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.

Objectives/Goals: The creatine (Cr) system is impaired in Alzheimer’s disease (AD). Data show that creatine monohydrate (CrM) supplementation may improve AD symptoms in AD mouse models, but no human studies have been reported. Thus, we investigated whether an eight-week CrM supplementation was feasible and associated with increased brain creatine in patients with AD. Methods/Study Population: Twenty participants with probable AD were allocated to an open-label, eight-week intervention of 20 g/day CrM. Fasting blood draws were taken at baseline, 4-, and 8-week visits to measure serum creatine (Quest Diagnostics). 1H magnetic resonance spectroscopy was performed at baseline and 8-week visits to measure brain Cr as a ratio to unsuppressed water. Self-reported compliance (with assistance from study partners) was assessed with daily CrM trackers. The mean compliance percentage across all participants was used to describe overall compliance with the intervention. We used paired t-tests to analyze the mean changes in serum Cr levels from baseline to 4- and 8-week visits and the mean change in brain Cr from baseline to 8-week visits. Statistical significance was set at p<0.05. Results/Anticipated Results: Participants were 65% male with a mean age of 73.1±6.3 years. All participants completed the study, with 19 out of 20 achieving the dose compliance target of ≥80%. The mean self-reported dose intake was 90%. Serum Cr levels were significantly increased at 4- and 8-week visits compared to baseline (0.6±0.4 mg/dL vs. 14.0±9.9 mg/dL and 15.0±13.6 mg/dL, respectively; p<0.001). Brain Cr levels also significantly increased (330.5±36.80 i.u. vs. 366.9±57.52 i.u., p<0.001). Discussion/Significance of Impact: We are the first to demonstrate that 20 g/day of CrM for eight weeks is feasible and associated with increased brain Cr in patients with AD. Our findings support further investigation of brain target engagement of CrM and its efficacy in AD. With AD cases expected to rise, CrM could serve as an effective, affordable therapeutic to slow AD progression.

Objectives/Goals: The study focuses on developing a wound patch that employs a biocompatible matrix which incorporates mesenchymal stem cells (MSCs) with wound healing and antimicrobial properties, along with antimicrobial metallic nanoparticles covered with keratinocytes derived from induced pluripotent stem cells to replicate the skin’s barrier function. Methods/Study Population: In vitro experiments will be conducted to combine bacteria with MSCs and metallic nanoparticles to assess whether bacterial killing is improved by this combination. The MSCs will then be evaluated in the presence of the nanoparticles to confirm that their functionality and phenotype are not altered. To verify the cells’ functional integrity, they will undergo trilineage differentiation, surface marker phenotypic testing, and evaluation of their capacity to inhibit lymphocyte proliferation in the presence of the nanoparticles. Subsequently, this living bandage will be created using a biomatrix embedded with induced pluripotent stem cell-derived keratinocytes and tested on a canine wound model to study the impact on healing. The model will assess the rate of healing and cellular response at weekly intervals until healed. Results/Anticipated Results: The combination of mesenchymal stem cells and antimicrobial nanoparticles works synergistically to enhance bacterial killing in vitro with S. aureus. The presence of the nanoparticles in combination with MSC did not affect the ability of the MSC to undergo trilineage differentiation. We anticipate that the surface phenotype will be similarly unaffected. In addition, we expect that the presence of the nanoparticles should not interfere with the ability of MSC to suppress lymphocyte proliferation. Utilization of the wound patch in the in vivo canine wound model is expected to enhance healing and prevent infection. We expect that we will observe a shift in the cellular composition of the wound with less inflammatory cells and more M2 or wound healing anti-inflammatory monocytes. Discussion/Significance of Impact: The incidence of resistant infections with no pharmacologic therapy available are on the rise. The development of an antimicrobial living bandage that increases the body’s ability to fight off infection, while providing a barrier to reinfection would provide a new way to treat infections regardless of their acquired antibacterial resistance.

