The Harvard Catalyst KL2/CMeRIT program is a 2-year mentored institutional career award that includes KL2 grants funded by National Institutes of Health (NIH) and CMeRIT grants funded by Harvard Catalyst nonfederal funds. The purpose of this study was to compare outcomes for early-stage investigators funded by the KL2/CMeRIT program to a group of applicants who were not chosen for support to assess the potential impact of the program on early career outcomes. Career data, including academic promotions, subsequent grant funding, and publication rates, from both successful and unsuccessful 2008–2018 KL2/CMeRIT applicants were compiled throughout the year 2020. Data were obtained directly through outreach to both groups and through assessment of online resources. The cohort comprised 487 individuals, 109 awardees, and 378 nonawardees. Awardees were more likely to be subsequently involved in clinical and translational research than nonawardees (92% vs 75%, p < 0.001). A higher proportion of awardees also had achieved academic promotion (81% vs 69%, p = 0.016) and subsequent NIH funding (72% vs 58%, p = 0.047), while there was no difference in publication rates (p = 0.555). Participants in the Harvard Catalyst KL2/CMeRIT program demonstrate greater early career success than nonparticipants though the nonparticipants also fared relatively well.

Cardiovascular disease (CVD) is largely preventable, and the leading cause of death for men and women. Though women have increased life expectancy compared to men, there are marked sex disparities in prevalence and risk of CVD-associated mortality and dementia. Yet, the basis for these and female-male differences is not completely understood. It is increasingly recognized that heart and brain health represent a lifetime of exposures to shared risk factors (including obesity, hyperlipidemia, diabetes, and hypertension) that compromise cerebrovascular health. We describe the process and resources for establishing a new research Center for Women’s Cardiovascular and Brain Health at the University of California, Davis as a model for: (1) use of the cy pres principle for funding science to improve health; (2) transdisciplinary collaboration to leapfrog progress in a convergence science approach that acknowledges and addresses social determinants of health; and (3) training the next generation of diverse researchers. This may serve as a blueprint for future Centers in academic health institutions, as the cy pres mechanism for funding research is a unique mechanism to leverage residual legal settlement funds to catalyze the pace of scientific discovery, maximize innovation, and promote health equity in addressing society’s most vexing health problems.

OBJECTIVES/GOALS: The objective of this research was to learn how the oncogenic transcription factor, ERG, is regulated in prostate cancer. If we could learn how ERG is regulated and which genes are important for its oncogenic phenotype in prostate cells, we could design new therapeutic strategies against ERG, which has proven to be difficult to target. METHODS/STUDY POPULATION: We conducted an shRNA screen in prostate cells to determine candidate genes and pathways that are important for ERG function. To validate the findings of the screen, we performed a variety of cell-based functional assays, including trans-well migration, wound healing, and clonogenic survival assays. To further investigate the mechanism between ERG and the genes revealed by the screen, we performed biochemical and molecular biology experiments such as Western blotting and qRT-PCR for protein and mRNA expression, co-immunoprecipitation assays to determine protein-protein interactions, and chromatin immunoprecipitation (ChIP-qPCR) to determine transcription factor binding to DNA sites. RESULTS/ANTICIPATED RESULTS: The screen revealed that genes involved in the toll-like receptor 4 (TLR4) pathway are important for ERG-mediated migration. We tested the effect of a TLR4 inhibitor on ERG function and observed decreased migration and clonogenic survival exclusively in ERG-positive cells. Expression of pMEK and pERG was reduced when TLR4 was inhibited, which suggests a mechanism in which TLR4 upregulates pMEK, leading to the phosphorylation and activation of ERG. This is supported by functional assays in which cells expressing a phosphomimetic ERG are resistant to the TLR4 inhibitor. We demonstrated that ERG drives the transcription of TLR4 and its endogenous ligands HSPA8 and BGN. Therefore, ERG can sensitize the cell to TLR4 activation by increasing the number of receptors as well as providing the ligands needed for stimulation. DISCUSSION/SIGNIFICANCE OF IMPACT: This research provides a new therapeutic pathway for treating ERG-positive patients through TLR4 inhibition. This can be beneficial because many patients become resistant to the standard therapy, leaving very few treatment options. TLR4-based therapies could provide an alternative for patients who have developed resistance.

