OBJECTIVES/GOALS: Super refractory status epilepticus (SRSE) is associated with high mortality, often due to withdrawal of life sustaining therapy (WLST) based on perceived poor neurological prognosis. Factors influencing decision making are underreported and poorly understood. We surveyed clinicians who treat SRSE to identify factors that influence WLST. METHODS/STUDY POPULATION: Health care providers (HCP), including physicians, pharmacists, and advanced practice providers, who treat SRSE answered a 51-question survey on respondent demographics, institutional characteristics and SRSE management that was distributed though professional societies. Respondents described approaches to prognostication and rated the importance of clinical factors in the management of two hypothetical clinical cases followed by their prediction of recovery potential for the same two cases. Neurointensivists and other HCP responses were compared using descriptive statistics to differentiate group characteristics; a p-value <0.05 was considered significant. Logistical regression models were employed to identify associations between clinician specific factors and prognostication. RESULTS/ANTICIPATED RESULTS: One-hundred and sixty-four respondents were included in the analysis. Compared to other HCPs (neurologists, epileptologists, neurosurgeons, other intensivists; n=122, 74%), neurointensivists (n=42, 26%) [Odds ratio (OR) 0.3, 95% confidence interval (CI) 0.14-0.68), p=.004)] were less likely to use prognostic severity scores and were less likely to prognosticate likelihood of good functional recovery (OR: 0.28 (95% CI: 0.13-0.62), p=.002) compared to non-neurointensivist HCPs, controlling for potential confounders including professional degree, years of experience, country of practice, and annual volume of SRSE cases. There was, however, significant overlap in factors deemed necessary for determining futility in care escalation. DISCUSSION/SIGNIFICANCE: Neurointensivists value similar clinical factors to other HCPs when evaluating medical futility in SRSE but are less likely to predict definitive outcomes. Pending final survey results, future studies aimed at understanding why neurointensivists may be less likely to decisively prognosticate (i.e. avoiding nihilism) in SRSE may be warranted.

OBJECTIVES/SPECIFIC AIMS: Our objective is to understand the influence of the features comprising metabolic syndrome (central obesity, raised fasting plasma glucose, triglycerides, blood pressure, and decreased HDL cholesterol) on brain structure in men and women. With the understanding that MetS is a strong predictor of gray matter volume loss in specific brain regions, in this study we sought to quantify the influence of each of the metabolic syndrome biometric variables on the structures involved in the neural signature of metabolic syndrome. METHODS/STUDY POPULATION: We conducted multiple linear regression analyses on a cross-sectional sample of 800 individuals from the Genetics of Brian Structure (GOBS) image archive (352 men and 448 women). GOBS is an offshoot of the San Antonio Heart Study involving an extended pedigree of Mexican Americans from the greater San Antonio area. Its goal is to localize, identify, and characterize genes/quantitative trait loci associated with variations in brain structure and function (Winkler, 2010). The archive has continuously added participants from approximately 40 families since 2006. Neuroanatomic (T1-weighted MRI scans obtained on a Siemens 3T scanner and processed using FSL), neurocognitive, and biometric phenotypes have been obtained for each subject (including blood lipids). Linear regressions were run using SPSS and incorporated biometric and gray matter volume values obtained from 800 GOBS participants. RESULTS/ANTICIPATED RESULTS: Linear regressions incorporating metabolic syndrome variables as dependent variables and gray matter volume from regions involved in the neural signature of metabolic syndrome as predictors show significant predictive patterns that are largely similar between men and women, with some differences. Another linear regression conducted with gray matter volume from the neural signature of metabolic syndrome as the dependent variable and metabolic syndrome variables as predictors show that waist circumference and triglycerides are the greatest predictors of gray matter volume loss in men, and fasting plasma glucose and waist circumference are the greatest predictors of gray matter volume loss in women. DISCUSSION/SIGNIFICANCE OF IMPACT: Significant sex differences in the relationships between metabolic syndrome variables and gray matter volume changes between brain regions comprising the neural signature of metabolic syndrome were identified. waist circumference, fasting plasma glucose, and triglycerides are the most reliable predictors of gray matter volume loss. The variance in gray matter volume of the neural signature of metabolic syndrome in men is more significantly explained by waist circumference and triglycerides (when accounting for age) and in women is more significantly explained by waist circumference and fasting plasma glucose (when accounting for age). A model of metabolic syndrome that emphasizes a risk of neurodegeneration should focus on waist circumference for both men and women and weigh the remaining variables accordingly by sex (triglycerides in men and fasting plasma glucose in women).

Objectives/Goals: Our research goal is to translate medical guidelines for adolescent-centered contraceptive counseling into improved clinical practice. Here, we describe the process of co-designing a training program for adolescent-serving primary care clinicians with teen, caregiver, and clinician advisory boards. Methods/Study Population: We recruited teens, caregivers, and clinicians residing in North Carolina to participate in three virtual advisory boards separated by role. Eligible teen advisors were assigned female at birth and 15–19 years old; eligible clinicians provided care for teen patients; and all groups were purposively sampled to reflect diverse identities and experiences. At each advisor meeting, we used human-centered design techniques to elicit participant priorities, generate training content and engagement strategies, and obtain feedback on the final training program. We conducted a focus group at our final meetings and used rapid qualitative analysis to understand our advisors’ experiences participating in program co-development. Results/Anticipated Results: We partnered with 20 advisors with diverse identities across geographic location, race and ethnicity, sexuality, and experiences with disability. During 15 meetings from January to May 2024 (five with each advisor group), we developed a 3-hour virtual, synchronous training for adolescent-serving primary care providers to improve their contraceptive counseling skills. The curriculum includes five interactive modules and a resource toolkit. Advisors described motivations to participate (e.g., chance to share their perspective, desire to make change), positive experiences with the advisory boards (e.g., opportunities to learn, to connect with others), and opportunities for improvement (e.g., better technology orientation). Discussion/Significance of Impact: We describe developing a successful longitudinal partnership with three community advisory boards and co-creating a training program that incorporates community-led priorities and perspectives, including youth. This approach can be adapted for other clinician training programs seeking to center community voices.

