATION: Peptide hydrazides are conjugated to the free carboxylic acid functional groups on dermatan sulfate using EDC chemistry. We added a 3 amino acid spacer (-Gly-Ser-Gly) to the C-terminus of the established QK sequence before the hydrazide functional group and refer to this modified QK as “VEGFp.” VEGFp, SILY, and N-terminal biotinylated versions were synthesized using standard Fmoc solid-phase peptide synthesis protocols and purified using reverse phase HPLC. Coupling efficiencies of peptides to dermatan sulfate were determined spectroscopically at 280 nm measuring the aromatic residues (Trp or Tyr) using a NanoDrop system. Dermatan sulfate with 1 or 4 VEGFp peptides coupled were termed DSV1 and DSV4, respectively. After further conjugation with SILY, we will blend this VEGFp-DS-SILY with unmodified DS-SILY to a total 10 μM to test increasing densities of VEGFp. To verify that the collagen-binding properties of VEGFp-DS-SILY are not compromised by the addition of VEGFp, we will use a streptavidin-HRP system to detect bound biotinylated VEGFp-DS-SILY on collagen-coated plates by established protocols. DSV1 and DSV4 were tested for their effects on endothelial VEGFR2 phosphorylation using an MSD ELISA-type assay and endothelial proliferation using an MTS assay. Cell migration was monitored using an ORIS assay where cells are grown to confluence around a silicone stopper that is then removed to allow cells to migrate inward. Tubulogenesis was evaluated by examining tubule formation on matrigel. Finally, in vivo angiogenesis will be evaluated using a chorioallantoic membrane assay. For extracellular VEGF release, hollow MMP-degradable thermoresponsive nanoparticles [NIPAM, 5 mol% 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1% Acrylic Acid (AAc), 2 mol% MMP-degradable peptide diacrylate, and potassium persulfate initiator] will be synthesized around noncross-linked polymer cores. The cores will then be diffused out through the shell by dialysis prior to drug loading. SILY (and some biotinylated SILY for visualization) will be conjugated with EDC chemistry for targeting nanoparticles to collagen. NPs size and zeta-potential will be measured on a Malvern Zetasizer. VEGF will be loaded into NPs by co-incubating a loading solution of 1 µg/mL VEGF with 1 mg of NPs, incubating overnight at 4°C. VEGF loading and release will be measured by ELISA. Biological activity of the released VEGF from particles will be determined on ECs using assays similar to those outlined previously. RESULTS/ANTICIPATED RESULTS: Preliminary data have verified the synthesis and purification of SILY and VEGFp (QK-Gly-Ser-Gly-hydrazide), as well as an N-terminal biotinylated version, through mass spectrometry and reverse-phase HPLC, respectively. For proof-of-concept, we have verified binding of VEGFp to the VEGF receptor 2 using a ForteBio Blitz interferometry instrument. In addition to support based on published reports showing retained bioactivity of QK after conjugation using other spacers, our preliminary data suggests that VEGFp still binds to VEGF receptor 2, albeit with decreased affinity like QK as compared with VEGF. Circular dichroism also shows that VEGFp has retained its α-helical structure necessary for bioactivity; however it appears that it has some uncoiling when conjugated to dermatan sulfate. We hypothesize that varying densities of VEGFp conjugated to the decorin mimetic (DS-SILY) will modulate the degree of angiogenic activity and synergy with VEGF. We determined that we can achieve ~70% VEGFp conjugation completion to dermatan sulfate after 3.5 hours. We have quantified VEGFR2 phosphorylation after 5 minute treatments by using phospho-specific antibodies and an ELISA-type