OBJECTIVES/GOALS: My long-term career objective is to become an established independent researcher focused on understanding and modulating immune responses to biologics in order to enhance their efficacy and understand the underlying mechanisms by which these interact with the immune system. METHODS/STUDY POPULATION: In this study we will evaluate the utility of cyanuric chloride based synthetic lipids in the presentation of peptide epitopes of the gene delivery vector, adeno-associated virus (AAV). The lipopeptide conjugates will be administered to mice via liposomal formulations to assess their ability to induce immune responses by ELISA as compared to mice treated with the AAV. The three-dimensional conformation of the peptides will be evaluated using nuclear magnetic resonance to determine their similarity with the natural conformation that these peptides adapt on the viral surface. Additionally, to assess the translatability of this conjugation strategy, we will test the ability of our lipopeptides to bind to serum antibodies from human subjects. RESULTS/ANTICIPATED RESULTS: We anticipate that peptide presentation using cyanuric chloride lipids will achieve a robust response in mice following immunization. Immunizations with our lipids should induce the production of antibodies targeting AAV, albeit a milder response that the virus itself, given the complexity of viral epitopes. Nuclear magnetic resonance will inform us on how to improve the synthetic lipids to optimize peptide presentation by altering the characteristics of the lipid anchors. Finally, by using human serum to test for the ability of our lipopeptides to bind to antibodies in serum from patients positive for AAV antibodies, we can become informed on whether our strategy has utility in human studies or whether our method is limited to animal models of human disease. DISCUSSION/SIGNIFICANCE OF IMPACT: The current work seeks to develop a strategy to present peptides from a well characterized biologic, AAV, on a liposome surface. Our ultimate purpose is to employ liposomal formulations as decoys that target AAV-specific lymphocytes to improve the in vivo efficacy of AAV.

OBJECTIVES/GOALS: Our objective is to address the gaps in our epidemiologic understanding of Echinococcosis, a neglected and emergent zoonotic parasite in the US. Comparing regional differences in human cases with known Echinococcus genotypes in canine reservoirs will reveal landscape-level transmission risks. METHODS/STUDY POPULATION: Retrospective analysis of de-identified diagnosis codes for human echinococcosis in the US from 2002-2022 in a federal multi-healthcare network database (TriNetX). Analyses included Kaplan-Meier survival and specific parasite species cohort comparisons with descriptive summaries of demography and co-morbidities, and odds ratios for interventions (surgery, antiparasitics, cyst aspiration) and complications (anaphylaxis, sepsis, etc.) Mitochondrial genomes of Echinococcus spp. from next-generation sequencing ova-positive archival animal scat (National Park Service), along with sequences from published sources in the US, will be used to compare regional genotypic and reservoir host differences. RESULTS/ANTICIPATED RESULTS: Over 36,000 US patients were diagnosed with Echinococcosis in the past twenty years. Most cases had an unspecified parasite species, primarily affecting pediatrics (average age 16y +/- 10y) in the South/Southeast US, with very few interventions or medical complications reported. Patients with specific parasite species diagnoses (E. granulosus or E. multilocularis, N=500) had higher rates of mortality (11.7% at 10y), surgical (8.8%) and medical (18.2%) interventions, and complications (2%-17.2%), with demographic differences noted. Sequencing data and analysis pending for 47 Echinococcus positive wolf samples, with 78 additional sequences available for two types of Echinococcus spp. in wildlife, and lacking data for domestic dog-adapted strains in the US. DISCUSSION/SIGNIFICANCE: This study revealed a higher-than-expected burden of Echinococcosis in the US, confirming it is an under-recognized neglected and emergent disease. Future studies will address gaps regarding the origin of unspecified infections, with targeted surveillance of domestic dogs in high-risk areas.

OBJECTIVES/GOALS: Since 2013, managers of community engagement (CE) programs across the Clinical and Translational Science Award (CTSA) consortium funded by the National Center for Advancing Translational Sciences (NCATS) have convened monthly to build connections, share knowledge, and enable collaboration. METHODS/STUDY POPULATION: Notable for focus on staff leadership, the CE Brokers group has been central to the ongoing success of CTSA community engagement partnerships and approaches to research. In early 2022, a survey of the 139-member group asked about their roles and responsibilities, the ways the CE Brokers network has contributed to their hubs adoption and development of best practices and innovations, resources and lessons learned, and the creation of opportunities for members to collaboratively conduct and disseminate original research, and research on the science of community engagement. The survey also asked CE Brokers if they or their community partners are part of an underrepresented community. RESULTS/ANTICIPATED RESULTS: These demographic data will be shared, along with analyses of data on growth of the group over time, evolving themes, and a SWOT analysis completed in 2021. DISCUSSION/SIGNIFICANCE: This will provide a platform to explore new avenues for the CE Brokers and their impact within the NCATS CTSA consortium, in line with the evolving direction of the clinical and translational research enterprise.