Objectives/Goals: Evaluate the impact of intrauterine growth restriction (IUGR) on neonatal brain development using magnetoencephalography (MEG) and correlate findings with NICU Network Neurobehavioral scale (NNNS) scores at 1 month Methods/Study Population: In this prospective cohort study, we will enroll 30 participants, consisting of 15 neonates diagnosed with IUGR and 15 healthy controls, matched by gestational age, from the University of Arkansas for Medical Sciences. We will perform MEG scans at three key developmental stages: during fetal life, at 1 month, and at 3 months of age and a Bayley IV exam at 12 months of age. The NNNS assessments will be conducted at the 1-month visit to evaluate neurobehavioral outcomes. All MEG data will be synchronized with clinical evaluations and maternal health records to ensure comprehensive analysis. Results/Anticipated Results: We anticipate that the study will reveal significant differences in brain maturation and neural activity patterns between IUGR-affected infants and healthy controls. Specifically, we expect to find altered neural connectivity and delayed maturation in the delta and theta frequency bands during the early neonatal period in the IUGR group. These anticipated neuroimaging findings will be correlated with NNNS scores to assess functional implications of the observed brain activity differences. If our hypotheses are confirmed, the study will provide robust biomarkers for early identification of neurodevelopmental delays in IUGR-affected infants, paving the way for targeted early interventions. Discussion/Significance of Impact: This study could significantly enhance early detection and intervention strategies for IUGR, potentially reducing long-term neurodevelopmental challenges and improving clinical outcomes

Objectives/Goals: Tacrolimus (TAC) is an immunosuppressant used after hematopoietic stem cell transplant (HSCT). Recently, TAC was found to be metabolized to a novel, less active metabolite by common gut microbiota. Our objective is to determine a microbiome signature that influences oral TAC pharmacokinetic (PK) and to develop a clinical tool to select the TAC dose. Methods/Study Population: This is an observational IRB approved microbiome-pharmacogenomic study using banked biospecimens and clinical data, TAC dose, and PK information from the electronic health record. Adult HSCT patients with pre-transplant DNA and stool specimens were included in this analysis if they received TAC in the first 100 days post-HSCT. A global diversity array was used for DNA pharmacogenomic (PGx) genotyping, and metagenomic shotgun sequencing was used for stool microbiome analysis. Spearman correlation will be used to identify potential stool microbiota associated with TAC PK. TAC trough concentrations at steady state will be modeled using nonlinear mixed effects (NLME) modeling to identify potential genetic and microbiota covariates that influence TAC clearance. Results/Anticipated Results: We identified 53 eligible patients who had available DNA and 90 stool samples. The majority (n = 49, 92.5%) were of European ancestry. These patients had 920 (oral  =  622, IV infusion  =  298) TAC trough blood concentrations. We expect that patients who have high abundance of bacteria that metabolize or reduce the absorption of TAC will have lower blood concentrations and will require a higher IV to oral dose conversion ratio than those with lower abundance. Those patients will also require higher oral TAC daily doses. Low stool microbial diversity is expected to be associated with high oral TAC trough intra-patient variability in the first 100 days post-transplant. In the NLME model, PGx when combined with potential bacterial signature will better explain variability in TAC clearance. Discussion/Significance of Impact: Combining PGx and microbiome biomarkers will provide a better understanding of the factors influencing TAC PK and lead to models for individualized dosing. To our knowledge, this is the first study to investigate the combined influence of microbiome and PGx on drug PK. The study is limited to the availability of samples in the biobank.

