Objectives/Goals: Common mamographic findings, breast arterial calcifications (BAC), are a sex-specific biomarker for cardiovascular disease in women. While less common, BAC is frequently encountered on mammograms in males. This study aims to investigate BAC and its association with cardiovascular risk factors in men. Methods/Study Population: A retrospective single-institution study of male patients with mammography studies was performed between September 1, 2018, and September 3, 2025. Diagnostic mammographic images of male patients from a high-volume breast imaging facility were evaluated for the presence of BAC. BAC severity will be assigned a score based on the Canadian Society of Breast Imaging’s grading system. Unique ICD-10 diagnosis codes from the electronic medical record that have been established as independent risk factors for cardiovascular disease will also be reviewed. Male patients who have a prior history of myocardial infarction, coronary revascularization, stroke, heart failure, peripheral vascular disease, breast cancer, dementia, or chronic dialysis/renal transplant were excluded. Results/Anticipated Results: The study is in process of pulling male mammograms from our institution. Current volume of male mammograms at the institution is about 160 male mammograms performed per month. Therefore, we anticipate having around 13,600 male mammograms to analyze the BAC presence. Anticipated results include that patients with independent cardiovascular risk factors such as increased systolic blood pressure, increased LDL levels, and current smokers will have a higher prevalence of BAC presence. Additionally, those with a presence of BAC will be older compared to those that have an absence of BAC on mammography. If BAC is shown to be a significant risk factor for the development of cardiovascular disease, perhaps a mammogram might be recommended in men purely for cardiovascular risk assessment. Discussion/Significance of Impact: A significant association between the presence of BAC and CVD risk factors may provide a supplementary assessment of CVD risk among men, offering a novel tool for risk stratification. Implementing a standardized BAC reporting model may allow for earlier identification of asymptomatic patients at high risk for adverse cardiac events.

Objectives/Goals: EEG neurofeedback is a promising neuromodulation approach for improving mental health outcomes but lacks standardized, neurophysiologically-grounded treatment protocols. This study uses high-density EEG (hdEEG) analyses to identify neural network patterns that inform development of evidence-based pediatric neurofeedback protocols. Methods/Study Population: This translational neuroscience project leverages resting-state hdEEG data and behavioral assessments from 600 six-year-old children in the Beginnings study (Arkansas Children’s Research Institute; Andres, NCT00616395). Analyses will identify functional and effective connectivity patterns across major brain networks – Default Mode (DMN), Salience (SN), and Central Executive (CEN) – using graph theoretical metrics, phase-locking values, and multivariate analyses. Identified network patterns will be linked with neuropsychological measures, including the Child Behavior Checklist (CBCL), to inform development of standardized neurofeedback protocol recommendations grounded in developmental neuroscience. Results/Anticipated Results: We anticipate identifying distinct patterns of functional connectivity that predict variation in children’s emotional and behavioral functioning. Findings are expected to highlight network patterns – particularly within emotion regulation and executive control circuits – that can guide neurofeedback protocol design. Anticipated products include neurophysiologically informed recommendations for standardized frequency bands, target networks, and protocol parameters to support future clinical trial development. This project represents a key T1 translational step toward establishing a neuroscience-based framework for EEG neurofeedback treatment protocols. Discussion/Significance of Impact: By integrating neurophysiological and behavioral data to guide protocol development, this work addresses a critical translational gap in the EEG neurofeedback literature. Findings will establish a foundation for standardized, evidence-based neurofeedback interventions that could improve accessibility and efficacy in clinical settings.

Objectives/Goals: Type 2 diabetes is a metabolic disease characterized by non-resolving inflammation. Our objective is to investigate how macrophages contribute to this dysregulation. We hypothesize that diabetic macrophages have impaired response mechanisms and are more sensitive to metabolic flux, limiting their ability to effectively resolve inflammation. Methods/Study Population: To study this, we isolated and cultured bone marrow-derived macrophages from WT and db/db mice in vitro and stimulated them with maturation, inflammatory, and resolving cues. We also isolated human monocytes from PBMCs from patients with type 2 diabetes and age-matched healthy controls and stimulated them ex vivo and cultured them in vitro. We assessed their timeline of maturation through changes in cell surface markers, their functional inflammatory response through phenotypic marker changes and cytokine release, and their sensitivity to metabolic flux by culturing in the presence or absence of extracellular glutamine. Together, these responses can allow us to understand intrinsic defects in diabetic macrophages’ ability to regulate inflammation. Results/Anticipated Results: Notably, diabetic and healthy macrophages display distinct maturation timelines. Overall, diabetic macrophages lag behind healthy macrophages in expression of CD45 and F4/80 but eventually acquire a similar level of mature cell surface markers by day 7-8 in culture. In addition, diabetic cells have a higher proportion of Ly6Chi cells earlier in culture, cells that are generally more poised to be pro-inflammatory. Both healthy and diabetic mature macrophages displayed similar levels of inflammatory response cytokines, inflammatory gene expression changes, and phenotypic marker expression. Interestingly, stimulating these macrophages in the absence of glutamine revealed underlying impairments in their response to inflammatory and resolving cues. Discussion/Significance of Impact: Patients with diabetes suffer significant complications, such as susceptibility to infections and non-healing wounds, driven by impairments in resolution of inflammation. This research begins to explore the cell-mediated inflammatory aberrations in type 2 diabetes and may reveal targetable mechanisms to improve patient outcomes.

