OBJECTIVES/SPECIFIC AIMS: To share the experience gained and lessons learned from a cross CTSA collaborative effort to improve the review process for Pilot Studies awards by exchanging external reviewers. METHODS/STUDY POPULATION: The CEREC process is managed by a web-based tracking system that enables all participating members to view at any time the status of reviewer invitations. This online tracking system is supplemented by monthly conference calls during which new calls for proposals are announced and best practices are identified. Each CTSA hub customized the CEREC model based on their individual pilot program needs and review process. Some hubs have supplemented their internal reviews by only posting proposals on CEREC that lack reviewers with significant expertise within their institutions. Other hubs have requested 1–3 external reviewers for each of their proposals or a selection of most promising proposals. In anticipation of potential scoring discrepancies, several hubs added a self-assessment of reviewer expertise and confidence at the end of each review. If a proposal is on the cusp of fundability, then the reviewers’ self-assessment may be taken into account. In addition to the tracking data collected by the online system, a survey of CEREC reviewers was conducted using Qualtrics. RESULTS/ANTICIPATED RESULTS: Across the 144 proposals submitted for reviews, CEREC members issued a total of 396 email invitations to potential reviewers. The number of invitations required to yield a reviewer ranged from 1 to 17. A total of 224 invitations were accepted, for a response rate of 56%. An external reviewer was unable to be located for 5 proposals (3%). Ultimately, 196 completed reviews were submitted, for a completion rate of 87%. The most common reasons for non-completion after acceptance of an invitation included reviewer illness and discovery of a conflict of interest. CEREC members found the process extremely useful for locating qualified reviewers who were not in conflict with the proposal being reviewed and for identifying reviewers for proposals related to highly specialized topics. The survey of CEREC reviewers found that they generally found the process easy to navigate and intellectually rewarding. Most would be willing to review additional CEREC proposals in the future. External reviewer comments and scores were generally in agreement with internal reviewer comments and scores. Thus, hubs could factor in external reviewer scores equally to internal reviewer scores, without feeling compelled to calibrate external reviewer scores. Overall, through CEREC external reviewers, mainly due to the stronger matching of scientific expertise and reduction of potential bias, the quality of reviews appear to be higher and more pertinent. DISCUSSION/SIGNIFICANCE OF IMPACT: Some aspects of the process emerged that will be addressed in the future to make the system more efficient. One issue that arose was the burden on the system during multiple simultaneous calls for proposals. Future plans call for harmonizing review cycles to avoid these overlaps. Efficiency also will be improved by optimizing the timing of reviewer invitations to minimize the probability of obtaining more reviews than requested. In addition to the original objective of CEREC, the collaboration has led to additional exchange of information regarding methods and processes related to running the Pilot Funding programs. For example, one site developed a method using REDCap to manage their reviewer database; an innovation that is being shared with the other CEREC partners. Another site has a well-developed process for integrating community reviewers into their review process and is sharing their training materials with the remaining CEREC partners.

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/GOALS: Lack of blood vessels remains a major obstacle in tissue regeneration. Angiopoietin 1 and 2 modulate angiogenesis through the Tie2 receptor tyrosine kinase. Ang1 activates pAKT to promote endothelial cell survival while Ang2 antagonizes these effects. We aim to dissect the Ang/Tie2 pathway to uncover the molecular basis for these opposing effects. METHODS/STUDY POPULATION: Ang1 and Ang2 bind Tie2 via nearly identical F-domains (Fd). To investigate the molecular basis regulating the Tie2 pathway, we generated a series of computationally designed self-assembling protein scaffolds presenting F-domains in a wide range of valencies and geometries using Rosette Molecular Modeling Suite. We examined the protein kinase activation, cell migration, and blood vessel formation produced by the designed proteins in human umbilical vein endothelial cells. RESULTS/ANTICIPATED RESULTS: Two phenotypic classes were demonstrated by the number of presented F domains: scaffolds presenting 3 or 4 Fd have Ang2 like activity, upregulating pFAK and pERK but not pAKT and failing to induce cell migration and tube formation; scaffolds presenting more than 6 Fd have Ang1 like activity, upregulating the three signaling branches and enhancing cell migration and tube formation. Scaffolds with 8 or more Fd show superagonist activity, producing significantly stronger phenotypes than Ang1. These results suggest that Fd valency largely determines Ang1 vs Ang2 signaling outcomes, and our designed superagonists can outperform Ang1 in vascularization and wound healing. In in vivo experiments, nanoparticles displaying 60 copies of Fd produce significant revascularization in hemorrhagic brains. DISCUSSION/SIGNIFICANCE OF IMPACT: Targeting the Tie2 pathway is a new paradigm in regenerative medicine. Our designed constructs will enable us to generate high-affinity Tie2 agonists and antagonists as drugs to control angiogenesis, enabling tissue regeneration that recapitulates the biological architecture of the native tissue physiology, improving organ transplant outcome.