protocol in a mesoscale discovery system. Preliminary data with human umbilical vein endothelial cells shows that VEGFp exhibits synergism with VEGF at levels similar to QK. DSV1 and DSV4 data suggests synergy with VEGF, although free-peptides and engineered compounds alone did not show effects similar to VEGF in the conditions tested. Prelminary data with 30 minute treatments suggests that the peptides and compounds may require longer exposures to induce activation, as they may have slower binding rates. In contrast, prolonged stimulation with VEGF causes a sharp increase in receptor activation, peaking around 10 minutes and decreasing significantly by 30 minutes. Peptides QK and VEGFp both slightly increased proliferation of dermal microvascular endothelial cells (HMVECs) after 60 hours incubation. However, incubation with dermatan sulfate and DSV caused significant cell death after 24 hours in reduced growth factor media, likely due to sequestering of growth factors. It is possible that VEGFp-DS-SILY may better stimulate proliferation since it would be presented as a surface bound proteoglycan mimic, rather than as a soluble factor. HMVECs migrated farther for all treatment groups (10 µM QK, 10 µM VEGFp, 1 µM DSV4, and 10 µM DSV4) than the 10 ng/mL VEGF positive control, although more cells migrated in response to VEGF. This may be accounted at least in part by the more pronounced proliferation induced by VEGF. Migration will also be tested in 3D culture within a collagen gel. We are currently testing a 2D matrigel system for tubulogenesis. We have found that 10 µM DSV4 forms qualitatively more well-defined tubules than the untreated control on reduced growth factor matrigel. However, we were not able to quantify the improved tubule formation and are still troubleshooting the tubule analysis. After seeding ECs and culturing for 4, 8, and 12 hours, cells will be fluorescently stained with anti-CD31 and imaged for 3D tubule formation. CAM assay angiogenesis growing into a collagen scaffold. In brief, fertilized chicken embryos are incubated for 2 days before exposing the CAM. VEGFp-DS-SILY bound to a collagen gel will be placed onto the CAM. Some treatment groups will receive additional VEGF to investigate synergistic effects. Light microscope images of angiogenesis into the collagen gel coated with VEGFp-DS-SILY, taken every day from days 10 to 13, will reflect the ability of the collagen scaffold to integrate into existing vasculature and 3D angiogenic potential of VEGFp-DS-SILY with or without VEGF. We expect that VEGFp-DS-SILY treatment will increase the number of vessels formed on the CAM. Preliminary data using a Fluoraldehyde assay indicates that loading of ~300 ng VEGF per mg of nanoparticles can be achieved. We expect that using an MMP-degradable peptide diacrylate crosslinker will allow nanoparticles to degrade in protease-rich environments like the chronic wound bed and release VEGF. Adjustments to the formulation, such as crosslinker density, may need to be modified to control the rate of VEGF release. DISCUSSION/SIGNIFICANCE OF IMPACT: We expect that our angiogenic decorin mimetic will lead to a novel treatment to accelerate healing of ischemic diabetic foot ulcers, thereby reducing the need for limb amputation and mortality rate of diabetic patients. We anticipate that the diabetes research and regenerative medicine communities will (1) gain a platform for targeted delivery of growth factors, (2) understand the dependence of vascularization within 3D collagen constructs on VEGFp densities and VEGF receptor activation in controlling the degree of angiogenesis, and (3) gain the benefits of controlled angiogenesis in ischemic diabetic wound healing.