Objectives/Goals: Colorectal cancer (CRC) disproportionately affects the U.S. Deep South. Socioeconomics, genetics, and healthcare access contribute to increased incidence and mortality. A next-generation endoscope with improved contrast and automatic detection may boost early detection and narrow expertise gaps between top centers and rural clinics. Methods/Study Population: We implemented excitation-scanning hyperspectral imaging (Ex-HSI) to discriminate normal and cancerous mouse colorectal tissues by detecting changes in autofluorescence. Ex-HSI permits detection of all emitted light above a cutoff wavelength. Ex-HSI reduces acquisition time and improves signal-to-noise ratio compared to traditional emission scanning. We used azoxymethane/dextran sodium sulfate (AOM/DSS) to induce colitis and nodule formation in the mouse CRC model. Ex-HSI data was complemented with transmitted light and confocal images. Histology sectioning with H&E staining was obtained for “gold standard” validation. Results/Anticipated Results: Inflammation and bleeding were observed, consistent with AOM/DSS treatment. Nodules were visible using 4x and 20x objectives. Background-subtracted Ex-HSI data was corrected to a flat spectral response. Excitation spectra were extracted from select regions within each field of view. Excitation spectra displayed multiple peak excitation wavelengths, suggesting multiple autofluorescent molecules present. Further investigation will utilize spectral unmixing, principal component analysis, and neural networks to interpret image data. Discussion/Significance of Impact: A hyperspectral endoscope that detects early-stage cancers and precancers would enhance CRC screening in the Deep South. This research is supported by NIH P01HL066299, NIH UL1TR00141, S10RR027535, S10OD02860, NSF MRI1725937, CCTS T32TR004767, and the Center for Lung Biology, University of South Alabama.

Objectives/Goals: This study evaluates the significance of PU.1 upregulation and the PU.1-mediated regulation of the anti-inflammatory miR-146a on pro-inflammatory NF-kB signaling in RCC-derived macrophages. The downstream effects of PU.1 targeting on key phenotypes related to RCC immunity and how this impacts RCC progression will also be examined. Methods/Study Population: Bioinformatics data identifying PU.1 upregulation within RCC tumors, correlation with prognostic clear cell RCC markers, and survival analysis were obtained using The Cancar Genome Atlas (TCGA) RNA-sequencing data. Murine RCC cell line Renca possessing a Vhl deletion are used to recapitulate a common deletion in human clear cell RCC tumors. This study utilizes two methods to target PU.1 in orthotopic RCC, a conditional PU.1 deletion model and a pharmacologic PU.1 inhibitor model. Quantitative real-time PCR, western blotting, and immunofluorescent staining will evaluate NF-kB signaling in PU.1-targeted macrophages and murine RCC tumors. Flow cytometry and single-cell RNA-sequencing will reveal the effects of PU.1 targeting on key immune cell phenotypes and key metrics of RCC outcomes. Results/Anticipated Results: PU.1 mRNA and protein expression are upregulated in human clear cell RCC tumors compared to normal kidney tissue. A 10-year overall survival analysis in clear cell RCC tumors indicates that increased PU.1 mRNA expression is unfavorable (p = 0.0298; HR = 1.24; N = 133/133). PU.1 inhibition in orthotopic RCC reduces tumor volume, compared to control (p = 0.0328; N = 5/5). NF-kB signaling effectors are upregulated in PU.1-inhibited macrophages, including canonical miR-146a targets. We anticipate that PU.1-targeted macrophages and RCC tumors will yield significant increases in NF-kB signaling, pro-inflammatory macrophage phenotypes, increased antitumor immunity, and reduced tumor volume. Discussion/Significance of Impact: This is the first study to address PU.1 upregulation in RCC and use multiple methods to examine a potential function of PU.1 in immunosuppressive macrophages and RCC immunity. This work also assesses the relevance of miR-146a on canonical, pro-inflammatory NF-kB signaling in the novel context of RCC tumor immunity.