Objectives/Goals: Germinal matrix hemorrhage (GMH) is a devastating disease of infancy that results in brain-related pathologies. Following rupture of vasculature in the brain, red blood cell (RBC) lysis, and hemoglobin breakdown results in heme/iron-related toxicities. We hypothesize that these cellular pathologies are mediated in part by the complement system. Methods/Study Population: Post-natal mice on day 4 (P4) were subjected to collagenase induced-GMH and treated with various complement inhibitors that function at different points in the complement pathway and differentially prevent the generation of specific complement activation products. The principle bioactive complement activation products are C3 opsonins (C3b, iC3b, and C3d), the proinflammatory anaphylatoxins (soluble C3a and C5a peptides), and the terminal cytolytic membrane attack complex (MAC). Experimental groups consisted of: Wild-type (WT) naïve mice, and WT GMH-mice treated with either PBS (vehicle), CR2-Crry (C3 inhibitor that blocks all activation products), anti-C5 mAb (blocks C5a and MAC), C5aRA (blocks C5a-C5a receptor interaction), or anti-C7 mAb (blocks MAC). Study endpoints were P7 or P14. Results/Anticipated Results: Following GMH, CR2-Crry treatment decreased MAC deposition on RBC and additionally decreased heme oxygenase-1 expression, heme deposition, and iron-induced inflammation measured at P7. In support of a specific role for the MAC, anti-C7 mAb treatment resulted in similar outcomes and was similarly protective. Anti-C7 mAb treatment also reduced hydrocephalus development at a later time point (P14). A similar result was obtained using C7 deficient mice and with anti-C5 mAb treatment. On the other hand, no protective effect was seen with C5aR blockade, and double knock out of C3aR/C5aR also did not provide protection, indicating no role for the anaphylatoxins C3a and C5a and their receptors expressed on leukocytes and endothelial cells in exacerbating deteriorating outcomes. Discussion/Significance of Impact: Our data indicate a key role for the MAC in RBC induced hemolysis after GMH which serves as a driver of inflammation and early GMH pathogenesis. We further show that we can effectively increase precision by targeting solely the MAC complex acutely. Future work will be undertaken to determine temporal roles of individual complement activation products.

Objectives/Goals: Short-chain fatty acids (SCFAs) exert protective effects against calcium oxalate (CaOx) urinary stone formation in experimental rodent models, yet these effects are not understood in natural stone formers. This study will define the impact of SCFAs on stone risk factors along the gut–kidney axis in a natural canine model of CaOx stone disease. Methods/Study Population: A randomized, placebo-controlled, clinical trial will be performed using a crossover study design. Twenty dogs that are natural CaOx stone formers will be fed a standardized diet and randomized to receive either a daily prebiotic fiber (inulin) that stimulates SCFA production or a placebo. We will perform fecal shotgun metagenomics and SCFA quantification before and after each intervention (four timepoints) to identify how inulin and SCFAs enrich or deplete pathways relevant to stone formation within the gut microbiome. RT-qPCR will be performed to determine the effects of SCFAs on intestinal oxalate transporter gene expression (SLC26A3 and SLC26A6). At each timepoint, urinary shotgun metagenomics and quantification of urine biochemical profiles used to predict stone risk will also be performed. Results/Anticipated Results: We anticipate that prebiotic stimulation of SCFAs with inulin will reduce stone risk factors along the gut–urinary axis in a natural canine model of urinary stone disease. Specifically, we anticipate that prebiotic stimulation of SCFAs will 1) modify gut and urinary microbial communities to promote pathways considered protective against stone formation, 2) alter the expression of oxalate transporters (SLC26A3, SLC26A6) to reduce net oxalate absorption, and 3) reduce stone-promoting metabolites (e.g., oxalate) in the urine. Discussion/Significance of Impact: By defining the impact of prebiotic fibers and SCFAs on the gut–urinary axis in a natural animal model of CaOx urolithiasis, we will lay the foundation for novel nutritional strategies to prevent CaOx stone disease in both humans and animals.

Objectives/Goals: The bromodomain PHD finger transcription factor (BPTF) is an oncogenic driver of neuroblastoma. Our objective is to pioneer the discovery of the first class of chemical compounds that engage the PHD finger of BPTF and inhibit its biological function in cellulo, thereby establishing first-in-class chemical probes for this epigenetic reader. Methods/Study Population: Our previous work has identified a collection of small molecules that engage BPTF PHD in vitro. Following structure–activity relationships analysis, candidates will be used in a neuroblastoma cell model to validate BPTF PHD interaction in cellulo and predict therapeutic potential. Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) will be used to confirm BPTF PHD engagement by compounds. Selective toxicity in neuroblastoma cells upon inhibitor treatment will be gauged by comparing cell growth and viability in the IMR-32 cell line against the HEK293 cell line. Treated HEK293 cells will be subjected to the assay for transposase-accessible chromatin (ATAC) and RNA sequencing methods to monitor changes in chromatin structure and transcriptional signatures against untreated cells. Results/Anticipated Results: We hypothesize that compounds with low micromolar potency for BPTF PHD in vitro will engage the target in cellulo and displace NanoLuciferase tagged protein from its HaloTagged® peptide binding partner. Additionally, we anticipate that our inhibitors will show cytotoxicity for IMR-32 cells with limited effects on HEK293 cells. We envision that inhibitor treatment in HEK293 cells will correlate with reduced chromatin exposure, suggesting that blocking the BPTF–histone interaction via PHD finger inhibition hinders the remodeling of transcriptionally silent heterochromatin into a transcriptionally active state. Finally, we expect that inhibitor treatment will result in diminished gene expression of oncogenic transcription factors, including N-Myc, a biomarker of neuroblastoma. Discussion/Significance of Impact: These first-in-class chemical probes for BPTF PHD will enable further investigation of BPTF and high-risk neuroblastoma progression, as well as its role in other diseases. In addition, these compounds will serve as a platform for the development of new anticancer agents that may improve outcomes for children that suffer from high-risk neuroblastoma.