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: To examine and compare the claims in Bzdok, Altman, and Brzywinski under a broader set of conditions by using unbiased methods of comparison. To explore how to accurately use various machine learning and traditional statistical methods in large-scale translational research by estimating their accuracy statistics. Then we will identify the methods with the best performance characteristics. METHODS/STUDY POPULATION: We conducted a simulation study with a microarray of gene expression data. We maintained the original structure proposed by Bzdok, Altman, and Brzywinski. The structure for gene expression data includes a total of 40 genes from 20 people, in which 10 people are phenotype positive and 10 are phenotype negative. In order to find a statistical difference 25% of the genes were set to be dysregulated across phenotype. This dysregulation forced the positive and negative phenotypes to have different mean population expressions. Additional variance was included to simulate genetic variation across the population. We also allowed for within person correlation across genes, which was not done in the original simulations. The following methods were used to determine the number of dysregulated genes in simulated data set: unadjusted p-values, Benjamini-Hochberg adjusted p-values, Bonferroni adjusted p-values, random forest importance levels, neural net prediction weights, and second-generation p-values. RESULTS/ANTICIPATED RESULTS: Results vary depending on whether a pre-specified significance level is used or the top 10 ranked values are taken. When all methods are given the same prior information of 10 dysregulated genes, the Benjamini-Hochberg adjusted p-values and the second-generation p-values generally outperform all other methods. We were not able to reproduce or validate the finding that random forest importance levels via a machine learning algorithm outperform classical methods. Almost uniformly, the machine learning methods did not yield improved accuracy statistics and they depend heavily on the a priori chosen number of dysregulated genes. DISCUSSION/SIGNIFICANCE OF IMPACT: In this context, machine learning methods do not outperform standard methods. Because of this and their additional complexity, machine learning approaches would not be preferable. Of all the approaches the second-generation p-value appears to offer significant benefit for the cost of a priori defining a region of trivially null effect sizes. The choice of an analysis method for large-scale translational data is critical to the success of any statistical investigation, and our simulations clearly highlight the various tradeoffs among the available methods.

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.

ABSTRACT IMPACT: My work is on the development of a novel tumor immunotherapy to treat various types of cancer OBJECTIVES/GOALS: As iNKT cells can have direct and indirect killing effects on tumor cells, we propose a novel strategy for activating iNKT cells, via a PLGA nanoparticle delivery platform, to promote anti-tumor immune responses. METHODS/STUDY POPULATION: Poly-lactic-co-glycolic acid (PLGA) nanoparticles can be reproducibly loaded with an iNKT cell glycolipid agonist, alpha-galactosylceramide (αGalCer), and a tumor associated antigen, ovalbumin (OVA). We then test our nanoP prophylactically and therapeutically against a murine model of melanoma, B16F10-OVA. RESULTS/ANTICIPATED RESULTS: These dual-loaded PLGA nanoparticles rapidly activate iNKT cells in vivo to produce IFNgamma. Furthermore, in an in vivo model of melanoma, using B16F10-OVA cells, both prophylactic and therapeutic administration of nanoparticles containing αGalCer and OVA led to decreased tumor cell growth and increased survival. We also show our nanoparticle therapy has synergistic potential with clinically used immune checkpoint blockade (ICB) therapies, anti-PD-1 and anti-CTLA-4, indicated by the significance increase in survival and lower tumor growth rate of ICB +nanoP treated mice compared to either ICB or nanoP alone. DISCUSSION/SIGNIFICANCE OF FINDINGS: This novel delivery system provides a platform with tremendous potential to harness iNKT cells for cancer immunotherapy purposes against many cancer types.