Objectives/Goals: Intratumoral B-cells are associated with better survival, but their antigenic targets are unclear. Here, I will delineate B-cell responses in human papillomavirus (HPV)-positive head and neck cancer (HNC) patients against all major HPV E proteins to identify immunodominant responses. Methods/Study Population: To profile tumor-infiltrating antibody-secreting cells (ASCs), I will analyze freshly prepared single-cell suspensions of lymphocytes from HPV+ HNC tumors using ELISpot assays. To profile circulating memory B-cells in circulation, peripheral blood mononuclear cells obtained at time of surgery will be differentiated into ASCs, followed by ELISpot analysis. To assess systemic antibody titers, ELISAs will be performed on matched plasma samples. Due to the reliance on fresh tumor samples, our study population consists mostly of stage I HPV+ HNC patients, for which surgical resection is often the primary treatment option. Notably, HPV+ HNC is known to disproportionally affect males. Results/Anticipated Results: I will identify the immunodominant targets of the HPV-specific B-cell response in HPV+ HNC. Based on my preliminary data, I expect that the HPV-specific responses will primarily target E1, E2, and E4, as opposed to the classical oncoproteins E6 and E7. I expect the magnitude of the intratumoral HPV-specific ASC response to be reflected in the systemic antibody level, with systemic antibodies, thus serving as viable surrogates for intratumoral HPV-specific responses. Additionally, I expect minimal presence of intratumoral B-cell response against antigens unrelated to the tumor, such as influenza or Epstein–Barr virus, in contrast to abundant systemic antibody titers and memory B-cells. Discussion/Significance of Impact: The majority of current immunotherapeutic treatment options for this malignancy focus solely on targeting E6 and E7. However, if other HPV E proteins are shown to be more immunogenic, targeting these antigens could enable more efficient immunotherapies.

Objectives/Goals: Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neuromodulation therapy FDA-cleared for adjunct treatment of major depressive disorder (MDD) in adolescents. Here, we describe a double-blind, randomized study that explores the use of anhedonia severity as a predictor of treatment response. Methods/Study Population: This study included 41 participants (25 females) aged 12–18 years who had a Children’s Depressed Rating Scale-Revised (CDRS-R) score greater or equal to 40. Participants underwent daily rTMS at either 1 Hz or 10 Hz (randomly assigned) 5 days/week, for 6 weeks. Anhedonia was measured via a composite score using certain items from the CDRS-R and Beck Depression Inventory (BDI)-II. Treatment response was assessed via the Clinical Global Impressions – Improvement (CGI-I) scale. A logistic regression approach was used to estimate the likelihood of clinical improvement from baseline. A linear mixed model of repeated measures was used to assess changes in anhedonia scores over the study duration, across both frequency groups. Results/Anticipated Results: A significant improvement in anhedonia scores was observed across both 1 Hz (p< 0.0001, d=0.8536) and 10 Hz (p<0.0001, d=0.9032) groups. However, no significant group differences (p=0.2559) were observed for 1 Hz (m=5.121, SE=0.450) versus 10 Hz (m=5.894, SE=0.486). Notably, our analysis revealed a significant inverse relationship between changes in anhedonia symptom severity and CGI-I response for both 1Hz (d= -1.1017, SE=0.5003, p=0.0277) and 10 Hz (d= -0.8581, SE=0.3371, p=0.0109) groups. No significant interaction between TMS treatment group and changes in anhedonia or CGI-I scores were observed (p=0.3204). TMS stimulation frequency (1 Hz vs 10 Hz) did not have specific effects on the change in anhedonia scores or CGI-I response. Discussion/Significance of Impact: This study shows that TMS (1 Hz or 10 Hz) can help with specific symptomology in depression via significant decreases in anhedonia and how anhedonia may moderate overall TMS response. Future research should further elucidate predictors or moderators of TMS treatment response to inform clinical decision-making and care for adolescents with MDD.

