Objectives/Goals: Novel therapeutics to control Staphylococcus aureus (S. aureus) infections are needed for people with cystic fibrosis (CF, PwCF). In this study, our objective is to determine if the pharmacologic MEK1/2 inhibitor compound ATR-002 can restrict the growth of S. aureus clinical isolates and modulate infection in a murine model of S. aureus infection. Methods/Study Population: To evaluate the anti-inflammatory effects of ATR-002 on human macrophages, cells were stimulated with TLR2 agonists FSL1 or Pam3CSK4 with a dose range of ATR-002, and secretion of proinflammatory cytokines were measured by ELISA. To determine the direct antibacterial effect of ATR-002, minimum inhibitory concentration (MIC) assays were performed with the community-associated methicillin-resistant S. aureus strain USA300 and 40 S. aureus isolates from PwCF. To validate our results in vivo, mice were provided i.p. treatment with either vehicle, the MEK1/2 inhibitor compound PD0325901 (20 mg/kg), or ATR-002 (10 mg/kg) prior to intranasal infection with 1x10^7 CFU of USA300. Bacterial burdens at 4- and 24-hour post-infection (p.i.) and inflammatory cell recruitment at 24 hours p.i. were quantified. Results/Anticipated Results: Macrophages treated with ATR-002 exhibited a dose-dependent decrease in secretion of proinflammatory cytokines TNF and IL-8 following TLR stimulation. Our studies identified that ATR-002, but not PD0325901 or other MEK1/2 inhibitors, had direct antibacterial effects, and ATR-002 had an MIC range of 8 to above 64 ug/mL on CF S. aureus isolates. In the murine pulmonary infection model, delivery of ATR-002 and PD0325901 significantly prevented infection-induced loss of body mass and decreased neutrophil inflammation. However, when bacterial burdens were quantified 4-hours p.i., only ATR-002 treatment reduced lung bacterial burden compared to vehicle or PD0325901-treated groups. Discussion/Significance of Impact: These results are the first demonstration of the in vivo anti-inflammatory and antibacterial effects of ATR-002. Our results further demonstrate that ATR-002 exhibits direct antibacterial effects across a collection of clinical isolates of S. aureus. Future studies will continue to investigate the therapeutic potential of ATR-002.

Objectives/Goals: This study aims to harmonize quality indicators (QIs) across University of Toronto-affiliated microbiology labs to establish universal benchmarks that enhance performance, patient safety, and health outcomes. Harmonized QIs will enable effective comparisons and enhance the consistency of care. Methods/Study Population: The study employed the Delphi method, a structured and iterative process to build consensus. An expert panel of clinical microbiology trainees, medical microbiologists, trainees, and site leads from five University of Toronto-affiliated microbiology labs was assembled. Initial insights were gathered through surveys and a comprehensive scoping review of the literature. The study involved two rounds of feedback, a SurveyMonkey-based survey, with a defined consensus of 75% agreement among participants. Followed by an implementation survey conducted through REDCap to assess how these QIs were adopted in practice and identify barriers to implementation. Results/Anticipated Results: The study achieved consensus on nine high-impact quality indicators, including blood culture volume and contamination rates, cerebrospinal fluid transport time, and turnaround times for Gram stain results. Blood culture contamination and positivity rates garnered the highest agreement, at 100% and 91%, respectively. While some indicators were widely accepted and implemented, others faced resistance due to feasibility concerns. The study also identified significant variability in the level of adoption across the participating laboratories, pointing to operational challenges and the need for further efforts to address these barriers. Discussion/Significance of Impact: This study highlights the importance of QI harmonization in improving lab services and patient safety. It reveals challenges in standardizing practices but promotes uniformity in QIs, laying the groundwork for better inter-lab collaboration, consistent outcomes, and improvements in microbiology.