OBJECTIVES/GOALS: Non-alcoholic fatty liver disease increases with aging and may be associated with MCI. Thus, adults >65 years with NAFLD have a greater risk of MCI. Our objective is to associate NAFLD with MCI and, therefore, optimize the management of MCI through prevention strategies and early interventions. METHODS/STUDY POPULATION: We will recruit 100 patients >65 of all sexes from the GI and Geriatrics clinics of the University of Puerto Rico (UPR). NAFLD prevalence in aging adults will be estimated via ICD-10 codes and definition will be liver fat accumulation or steatosis (AASLD) without secondary causes. We will exclude patients RESULTS/ANTICIPATED RESULTS: We expect to find a higher NAFLD prevalence in aging adults >65 years old compared to younger ones. Furthermore, we aim to elucidate an association of NAFLD with MCI in aging adults. Besides, as the literature has shown, we anticipate that the main cognitive domains affected by NAFLD will be the visuospatial and executive functions. As the population ages, this study will help identify future targets for early interventions in people diagnosed with NAFLD that could have a greater risk for cognitive impairment. DISCUSSION/SIGNIFICANCE: To our knowledge, no study has determined the association of NAFLD with MCI in Hispanics >65 in PR. Raising awareness of NAFLD as a possible treatable or preventable risk factor for MCI by screening NAFLD patients for MCI may improve not only their global health but their quality of life as well.

OBJECTIVES/GOALS: We aim to develop an understanding of how polysubstance use (PSU) relates to the general psychopathology factor (p-factor), as well as to individual components of the Hierarchical Taxonomy of Psychopathology (HiTOP) model (e.g., fear, distress). This insight can help identify treatment targets related to substance use and psychopathology. METHODS/STUDY POPULATION: Psychopathology and substance use data, collected at a Baltimore treatment center over several years, will be analyzed. The center aids about 6000 underserved clients per year, and the population is primarily African American clients of all genders. Structural equation modeling (using Mplus software) will be used to develop the latent models and identify relationships between psychopathology and PSU (i.e., direct and indirect pathways). The current latent HiTOP model was developed from symptom checklists completed upon entry at the treatment center. The PSU latent factor will be developed from a biopsychosocial assessment where clients list their drug of choice. Due to the varying organizations of the datasets, smaller-scale preliminary models will be developed to ensure an accurate large-scale final model. RESULTS/ANTICIPATED RESULTS: Current models being tested are derived from January to September 2023 data (i.e., completed months' data), with an N of 1,564. From symptom checklist data collected at the treatment center, a preliminary HiTOP model was derived with reasonable fit (χ2 = 4532.35 (df = 321, p<.001), CFI = .77, SRMR = .07, RMSEA = .09 (.089, .094)). Data analysis is being conducted to derive the PSU factor before relating PSU to the HiTOP model. Given previous work at a local treatment center (Pavuluri etal., 2022) and with the National Comorbidity Survey-Replication data, we expect all positive direct relationships, negative indirect relationships between internalizing factors (fear and distress) and PSU when accounting for p-factor, and a positive indirect relationship between antagonism and PSU when accounting for p-factor. DISCUSSION/SIGNIFICANCE: Given our previous work to develop such models, we want to establish proof of concept in alarger treatment center population. This confirmation will help provide a path towards conducting therapeutic trials to target psychopathology when treating substance use given the shared relations, some of which are less understood (e.g., fear and PSU).

