OBJECTIVES/SPECIFIC AIMS: Our objective is to study the role of soluble adenylyl cyclase in the melanocyte regulation of pigment in response to ultraviolet radiation. Melanocytes are specialized cells that produce melanin in organelles called melanosomes, and melanin determines the pigmentation of hair and skin. cAMP is a master regulator of pigmentation and transmembrane class of adenylyl cyclases are essential for expression of important enzymes involved in melanogenesis. However, pigmentation is also controlled by melanosomal pH, which regulates melanogenesis, tyrosinase activity, and melanosome maturation. The relationship between melanosomal pH and cAMP has been elusive. Soluble adenylyl cyclase is a noncanonical source of cAMP that is not responsive to G proteins but rather functions as a pH sensor. We recently demonstrated that loss of soluble adenylyl cyclase (sAC) activity leads to increased melanosomal pH as well as increased pigmentation in cells and hair. We expanded our research to investigate the role of sAC in the intrinsic response of melanocytes to ultraviolet radiation. METHODS/STUDY POPULATION: We utilized sACfl/fl (wild type) and sACKO mouse melanocytes and compared their change in pigmentation in response to ultraviolet radiation. Melanin was used as a measure of pigmentation. We irradiated these cells at differing doses of UVB (0, 1, 2, or 3 mJ/cm2) daily for 3 days. After UVB treatment, cells were observed and the surviving cell numbers were determined. Cells were then analyzed for melanin content using spectroscopy. RESULTS/ANTICIPATED RESULTS: We found that while both sACfl/fl and sACKO cells had increased melanin content in response to UVB, the melanin content of sACKO cells increased more compared with sACfl/fl cells (p=0.001 at daily dose of 3 mJ/cm2). In addition, sACKO cells required less UVB dose to induce a response. We also observed that sACKO cells show increased cell death compared with sACfl/fl cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Although both sACfl/fl and sACKO cells can induce melanin production in response to UV, our results suggest that sACKO cells are more sensitive. We believe that this increased response in sACKO cells is due to increased melanosomal pH. In addition, sACKO cells show increased cell death, suggesting that sAC is important in the damage response secondary to UV exposure. UV plays a wide range of roles in skin biology such as contributing to cancer risk and pigmentation. Since pigmentation is essential for the protection of the skin from UV insult, further investigation of possible mechanisms in which sAC can influence pigmentation in response to UV is warranted.

OBJECTIVES/SPECIFIC AIMS: Our research strives to understand the pathophysiology of doxorubicin cardiotoxicity, focusing on the understudied nonmyocyte cardiac cells. Our understanding will enable researchers to develop protective or alternative therapies for cancer patients and treatments for cancer survivors. METHODS/STUDY POPULATION: Early studies have been carried out in isolated primary cardiac fibroblasts. Cells were treated with varying doses of doxorubicin. Cell viability, proliferation, and reactive oxygen species generation have all been studied. Future studies will focus on mitochondrial assessment in treated cells and confirmation of findings in animal models. Potential therapies discovered in these studies will also be conducted in animal models. RESULTS/ANTICIPATED RESULTS: Our results show a direct effect of doxorubicin on cardiac fibroblasts in vitro. Treated cells show a decreased rate of proliferation and increased production of reactive oxygen species. Similarly to cardiomyocytes, we hypothesize that reactive oxygen species damage the mitochondria of cardiac fibroblasts thereby altering their function and playing a role in doxorubicin cardiotoxicity. DISCUSSION/SIGNIFICANCE OF IMPACT: Current therapies have not been able to adequately protect patients from the cardiotoxicity of doxorubicin and other anthracyclines. A complete understanding of how doxorubicin damages cardiac tissue will only be possible by studying all cell types of the heart. With a better understanding, alternative therapies can be developed to prevent or treat doxorubicin cardiotoxicity without sacrificing the efficacy of doxorubicin in treating cancer.