Objectives/Goals: People with insulin-treated diabetes face hypoglycemia risk due to imperfect insulin replacement and impaired counterregulation. We identified the dopamine antagonist, metoclopramide, as a potential treatment. Hypothesis: Treatment with metoclopramide will prevent the development of impaired counterregulatory response to hypoglycemia. Methods/Study Population: In a pre-clinical model, diabetes was induced in 10-week-old Sprague-Dawley rats with streptozotocin (STZ, 65 mg/kg IP). Rats were divided into three groups: 1) diabetic controls (STZ+RS, n = 6), 2) recurrent hypoglycemia (STZ+RH, n = 7), and 3) recurrent hypoglycemia + metoclopramide (STZ+RH+MET, 3 mg/kg IP, n = 7). After 3 days, all rats underwent a hyperinsulinemic (50 mU/kg/min) and hypoglycemic (~45 mg/dl) clamp. In the clinical trial, adults with Type 1 diabetes (age 20–60, ≥5 years duration) were enrolled in a phase II, double-blinded, placebo-controlled trial. Awareness status was assessed via Gold score, and subjects maintained drug regimens and underwent two hyperinsulinemic-hypoglycemic clamps (where blood glucose was lowered to 100, 65, 55, and 45 mg/dl) to assess counterregulation. Results/Anticipated Results: In the pre-clinical model, glucose infusion rates (GIR) to maintain hypoglycemia were higher in STZ+RH (27±0.9 mg/kg/min) than STZ+RS (19±0.8 mg/kg/min, p Discussion/Significance of Impact: Metoclopramide improves glucoregulatory, sympathoadrenal, and counterregulatory responses to hypoglycemia in pre-clinical models, suggesting dopaminergic regulation. While clinical data are still blinded, increased epinephrine and growth hormone responses suggest treatment may preserve or restore counterregulation.

Objectives/Goals: The goal of this study is to investigate the peri-metastatic rims of peritoneal lesions to determine features that predict malignancy using both imaging processing techniques and machine learning. This information will subsequently be added to our existing knowledge of peritoneal lesions to improve classification accuracy as benign or malignant. Methods/Study Population: The study population consists of 521 imaged lesions from 163 subjects with cancers of GI-origin with biopsy results as well as the clinical subject information and follow up. All images were obtained during staging laparoscopy by the senior author (TS). On the images, the central lesion as well as the surrounding peri-metastatic rim will be segmented as regions of interest (ROIs). Image processing will be used to calculate a variety of metrics for these two regions. A general estimating equation approach will be used to determine significance of these metrics compared to the dependent outcome of malignancy determined on the pathology report as the ground truth. These ROIs and significant metrics will then be used to improve the accuracy of a machine learning model to classify these lesions as benign or malignant. Results/Anticipated Results: Our previous research showed that experts performed this task at only a 52% accuracy rate (classifying lesions as malignant or benign based on imaging). A previous machine-learning model on a much smaller dataset was able to achieve by contrast an area under the curve of 0.78. We anticipate that by including a larger dataset in addition to including the peri-metastatic rim, we will be able to improve the accuracy of the the model in this task while uncovering significant biomarkers as well that can be used in future studies. Discussion/Significance of Impact: Classifying peritoneal lesions determines the correct treatment for cancer patients whether chemo-radiation, definitive surgery or palliative surgery. This project aims to develop an improved model that can perform this task using nonlabeled laparoscopic imaging with a particular focus on the diagnostic value of the peri-metastatic rim.

Objectives/Goals: • To determine the impact of liraglutide on inflammation and organ injury during sepsis. • To investigate the protective effects of liraglutide on microvascular dysfunction in a clinically relevant model of sepsis. • To provide evidence for the potential therapeutic use of GLP-1 receptor agonists in endothelial dysfunction in sepsis. Methods/Study Population: Sepsis was induced in mice (N = 34) by intraperitoneal injection of cecal contents (1.8 mg/g body weight) and 24-hour hyperoxia (FiO2 90–95%). Mice received saline or liraglutide (0.1 mg/kg) at 6 and 18 hours post-injection and fluids and antibiotics at 12 hours. At 24 hours, mice were euthanized for plasma, bronchoalveolar lavage (BAL), and tissue collection. Plasma inflammatory markers, organ injury markers, and BAL components were measured. In vitro, primary human lung microvascular endothelial cells (HLMVECs) were treated with saline or liraglutide for 24 hours before exposure to saline or LPS (100 ng/mL). HLMVEC barrier dysfunction was evaluated using express permeability testing (XPerT) and electric cell-substrate impedance sensing (ECIS) to measure transendothelial electrical resistance (TER). Results/Anticipated Results: In murine sepsis, illness severity scores and lung injury were improved in mice pretreated with liraglutide (N = 10). Plasma blood urea nitrogen (BUN; P = 0.0036), alanine transaminase (ALT; P = 0.0311) and vascular inflammatory markers MCP-1 (P = 0.0172), ICAM-1 (P = 0.0356), and Pecam-1 (P = 0.0493) in plasma were reduced in mice treated with liraglutide. In HLMVECs, liraglutide (1.5 nM) significantly reduced LPS-induced barrier dysfunction measured by XPerT assay (P = 0.0030) and ECIS (P = 0.0075). Discussion/Significance of Impact: Liraglutide reduces illness severity, vascular inflammation, and organ injury in a two-hit model of sepsis. Liraglutide has direct effects in the microvascular endothelium, limiting LPS-mediated barrier dysfunction. These findings support a protective role for GLP-1 receptor agonism in sepsis, mediated through the microvasculature.