ABSTRACT IMPACT: Human exhaled breath is rich in metabolomic content that represents pulmonary function and gas exchange with blood, which can provide insights into an individual’s state of health. OBJECTIVES/GOALS: Human exhaled breath is rich in metabolomic content that represents pulmonary function and gas exchange with blood. It contains a mixture of compounds that offer insight into an individual’s state of health. Here, we present two novel non-invasive breath sampling devices for use in basic medical practice. METHODS/STUDY POPULATION: The two breath samplers have a disposable mouthpiece, a set of inhale and exhale one-way flap valves to allow condensation of exhaled breath only, and a saliva filter. The housing is constructed out of Teflon®, a chemically inert material to reduce chemical absorbance. The first device condenses exhaled breath into a frozen condensate using dry ice pellets and the other is a miniaturized design that liquifies exhaled breath on a condenser surface with micropatterned features on a cooling plate. Both designs have individual strategic and analytical advantages: frozen exhaled breath condensate (EBC) has high retention of analytes and sample volume; EBC collected in liquid phase offers facilitated sample collection and device portability. RESULTS/ANTICIPATED RESULTS: We investigated if breath aerosol size distribution affects the types or abundances of metabolites. We modified the geometry of the first device to redirect aerosol trajectories based on size. The trapping of larger aerosols increases with filter length, thus altering the aerosol size distribution although no significant changes in the metabolite profiles were found. With the miniaturized device, metabolite abundances were measured in a small cohort of healthy control and mild asthmatic subjects. Differences among subjects were found, as well as main differences between control and asthmatic groups. All analyses of EBC were performed with liquid chromatography - mass spectrometry. Inflammatory suppression found in asthmatic subjects can be explained by prescribed daily use of inhaled corticosteroids. DISCUSSION/SIGNIFICANCE OF FINDINGS: Breath collection devices can be used in intensive care units, outpatient clinics, workplaces, and at home. EBC analysis has been used to monitor asthma and chronic obstructive pulmonary disease. It can be applied to infectious respiratory diseases (e.g. influenza, COVID-19) and for monitoring environmental and occupational chemical exposures.

OBJECTIVES/GOALS: Difficult-to-heal wounds of the skin are a common and costly medical problem. Dermal replacement strategies have emerged as a solution, but a challenge is identification of optimal scaffold parameters. We present a model for assessment of clinical potential of collagen scaffolds for wound healing. METHODS/STUDY POPULATION: In previous animal experiments, we evaluated dermal replacement scaffolds custom-fabricated from fibril-forming collagen oligomer with controlled fibril density (4, 20, 40mg/cm3) and spatial gradients in rat excisional wounds. Wound contraction and cellularization were monitored by gross and histological image analysis for comparison with model outcomes. We now parameterize the scaffold parameters for use in the mathematical model of wound healing with nonlinear curve fitting. A preliminary chemo-bio-mechanical finite element model including collagen, cells, and an inflammatory signal was adapted to simulate wound healing results. RESULTS/ANTICIPATED RESULTS: Collagen oligomer microstructure was quantified from scanning electron micrographs. A constitutive law for collagen mechanics was fit to experimental uniaxial tensile tests. We have conducted preliminary three-dimensional finite element model simulations to be validated against experimental wound contraction, recellularization, and collagen remodeling data collected from each experimental group. We show the effects of collagen density and stiffness on wound contraction by altering early wound mechanical properties. We anticipate future work to further improve the model of mechanotransduction, inflammation, and recellularization. DISCUSSION/SIGNIFICANCE OF IMPACT: This work represents the first step towards a computational model of wounds treated with collagen scaffold dermal replacements. In turn, the model will be used to explore cell-scaffold interactions for purposes of prediction and optimization of tissue regeneration outcomes.

OBJECTIVES/GOALS: Xylazine is a strong sedative and fentanyl contaminant which has been increasingly detected in drug overdose deaths in Maryland. The goal of this project is to analyze the demographic characteristics and time trends of xylazine-related overdose deaths (XROD) in Maryland from 2020-2022. METHODS/STUDY POPULATION: This cross-sectional study utilizes the Maryland medical examiner's autopsy reports from 2020-2022. These reports include every death in the state that was investigated by the medical examiner, with demographic and toxicological data showing the presence of various substances at the time of death. An XROD was defined as someone who died from drug overdose and had a positive serum xylazine test at time of death. Demographic characteristics and time trends for XROD were analyzed. Multivariable logistic regression modeled associations between demographic variables and the presence of other substances with XROD. RESULTS/ANTICIPATED RESULTS: A total of 1,509 people died from XROD, of which the mean age was 44.4 years and 73.3% were male. The majority were White (57.6%), 39.2% were Black, and 3.2% identified as another race. Over 99.9% of individuals who died from XROD tested positive for fentanyl. XROD peaked in January 2021 and has been trending downwards since then. Adjusted multivariable logistic regression revealed that White individuals had greater odds of XROD relative to Black individuals (OR=1.22, 95% CI=1.07-1.37), and adults aged 30-45 years had higher odds of XROD relative to adults over age 60 (OR=1.26, 95%CI=1.04-1.54). Individuals who used fentanyl had higher odds of XROD relative to those who did not use fentanyl (OR=327.4, 95%CI=46.0-2331.3). DISCUSSION/SIGNIFICANCE: This study demonstrates that middle age, White race, and fentanyl use are associated with xylazine-related overdose deaths in Maryland. Efforts to reduce xylazine-related mortality in the state should address the unique social and geographic factors that influence substance use in this population.