OBJECTIVES/GOALS: The prevalence of patients with end-stage kidney disease (ESKD) in PR has increased in the last decade along with an aging population, while waitlisted patients have decreased. Our objective is to compare clinical and sociodemographic characteristics of patients with ESKD on dialysis who are waitlisted from those who are not waitlisted. METHODS/STUDY POPULATION: Retrospective study of all patients with ESKD who received dialysis in PR from 2013-2019, based on the United States Renal Data System data. Variables: (1)waitlisting status (yes or no; waitlisting date); (2)sociodemographics (age at dialysis initiation, sex, zip-code area, health insurance type, and employment status); and (3)clinical characteristics (dialysis initiation date, primary diagnosis of renal disease, comorbidities including obesity, diabetes, peripherovascular/cardiovascular disease, cancer, etc.). Statistics: (1)descriptive statistics (continuous - central tendency [mean, median] and dispersion [standard deviation, interquartile range]; and categorical [frequencies and percentages]); (2)Chi-square test, Students t-test, and Mann Whitney U test to evaluate differences. RESULTS/ANTICIPATED RESULTS: We expect patients on dialysis not waitlisted will be older and have more comorbidities than those who are on dialysis and waitlisted. DISCUSSION/SIGNIFICANCE: Kidney transplant is the choice of therapy for ESKD and its benefits are better quality of life, improved survival, and best long term-cost effectiveness. If our hypothesis is true, findings will highlight the importance of characterizing which patients with ESKD on dialysis are kidney transplant candidates when considering barriers to waitlisting.

ABSTRACT IMPACT: This work may provide new targets for vaccine and immunotherapeutic development against MRSA infections. OBJECTIVES/GOALS: Staphylococcus aureus is the leading cause of skin and skin structure infection (SSSI), a primary portal of entry for invasive infection. Patients with SA SSSI have a high 1-year recurrence. We have shown innate memory protects mice against SA SSSI. The goal of this project is to determine epigenetic mechanisms of protective memory against SA SSSI. METHODS/STUDY POPULATION: We have shown macrophages (Mf) afford protective memory against recurrent SA SSSI in mice. Priming by prior infection reduced skin lesion size and MRSA burden, which correlated with increased Mf in abscesses and lymph nodes. Priming potentiated the opsonophagocytic killing of SA by bone-marrow derived Mf (BMDM) in vitro, and their adoptive transfer into naive skin afforded protective efficacy in vivo. Here, we investigated epigenetic mechanisms of anti-SA efficacy in BMDMs. BMDM from naive (uninfected) or primed (SA SSSI) wild-type C57Bl/6 mice were cultured ex vivo. DNA from BMDM groups were isolated and analyzed for methylation changes using reduced representation bisulfite sequencing (RRBS). Pathway analyses of methylation changes were determined with Panther. RESULTS/ANTICIPATED RESULTS: Present findings indicate the protective memory afforded by BMDM was mediated by epigenetic modifications of the DNA. Using RRBS, we profiled differentially methylated regions (DMR) in DNA from naive vs. primed BMDM. Primed BMDM exhibited significantly different DMRs as compared to naive BMDM. Proximity to known genes were mapped using GREAT. Pathway analyses revealed DMRs predominant in genes integral to immune modulation, such as integrin signaling, cytokine/chemokine networks, and growth regulation. For example, SA-primed BMDM were hypermethylated proximate to GIMAP8 versus naive BMDM, suggesting repression of this protein. Gimap family ligands are small GTPase immune-associated proteins expressed in immune cells known to regulate macrophage lysosomal fusion during parasite infection. DISCUSSION/SIGNIFICANCE OF FINDINGS: These findings reveal epigenetic mechanisms of macrophage innate memory against recurrent MRSA infection. Functional testing of these genes in response to SA infection is needed to confirm their protective role. These insights may provide new targets for vaccine and immunotherapeutic development against MRSA.

