bluuebunny/crossref_metadata_2025 huggingface dataset:
337 Cellular senescence contributes to inflammation and disease progression in an animal model of multiple sclerosis
Authors: Manavi Zeeba , Gross Phillip , Baydyuk Maryna , Huang Jeffrey
Published: April 2024

OBJECTIVES/GOALS: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. MS affects more than two million people worldwide, resulting in physical disability, cognitive impairment, and decreased quality of life. We are investigating the role of senescent cells, a hallmark of aging, in inflammation and disease progression in MS. METHODS/STUDY POPULATION: Female mice on a C57BL/6 background were subjected to the Hooke Laboratory EK-2110 experimental autoimmune encephalomyelitis protocol (EAE; n=10) to induce hind limb paralysis, and matching control mice received no injections (naīve; n=10). Immunofluorescent staining was used to visualize senescence cells and their localization in spinal cord tissue sections from naīve and EAE mice based on antibody detection of cell surface or intracellular proteins. Tissue sections from each group were analyzed in duplicates for each antibody (n=3-4/group). Flow cytometry was performed to immunophenotype the senescence cells using conjugated fluorescent antibodies to cell surface or intracellular proteins (n=5/group). RESULTS/ANTICIPATED RESULTS: Immunostaining demonstrated an increase in the cell senescence markers p16 (15-fold; unpaired t-test, p=0.01; n=3) and p21 (15-fold; unpaired t-test, p=0.003; n=3) in the spinal cord of EAE mice compared to naīve mice. Next, we showed that p16+ and p21+ cells increase with disease progression in the meninges adjacent to the ventral demyelinating lesions (one-way ANOVA, p=0.0009; n=3/group). We further found that 30±9.7% of M2 macrophages, a subset of myeloid cells, express p21 in the spinal cord of EAE mice by using flow cytometry analysis compared to only 5±1.6% in naīve mice (unpaired t-test, p=0.002; n=5/group). DISCUSSION/SIGNIFICANCE: Our findings demonstrate that senescent cells accumulate in the meningeal compartment following EAE induction, suggesting that decreasing senescent cell burden is a promising avenue in delaying disease progression and preventing neuroinflammation in MS.
358 In Vitro Uptake of Harmful Algal Bloom Toxin Microcystin-LR in Human Placental Cells
Authors: Campbell Michael , Patel Mokshitkumar , Xiao Shuo , Wen Xia , Aleksunes Lauren M.
Published: April 2024

OBJECTIVES/GOALS: Harmful algal blooms (HABs) are increasing in both frequency and intensity due to climate change. HABs release the toxin microcystin-LR (MC-LR) which enters cells via organic anion-transporting polypeptides (OATPs). In this study, we sought to assess the ability of MC-LR to accumulate in trophoblasts, potentially disrupting placental functions. METHODS/STUDY POPULATION: Intracellular accumulation of MC-LR at exposure concentrations of 0.1, 1, and 10 µM over 6 hrs was evaluated in immortalized JAR placental cytotrophoblasts. Western blotting was used to evaluate protein-bound MC-LR accumulation in JAR cells. The function of OATP transporters in JAR cells was determined by pre-incubating cells with 10µM cyclosporin A, a general OATP inhibitor for 1 hr, and then incubated with 1µM OATP substrate fluorescein for up to 40 min. Fluorescence of fluorescein was measured at Ex/Em: 494nm/515nm by spectrophotometry. RESULTS/ANTICIPATED RESULTS: A concentration-dependent increase of MC-LR bound proteins in JAR cells was observed at 6 hrs with the greatest intracellular accumulation of MC-LR at 10µM. In the transporter experiments, a significant decrease of fluorescein uptake by up to 45% into JAR cells was observed following cyclosporin A inhibition of OATPs. These findings are consistent with the functional expression of OATP transporters in JAR placenta cells. Ongoing studies are evaluating whether the cyclosporin A-mediated inhibition of OATPs also inhibits the uptake of MC-LR. DISCUSSION/SIGNIFICANCE: Although MC-LR is well-known for its hepatotoxic and neurotoxic effects, there is growing interest in examining its potential adverse impacts on female reproductive health, particularly during pregnancy. Active uptake of MC-LR into the placenta could interfere with placental and fetal development.
362 Examining Temporal Links Between Distinct Negative Emotions and Tobacco Lapse During A Cessation Attempt
Authors: Jones Dusti , Potter Lindsey N. , Lam Cho Y. , Wetter David W.
Published: April 2024