OBJECTIVES/SPECIFIC AIMS: Obstructive lung disease following particulate matter (PM) exposure is a major health concern. Coexisting metabolic syndrome (MetSyn) often occurs. Receptor for advanced glycation end products (RAGE) is highly expressed in the lung, is a strong predictor of FEV1, and is a key mediator of MetSyn. To determine if the loss of RAGE protects from the persistence of effects of particulate associated lung injury in a murine model. METHODS/STUDY POPULATION: Wild type (WT) and RAGE knockout (RKO) mice were exposed to 100 μg of PM (WTC-Aggregate, PM53) or PBS control by oropharyngeal aspiration. Lung function, methacholine challenge, and bronchoalveolar lavage (BAL) were quantified 28 days after PM exposure using flexiVent (Scireq Montreal, QC). BAL was obtained and cell differentials, cytokines and transcription factors were assayed. Bio-volume to airspace ratio and mean chord length were measured (Image J and Adobe Photoshop). RESULTS/ANTICIPATED RESULTS: WT mice were hyper-reactive to methacholine compared with their PBS controls 28 days after a single exposure to PM. They recovered from increased neutrophilia, loss of FEV, decreased compliance, and increased resistance, which were previously observed 24-hours after exposure. RKO were not hyper-reactive when compared with their own PBS controls. Lung histology shows persistence of loss of alveolar space in WT mice exposed to PM after 28 days. Area fraction was significantly higher in PM exposed WT mice after 28 days which was not significant after 24 hours. Mean chord length was significantly shorter for PM exposed at both time points for WT mice. The relative expression of phosphorylated to total CREB and ERK1/2 proteins was lower in RKO PM exposed mice compared with WT PM while STAT3 expression was lower in WT PM compared with WT PBS. PM induced a lower fold change of total proteins from the PBS controls in RKO for CREB, p38, ERK1/2, STAT3, and STAT5. JNK and p70S6k total proteins expressed a decreased fold change in WT PM exposed mice compared with WT PBS controls. DISCUSSION/SIGNIFICANCE OF IMPACT: A single dose of PM can produce persistent airway hyper-reactivity after 28 days of exposure. This PM induced injury is alleviated in the absence of RAGE, similar to what was seen at 24 hours. Inhibiting RAGE may be key to limiting the persistent inflammatory effects of high intensity PM exposure.

OBJECTIVES/SPECIFIC AIMS: Several clinical studies have established a correlation between changes in relative bacterial populations in the gut and Parkinson disease. However, few published experiments have been able to parse out whether these associations are causative or correlative. Our aim is to determine how bacteria in the gut may impact the health and resilience of dopaminergic signaling. Our experiment is designed to serve as a proof-of-principle that controlled alterations to the gut microbiome alters mechanisms in dopamine homeostasis in the midbrain. METHODS/STUDY POPULATION: Bacterial inoculation 8–10-week-old germ-free male mice (C57BL/6) were exclusively used in this experiment. Mice were orally gavaged every 3 days (D0, 3, 6, and 9) with 100 µL novel bacterial suspension (~108 CFU resuspended in PBS with 1.5% NaHCO3) or vehicle and were sacrificed on D11. Tissue preparation—brains were quickly extracted and the striatum was isolated and homogenized in either RIPA buffer with protease inhibitors (for Western blot analysis) or in 0.1 N HClO4 (for HPLC processing). The homogenates were processed through fractional centrifugation to remove cellular debris. Lysate samples were frozen at −80°C until ready for analysis. Protein expression quantification—expression of proteins were measured using intensity of bands from Western blots. Lysates were denatured prior to loading with LB with 10% β-mercaptoethanol and 30-minute incubation at 37°C. All immunoblots were normalized to immunoreactivity to α-tubulin. Immunoblot intensity was determined using the ImageJ software. Dopamine/dopamine metabolite quantification HPLC analysis was used to determine dopamine and dopamine metabolite concentration. Aliquots of the lysate were injected onto a C18 column using a mobile phase consisting of 50 mM H2NaO4P·H2O, 0.72 mM sodium octyl sulfate, 75 µM Na2 EDTA, and 10% acetonitrile (pH 3.0). The mobile phase was pumped through the system at 0.3 mL/minute. RESULTS/ANTICIPATED RESULTS: Measured total dopamine concentration through HPLC analysis in the striatum showed no significant differences in the bacteria-treated group relative to the control group. The metabolites DOPAC and HVA had an elevated measured concentration in the bacteria-treated group relative to the control group. Western blot analysis showed decreased immunoreactivity for DAT and TH in the bacteria-treated group compared with the control group. There was no significance difference in the immunoreactivity for VMAT2. DISCUSSION/SIGNIFICANCE OF IMPACT: This study demonstrates that dopamine signaling dynamics in the midbrain can be altered by changes in the gut flora in mice. These results further substantiate the impact of the gut-brain axis and may even point to a potential avenue of bolstering the resilience of dopaminergic neurons in preventing the onset of PD. Further experiments must be performed to understand the mechanism of the observed changes and to determine if these changes have any salutary effect.

