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: 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.

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/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.

Objectives/Goals: The UCLA Clinical and Translational Science Institute (CTSI) training programs have been optimized by clinical research experts since 2013. They inculcate an interest in clinical and translational research careers. The acquisition of new skillsets and early exposure to potential career opportunities often influence lifetime decision-making. Methods/Study Population: The first program, in 2013, was the CTSI Research Associates Program (CTSI-RAP), which exposes undergraduate students to clinical research opportunities. RAP students are now mentoring high school students in the Mentoring and Advocacy in Teaching Clinical and Health-Related Research (MATCH) program. The Fiat Lux seminar is a research course designed to allow freshman students to explore diverse interests. The Leveraging Amazing Undergraduates in Clinical Research at UCLA Health (LAUNCH) program continues the workforce development pipeline by recruiting and training recent graduates to enter clinical research study coordinator careers. Each of these programs has their own stellar track records in terms of high interest and satisfaction and are assessed by annual evaluations from stakeholders. Results/Anticipated Results: CTSI-RAP is in its 11th year and a recent 10-year impact survey demonstrated the value of the program to students and their career decision-making especially those who are underserved and/or disadvantaged. The MATCH program arose from the interest of RAP students to mentor STEM high school students from local disadvantaged schools and is now in its 4th year across the state. The Fiat Lux freshman seminar began with a clinical research essentials emphasis, followed by an FDA/regulatory focus, and is now evolving to highlighting specific innovative areas of research with this year’s course spotlighting Cellular & Gene Therapy/Regenerative Medicine. LAUNCH is now in its third year, having been inspired by graduating RAP students wishing to continue in clinical research and feedback from their own focus groups. Discussion/Significance of Impact: The UCLA CTSI has supported these highly successful workforce development pipeline programs, which have had a demonstrated impact on students and the overall institutional clinical research infrastructure. Their stellar reputations generate high interest at UCLA and serve as model programs for implementation at other academic medical centers.

People with disabilities in the US are now a health disparities population. Though 25% of US adults have a disability, only 5% of medical research grants are disability related. Knowledge about researchers’ perceived barriers to including people with disabilities in research has focused on a single disability/condition and thus has limited translational science applications. Our CTSA’s Disability as Difference: Reducing Researcher Roadblocks (D2/R3) project examined such roadblocks towards inclusion of people with intellectual and developmental disabilities (I/DD). I/DDs are broad, heterogeneous conditions that originate in childhood, have varying impact and function, and persist throughout the lifespan. Strategies that mitigate their under-representation in research will likely have general applicability to all disabilities. In D2/R3’s first phase we conducted semi-structured interviews with translational science and I/DD program leaders at ten US institutions about perceived barriers and facilitators to including people with I/DD in research. Interviews were held with 25 individuals from partnering Intellectual and Developmental Disabilities Research Centers, University Centers for Excellence in Developmental Disabilities, and Clinical and Translational Science Award programs. Collaborative thematic coding identified key themes as: attitudinal barriers (e.g., assumptions about consent capacity), logistical barriers (e.g., accommodation costs), health disparities, and generalizability concerns. Findings informed development of a survey based on Prosci’s ADKAR® model of change management’s five components: Awareness, Desire, Knowledge, Ability and Reinforcement. Exclusion appears to stem from researchers’ lack of awareness, misconceptions, and knowledge gaps rather than insurmountable obstacles.

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.

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

OBJECTIVES/SPECIFIC AIMS: This study aims to test the hypothesis that bacterial biotransformation of chemotherapeutics promotes gut microbial diversity by enhancing persistence of drug-sensitive taxa. METHODS/STUDY POPULATION: The impacts of doxorubicin on a model community of gut bacteria was investigated in vitro in anaerobic batch culture. The synthetic community was composed of specific members predicted by genomic analysis to be sensitive to the therapeutic (i.e., Clostridium innocuum, Lactobacillus sp.), resistant via putative biotransformation (i.e., Escherichia coli, Klebsiella pneumoniae), or resistant via putative efflux (i.e., Enterococcus faecalis). Bacterial growth was monitored in monocultures by measuring OD600 and standard plate counts, and in mixed cultures by strain-targeted qPCR. Doxorubicin concentration was detected via absorbance assay. RESULTS/ANTICIPATED RESULTS: Strains with predicted resistance to doxorubicin by drug biotransformation significantly lowered concentrations of the drug in culture media. In contrast, E. faecalis proved resistant without evidence of drug transformation. Predicted sensitive strains were growth-repressed by the doxorubicin, but able to grow in spent media where biotransformation had occurred. However, they remained growth-repressed in spent media from E. faecalis where drug transformation had not been observed. Bacterial growth kinetics in mixed batch culture were dependent on starting bacterial concentrations and timing of drug exposure. DISCUSSION/SIGNIFICANCE OF IMPACT: This work will be extended to model microbial community responses to doxorubicin as a factor of microbial interactions and extent of drug transformation prior its exposure to sensitive strains. The resulting model will have translational implications for mitigating health risks during pediatric cancer treatment.