OBJECTIVES/GOALS: Negative emotions (NE) play a pivotal role in addiction-related processes, including tobacco lapse during a quit attempt. Some NEs (e.g., shame, guilt) are posited to lead to a spiraling effect, whereby lapse predicts increased NEs leading to further lapse. This study goal is to examine associations between NEs and lapse. METHODS/STUDY POPULATION: This study examined associations between tobacco lapse and 13 distinct NEs among people who use tobacco and are trying to quit in two tobacco cessation studies. In Study 1, 220 adult (ages 18-74) cigarette users who identified as Black (50% female) participated in a 14-day study where ecological momentary assessment (with assessments approximately every 4 hours) was used to assess emotions and lapse in real-time and real-world settings. In Study 2, 288 adult (ages 18-71) cigarette users who were low socioeconomic status (51% White, 14% Black, 10% Hispanic, 49% female) participated in a 14-day study with the same study protocol as Study 1. Between and lagged within-person associations testing links between distinct NEs and lapse were examined with multilevel modeling with logistic links for binary outcomes. RESULTS/ANTICIPATED RESULTS: Results from Study 1 suggested that at the between-person level, disgust (OR =1.22, CI: 1.05, 1.42), nervousness (OR=1.23, CI:1.05,1.43), guilt (OR=1.40, CI: 1.16,1.69), and sadness (OR=1.18, CI:1.02,1.36) were predictive of higher odds of lapse, and at the within-person level, shame (OR=1.23, CI:1.04,1.45) was associated with higher odds of lapse. Results from Study 2 were similar and suggested that at the between-person level, disgust (OR=1.35, CI: 1.16, 1.56) and guilt (OR=1.88, CI:1.07,3.30), and at the within-person level, shame (OR =1.31, CI:1.10,1.55), were associated with higher odds of lapse. DISCUSSION/SIGNIFICANCE: The present study uses real-time, real-world data to demonstrate the role of distinct NEs on momentary tobacco lapse and helps elucidate specific NE that hinder the ability to abstain from tobacco use during a quit attempt. Results suggest that disgust, guilt, and shame play consistent roles in predicting lapse among diverse samples of tobacco users.
363 A CRISPR/dCas9 Epigenetic Therapuetic Approach for CASK-Related MICPCH
Authors: Gonzalez Casiana , Halmai Julian , Fink Kyle
Published: April 2024

OBJECTIVES/GOALS: CASK-related microcephaly with pontine and cerebellar hypoplasia (MICPCH) is a rare X-linked neurodevelopmental disorder caused by mutations in calcium/calmodulin-dependent serine protein kinase (CASK). We aim to rescue CASK expression via an CRISPR/dCas9 epigenetic therapeutic and create iPSC-based CASK relevant in vitro model systems. METHODS/STUDY POPULATION: As females have two X-chromosomes, disease causing mutations present with a 50/50 expression of mutant and wildtype, due to the mosaicism caused by random X-chromosome inactivation (XCI). This project will adapt an established CRISPR/dCas9 epigenetic approach to rescue expression from the silenced, wild-type CASK allele. We aim to accomplish this through testing different dCas9 orthologues and a guide RNA screen targeting the CASK promoter. Constructs will be tested for optimal targeting efficacy in vitro and assessed via RT-qPCR. Additionally, epigenetic modifications from our approach will be analyzed through bisulfite sequencing. We also aim to apply this epigenetic rescue technology in disease relevant cell lines and eventually in engineered patient mutation iPSC-derived neurons. RESULTS/ANTICIPATED RESULTS: Our results show the ability to target CASK and assess gene expression changes with CRISPR/dCas9 paired with an epigenetic modifier and transcriptional activator. Additionally, our fibroblast model with nonpathogenic single nucleotide polymorphisms within CASKallow for allele specific analysis of our targeted reactivation. We anticipate that following an increase of CASK expression, there would be a decrease in region specific promoter methylation. Further, with the identification of clinically described disease-causing point mutations that result in a loss of function of CASK protein, induction of the mutant sequence onto a healthy cell background will result in a similar reduction of CASK protein in our cell model. DISCUSSION/SIGNIFICANCE: This project will demonstrate the first therapeutic avenue for CASK-related MICPCH, and the potential to utilize targeted X-reactivation as a platform approach for X-linked disorders. Further, investigation of smaller dCas9 orthologues prepares our approach for future translational applications such as packaging into AAV for delivery.
364 Clinical and Translational Research at The University of Florida College of Veterinary Medicine
Authors: Deabold Kelly A. , Milner Rowan J. , Fagman Lana I. , Sayour Elias J. , Ligon John A. , Lee Ji-Hyun
Published: April 2024

OBJECTIVES/GOALS: To demonstrate a successful example of clinical and translational research at a busy veterinary teaching hospital and highlight a collaborative effort in Comparative Oncology between the University of Florida’s (UF) Colleges of Medicine and Veterinary Medicine. METHODS/STUDY POPULATION: The UF College of Veterinary Medicine (CVM) is a full-time teaching hospital with multiple departments actively recruiting patients for clinical trials. These departments include but are not limited to Oncology, Internal Medicine, Dermatology, Cardiology, and Emergency and Critical Care. The Oncology department collaborates with the doctors at the UF Health Cancer Center (UFHCC) as part of a Comparative Oncology Initiative, which has many ongoing canine and feline trials focusing on immunotherapy. RESULTS/ANTICIPATED RESULTS: As of August 2023, there are 60 clinical trials actively recruiting and enrolling patients at the UF CVM. 57% of these trials are interventional studies, while the other 43% are observational studies. The UFHCC Comparative Oncology Initiative has successfully completed one clinical trial focusing on canine gliomas; has 4 clinical trials that are actively recruiting patients, and 6 trials that are opening for enrollment in the near future. These studies focus on osteosarcoma, melanoma, and squamous cell carcinoma. It is anticipated that with continued successful collaborations, more clinical trials will be possible, and new treatment options will become available for not only veterinary patients but human patients as well. DISCUSSION/SIGNIFICANCE: Clinical and translational research is an important part of veterinary medicine to further patient care. Due to ongoing collaborative efforts, not only veterinary patients but also human patients will benefit from the research being conducted at the UF CVM.
12 Time to Sustained Recovery between Oral Tablet and Inhaler Placebos in the ACTIV-6 Platform Clinical Trial
Authors: Gao Yue , Mourad Ahmad , Lindsell Chris , Stewart Thomas
Published: April 2024