OBJECTIVES/SPECIFIC AIMS: Engaging Richmond is a community-university partnership, made up of local residents and university faculty and staff that was established in 2011 with an NIH supplement to a Clinical and Translational Science Research Award at Virginia Commonwealth University (VCU). The primary aims of the supplement were to (1) to conduct community-based participatory research (CBPR) on the leading causes of health disparities perceived by the Richmond community and (2) to thereby highlight community needs and assets and build capacity for future community-engaged research (CEnR). The goal was to prepare a community-focused, community-prioritized, health equity report while building capacity, strengthening relationships, and discovering local barriers to CEnR, and therefore to stimulate, facilitate, and inform future CEnR at VCU. METHODS/STUDY POPULATION: This is a case study exploring the impact of 1 community-university partnership on investigator-initiated research using historical and qualitative data. RESULTS/ANTICIPATED RESULTS: Although Engaging Richmond received only 12 months of support from the NIH supplement that provided its initial funding, the community-university partnership has worked continuously since its formation in 2011. This work has not only helped to build connections with the community and key stakeholders, it has also contributed substantially to the resources available to university faculty pursuing CEnR. Specifically, we find that Engaging Richmond has contributed to investigator initiated research in the following ways, either working as co-investigators or in a consultative capacity: consultation on proposal development (5 projects); assisted with instrument development (4 projects); participant recruitment (7 projects); data collection and analysis (6 projects); dissemination (5 projects). In addition to collaboration on projects, Engaging Richmond has increased institutional capacity for CEnR through its contributions to the Annual Community Engaged Institute at the university and the Center of Clinical and Translational Science’s Community Review Board (CRB). The CRB helps researchers work successfully in a community setting, enhance the research design, help to improve study implementation and assist with translation and dissemination of findings. DISCUSSION/SIGNIFICANCE OF IMPACT: Although community-university partnerships have become much more common over the past several decades, there remains a gap in research evidence on the impact of these partnerships. In their 2004 review, Viswanathan et al. note that community-based participatory research studies infrequently document improved capacity of researchers and research organizations as an outcome, despite the expectation that such improvement will accrue through investment in CEnR. A more recent study assessing the range of community-university partnerships across a research university also noted the lack of processes in place to assess impacts (Holton et al., 2015). While assessments of CEnR impact on communities have become increasingly common as demand for evidence about the effectiveness of community-engaged partnerships has mounted, there does not appear to be a similar trend in assessing the impact of these efforts on faculty research and institutional capacity. By focusing on the impact of 1 community-university partnership that has been sustained for over 5 years, we highlight the ways in which having ongoing partnerships in place can support and strengthen investigator-initiated research, reflecting the flexible, “2-way approach” (Weerts and Sandmann, 2010) at the heart of CEnR.

OBJECTIVES/SPECIFIC AIMS: The objectives of this study are to determine whether high-frequency ipsi-lesion or low-frequency contra-lesion ECS improves forelimb function following experimental stroke in aged animals with focal and large strokes. We also want to investigate whether ECS-induced improvements in motor function are related to an enhancement of neural structural plasticity (dendrites and synapses) and changes in growth promoting (BDNF) and growth inhibiting (NOGO-A) expression in the infarcted motor cortex in young and aged animals. METHODS/STUDY POPULATION: We will investigate whether excitatory ECS of the infarcted cortex or inhibition of the noninfarcted cortex combined with daily impaired-forelimb rehabilitative training (RT) results in greater motor functional recovery compared to RT alone. Immunohistochemical (IHC) analyses and unbiased stereological techniques will be performed to investigate changes in proteins associated with dendritic restructuring (MAP2), synaptic plasticity (PSD95 and synaptophysin), and alteration in the expression of BDNF and NOGO-A. RESULTS/ANTICIPATED RESULTS: We expect that inhibitory ECS of the noninfarcted motor cortex will improve behavioral outcomes in moderate to severe stroke animals compared with excitatory ECS or no stimulation (RT alone) animals. We predict that the ECS condition that improves motor performance most significantly compared with RT alone will have a corresponding greater increase in remaining ipsi-infarct motor cortical dendritic and synaptic plasticity (demonstrated by a greater density of MAP2, synaptophysin, and PSD-95 immunoreactivity), and greater expression of BDNF. It is unknown, but also expected that better behavioral recovery will coincide with a greater reduction in NOGO-A in the injured motor cortex. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will aid in creating a model that will allow for a better understanding of the relationship between brain stimulation, severity of injury and, in future studies, aging. These studies will also help clarify previous conflicting brain stimulation results.

OBJECTIVES/SPECIFIC AIMS: Joint injury is a common cause of premature retirement for many equine athletes. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) offer a particularly attractive cell source for cartilage engineering. Although bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine superior equine MSC source for cartilage engineering via a self-assembling process (SAP). METHODS/STUDY POPULATION: MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis via 3D culture and then used to generate cartilage via SAP. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring biochemical, mechanical, and histological properties. RESULTS/ANTICIPATED RESULTS: We found that while BM-MSCs possessed higher tensile properties and collagen content, CB-MSCs had superior compressive properties and GAG content. Moreover, CB-MSCs had lower alkaline phosphatase activity and higher collagen type II, suggesting a more hyaline cartilage-like phenotype. DISCUSSION/SIGNIFICANCE OF IMPACT: In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage, and is therefore the superior MSC source for purposes of cartilage self-assembly.