OBJECTIVES/SPECIFIC AIMS: Focal cartilage injuries of the knee joint are common and present a treatment challenge due to minimal intrinsic repair. Cartilage tissue engineering techniques currently used in clinical practice are expensive, cumbersome, and often ineffective in patients with mechanical or medical comorbidities. To address these issues, we developed an acellular nanofibrous scaffold with encapsulated growth factors designed to enhanced articular cartilage repair. Our goal is to evaluate this technology in vitro and pilot a large animal model for eventual translation into human subjects. METHODS/STUDY POPULATION: Hyaluronic acid (HA, 65 kDa) will be methacrylated (~40% modification, MeHA) and conjugated with cell-adhesive (RGD) groups. A solution of 4% wt/vol MeHA, 2% wt/vol polyethylene oxide (900 kDa), 0.05% wt/vol Irgacure 2959, and 0.005% wt/vol stromal cell-derived factor-1α (SDF-1α) and/or transforming growth factor-β3 (TGF-β3) will be prepared in ddH2O. The solution will be electrospun onto a rotating mandrel to achieve a dry scaffold thickness of 0.5 mm. The scaffold matt will be UV cross-linked and 5 mm-diameter samples will be cut out. Four groups of scaffolds will be prepared: MeHA, MeHA+SDF, MeHA+TGF, MeHA+SDF+TGF. All groups will be evaluated for fiber diameter, swell thickness, equilibrium compressive modulus, degradation rate, and growth factor release rate over 4 weeks (n=10). Scaffolds will also be seeded with juvenile porcine MSCs (5×104) in 200 μL of medium incubated for 24 hours. Seeded scaffolds will be evaluated for equilibrium compressive modulus, cell infiltration, and chondrogenesis at 4 and 8 weeks (n=10). Scaffolds will then be evaluated in a juvenile Yucatan minipig cartilage defect model. In total, 6 animals will undergo bilateral knee surgery to create four 4 mm-diameter full-thickness cartilage defects in each trochlear grove. All defects will receive microfracture to release marrow elements. Each knee will receive 2 scaffolds of the same group (replicates) with paired microfracture controls, resulting in a sample size of 3. Animals will be sacrificed at 12 weeks and defects will be evaluated via non-destructive indentation testing for mechanical properties, microCT for defect fill and subchondral bone morphology, and histology for ICRS II Visual Histological Assessment Scoring. RESULTS/ANTICIPATED RESULTS: Our preliminary studies have shown reliable replication of electrospun MeHA scaffolds. We anticipate cross-linking density to correlate positively with compressive modulus, and negatively with swell thickness, degradation rate, and growth factor release rate. We anticipate the addition of SDF-1α and TGF-β3 to increase cell infiltration and chondrogenesis, respectively, within seeded scaffolds. Similarly, we expect minipig defects treated with growth factor-releasing scaffolds to show greater mechanical properties, defect fill, and ICRS II score compared with MeHA scaffolds without growth factor. DISCUSSION/SIGNIFICANCE OF IMPACT: This study has the potential to show how an HA-based cell-free scaffold can be augmented with 2 growth factors that act synergistically to improve cartilage repair in a large animal model. This technology would improve upon the cell-free scaffolds already used clinically for autologous matrix-induced chondrogenesis and is directly translatable.