Objectives/Goals: We recently showed that a single dose of psilocybin, a serotonergic psychedelic, attenuated mechanical hypersensitivity for up to 28 days in a rat model of formalin-induced chronic centralized pain. The goal of this study is to determine whether psilocybin can alleviate chronic pain in a rat model of reserpine-induced chronic centralized pain. Methods/Study Population: Adult Sprague-Dawley rats (male=12, female=12) will be surgically equipped with a chronic indwelling catheter in jugular vein for the delivery of psilocybin. After 14 days of post-surgical recovery, rats will undergo Randall–Selitto testing to assess their baseline muscle pressure threshold. Thereafter, the rats will receive subcutaneous reserpine (1mg/kg) for three consecutive days, which depletes biogenic amines that have been reported to be reduced in fibromyalgia patients. The rats will be tested again on the Randall–Selitto assay on post-reserpine days 1, 3, 5, 7, 14, 21, and 28. One subgroup of the rats will receive intravenous psilocybin (1mg/kg) on post-reserpine day 4, while the other subgroup will receive intravenous saline. Results/Anticipated Results: We have completed data collection from four female rats which shows that compared to baseline, reserpine reduced muscle pressure threshold on post-reserpine days 1–21 (mean ± SD, day 1: -18.33%±7.26%, day 3: -15.05%±7.2%, day 5: -29.19%±3.75%, day 7: -19.64%±14.67%, day 14: -30.02%±10.14%, day 21: -14.95%±12.9%). On day 28, muscle pressure thresholds approached baseline levels (-8.12%±14.73%). These data show that we have successfully reproduced the reserpine model in our laboratory. Based on our previous study showing alleviation of mechanical hypersensitivity in formalin-treated rats after a single dose of intravenous psilocybin, we expect that psilocybin will shorten the duration of reserpine-induced mechanical hypersensitivity as compared to the rats receiving 0.9% saline as vehicle control. Discussion/Significance of Impact: These studies are expected to provide a neuroscientific foundation for the emerging use of serotonergic psychedelics as a potential treatment for chronic pain conditions, including fibromyalgia.

Objectives/Goals: The specialized riboflavin (vitamin B2) biosynthetic enzymes are targets for novel antibiotics. This project provides direct evidence for the chemical mechanism of Mycobacterium tuberculosis lumazine synthase, the penultimate enzyme of the riboflavin production and target for therapeutic intervention. Methods/Study Population: Lumazine synthase catalyzes the condensation of 3,4-dihydroxy-2-butanone-4-phosphate and 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione forming 6,7-dimethyl-8-ribityllumazine. To determine the chemical mechanism of Mycobacterium tuberculosis lumazine synthase, we performed transient state kinetics to monitor the formation and disappearance of intermediates and products over time. These traces indicated time points to flash-freeze the reaction to isolate intermediates that are analyzed with 13C-NMR to determine the chemical structure. Time resolved X-ray crystallography is used as a complementary method to determine the structures of the reaction intermediates in the context of the active site. Results/Anticipated Results: The NMR data provide direct evidence of the chemical mechanism of lumazine synthase. Complementary crystal structures of lumazine synthase with intermediates bound to the active site will corroborate the chemical mechanism determined by NMR and model key residues in the active site that facilitate catalysis. These data will inform steps of the chemical mechanism that can be targeted for therapeutic intervention. Discussion/Significance of Impact: This work informs drug design to address the growing threat of antibiotic resistance. The riboflavin biosynthetic enzymes do not have human homologs, so drugs designed to specifically target these enzymes will minimize off target effects.

Objectives/Goals: Opioids treat pain but with the risk of severe adverse effects. The opioid positive allosteric modulator BMS-122 has been shown to enhance opioid antinociception in animal models without increasing adverse effects. However, whether BMS-122 is effective in chronic neuropathic pain is unknown. Methods/Study Population: We used two animal models: spared nerve injury (SNI) and tibial neuroma. Injuries were performed on male and female Sprague-Dawley rats. Animals developed robust sensitivity to tactile stimulation similar to that seen in patients. Animals with SNI received the mu-opioids methadone, morphine, and buprenorphine, or gabapentin (30 mg/kg, s.c.), with or without BMS-122 (10 mg/kg, s.c.). Tactile hypersensitivity was measured using von Frey filaments. Animals with tibial neuroma received methadone (s.c.) with or without BMS-122 (10mg/kg s.c.), and tactile hypersensitivity was measured using a modified Tinel’s test, in which the neuroma was directly stimulated. Results/Anticipated Results: Hypersensitivity following SNI was reversed with administration of high-dose opioids or gabapentin. Following pre-treatment with BMS-122 low, ineffective doses of opioid or gabapentin significantly reversed the hypersensitivity. Buprenorphine was unable to fully reverse the hypersensitivity alone but was fully efficacious in the presence of BMS-122. Animals with tibial neuroma developed hypersensitivity at the neuroma site, but the doses of methadone required to reverse this sensitivity also induced respiratory depression. In the presence of BMS-122, a low, non-effective, dose of methadone reversed hypersensitivity without depressing breathing. No sex differences were observed in either condition. Discussion/Significance of Impact: These data show that positive allosteric modulators like BMS-122 provide an opioid-sparing effect in chronic neuropathic pain. Furthermore, BMS-122 appears to enhance endogenous opioid peptides to increase the effectiveness of non-opioid analgesics. Overall, these data support the continued development of modulators as therapeutics.