Objectives/Goals: Congenital cytomegalovirus (cCMV) continues to be the primary infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells in response to cCMV remains largely unexplored. This study seeks to investigate how fetal NK cells respond to human cytomegalovirus (HCMV) during gestation. Methods/Study Population: Umbilical cord blood and corresponding umbilical cord tissues were collected from fetuses that had no complications during gestation. These samples, provided by the Medical College of Wisconsin Tissue Bank, were processed within 24 hours after live birth. Single-cell suspensions were prepared from the samples, and fetal NK cells were isolated and exposed to HCMV antigen peptides VMAPRTLFL, VMAPRTLIL, VMAPQSLLL, and the human self-peptide ALALVRMLI. These peptides were presented on HLA-E*01:03 BV421-conjugated tetramers produced by the National Institutes of Health Tetramer Core Facility. Additionally, fetal NK cells were also prepared for single-cell RNA sequencing (scRNA-seq), and cells were filtered and clustered based on the number of uniquely expressed genes. Results/Anticipated Results: Through unbiased clustering, our scRNA-seq analysis identified five unique fetal NK cell subsets in umbilical cord blood and four in the corresponding umbilical cord tissue. Notably, fetal NK cells exposed to HCMV during gestation were primarily mature NK cell subsets, while those from unexposed fetuses were mostly immature subsets. Additionally, HCMV-exposed fetal NK cells exhibited a strong recall response to the HCMV antigen, with a notably higher frequency and elevated production of IFN-γ. Conversely, naïve fetal NK cells from fetuses unexposed to HCMV produced significantly lower levels of IFN-γ. Finally, we identified a distinct subset of fetal NK cells that emerge following exposure to the HCMV antigen. Discussion/Significance of Impact: In this study, we show that HCMV infection can influence the formation of specific NK cell subsets and re-exposure to the HCMV antigen can trigger a recall response. These insights could pave the way for the development of innovative NK cell-based immunotherapies aimed at preventing fetuses from developing symptomatic cCMV.

Objectives/Goals: Our goal was to investigate the associations between parental resilience and parenting behaviors, and their relationship to their offspring’s reward neurocircuitry function; in particular, the reward prediction error (RPE) circuit, a transdiagnostic marker of psychopathology. Methods/Study Population: N = 26 parent–child dyads (children ages 10–14) were recruited. Parents reported on parenting behaviors using the Alabama Parenting Questionnaire (APQ), and resilience using the Connor-Davidson Resilience Scale (CD-RISC). Children performed the Novelty task, a reward learning task, during fMRI scanning. Trial-by-trial RPEs were calculated based on a reinforcement learning model. Brain regions of interest (ROIs) including the nucleus accumbens, anterior putamen, and ventromedial prefrontal cortex were created (regions implicated in RPE representation). Results/Anticipated Results: The APQ parental involvement subscale was associated with increased negative affect tolerance (r = 0.40, p Discussion/Significance of Impact: Findings suggest that parental factors may impact neurocircuitries underlying psychopathology in offspring, and consequently, risk for offspring psychopathology. Interventions designed to increase parental resiliency may reduce risk for psychopathology in offspring, perhaps by increasing parental involvement and neural RPE sensitivity.