OBJECTIVES/SPECIFIC AIMS: Alcohol consumption perturbs the normal intestinal microbial communities (alcohol dysbiosis). To begin to investigate the relationship between alcohol-mediated dysbiosis and host defense we developed an alcohol dysbiosis fecal adoptive transfer model, which allows us to isolate the host immune response to a pathogenic challenge at a distal organ (ie, the lung). This model system allowed us to determine whether the host immune responses to Klebsiella pneumoniae are altered by ethanol-associated dysbiosis, independent of alcohol use. We hypothesized that alcohol-induced changes in intestinal microbial communities would impair pulmonary host defenses against K. pneumoniae. METHODS/STUDY POPULATION: Mice were treated with a cocktail of antibiotics daily for 2 weeks. Microbiota-depleted mice were then recolonized by gavage for 3-days with intestinal microbiota from ethanol-fed or pair-fed animals. Following recolonization groups of mice were sacrificed prior to and 48 hours post respiratory infection with K. pneumoniae. We then assessed susceptibility to Klebsiella infection by determining colony counts for pathogen burden in the lungs. We also determined lung and intestinal immunology, intestinal permeability, as well as, liver damage and inflammation. RESULTS/ANTICIPATED RESULTS: We found that increased susceptibility to K. pneumoniae is, in part, mediated by the intestinal microbiota, as animals recolonized with an alcohol-induced dysbiotic intestinal microbial community have significantly higher lung burdens of K. pneumoniae (5×104 CFU vs. 1×103 CFU) independent of EtOH. We also found that increased susceptibility in alcohol-dysbiosis recolonized animals was associated with a decrease in the recruitment and/or proliferation of CD4+ and CD8+ T-cells (1.5×109 cells vs. 2.5×109 cells) in the lung following Klebsiella infection. However, there were increased numbers of T-cells in the intestinal tract following Klebsiella infection, which may suggest that T cells are being sequestered in the intestinal tract to the detriment of host defense in the lung. Interestingly, mice recolonized with an alcohol-dysbiotic microbiota had increased intestinal permeability as measured by increased levels of serum intestinal fatty acid binding protein (55 vs. 30 ng/mL). Alcohol-dysbiotic microbiota also increased liver steatosis (Oil Red-O staining) and liver inflammation (>2-fold expression of IL-17 and IL-23). DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest that the commensal intestinal microbiota support mucosal host defenses against infectious agents by facilitating normal immune responses to pulmonary pathogens. Our data also suggest that increased intestinal permeability coupled with increased liver inflammation may impair the recruitment/proliferation of immune cells in the respiratory tract following infection. The role of the microbiota during host defense will be important areas of future research directed at understanding the effects of microbial dysbiosis in patients with AUDs.

OBJECTIVES/SPECIFIC AIMS: This study will assess the effect of essential amino acid (EAA) supplementation on plasma triglyceride (TG) in elderly adults. We will also explore the mechanisms mediating EAA mediated changes in fat metabolism and to suggest promising routes to refine therapy of hypertriglyceridemia. METHODS/STUDY POPULATION: In total, 7 nondiabetic male and female subjects ages 50–75 years with elevated plasma TG levels (130–500 mg/dL) were recruited to participate in an acute (5 h) and long-term (8 wk) EAA supplementation study. We measured changes in regional and whole body fat metabolism, including changes in body composition, plasma TG levels, whole body fat metabolic rates, tissue mitochondrial respiratory capacity, and metabolomic profiles before and after supplementation. RESULTS/ANTICIPATED RESULTS: Long-term EAA supplementation decreased fasted plasma TG levels by 19% (p<0.01). Metabolomics of skeletal muscle found acute EAA supplementation resulted in increased EAA metabolic products while long-term supplementation resulted in increased anaplerosis [flux into the tricarboxylic acid cycle (TCA) intermediate pool] and anaplerotic substrates [propionyl (p<0.01) and succinyl (p<0.01) carnitine] and intermediates of long-chain fatty acid metabolism [stearoyl (p<0.01) and myristoyl (p<0.05) carnitine]. However, tissue level respiratory capacity appeared to be unaffected by EAA supplementation. DISCUSSION/SIGNIFICANCE OF IMPACT: EAA supplementation has potential to improve lipid metabolism and plasma TG levels in non-diabetic older adults. Mitochondrial metabolomics suggest that insufficient TCA pool size may limit tissue fatty acid oxidation and may provide an additional route for nutritional therapy.