OBJECTIVES/SPECIFIC AIMS: Our long-term goal is to elucidate the molecular mechanisms and virulence factors that control the differential presentation of infection with Salmonella typhimurium and Salmonella typhi. The objectives of this study are to study the mechanisms that enable S. typhi to trigger a decreased inflammatory response in comparison with S. typhimurium and evade detection by the immune system, leading to the development of asymptomatic chronic carriage of S. typhi. METHODS/STUDY POPULATION: A loss of function eptB mutant S. typhimurium strain was generated. Lipopolysaccharide (LPS) was isolated from wild-type and eptB mutant S. typhimurium and wild-type S. typhi. Binding of LPS to recombinant intelectin was tested by dot blot and enzyme-linked immunosorbant assay (ELISA). C57BL/6 mice were infected with wild-type or eptB mutant S. typhimurium by oral gavage and inflammatory cytokines in the spleen, liver, and Peyer’s patches were measured by qPCR. RESULTS/ANTICIPATED RESULTS: LPS isolated from wild-type S. typhimurium is not bound by intelectin, a protein that has been proposed to function in innate immunity and that is known to be able to bind certain moieties within LPS. Conversely, LPS isolated from eptB mutant S. typhimurium and wild-type S. typhi, which lacks a functional eptB, is bound by intelectin. Mice infected with an eptB mutant S. typhimurium exhibit decreased expression of inflammatory cytokines in the spleen compared to mice infected with the wild type S. typhimurium, suggesting that loss of eptB function allows a nontyphoidal Salmonella serovar to mimic the stealth phenotype of typhoidal serovars. Together, these results suggest that loss of eptB function allows intelectin to bind to and detoxify Salmonella LPS, leading to decreased systemic inflammation during infection. DISCUSSION/SIGNIFICANCE OF IMPACT: These results have broad implications for how pathogens such as S. typhimurium induce systemic shock during infection and may also help to explain a mechanism for how S. typhi is able to evade immune detection and enhance dissemination to systemic sites, leading to development of the asymptomatic chronic carrier state. Further investigation of this novel virulence mechanism will mark a decisive step forward in understanding the mechanisms underlying the differential pathogenesis of S. typhimurium-induced gastroenteritis and S. typhi-induced typhoid fever. Additionally, these results contribute to our understanding of the interactions between host and pathogen in affecting disease presentation, which will have wide appeal among researchers interested in microbial pathogenesis and the contribution of host-pathogen interactions to health and disease.

OBJECTIVES/SPECIFIC AIMS: Immune-based therapies hold great promise for treatment of refractory tumors. However, development is limited by a lack of identified immune correlates to vaccination. We recently showed that dendritic cells (DCs) prolong progression-free survival (PFS) and overall survival (OS) in patients with glioblastoma, and that DC migration to site draining lymph nodes robustly correlates with both PFS and OS. While this appears to be a reliable immune correlate, the complexity of routine labeling for PET and SPECT prohibits validation in a large clinical study. We therefore seek to develop a safe, translatable reporter that can be imaged with a widely available imaging modality. METHODS/STUDY POPULATION: The cationic liposome 1,2-doleoyl-3-trimethylammonium-propane (DOTAP) was loaded with MRI-imageable iron oxide nanoparticles (IONPs) with or without the neutral molecules PEG and cholesterol. The resulting nanoparticles were loaded with RNA to form RNA-NPs that were characterized with transmission electron microscopy (TEM) and used to transfect DCs in vitro; 4.7 T MRI was then used to image particles or cells in agarose gel phantoms. RESULTS/ANTICIPATED RESULTS: TEM images of RNA-NPs indicate the presence of IONP-loaded liposomes. In vitro transfection experiments demonstrate that iron oxide does not reduce RNA-NP-mediated transfection of DCs. Additionally, small amounts of either PEG or cholesterol within RNA-NPs increased transfection of DC2.4s and enhanced T-cell priming by bone marrow-derived dendritic cells. A series of 4.7 T MRI images of particles in cells, spleens, and LNs demonstrated quantifiable differences in particle density between groups. DISCUSSION/SIGNIFICANCE OF IMPACT: This data suggests that IONP-loaded RNA-NPs can be imaged with MRI and manipulated to augment DC function. Future work will include in vivo imaging in mice and safety studies to facilitate translation into first-in-human studies. Successful completion of this project would provide a powerful clinical tool to improve and track patient responses to immune therapy.