Objectives/Goals: The aging population faces unique health issues, many of which are exacerbated by aging-associated immune function decline. However, the driving mechanisms behind this decline are poorly understood. We use a mouse model to study the relationship between extracellular matrix (ECM) stiffness and cell mobility within the lymph node (LN) as one potential driving mechanism. Methods/Study Population: We will collect LN from young (6 weeks old) mice and acquire LN from aged (11 month old) mice from a collaborator. We will section LN for ex vivo analysis, including quantification and localization of collagen I using immunofluorescent staining, analysis of microrheological properties using multiparticle tracking (MPT) with PEGylated fluorescent nanoparticles, and migration assays to track the movement of B and T cells. Results/Anticipated Results: We hypothesize a positive correlation between collagen deposits and stiffness within murine LNs due to known mechanisms underlying age-related fibrosis. We also hypothesize that areas of increased stiffness (as revealed by MPT) will exhibit decreased cell migration due to physical hindrance to B and T cell mobilization. Furthermore, we hypothesize that aged murine LN will exhibit a significant increase in stiffness and resultant decreased cell mobility when compared to young murine LN, particularly in areas with increased collagen localization. Discussion/Significance of Impact: These studies will elucidate structure–function relationships driving age-associated LN fibrosis and stiffness, and the resultant impedance to cell migration, thus clarifying some of the potential driving mechanisms behind immune aging and providing data capable of informing the development of relevant models and interventions.

Objectives/Goals: The never in mitosis kinase (NEK) family regulates vital processes, namely cell cycle progression, but their potential as therapeutic targets in TNBC has not been fully explored. Our studies aim to develop a toolkit to investigate the functional roles of NEKs in pathologies including carcinogenesis. Methods/Study Population: To assess differential NEK expression in normal and tumor tissues and correlation of gene expression with patient survival, we used Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan–Meier Plotter (KMPlot) pan-cancer analysis, respectively. Basal NEK protein levels were determined by immunoblot across a panel of cell lines, including breast cancer, osteosarcoma, hepatocellular carcinoma, and non-cancerous cells, to identify appropriate systems for evaluation of NEK function. Doxycycline-inducible cell lines were generated by transduction with lentiviral stocks of NEK shRNA and overexpression constructs and antibiotic selection. Expression was analyzed by qPCR and immunoblot. Results/Anticipated Results: Expression of NEK2, 4, 5, 6, 8, and 11 was higher in breast tumors compared to normal tissue by GEPIA analysis. Further examination using KMPlot showed a correlation between elevated NEK6 expression and decreased overall survival in patients with aggressive cancers. As an initial proof-of-concept study, we analyzed NEK6 protein expression in breast cancer cells. Levels of NEK6 were elevated in TNBC cells (MDA-MB-231) compared to hormone receptor positive (HR+) breast cancer cells (MCF7). Using complementary approaches to investigate the functional role of NEK6 in breast cancer, we depleted NEK6 expression using shRNAs in TNBC cells and expressed NEK6 in HR+ cells Discussion/Significance of Impact: Because kinase dysregulation promotes oncogenesis and metastasis, targeting kinases is a key strategy in therapeutic development. A NEK-specific molecular toolkit allows researchers to elucidate NEK functions and contributions to carcinogenesis, promoting advancement of novel therapies.