Objectives/Goals: The WF DICQI team uses targeted messaging across many platforms to inform investigators across the Advocate Health (AH) enterprise of available programs and services, turning research into action. Translational science is valued at Wake Forest University School of Medicine, the academic core of AH. Methods/Study Population: The DICQI team partners with all Wake Forest CTSI teams to create tailored communication and dissemination plans for their resources and initiatives, designed to help investigators translate research into practice. Participants include program leaders, staff, and high users of CTSI services. Efforts focus on interviewing previous program participants and producing related articles, alongside developing targeted plain language messaging for internal newsletters, social media, and providing customized flyers and email content. This initiative enhances awareness among investigators along with driving engagement and collaboration. Results/Anticipated Results: These dissemination strategies motive investigators to use CTSI resources and engage in continuous learning. An increase in CTSI service usage is expected after implementing the dissemination plans. Plain language messaging and visually appealing graphics make scientific findings more accessible to a broader audience, so increased engagement on social media is also anticipated. These efforts make it easier for readers to understand research findings, apply them, and encourage collaboration. We will work with more AH centers and programs to create tailored plans and amplify the impact of WF CTSI. We plan to share our approach with other institutions, fostering collaboration nationwide. Discussion/Significance of Impact: The WF CTSI DICQI’s dissemination efforts are designed to increase awareness and utilization of available resources, which will drive more robust research from investigators, and more engagement from a broader audience, bridging practice–research collaboration across the AH enterprise.

Objectives/Goals: The Human Immune Dysregulation Evaluation Framework (Hi-DEF) quantifies myeloid dysregulation in infectious and non-infectious illness using peripheral blood gene expression (Moore, Nat Med). We evaluated immune dysregulation in obesity and quantified the changes in dysregulation due to lifestyle and medical interventions. Methods/Study Population: We assessed the association of obesity with myeloid dysregulation in the Framingham Cohort using linear regression. In two additional datasets (GSE66175, GSE19790), we assessed the effect of dietary interventions and bariatric surgery on myeloid dysregulation using paired Wilcoxon rank sum test pre- and post- intervention and Pearson’s correlation. Results/Anticipated Results: In the Framingham cohort (n=5321), myeloid dysregulation score was associated with BMI (p=2.2e-6) after correcting for age and sex. Patients assigned to diet/exercise experienced significant reductions in myeloid score after 3 months (n=63, p=1.2e-7), which was not seen in controls (n=63, p=0.29). Change in myeloid score was significantly correlated with change in weight loss (r=0.32, p=0.006). This reduction in myeloid dysregulation was also seen post-bariatric surgery (n=11, p=0.0001). Discussion/Significance of Impact: Patients with obesity have significantly higher myeloid dysregulation compared to healthy subjects, which is modifiable with lifestyle and medical interventions. These results suggest that we may be able to quantify and monitor immune dysregulation from obesity which may place these patients at risk for worse outcomes.

Objectives/Goals: The impact of genetic alterations on survival in patients with neuroendocrine prostate cancer (NEPC) is unclear. This study assessed how genetic alterations and tumor mutational burden (TMB) affect overall survival (OS) in patients with NEPC treated at Mayo Clinic. Methods/Study Population: Demographic, clinicopathologic, and genomic data from patients with NEPC treated at Mayo Clinic (2015–2025) were abstracted from electronic medical records. Patients had consented to research and received at least one cycle of therapy. Descriptive statistics summarized patient characteristics, and survival was estimated with Kaplan–Meier analysis. OS was compared by disease type (de novo vs treatment-emergent NEPC), TMB (high vs low), and RB1, TP53, and PTEN alteration status. Results/Anticipated Results: Among 66 patients in the study, the average age at NEPC diagnosis was 67.5 years. There were no significant differences between the age at diagnosis of de novo and treatment emergent NEPC (t-NEPC). t-NEPC made up 77.3% of the cohort. Median follow-up was 46.5 months (IQR 28–120). The most common alteration was TP53. RB1 alterations were associated with significantly shorter OS compared to patients without RB1 alterations (mOS Not Reached (NR) vs 13.8 months; p = 0.0056). Similarly, patients with high TMB had worse overall survival (mOS NR vs 14.9 months; p = 0.077). We did not observe worse OS in patients with alterations in TP53 or PTEN. RB1 alteration and high TMB were the strongest predictors of worse survival. Discussion/Significance of Impact: RB1 alterations and high TMB predict poor survival in patients with NEPC, highlighting potential biomarkers for risk stratification. TP53 and PTEN did not significantly affect OS. Early genomic profiling and targeted management of actionable alterations such as BRCA and ATM may improve survival and inform future translational studies.