OBJECTIVES/SPECIFIC AIMS: The goal of this study is to examine bioenergetic phenotype of retinoic acid receptor responder 1 (RARRES1)-depleted epithelial cells and to facilitate the discovery of personalized metabo-therapeutics in the context of cancers characterized with loss of or low expression of RARRES1. METHODS/STUDY POPULATION: Anoikis assay and annexinV labeling were used to assess drug resistance and apoptotic phenotype in RARRES1-depleted epithelial cells. Metabolomics, AMP kinase activity, mito-tracker, and extracellular flux assays were used to examine the bioenergetic profile of RARRES1-depleted epithelial cells. Extracellular flux assays were used to assess the phenotype of RARRES1-depleted epithelial cells treated with or without metformin. RESULTS/ANTICIPATED RESULTS: RARRES1 is a major regulator of mitochondrial function. Its depletion in tumors induces an oxidative phosphorylation dependent phenotype and subsequently increases ATP abundance in the cell, enhances anabolic pathways and increases survival. Treatment with FDA approved mitochondrial respiration inhibitor, metformin, reversed the metabolic phenotype of RARRES1 depleted-epithelial cells. Metformin could be the ideal therapeutics to reduce tumor burden in cancers with loss of or low expression of RARRES1. DISCUSSION/SIGNIFICANCE OF IMPACT: Bioenergetic dynamics are emerging as a basis for understanding the pathology of cancer. The malignancy progresses as its metabolic pattern and mitochondrial respiration become more dysfunctional. The regulatory pathways of bioenergetic dynamics are currently poorly understood, and the characterization of proteins implicated in those processes must be assessed. One understudied protein and tumor suppressor is RARRES1. RARRES1 is induced by retinoic acid (a major metabolic regulator) and functions as a putative carboxypeptidase inhibitor. Understanding the connection between this carboxypeptidase inhibitor and intermediary metabolism will enlighten our understanding of the bioenergetic profile of cells and facilitate the discovery of personalized metabo-therapeutics in the context of cancer.

OBJECTIVES/SPECIFIC AIMS: Middle ear volume (MEV) is a clinically relevant parameter in the treatment of many common conditions including otitis media, tinnitus, and conductive hearing loss. A growing number of studies have shifted from using tympanometry to 3-dimensional volume reconstruction (3DVR) to calculate MEV; however, MEV values between these methodologies have never before been directly compared. Here, our objective is to characterize agreement between MEV measurement methods across disease states and middle ear sizes. METHODS/STUDY POPULATION: Middle ears were identified from 36 patients ranging 18–89 years of age who underwent tympanometry testing during preoperative workup for tympanic membrane (TM) perforation, up to 1 month prior to a standard-of-care temporal bone computed tomography (CT) between October 15, 2005 and October 15, 2015. MEV values calculated by both tympanometry and 3DVR were analyzed for agreement using Bland and Altman plots. A correction factor was calculated where ear canal volumes were available for contralateral middle ears without TM perforation (n=12), and was applied to a second Bland and Altman plot in the corresponding patient subgroup. MEV agreement was characterized across MEV quartiles (1=smallest; 4=largest) and across increasing states of middle ear disease using Kruskal-Wallis and Wilcoxon testing with Bonferonni correction. RESULTS/ANTICIPATED RESULTS: A Bland Altman plot demonstrated significant disagreement of MEV differences as compared to a priori clinical thresholds. Absolute MEV difference was significantly greater in the average MEV fourth to first quartile (p=0.0024), fourth to second quartile (p=0.0024), third to first quartile (p=0.0048), and third to second quartile (p=0.048). Absolute MEV difference was not significantly different across varying states of middle ear disease (p=0.44). DISCUSSION/SIGNIFICANCE OF IMPACT: Statistically evident and clinically significant disagreement was demonstrated across tympanometric and 3DVR MEV estimates. This lack of agreement was most pronounced at higher average MEV and was persistent yet not appreciably different across varying severities of middle ear disease. These findings may limit the generalizability of studies of the middle ear that differ in MEV estimation methodology, particularly in pathophysiological states where MEV is increased.

OBJECTIVES/SPECIFIC AIMS: Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies to the keratinocyte adhesion protein desmoglein 3. Several other autoimmune diseases have defective B cell tolerance checkpoints, resulting in the accumulation of self-reactive and polyreactive B cells. METHODS/STUDY POPULATION: The present work aims to determine whether PV patients develop normal tolerance to self-antigens other than desmoglein 3, as a potential “first hit” in the development of autoimmunity. We use FACS to isolate single B cells at 4 developmental stages from 8 PV patients. We perform single-cell RT-PCR to amplify each B cell receptor, produce monoclonal antibodies, and screen these for autoreactivity using ELISA/IF to several self-antigens. At each B cell stage, we compare the frequencies of self-reactive and polyreactive B cells to those found in healthy controls. RESULTS/ANTICIPATED RESULTS: We anticipate similar frequencies between PV patients and controls, suggesting that the B cell repertoire in PV patients develops normally at early checkpoints. DISCUSSION/SIGNIFICANCE OF IMPACT: The absence of generalized reactivity would distinguish PV from other autoimmune diseases and would show that PV arises from a specific break in tolerance to a single self-antigen (desmoglein 3) during late B cell maturation. Such a result would further support PV as an ideal candidate for targeted immunotherapy.