Objectives/Goals: To develop and deploy an academic learning health system (aLHS) Bridge Program to capitalize on our unique organizational strengths in Implementation Science (IS) and to overcome the gap between science and practice that threatens the success of an aLHS. The aLHS Bridges includes an IS Shared Resource, intended to advance IS to practice. Methods/Study Population: The new IS Shared Resource is built on our expertise in dissemination and implementation science, pragmatic, and adaptive trials and the CTSI’s prior success in integrating academic and clinical missions. We also leveraged our existing experts to co-lead the aLHS Bridge including Kristie Foley, PhD, inaugural Chair of the Department of Implementation Science, and Gary Rosenthal, MD, Chair of the Department of Internal Medicine. Specifically, the new IS Shared Resource builds on the capacity of the Department of IS, comprised of 36 faculty members (19 primary and 17 secondary/adjunct appointments) with expertise in qualitative and mixed-methods research, stakeholder engagement, participatory research, digital health, and organizational theory. Results/Anticipated Results: The IS Shared Resource is primed to aid faculty with dissemination and implementation needs, including shortening the time of intervention adoption and using implementation science to inform sustainable and effective implementation practice. The IS Shared Resource is equipped to provide consultation services to faculty members to understand their specific request and match IS faculty members who are expertly trained in specific strategies or contexts. Discussion/Significance of Impact: Leveraging current resources and our first-of-its-kind Department of Implementation Science, our CTSI was able to stand up the IS Shared Resource to support the goals of the CTSA and our greater institution mission. Using a multidisciplinary approach was essential to the success of the IS Shared Resource.

Objectives/Goals: The objectives of the study were to evaluate end-user feedback regarding usefulness and compliance with the revised DoD PRA-CR. The PRA-CR utilizes symptom-guided management strategies to advance service members with acute concussion through 6 stages of gradually increased activity prior to their Return to Duty (RTD). Methods/Study Population: Clinical providers previously trained on the PRA-CR were invited via email to participate in an online survey-based study to examine their opinions and utilization of the revised PRA-CR. Participants who responded to the initial email invitation were provided an electronic Microsoft Forms based survey. Of the 83 total responders, 36 met inclusion criteria and advanced to the end-user survey. Six items were designed to assess inclusion–exclusion criteria (i.e., credentialed medical provider trained in the PRA-CR with experience treating concussion over the previous 2 years). Four items gauging utilization required yes/no responses; 20 opinion items on a 7-point Likert scale ranged from strongly disagree to strongly agree; 5 explanatory items were multi-select; and 1 item allowed free text responses. Results/Anticipated Results: Overall, 87% of respondents who had used the revised CR indicated that it helped them treat patients with acute concussion and 73% rated, “ease of use” favorably. Of the newly added elements to the CR, utilization of the Patient Leadership Guide (PLG) was the highest at 78%, with the majority of the providers rating the PLG as useful in communicating with patients and command. In contrast, only 35% of participants reported using the Physical RTD screening section and 22% indicated using the Cognitive RTD screening tool. Those not utilizing the Physical screening identified a lack of support staff (67%) or setting barriers (47%) as the primary reasons. Those not utilizing the Cognitive RTD screening tool identified multiple barriers to use including availability (72%), inexperience (39%), and baseline data access (33%). Discussion/Significance of Impact: This study sought end-user (provider) feedback regarding the revised PRA-CR’s usability and utility, in addition to their confidence in the tool itself. Overall results were generally positive, except for the updated Physical RTD and newly introduced Cognitive RTD screenings.

Objectives/Goals: This study assesses the feasibility of a human-to-swine lung transplant model for the evaluation of bioengineered organs. Given the critical organ shortage, this research explores bioengineered organs as a potential solution by evaluating early lung function, immune responses, and technical aspects to develop a model for bioengineered lungs. Methods/Study Population: The study employs a non-survival human-to-swine left lung transplant model in an immunosuppressed Yorkshire swine. A combination of cobra venom factor pretreated with methylprednisolone and Benadryl with scheduled dosing of tacrolimus and mycophenolate over a 24-hour period will be administered. The transplanted human lung is assessed over a 24-hour post-transplant period, with hourly pulmonary vein gas sampling and lung tissue resections. The proposed model will assess the immunological response of swine to human lung tissue as well as the efficacy of the immunosuppression model. Tissue samples are taken at intervals to evaluate for signs of rejection, cellular damage, and the overall function of the transplanted lung. All tissues are preserved in formaldehyde for subsequent immunohistology evaluation. Results/Anticipated Results: We anticipate a successful non-survival swine transplant model with pulmonary function sustained for the full 24-hour study using our proposed immunosuppression regimen. Initial testing with a standard human lung will lay the groundwork to assess the effectiveness of the human-to-swine transplant model. Hourly pulmonary vein gas analyses and tissue biopsies are expected to show minimal immune rejection, supported by the preoperative immunosuppression regimen. Early data indicate that the swine tolerates both the surgical procedure and immunosuppressive therapy well, with manageable hemodynamic stability. This model is expected to yield critical insights into lung viability and will identify areas for optimization for long-term survival studies to test the efficacy of bioengineered organs. Discussion/Significance of Impact: This non-survival swine model offers valuable insights into the acute-phase immune response and functional viability of human-to-swine lung transplant model. The findings will support the development of long-term survival models that will allow the evaluation of bioengineered organs based solely on their functionality as engineered organs.