OBJECTIVES/GOALS: Environmental factors may significantly increase the risk of or buffer against Alzheimer’s disease and related dementias, yet strategies to address cognitive decline and impairment to date largely overlook the role of neighborhoods. This mixed-methods study is the first to examine potential links between access to eateries and cognitive function. The goal is to inform place-specific interventions to help aging individuals reduce risk for cognitive impairment through neighborhood community and design. METHODS/STUDY POPULATION: Following an exploratory sequential mixed-methods design, seated and mobile interviews with 125 adults aged 55-92 (mean age 71) living in the Minneapolis (Minnesota) metropolitan area suggest that eateries, including coffee shops and fast-food restaurants, represent popular neighborhood destinations for older adults and sources of wellbeing. To test the hypothesis that these sites, and the benefits they confer, are associated with cognitive welfare, we analyzed data from urban and suburban dwelling participants in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study, a national racially diverse sample of older Americans followed since 2003 (n = 16,404, average age at assessment 72 years). RESULTS/ANTICIPATED RESULTS: Qualitative thematic analysis of how older adults perceived and utilized local eateries include sites of familiarity and comfort; physical and economic accessibility; sociability with friends, family, staff, and customers; and entertainment (e.g., destinations for outings and walks, free newspapers to read). Quantitative results from multilevel linear regression models demonstrate a positive association between density of eateries and cognitive functioning. Taken together, these results complicate our understanding of fast-food settings as possible sites of wellbeing through social interaction and leisure activities. DISCUSSION/SIGNIFICANCE OF IMPACT: The results contribute new evidence towards an emerging ecological model of cognitive health. Understanding whether and how retail food environments can help buffer against cognitive decline among older adults provides novel opportunities to promote wellbeing in later life through community interventions and neighborhood design.

OBJECTIVES/SPECIFIC AIMS: Negative symptoms of schizophrenia, including motivational deficits, social withdrawal, poverty of speech, decreased emotional reactivity, and psychomotor retardation, have been shown to be most predictive of functional impairment and poor outcome in patients with schizophrenia. Furthermore, these symptoms tend not to be responsive to antipsychotic medications. Inflammation could be one mechanism underlying these difficult to treat symptoms. METHODS/STUDY POPULATION: Three cohorts of patients, reflecting different phases of disease, were studied. One cohort was comprised of a sample of patients with deficit schizophrenia (characterized by primary and enduring negative symptoms; n=17), nondeficit patients (n=39), and healthy controls (n=28). ANOVA and multivariate general linear models were used to compare groups, and linear regression models were used to examine relationships between inflammatory cytokines and negative symptoms. The second cohort was comprised of 80 individuals at clinical high risk for psychosis from the North American Prodromal Longitudinal Study. Linear regression models examined the relationship between baseline inflammatory markers and subsequent negative symptoms at follow-up visits up to 2 years. The third cohort consisted of patients with treatment-resistant schizophrenia (TRS) on clozapine (n=10). Correlations were performed to examine relationships between inflammatory markers and negative symptoms. In a subgroup of patients from this third sample, resting state functional connectivity analyses were performed on fMRI data to explore relationships between inflammatory markers and connectivity in brain reward circuitry. RESULTS/ANTICIPATED RESULTS: In a sample of patients with the deficit syndrome of schizophrenia (n=17), a subtype of the disorder characterized by primary and enduring negative Symptoms, tumor necrosis factor (TNF) was significantly increased relative to nondeficit patients (n=39) and healthy controls (n=28; F2,57=3.51, p=0.036), and predicted total negative symptoms (β=0.31, p=0.012), alogia (β=0.30, p=0.024), and blunted affect (β=0.31, p=0.018) items of the Positive and Negative Symptom Scale in linear regression models while controlling for antipsychotics. In another sample of individuals at clinical-high risk for psychosis (n=80), baseline concentrations of TNF significantly predicted negative symptoms, including anhedonia, apathy, and loss of interest in linear regression models, at the 6-month (β=0.25, p=0.011) and 12-month follow-up (β=0.39, p=0.001). Interleukin (IL)-1 receptor antagonist also predicted these symptoms at the 6-month follow-up (β=0.21, p=0.037). In a third sample (n=10) of patients with TRS treated with clozapine, IL-1β was correlated with passive/apathetic social withdrawal (r=0.657, p=0.039) and disturbance of volition (r=0.686, p=0.029) items of the Positive and Negative Symptom Scale and the global avolition-apathy scores of the Scale for the Assessment of Negative Symptoms (r=0.751, p=0.012). Finally, in a small subsample (n=5) of patients from this TRS cohort for whom we collected fMRI data, we found resting-state functional connectivity from a right nucleus accumbens seed to a cluster in medial prefrontal cortex. We found relationships between higher inflammation and decreased connectivity for TNF (r=−0.64) and CRP (r=−0.89). DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, these preliminary data show the predicted relationship between inflammatory markers and negative symptoms and demonstrate the reproducibility of TNF and other monocytic-derived cytokines as reliably elevated in schizophrenia and associated with negative symptoms across samples of patients with schizophrenia and individuals at high risk for psychosis. Cytokines may exert their effects via their impact on brain reward circuitry, and could represent novel treatment targets for motivational deficits and negative symptoms of schizophrenia.