Objectives/Goals: The identification of the cascade of molecular and cellular events occurring during the progression of focal segmental glomerulosclerosis in human kidney biopsies from kidney transplant (KTx) recipients (KTR) with normal function or recurrent FSGS to determine potential targets of intervention and therapy. Methods/Study Population: In this study, we evaluate the molecular and cellular events associated with primary FSGS in both native and transplant kidneys. We collected biopsy samples from the native normal kidney (nNK, n = 3), normal functioning allografts (NKTx, n = 3), primary FSGS in the native kidney (nFSGS, n = 1), recurrent FSGS (KTxFSGS, n = 5). KTxFSGS comprises a collection of longitudinal samples with biopsy also collected at the subsequent recurrence. Blood samples were collected during biopsy collection. Biopsies were preserved in RNAlater at the time of collection. 10X genomics chromium single nuclei RNA sequencing (snRNAseq) was performed using isolated nuclei. Data was analyzed using Seurat on R. Conditionally immortalized podocytes were treated with a patient serum to determine the change in expression observed in snRNAseq data. Results/Anticipated Results: Recurrence rates of primary FSGS are high in kidney allograft recipients up to 25–50% in first, and up to 80% in second transplants, often leading to graft loss. Our findings reveal that podocyte detachment is driven by metabolic and structural dysregulation rather than cell death, increasing VEGFA expression and disrupting glomerular endothelial cell growth and permeability. Parietal epithelial cells initially compensate by dedifferentiating toward podocytes but later increase collagen deposition, contributing to glomerular sclerosis. Increased interactions of glomerular cells with B cells exacerbate extracellular matrix deposition and scarring. We also observed tubular sclerosis and disruption of the regenerative potential of proximal tubular cells, with increased interaction with T cells. Discussion/Significance of Impact: These findings offer new insights into the pathogenesis of recurrent FSGS and suggest potential therapeutic targets and establishes a foundation for future studies to further evaluate the role of metabolic dysfunction as the cause of podocyte injury and loss.

Objectives/Goals: Injury to the medial knee ligaments (sMCL, dMCL, POL) and anterior cruciate ligament (ACL) can cause anteromedial rotatory instability (AMRI). AMRI can cause knee instability and ACL graft failure, but it is unclear how the sMCL, dMCL, POL, and ACL resist AMRI. We aimed to characterize the in-situ forces of the sMCL, dMCL, POL, and ACL under loading conditions involved with AMRI. Methods/Study Population: We characterized the in situ forces of the sMCL, dMCL, POL, and ACL under 1) isolated external tibial rotation torque (ER), 2) isolated anterior tibial force (Ant), and 3) combined ER+Ant loading. Twenty-eight human cadaveric knees (18 male; mean age, 48±13; 21–65 years) were tested on a robotic manipulator with force sensing. Tibiofemoral kinematics were recorded under isolated ER (4Nm, 0–90°), isolated Ant (134N at 0–90°), and combined ER+Ant (4Nm+100N at 15, 30, 90°). The sMCL, dMCL, POL, and ACL were dissected in random order. The in situ force (N) in the sMCL, dMCL, POL, and ACL at the peak applied load for each loading condition was calculated using superposition and compared with Kruskal–Wallis tests with post hoc pairwise testing using a Bonferroni–Holm correction for multiple comparisons (α = 0.05). Results/Anticipated Results: Under isolated ER, the force in the sMCL (32–52N) from 0°-90° exceeded that of the ACL, dMCL, and POL at each flexion angle (p0.05). Force in the ACL was the second highest (26–6N from 0°-90°). Force in the dMCL and POL was low (≤12N). Under isolated Ant, the ACL carried the highest force at all flexion angles (≥113N) (p0.05), but at 90° the sMCL carried the highest force of all ligaments (p0.05). At 90°, force in the dMCL diminished (Discussion/Significance of Impact: We show that the sMCL is the major stabilizer to external rotation torques and combined anterior and external loading conditions related to anteromedial rotatory instability across the arc of knee flexion, while the dMCL, POL, and ACL play a less prominent role, with the exception of the ACL and dMCL near full extension.