Objectives/Goals: Colorectal cancer risk varies across populations. Bile acid (BA) composition and the gut microbiome can impact CRC risk, but their differences across populations is poorly understood. We characterized serum BA and gut microbiome profiles in non-Hispanic Black and White Americans and examined associations with diet and social determinants of health. Methods/Study Population: To characterize BA composition, participants from the Chicago Multiethnic Prevention and Surveillance Study with a banked serum sample (n=100) were selected, with a subset of participants having completed a diet recall survey (ASA24; n=23). Sixty-two BA (primary, secondary, and glyco/tauro-conjugated BA) from serum samples were analyzed using liquid chromatography-mass spectrometry. For microbiome profiling, normal colonic biopsy samples from non-cancer participants were obtained from the Digestive Disease Research Core Center’s Integrative Clinical and Biospecimens Core (University of Chicago; n=109). Following DNA extraction, the mucosally adherent microbial community was assessed by 16S rRNA sequencing. Results/Anticipated Results: Of the BA detected in over 80% of the samples, glycochenodeoxycholic acid (GCDCA) and deoxycholic (DCA) had the highest median concentrations (56.7 and 55.0 µg/mL, respectively). Moderate positive correlations between DCA/isoDCA and calories, fat, and cholesterol were observed. The linear regression model employed to understand the relationship between log-transformed DCA and race, age, sex, and area deprivation index highlighted age as a key predictor of DCA level when controlling for other covariates. Microbiome analysis revealed that diversity was not different when comparing sexes, races, or colonic anatomic locations. At the phylum level, taxa belonging to the Bacillota and Bacteroidota phyla were common across samples. Discussion/Significance of Impact: These data and subsequent analysis, including additional BA analysis, improvement of the regression model, and deeper analysis of microbiome community related to BA metabolism, will provide additional context to the role of social and biological factors in better understanding differences in CRC risk and pathogenesis across human populations.

Objectives/Goals: There remains an urgent need for the development of safe and effective therapeutics for the long-term management of pain. We evaluated our novel analgesic in multiple pre-clinical models to assess its abuse liability, analgesic efficacy in models of chronic pain, and its mechanism of action in ex vivo spinal cord slices. Methods/Study Population: We evaluated SSA3’s abuse liability by utilizing an IV self-administration paradigm. In this experiment, rats are placed in a chamber and have the option to press a drug-infusion lever or a control lever. Abuse liability was determined based on extent of lever pressing on the drug-infusion lever. To evaluate chronic pain efficacy, mice underwent a spared nerve injury (SNI) which induces sustained hypersensitivity in their hind paw. Mice were then treated with SSA3, and their hypersensitivity was assessed. Finally, the mechanism of action was investigated through patch clamp electrophysiology recordings taken from excised spinal cord slices in mice. We recorded currents from neurons involved in the pain signaling pathway before and after application of our compound to observe drug efficacy at the cellular level. Results/Anticipated Results: Our results demonstrated that in a pre-clinical IV self-administration paradigm, SSA3 exhibited no abuse liability, indicated by a lack of drug intake or escalation of drug intake. Results from our SNI model of chronic pain reveal that when SSA3 is administered directly onto the spinal cord via intrathecal injection, it is efficacious in reversing the pain hypersensitivity established by the model, suggesting a spinally mediated mechanism of action. Finally, we observed that SSA3 effectively reduced the amplitude of NMDA receptor-mediated excitatory postsynaptic currents in a concentration-dependent manner. This reduction in amplitude suggests our compound’s analgesic efficacy comes from the inhibition of spinally mediated NMDA receptor signaling. Discussion/Significance of Impact: This investigation into the therapeutic potential of substituted agmatine for the management of chronic pain revealed several key insights. Our drug is efficacious in models of chronic pain and, importantly, lacks the abuse liability commonly seen with analgesic drugs. Our drug’s mechanism of action involves inhibition of spinal NMDA receptors.