OBJECTIVES/SPECIFIC AIMS: Accumulation of cholesterol-laden macrophages in arterial walls leads to atherosclerosis. LXRs induce expression of genes that are atheroprotective in macrophages including CCR7, a chemokine receptor that promotes their emigration from the plaque. CCR7 expression has been shown to be negatively regulated by phosphorylation of LXRα at S198 and is reduced in diabetic mice that show impaired plaque regression. I hypothesized that LXRα phosphorylation at S198 diminishes macrophage emigration from atherosclerotic plaque and contributes to impaired regression in diabetes. METHODS/STUDY POPULATION: Inducible LXRα S198A phosphorylation deficient knock in mouse were used as donors for bone marrow transplantation into mice prone to develop atherosclerosis. Plaques were developed by placing mice on western diet; and regression was induced by lowering their lipid levels. Aortic plaques were then analyzed by using morphometric, histological, and molecular analyses in control and diabetic mice expressing either LXRα WT or LXRα S198A during regression. RESULTS/ANTICIPATED RESULTS: Surprisingly, lack of phosphorylation increased plaque macrophage content and impaired regression under normoglycemic condition; however, it did not exacerbate diabetic regression. Plaques in diabetic mice were associated with increased LXRα S198 phosphorylation. Consistent with this, LXRα phosphorylation is enhanced in macrophages cultured under hyperglycemic conditions indicating glucose-dependent regulation of LXRα phosphorylation. Monocyte trafficking studies reveal that lack of phosphorylation and diabetes independently increase recruitment of monocytes in the plaque that might contribute to increased macrophage content. Importantly, I found that diabetes also increases macrophage retention in the plaque, which is reversed in the absence of phosphorylation. We predict that this increased retention results from inhibition of emigration of plaque macrophages through enhanced phosphorylation in diabetes. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that inhibiting LXRα phosphorylation could be beneficial in diabetic atherosclerosis to reverse the accumulation of macrophages in the plaque. This study imparts insight on regulation of plaque macrophage trafficking through LXRα S198 phosphorylation.

OBJECTIVES/SPECIFIC AIMS: Hematopoietic stem and progenitor cells (HSPCs) function to maintain steady state production of new blood cells and to rapidly respond to blood cell loss. Little is known regarding how HSPCs develop the ability to sense and respond to blood cell loss during embryogenesis. Gaining insight into the robust ability of HSPCs to regenerate blood during early development may allow us to develop therapies to rejuvenate this capacity at any stage. METHODS/STUDY POPULATION: We generated a new hematopoietic-specific and inducible cell ablation zebrafish model to uncover the origins of regenerative capacity in HSPCs during development. These transgenic zebrafish express a cyan fluorescent protein (CFP)-nitroreductase (NTR) fusion construct under the control of the draculin (drl) promoter (drl:CFP-NTR), which restricts NTR expression to blood cells. Co-expression analyses of drl:CFP-NTR with known markers of other blood types including HSPCs (runx1+23:mCherry), erythroid cells (gata1:dsRed), and lymphoid cells (rag2:RFP), revealed drl:CFP-NTR was restricted to HSPCs and erythrocytes. To delineate the regeneration potential of embryonic HSPCs, we exposed drl:CFP-NTR transgenic zebrafish embryos to Metronidazole (MTZ), which results in selective ablation of only NTR-expressing blood cells. Embryos were treated from 2 to 3 days postfertilization and recovery of drl+ and gata1+ cells was evaluated over a 7-day recovery period. RESULTS/ANTICIPATED RESULTS: Following MTZ exposure, the nadir of drl+ cell ablation occurs at 2 days post MTZ (dpM) treatment. During the renewal phase of blood regeneration, we first observe recovery of drl+ cells by about 4 dpM, while more mature blood cells like gata1+ erythrocytes show a delayed recovery at about 6 dpM. Our results suggest that HSPCs can respond to injury as early as 2 days of life and that the HSPC-driven regeneration of embryonic blood cells occurs in a hierarchical fashion, similar to regeneration of the adult blood system. DISCUSSION/SIGNIFICANCE OF IMPACT: We have established a quantitative method for in vivo real-time monitoring of embryonic and larval blood regeneration. A significant advantage of our system is that we can use these insights to guide an in-vivo drug screen for factors that accelerate HSPC-driven blood regeneration in a complex biological environment.