Objectives/Goals: We describe the prevalence of individuals with household exposure to SARS-CoV-2, who subsequently report symptoms consistent with COVID-19, while having PCR results persistently negative for SARS-CoV-2 (S[+]/P[-]). We assess whether paired serology can assist in identifying the true infection status of such individuals. Methods/Study Population: In a multicenter household transmission study, index patients with SARS-CoV-2 were identified and enrolled together with their household contacts within 1 week of index’s illness onset. For 10 consecutive days, enrolled individuals provided daily symptom diaries and nasal specimens for polymerase chain reaction (PCR). Contacts were categorized into 4 groups based on presence of symptoms (S[+/-]) and PCR positivity (P[+/-]). Acute and convalescent blood specimens from these individuals (30 days apart) were subjected to quantitative serologic analysis for SARS-CoV-2 anti-nucleocapsid, spike, and receptor-binding domain antibodies. The antibody change in S[+]/P[-] individuals was assessed by thresholds derived from receiver operating characteristic (ROC) analysis of S[+]/P[+] (infected) versusS[-]/P[-] (uninfected). Results/Anticipated Results: Among 1,433 contacts, 67% had ≥1 SARS-CoV-2 PCR[+] result, while 33% remained PCR[-]. Among the latter, 55% (n = 263) reported symptoms for at least 1 day, most commonly congestion (63%), fatigue (63%), headache (62%), cough (59%), and sore throat (50%). A history of both previous infection and vaccination was present in 37% of S[+]/P[-] individuals, 38% of S[-]/P[-], and 21% of S[+]/P[+] (P<0.05). Vaccination alone was present in 37%, 41%, and 52%, respectively. ROC analyses of paired serologic testing of S[+]/P[+] (n = 354) vs. S[-]/P[-] (n = 103) individuals found anti-nucleocapsid data had the highest area under the curve (0.87). Based on the 30-day antibody change, 6.9% of S[+]/P[-] individuals demonstrated an increased convalescent antibody signal, although a similar seroresponse in 7.8% of the S[-]/P[-] group was observed. Discussion/Significance of Impact: Reporting respiratory symptoms was common among household contacts with persistent PCR[-] results. Paired serology analyses found similar seroresponses between S[+]/P[-] and S[-]/P[-] individuals. The symptomatic-but-PCR-negative phenomenon, while frequent, is unlikely attributable to true SARS-CoV-2 infections that go missed by PCR.

ABSTRACT IMPACT: Our proposed jaw-specific control mechanism of tooth development is expected to address the site-specific prevalence of tooth agenesis in humans. OBJECTIVES/GOALS: To determine the molecular mechanisms that control jaw-specific tooth development. To identify the molecular basis of the site-specific prevalence of humans tooth agenesis cases. METHODS/STUDY POPULATION: We used three different genetically engineered mouse lines: Msx1 ^’/ ^’, Dkk2 ^’/ ^’, and Sostdc1 ^’/ ^’ mice. We used developmental mouse genetics approaches, basically generating different combinations of compound mutant mice. We examined their tooth development by using gross, histology, and mRNA expression analyses. RESULTS/ANTICIPATED RESULTS: We identified that Sostdc1, a secreted Wnt inhibitor, also plays an important role in regulating the Msx1-dependent odontogenic pathway. Sostdc1 mRNA showed similar expression patterns in the developing tooth germs between control and Msx1-null molar buds. Remarkably, by deleting the Sostdc1 gene, as well as the Dkk2 gene, in the Msx1-null background mouse, molar tooth development was rescued in the maxillary jaw, but not in the mandibular jaw. Furthermore, tooth developmental rescue could be achieved in both the maxillary and mandibular molars by combinedly deleting Dkk2 and Sostdc1 in Msx1-null mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our study demonstrates that secreted Wnt inhibitors Dkk2 and Sostdc1 synergistically regulate the Msx1-dependent odontogenic pathway and further control early tooth morphogenesis. These mouse model will be used to further address the site-specific prevalence of tooth agenesis in humans.