OBJECTIVES/GOALS: Reactive gliosis is a hallmark of neurodegenerative disease and is characterized by the release of pro-inflammatory cytokines and physiologic changes to glial cells. Our work identifies a novel inflammatory glial-glial cell interaction and role for mGluR5 that has the potential to provide novel insight into the mechanisms of neurodegeneration. METHODS/STUDY POPULATION: Cell Culture: Mouse primary astrocytes and microglia were isolated from P0-P3 C57BL/6 or Cx3cr1GFP/+ mice.1Treatment: Glia were treated with oligomeric α-synuclein 1μg/mL or mGluR5 agonist CHPG 100 μM.2,3ELISA: Glia culture media was collected and analyzed according to the manufacturer. qRT-PCR: TaqMan™ probes were used according to manufacturer on extracted glia mRNA. ICC: Microglia were labeled with 1:750 Rb x Iba1 (Wako) and 1:500 Alexa Fluor 488 Gt x Rb. Phagocytosis Assay: Primary glia were treated with α-synuclein or astrocyte-conditioned culture media for 24-48hrs. For treatment of microglia with conditioned media, astrocytes were washed with PBS and fresh media was added to prevent carry over of α-synuclein to microglia. The number of fluorescent microbeads per microglia was quantified. RESULTS/ANTICIPATED RESULTS: Mouse primary cortical astrocytes simulated with α-synuclein aggregates adopt a reactive A1 phenotype independent of microglial stimulation. This A1 phenotype is characterized by release of pro-inflammatory cytokines including Complement Component 3 and the monocyte chemoattractant CCL2. Reactive astrocyte media induces a phagocytic phenotype in primary mouse microglia. Along with this, α-synuclein-directed microglial phagocytosis was attenuated with the addition of the mGluR5 agonist CHPG. DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest that oligomeric α-synuclein is capable of inducing a reactive phenotype in astrocytes independent of microglia and implicate crosstalk between glia as an important mediator of inflammation and microglial phagocytosis in synucleinopathies.

OBJECTIVES/GOALS: The goal of this presentation is to highlight the role of and challenges to the Biostatistics, Epidemiology and Research Design (BERD) Core as it transitions into (i) the resources and services module (ii) and also possibly a regional center of translational science. METHODS/STUDY POPULATION: BERD cores play key roles in translational research missions, often in the form of long-term collaborative relationships focusing on project conception and design, interim monitoring and review, analysis and dissemination. The resources and services module encourages consolidation of all resources and services under the single modular umbrella, which can pose challenges to BERD autonomy and function. CTSAs transitioning from local to regional centers can also threaten to overwhelm BERD workloads and resources. Our CTSA planned an outsource-insource model to make the BERD a central feature of the new module and transplant its functionality to partner institutions. RESULTS/ANTICIPATED RESULTS: The team has planned a centralized, web-based entry-point for both self-guided inquiry and electronic requests of all resources and services, which aides project database creation and provides notification for review. The review process adds two new approval types: guided assistance to help navigate connections to relevant resources, and triaged referrals to training modules for remediation and skills development. Our BERD has added a Consulting Laboratory for projects of sufficient but non-priority merit that would otherwise be waitlisted, and established connections with regional partner institutions to whom they can refer investigators. DISCUSSION/SIGNIFICANCE: We have consolidated all resources and services into a single, accessible location, emphasizing tailored guidance to maximize limited resources. We also planned a transportable regional model that accounts for local resources and capacity to keep from overstretching the BERD and other hubs.

OBJECTIVES/GOALS: This study models a framework for integrating epidemiological and experimental approaches to investigate the effect of prenatal polycyclic aromatic hydrocarbon (PAH) exposure on mitochondrial function (mtDNAcn, superoxide production and membrane potential) as a potential mechanism of toxicity. METHODS/STUDY POPULATION: The epidemiological aim of this study characterizes mitochondrial outcomes in samples of umbilical cord tissue and blood from two Manhattan based birth cohorts. Prenatal PAH exposure is quantified using silicone wristbands worn for 48 hours during the third trimester of pregnancy. Experimentally, we are applying a PAH mixture designed to emulate the exposure profile of the human cohorts to mouse preimplantation embryos on various dosing schedules and quantifying the same mitochondrial outcomes. mtDNAcn is quantified using rtPCR while superoxide production and membrane potential are measured using fluorescence microscopy. The goal of this study design is to leverage the strengths of each approach to draw more robust conclusions than could be derived from either alone. RESULTS/ANTICIPATED RESULTS: Preliminary results of this study have found associations between higher levels of PAH exposure and increased mitochondrial superoxide production and hyperpolarization of the mitochondrial membrane potential in mouse preimplantation embryos. We anticipate these findings to persist across dosing schedules. We furthermore expect a decrease in mtDNAcn in association with higher PAH exposure in umbilical cord tissue samples and decreased mtDNAcn with exposure to the PAH mixture in mouse embryos. DISCUSSION/SIGNIFICANCE: Characterizing the effect of prenatal PAH exposure on the mitochondria is a critical step in understanding the mechanisms that underlie the toxicity of this exposure. By employing a similar exposure mixture and mitochondrial outcomes across epidemiological and experimental approaches, we offer a model of true interdisciplinary research design.