Objectives/Goals: Motivational deficits are associated with depression and poorly treated by current therapeutics. We sought to identify more effective therapeutics for these deficits using a mouse model of early-life exposure to SSRIs, a developmental risk factor identified for depression in humans. Methods/Study Population: Mice were administered the SSRI, fluoxetine (FLX), or a vehicle control from postnatal day P2-P11, a window that mimics brain development occurring during the third trimester in a human pregnancy. Motivation and hedonic perception were assessed in adulthood using the progressive ratio and lickometer tasks, respectively. Behavioral testing was repeated after chronic adult administration of either an SSRI (fluoxetine), an atypical antidepressant and mu-opioid receptor agonist (tianeptine), or a mu-opioid receptor antagonist (methocinnamox). Results/Anticipated Results: Mice administered FLX in early-life showed motivational deficits in the progressive ratio task, while hedonic perception, as measured by the lickometer task, remained intact. Chronic administration of FLX in adulthood did not improve motivational deficits and did not alter hedonic perception. Chronic administration of tianeptine (TIA) slightly improved motivational behavior without altering hedonic perception. In contrast, chronic administration of the mu opioid antagonist methocinnamox (MCAM) markedly improved motivational deficits in mice, even while blunting hedonic perception. The ability of MCAM to enhance motivation was selective to early-FLX exposed mice Discussion/Significance of Impact: Our results reveal that unexpectedly opioid receptor antagonism is effective at improving motivation in mice exposed to SSRIs in early life. This suggests potential novel treatment approaches in individuals with motivational impairments and a history of in utero exposure to SSRIs.

Objectives/Goals: Our research addresses critical gaps by examining the prevalence, blood concentrations, and health implications of xylazine abuse, with a focus on its cardiotoxic effects. We aim to map the geographic distribution of xylazine use across Puerto Rico and characterize its impact on cardiomyocytes at relevant exposure levels. Methods/Study Population: To accurately detect and quantify xylazine in blood samples, we will employ chromatographic techniques coupled with mass spectrometry (UPLC/MS or GC/MS). The xylazine prevalence across the island will be mapped based on health system classifications. Samples will be categorized according to the eight healthcare regions of Puerto Rico, as defined by the “Administración de Seguros de Salud” (ASES), ensuring comprehensive geographic representation. We will investigate the expression profiles of proteins associated with cardiac injury and dysfunction in human cardiomyocytes and in the blood of drug users. Blood samples will be provided by “Iniciativa Comunitaria.” We will assess the xylazine effects on human cardiomyocyte viability and identify key biomarkers of cardiotoxicity induced by xylazine exposure. Results/Anticipated Results: In previous research, we demonstrated that xylazine induces cell death in endothelial cells through both extrinsic and intrinsic pathways. We also observed an increase in reactive oxygen species (ROS) levels after drug exposure, indicating oxidative stress as a potential mechanism of toxicity. Additionally, DNA damage was detected. Given the known relationship between endothelial damage and cardiomyocyte dysfunction in drug-induced cardiotoxicity, we hypothesize that xylazine concentrations vary regionally within Puerto Rico and that chronic xylazine abuse will elevate markers of cardiac injury and dysfunction at common user doses. Discussion/Significance of Impact: The increasing xylazine abuse, particularly in Puerto Rico, represents a critical public health challenge. Our study will fill a knowledge gap by providing crucial data on xylazine’s cardiotoxicity and mapping its geographic prevalence, with the potential to advise healthcare approaches and improve care for drug-using Hispanic populations.