Objectives/Goals: Returning genetic research results to participants can improve community engagement and enhance health equity. Providing research investigators with a convenient and cost-effective pathway for returning genetic findings, along with ensuring the necessary criteria for validity and utility, may reduce the barriers to returning results. Methods/Study Population: The ICTS Precision Health Genomic Return of Research Service Core (ICTS PH gROR) developed a process of returning genetic findings to participants who have indicated their preference to be notified of any findings that may impact their health. The service includes returning primary findings (actionable results discovered as part of the Investigator’s approved IRB study) and/or secondary findings [clinically actionable genes by the American College of Medical Genetic and Genomics (ACMG)]. Participants with positive findings will receive a written report, generated by a board-certified clinician specializing in genetics or molecular genetic pathology. A visit with a genetic counselor provides additional resources and guidance on the identified health risks. Results/Anticipated Results: Centralizing a service for the return of genetic results will ensure best-practices and minimize the burden. By offering results at no cost to the participant or their family, the service promotes accessibility and removes financial barriers that could otherwise prevent individuals from benefiting from genetic insights. Furthermore, by involving expert oversight committees and genetic counselors in the process, participants will receive accurate information and appropriate guidance, enhancing their understanding of the results and empowering them to address any potential health risks. Subsidizing the service with the CTSA grant keeps the costs predictable and manageable for investigators. Discussion/Significance of Impact: This approach recognizes the importance of informed consent, ethical considerations, and the potential social implications associated with genetic findings. Through open communication, participants are actively involved in the decision-making process and have the opportunity to seek further resources and support.

Objectives/Goals: The objective of this research is to characterize the role of anemia and its treatment with iron in the development of oxygen-induced retinopathy and to identify possible mechanisms of action for future investigation. Methods/Study Population: Newborn rats were exposed to cyclic hyperoxia (50%) hypoxia (10%) for 14 days to induce oxygen-induced retinopathy (OIR). Half of the pups were phlebotomized to induce anemia. Half of anemic and control pups received high-dose iron, while the other half received standard, low-dose iron. Retinas were dissected at postnatal day 20, and vascular outcomes were measured to determine phenotype. Comparisons will be made by two-way ANOVA. Additional future studies are planned: 1) electroretinogram to measure retinal function, 2) measure of retinal hypoxia and angiogenic protein levels to evaluate the effect on hypoxia-sensitive angiogenesis, 3) serum and retinal iron measures to determine iron status, 4) flow cytometry and cytokine array to evaluate immune cell response, and 5) exploratory single-cell RNA sequencing. Results/Anticipated Results: Preliminary vascular outcomes are pending. We anticipate that retinas of anemic pups treated with low-dose (standard of care) iron will exhibit iron deficiency and hypoxia and reduced severity of OIR. Retinas of anemic and non-anemic pups treated with high-dose iron will exhibit iron excess and increased severity of OIR. However, retinal function will be reduced in anemia and rescued with treatment with high-dose iron. We further anticipate that anemia and iron-deficiency will down-regulate overall immune cell fractions, which will correlate with reduced cytokine and chemokine levels, suggesting a reduced immune response. Finally, we expect vascular endothelial cells and pericytes in anemic pup retinas to show increased angiogenic gene expression and neurons to show signs of impaired development. Discussion/Significance of Impact: We expect that anemia has a differential impact on retinal vascular anatomy and neuronal function. While retinopathy severity may be improved, overall retinal function will be dampened. These findings and the mechanistic work of this study will generate targets for intervention to preserve improved retinopathy outcomes but rescue retinal function.