OBJECTIVES/SPECIFIC AIMS: To investigate the role of platelet factor-4 (PF4) in B cell differentiation and develop strategies to better modulate B cell differentiation in vitro and in vivo. METHODS/STUDY POPULATION: We use tissue culture and flow cytometry to examine the role of PF4 in B cell differentiation. We use wild type (WT) and PF4−/− mice on a C57Bl6/J background. PF4−/− mice have reduced in vivo B cell differentiation responses. RESULTS/ANTICIPATED RESULTS: We anticipate that our studies will demonstrate that PF4 promotes B cell differentiation in the bone marrow microenvironment. DISCUSSION/SIGNIFICANCE OF IMPACT: The significance of this project may be valuable in developing efficient methods and strategies to increase or limit B cell numbers in vitro and in human disease.

OBJECTIVES/SPECIFIC AIMS: High-sensitivity diagnostics for early infant diagnosis (EID) of HIV at the point of care (POC) are not widely available. Lateral flow immunoassays (LFA) can detect HIV-p24, but are not sensitive enough in practice. With improvements, LFA are a compelling platform for POC in EID. We used functionalized magnetic beads and immunocomplex dissociation to improve sensitivity of HIV-p24 LFA. Here, we evaluate the utility for LFA to quantitatively report HIV-p24 concentration and estimate HIV viral load. Using purified p24 protein and virion constructs, we determined the limits of detection for HIV-p24 using LFA rapid tests. Using measurements from HIV-p24 ELISA, laboratory-developed RT-qPCR, droplet digital PCR, and gold standard clinical viral load, we further characterized the relationship between HIV-p24 concentration, HIV genomic RNA, and LFA test line signal. METHODS/STUDY POPULATION: We measured HIV-p24 concentration by ELISA (R&D Systems) and LFA (Alere Determine HIV-1/2 Ab/Ag Combo). An LFA reader instrument was used to image test lines and measure test line signal on the LFA. HIV viral loads were measured using RT-qPCR and droplet digital RT-PCR protocols adapted in our lab. We obtained gold standard viral load measurements using the Roche Cobas TaqMan system at Vanderbilt University Medical Center. Data analysis was performed using Prism 7 and Stata 14. RESULTS/ANTICIPATED RESULTS: LFA test line signal increases in a predictable, dose-dependent manner and correlates with concentration of purified HIV-p24 with a linear range between 50 and 1000 pg/mL (Spearman r=1; p=0.0004). We compared p24 concentration (ELISA). We evaluated the utility of LFA to quantify HIV-p24 from virions suspended in human plasma, which increased the limit of detection for HIV-p24 to 100 pg/mL and shifted the linear range 100–10,000 pg/mL (Spearman r=0.77; p<0.001). To evaluate the relationship between HIV-p24 concentration and concentration of HIV RNA, we employed 3 molecular techniques. The LFA is capable of detecting HIV-p24 concentrations that correspond to a range of viral loads between 653,000 and 1655 copies of viral RNA/mL. DISCUSSION/SIGNIFICANCE OF IMPACT: Our preliminary results are very promising, indicating that commercially available LFA can quantitatively measure HIV-p24 concentration to low levels. When coupled with our analysis of the relationship between HIV-p24 concentration and HIV RNA concentration, LFA may be a potential platform allowing us to estimate HIV viral burden at clinically relevant levels. Our next steps will be to evaluate this relationship in primary, clinical specimens in collaboration with the Tennessee Center for AIDS Research. We will incorporate technologies to improve the sensitivity of these LFA and evaluate their performance in field settings in Zambia. Our findings are broadly applicable for use in HIV care and treatment programs and early infant diagnosis programs around the world.

OBJECTIVES/SPECIFIC AIMS: Obesity is a rapidly growing epidemic and long-term interventions aimed to reduce body weight are largely unsuccessful due to an increased drive to eat and a reduced metabolic rate established during weight loss. Previously, our lab demonstrated that exercise has beneficial effects on weight loss maintenance by increasing total energy expenditure above and beyond the cost of an exercise bout and reducing the drive to eat when allowed to eat ad libitum (relapse). We hypothesized that exercise’s ability to counter these obesogenic-impetuses are mediated via improvements in skeletal muscle oxidative capacity, and tested this using a mouse model with augmented oxidative capacity in skeletal muscle. METHODS/STUDY POPULATION: We recapitulated the exercise-induced improvements in oxidative capacity using FVB mice that overexpress lipoprotein lipase in skeletal muscle (mLPL). mLPL and wild type (WT) mice were put through a weight-loss-weight-regain paradigm consisting of a high fat diet challenge for 13 weeks, with a subsequent 1-week calorie-restricted medium fat diet to induce a ~15% weight loss. This newly established weight was maintained for 2 weeks and followed with a 24-hour relapse. Metabolic phenotype was characterized by indirect calorimetry during each phase. At the conclusion of the relapse day, mice were sacrificed and tissues were harvested for molecular analysis. RESULTS/ANTICIPATED RESULTS: During weight loss maintenance, mLPL mice had a higher metabolic rate (p=0.0256) that was predominantly evident in the dark cycle (p=0.0015). Furthermore, this increased metabolic rate was not due to differences in activity (p=0.2877) or resting metabolic rate (p=0.4881). During relapse, mLPL mice ingested less calories and were protected from rapid weight regain (p=0.0235), despite WT mice exhibiting higher metabolic rates during the light cycle (p=0.0421). DISCUSSION/SIGNIFICANCE OF IMPACT: These results highlight the importance of muscular oxidative capacity in preventing a depression in total energy expenditure during weight loss maintenance, and in curbing overfeeding and weight regain during a relapse. Moreover, our data suggest that the thermic effect of food is responsible for the differences in metabolic rate, because no differences were found in activity or resting metabolic rate. Additional studies are warranted to determine the molecular mechanisms driving the ability of oxidative capacity to assist with weight loss maintenance.