OBJECTIVES/GOALS: We investigated the association between gut microbiota features in newly admitted nursing facility (NF) patients and the acquisition of vancomycin-resistant Enterococcus (VRE) and/or resistant Gram-negative bacteria (rGNB) within 14 days. METHODS/STUDY POPULATION: Patients were recruited at 6 Michigan NFs from 09/16-08/18. VRE or rGNB colonization status was determined by culture swabs collected from multiple body sites at enrolment, day 7, and day 14. Our analysis focused on patients with no colonization at baseline, a perirectal swab collected at baseline, and at least one follow-up visit. The V4 hypervariable region of the 16S rRNA gene from bacterial DNA in each sample was PCR-amplified and sequenced on the MiSeq platform. Sequencing results were then processed with the mothur bioinformatics pipeline to classify bacterial taxa present in each sample. Taxa typically associated with the skin microbiota were removed. The primary outcome was acquisition of VRE and/or rGNB within 14 days. Exposures of interest included patient and microbiota characteristics. RESULTS/ANTICIPATED RESULTS: Among 61 patients, 18 (30%) acquired AROs within 14 days of enrolment (3 VRE, 13 rGNB, 2 both) (Table 1). The baseline microbiota features differed significantly in those who acquired a new ARO. Of the major 8 phyla found across samples, patients who acquired an ARO were depleted in the number of phyla present (5.74 ± 1.20 vs 5.06 ± 1.43; p = 0.037) (Fig. 1). The log10-transformed relative abundance of Enterococcus was enriched in patients who acquired an ARO (−0.32 ± 1.47) compared to those who did not (−1.68 ± 1.76; p = 0.021) (Fig. 2). Patients who did not acquire an ARO tended to harbour more butyrate-producing bacterial taxa and strict anaerobes, although the differences were not statistically significant (relative abundance of butyrate producer: 29.49 ± 22.09 vs 22.05 ± 17.76; anaerobes: 64.78 ± 23.54 vs 53.68 ± 27.61). DISCUSSION/SIGNIFICANCE OF IMPACT: Microbiota metrics calculated from perirectal samples are predictive of ARO acquisition. The clinical utility of perirectal samples thus warrants further assessment.