Objectives/Goals: The goal of this study is to determine the function of the rete ovarii (RO), an uncharacterized secretory epithelial appendage to the ovary. I am testing the hypothesis that the RO is critical for the maintenance of the ovarian reserve and fertility, and progesterone signaling plays a role in the function of the RO. Methods/Study Population: For this project, I am utilizing a mouse model. To visualize the rete ovarii (RO) in vivo, I am using a Pax8rtTA; TRE-H2B-Gfp (PTG) reporter mouse, which expresses green fluorescent protein in the RO. To determine the role of the RO in fertility and maintenance of the ovarian reserve, I will surgically ablate the RO or perform a sham surgery on adult female PTG mice. Then, I will follow-up with quantification of the ovarian reserve and long-term fertility tracking studies. To determine how the RO responds to progesterone, the RO will be cultured ex vivo in the presence and absence of progesterone. I will perform a morphometric analysis of the RO, as well as collect secreted proteins from the media for proteomic analysis. Results/Anticipated Results: If the RO is important for ovarian homeostasis, I expect that in the absence of the RO, ovarian functions such as maintenance of the ovarian reserve and fertility will be impaired. Additionally, because the RO expresses progesterone receptors, I anticipate that the RO will be responsive to progesterone as shown in changes in the morphometric analysis and in changes in secreted proteins in the presence of progesterone. Discussion/Significance of Impact: A major gap in knowledge regarding female physiology and reproductive health is the role of the RO. We expect this work to reveal that progesterone signaling in the RO is important for regulating ovarian functions and to show that the RO is a critical modulator of female fertility and reproductive function.

Objectives/Goals: A key strategy in generating a protective HIV vaccine is the elicitation of broadly neutralizing antibodies (bnAbs), capable of neutralizing a large diversity of HIV-1 isolates. The goal of this study is to identify molecular signatures of HIV bnAb development early in life, to guide the development of a successful pediatric HIV vaccine strategy. Methods/Study Population: We previously defined HIV neutralization breadth in 40 ART-naive children living with HIV. Single-cell RNAseq was performed utilizing peripheral blood mononuclear cells (PBMCs) from the top 5 children with highest neutralization breadth scores and compared their transcriptome to that of PBMCs from 5 children that did not develop neutralization breadth within the first three years of life. Additionally, we incorporated analysis of PBMCs from 5 healthy uninfected children, matched to our experimental groups by race, ethnicity, and gender. Results/Anticipated Results: We expect that a distinct host transcriptional profile is associated with the development of HIV-specific antibody neutralization breadth in early life. Discussion/Significance of Impact: Identifying immune cell transcriptional profiles associated with neutralization breadth will lead to more targeted vaccine approaches for eliciting the appropriate B cell responses and provide an invaluable screening tool allowing early identification of vaccine candidates with the potential to induce bnAbs.

Objectives/Goals: Cerebral amyloid angiopathy-related inflammation (CAA-ri) is a spontaneous inflammatory cerebral vasculopathy that mimics complications of Alzheimer’s disease immunotherapies. Our objective is to evaluate imaging and cerebrospinal fluid (CSF) markers of blood–brain barrier (BBB) impairment and inflammation in CAA-ri. Methods/Study Population: We plan to enroll 20 patients total: 1) 10 patients with CAA-ri as defined by Auriel et al (JAMA Neurology 2016) (exposure group). 2) 10 patients with non-inflammatory CAA defined using Boston criteria 2.0 that do not also meet criteria for CAA-ri (control group). The primary outcome will be Ktrans, a parameter of BBB impairment calculated from dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of the brain. Secondary outcomes will include DCE-MRI parameter VL, CSF albumin index, CSF fibrinogen, CSF sPDGFR-β, CSF MMP-2, CSF MMP-9, CSF C3, CSF IL1β, CSF IL6, IL8, and TNFα. Statistical comparisons between the exposure and control groups will be made using Wilcoxon rank sum test. Results/Anticipated Results: We anticipate significantly higher levels of BBB impairment and inflammatory biomarkers from DCE-MRI and CSF in subjects with CAA-ri relative to control subjects with non-inflammatory CAA. Discussion/Significance of Impact: Biomarkers are essential to characterize risk factors, pathophysiology, and possible treatment targets in CAA-ri. We plan to use the results of the current study to inform longitudinal studies that will test whether these markers are useful in identifying not only the presence of CAA-ri but also severity, progression, and response to treatment.