OBJECTIVES/SPECIFIC AIMS: To study the rate of recruitment to the Pulmonary Research Registry (PRR) at the University of Chicago using HealtheRx recruitment Versus usual practice. METHODS/STUDY POPULATION: CommunityRx is a health information technology, integrated with electronic medical record (EMR) platforms, that generates personalized referrals (“HealtheRxs”) for community-based programs and services that address basic and other health-related self-care needs. The target population included people ages 18 and older, English speaking, living in 1 of 16 ZIP codes on Chicago’s south and west sides (106 mi2) who received care at ≥1 of 28 CommunityRx partner sites and had a diagnosis of asthma or COPD. Between December 2015 and December 2016, information about pulmonary research participation opportunities was included on the HealtheRxs of eligible patients contemporaneously with usual registry recruitment methods. Usual methods, used since 2010 by the PRR group, included public advertisements requiring the patient to call the research team for more information and education of eligible patients identified during routine clinical care with a Pulmonary/Critical Care clinician or when enrolling in a pulmonary clinical trial. We hypothesized that, compared with usual recruitment practices, the HealtheRx recruitment strategy would increase the rate and decrease the per subject cost of patient recruitment to a prospective registry. Total annual recruitment costs for each method were calculated and divided by the number of consented PRR enrollees per method. RESULTS/ANTICIPATED RESULTS: Between December 22, 2015 and December 15, 2016 13,437 HealtheRxs (8762 for asthma, 3842 for COPD, and 833 for both asthma and COPD) were generated with the recruitment advertisement. In total, 41 patients responded to the ad and participated in the phone survey. In which 15 (36.5%) participants self-reported a diagnosis of asthma only (65% of all HealtheRxs with advertisement were for asthma only), 9 (22%) reported a diagnosis of COPD only (28.5% of all HealtheRxs with advertisement were for COPD only), and 17 (41.5%) reported diagnoses of both asthma and COPD (6.2% of all HealtheRxs with advertisement were for asthma and COPD). Most participants were female (n=28), non-Hispanic black (n=37), and not employed (n=39). The median age was 57. The majority (n=31) had never participated in health or medical research and was not aware of current opportunities to participate in research (n=25). All 41 participants expressed interest in joining PRR and were mailed a blank PRR consent form and a prepaid return envelope with their incentive check for the telephone survey. To date, 5 participants returned a signed consent form via mail and were enrolled in PRR. During the same period, 4 patients enrolled in PRR via usual recruitment methods. The cost per subject to enroll in PRR was $364.40 using the HealtheRx recruitment and $4590 using usual practice. DISCUSSION/SIGNIFICANCE OF IMPACT: NIH has called for innovation in research recruitment methodologies to increase enrollment especially of people who are under-represented in clinical trials research. This study demonstrates the feasibility and efficiency of using an EMR-integrated recruitment method to enroll people of under-represented minority groups to a research registry.

OBJECTIVES/SPECIFIC AIMS: Prostate cancer is the second most common cancer in the world for men. For reasons still unclear, aggressive PCa disproportionately affects males of African ancestry (MoAA). Incidence and mortality rates are highest in MoAA as they have consistently shown a 2.3–3.0-fold higher risk of mortality compared with Caucasian men. This aggressiveness of PCa may be due to specific biological factors. Studies have established that microRNAs (miRNAs), essential in post-transcriptional gene regulation, are dysregulated in PCa. miR-1207-3p is encoded at the PVT1 gene locus, which is located downstream of MYC on the 8q24 PCa susceptibility locus. The chromosomal region 8q24 is associated with aggressive PCa. Yet miR-1207-3p in PCa in MoAA has never been investigated. Moreover, studies have shown that PVT1/MYC co-operation is a fundamental feature in all cancers with 8q24 amplification and 98% of the 8q24 amplicons contained concurrent amplification of the MYC and PVT1 loci. Moreover, MYC has been linked to PCa aggressiveness, and has been reported to be downstream of androgen receptor (AR) in some PCa. However, the mechanisms regulating c-MYC have never been studied in MoAA. We have recently demonstrated that miR-1207-3p has prognostic value in PCa, and directly binds to the 3′ UTR of Fibronectin type III domain containing 1 (FNDC1) to regulate a novel FNDC1/fibronectin (FN1)/AR pathway upregulated in metastatic PCa. However, the relevance of this novel and clinically significant miR-1207-3p molecular pathway in PCa in MoAA is unknown. Therefore, we