OBJECTIVES/SPECIFIC AIMS: To compare methods of ascertaining prevalence for adrenal incidentalomas METHODS/STUDY POPULATION: Retrospective electronic medical record study using Looking Glass Clinical Analytics (Streamline Health, Atlanta, GA, USA) at an urban university medical center. All patients with CT or MR imaging of the abdomen between 1997 and 2014 were identified. Patients with a documented diagnosis (ICD-9 code or problem list) for any history of adrenal disease were excluded. The prevalence of adrenal incidentalomas was ascertained by 2 different detection strategies: (1) documented diagnosis of adrenal incidentaloma or (2) imaging reports containing in the same sentence “adrenal” and “nodule*,” “adenoma*,” or “mass*,” and not containing “no” and “adrenal” in the same sentence. Adrenal pathology surprise was further established in the second approach by excluding patients having previously undergone adrenal lab testing (cortisol, aldosterone, catecholamines, adrenocorticotropic hormone, renin) or having been registered in the cancer registry for any cancer excluding superficial skin cancers. RESULTS/ANTICIPATED RESULTS: In total, 194,624 individuals were identified in our initial search, from which 1056 were excluded for past adrenal disease (Table 1). Detection by the documented diagnosis method yielded 1578 cases (0.8%), compared with 13,697 cases (7.1%) by the imaging report method (Figure 1). Further restricting detection to true “Adrenal Surprise” by excluding those with any past adrenal lab testing and cancer history yielded 10,568 cases (6.1%). Validation studies for the 7.1% prevalence with 100 records revealed an adrenal incidentaloma positive predictive value (PPV) of 98%. When restricted to size ≥1 cm the PPV was 84%. DISCUSSION/SIGNIFICANCE OF IMPACT: Comparing our first strategy using documented diagnoses as criterion for incidentaloma as used in a recent paper by Lopez D (Annals of Internal Medicine 2016; 165: 533–542), we found a prevalence of 0.8% in our population similar to her 0.6%. However, when searching at the level of radiology report text, we found a prevalence ten-fold greater at 7.1%. Therefore, adrenal incidentalomas are more robustly identified by searching radiologic reports.

OBJECTIVES/SPECIFIC AIMS: Describe (1) the features of the first release of Duke’s myRESEARCHhome portal for researchers, and 2) the methods and results of adoption strategies METHODS/STUDY POPULATION: Through methods described previously (cite ACTS poster, 2016), the myRESEARCHhome portal team conducted a needs assessment to determine priorities for inclusion in the tool. Based on results of that assessment, the “minimal viable product” launched in June 2016 included the following features, organized into 9 distinct widgets: Access to all web-based research applications; ability to find and request research services; at-a-glance view of financial, protocol, and salary distribution information; access to financial and personnel reports; access to status of agreements and patents; access to CTSA-supported navigation services; visibility into required training and expiration dates; listing of announcements relevant to researchers; customized links area; ability to customize portal. The portal was developed using Ruby on Rails™, with a REACT grid framework. The development team, internal to Duke University, followed industry-standard best practices for development. After the initial release, the team employed several strategies to ensure awareness and adoption. Although written communications were an important factor for awareness, the presentations and hands-on studios proved most important. RESULTS/ANTICIPATED RESULTS: Use of the portal was directly related to in-person outreach efforts. There were small spikes after written communications, but strategies such as presentations, hands-on demonstrations, training sessions, and faculty meetings garnered the steadiest adoption rates. As of early January, 2017, almost 3000 users have interacted with the portal, with numbers rising steadily. There are an estimated 10,000+ faculty, staff, and trainees engaged in research at Duke. DISCUSSION/SIGNIFICANCE OF IMPACT: To maintain high adoption rates with the research community, engagement strategies must be ongoing. In addition to frequent in-person demonstrations, updates via written communications, and attendance at events, the portal team will employ a key adopt strategy—engaging the researchers in ongoing needs assessments. By maintaining the portal’s relevance to the needs of the research community, the tool can better improve the efficiency of research at a large academic medical center.