OBJECTIVES/GOALS: Platform trials gain efficiency by sharing placebo controls among different study arms. However, the varying routes of administration make it unclear whether participants exposed to different placebos have similar outcomes. As such, we seek to compare outcomes between participants receiving tablet and inhaler placebos in the ACTIV-6 trial. METHODS/STUDY POPULATION: ACTIV-6 is a large, decentralized platform trial exploring repurposed drugs for the treatment of adults with mild to moderate COVID-19. Enrolled participants were randomly assigned to a study arm vs. placebo and then mailed the study drug. They were monitored until symptom resolution or Day 28. Here, we compare outcomes for control participants contributing to the fluticasone furoate study arm, in which 251 were assigned to a tablet placebo and 370 an inhaler placebo. Time to sustained recovery and time to resolution of individual symptoms are compared between groups using Kaplan-Meier curves and unadjusted log-rank tests. A step-down procedure is applied to control the false discovery rate. RESULTS/ANTICIPATED RESULTS: Control participants assigned to tablet placebos had shorter time to sustained recovery (adjusted hazard ratio (HR) 1.34 (95% CI 1.11, 1.62)). When examining each of the eleven individually reported symptoms on study Day 14, nasal symptoms (adjusted odds ratio (OR) 0.44 (0.27, 0.72), p<0.01), dyspnea (OR 0.44 (0.22, 0.87), p = 0.02), and cough (OR 0.54 (0.35, 0.83), p<0.01) were identified as symptoms in which the tablet-placebo group performed notably better than those who received inhaler-placebos. In the follow-up, longitudinal analysis, we anticipate similar results. DISCUSSION/SIGNIFICANCE: Among ACTIV-6 control participants, those receiving a tablet placebo had a significantly shorter time to sustained recovery than those receiving an inhaler placebo. Platform trials using shared controls should consider efficiency in the context of the additional variability when sharing controls with a different route of administration.
376 Pathophysiology of voluntary motor commands in patients with multiple sclerosis identified using reverse engineering of motor unit population discharge.
Authors: McPherson Laura , Reece Tanner , Simon Skyler , Lohse Keith , Negro Francesco , Lang Catherine , Naismith Robert , Cross Anne
Published: April 2024

OBJECTIVES/GOALS: Our objective is to characterize excitatory, inhibitory, and neuromodulatory components of the voluntary motor command at the level of the spinal motoneuron in people with multiple sclerosis (MS). This information will provide insight into neural mechanisms of motor dysfunction and their heterogeneity among patients with MS. METHODS/STUDY POPULATION: Due to advances in high-density surface EMG (HDsEMG) decomposition and the recent development of a paradigm for reverse engineering of motor unit population discharge, we can feasibly estimate aspects of excitatory, inhibitory, and neuromodulatory components of the voluntary motor command in humans on a person-specific basis. We tested 11 ambulatory patients with MS and mild-moderate disability. We recorded HDsEMG from tibialis anterior (TA) and soleus (SOL) during isometric plantarflexion and dorsiflexion, performed as slow triangle contractions. EMG was decomposed into motor unit spike trains using blind source separation. We calculated a number of motor unit variables, most notably delta-F, which estimates motoneuron excitability and the balance of neuromodulatory and inhibitory inputs. RESULTS/ANTICIPATED RESULTS: There were consistent differences in MS patients vs. controls. For TA, values were decreased for delta-F (3.9 vs. 5.9 pps), initial firing rate acceleration (5.8 vs. 7.1 pps), firing rate range (9.3 vs. 11.9 pps), and max firing rate (12.3 vs. 15.0 pps). SOL had more modest decreases in delta-F (3.0 vs. 3.8 pps) and firing rate range (4.8 vs. 5.6 pps). Self-sustained firing was longer for MS patients. Within a patient, abnormalities in motor unit variables were not consistent across muscles and legs. Interestingly, there were several abnormalities in the patients with a normal clinical motor exam, indicating that perhaps our measures are sensitive to subclinical changes in processing of voluntary motor commands. DISCUSSION/SIGNIFICANCE: Excitatory, inhibitory, and neuromodulatory components of the voluntary motor command must be appropriately balanced for skilled motor output. This study is the first to characterize how they are disrupted in MS, providing foundational information to inform the development of mechanistically-based rehabilitation interventions.
395 Systemic Administration of miR-451 Improves Autophagy Response in an Accelerated Mouse Model of Diabetic Kidney Disease
Authors: Ghaderzadeh Sadaf , Agbor-Baiyee Baiyeendang , Obiwuma Chidera , Mohit Neal , Gambhir Kanwal K. , Ecelbarger Carolyn M. , Fluitt Maurice B.
Published: April 2024

