OBJECTIVES/GOALS: 30,000,000 people in the U.S. have hearing loss, negatively impacting quality of life and work. Understanding auditory axon guidance for spiral ganglia neurons (SGNs) will aid development of new therapies. I study role of Eph/Ephrin signaling in mediating type II SGN turning events, and how planar cell polarity (PCP) signaling modulates this process. METHODS/STUDY POPULATION: This quantitative study was conducted on Efna3 and Vangl2 null mice possessing Neurog1CreERT2 and R26RtdTomato mutations. Spontaneous Cre activity within the Neurogenin 1 CreERT2 line causes recombination and expression of fluorescent Rosa26 Reporter (R26R)tdTomato in a restricted number of SGNs, including type IIs. Together these lines permit SGN sparse labeling. Bulk-labeling was used for Efna3;Vangl2 double knockout (DKO) mutants. Immunostaining and confocal imaging was used to analyze dsRed in Efna3; Vangl2 and NF-200 in DKOs to quantify type II SGN turning. In combination, 3D rendering in Imaris software was used to quantify type II SGN turning, branching and other growth and navigation characteristics. 5-6 cochleae per genotype were analyzed and compared by t-test to wildtype controls. RESULTS/ANTICIPATED RESULTS: EPHRIN-A3 is expressed on the membranes of outer pillar and Deiters’cells of the cochlear epithelium. Efna3 nulls showed a small rise in type II SGNs incorrectly turning toward the apex at an error frequency of 16.9% compared to controls (n=6; p=0.05). Efna3 nulls had reduced branch number/fiber compared to controls, 4.14 and 7.22, respectively (n=129; p DISCUSSION/SIGNIFICANCE: Our results suggest that Eph/Ephrin signaling acts parallel of PCP signaling to mediate type II SGN guidance during development. The clinical implications of these findings are that therapeutics targeting EPHRIN-A3 and/or VANGL2 in this pathway could stimulate new outer hair cell innervation by type II SGNs following auditory damage.

ABSTRACT IMPACT: We present a minimally-invasive approach to monitoring heavy metal exposures in geographically disperse populations; this framework may encourage and facilitate greater participation in a broad range of related clinical studies that require biosampling. OBJECTIVES/GOALS: We hypothesize that microneedle array (MA) extraction of interstitial fluid (ISF) will enable minimally-invasive quantitation of heavy metal (HM) exposure. We establish analytical parameters for ICP-MS analysis of HMs in ISF, quantify baseline HM content in ISF vs other fluids, and characterize a mixed HM exposure model. METHODS/STUDY POPULATION: Recent advances in ISF extraction and analysis suggest a minimally-invasive to monitor HM exposure longitudinally in both urban and dispersed communities. ISF can be collected with MAs and is a rich source of disease and exposure biomarkers. To refine analytical methods, Human subjects with no underlying skin disease were recruited into the IRB-approved study. Each subject had blood and urine collected. ISF was also collected using 3D-printed MA-holders. The fluids were analyzed, using ICP-MS, to quantify the levels of uranium (U), cadmium (Cd), vanadium (V), and arsenic (As). Additionally, we analyzed 2,770 subjects from the public NHANES dataset from 2018-2019. Python and Scikit-learn were used to analyze the demographics, survey responses, and metal concentrations for these individuals. RESULTS/ANTICIPATED RESULTS: While several studies have described the surface water and sediment content of toxic metals, determining biological loads remains challenging due to the need to collect blood or urine from a dispersed rural population over time. Our preliminary results suggest similar HM concentrations in ISF, compared with blood in a small unexposed population. Analysis of subjects from the NHANES public datasets suggest similar baseline blood HM concentrations in diverse subject populations with some differences in Cd depending on smoking and e-cigarette usage. Correlation maps also suggest possible synergy between different metals with cobalt and chromium showing the highest correlation. The initial results from this study have been applied to develop a mixed HM exposure model in rats for further translational testing. DISCUSSION/SIGNIFICANCE OF FINDINGS: We present a minimally-invasive HM monitoring approach. Exposure to multiple HMs is suspected to have additive or synergistic harmful health effects. We ultimately envision a wearable microneedle patch that could be mailed to individuals or distributed through community centers, worn for a few hours, and returned to a central laboratory.