Objectives/Goals: SARS-CoV-2 infection has been shown to impact multiple organ systems, including the brain, and is associated with increased cognitive decline in vulnerable populations. The gut microbiome may play a significant role in modulating these effects, as shifts in microbiota composition have been linked to inflammation and systemic disease processes. Methods/Study Population: To explore these interactions, we conducted an acute COVID-19 study using 12-week-old C57 mice intranasally inoculated with 1x10^4 PFU of the Mouse Adapted 10 (MA-10) strain of SARS-CoV-2. On day 5 postinfection, we performed 16S ribosomal RNA sequencing on fecal samples, analyzed using QIIME2 and DeSeq2, to assess microbiome alterations and identify changes in metabolic pathways associated with inflammatory responses. Results/Anticipated Results: Analysis revealed notable shifts in the gut microbiome during the acute phase of COVID-19 infection, with significant alterations in metabolic pathways related to inflammation and immune regulation. These changes suggest that early gut microbiome disruptions may contribute to the host response in acute COVID-19. Discussion/Significance of Impact: Our findings indicate that acute SARS-CoV-2 infection leads to rapid alterations in the gut microbiome, which could underlie inflammatory and immune pathways impacting disease progression. These insights highlight the potential for microbiome-targeted therapies to mitigate acute COVID-19 morbidity and warrant further investigation.

Objectives/Goals: The gut microbiome and its metabolites, such as short-chain fatty acids (SCFA), are dysregulated in rheumatoid arthritis (RA); however, the significance of this observation and its implications in pathogenesis and therapeutics is unclear. Here, we explore the role of the SCFA, butyrate, in treatment efficacy in new-onset rheumatoid arthritis. Methods/Study Population: We designed a proof-of-principle study to determine the effects of butyrate supplementation in new-onset RA (NORA) patients that fulfilled 2010 ACR/EULAR RA criteria. We evaluated the effects of methotrexate (MTX) plus butyrate in NORA (n = 17; 1 gm butyrate, 3 times daily) compared to MTX alone (n = 19) over 4 months. MTX responders were defined by a change in disease activity score (DAS)-28 ESR of > 1.8 at 4 months. Fecal samples were collected at baseline and followed up for 16s rRNA sequencing and metabolite quantification by 1H NMR spectroscopy. Unpaired-t, paired-t, Wilcox and Fisher’s exact test were performed as appropriate. Results/Anticipated Results: MTX responders in the MTX-only group had a higher concentration of fecal butyrate than nonresponders at baseline (p = 0.045). Fecal butyrate concentration decreased over time in treatment responders in MTX group (p = 0.05), whereas butyrate concentration remained similar in MTX/butyrate group. Prior to treatment, both MTX and MTX/butyrate groups demonstrated similar levels of gut bacterial alpha diversity (Shannon index), yet only the MTX/butyrate group demonstrated a significant increase in alpha diversity by 4 months (p = 0.022). LefSe analysis demonstrated increased abundances of Bacteroides, Clostridium, and Phascolarctobacterium in responders in the MTX/butyrate group by 4 months. Ten (52.6%) patients in MTX and 11 (64.7%) in MTX/butyrate group were considered MTX responders by 4 months (p = 0.516). Discussion/Significance of Impact: Butyrate supplementation increased gut microbial diversity in patients and led to increased abundance of Bacteroides, which has been implicated in efficacy of methotrexate, a first line medication in rheumatoid arthritis. Butyrate may have implications for the maximization of therapeutic effectiveness in rheumatoid arthritis.

Objectives/Goals: Early childhood obesity is a major concern for Latin American children in the U.S., with gut barrier dysfunction as a key risk factor. Diet plays a role in gut development, but few studies have focused on Latin American infants. Our objective is to identify culturally relevant introductory foods that promote in vitro gut barrier development and function. Methods/Study Population: Pooled human milk (2.5 mL) from 6-month postpartum Hispanic mothers was combined with fruit and vegetable baby food products (2.5 g) and subjected to a 3-phase in vitro digestion system that simulates oral, gastric, and intestinal digestion. Digesta products were then anaerobically fermented for 24-hours using human stool inoculum, centrifuged, and filter sterilized. Intestinal epithelial cells (Caco-2, ATCC) were grown to confluence on 0.4 μm polystyrene transwell inserts using a DMEM + 10% FBS medium and allowed to differentiate for 21-days. Highly differentiated monolayers were treated with a 1:4 dilution of fermenta with medium in triplicate. The cell experiment was conducted twice. Cell layer integrity was measured using transepithelial electrical resistance (TEER) 24- and 48-hours after treatment. Results/Anticipated Results: Dietary intake data from the What We Eat in America database indicated that the top 3 fruit and vegetable exposures for infants with Mexican or Hispanic ethnicity were banana, apple, and carrot. Commercial baby food purees of these fruits and vegetables, in addition to baby foods with blueberry and spinach (Natural for Baby, Gerber Products Company) were acquired for digestion and fermentation experiments. Caco-2 cell experiments with these foods are ongoing. We expect Caco-2 monolayer incubated with fermenta from human milk and fruit or vegetables will have greater TEER values due to increased integrity of the cell layer as compared to those with breast milk alone. We also expect that exposure to fruit and vegetable fermenta will increase gene expression of tight junctions compared to exposure to media and human milk. Discussion/Significance of Impact: Using an in vitro digestion and fermentation system coupled with cell culture studies, we are identifying cellular mechanisms that link individual fruits and vegetables to gut barrier function. This will support translational work focused on mitigating obesity development in vulnerable populations.