Objectives/Goals: Men and women with opioid use disorder (OUD) show differences in their initiation, use patterns, and outcomes that may have biological underpinnings. Here we present data directly comparing adult male and female rats across heroin-induced behaviors in order to provide insight into the nuances of sex differences in OUD. Methods/Study Population: We first used a 6-hour intermittent access heroin self administration paradigm to quantify six distinct drug-taking and drug-seeking behaviors. Based on the sum of the z-scores for each behavior, we classified rats as having high- or low-severity phenotypes. In a separate group of rats, we adapted this classification system to a 10-minute continuous access self-administration paradigm to better represent the timeframe of use common in people. Finally, we examined locomotor sensitization following daily heroin injections in two groups of rats. The first were given 2 mg/kg/day i.p. heroin for 10 days and the second were given 0.55 mg/kg/day i.v. heroin for 20 days. Results/Anticipated Results: In the 6-hour intermittent access, both sexes showed variability across individuals, but a greater proportion of females were classified as having a high-severity phenotype compared to males. This difference in severity distributions was also found in the 1-hour continuous access experiment. Consistent with the literature, in our sensitization experiments, we found that males had a lower baseline level of locomotion compared to females. Across sex and route of administration, rats treated with heroin initially decreased locomotion, but returned to baseline over the course of treatment. Females given i.v. infusions showed a rapid escalation of locomotion past baseline that was not seen in males or following i.p. injections. Discussion/Significance of Impact: Our results indicate a consistent pattern of females having a greater behavioral response to heroin compared to males. This suggests a sex-based effect on OUD that may interact with gender-based influences. As such, future research needs to consider sex in the development of treatments for OUD and other substance use disorders.

Objectives/Goals: This study aims to uncover immuno-endotypes in sepsis-associated pediatric acute respiratory distress syndrome (SA-PARDS) by using single-cell RNA sequencing (scRNA seq) to analyze the immune cell populations of the lower respiratory tract of intubated pediatric subjects with SA-PARDS. Methods/Study Population: Inclusion criteria are age less than 18 years, admission to the PICU, diagnosis of SA-PARDS, and intubation. Both sepsis and PARDS will be defined using the most recent consensus definitions. Exclusion criteria include an order of limited resuscitation and clinician discretion. After informed consent is obtained, a tracheal aspirate and blood sample will be obtained on days 1, 3, and 7. Both samples will be processed for single-cell RNA seq via the HIVE platform per manufacturer protocol. cDNA libraries will then be sent for 150 base pair paired-end sequencing. Sequences will be aligned to a reference genome, and count matrices will be generated. The Seurat package in R will be used for cell-type annotation and analysis of differential gene expression. Clinical variables, labs, and outcomes will be recorded in REDCap. Results/Anticipated Results: We expect to find that subjects with nonresolving SA-PARDS, defined as intubation and mechanical ventilation, will have a monocyte/macrophage transcriptome characterized by continued hyper-inflammation (M1-like phenotype) that does not transition over time to an anti-inflammatory and pro-repair phenotype (M2-like). Additionally, we expect to see that subjects with non-resolving SA-PARDS will have evidence of continued inflammation driven by hyper-inflammatory neutrophils. Finally, we expect that subjects with non-resolving SA-PARDS will have epithelial cells characterized by continued upregulation of canonical pathways of innate immunity including interferon signaling and the damage associated molecular pattern recognition pathway. Discussion/Significance of Impact: The discovery of immuno-endotypes in SA-PARDS would represent a major step toward developing precision medicine therapies for this group of patients. It would simultaneously provide a strategy to reduce biological heterogeneity and identify novel pathways and targets for therapy.