Objectives/Goals: People experiencing homelessness (PEH) face excess cervical cancer burden and unique barriers to screening. As part of a broader study addressing cervical cancer disparities in homeless populations in Indiana, our goal was to engage unhoused women in a human-centered design process to inform a homeless shelter-based self-sampling intervention. Methods/Study Population: An established community-academic partnership enabled meaningful engagement of homeless communities in Indiana and informed the need to understand and address cervical cancer disparities in this population. Rapid assessment surveys (n = 202) and in-depth interviews (n = 30) were conducted with PEH at two major shelters in Indianapolis and Lafayette to understand cervical cancer screening coverage, knowledge, attitudes, and practices; barriers and facilitators; and acceptability of human papillomavirus (HPV) self-sampling for onsite shelter-based screening. A human-centered design session with (n = 12) unhoused women further explored motivators and concerns regarding self-sampling and informed key messages and informational materials to encourage uptake of screening. Results/Anticipated Results: At least 37% were overdue for screening (last screened >5 years ago; 50% were last screened >3 years ago), far greater than national (22%) or state (24%) averages. Despite common misconceptions regarding indifference toward preventive healthcare among homeless populations, most (87%) wanted to be screened and believed it is important for their health. Competing priorities for daily survival, transportation, cost, provider mistrust, stigma, and related trauma were common barriers to screening. Enthusiasm for HPV self-sampling centered on convenience, privacy, and comfort in taking one’s own sample at the shelter. Notable concerns included lack of confidence regarding ability to self-sample correctly, unhygienic conditions in shelter restrooms, preference to be seen by a doctor, and the need for education. Discussion/Significance of Impact: The unique challenges of PEH require human-centered strategies to improve cervical cancer screening access. Willingness to be screened and acceptability of HPV self-sampling is high. Identified concerns and preferences will guide implementation of HPV self-sampling delivered by trusted community health workers in homeless shelters in Indiana.

OBJECTIVES/GOALS: Understanding how SARS-CoV-2 is evolving as well as spreading within and between communities is vital for the design of rational, evidence-based control measures. Continuous genomic surveillance is imperative to identify and track variants and can be paired with clinical data, to identify associations with severity or vaccine breakthroughs. METHODS/STUDY POPULATION: In June of 2021, we established UNM as a CDC-funded hub for genomic surveillance of SARS-CoV-2 for New Mexico and 3 other Rocky Mountain region states (Wyoming, Idaho, Montana). Through our Rocky Mountain COVID Consortium (RMCC), we have sequenced over 6,000 genomes of SARS-CoV-2 from RMCC partners. For New Mexico we integrate county and zip code data to provide more granular insights into how SARS-CoV-2, and particular variants, are transmitting within the state. We also pair this data with vaccine breakthrough cases identified by the NMDOH, as well as with clinical outcome data. RESULTS/ANTICIPATED RESULTS: We sequenced over 6,000 SARS-CoV-2 genomes from New Mexico (n=3091), Idaho (n=1538), Arkansas (n=1101), Wyoming (n=251), and Montana (n=33). We used this data to infer the transmission dynamics, identify variants, and map the spread of the virus. We identified a novel local variant that spread across New Mexico in early 2021, but was quickly replaced by the Alpha variant. In all RMCC states, the Delta variant overtook Alpha and has become nearly the only variant currently circulating in these states. We identified sequenced isolates from vaccine breakthrough cases in NM and demonstrate their role in onward transmission. We can identify shifts at a county or zip-code level in circulating lineages which may correspond to clinical outcomes or fluctuating case counts. DISCUSSION/SIGNIFICANCE: This integrated genomic data can be used by policy and decision makers within the New Mexico Department of Health and our RMCC partners to guide their public health response to the COVID-19 pandemic.

OBJECTIVES/GOALS: The purpose of our study was to assess the knowledge, attitudes and practices of nursing students during the COVID-19 pandemic. METHODS/STUDY POPULATION: Data were collected using an online questionnaire consisted of demographic characteristics and 24 items about COVID-19-related knowledge, attitudes and practices. RESULTS/ANTICIPATED RESULTS: A total of 1,216 nursing students participated in this study. About 82% of the participants reported that the COVID-19 virus spreads via respiratory droplets of infected individuals. The most clinical symptoms of COVID-19 correctly identified by participants were fever (97.6%), dry cough (92.4%), dyspnoea (82%) and fatigue (74.9%). More than 56.6% of the participants were afraid of being affected by COVID-19. Almost all participants reported that they avoid crowded places frequently. About 93.4% of the participants declared frequently wearing face mask when leaving home, and 85.5% maintained social distancing frequently. However, only 47.4% reported that they frequently washed their hands. About 51% stated that coronavirus outbreak has considerably changed their daily routines. DISCUSSION/SIGNIFICANCE: Sensitization and education campaigns are needed to improve their preventative practices, such as hand hygiene and wearing face mask. In addition, it may be of importance to incorporate competences into curricula to improve knowledge, attitudes and practices of future health professionals and to prepare them for emergencies and outbreaks.

OBJECTIVES/GOALS: The purpose of this study is to compare the mental health of African Americans to Whites during 2019 and 2020 using the a National Survey on Drug Use and Mental Health (NSDUMH). METHODS/STUDY POPULATION: Secondary data analysis from the National Survey on Drug Use and Health. The data consisted of 55,772 observations, 3,090 variables. This study will consist of the United State adult data population from 2019 and 2020 using the National Survey on Drug Use and Mental Health (NSDUMH). RESULTS/ANTICIPATED RESULTS: The results of this research will be produced from the following analysis. The analysis will consist of a secondary data analysis from the National Survey on Drug Use and Mental Health (NSDUMH). The primary independent variable of interest is race. All the indicator (race, sex, insurance, etc). Dependent variable is the mental health of African Americans and Whites. This is the variable in the NSDUMH labeled as the Major Depressive Episodes (MDE). The data analysis will be conducted using univariate analysis describing the study population. Bivariate analysis will be performed using chi-square. Since our dependent variable will be dichotomous we will be using several logistic regressions. DISCUSSION/SIGNIFICANCE: Strengthen mental health and psychosocial support services as part of strengthening preparedness, response and resilience to COVID-19 and future public health emergencies. Also adopt the updated Comprehensive Mental Health Action Plan for the future.