OBJECTIVES/SPECIFIC AIMS: This study aims to evaluate an injectable sulfonated reserve thermal gel (SPSHU-PNIPAM) for angiogenic growth factor delivery by examining the vascularization and cardioprotective properties of the polymer system. This study could lead to clinical translation by moving into larger animal studies and eventually clinical trials. The success of this study was determined by analyzing the results of echocardiography data on cardiac function (ejection fraction, fractional shortening, and left ventricle inner diameter) and assessment of histological staining on cardiac tissue (fibrotic tissue formation, infarct size, wall thinning, blood vessel cell counts, and vessel size quantification) after MI. Five groups were compared for this study: saline, VEGF, SPSHU-PNIPAM, SPSHU-PNIPAM loaded with VEGF, and no injection (sham). Significant statistical differences between control groups and polymer injection groups, when p < 0.05, indicates successful outcomes from this study. METHODS/STUDY POPULATION: SPSHU-PNIPAM Polymer Synthesis: SPSHU-PNIPAM was synthesized as previously described. Briefly, PSHU was synthesized with N-BOC serinol, urea, and HDI at 90 °C for 7 days. PSHU was deprotected in DCM and TFA at room temperature for 45 min. PNIPAM was conjugated to the deprotected PSHU using EDC and NHS at room temperature for 24 h. PSHU-PNIPAM was sulfonated with 1,3-propanesultone and potassium tert-butoxide at 60 °C for 3 days. Surgical Procedure: Male C57BL/6 mice weighing 24-28 g were anaesthetized using isoflurane and artificial ventilation provided. A small left thoracotomy incision was made at the left fourth intercostal space to expose the heart, and the proximal left anterior descending coronary artery was ligated for 45 min. The coronary artery was then released and 30 μl injections of saline, SPSHU-PNIPAM (1% w/v), bolus VEGF (200 ng), or SPSHU-PNIPAM + VEGF (1%, 200 ng) were injected intramyocardially at the infarcted site and the incision closed. Echocardiography and Histological Staining: Standard serial transthoracic echocardiography was performed while simultaneously recording ECG to assess cardiac morphology and left ventricular function. Immunohistochemistry and histology staining procedures were used to identify: fibrotic tissue formation, infarct size, wall thinning, blood vessel cell counts, and vessel size quantification. These were performed according to manufacturer instructions or by previously published criteria. Statistical Analysis: Two-tailed t-test assuming unequal variances was used to determine significant differences between two groups. Analysis of variance (ANOVA) was used to determine significant differences between three or more groups followed by Tukey-Kramer to determine significant differences between two groups as appropriate. Statistical significance was considered when p < 0.05. References: Lee, D. J., Rocker, A. J., Bardill, J. R., Shandas, R. and Park, D. (2018), A sulfonated reversible thermal gel for the spatiotemporal control of VEGF delivery to promote therapeutic angiogenesis. J Biomed Mater Res. doi:10.1002/jbm.a.36496. RESULTS/ANTICIPATED RESULTS: Echocardiography results: Ejection fraction improved for the SPSHU-PNIPAM groups compared to the saline, VEGF, and no injection controls (Figure 1). SPSHU-PNIPAM either loaded with or without VEGF seemed to have very similar treatment effects for ejection fraction and fractional shortening. This indicates that the more significant component of the cardioprotective effects of the hydrogel system is the biomaterial itself rather than the release of VEGF (Figure 1). However, the only statistically significant improvement for ejection fraction, fractional shortening, and left ventricular inner diameter that was observed compared to the saline, VEGF, and no injection controls was the SPSHU-PNIPAM + VEGF group (Figure 1). Histology Results: After analyzing Masson trichrome staining, SPSHU-PNIPAM + VEGF demonstrated the smallest infarct size after MI reperfusion injury and was statistically reduced compared to the saline, VEGF, and no injection controls (Figure 2). Furthermore, left ventricular wall thickness showed that the SPSHU-PNIPAM + VEGF treatment group reduced the wall thinning resulting from MI. The SPSHU-PNIPAM group without VEGF displayed a thicker ventricular wall as well, which may be attributed to the increased mechanical stability with the intramyocardial injection of the biomaterial (Figure 2). The immunohistochemical results for vascularization show that the SPSHU-PNIPAM + VEGF group significantly increased the number of functional vascular endothelial cells compared to the saline, VEGF, SPSHU-PNIPAM, and no injection controls (Figure 3). Additionally, the SPSHU-PNIPAM + VEGF group showed a significant increase in total vessel formation compared to the control groups, although there was no significant difference compared to SPSHU-PNIPAM without VEGF (Figure 3). The promotion of angiogenesis, without the delivery of VEGF, may be attributed to inflammation induced vascularization, including VEGF dependent vascularization that is initiated via signal transducer and activator of transcription 3 (STAT3) pathway that is induced by the pro-inflammatory cytokine interleukin 6. DISCUSSION/SIGNIFICANCE OF IMPACT: The SPSHU-PNIPAM loaded with VEGF was evaluated for therapeutic angiogenesis to protect cardiac function after MI. Treatment with SPSHU-PNIPAM showed improved cardiac function and vascularization; however, the additional delivery of VEGF showed inadequate additional therapeutic benefits. Further investigation will include optimizing VEGF release characteristics including both loading amount and release rate. The decline of ejection fraction and fractional shortening after MI were reduced, while left ventricular internal diameter showed reduced ventricular dilation. Both infarct size and left ventricular wall thinning decreased while an increase in the vessel formation was observed. These results demonstrate the SPSHU-PNIPAM biomaterial has cardioprotective and increased vascularization properties for the treatment of MI.