Objectives/Goals: * Examination of the acute glucose-lowering response to physical activity within a comprehensive behavioral weight loss intervention in adults without T2DM. * Explore whether the acute response to yogadiffers from the acute response to brisk walking and to examine whether these responses vary across the intervention period. Methods/Study Population: Participants in the behavioral weight loss and aerobic exercise group will start with 100 minutes per week of moderate-intensity aerobic activity, increasing every four weeks to 250–300 minutes, spread over five days. Activities will be self-selected, such as walking. Participants in the combined aerobic exercise and yoga group will do aerobic exercise three days a week and yoga two days a week, also progressing from 100 to 250–300 minutes weekly. All participants will follow an energy-restricted diet (1200–1800 kcal/day) and participate in weekly education sessions to learn lifestyle modification skills for successful weight loss. The study will explore differences in acute responses to yoga versus walking and how these vary during the intervention, controlling for initial and changing weight status. Results/Anticipated Results: Primary and secondary outcomes from the parent study will include body weight, BMI, body composition (via DXA), cardiorespiratory fitness, energy intake, and physical activity. Glucose and insulin levels will be measured pre- and post-exercise, with HOMA-IR computed. Continuous glucose monitoring (CGM) will be used to track glucose responses during each session, with the area under the curve (AUC) as the primary metric. The study will also explore advanced CGM analytics in collaboration with the KUMC Diabetes Institute that will include in-depth analyzation of peak and trough change velocity as well as novel correlations betwen glucose dynamics and physical activity patterns with an aim to uncover insights that transcend conventional CGM analyses. Discussion/Significance of Impact: This study uses advanced CGM analytics to examine glucose control during physical activity, collaborating with experts to create comprehensive models for glucose fluctuations. It compares acute responses to walking and yoga, addressing a key gap in research, with potential clinical insights for managing glucose in obesity.

Objectives/Goals: Diamond Blackfan anemia (DBA) is caused by loss of ribosomal proteins leading to death of red blood cell progenitors. We identified a novel heterozygous variant (c.167+769C>T) in RPL30 in a patient with DBA. We hypothesized that this variant, in a gene not previously studied in DBA, would demonstrate DBA phenotype and reveal early drivers of disease. Methods/Study Population: To study the role of our novel variant, we developed an induced pluripotent stem cell (iPSC) model, including wild type (WT) and CRISPR-edited RPL30 mutant clones. We differentiated the iPSC into hematopoietic stem cells, identified cell populations with flow cytometry, and applied single-cell RNA sequencing. We identified erythroid clusters for differential gene expression analysis, using R Studio DESeq followed by Gene Ontology (GO) enrichment analysis. We are differentiating cells into red blood cells for further comparison with flow cytometry, bulk RNA sequencing, protein analysis, and hemoglobin staining. Our approach has relied on multidisciplinary expertise in clinical hematology and genetics, basic science study of ribosomes, computational biology, stem cell, and hematopoietic biology. Results/Anticipated Results: Compared to WT hematopoietic stem cells, RPL30mutant cells had significantly decreased expression of RPL30. Analysis of top differentially expressed genes revealed downregulation of HSPA1A which encodes heat shock protein 70 (HSP70), chaperone of a critical red blood cell transcription factor. Loss of HSP70 protein has been implicated in RPL-mutated red blood cells previously as a potential modulator of severe DBA phenotype. Upon GO enrichment analysis of downregulated genes, biologic process terms GO:0042254 ribosome biogenesis, GO:1903708 positive regulation of hemopoiesis, and GO:0045646 regulation of erythrocyte differentiation were all highlighted as driver terms. We expect further differentiation to reveal early death of RPL30mutant cells with associated downregulated HSP70. Discussion/Significance of Impact: Our results support our hypothesis that the RPL30 variant downregulates erythropoiesis, with a potential early role of HSP70 protein. Upon completion of our study, we will demonstrate the role of RPL30in DBA pathogenesis as well as provide understanding of its drivers, which is critical for improved management of this disease.

Objectives/Goals: Explore and compare the functional mechanisms of song-based exercises compared to speech-language pathology exercises for dysphagia. The long-term goal is to increase patient outcomes through song-based programs that are accessible, enjoyable, and personalizable. Methods/Study Population: We will pilot the use of combined electroencephalography (EEG) and electromyography (EMG) technologies to analyze both central and peripheral contributors to laryngeal control in a cohort of healthy individuals. This approach provides detailed insight into the coordination between neural and muscular activity, which will serve as a baseline for future studies in clinical populations. Song-based vocal exercises will be compared with standard dysphagia exercises prescribed by speech-language pathologists to assess their mechanistic differences. Results/Anticipated Results: We anticipate identifying specific song-based tasks, such as variations in pitch, rhythm, and intensity, which differentially impact laryngeal musculature. Additionally, we will localize neural activation hotspots using EEG during these tasks, providing a more comprehensive understanding of how song-based therapy influences both peripheral and central mechanisms. Discussion/Significance of Impact: This project will lay the groundwork for developing evidence-based song-based therapies for dysphagia, providing an alternative to traditional SLP exercises. By creating an engaging therapeutic program, we aim to reduce dysphagia’s healthcare burden, including aspiration events, healthcare costs, and related mortality.