OBJECTIVES/GOALS: Diabetic Kidney Disease (DKD) is a common diabetes complication, often linked to end-stage renal disease in the United States (US). While autophagy and miRNAs are pivotal, miR-451’s specific role remains understudied. Our study explores its renoprotective effects in an accelerated DKD mouse model. METHODS/STUDY POPULATION: We assessed the effect of miR-451 mimic treatment on Diabetic Kidney Disease (DKD) in BTBR ob/ob mice, known for their rapid DKD-like renal lesions. Mice were divided into four groups: WT (wild-type), BTBR ob/ob, WT+miR-451 (wild-type with miR-451 mimic), and BTBR ob/ob+miR-451 (BTBR ob/ob with miR-451 mimic). MiR-451 mimics were administered at 2mg/kg body weight once weekly for three consecutive weeks. We collected spot urine and monitored blood glucose levels at each time point. After the treatment period, mice were euthanized for kidney and blood samples. Western blot analysis assessed autophagy-related protein markers. Statistical analysis included Student’s t-test and ANOVA (p<0.05). RESULTS/ANTICIPATED RESULTS: The study assessed the impact of miR-451 mimic treatment in BTBR ob/ob mice. Albumin:creatinine ratio increased fourfold (p=0.01) in BTBR ob/ob mice at 5 weeks. MiR-451 mimic treatment had no impact on body weight. Blood glucose levels were notably higher in both treated and untreated BTBR ob/ob mice at 12 (425±33.1 mg/dL; p=0.04) and 13 weeks (383±25.3 mg/dL; p=0.007). However, a significant drop occurred from week 13 (554.7±10.8 mg/dL) to week 14 (289±13.3 mg/dL; p=0.0002) in BTBR ob/ob miR-451 treated mice. Western blot analysis in whole kidney homogenates showed a 91% reduction (p=0.02) in YWHAZ, a predicted miR-451 target, in treated BTBR ob/ob mice and a 95% reduction (p=0.01) in WT mice. Furthermore, miR-451 mimic treatment led to a 68% increase (p=0.01) in ATG101 and a 44% increase in Beclin-1 in BTBR ob/ob mice. DISCUSSION/SIGNIFICANCE: The study uncovers miR-451-based interventions as a promising avenue to counter Diabetic Kidney Disease by modulating autophagy, potentially introducing novel therapies for at-risk individuals. However, practical DKD treatments will require further research and rigorous clinical validation to harness the full potential of these insights.
403 Epithelial hypoxia maintains colonization resistance against Candida albicans
Authors: Bays Derek J. , Savage Hannah P. , Tiffany Connor , Gonzalez Mariela A. F. , Bejarano Eli. J. , Nguyen Henry , Masson Hugo L. P. , Carvalho Thaynara P. , Santos Renato L. , Tritt Andrew , Noble Suzanne M. , Thompson George R. , Bäumler Andreas J.
Published: April 2024

OBJECTIVES/GOALS: Antibiotic treatment sets the stage for intestinal domination by Candida albicanswhich is necessary for development of invasive disease, but the resources driving this bloom remain poorly defined. We sought to determine these factors in order to design novel prophylaxis strategies for reducing gastrointestinal (GI) colonization. METHODS/STUDY POPULATION: We initially developed a generalizable framework, termed metabolic footprinting to determine the metabolites C. albicanspreferentially uses in the mouse GI tract. After identifying the metabolites C. albicansutilizes, we usedin vitro growth assays in the presence and absence of oxygen to validate out metabolomics findings. We next determined if a probiotic E. coli that utilizes oxygen would reduce C. albicanscolonization compared to a mutant E. coli that could not respire oxygen. Finding that oxygen was a necessary resource, we utilized germ-free mice to determine if Clostridiaspp. known to reduce GI oxygen would prevent C. albicanscolonization. Lastly, we sought to see if 5-aminosalicylic acid (5-ASA) could prevent C. albicanscolonization. RESULTS/ANTICIPATED RESULTS: We found that C. albicans preferentially utilizes simple carbohydrates including fructo-oligosaccharides (e.g., 1-kestose), disaccharides (e.g., β-gentiobiose), and alcoholic sugars (e.g., sorbitol) and is able to grow in vitro on minimal media supplemented with either of these nutrients. However, in the hypoxic environment that is found in the “healthy” colon, C. albicans cannot utilize these nutrients. We next found that pre-colonization in a mouse model with a probiotic E. coli significantly reduced C. albicanscolonization, but the mutant E. coli had no effect on colonization. We next showed that Clostridia supplementation restored GI hypoxia and reduced C. albicanscolonization. Remarkably, we found that 5-ASA significantly reduced GI colonization of C. albicans. DISCUSSION/SIGNIFICANCE: We have shown that C. albicans requires oxygen to colonize the GI tract. Importantly, we found that 5-ASA can prevent an antibiotic mediated bloom of C. albicans by restoring GI hypoxia, which warrants additional studies to determine if 5-ASA can be used as an adjunctive prophylactic treatment in high risk patients.
15 Discrepancies in Medication Usage and Lifestyle Modification Referrals in Metabolic Syndrome is Dependent on how the Syndrome is Coded: A TriNetX Study
Authors: Brinkerhoff Annabelle N. , Gosalia Jigar , Qiu Juan J. , Pawelczyk James A. , Proctor David N.
Published: April 2024

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.
417 Identifying Biomarkers of Social Threat Sensitivity Associated with Social Anxiety and Depressive Symptoms in Adolescents
Authors: Politte-Corn Madison , Buss Kristin A.
Published: April 2024