OBJECTIVES/SPECIFIC AIMS: Allostatic load, the chronic stress-induced wear and tear on the body, has a cumulative deleterious effect in individuals over their lifetime. Recent studies have suggested that socio-economic status, psychological determinants, and biomedical health cumulatively contribute to allostatic load in young adults. Although these finding individually suggest that African American children may be particularly susceptible to the effects of allostatic loading due to racially-based discrimination and economic instability, few studies have shown the effect of exposure to violence on the allostatic load carried by young African Americans. METHODS/STUDY POPULATION: The Biological and Social Correlates of Drug Use in African American Emerging Adults (BADU) data set is composed of young African Americans (n=557 individuals) living in the Washington, DC area, collected from 2010 to 2012. Study participants were sought equally between males and females (n=283, n=274, respectively). This data set provides a rich source of information on the behavioral, mental, and physical health of African American young adults (18–25 year olds) living in the Washington, DC area. Analysis of 6 biomedical markers were measured in BADU study participants: C-reactive protein, cortisol, Epstein-Barr virus IgG, IgE, IgA, and IgM, known to be markers of immune stress and allostatic load. Naive Bayes was used to identify participant responses that were correlated to elevated stress biomarker levels. RESULTS/ANTICIPATED RESULTS: Violence was most closely correlated to elevated EBVVCA IgM and IgE levels. Elevated IgE levels correlated to increased experience of familial violence and sexual abuse; familial drug abuse and depression; violence and community violence. Cortisol is positively correlated to reported emotional state (R=0.072) and perceived individual discrimination (R=0.059). DISCUSSION/SIGNIFICANCE OF IMPACT: Allostatic load appears to be high in individuals who self-report exposure to violence. Both perceived mental health and violence were correlated to elevated stress biomarkers. When Epstein-Barr virus viral capsid antigen IgM was compared with violence features characterized in the data set, we found that internalization of environmental stressors were most strongly correlated to elevated allostatic load markers. This work suggests that internalization of experienced violence may be as important as the actual violence experience.

OBJECTIVES/GOALS: COVID-19 has taken a big toll on health, mental health and community well-being. COVID-19 has also presented unique opportunities for community engagement programs to sustain their work within communities. Low-tech methods of engagement might provide opportunities for success. METHODS/STUDY POPULATION: When we could not be in the field due to COVID-19, we recontacted our 12.4k HealthStreet members by phone and in 12 months successfully contacted 3.2k. By contrast, over the two COVID affected years (2019 & 2020), our in-person recruitment efforts in the community fell to 832 from 1817 in the two prior years (2017 & 2018). Reconnecting with existing members is an important aspect of sustainable community engagement. RESULTS/ANTICIPATED RESULTS: While 8/10 of the 3.2 K sample had internet access in their homes, 9/10 said having it was somewhat to very important. Importantly, 1 in 3 people who had additional needs or suggestions were looking for better COVID-19 knowledge or ways to reduce the threat of illness, 1 in 4 wanted resources; and 1 in 5 encouraged us to keep doing what we were doing. Among recontacted members, 30.2% indicated a high stress level (8 & over on a 10 point scale) while 15.2% reported a high level of loneliness (8 & over on a 10 point scale). High stress was significantly more likely among those identifying as African American (23.1%) compared to non-African American (16.6%). Loneliness did not vary (10.1% among both). DISCUSSION/SIGNIFICANCE: Sustainable community engagement was important to reach community members during the pandemic, especially given the high rates of stress and loneliness among members contacted. In-person connections were reduced, but phone contact provided an opportunity for successful engagement.

OBJECTIVES/GOALS: As of 2021, the lung transplantation waiting list has a mortality rate of 7.6 deaths per 100 patient-years. Bioengineered human organs is an emerging field of tissue engineering with a goal of developing suitable organs for transplantation. The focus of the project is to evaluate the efficacy of bioengineered lungs using a human-to-swine model. METHODS/STUDY POPULATION: This project will involve designing and assessing the bioengineered lung by establishing a human-to-pig xenotransplantation survival model. The project aims to evaluate how well the bioengineered lung functions within a living model. The bioengineered lung is constructed using swine connective tissue scaffolding, which has been recellularized with human cells. Anatomically, the lung will resemble a swine lung but will possess the immunological and cellular markers of human tissue. The proposed model will initially assess the immunological response of swine to human lung tissue. Lung function will be assessed during surgery using pulmonary vein gas samples and tissue sampling. Following the end of the study, additional tissues samples will be taken to evaluate the immunological response to the tissue. RESULTS/ANTICIPATED RESULTS: Xenotransplantation and bioengineered organs are two new emerging fields of research that have just begun to enter the large animal testing phase. This model will provide a novel human-to-pig xenotransplant survival model that will be used to test the efficacy of bioengineered lungs function in a dynamic living organism. The design has taken the principles of immunology learned from the current clinical and xenotransplant research and has incorporated this knowledge into the known pig-to-pig transplant models. We anticipate that this model design will be easily reproducible and can be expanded to other bioengineered organs as an effective means to test functionality. DISCUSSION/SIGNIFICANCE: The COVID-19 pandemic’s aftermath may lead to an increased demand for lung transplants. Bioengineered lungs could provide an additional source of organs to supplement current availability. This novel approach has the potential to offer a means to test several different types of bioengineered organs in the future.