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/GOALS: To examine the relationship between chronic cadmium exposure and cognitive function in later life, we estimated the association of urinary cadmium concentration on composite cognitive score, an important marker of progression toward dementia, while accounting for diet and key co-pollutants tobacco use and lead exposure. METHODS/STUDY POPULATION: After excluding those missing cognitive data (141) or covariate data (190), we included 760 persons >/= 60 years of age from the National Health and Nutrition Examination Survey (NHANES), 2011-2014. Urinary cadmium reflects prolonged exposure: mean=0.41μg/L (standard deviation (SD)= 0.46μg/L). Concurrent with urine sampling, neuropsychological tests tapping memory, executive function, sustained attention and working memory were combined into a standardized z-score (mean 0, SD 1, 25th and 75th percentiles −0.68 and 0.72, respectively). We used linear models to estimate change in cognition per cadmium interquartile range, incorporating NHANES sampling weights, adjusting for demographic characteristics, diet, lead, and active tobacco use (classified by self-report or serum cotinine levels >10ng/mL). RESULTS/ANTICIPATED RESULTS: A baseline model showed that an IQR (0.38 μg/L) increase in urinary cadmium exposure was associated with a 13% standard deviation lower cognitive z-score (95%CI: −0.19, −0.06), after adjusting for sampling weight and urinary creatinine (measure of urine dilution). This association was attenuated to 7% standard deviation lower cognitive z-score (95% CI −0.13, −0.02) after adjusting additionally for demographic characteristic of sex, age, age^2, race/ethnicity, marital status, education level, and poverty income ratio. Models further adjusted for smoking status (active/former/never), blood lead concentration, and key dietary sources of cadmium showed IQR increase in urinary cadmium exposure associated with 7% standard deviation lower cognitive z-score (95%CI: −0.14, −0.01). DISCUSSION/SIGNIFICANCE: Our findings suggest cadmium exposure is associated with lower cognitive scores even after accounting for confounding influence of diet, tobacco use and lead exposure. Alternate explanations include selection bias due to dropping persons missing needed variables and using concurrent cognitive measures rather than cognitive measures of over time.

ABSTRACT IMPACT: Understanding the influence of genetic variation and smoking on alcohol consumption helps in improving the treatment strategies for alcohol addiction OBJECTIVES/GOALS: Variation in the nicotinic receptor gene CHRNA5 (rs16969968) is associated with nicotine use and dependence, however its role in alcohol consumption is unclear. This study examined the effects of rs16969968 and smoking on alcohol related phenotypes in people without alcohol use disorder (AUD). METHODS/STUDY POPULATION: The study included 1,037 healthy adult drinkers without AUD (201 smokers, 836 non-smokers). A subset (n=161) participated in an Intravenous Alcohol Self-Administration (IV-ASA) laboratory session. Alcohol-related measures included Timeline Followback (TLFB), which measures drinking quantity and frequency in the past 90 days, and the Alcohol Use Disorders Identification Test (AUDIT), which measures alcohol use and consequences. IV-ASA measures included average and peak breath alcohol concentration (BrAC). The effect of rs16969968 was tested using a dominant model based on the presence of the A allele, and the influence of the rs16969968 polymorphism and smoking on alcohol phenotypes was assessed using t-tests and two-way ANOVA. RESULTS/ANTICIPATED RESULTS: There was a main effect of rs16969968 genotype with A-allele carriers (AA/AG) showing higher AUDIT-Dependence scores compared to the GG group. A main effect of smoking was observed on all the TLFB and AUDIT measures, with smokers showing greater alcohol consumption and problems compared to non-smokers. In the rs16969968 AA/AG group, smokers reported significantly more drinking days (p<0.0001), and greater number of drinks (p<0.0001), as well as higher AUDIT scores than non-smokers. IV-ASA measures did not show any difference between genotype groups or between smokers and non-smokers. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study identifies both independent and interactive effects of CHRNA5 gene variation and smoking on alcohol drinking measures and provides strong evidence for the effect of smoking on alcohol drinking and its consequences.