OBJECTIVES/GOALS: Increases in anxiety and depression during adolescence may be related to increased biological reactivity to negative social feedback (i.e., social threat sensitivity). Our goal was to identify biomarkers of social threat sensitivity, which may provide unique etiological insight to inform early detection and intervention efforts. METHODS/STUDY POPULATION: Adolescents aged 12-14 (N=84; 55% female; 80% White; 69% annual family income <$70,000) were recruited. Youth viewed a series of happy, neutral, and angry faces while eye-tracking and electroencephalogram (EEG) data were recorded to capture cognitive and neural markers of sensitivity to social threat (i.e., an angry face). Fixation time and time to disengage from angry faces were derived from eye-tracking and event-related potentials were derived from EEG, which index rapid attention capture (P1), attention selection and discrimination (N170), and cognitive control (N2). Adolescents also completed a social stress task and provided salivary cortisol samples to assess endocrine reactivity. Social anxiety and depressive symptoms were self-reported concurrently and one year later. RESULTS/ANTICIPATED RESULTS: Latency to disengage from threatening faces was associated with lower N2 amplitudes (indexing poor cognitive control; r= -.24, p = .03) and higher concurrent social anxiety (r = .28, p = .01). Higher N170 amplitudes, reflecting attentional selection and discrimination in favor of threatening faces, predicted increases in depressive symptoms one year later (b= .88, p = .02). No other neurophysiological measures were associated with each other or with concurrent or prospective symptomatology. DISCUSSION/SIGNIFICANCE: Eye-tracking and EEG measures indexing difficulty disengaging from social threat and poor cognitive control may be biomarkers of social anxiety, which could be utilized as novel intervention targets. High N170 amplitudes to social threat, derived from EEG, may have clinical utility as a susceptibility/risk biomarker for depressive symptoms.
419 Intramuscular immunization with rVCG-MECA vaccine elicits stronger chlamydial specific immune response than intranasal immunization
Authors: FNU Medhavi , Tayhlor Tanner , Shakyra Richardson , Leandra Jones , Counrtnee Bell , Igietseme Josesph U. , Yusuf Omosun , Francis Eko
Published: April 2024

OBJECTIVES/GOALS: We prioritize Chlamydia’s public health impact, aim to develop rVCG-MECA for practical use, study robust immunity for effective strategies, and assess animal models for human vaccination adaptation. Our work highlights rVCG-MECA’s translational significance in public health. METHODS/STUDY POPULATION: Female Mice C57BL/6J mice (N=8) were immunized intramuscularly(IM) and intranasally(IN) and boosted twice, two weeks apart, with rVCG-MECA, once with live Chlamydia (C. trachomatis serovar D elementary bodies) and PBS. Specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined from chlamydia shedding following the transcervical challenge. Additionally, Chlamydia-specific cytokine (IFN-γ and IL-4) production by splenic and ILN T cells was assessed after 16 weeks RESULTS/ANTICIPATED RESULTS: Immunization with rVCG-MECA via intramuscular and intranasal routes triggered notable humoral responses in systemic and mucosal tissues. Intramuscular vaccination produced higher IgG2c levels in both tissues, while intranasal vaccination led to elevated IgA levels in mucosal tissues. rVCG-MECA-immunized mice exhibited significantly higher IFN-γ (Th1) secretion compared to IL-4 (Th2), with intramuscular immunization showing the highest IFN-γ levels. These findings anticipate robust immune responses, promising protection against Chlamydia, particularly through the intra muscular route. Overall, our results support rVCG-MECA as a promising Chlamydia vaccine, aligned with public health goals. DISCUSSION/SIGNIFICANCE: This study suggests that IM and IN immunization with rVCG-MECA induces immune effectors such as IFN-gamma and IgG2c that mediate chlamydial clearance in the genetical tract.
423 Innovation in MS Patient Care: Linking Cognitive Health and Myelin Integrity
Authors: Pike Ashley , James GA , Calvert ML , Kilts CD , Archer RL , Wolfe T
Published: April 2024

OBJECTIVES/GOALS: Our objective is to develop a patient-friendly application addressing the progression of cognitive impairments in multiple sclerosis (MS) patients. This initiative aims to augment individualized care and precision management of a major MS comorbidity by generating a cognitive health brain map for each patient. METHODS/STUDY POPULATION: Using the UAMS COMS Database, featuring high-resolution multi-contrast MRIs, and a comprehensive clinical, behavioral, and demographic dataset, we are developing a hierarchical learning-based software tool to compute maps correlating brain structure-function and individual cognitive function. Our MRI analysis employs a three-compartment model (NNLS>0.96). Functional scores are defined by individualized accuracy during the modified information processing speed task (e.g., m-SDMT). We utilize a Bayesian classifier with explicit Pearson’s correlation for tissue classification (BF10>100) to compute an index of the likelihood of correlation with cognitive impairment throughout brain tissue. RESULTS/ANTICIPATED RESULTS: This approach allows us to reveal subtle cognitive changes and their potential links to myelin integrity, offering vital insights into disease progression and management. The m-SDMT strongly correlates with the standard SDMT (r=0.79, p<0.001), confirming reliability as a cognitive assessment tool in clinical and research contexts. Analysis of the COMS dataset emphasized insights into the role of fine myelin structure in MS patients' cognitive functionality. Our findings heightened the pivotal significance of myelin integrity in preserving cognitive abilities and identify disruptions in myelin synthesis and homeostasis as primary contributors to cognitive decline. This discovery stresses the critical role that specialized brain pathways, influenced by myelin integrity, play in the pathology of MS. DISCUSSION/SIGNIFICANCE: This development bridges advanced neuroimaging techniques with practical clinical applications, emphasizing the nuanced role of myelin integrity in MS-related cognitive deficits. Our findings advocate for a multidisciplinary approach to MS management, demanding collaborative workforce development and education in translational science.
441 Transcriptomic analysis of Influenza A infected lung organoids reveals Warburg-like phenotype
Authors: Hartsell Emily , Roberts Justin T. , Haastrup Adeyeye I. , Langley Raymond J.
Published: April 2024