OBJECTIVES/GOALS: The goal of this project is to identify new therapeutic targets and biomarkers to treat or prevent cocaine toxicity by investigating proteomic, transcriptomic and epigenetic signatures of cocaine exposure in human subjects. METHODS/STUDY POPULATION: Cocaine is a highly addictive neurotoxic substance, and it is estimated that 1.9 million Americans are current users of cocaine. To study the molecular effects of cocaine, we generated preliminary proteomics and next-generation RNA sequencing (RNAseq) data from human postmortem dorsolateral prefrontal cortex (Broadmann area 9 or BA9) of 12 cocaine-exposed subjects and 17 controls. Future directions for this project include RNAseq and DNA methylation analysis of neuronal nuclei sorted from human postmortem BA9 and a human induced pluripotent stem cell-derived neuron (hiPSN) model of cocaine exposure from the same postmortem subjects from whom we have brain samples. RESULTS/ANTICIPATED RESULTS: We found alterations in neuronal synaptic protein levels and gene expression, including the serotonin transporter SLC6A4, and synaptic proteins SNAP25, SYN2, SYNGR3. Pathway analysis of our results revealed alterations in specific pathways involved with neuronal function including voltage-gated calcium channels, and GABA receptor signaling. In the future, we expect to see an enhancement in neuron-specific gene expression signatures in our sorted neuronal nuclei and our hiPSN model of cocaine exposure. The hiPSN model will help elucidate which effects are due to acute versus chronic exposure of cocaine. DISCUSSION/SIGNIFICANCE OF IMPACT: Neuronal signatures found with this analysis can help us understand mechanisms of cognitive decline in long-term cocaine users as well as the acute effects on the brain of cocaine taken in overdose. With this work and future proposed studies, we can discover novel clinical biomarkers for cocaine neurotoxicity in patients with cocaine use disorder and determine readouts for future therapeutic development on cocaine addiction and overdose.

OBJECTIVES/SPECIFIC AIMS: Promote knowledge translation and evidence-informed decision-making by assessing barriers and facilitators to balancing cost and quality of care within the US state of Maryland and nation of Denmark. METHODS/STUDY POPULATION: Open-ended and semi-structured key-informant interviews were conducted in 2016 and 2017 among high level decision-makers in Maryland (N=21) and the Danish (N=17) healthcare systems, including hospital, local, regional, and cross-organizational administrators and elected officials. The interviews consisted of questions related to: (1) currently practiced and preferred approaches to resource allocation and development and use of quality performance measures, and (2) preferred sources, formats/styles, modes of information, and decision-making strategies based on a shift from volume to quality-driven care. RESULTS/ANTICIPATED RESULTS: Decision-makers in Maryland expressed the need for collaboration in a changing environment, yet increasingly rely on cost and quality outcomes data to drive decisions and note the struggle to identify credible and useful information. Maryland decision-makers also face challenges in regulating utilization and costs without mandated participation of physician practices within the global budget cap model, which is perceived to be a primary driver of healthcare utilization in the hospital sector. Similarly, decision-makers in Denmark conveyed the importance of quantitative data to aid decisions, however, stress collaboration and dialogue as driving factors and important sources of information. Danish decision-makers also express challenges to wide-spread adoption of a quality-driven approach due to unsustained quality assurance regulatory bodies. DISCUSSION/SIGNIFICANCE OF IMPACT: The findings suggest implementation of value-based healthcare is highly driven and influenced by availability of credible data, which may significantly impact development of policies and innovative cost control strategies, and regulatory oversight to promote adoption of quality measures in decision-making. Furthermore, collaboration within and across healthcare organizations remains a key component to health system improvement as it fosters dialogue and sharing of best practices among stakeholders.

OBJECTIVES/SPECIFIC AIMS: For this reason, our overall objectives are to determine (i) whether natural disaster-related PNMS alters infants’ EC at two years of age, and (ii) if the timing of exposure moderates its effects on toddlers EC. METHODS/STUDY POPULATION: We propose a longitudinal study with 50 mother-toddler dyads. Natural disaster-related PNMS would be measured at 12-18 and 24-30 months of age and will include: objective exposure and maternal distress. EC will be measured with a questionnaire and a Laboratory Temperament Assessment Battery at two years of age. To accomplish our objectives, we will conduct regression and moderation analyses. RESULTS/ANTICIPATED RESULTS: We anticipate that children exposed to Hurricane-related PNMS would present low EC levels compared to those with low prenatal exposure. DISCUSSION/SIGNIFICANCE OF IMPACT: These results are expected to provide evidence for further promoting early intervention and ameliorating adverse effects of PNMS on child outcomes.