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

OBJECTIVES/GOALS: The primary objective of this study was to apply design sprint methodology to develop a proactive outreach tool prototype for patients with chronic obstructive pulmonary disease (COPD). METHODS/STUDY POPULATION: We utilized a 3-day process to align our team and key stakeholders behind answering the following question: “how might we empower COPD patients to understand their healthcare information, make decisions in partnership with their providers, and more easily manage their daily health?” On Day 1, we focused on understanding and defining the problem, and mapping the patient experience. On Day 2, we quickly brainstormed potential solutions, sketched our top ideas, and listed the solutions’ inherent assumptions. On Day 3, we created a prototype of our top solution and storyboarded each step of the prototype experience to review its potential usability and comprehensibility with patients. RESULTS/ANTICIPATED RESULTS: At the end of the design sprint, our team developed a prototype centered around personalized communication between COPD patients and providers. The prototype focuses on augmenting the current transitional care management (TCM) workflow in the post-discharge period. We are working to further develop the prototype prior to formal testing with care coordinators and patients. We anticipate that our prototype will assist in automating the current TCM workflow and facilitate contact with more patients post-discharge. DISCUSSION/SIGNIFICANCE OF IMPACT: Contact with patients is currently challenging due limited resources and the time sensitive nature of the TCM requirements. Automated patient outreach may be especially effective in engaging patients on a large scale, while also minimizing time and resources needed from healthcare staff.

OBJECTIVES/GOALS: While the current management of single ventricle repairs has drastically prolonged life expectancy, the repair fails over time primarily through pathologic inflammation and fibrosis. Our goal is to demonstrate that cardio-omentopexy can decrease inflammation and fibrosis in swine after cryoinjury. METHODS/STUDY POPULATION: A cryoinjury is created using a liquid nitrogen cooled probe to the right ventricle of 20kg swine. In half the groups the omentum is attached to the heart over the area of the injury. The swine are recovered and monitored for 4 or 8 weeks at which time they are euthanized. The injured area is evaluated via histological and immunohistochemical testing for markers of inflammation and scarring including collagen type, scar area, macrophage activity. RESULTS/ANTICIPATED RESULTS: We anticipate that the addition of omentopexy to cryoinjury will decrease scar area, fibrosis and markers of chronic inflammation. Additionally, we expect an increase in myocytes in the area of injury. We expect that this will occur through the anti-inflammatory and protective mechanism of the omentum. DISCUSSION/SIGNIFICANCE: Cardio-omentopexy, if able to decrease fibrosis and preserve myocytes, may provide a useful adjunct to the treatment of single ventricle repair by prolonging the longevity of the repair. Additionally, as these repairs often require a ventriculotomy, decreasing the operative scar may preserve myocardial function.

OBJECTIVES/GOALS: The limited treatment options for ischemic stroke patients have resulted in stroke being a leading cause of death and the primary cause of long-term disability in the U.S. Finding effective treatment options requires a better fundamental understanding of the ongoing processes that contribute to poor long-term outcome. METHODS/STUDY POPULATION: Expression of Apolipoprotein E4 predisposes stroke patients to poor long-term outcome. This study aims to test one possible mechanism by which ApoE4 contributes to cognitive decline after stroke. Here, we examine the effect of a major ApoE4 receptor, low-density lipoprotein receptor related protein 1 (LRP1) on sensitivity to stress in astrocytes. LRP1 binds and moves extracellular ligands and plasma membrane proteins into the endocytic system. Others have shown that LRP1 regulates cell-surface TNF receptor (TNFR1) in non-astrocytic cells. We propose That LRP1 similarly regulates TNFR1 in the central nervous system to attenuate inflammatory response after stroke. Studies have shown that ApoE4 slows the recycling of endocytic LDL receptors. We hypothesize that ApoE4 inhibits the ability of LRP1 to remove TNFR1 from the plasma membrane. This is expected to increase cytokine sensitivity, resulting in worse outcome after stroke. We investigated the effect of LRP1 on astrocyte TNFα signaling and response in immortalized ApoE null mouse astrocytes subjected to lentiviral-mediated knockdown of LRP1. The astrocyte response to TNFα stimulation was tested in a time dependent manner using Western blotting of NFkB pathway components, which are the downstream mediators of TNFα signaling. We also tested astrocyte viability after prolonged TNFα stimulation using Alamar Blue reagent. We found that LRP1 deficient cells have increased phosphorylation of NFkB upon TNFα stimulation, and that loss of LRP1 resulted in significant loss of astrocyte viability after prolonged stimulation. RESULTS/ANTICIPATED RESULTS: Altogether, our results indicate that loss of LRP1 renders astrocytes more sensitive to TNFα. Future experiments will focus on testing the influence of LRP1 on recovery after middle cerebral artery occlusion in mice. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will elucidate how astrocyte-LRP1 contributes to outcome after stroke, and helps us to understand one potential way that ApoE4 exerts pathological effects. A better understanding of the long-term processes after stroke will allow identification of therapies which improve the morbidity and mortality associated with stroke. CONFLICT OF INTEREST DESCRIPTION: NA.