OBJECTIVES/GOALS: The CDC estimates that Influenza infections account for an average of 420,000 hospitalizations and 34,700 deaths in the U.S. each year. This project explores the underlying mechanisms of the infectious process of Influenza A in human lung organoids by examining the differential transcriptomic expression compared to uninfected controls. METHODS/STUDY POPULATION: Lung organoids were cultured from differentiated human bronchial epithelial cells from lung transplant donors on an air-liquid interface until they were confirmed to contain both mucous producing and ciliated cells. Lung organoids are ideal models in translational science due to their structural and functional characteristics which closely mimic those of in vivo human epithelial tissue. Half the organoids were exposed to Influenza A pH1N1 for 72h; the other half served as uninfected controls. RNA was isolated from both groups and sequenced using the Oxford Nanopore MinION which generates full length reads. Reads were aligned to the human reference genome (GRCh38.p14) using Minimap2. RESULTS/ANTICIPATED RESULTS: The MinION sequenced an average of 3.24m reads per sample and a total of 13,128 genes were relevantly expressed (defined as greater than 1 read per million in at least half the samples). ANOVA with a 5% false discovery rate (Benjamini and Hochberg correction) revealed 5,417 differentially expressed genes between infected and control groups. Within this subset, we identified downregulation of mucociliary clearance, mitochondrial and ÃŸ-oxidation, peroxisome, and glutathione replenishment genes. We further identified upregulation in inflammatory markers, lactate dehydrogenase enzymes, and several s100 proteins. The downregulation of mitochondrial and β-oxidation markers and the upregulation of lactate dehydrogenase enzymes revealed a Warburg-like phenotype which has not previously been reported. DISCUSSION/SIGNIFICANCE: This study reveals a novel Warburg-like phenotype in Influenza A infection alongside downregulated mucociliary clearance and upregulated inflammatory processes. These findings improve our understanding of Influenza A infection and point to potential therapeutic targets to advance precision medicine approaches to treatment.
444 Deciphering the role of IL-4 in post-colitis repair
Authors: Moreno Nicolas F , Yang Yang , Jeong Jong-Min , Tran Vivian , Wen Yankai , Atkins Constance , Zhao Jie , Fan Yuanyuan , Gao Junda , Ju Cynthia
Published: April 2024

OBJECTIVES/GOALS: Incomplete mucosal healingand dysbiosis prevent long-term remission after colitis. IL4 may restore colon homeostasis through its action on immune cells and the microbiome. We will demonstrate this mechanism using genetically modified mice and molecular tools. This may result in target therapies that prolong remission in patients with IBD. METHODS/STUDY POPULATION: Mice were treated with 3% dextran sulfate sodium (DSS) in drinking water for 5 days to induce colitis. Mice were monitored daily for changes in body weight, and to monitor colitis severity. At each endpoint, mice were sacrificed and colon length was measured. For disease severity assessment, mouse colons were prepared in paraffin sections by the 'swiss-rolling' method. For flow cytometry, lamina propria mononuclear cell isolation was performed and cellular populations were stained with fluorophore-conjugated antibodies. IL4-eGFP-expressing (4get) mice were used to analyze the cellular expression of IL4 after colitis. Cell-specific IL4 deletion mice were generated using the cre-lox system. RESULTS/ANTICIPATED RESULTS: IL4-deficient mice had worse colitis compared with wild-type controls. Flow cytometry of lamina propria cells from 4get mice showed that most IL4-producing cells after colitis are eosinophils (CD11b+SiglecF+). Flow cytometry of C57bl6 mice showed an influx of IL4Ra+ monocytes (CD11b+Ly6C+) and macrophages (CD11b+F480+). IL4-stimulated bone marrow-derived macrophages demonstrated an increase in HB-EGF mRNA transcription. Myeloid-specific IL4R deleted mice had no difference in colitis severity compared with controls. Neutrophil-specific IL4R-deleted mice had increased colitis severity and mortality. Co-housing of littermate mice rescued recovery after DSS in IL4 deficient mice. DISCUSSION/SIGNIFICANCE: IL4 appears to play a role in restoring homeostasis after colitis. The mechanism depends on eosinophil-derived IL4, and action through neutrophils. However, the reparative function of IL4 can be shared with deficient mice through the microbiome. I will study the cellular and microbial mechanism by which IL4 restores homeostasis after colitis.
453 Ketamine Promotes Sustained Brain-Derived Neurotrophic Factor Levels in the Corticoaccumbens Circuit
Authors: Chapman Holly L. , Anastasio Noelle C.
Published: April 2024

OBJECTIVES/GOALS: Neuropsychiatric disorders classified as synaptopathies are marked by a glutamate-associated hypofrontality which impacts decision making and impulsivity. We hypothesized that behavioral efficacy of the psychoplastogen ketamine is mediated in part through lasting promotion of markers of synaptic strength in corticoaccumbens circuit. METHODS/STUDY POPULATION: Male, Sprague-Dawley rats received an intraperitoneal (i.p.) injection of saline, single ketamine (10 mg/kg; 1x/day), or repeated ketamine (10 mg/kg; 1x/day for three days). Twenty-four hrs following the dosing regimen, animals were euthanized, and brains dissected to harvest corticoaccumbens structures including the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). mRNA was extracted and converted to cDNA. Levels of brain derived neurotrophic factor (BDNF) exon II mRNA were quantified using reverse transcriptase polymerase chain reaction (RT-PCR); cyclophilin A (PPIA) was used as a loading control. Gene expression differences in ketamine-treated rats were identified versus saline-treated rats. BDNF protein levels were quantified using capillary-electrophoresis immunoblotting. RESULTS/ANTICIPATED RESULTS: Repeated, but not single, ketamine administration decreased mPFC, but increased NAc, BDNF exon II mRNA levels versus saline (p<0.05). Single and repeated ketamine administration increased NAC BDNF protein (p<0.05), while both dosing paradigms induced a trend towards an increase in mPFC BDNF levels. DISCUSSION/SIGNIFICANCE: We discovered a dosing regimen-dependent and sustained effects of ketamine administration on BDNF levels in the rodent brain. Taken together, ketamine-mediated BDNF levels may sustain synaptic strengthening mechanisms supporting future investigation into the utility of ketamine for diseases characterized by synaptopathies.
454 The Role of bHLHe40 in Systemic Sclerosis-associated Pulmonary Fibrosis
Authors: Adewale Adegboyega "Tim" , Feghali-Bostwick Carol
Published: April 2024