OBJECTIVES/SPECIFIC AIMS: Purpose - The goal of this study is to understand how loss of the membrane protein SLC4A11 alters endothelial cell metabolism thereby producing Corneal Endothelial Dystrophy. Studies from our lab indicated that glutamine-dependent mitochondrial dysfunction is one of the outcomes of SLC4a11 loss. In the current study, we ask if autophagy and mitophagy pathways and the signaling pathways that regulate these processes are altered in SLC4a11 KO cells. METHODS/STUDY POPULATION: Methods – Immortalized mouse WT and SLC4a11 KO cell lines were incubated in DMEM with and without 0.5mM glutamine for 6 hours. In order to assess mitophagy, cells were stained using Lysotracker Red and Mitotracker Green. Colocalization co-efficients of red and green channels were obtained for at least 35 cells using Zeiss-Zen Pro software. Student’s t-test was used to determine statistical significance. For Western Blots, antibodies against LC3b, AMPK, pAMPK, and b-actin were used to examine autophagy flux and potential signaling pathways that regulate autophagy. RESULTS/ANTICIPATED RESULTS: Results – In the presence of glutamine, the colocalization co-efficient of Lysotracker Red and Mitotracker Green channels was significantly increased in KO cells (0.74 ±0.18) relative to WT (0.58±0.20) with a p-value ≤0.0024. In the absence of glutamine, the colocalization co-efficient was reversed, for KO cells 0.54 ±0.14 and for WT cells 0.77±0.0.16 with a p-value ≤0.0001, suggesting increased mitophagy by glutamine in KO cells. Western Blots indicated that glutamine increased autophagy flux, as indicated by increased levels of LC3b following bafilomycin A treatment in KO cells. Concomitantly, there was an increase in pAMPK/AMPK levels suggesting a potential mechanism for increased mitophagy. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion and Future studies –Our data indicates enhanced mitophagy as well as autophagy in SLC4a11 KO cells. Future studies will determine whether these processes regulate cell survival in mouse models of corneal endothelial dystrophies.

OBJECTIVES/SPECIFIC AIMS: This project aims to synthesize an angiogenic decorin mimic (VEGFp-DS-SILY) with varying densities of QK and characterize its angiogenic potential and synergism with VEGF by evaluating (1) endothelial cell (EC) migration and proliferation, (2) EC VEGF receptor activation, (3) EC tubule formation in collagen scaffolds, and (4) angiogenesis from a chick chorioallantoic membrane (CAM assay) growing into the scaffold, reflecting the ability of the collagen scaffold to integrate into existing vasculature. The next main goal is to develop and characterize an MMP-degradable nanoparticle system for controlled release of VEGF. Future work will evaluate in vivo effects of VEGFp-DS-SILY bound to a 3D collagen scaffold on ischemic wound repair in a combined excisional wound/bipedical dorsal skin flap rat model. METHODS/STUDY POPULATION: Peptide hydrazides are conjugated to the free carboxylic acid functional groups on dermatan sulfate using EDC chemistry. We added a 3 amino acid spacer (-Gly-Ser-Gly) to the C-terminus of the established QK sequence before the hydrazide functional group and refer to this modified QK as “VEGFp.” VEGFp, SILY, and N-terminal biotinylated versions were synthesized using standard Fmoc solid-phase peptide synthesis protocols and purified using reverse phase HPLC. Coupling efficiencies of peptides to dermatan sulfate were determined spectroscopically at 280 nm measuring the aromatic residues (Trp or Tyr) using a NanoDrop system. Dermatan sulfate with 1 or 4 VEGFp peptides coupled were termed DSV1 and DSV4, respectively. After further conjugation with SILY, we will blend this VEGFp-DS-SILY with unmodified DS-SILY to a total 10 μM to test increasing densities of VEGFp. To verify that the collagen-binding properties of VEGFp-DS-SILY are not compromised by the addition of VEGFp, we will use a streptavidin-HRP system to detect bound biotinylated VEGFp-DS-SILY on collagen-coated plates by established protocols. DSV1 and DSV4 were tested for their effects on endothelial VEGFR2 phosphorylation using an MSD ELISA-type assay and endothelial proliferation using an MTS assay. Cell migration was monitored using an ORIS assay where cells are grown to confluence around a silicone stopper that is then removed to allow cells to migrate inward. Tubulogenesis was evaluated by examining tubule formation on matrigel. Finally, in vivo angiogenesis will be evaluated using a chorioallantoic membrane assay. For extracellular VEGF release, hollow MMP-degradable thermoresponsive nanoparticles [NIPAM, 5 mol% 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS), 1% Acrylic Acid (AAc), 2 mol% MMP-degradable peptide diacrylate, and potassium persulfate initiator] will be synthesized around noncross-linked polymer cores. The cores will then be diffused out through the shell by dialysis prior to drug loading. SILY (and some biotinylated SILY for visualization) will be conjugated with EDC chemistry for targeting nanoparticles to collagen. NPs size and zeta-potential will be measured on a Malvern Zetasizer. VEGF will be loaded into NPs by co-incubating a loading solution of 1 µg/mL VEGF with 1 mg of NPs, incubating overnight at 4°C. VEGF loading and release will be measured by ELISA. Biological activity of the released VEGF from particles will be determined on ECs using assays similar to those outlined previously. RESULTS/ANTICIPATED RESULTS: Preliminary data have verified the synthesis and purification of SILY and VEGFp (QK-Gly-Ser-Gly-hydrazide), as well as an N-terminal biotinylated version, through mass spectrometry and reverse-phase HPLC, respectively. For proof-of-concept, we have verified binding of VEGFp to the VEGF receptor 2 using a ForteBio Blitz interferometry instrument. In addition to support based on published reports showing retained bioactivity of QK after conjugation using other spacers, our preliminary data suggests that VEGFp still binds to VEGF receptor 2, albeit with decreased affinity like QK as compared with VEGF. Circular dichroism also shows that VEGFp has retained its α-helical structure necessary for bioactivity; however it appears that it has some uncoiling when conjugated to dermatan sulfate. We hypothesize that varying densities of VEGFp conjugated to the decorin mimetic (DS-SILY) will modulate the degree of angiogenic activity and synergy with VEGF. We determined that we can achieve ~70% VEGFp conjugation completion to dermatan sulfate after 3.5 hours. We have quantified VEGFR2 phosphorylation after 5 minute treatments by using phospho-specific antibodies and an ELISA-type protocol in a mesoscale discovery system. Preliminary data with human umbilical vein endothelial cells shows that VEGFp exhibits synergism with VEGF at levels similar to QK. DSV1 and DSV4 data suggests synergy with VEGF, although free-peptides and engineered compounds alone did not show effects similar to VEGF in the conditions tested. Prelminary data with 30 minute treatments suggests that the peptides and compounds may require longer exposures to induce activation, as they may have slower binding rates. In contrast, prolonged stimulation with VEGF causes a sharp increase in receptor activation, peaking around 10 minutes and decreasing significantly by 30 minutes. Peptides QK and VEGFp both slightly increased proliferation of dermal microvascular endothelial cells (HMVECs) after 60 hours incubation. However, incubation with dermatan sulfate and DSV caused significant cell death after 24 hours in reduced growth factor media, likely due to sequestering of growth factors. It is possible that VEGFp-DS-SILY may better stimulate proliferation since it would be presented as a surface bound proteoglycan mimic, rather than as a soluble factor. HMVECs migrated farther for all treatment groups (10 µM QK, 10 µM VEGFp, 1 µM DSV4, and 10 µM DSV4) than the 10 ng/mL VEGF positive control, although more cells migrated in response to VEGF. This may be accounted at least in part by the more pronounced proliferation induced by VEGF. Migration will also be tested in 3D culture within a collagen gel. We are currently testing a 2D matrigel system for tubulogenesis. We have found that 10 µM DSV4 forms qualitatively more well-defined tubules than the untreated control on reduced growth factor matrigel. However, we were not able to quantify the improved tubule formation and are still troubleshooting the tubule analysis. After seeding ECs and culturing for 4, 8, and 12 hours, cells will be fluorescently stained with anti-CD31 and imaged for 3D tubule formation. CAM assay angiogenesis growing into a collagen scaffold. In brief, fertilized chicken embryos are incubated for 2 days before exposing the CAM. VEGFp-DS-SILY bound to a collagen gel will be placed onto the CAM. Some treatment groups will receive additional VEGF to investigate synergistic effects. Light microscope images of angiogenesis into the collagen gel coated with VEGFp-DS-SILY, taken every day from days 10 to 13, will reflect the ability of the collagen scaffold to integrate into existing vasculature and 3D angiogenic potential of VEGFp-DS-SILY with or without VEGF. We expect that VEGFp-DS-SILY treatment will increase the number of vessels formed on the CAM. Preliminary data using a Fluoraldehyde assay indicates that loading of ~300 ng VEGF per mg of nanoparticles can be achieved. We expect that using an MMP-degradable peptide diacrylate crosslinker will allow nanoparticles to degrade in protease-rich environments like the chronic wound bed and release VEGF. Adjustments to the formulation, such as crosslinker density, may need to be modified to control the rate of VEGF release. DISCUSSION/SIGNIFICANCE OF IMPACT: We expect that our angiogenic decorin mimetic will lead to a novel treatment to accelerate healing of ischemic diabetic foot ulcers, thereby reducing the need for limb amputation and mortality rate of diabetic patients. We anticipate that the diabetes research and regenerative medicine communities will (1) gain a platform for targeted delivery of growth factors, (2) understand the dependence of vascularization within 3D collagen constructs on VEGFp densities and VEGF receptor activation in controlling the degree of angiogenesis, and (3) gain the benefits of controlled angiogenesis in ischemic diabetic wound healing.