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values <0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P<0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.

OBJECTIVES/GOALS: Our goal is to explore the extent to which organoids can serve as models for the protective mechanisms of the stomach–the mucus barrier and the pH gradient across it. We aim to first optimize and validate an organoid-based model of the gastric mucus layer, and then define the cellular mechanisms by which the gastric pH gradient is maintained across it. METHODS/STUDY POPULATION: We have developed a method for the in vitro engineering of gastric mucus by growing epithelial cells at the air-liquid interface (ALI). We use microrheology with fluorescent microspheres to define and compare the biophysical and viscoelastic properties of our lab-grown mucus to those of native mucus. We will perform CryoFE-SEM to compare the internal heterogeneity of our lab-grown mucus to fresh mucus obtained from patient tissue. For our mechanistic studies, we will use a pH-sensitive dye (methyl red) to assess the ability of our lab-grown mucus to maintain an artificial pH gradient in a microfluidic device. Next, we will use a pH microelectrode to measure proton flux through our mucus in vitro, investigating the potential for a physiological gradient in both 2D and 3D organoid models. RESULTS/ANTICIPATED RESULTS: Here we show that gastric organoids and their corresponding epithelial monolayers produce a mucus gel that does indeed mimic in vivo functions. Immunohistochemical staining, electron microscopy, microrheology, and particle tracking analyses revealed that our gastric organoid mucus is viscoelastic and structurally heterogeneous–both properties that are crucial to the stomach’s mucosal first line of defense. Mechanically similar mucus was also engineered using two-dimensional air-liquid interface cultures of the same epithelia. Lastly, live confocal imaging revealed that H. pylori motility–an important virulence factor–was drastically hindered by our lab- grown mucus. DISCUSSION/SIGNIFICANCE: We describe a novel method for the in vitro engineering of gastric mucus and highlight biophysical properties that contribute to our stomach’s defense against pathogens. This work will lead to an improved understanding of gastric physiology and may contribute to the development of novel drug delivery systems to tackle diseases of the gastric mucosa.

OBJECTIVES/GOALS: The goal of this study is to better understand the homicide victim population who were institutionalized within 30 days prior to their death. Improved knowledge of this population can potentially prevent these future homicides. METHODS/STUDY POPULATION: A retrospective analysis of the 36 states included in the 2003-2017 National Violent Death Reporting System was performed. Demographics of recently institutionalized homicide victims (RIHV) in the last 30 days were compared to homicide victims who were not recently institutionalized. Circumstances of the homicide, such as suspected gang involvement, were also compared. Parametric and non-parametric statistical analyses were performed. Significance was set at p<0.05. RESULTS/ANTICIPATED RESULTS: There were 81,229 homicides with 992 (1.2%) RIHV. The majority of RIHV were Black (49.6%) and older than victims who were not recently institutionalized (37.2 vs. 34.8, p<0.001). RIHV had a high school degree or higher in 54.8% of cases and the primary homicide weapon was a firearm in 67% of the deaths. They were more likely to be homeless (3.1% vs. 1.5%), have a mental health diagnosis (9.2% vs. 2.3%), abuse alcohol (6.1% vs. 2.2%), or abuse other substances (15.2% vs. 5.8%) [all p <0.001]. These victims were most commonly institutionalized in a correctional facility or a hospital compared to other facilities such as nursing homes. Homicide circumstances for RIHV were more likely to involve abuse/neglect (4.3% vs. 2.2%, p<0.001), gang violence (7.6% vs. 5.6%, p = 0.002), or a hate crime (1.0% vs. 0.1%. p<0.001). DISCUSSION/SIGNIFICANCE OF IMPACT: Contact with an institution such as a hospital or prison provides high-risk patients the opportunity to potentially participate in violence intervention programs. These institutions should seek to identify and intervene on this population to reduce the risk of violent homicides.