OBJECTIVES/SPECIFIC AIMS: Neurological injury remains as the main limiting factor for overall recovery after cardiac arrest (CA). Currently available indicators of neurological injury are inadequate for early prognostication after return of spontaneous circulation (ROSC). High diversification of brain mitochondrial cardiolipins (CL) makes them unique candidates to quantify brain injury and to predict prognosis early after ROSC. METHODS/STUDY POPULATION: CL content in plasma in 39 patients within 6 hours of ROSC and 10 healthy subjects as well as CL content in human heart and brain specimens were quantified using a high-resolution liquid chromatography mass spectrometry method. The quantities of brain-type CL species were correlated with clinical parameters of brain injury severity permitting derivation of a cerebral CL score (C-score) using linear regression. C-score and a single CL species (70:5) were evaluated in patients with varying neurological injury and outcome. Using a rat model of CA, CL was quantified in the plasma and brain of rats using similar methods and results compared with the controls. RESULTS/ANTICIPATED RESULTS: We found that brain and the heart fell on extreme ends of the CL diversity spectrum with 26 species of CL exclusively present in human brain not heart. Nine of these 26 species were present in plasma within 6 hours of ROSC with quantities correlating with greater brain injury. The C-score correlated with early neurologic injury and predicted discharge neurologic/functional outcome. CL (70:5) emerged as a potential point-of-care marker that alone was predictive of injury severity and outcome nearly as well as C-score. Using a rat CA model we showed a significant reduction in hippocampal CL content corresponding to CL released from the brain into systemic circulation. C-score was significantly increased in 10 minute Versus 5 minute no-flow CA and naïve controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CA results in appearance and accumulation of CL in plasma, proportional to injury severity. Quantitation of brain-type CL species in plasma can be used to prognosticate neurological injury within 6 hours after ROSC.

OBJECTIVES/GOALS: SalmonellaTyphi primarily persists in human chronic carriers by forming biofilms on gallstones in the gallbladder (GB). We developed a mouse model of GB chronic carriage, and using this model, aim to detect Salmonella in living mice and track the progression of GB carriage with bioluminescent S.Typhimurium and in vivoimaging. METHODS/STUDY POPULATION: S.Typhimurium 14028 (WT) was transduced with the lux operon from the S. Typhimurium Xen33 strain from Perkin Elmer©, creating 14028lux. 129X1/SvJ mice were fed a lithogenic diet for 6 weeks to induce gallstone formation. After cessation of diet, these mice were infected with 5x103-1x104 colony forming units (CFU) of either the 14028lux isolate, WT (non-luminescent) isolate, or an equal volume of sterile saline. Mice were serially imaged (IVIS SpectrumCT) every 2-3 days for up to 63 days. Images were quantified by measuring average radiance over selected regions of interest. The presence of bioluminescent bacteria in specific organs was confirmed by imaging the abdominal cavity post-mortem. Organs were homogenized and CFUs per mg of tissue were quantified and compared between each group. RESULTS/ANTICIPATED RESULTS: Compared to the controls, mice infected with 14028lux showed luminescence in the abdomen as early as three days post-infection. Within 15 days, the resolution was sufficient to discriminate signal in specific organs, notably the gallbladder, liver, spleen, and cecum. The presence of bacteria was confirmed in these organs via direct imaging and by quantifying CFUs in the tissues. At 63 days post-infection, we identified >103 CFUs and significant luminescence in the GB of a portion of 14028lux-infected mice. For all days post-infection, 14028lux-infected mice that lacked observable luminescence had <100 CFUs/mg tissue. DISCUSSION/SIGNIFICANCE: We have developed a technique using bioluminescent S.Typhimurium and in vivo imaging that, without sacrificing infected mice, enables us to reliably distinguish between mice that have maintained gallbladder chronic carriage >60 days and those that have cleared infection.