OBJECTIVES/SPECIFIC AIMS: QuantiFERON-TB Gold In-Tube (QFT) conversion from negative to positive, is regarded as a marker of recent latent tuberculosis infection and may be predictive of incident active tuberculosis (TB) disease. However, it remains unclear how conversion is influenced by individual and environmental factors, including the infectiousness of the source case to whom the contact was exposed. We aimed to examine the effect of infectiousness of TB in the source case, as measured by presence of cavitation on chest X-ray, on the incidence of QFT conversion among close contacts of the pulmonary TB index case, after adjusting for potential confounding by contact and source case characteristics. METHODS/STUDY POPULATION:The Regional Prospective Observational Research for Tuberculosis (RePORT)-Brazil is an ongoing prospective cohort study that enrolls close contacts of culture-confirmed pulmonary TB patients and follows them for 24 months for development of active TB. Demographic, clinical, and diagnostic information are obtained at baseline and during follow-up at clinical visits and by telephone. QFT testing is performed at baseline and repeated after 6 months if the baseline QFT is negative. A positive IFN-γ value is defined as >0.35 IU/mL, as recommended by the manufacturer and the CDC, and QFT conversion is defined as a negative QFT at baseline followed by a positive QFT at 6 months. RESULTS/ANTICIPATED RESULTS: Among 260 enrolled contacts with nonpositive baseline QFT results and 6 months of follow-up, 198 (76%) were retested with QFT 6 months after enrollment. Of those retested, 26 (13%) converted to positive. Presence of any cavitation in the source case, based on chest radiography, was significantly associated with QFT-conversion (ORunadjusted=2.4, 95% CI: 1.0–5.7). Additional univariate analyses revealed that QFT conversion was associated with black and brown race (compared with white race) of the contact, current smoking and current alcohol use in the source case. After adjusting for potential confounders (age, sex, and race of the contact and current smoking of the source case), the association between source case cavitation and QFT conversion remained (ORadjusted=2.5 95% CI: 1.0–6.2). As of December 6, 2017, none of the QFT-retested contacts had developed active TB, with a median follow-up of 12.3 months (IQR: 7.1–13.1). We anticipate that ongoing enrollment and follow-up may yield cases of active TB; future analyses will provide greater precision for examining predictors of QFT-conversion and its association with incident TB. DISCUSSION/SIGNIFICANCE OF IMPACT: Our preliminary results agree with published literature suggesting the infectiousness of TB in the index case is a predictor of incident LTBI. Along with recent LTBI, immune suppression, HIV co-infection, and type 2 diabetes are considered risk factors for progression to active TB disease. Because only a small proportion of persons progress from LTBI to active TB disease, it is not appropriate to treat all persons with LTBI. Thus, more research is needed to identify groups at highest risk for QFT-conversion and incident TB disease, so these groups can be targeted for TB prevention, interventions, and facilitate a decline in TB incidence and mortality.