Objectives/Goals: Increased numbers of senescent cells have been detected in both traumatic brain injury and epilepsy, suggesting them as targets for therapeutic intervention for treating posttraumatic epilepsy (PTE) and underscoring the need for innovative methods to identify and target senescent cells as a means of alleviating pathology. Methods/Study Population: C57BL/6 mice will receive a single controlled cortical impact (CCI) before having their brains removed at 1 week, 2 weeks, 4 weeks, 1 month, 2 months, and 4 months post injury (n = 5 per time point). Brain sections will then be co-labelled for glial and senescent markers to observe which cells begin to express senescent markers at various time points. We will also perform single-cell RNA sequencing to observe genetic changes associated with both TBI and epileptogenesis. Mice will also be treated with navitoclax, a BCL2 inhibitor being investigated as a senolytic agent, to determine if treatment results in decreased senescence and epileptogenesis, as well as improved behavioral outcomes. Results/Anticipated Results: Preliminary data revealed that senescent microglia begin to arise in the mouse hippocampus as early as 1 week post injury and continue to increase in concentration over the course of the following month, with up to 25% of microglia expressing p16, a known marker of senescence. We anticipate that further staining will reveal senescent astrocytes and neurons in a similar time-dependent manner. Further, we hypothesize that the single-cell sequencing of microglia from injured mice will reveal alterations to the expression of genes associated with neuronal excitability, inflammation, and/or synaptic modeling, features known to be associated with epilepsy. Finally, we anticipate treatment with navitoclax will alleviate the senescent phenotype, resulting in decreased epileptogenesis and improved behavioral outcomes. Discussion/Significance of Impact: Considering the lack of any studies examining senescent cell prevalence in PTE, these data will be the first to identify these cells as etiological factors in PTE onset, as well as druggable targets for improving pathological outcomes in PTE patients.

Objectives/Goals: Currently, a lack of screening markers and targeted therapies prevent clinicians from successfully treating PDAC. Precision medicine may allow oncologists to better combat this disease. To personalize care, knowledge of tumor protein posttranslational modifications, extracellular matrix makeup, and infiltrating immune cells is imperative. Methods/Study Population: Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) was employed to characterize the N glycosylation state, the ECM composition, and immune cell populations present within 10 formalin fixed paraffin embedded PDAC patient samples. Molecular dry spray of PNGase F and Collagenase III followed by enzymatic digestion allowed for the release of N glycans and ECM peptides from the tissue. Multiplex immunohistochemistry with photocleavable, mass-tagged probes was also performed on each tissue. This analysis produced a spatial map of N glycans, ECM peptides and immune cells with their distribution and abundance color-coded as a heat map of each tissue. Results/Anticipated Results: This analysis produced a unique N-glycan signature associated with specific tumor regions (necrosis, invasive margin, etc.) and immune cell clusters. Additionally, immune cells within the PDAC tumor microenvironment were found to be organized into immature tertiary lymphoid structures composed primarily of CD20+ B cells. Finally, a distinct distribution of ECM peptides within and surrounding tumor tissue was visualized, and putative identifications have been assigned to these stromal elements. Discussion/Significance of Impact: In the future, insights from this hypothesis-generating study may be leveraged to identify diagnostic and prognostic biomarkers for PDAC to improve early diagnosis and treatment response rates. The N glycan signature, ECM composition, and immune activation state in liquid biopsies including serum and PBMCs will be compared to data from this study.