hypothesized that miRNA 1207-3p, encoded at the 8q24 PCa susceptibility locus, is a PCa biomarker with clinical applications in MoAA. Our specific aim was to assess the clinical relevance of miR-1207-3p, FNDC1, FN1, AR, and MYC expression in aggressive PCa in a cohort of West African Black males. METHODS/STUDY POPULATION: Consequently, we aimed to determine the expression profile of miRNA-1207-3p, FNDC1, FN1, AR, and MYC in histologically confirmed normal prostate (n=24), benign prostate (n=44) and malignant prostate tissue (n=29) from prostatectomy or transrectal ultrasound-guided biopsies in patients recruited at the University College Hospital, Ibadan, Nigeria, a sub-Saharan Black African population. In total, 17 patients had tumor tissues with Gleason score ≥8. Tissues were collected in compliance with Institutional Ethics Board approved protocols. RNA extraction, cDNA synthesis, and quantitative-PCR were performed to analyze mRNA expression of miRNA-1207-3p, FNDC1, FN1, AR, and MYC. Statistical analysis were performed using SPSS software. All data were analyzed by the 1-way ANOVA test. Tukey post-hoc test was performed to determine the differences in mean expression between normal and tumor prostate tissues. p<0.05 were considered significant. RESULTS/ANTICIPATED RESULTS: We discovered that miR-1207-3p is significantly underexpressed in prostate tumor tissues [0.09±0.02, 95% CI (0.04, 0.136), p=0.000] in comparison with normal prostate tissue in MoAA [0.92±0.15, 95% CI (0.60, 1.244), p=0.000]. This is the first description of miR-1207-3p differential expression in human clinical PCa in MoAA. In contrast, FNDC1 was significantly overexpressed in prostate tumor tissues [21.93±8.21, 95% CI (4.97, 38.89), p=0.003] in comparison with normal prostate tissues in MoAA [1.57±0.45, 95% CI (0.625, 2.51), p=0.003]. The same positive correlation with advanced disease held true for FN1 [tumor: 13.66±3.53, 95% CI (6.38, 20.93), p=0.000; normal: 1.07±0.235, 95% CI (0.58, 1.56), p=0.000], AR [tumor: 20.49±6.74, 95% CI (6.50, 34.48), p=0.000; normal: 0.94±0.20, 95% CI (0.52, 1.36), p=0.000], and c-MYC [tumor: 33.93±8.43, 95% CI (16.53, 51.33), p=0.000; normal: 1.94±0.36, 95% CI (1.18, 2.70)]. The significantly increased mean expression for FNDC1, FN1, AR, and c-MYC in prostate tumor tissues in comparison with normal prostate tissues indicate that their overexpression is associated with increased risk of cancer progression in MoAA. DISCUSSION/SIGNIFICANCE OF IMPACT: These results show that the underexpression of miR-1207-3p and the overexpression of FNDC1, FN1, AR and MYC is associated with aggressive PCa in MoAA. miR-1207-3p, and it molecular target FNDC1, may be useful biomarkers for prognostication of PCa in MoAA.

OBJECTIVES/SPECIFIC AIMS: Recent evidence from resting-state fMRI studies have shown that brain network connectivity is altered in patients with neurodegenerative disorders. However, few studies have examined the complete connectivity patterns of these well-reported RSNs using a whole brain approach and how they compare between dementias. Here, we used advanced connectomic approaches to examine the connectivity of RSNs in Alzheimer disease (AD), Frontotemporal dementia (FTD), and age-matched control participants. METHODS/STUDY POPULATION: In total, 44 participants [27 controls (66.4±7.6 years), 13 AD (68.5.63±13.9 years), 4 FTD (59.575±12.2 years)] from an ongoing study at Indiana University School of Medicine were used. Resting-state fMRI data was processed using an in-house pipeline modeled after Power et al. (2014). Images were parcellated into 278 regions of interest (ROI) based on Shen et al. (2013). Connectivity between each ROI pair was described by Pearson correlation coefficient. Brain regions were grouped into 7 canonical RSNs as described by Yeo et al. (2015). Pearson correlation values were then averaged across pairs of ROIs in each network and averaged across individuals in each group. These values were used to determine relative expression of FC in each RSN (intranetwork) and create RSN profiles for each group. RESULTS/ANTICIPATED RESULTS: Our findings support previous literature which shows that limbic networks are disrupted in FTLD participants compared with AD and age-matched controls. In addition, interactions between different RSNs was also examined and a significant difference between controls and AD subjects was found between FP and DMN RSNs. Similarly, previous literature has reported a disruption between executive (frontoparietal) network and default mode network in AD compared with controls. DISCUSSION/SIGNIFICANCE OF IMPACT: Our approach allows us to create profiles that could help compare intranetwork FC in different neurodegenerative diseases. Future work with expanded samples will help us to draw more substantial conclusions regarding differences, if any, in the connectivity patterns between RSNs in various neurodegenerative diseases.