OBJECTIVES/GOALS: The objective of this research was to determine the associations of candidate genetic variants withdrug-induced long QT syndrome (diLQTS) risk, an adverse effect of over 150 FDA-approved drugsthat can lead to cardiac arrhythmias and sudden cardiac death. METHODS/STUDY POPULATION: This was a retrospective observational study of the genomic biobank at the University of Michigan Health System. Patients treated with a high-risk QT-prolonging drug and ECG measurements were included. The primary outcome was exaggerated prolongation of the QTc interval (i.e., >60 ms change from baseline and/or >500 ms absolute value) corrected using Bazett. We analyzed 3 genetic variants: KCNE1-D85N (rs1805128), SCN5A-G615E (rs12720452) and KCNE2-I57T (rs7415448) in the dominant genetic model. A Bonferroni-corrected p-value of 0.017 was considered statistically significant using logistic regression adjusted for clinical covariates. RESULTS/ANTICIPATED RESULTS: In total 6,083 self-reported white patients were included (12% event rate). The adjusted odd ratio for KCNE1-D85N was 2.24 (95%CI: 1.35-3.57; p=0.0011). The adjusted odds ratio forKCNE2-I57T was 1.40 (95%CI: 0.26-5.78, p=0.662). Only 4 total patients carried the SCN5A-G615E variant, and none of the carriers had prolonged QTc. DISCUSSION/SIGNIFICANCE: This is the largest study of candidate genetic variants in cardiac ion channels associated with the diLQTS risk. KCNE1-D85N was associated with diLQTS risk, while KCNE2-I57T was suggestive of a potential association. KCNE1-D85N should be considered in clinical guidelines as a risk factor of diLQTS.

OBJECTIVES/GOALS: The goals of this project are to: i) investigate the cargo such as immune mediators (cytokines) and small non coding RNAs (sncRNAs) of EVs derived from nasopharyngeal secretions (NPS) of children with episodes of viral infections and exposed to SHTS; ii) test the biological activity of EVs released from upper airway mucosa on target/recipient lung cells METHODS/STUDY POPULATION: EVs were isolated from ten NPS samples of children with episodes of acute respiratory infections. EVs were characterized by particle sizing (size and concentration), EV markers, and protein arrays for interferons, cytokines, and other immune mediators content. RNA was extracted from ten samples of NPS-derived EVs by column for next generation high throughput sequencing (NGS) to identify sncRNAs in EVs. In studies currently in progress, EVs will be isolated from RSV-infected human nose organoids (HNOs) cells in air liquid interface (ALI) culture, with or without pre-exposure to tobacco smoke. EVs will be then tested for antiviral activity on recipient RSV-infected lower airway cells. Viral titers will be measured in recipient infected lung cells. RESULTS/ANTICIPATED RESULTS: We isolated EVs from NPS samples and confirmed by immunoblot EV markers CD63 and Alix. The average size of NPS-derived EVs of virus positive and negative patients was 170 nm and 145 nm, respectively. We determined the particles number of EVs, concentrations of IFN-βand IFN-λin NPS and NPS-derived EVs of these children. While IFN-βlevels were below the limit of detection in both NPS and NPS-derived EVs of all children, IFN-λwas detected in NPS and NPS-derived EVs from infected patients, except for the two patients with no viral infections. We extracted RNA from control-, virus infected- and/or SHTS- EVs and found that piR-36511, piR-40926, piR-49645, piR-32679 and piR- 53263 were all significantly enriched in EVs derived from NPS of children exposed to TS compared to those not exposed. DISCUSSION/SIGNIFICANCE: RSV leads to approximately 22,000 hospitalizations of children due to second-hand smoke. A vaccine is not currently available for RSV infection. EVs represent a novel translational approach to target undruggable. Airway mucosa EVs in viral respiratory infection function as antiviral messengers and tobacco smoke impairs the EV antiviral activity.