Objectives/Goals: Heart rate variability (HRV) is the variation in time between each heartbeat and an important biomarker of autonomic nervous system function, with higher HRV generally associated with improved parasympathetic tone and cardiac health. The most reliable measurement is during sleep, but sleep and HRV are both understudied in cirrhosis and HE. Methods/Study Population: We longitudinally assessed sleep and resting cardiovascular biomarkers in a cohort (n=119) of patients with cirrhosis using Oura, a commercially available fitness tracker. Baseline clinical data, psychometric hepatic encephalopathy score (PHES) and 6 months of passive sleep data collection were obtained. 117 participants enrolled, 6 collected no sleep data, 13 did not complete PHES; 10,065 nights (n=98) were available, and 9,692 were analyzed after outlier trimming (top and bottom 1% of data). Mixed effects modeling (MEM) assessed associations. Pre-specified covariates included age, gender, sodium, albumin, bilirubin, INR, and ascites. Missing data was not imputed. Results/Anticipated Results: In univariable analysis, impaired cognition by PHES was associated with increased average HRV (B 5.2 95%CI 0.25, 10.1 p=0.04) but not respiratory rate, heart rate, or lowest measured heart rate. CTP Score (B -1.8 95%CI -3.0, -0.6 p<0.01) and INR (B -6.1 95%CI -11.6, -0.5 p=0.03) were inversely associated with HRV. Higher baseline albumin (B 3.9 95%CI 0.34, 7.4 p=0.03) and sodium (B 1.0 95%CI 0.18, 1.75 p=0.02) were associated with increased HRV, while age, gender, and creatinine did not significantly associate with HRV. In a MEM adjusting for the pre-specified covariates, the association was greater (B 8.85 95%CI 3.85, 13.9 p<0.01), remaining significant after log-normalizing HRV. In a sensitivity analysis adjusting for current alcohol use or prior variceal bleed, the results were unchanged. Discussion/Significance of Impact: In a large cohort in cirrhosis, cognitive impairment was associated with increased HRV, in contrast to other markers of liver disease, where HRV was reduced and unrelated to other cardiovascular measures. This paradoxical relationship, if validated, could be developed as a longitudinal digital biomarker for the detection of cognitive impairment in HE.

Objectives/Goals: To define how stress alters dopamine system function in PTSD with comorbid psychosis and determine whether orexin receptor antagonism with suvorexant can normalize dopamine neuron activity by targeting the insular cortex–orbitofrontal cortex (IC–OFC) circuit. Methods/Study Population: We used in vivo electrophysiology to record ventral tegmental area dopamine neuron population activity in rats following restraint stress. Chemogenetic and pharmacological manipulations targeted the IC–OFC pathway to assess its role in stress-induced dopamine dysregulation. Suvorexant, a dual orexin receptor antagonist, was administered systemically or directly into the OFC. Fiber photometry recordings measured IC-evoked glutamatergic transmission within the OFC to determine how suvorexant modulates circuit-specific activity contributing to altered dopamine function. Results/Anticipated Results: Foot shock stress produced robust increases in dopamine neuron population activity. Intra-OFC suvorexant infusion reversed these effects, restoring dopamine system function to baseline levels. Chemogenetic inhibition of the IC–OFC circuit produced a similar normalization, confirming this pathway’s role in stress-induced dopamine dysregulation. Fiber photometry revealed that suvorexant attenuated IC-evoked glutamatergic signaling in the OFC, indicating that orexin receptor antagonism modulates excitatory input within this cortical circuit to restore balanced dopamine activity. Discussion/Significance of Impact: These findings identify the IC–OFC circuit as a key mediator of stress-induced dopamine dysfunction and reveal orexin receptor antagonism as a promising strategy for treating PTSD with comorbid psychosis. Targeting cortical orexin signaling may enable circuit-specific precision therapies.

Objectives/Goals: The goal of this study is to explore the lysogenized bacteriophages of clinical isolates of Pseudomonas aeruginosa (Pa) for their capacity to affect intraspecies competition. This study also aims to explore the capacity of using clinical isolates as a source of new candidates for phage therapy. Methods/Study Population: We compiled a collection of 207 Pa isolates and screened them for the presence of lysogenic phages using polymerase chain reaction (PCR). A subset of 96 phage containing clinical isolates was tested for intra-strain infectivity. Using a representative subset of isolates producing infective and non-infective phage, we evaluated competitive dynamics in liquid co-cultures, bacterial biofilms and in a murine chronic wound model. Results/Anticipated Results: Among all isolates, 74.3% contained detectable phages in their genome, and 63.7% contained detectable phage in their supernatants. Among those tested for intra-strain infectivity, 82.2% of strains demonstrated inhibition of growth on lawns of at least one other strain, while 94.8% of these strains were susceptible to at least one other supernatant. In in vitro co-culture competition experiments, strains producing infective phages outcompeted competitors more effectively than strains producing non-infective phage. Infective phage producing strains were more resistant to biofilm invasion than non-infective counterparts. In vivo competition experiments showed that PAO1 was significantly reduced when co-infecting with an infective phage-producing strain compared to a non-infective one. Discussion/Significance of Impact: Collectively, these data demonstrate that clinical isolates of Pa frequently encode active bacteriophages that can mediate intraspecies competition and confer a competitive advantage during infection. Future work will center on combining these phages as therapeutic cocktails targeting acute Pa infections.