OBJECTIVES/GOALS: The dominant complication of Systemic Sclerosis (SSc) is clinically severe and commonly fatal pulmonary fibrosis (PF). We sought to determine the downstream regulatory role of the basic Helix-Loop-Helix protein 40 (bHLHe40), in response to Insulin-like Growth Factor II (IGF-II) on Pro-Lysyl Oxidase cleavage products. METHODS/STUDY POPULATION: We examined the response of primary pulmonary fibroblasts cultured from the lungs of control donors and SSc lung explants to IGF-II as well as human recombinant Lysyl Oxidase Propeptide (LOX-PP). In addition, we utilized an experimentally-induced model of lung fibrosis with intratracheal bleomycin administration. We used qPCR and immunoblotting to quantify mRNA and protein levels, respectively. We used sequence-specific small-interfering RNA to silence targeted genes. Immunoblots were quantified in ImageJ (NIH) and statistical analyses were performed in GraphPad Prism. RESULTS/ANTICIPATED RESULTS: IGF-II regulates levels of Pro-LOX, active LOX, and LOX-PP, as well as isoforms of proteases Bone Morphogenetic Protein 1 (BMP1) and Tolloid-like 1 (TLL1). The transcription factorbHLHe40 localizes to the nucleus in response to IGF-II. bHLHe40 silencing downregulated TLL1, abrogating the enzymatic cleavage of Pro-LOX. SSc lungs have higher baseline levels of the total (N-glycosylated/unglycosylated) LOX-PP than normal lung tissues, and baseline levels of LOX-PP correlated with TLL1 Isoform 2 in SSc lungs. LOX-PP contributes to the development and progression of SSc-PF by mediating changes consistent with the extracellular matrix deregulation implicated in SSc-PF: elevated levels of Collagen 3A1 (COL3A1), Fibronectin-1 (FN1), and Plasminogen Activator Inhibitor-1 (PAI1). DISCUSSION/SIGNIFICANCE: Our findings indicate that bHLHe40, TLL1, and LOX-PP may serve as targets of therapeutic intervention to stop the progression of SSc-PF. Since activation of common fibrotic pathways are involved in different diseases characterized by lung fibrosis such as IPF, our findings may have wider implications for lung fibrosis associated with other diseases.
20 Characteristics of Medicare patients receiving peripheral vascular interventions for peripheral artery disease differ by outpatient site of service
Authors: Tsou Terrence , Dun Chen , Hicks Caitlin
Published: April 2024

OBJECTIVES/GOALS: Endovascular peripheral vascular interventions (PVIs) are increasingly utilized for the treatment of peripheral artery disease (PAD). We aimed to assess characteristics of patients receiving PVI at ambulatory surgical centers and office-based labs (ASC/OBL) versus the outpatient hospital (hospital) site of service. METHODS/STUDY POPULATION: We performed a retrospective analysis using 100% Medicare fee-for-service claims data between January 1, 2017 and December 31, 2022. We used Current Procedural Terminology (CPT) codes to identify patients undergoing angioplasty, stenting, or atherectomy. Patient demographics were collected from the Medicare Master Beneficiary Summary File and associated comorbidities and PVI indications were identified using International Classification of Disease (ICD)-10 codes. We used patient ZIP codes to determine patients’ residence densities and regions. We used site of service codes to determine whether PVI were performed in the ASC/OBL versus hospital. Results were analyzed with descriptive statistics. RESULTS/ANTICIPATED RESULTS: Of 817,241 patients undergoing PVI for PAD, 461,068 (56.4%) were treated in an ASC/OBL. Compared to patients treated in the hospital, patients receiving PVI at ASC/OBLs were more likely to be older, female, non-white race, with fewer comorbidities (end stage renal disease, diabetes, hypertension, and any history of tobacco use) (all, P<0.001). Patients treated in ASC/OBLs more frequently resided in urban (vs. rural) locations, and in the South and West (both, P<0.001). Indication for PVI was predominately chronic limb-threatening ischemia, and clinically similar between groups (77.1% vs. 76.2%). There was a significant change in site of service over time: a minority (47.6%) of PVIs were performed in the ASC/OBL in 2017, whereas the majority (64.7%) of PVIs were performed in the ASC/OBL in 2022 (P<0.001). DISCUSSION/SIGNIFICANCE: Patients treated in ASC/OBLs were less medically complex compared to those treated in the outpatient hospital setting. Further study is needed to examine whether differences in patient characteristics versus other factors (e.g. reimbursement) are driving the increase in PVIs performed in the ASC/OBL over time.
473 Application of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for modeling of Ankyrin-2 p.R990Q variant-induced ventricular arrhythmia and personalized medicine
Authors: Joshi Jyotsna , Schwieterman Neill , Smole Nate , Guo Shuliang , Wan Xiaoping , Ramirez-Navarro Angelina , Lane Cemantha , Deschenes Isabelle , Hund Thomas , Wold Loren , Smith Sakima
Published: April 2024