OBJECTIVES/GOALS: In the United States, oropharyngeal cancer (OPC) is the leading human papilloma-virus (HPV) related malignancy, and OPC rates are increasing among the US veteran population. The purpose of this study is to identify demographic and regional factors that may be associated with low HPV-vaccination rates among the US veteran population. METHODS/STUDY POPULATION: This study will use Veterans Health Administration (VHA) administrative data to create a national cross-sectional cohort of veterans ages 18-45 with a VHA primary care visit from 2018-2020. HPV-vaccination status of each subject (initiation of vaccination series, completion of vaccination regimen, and age vaccinated) will be determined. Differences in the prevalence of HPV-vaccination by smoking status, geographic location, socioeconomic status, race/ethnicity, and rural-urban context will be examined in the unadjusted analysis. Factors associated with low HPV-vaccination rates in the VHA will be identified using multivariable logistic regression to model no (vs any) HPV-vaccination, no completion (vs completion) of HPV-vaccination recommendations, and non-routine (vs routine) HPV-vaccination: RESULTS/ANTICIPATED RESULTS: In 2019, HPV-vaccination guidelines were expanded to include some adults between the ages of 26-45, making many young veterans in the VHA eligible for vaccination. From this study’s recently generated dataset, more than 1.2 million subjects (n=1,219,896) met the study inclusion criteria. Extrapolating from trends in the civilian population, it is anticipated that HPV-vaccination rates will be lower among African Americans compared to non-Hispanic Caucasian Americans, within the South Central and Southeastern regions of the US, and in rural communities. This study will define a veteran’s geographic location by their associated Veteran Affairs Integrated Service Network (or VISN), which are groups responsible for healthcare planning and resource allocation in particular regions of the US: DISCUSSION/SIGNIFICANCE: Identifying factors associated with low HPV-vaccination rates within the VHA will be the first step to reducing future incidence of HPV-related cancer burden among US veterans. Assessing geographic variability in vaccination rates by VISNs will create the potential to generate targeted interventions within an existing VHA framework.

OBJECTIVES/SPECIFIC AIMS: The purpose of this research was to investigate how chronic inflammation promotes the generation of proinflammatory intestinal macrophages and if macrophages contribute to intestinal inflammation through Notch activation. METHODS/STUDY POPULATION: We utilized two animal models of chronic colitis, the chronic DSS-induced colitis mouse model and the spontaneous enterocolitis development in IL-10-deficient mice to investigate the role of chronic inflammation in the generation of proinflammatory intestinal macrophages and its influence in notch signaling. Bone marrow-derived monocytes were collected from each group and differentiated into macrophages (BMM) for gene and protein analysis. Ex vivo phenotypical and functional analysis of colonic macrophages was assessed as was the presence of goblet cells and mucosal T cells. In addition, we analyzed the development of goblet cell differentiation in colonoids in a co-culture system with proinflammatory macrophages. RESULTS/ANTICIPATED RESULTS: Our chronic inflammation models revealed an increase in proinflammatory macrophages present in the lamina propria and that these cells expressed significantly higher levels of notch ligand, Jagged1. Jagged 1 has been shown to enhance TH1 differentiation and T cells isolated from the mucosa of both chronic colitis models display strong TH1 skewing compared to controls. Chronic inflammation also contributes to intestinal barrier defects, enhanced permeability and bacterial translocation. We believe this enhanced intestinal permeability and subsequent bacterial translocation promote Jagged1 expression in intestinal macrophages. To support this concept, we show TLR stimulation induces the upregulation of Jagged1 in BMM. Additionally, the generation of BMM from our chronic DSS-induced colitis mice or age matched controls, revealed BMM derived from a host of chronic inflammation were skewed to a proinflammatory state prior to stimulation showing increased gene expression of several proinflammatory molecules including IL-1α, IL-1β, IL-12 and TNF-α. This would suggest monocytes migrating to the intestinal mucosa have more potential to become proinflammatory instead of traditional anti-inflammatory macrophages. Furthermore, proinflammatory notch ligand-positive macrophages co-cultured with colonoids, derived from unperturbed mice, significantly decreased the number of mucus producing goblet cells. In support of this observation, notch activation in intestinal stem cells promote absorptive (i.e. colonocytes) cell differentiation and prevents secretory cell (i.e. goblet cells) differentiation. DISCUSSION/SIGNIFICANCE OF IMPACT: Taken together, our results strongly suggest chronic inflammation modulates macrophages role in maintaining intestinal homeostasis through possible notch activation in both T cells and the intestinal epithelial barrier.