Objectives/Goals: We evaluated the associations of urine uranium with incident chronic kidney disease (estimated glomerular filtration rate <60 mL/min/1.73m2), incident albuminuria (albumin-to-creatinine ratio >30 mg/g), and baseline and follow-up plasma metabolite levels in participants without diabetes across the Great Plains and Southwest. Methods/Study Population: We evaluated participants with available urine uranium estimates, eGFR, uACR, and other variables of interest. In incident case analyses, participants were free of CKD (N=1,845) and albuminuria (1,693) at baseline (2001-2003). We used generalized estimating equations, clustering on family identifier, to estimate risk ratios (RRs) of incident CKD and albuminuria per doubling of urine uranium adjusted for sex, age, eGFR or uACR, study center, educational level, smoking status, BMI, and arsenic exposure. Nontargeted metabolomic profiling was analyzed by gas chromatography-mass spectrometry. In metabolome-wide association analyses, we used adjusted linear mixed models to estimate the association of urine uranium (N=1,223) with relative changes in metabolite concentrations over time. Results/Anticipated Results: Overall, the median (IQR) age was 35.9 (23.7, 46.8) years and 60% participants were female. The number of incident CKD cases was 28 (mean follow-up 5.6 years), and the number of albuminuria cases was 140. The adjusted RR (95% CI) per doubling of urine uranium was 0.90 (0.71, 1.15) for incident CKD and 1.00 (0.92, 1.08) for incident albuminuria. In metabolome-wide analyses, urine uranium was associated with a relative change in 8 unique metabolites and 31 unknown metabolomic features (p-value <0.05), with 5 unique metabolites (threonic acid, N-acetylornithine, isocitric acid, cellobiose, and 6-deoxyglucose) and 7 unknown metabolomic features reaching false discovery rate significance (BH-adjusted p-value <0.05). Discussion/Significance of Impact: Urine uranium was not associated with incident CKD nor with albuminuria. We identified 5 unique metabolites associated with urine uranium. Our findings are relevant for uranium-related kidney disease in people without diabetes, but require additional confirmation due to potential reverse causality and lack of drinking water uranium exposure.

OBJECTIVES/GOALS: Study the regulatory role of sodium glucose co-transporter 1 (SGLT1) in cardiomyocytes and the therapeutic potential of sotagliflozin in hypertrophic cardiomyopathy (HCM) by (a) quantifying SGLT1 expression in HCM and (b) examining the impact of sotagliflozin on cardiac mechanics. METHODS/STUDY POPULATION: * Use Western Blot in cardiac tissue from HCM and non-HCM patients and pre-existing RNA seq and proteomics datasets to quantify SGLT1 levels in HCM. Hypothesis: SGLT1 is upregulated in HCM * Determine how SGLT1/2 inhibition by sotagliflozin will affect cardiac mechanics using living myocardial slice (LMS) preparations. A vibratome creates 200um-thick slices from (a) failing HCM heart explants, (b) septal myectomy samples from HCM patients, and (c) nonfailing rejected donor hearts. LMS are mounted on a force transducer and work-loops are stimulated under varying pre- and after-loads. Collecting baseline and post-drug work loops allows each slice to function as its own control. Hypothesis: sotagliflozin will improve diastolic mechanics by reducing stiffness in the end-diastolic pressure-volume relationship RESULTS/ANTICIPATED RESULTS: * Preliminary results from RNA seq data indicate that SLC5A1 mRNA (encoding gene for SGLT1) is significantly decreased in HCM. No proteomics study examined thus far has detected SLC5A1, indicating that overall SGLT1 levels in cardiac tissue are quite low. We will examine SGLT1 levels in our own HCM and non-HCM tissue samples with both mass-spectrometry and Western Blot. * We analyze six slices from each heart and expect 15 donor hearts and 15 HCM hearts/myectomy samples. We visualize the work loop by plotting stress/strain. Stress/strain at mitral valve closure represents exponential end diastolic pressure-volume relationship; Stress/strain at aortic valve closure represents linear end systolic pressure volume relationship. A two-sample paired t-test will compare change in stiffness and elastance. DISCUSSION/SIGNIFICANCE: This project contributes to a growing body of research surrounding the currently unknown cardioprotective mechanism of SGLT 1/2 inhibitors, furthers the technique of using living myocardial slices to study cardiac mechanics, and supports a trial examining sotagliflozin in HCM, for which disease modifying therapy remains a prevailing unmet need.