Objectives/Goals: There remains an urgent need for the development of safe and effective therapeutics for the long-term management of pain. We evaluated our novel analgesic in multiple pre-clinical models to assess its abuse liability, analgesic efficacy in models of chronic pain, and its mechanism of action in ex vivo spinal cord slices. Methods/Study Population: We evaluated SSA3’s abuse liability by utilizing an IV self-administration paradigm. In this experiment, rats are placed in a chamber and have the option to press two different levers. Abuse liability was determined based on extent of lever pressing on the drug-infusion lever. To evaluate chronic pain efficacy, mice underwent a spared nerve injury (SNI) which induces sustained hypersensitivity in their hind paw. Mice were then treated with SSA3, and their tactile hypersensitivity was assessed. Finally, the mechanism of action was investigated through patch clamp electrophysiology recordings taken from excised spinal cord slices in mice. We recorded currents from neurons involved in the pain signaling pathway before and after application of our compound to observe drug efficacy at the cellular level. Results/Anticipated Results: Our results demonstrated that in a pre-clinical IV self-administration paradigm, SSA3 exhibited no abuse liability, indicated by a lack of drug intake or escalation of drug intake. Results from our SNI model of chronic pain reveal that when SSA3 is administered directly onto the spinal cord via intrathecal injection, it is efficacious in reversing the pain hypersensitivity established by the model, suggesting a spinally mediated mechanism of action. Finally, we observed that SSA3 effectively reduced the amplitude of NMDA receptor-mediated excitatory postsynaptic currents in a concentration-dependent manner. This reduction in amplitude suggests that our compounds analgesic efficacy comes from the inhibition of spinally mediated NMDA receptor signaling. Discussion/Significance of Impact: This investigation into the therapeutic potential of substituted agmatine for the management of chronic pain revealed several key insights. Our drug is efficacious in models of chronic pain and, importantly, lacks the abuse liability commonly seen with analgesic drugs. Our drug’s mechanism of action involves inhibition of spinal NMDA receptors.

Objectives/Goals: Cholangiocarcinoma is an aggressive cancer with a poor prognosis. Fibroblast Growth Factor Receptor 2 fusions are therapeutic targets in ~20% of cholangiocarcinoma, which restricts their benefit to only a subset of patients. We investigate reverse phase protein array, as a novel assay for biomarker identification in cholangiocarcinoma. Methods/Study Population: Tumor samples were obtained via our registry (OSU-13053, NCT02090530). RPPA analysis was performed on tumor samples from 24 cholangiocarcinoma patients. Of these, 12 had FGFR2 fusions, 4 had FGFR2 SNVs, and 8 were FGFR wildtype. Tumor content was enriched using laser capture microdissection prior to analysis by reverse phase protein array. RPPA signal was measured by antibody binding to the phosphorylated tyrosine residue at location Y653.654 of FGFR2. Additional analysis was done for the following proteins: CD3, RSK, SGK1, STAT3, VEGFR2, 4EBP1, AKT, EGFR, eIF4G, ERK, FSR2, HLA, mTOR, p38 MAPK, p70S6K, p90RSK, PD-L1, PDGFR, RET, SHC, STAT, and ZAP70. Results/Anticipated Results: There was not a significant difference between FGFR2-phosphorylation in tumors with FGFR2 genomic alterations and tumors with wildtype FGFR. On the other hand, CD3 protein abundance was significantly lower in FGFR-altered tumors compared to wildtype, potentially indicating a cold tumor immune microenvironment in these patients. We plan to perform RPPA analysis on 61 more cases (17 FGFR-altered and 44 FGFR-wildtype) to generate a more robust sample size for subsequent analysis. Furthermore, we have access to 580 additional archival cholangiocarcinoma tumors for RPPA analysis (IRB approved protocol OSU-15030). We plan to sequence these tumors to determine FGFR status. We predict approximately 116 (20%) of these cases to be FGFR-altered. Discussion/Significance of Impact: Our results suggest that FGFR wildtype tumor may have increased FGFR pathway activation and could respond to FGFR inhibitors. Additionally, the cold tumor microenvironment in FGFR-altered tumors provide rationale for combination of FGFR inhibitors and immunotherapy. These results will be confirmed by RPPA analysis on additional cases.