OBJECTIVES/SPECIFIC AIMS: The overall goal of this project is to understand the genetic and clinical differences in POAG that specifically increase risk in individuals of African genetic ancestry. We will approach this goal by completing the following objectives: (i) localize a genetic signal that accounts for the significantly increased risk for primary open-angle glaucoma in African Americans and (ii) utilize electronic health records (EHR) data to expand our understanding of risk to incorporate endophenotypes of glaucoma and other clinically recorded variables that may influence disease risk. METHODS/STUDY POPULATION: We will genotype at least 200 available African American samples with glaucoma on the Illumina Infinium® Expanded Multi-Ethnic Genotyping Array (MEGAEX) and perform admixture mapping. We will then access EHR data to expand our analysis beyond glaucoma to encompass other relevant risk modifiers captured in the clinical record. RESULTS/ANTICIPATED RESULTS: We anticipate localizing a genetic signal or signals that may account for the increased POAG risk in African Americans. Our calculations indicate that we have ~81% power to detect association at a LOD score of 2 and a risk ratio of 2. Thus, we are well-powered to detect a true signal at this modest level of association DISCUSSION/SIGNIFICANCE OF IMPACT: This project will not only help to achieve precision medicine by filling in the gaps in knowledge regarding glaucoma in African Americans, but it will also address health disparities and aid in the realization of the full potential of “big data” so that all of these elements can be incorporated into a better understanding of health disparities.

OBJECTIVES/SPECIFIC AIMS: (i) Digitally quantify pathologically relevant glomerular microcompartmental structures in murine renal tissue histopathology images. (ii) Digitally model disease trajectory in a mouse model of diabetic nephropathy (DN). METHODS/STUDY POPULATION: We have developed a computational pipeline for glomerular structural compartmentalization based on Gabor filtering and multiresolution community detection (MCD). The MCD method employs improved, efficient optimization of a Potts model Hamiltonian, adopted from theoretical physics, modeling interacting electron spins. The method is parameter-free and capable of simultaneously selecting relevant structure at all biologically relevant scales. It can segment glomerular compartments from a large image containing hundreds of glomeruli in seconds for quantification—which is not possible manually. We will analyze the performance of our computational pipeline in healthy and streptozotocin induced DN mice using renal tissue images, and model the structural distributions of automatically quantified glomerular features as a function of DN progression. The performance of this structural-disease model will be compared with existing visual quantification methods used by pathologists in the clinic. RESULTS/ANTICIPATED RESULTS: Computational modeling will reveal digital biomarkers for early proteinuria in DN, able to predict disease trajectory with greater precision and accuracy than manual inspection alone. DISCUSSION/SIGNIFICANCE OF IMPACT: Automated detection of microscopic structural changes in renal tissue will eventually lead to objective, standardized diagnosis, reflecting cost savings for DN through discovery of digital biomarkers hidden within numerical structural distributions. This computational study will pave the path for the creation of new digital tools which provide clinicians invaluable quantitative information about expected patient disease trajectory, enabling earlier clinical predictions and development of early therapeutic interventions for kidney diseases.

OBJECTIVES/SPECIFIC AIMS: This poster presents a detailed characterization of the distribution of semantic concepts used in the text describing eligibility criteria of clinical trials reported to ClincalTrials.gov and patient notes from MIMIC-III. The final goal of this study is to find a minimal set of semantic concepts that can describe clinical trials and patients for efficient computational matching of clinical trial descriptions to potential participants at large scale. METHODS/STUDY POPULATION: We downloaded the free text describing the eligibility criteria of all clinical trials reported to ClinicalTrials.gov as of July 28, 2015, ~195,000 trials and ~2,000,000 clinical notes from MIMIC-III. Using MetaMap 2014 we extracted UMLS concepts (CUIs) from the collected text. We calculated the frequency of presence of the semantic concepts in the texts describing the clinical trials eligibility criteria and patient notes. RESULTS/ANTICIPATED RESULTS: The results show a classical power distribution, Y=210 X(−2.043), R2=0.9599, for clinical trial eligibility criteria and Y=513 X(−2.684), R2=0.9477 for MIMIC patient notes, where Y represents the number of documents in which a concept appears and X is the cardinal order the concept ordered from more to less frequent. From this distribution, it is possible to realize that from the over, 100,000 concepts in UMLS, there are only ~60,000 and 50,000 concepts that appear in less than 10 clinical trial eligibility descriptions and MIMIC-III patient clinical notes, respectively. This indicates that it would be possible to describe clinical trials and patient notes with a relatively small number of concepts, making the search space for matching patients to clinical trials a relatively small sub-space of the overall UMLS search space. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results showing that the concepts used to describe clinical trial eligibility criteria and patient clinical notes follow a power distribution can lead to tractable computational approaches to automatically match patients to clinical trials at large scale by considerably reducing the search space. While automatic patient matching is not the panacea for improving clinical trial recruitment, better low cost computational preselection processes can allow the limited human resources assigned to patient recruitment to be redirected to the most promising targets for recruitment.