OBJECTIVES/GOALS: Our goal was to assess the ability of a 3D-Printed dual cover-core design alveolar ridge bone graft, to withstand the average maximum masticatory force of a healthy person. To this end, we characterized the materials, ran a finite element analysis (FEA) model, and validated it using a resin 3D-printed version tested under compression with strain gauges. METHODS/STUDY POPULATION: A tricalcium-phosphate/hydroxyapatite paste and mixed methacrylated alginate-gelatin were used for the core, and polycaprolactone for the cover. These were characterized using ASTM standards D695 and D638 for compression, tensile, and rheological testing. Then we converted cone CT-scan images of a mandibular alveolar ridge defect to an .stl file, and designed the cover and core in Meshmixer. The model was then imported into ANSYS 11.0, and a downward compression force of 500 N, the maximum masticatory force of a healthy adult, was applied on the graft and mandible’s top ridge. The different models included solid and porous covers and cores, as well as comparing screws on one or both sides of the cover, then validated by compressing a resin 3D-printed versions. RESULTS/ANTICIPATED RESULTS: The FEA model provided maximum displacements, Von Mises stress (VMS), and stress/strain values for each model. The highest maximum displacement was found on the solid covers with a combination of both buccal and lingual screws, at 0.162 mm. The lowest maximum displacement was found in the porous cover at 0.085 mm. All VMS values were below the tensile yield strength, meaning that the materials would not yield. The highest maximum stress was found on the porous cover at 13.52 MPa, the lowest was 1.06 MPa on the cover with no screws. The highest strain was found on the porous model at 0.010, which was 5.6x higher than the solid cover. The porous cover also showed less stress shielding, thus allowing a beneficial mechanical stimulation of the bone, and the lowest maximum displacement, possibly due to flexion through the pores. DISCUSSION/SIGNIFICANCE: Preliminary FEA models demonstrated that for the considered materials, a cover-core design of the mandibular implant would sustain the desired 500 N of force without yielding. The porous cover provides the most benefits, causing the least stress shielding and allowing diffusion of biological factors to support the osteoinductive role of the core.

OBJECTIVES/SPECIFIC AIMS: The objective of this study is to determine the degree to which the use of a contralesionally-controlled brain-computer interface for stroke rehabilitation drives change in interhemispheric motor cortical activity. METHODS/STUDY POPULATION: Ten chronic stroke patients were trained in the use of a brain-computer interface device for stroke recovery. Patients perform motor imagery to control the opening and closing of a motorized hand orthosis. This device was sent home with patients for 12 weeks, and patients were asked to use the device 1 hour per day, 5 days per week. The Action Research Arm Test (ARAT) was performed at 2-week intervals to assess motor function improvement. Before the active motor imagery task, patients were asked to quietly rest for 90 seconds before the task to calibrate recording equipment. EEG signals were acquired from 2 electrodes—one each centered over left and right primary motor cortex. Signals were preprocessed with a 60 Hz notch filter for environmental noise and referenced to the common average. Power envelopes for 1 Hz frequency bands (1–30 Hz) were calculated through Gabor wavelet convolution. Correlations between electrodes were then calculated for each frequency envelope on the first and last 5 runs, thus generating one correlation value per subject, per run. The chosen runs approximately correspond to the first and last week of device usage. These correlations were Fisher Z-transformed for comparison. The first and last 5 run correlations were averaged separately to estimate baseline and final correlation values. A difference was then calculated between these averages to determine correlation change for each frequency. The relationship between beta-band correlation changes (13–30 Hz) and the change in ARAT score was determined by calculating a Pearson correlation. RESULTS/ANTICIPATED RESULTS: Beta-band inter-electrode correlations tended to decrease more in patients achieving greater motor recovery (Pearson’s r=−0.68, p=0.031). A similar but less dramatic effect was observed with alpha-band (8–12 Hz) correlation changes (Pearson’s r=−0.42, p=0.22). DISCUSSION/SIGNIFICANCE OF IMPACT: The negative correlation between inter-electrode power envelope correlations in the beta frequency band and motor recovery indicates that activity in the motor cortex on each hemisphere may become more independent during recovery. The role of the unaffected hemisphere in stroke recovery is currently under debate; there is conflicting evidence regarding whether it supports or inhibits the lesioned hemisphere. These findings may support the notion of interhemispheric inhibition, as we observe less in common between activity in the 2 hemispheres in patients successfully achieving recovery. Future neuroimaging studies with greater spatial resolution than available with EEG will shed further light on changes in interhemispheric communication that occur during stroke rehabilitation.