OBJECTIVES/GOALS: A particularly debilitating consequence of stroke is alexia, an acquired impairment in reading. Cognitive models aim to characterize how information is processed based on behavioral data. If we can concurrently characterize how neural networks process that information, we can enhance the models to reflect the neuronal interactions that drive them. METHODS/STUDY POPULATION: There will be 10 unimpaired adult readers. Two functional localizer tasks, deigned to consistently activate robust language areas, identify the regions of interest that process the cognitive reading functions (orthography, phonology, semantics). Another task, designed for this experiment, analyses the reading-related functional-connectivity between these areas by presenting words classified along the attributes of frequency, concreteness, and regularity, which utilize specific cognitive routes, and a visual control. Connectivity is analyzed during word reading overall vs. a control condition to determine overall reading-related connectivity, and while reading words that have high vs. low attribute values, to determine if cognitive processing routes bias the neural reading network connectivity. RESULTS/ANTICIPATED RESULTS: The localizer analysis is expected to result in the activation of canonical reading areas. The degree of functional connectivity observed between these regions is expected to depend on the degree to which each cognitive route is utilized to read a given word. After orthographic, phonologic, and semantic areas have been identified, the connectivity analysis should show that there is high correlation between all three types of areas during reading compared to the control condition. Then the frequency, regularity, and concreteness of the words being read should alter the reliance on the pathways between these area types. This would support the hypothesized pattern of connectivity as predicted by the cognitive reading routes. Otherwise, it will show how the neural reading network differs from the cognitive model. DISCUSSION/SIGNIFICANCE OF IMPACT: The results will determine the relationship between the cognitive reading model and the neural reading network. Cognitive models show what processes occur in the brain, but neural networks show how these processes occur. By relating these components, we obtain a more complete view of reading in the brain, which can inform future alexia treatments.

OBJECTIVES/SPECIFIC AIMS: The objective of this study was to prospectively assess caregiver-perceived barriers to accessing post-acute care for their injured child and determine if caregivers report ongoing, unmet health needs for their children after trauma. METHODS/STUDY POPULATION: This was a prospective cohort study that followed 50 participants for 6 months and administered surveys to parents of children who are admitted to a pediatric level 1 trauma center for injury. Surveys were given bi-weekly regarding care children received after hospital discharge. At 3 months, parents were surveyed over the phone on whether they were able to access all needed health services and if there were any perceived barriers to obtaining or providing at-home care. At 6 months, parents were given the Child & Family Follow-up Survey to assess ongoing physical, mental, social, and scholastic needs. Free responses and transcribed interviews were analyzed using thematic content analysis and frequencies are reported for discrete data. RESULTS/ANTICIPATED RESULTS: Out of 50 families recruited, 47 completed follow-up assessments. At 3 months, common themes regarding challenges after hospital discharge included difficulty scheduling specialist care; uncertainty in managing their child’s pain; transitioning home without enough knowledge to meet their child’s medical needs; lack of communication between multiple providers; distress at having providers release children to full activities before caregivers were comfortable. At 6 months, approximately 24% of parents reported children had ongoing cognitive limitations, 29% reported emotional problems, 19% reported physical limitations, 33.3% reported difficulty in school, and 15% reported play/social difficulties. DISCUSSION/SIGNIFICANCE OF IMPACT: Evidence suggests families face significant barriers in accessing follow-up care, despite nearly universal health insurance coverage for children. Further, a large percentage of parents report ongoing health needs, despite the majority of the cohort having only mild or moderate severity injuries. Making follow-up care more patient-centered for families of traumatically injured children may improve compliance with medical regiments and reduce the likelihood of future disability. Examples of this may be coordinating care among multiple specialty providers, so that patients with multiple injuries can schedule multiple follow-up appointments on the same day. Additionally, more caregiver education on administering pain medication, caring for wounds, and safe practices for returning to full activities would be beneficial for families.

ABSTRACT IMPACT: Implementation of proteomics as a diagnostic tool for Nontuberculous mycobacteria (NTM) infection can provide a more accurate, efficient and cost-effective means for effectively diagnosing disease and enacting timely management decisions which can revolutionize patient care. OBJECTIVES/GOALS: Proteomic analysis is a proven diagnostic modality enabling rapid identification of microorganisms. We sought to apply proteomics to detect proteins unique to the most clinically relevant NTM. We then determined whether these unique proteomes could be used to successfully identify NTM species from in vitro cocktail preparations. METHODS/STUDY POPULATION: NTM reference strains for M. avium, m. intracellulare, m.chimaera, m. abscessus abscessus, m. abscessus massiliense and m. abscessus boletti were cultured in vitro and subjected to proteomic analysis using Liquid Chromatography tandem-Mass Spectrometry (LCMS). Tandem Mass Tag (TMT) data acquisition utilized an MS3 approach for data collection using Proteome Discoverer 2.4.