OBJECTIVES/SPECIFIC AIMS: The preliminary analysis sought to retrospectively characterize the role of hypocretin receptor 2 (HCRTR2) in the development and prognosis of AD along with associated behavioral measures including smoking, self-reported drinking history, and neuroticism. Given the results in this study along with the paucity of information regarding the functional significance of rs2653349, we intend to comprehensively characterize HCRTR2 using haplotype analyses. We will then identify relationships between our haplotype analysis and IV alcohol self-administration using the Computer-Assisted Infusion System, and phenotypes identified in a sleep study. Furthermore, we aim at identifying functional loci in the hypocretin/orexin system by investigating differential allele expression in the orexin receptors in hippocampus tissue obtained from postmortem human brains. METHODS/STUDY POPULATION: This study examined 1569 European American and African American individuals between 18 and 65 years old, 922 of whom with a current diagnosis of AD. Participants were genotyped for HCRTR2 rs2653349 and ancestry was determined via a genome-wide panel of ancestry informative markers. AD was diagnosed using the Structured Clinical Interviews for DSM-IV (SCID-IV) for psychiatric disorders and recent alcohol use was assessed by 90-day Timeline Follow-back (TLFB) interviews. Smoking was assessed using the Fagerström Test for Nicotine Dependence and neuroticism was measured using the NEO Personality Inventory. RESULTS/ANTICIPATED RESULTS: In European Americans, a significant difference was found in current AD diagnosis between AX carriers and GG carriers (z=−2.390, p=0.017). This relationship remained significant in a logistic regression model controlled for age and gender (R2=0.269, p=0.015). TLFB drinking measures were compared based on the median values to correct for the ceiling effect resulting from the assessment covering the past 90 days. Total drinks (U=8.280, p=0.004), number of drinking days (U=6.983, p=0.008), and average drinks per days (U=7.221, p=0.007) were all noted to significantly differ between the two allele groups among Caucasians. The associations between rs2653349 and total drinks (R2=0.115, p=0.023) and heavy drinking days (R2=0.190, p=0.015) remained significant in linear regressions controlled for age and gender. Furthermore, Caucasian AX carriers had a higher median number of drinking days relative to GG homozygotes among current AD positive subjects (U=6.937, p=0.012) and a lower median number of drinking days among current AD negative subjects (U=4.430, p=0.035). Among Caucasian AD negative subjects, there was a significantly greater frequency of smokers (χ2=3.550, p=0.046). In African American participants, there were no significant differences in AD diagnosis and in measures of AD severity by genotype. African American males diagnosed with current AD had higher rates of smoking in the AX group (χ2=4.969, p=0.017). No significant associations were found between rs2653349 and neuroticism in any of the cohorts analyzed in this sample. DISCUSSION/SIGNIFICANCE OF IMPACT: The results suggest that, among Caucasians, AX carriers have an increased risk to develop AD independently of their age and gender. In addition, among individuals with a diagnosis of AD, AX carriers reported a greater number of drinking days, as measured by the TLFB, suggesting that this polymorphism also exerts an effect on the severity of the disease. This effect on increased alcohol consumption was absent in Caucasian AX carriers without current AD diagnosis. In future analysis, we will explore how different genetic profiles in HCRTR2, and also HCRTR1, may alter the orexin signaling pathway and how such alterations may predispose patients to develop AD and exacerbate AD once it develops.