OBJECTIVES/SPECIFIC AIMS: The goal for this project is to determine the feasibility of using a novel multi-photon fiber-coupled microscope to aid surgeons in localizing STN during surgeries. In order to accomplish this goal, we needed to identify the source of a strong autofluorescent signal in the STN and determine whether we could use image classification methods to automatically distinguish STN from surrounding brain regions. METHODS/STUDY POPULATION: We acquired 3 cadaveric brains from the University of Colorado Anschutz Medical Campus, Department of Pathology. Two of these brains were non-PD controls whereas 1 was diagnosed with PD. We dissected a 10 square centimeter region of midbrain surrounding STN, then prepared this tissue for slicing on a vibratome or cryostat. Samples were immuno-labeled for various cellular markers for identification, or left unlabeled in order to observe the autofluorescence for image classification. RESULTS/ANTICIPATED RESULTS: The border of STN is clearly visible based on the density of a strong autofluorescent signal. The autofluorescent signal is visible using 2-photon (850–1040 nm excitation) and conventional confocal microscopy (488–647 nm excitation). We were also able to visualize blood vessels with second harmonic generation. The autofluorescent signal is quenched by high concentrations of Sudan-black B (0.5%–5%), and is primarily localized in microtubule-associated protein-2 (MAP2)+ cells, indicating that it is likely lipofuscin accumulation in neurons. Smaller lipofuscin particles also accumulate in microglia, identified based on ionized calcium binding adopter 1 (Iba1)+ labeling. We anticipate that colocalization analysis will confirm these qualitative observations. Using 2-photon images of the endogenous autofluorescent signal in these samples, we trained a logistic regression-based image classifier using features derived from gray-level co-occurrence matrices. Preliminary testing indicates that our classifier performed well, with a mean accuracy of 0.89 (standard deviation of 0.11) and a Cohen’s Kappa value of 0.76 (standard deviation of 0.24). We are currently using coherent anti-Stokes Raman scattering and third harmonic imaging to identify different features of myelin that can be used to distinguish between these regions and expect similar results. DISCUSSION/SIGNIFICANCE OF IMPACT: Traditional methods for localizing STN during DBS surgery include the use of stereotactic coordinates and multi-electrode recording (MER) during implantation. MERs are incredibly useful in DBS surgeries, but require penetration of brain structures in order to infer location. Using multi-photon microscopy techniques to aid identification of STN during DBS surgeries offers a number of advantages over traditional methods. For example, blood vessels can be clearly identified with second harmonic generation, something that is not possible with MER. Multi-photon microscopy also allows visualization deep into tissue without actually penetrating it. This ability to look within a depth of field is useful for detection of STN borders based on autofluorescent cell density. When combined with traditional stereotactic information, our preliminary image classification methods are a fast, reliable way to provide surgeons with extra information concerning their location in the midbrain. We anticipate that future advancements and refinements to our image classifier will only increase accuracy and the potential applications and value. In summary, these preliminary data support the feasibility of multi-photon microscopy to aid in the identification of target brain regions during DBS surgeries. The techniques described here complement and enhance current stereotactic and electrophysiological methods for DBS surgeries.

There is an urgent need for vaccines to the 2019 coronavirus (COVID19; SARS-CoV-2). Vaccine development may not be straightforward, due to antibody-dependent enhancement (ADE). Antibodies against viral surface proteins can, in some cases, increase infection severity by ADE. This phenomenon occurs in SARS-CoV-1, MERS, HIV, Zika, and dengue virus infection and vaccination. Lack of high-affinity anti-SARS-CoV-2 IgG in children may explain the decreased severity of infection in these groups. Here, we discuss the evidence for ADE in the context of SARS-CoV-2 infection and how to address this potential translational barrier to vaccine development, convalescent plasma, and targeted monoclonal antibody therapies.

Temporal challenges are not only contextual in nature but manifest internally in teams when members enter the team with different temporal orientations (e.g., time urgency and pacing style). Researchers have demonstrated that temporal diversity has important implications for key team outcomes (performance, timeliness, and team conflict) across a range of samples and countries. Unfortunately, the practical implications of this research have yet to be unpacked. We respond to this need by developing an approach to translate temporal diversity research studies into actionable, evidence-based team interventions. Because journal articles are often deficient on actionable steps, whereas practitioner-friendly outlets tend to be deficient on scientific rigor, incorporating both criteria necessitates merging these literatures. Specifically, we delineate four main steps: (1) identify significant moderators, (2) match the moderators to scientifically based interventions, (3) design intervention tools with specific, actionable procedures, and (4) evaluate the effectiveness of the intervention tools by designing research studies. We believe the process we outline to marry actionable and evidence-based benchmarks is applicable to other research domains in team science beyond temporal research. It is our hope that this research will be a catalyst for further exploration of interventions that can help team members navigate temporal differences.