OBJECTIVES/GOALS: The cytoskeletal protein α²II spectrin interacts with actin and ankyrin-2 in cardiomyocytes which is essential to orchestrate ion channels and membrane proteins in the cardiac dyad. Our goal is to understand molecular mechanism causing severe ventricular arrhythmias due to spectrin dysfunction and explore novel therapies to treat such conditions. METHODS/STUDY POPULATION: We previously published a case of a 36-year-old woman with an ankyrin-2 p.R990Q (ANK2) variant, presented with severe ventricular arrhythmias and sudden cardiac arrest, caused by a novel mutation in the ankyrin-B gene (c.2969G>A) that disrupts the interaction of ankyrin-B/βII spectrin. To model the condition, we will use human induced pluripotent stem cell (DF 19-9-7T, WiCell)-derived ventricular cardiomyocytes (iPSC-CMs) having ANK2 variant, engineered using CRISPR/Cas9 method (Synthego Corp.). We will validate the differentiation of iPSCs into ventricular lineage and characterize the ANK2 ventricular phenotype. Next, we will express light-gated cation channel Channelrhodopsin (ChR2) in the ANK2 iPSC-CMs and investigate the potential role of optogenetics in treating such severe arrhythmias. RESULTS/ANTICIPATED RESULTS: Immunostaining shows 87.339% of iPSC-CMs, treated with All-trans retinoic acid (RA) (1 uM) on days 7 and 12 [RA 7,12], and 23.84% of those, treated on days 3 and 5 [RA 3,5], expressed MLC-2V (p<0.001). Calcium reuptake (τ) is 0.5914 s in RA 7, 12 while 0.2247s in RA 3, 5 (p<0.001). APD90 and APD50 of RA 7, 12 are 2- and 5-fold higher than RA 3, 5, showing distinct ventricular and atrial phenotypes. Protein expression of βII-spectrin and ankyrin-2 and their co-localizations were reduced in the ANK2 phenotype compared to the healthy phenotype. We found prolongation of Ca2+ waves and τ with blue light on iPSC-CMs, expressing ChR2. We anticipate that such prolongation of calcium transients would prevent aberrant calcium spikes, rescue Ca2+/calpain-induced βII-spectrin loss and provide electrical stability. DISCUSSION/SIGNIFICANCE: Animal models cannot accurately recapitulate human cardiac electrophysiology. The proposed human iPSC-CM-based ANK2 model would provide better mechanistic insights of severe ventricular arrhythmias. Also, the proposed optogenetic cardioversion has the potential to provide safe, targeted and painless cardioversion to manage arrhythmias.
474 An Investigation of Novel Urinary Cell mRNA Profiles for Noninvasive Diagnosis of Acute Rejection in Kidney Transplant Recipients
Authors: Salinas Thalia , Li Carol , Snopkowski Catherine , Stryjniak Gabriel , Albakry Shady , Ding Ruchuang , Salvatore Steven P. , Seshan Surya V. , Dadhania Darshana M. , Muthukumar Thangamani , Suthanthiran Manikkam
Published: April 2024

OBJECTIVES/GOALS: RNA-seq of urine and kidney allograft biopsies (bx) found that urinary cell immune landscape reflects intragraft molecular events and we discovered a shared set of 127 mRNAs in urine matched to T cell mediated and antibody mediated rejection bx. We prioritized ITM2A, SLAMF6 and IKZF3 mRNAs and herein investigate if these accurately predict rejection. METHODS/STUDY POPULATION: We collected urine samples from adult kidney allograft (KA) recipients at the time of KA bx. KA bx were classified by pathologists by Banff criteria. Total RNA was isolated from KA bx-matched urine samples. Absolute copy numbers of ITM2A, SLAMF6, and IKZF3 mRNAs and 18S rRNA were measured using our customized RT-qPCR assays. Logistic regression used to derive an equation for a combined signature score of 18S-normalized urinary cell mRNA levels of ITM2A, IKZF3, and SLAMF6 that best predicts Acute Rejection (AR= both T cell mediated rejection and antibody mediated rejection). Area under the ROC curve (AUC) was calculated to discriminate between AR and No Rejection (NR) biopsies for 18S-normalized urinary cell levels of ITM2A, IKZF3 and SLAMF6 and the composite signature score. AUCs were compared by DeLong Method. RESULTS/ANTICIPATED RESULTS: Urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNAs in urine discriminated KA recipients with AR biopsies (n=95) from those with NR biopsies (n=160) (All P values <0.05, Mann-Whitney test) and the AUC was 0.69 (95%CI, 0.62 to 0.76) for ITM2A, 0.61 (95%CI, 0.53 to 0.68) for IKZF3, and 0.60 (95%CI, 0.53 to 0.68) for SLAMF6. The derived combination signature score of urinary cell 18S-normalized levels of ITM2A, IKZF3, and SLAMF6 mRNA discriminated KA recipients with AR from those with NR (P<0.0001, Mann Whitney test) and the combined signature score AUC was 0.72 (95%CI, 0.65 to 0.79). The combination signature score discriminated AR vs NR better than IKZF3 and SLAMF6 alone, but was not significantly different than ITM2A alone (DeLong method). (Additional results/figures to be included in poster) DISCUSSION/SIGNIFICANCE: Our RNA-seq offered a unique opportunity to diagnose AR by measuring the mRNAs in urine. We now found that urinary cell mRNA levels of ITM2A, IKZF3, SLAMF6 and the combined signature are diagnostic of AR, a major and serious post-transplant complication. This allows for much-needed KA molecular surveillance and personalization of immunosuppression.