A comparative analysis of the proteome of each of these six species was performed quantitatively using LCMS. The process was repeated for three technical replicates and analyzed using the SEQUEST algorithm. Only high scoring peptides were considered utilizing a false discovery rate (FDR) of 1%. Once species-specific proteins were identified, we validated detection in individual and mixed samples of the six reference strains. RESULTS/ANTICIPATED RESULTS: The proteomic profiling of the six NTM reference strains successfully demonstrated proteins unique to each of the MAC species and MABC subspecies. Proteomic MAC species analysis produced between 327 to 2,540 unique peptides for each of the 3 species. MABC proteomic analysis identified between 17-74 unique peptides for each of the 3 subspecies. Fifteen different mixed preparations of MAC and MABC were then subjected to LCMS analysis and compared against the proteome profiles already curated for the six strains. We accurately identified at least one NTM in the majority of the samples (10/15). In three samples (3/15), the NTM was not correctly identified; in two of the samples (2/15) we were unable to determine the identity of NTM within the preparation. Further database curation will be performed to hone these results. DISCUSSION/SIGNIFICANCE OF FINDINGS: Proteomic analysis of in vitro reference strains successfully demonstrated protein fingerprints specific to six common disease-causing strains of NTM. Such findings can be used to evaluate clinical samples enabling more efficient diagnostic specificity. Further research will focus on identification of NTM in sputum samples of infected patients.

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p<0.001), as well as higher levels of IFN-γ (all p<0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p<0.01) and CXCL10 (p<0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p<0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

Project management expertise is employed across many professional sectors, including clinical research organizations, to ensure that efforts undertaken by the organization are completed on time and according to specifications and are capable of achieving the needed impact. Increasingly, project leaders (PLs) who possess this expertise are being employed in academic settings to support clinical and preclinical translational research team science. Duke University’s clinical and translational science enterprise has been an early adopter of project management to support clinical and preclinical programs. We review the history and evolution of project management and the PL role at Duke, examine case studies that illustrate their growing value to our academic research environment, and address challenges and solutions to employing project management in academia. Furthermore, we describe the critical role project leadership plays in accelerating and increasing the success of translational team science and team approaches frequently required for systems biology and “big data” scientific studies. Finally, we discuss perspectives from Duke project leadership professionals regarding the training needs and requirements for PLs working in academic clinical and translational science research settings.

Neonatal sepsis is a major cause of death and disability in newborns. Commonly used biomarkers for diagnosis and evaluation of treatment response lack sufficient sensitivity or specificity. Additionally, new targets to treat the dysregulated immune response are needed, as are methods to effectively screen drugs for these targets. Available research methods have hitherto not yielded the breakthroughs required to significantly improve disease outcomes, we therefore describe the potential of zebrafish (Danio rerio) larvae as preclinical model for neonatal sepsis. In biomedical research, zebrafish larvae combine the complexity of a whole organism with the convenience and high-throughput potential of in vitro methods. This paper illustrates that zebrafish exhibit an immune system that is remarkably similar to humans, both in terms of types of immune cells and signaling pathways. Moreover, the developmental state of the larval immune system is highly similar to human neonates. We provide examples of zebrafish larvae being used to study infections with pathogens commonly causing neonatal sepsis and discuss known limitations. We believe this species could expedite research into immune regulation during neonatal sepsis and may hold keys for the discovery of new biomarkers and novel treatment targets as well as for screening of targeted drug therapies.

OBJECTIVES/SPECIFIC AIMS: The aim of this study is to understand how porosity and collagen filling impact cell proliferation and differentiation in 3D printed scaffolds. METHODS/STUDY POPULATION: 3 groups of scaffolds will be 3D printed using FDM: solid scaffold, porous scaffold and porous scaffold with collagen gel (n=10 for each group) Internal geometries and surface structure will be analyzed using micro CT and Scanning Electron Mi RESULTS/ANTICIPATED RESULTS: We hypothesize that porosity and collagen filler will increase signal from Picogreen assay and ALP assay when normalized to scaffold surface area, indicating enhanced cell proliferation and differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: 3D printing PCU is a relatively new technique with very little published in the literature. Previous work has focused on the mechanical properties and not the biological response to the polymer. Understanding how to optimize cellular proliferation and differentiation can lead to the development of better implants that will integrate into the host’s structure and facilitate tissue regeneration.