OBJECTIVES/SPECIFIC AIMS: Alcohol consumption perturbs the normal intestinal microbial communities (alcohol dysbiosis). To begin to investigate the relationship between alcohol-mediated dysbiosis and host defense we developed an alcohol dysbiosis fecal adoptive transfer model, which allows us to isolate the host immune response to a pathogenic challenge at a distal organ (ie, the lung). This model system allowed us to determine whether the host immune responses to Klebsiella pneumoniae are altered by ethanol-associated dysbiosis, independent of alcohol use. We hypothesized that alcohol-induced changes in intestinal microbial communities would impair pulmonary host defenses against K. pneumoniae. METHODS/STUDY POPULATION: Mice were treated with a cocktail of antibiotics daily for 2 weeks. Microbiota-depleted mice were then recolonized by gavage for 3-days with intestinal microbiota from ethanol-fed or pair-fed animals. Following recolonization groups of mice were sacrificed prior to and 48 hours post respiratory infection with K. pneumoniae. We then assessed susceptibility to Klebsiella infection by determining colony counts for pathogen burden in the lungs. We also determined lung and intestinal immunology, intestinal permeability, as well as, liver damage and inflammation. RESULTS/ANTICIPATED RESULTS: We found that increased susceptibility to K. pneumoniae is, in part, mediated by the intestinal microbiota, as animals recolonized with an alcohol-induced dysbiotic intestinal microbial community have significantly higher lung burdens of K. pneumoniae (5×104 CFU vs. 1×103 CFU) independent of EtOH. We also found that increased susceptibility in alcohol-dysbiosis recolonized animals was associated with a decrease in the recruitment and/or proliferation of CD4+ and CD8+ T-cells (1.5×109 cells vs. 2.5×109 cells) in the lung following Klebsiella infection. However, there were increased numbers of T-cells in the intestinal tract following Klebsiella infection, which may suggest that T cells are being sequestered in the intestinal tract to the detriment of host defense in the lung. Interestingly, mice recolonized with an alcohol-dysbiotic microbiota had increased intestinal permeability as measured by increased levels of serum intestinal fatty acid binding protein (55 vs. 30 ng/mL). Alcohol-dysbiotic microbiota also increased liver steatosis (Oil Red-O staining) and liver inflammation (>2-fold expression of IL-17 and IL-23). DISCUSSION/SIGNIFICANCE OF IMPACT: Our findings suggest that the commensal intestinal microbiota support mucosal host defenses against infectious agents by facilitating normal immune responses to pulmonary pathogens. Our data also suggest that increased intestinal permeability coupled with increased liver inflammation may impair the recruitment/proliferation of immune cells in the respiratory tract following infection. The role of the microbiota during host defense will be important areas of future research directed at understanding the effects of microbial dysbiosis in patients with AUDs.

ABSTRACT IMPACT: Characterizing the cellular composition of human adipose tissue may contribute to the prevention and/or treatment of obesity-associated metabolic diseases. OBJECTIVES/GOALS: Our aims in this study were to use single-cell techniques 1. to characterize cell types within the stromavascular fraction of human adipose tissue, 2. to identify subsets of cells within each type (sub-clustering), 3. to identify gene sets and pathways that may provide information on the function and significance of each cell cluster. METHODS/STUDY POPULATION: Abdominal subcutaneous adipose tissue samples from n=6 healthy volunteers (1M, 5F, age 28-38 y, BMI 24.5-63.0 kg/m2) were collected by aspiration or during surgery. In 3 subjects, all females, paired femoral samples were also collected. After collagenase digestion approximately n=10,000 cells/sample were used for single-cell RNA sequencing using the 10X Genomics platform. After QC and downstream analysis, data were analyzed in Seurat v.3.1.5. We identified first different cell types and then subclusters in an unbiased fashion. Gene Set Enrichment Analysis (GSEA) was used for pathway analysis. RESULTS/ANTICIPATED RESULTS: Progenitor markers are related to extracellular matrix, eg DCN (logFC progenitors vs other cells types=3.17, expressed in 99.7% (pct.1) of progenitors, 26.1% (pct.2) of others). Endothelial and pericytes shared markers like RBP7 (logFC=1.67, pct.1 0.88, pct.2 0.39); pericytes also showed unique markers, eg RGS5 (logFC=2.29, pct.1 0.89, pct.2 0.17). Progenitors are further divided into 11 sub-clusters, one of which showed enrichment of CD36 (high proliferation potential), FABP4 (differentiation), and of the novel marker PALMD (logFC=7.13, pct.1 0.94, pct.2 0.48). All p<10E-5. GSEA analysis suggests that inflammatory pathways are downregulated in both adipose progenitors and endothelial/pericyte cells in the femoral compared to the abdominal depot. DISCUSSION/SIGNIFICANCE OF FINDINGS: Single cell RNA sequencing provides unique insights into the molecular profile of cell types and the identification of novel subsets of cells within the human adipose tissue. Such cellular heterogeneity may explain differences in adipose function between individuals and eventually in the risk of obesity-associated metabolic diseases.

OBJECTIVES/GOALS: ICD-10 coding inconsistencies hinder timely recognition and treatment of metabolic syndrome (MetS), posing a significant risk for cardiometabolic disease progression. This study employed a digital phenotype for MetS and compared odds for medication and lifestyle intervention compared to those coded for MetS. METHODS/STUDY POPULATION: MetS is a cluster of cardiometabolic risk factors that increase risk for numerous adverse clinical outcomes. Patients with MetS were identified through electronic medical records on TriNetX LLC using the standard ICD-10 code or through a digital phenotype, involving grouping codes for the individual components. Percentage of patients with MetS not captured with the standard code was identified. In addition, disparities in blood pressure, glucose, lipid-lowering medication, and lifestyle intervention between the coding schemas were assessed, shedding light on healthcare inequities and informing targeted interventions. Odds ratios (RR) were presented for all outcomes. RESULTS/ANTICIPATED RESULTS: Patient demographics and lab values were similar between the standard code and digital phenotype cohorts. Of the 4.3 million individuals aged 50 to 80 identified as having MetS using the digital phenotype in the TriNetX research network, only 1.78% of participants shared the standard code. Individuals with the digital phenotype for MetS were at lower odds in receiving glucose lowering medication (OR: 2.11, 95% CI: 1.98–2.13, p <0.001) and exercise or nutrition-based intervention advice (OR: 1.76, 95% CI: 1.55–1.96, p <0.001) after controlling for demographics and lab values for each MetS component. DISCUSSION/SIGNIFICANCE: This project utilized TriNetX to create a digital phenotype for MetS, and suggests most patients are not coded for it using the standard ICD-10 system. This is troublesome given those with the standard code are less likely to receive certain interventions.