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: 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: 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.

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: This research aims to identify genetic alterations influencing congenital anomalies of the kidney and urinary tract (CAKUT) and bridge a fundamental gap in understanding the cellular mechanisms underlying kidney development, with the long-term goal of enhancing treatments for congenital renal anomalies. Methods/Study Population: We will use a loss-of-function approach in combination with immunofluorescent microscopy techniques to determine the influence of Dnmbp perturbation on Daam1 localization, actin assembly, and junctional turnover. Additionally, to establish a foundation for delineating the molecular mechanism of DNMBP during kidney development, we will utilize clinical whole exome sequencing data to identify human DNMBP mutations associated with urogenital anomalies. Furthermore, we will determine whether human DNMBP mutations linked to CAKUT lead to disruptions in nephron development through loss-of-function rescue experiments in Xenopus. Results/Anticipated Results: Here, we evaluate the dynamics of Dnmbp-mediated transport of Daam1 within the developing kidney and show preliminary data suggesting that Dnmbp and Daam1 directly interact to promote adhesive contact formation between nephron progenitor cells. Furthermore, we propose a model in which Dnmbp functions as a critical regulator of epithelial tissue morphogenesis and provides a functional link between the dynamic processes of actin cytoskeleton regulation, intracellular adhesion, and vesicular transport. Future studies will determine whether Dnmbp interaction with Daam1 facilitates junctional actin assembly by directing Daam1 to cell–cell contact sites via Dnmbp-associated vesicle targeting, enhancing our understanding of the cellular mechanisms influencing tubule morphogenesis. Discussion/Significance of Impact: This research will establish a previously unknown role for DNMBP in kidney development and provide a comprehensive understanding of the impacts of simultaneously regulating vesicular transport and actin dynamics in nephrogenesis.

Objectives/Goals: Monensin is FDA approved for use in veterinary medicine. Recent studies pointed to its potent anticancer activity. Since de novo drug discovery process typically takes 10 to 15 years and requires an investment of approximately $1.3 to $3 billion, drug repositioning can bypass several steps in this process and increase the potential for success. Methods/Study Population: Cell viability assays were conducted on human MDA-MB-231, MDA-MB-468, and MCF10A breast cancer cell lines and mouse EO771 and 4T1 breast cancer cell lines. MDA-MB-231 cell line was used in all the studies unless specified otherwise. Time course levels of Bcl-2, Bak, p62, and LC3II were assessed via Western blotting with GAPDH as a loading control. Proteomics analysis was conducted by the IDEA National Resource for Quantitative Proteomics. Time course levels of major histocompatibility complex (MHC) I and II and calreticulin were evaluated using flow cytometry. At least three biological replicates have been conducted for each experiment. Results/Anticipated Results: Monensin and several of its novel analogs were potent toward human and mouse breast cancer cell lines. Furthermore, they induced apoptotic cell death as evidenced by Annexin V/PI assay, downregulation of Bcl-2, and upregulation of Bak in MDA-MB-231 cells. Proteomics analysis revealed that several molecular pathways related to MHC class I and II antigen presentation were significantly altered following treatment with these compounds. Additionally, monensin and its analogs significantly increased the expression of MHC class I and II. Our studies also showed that monensin and its analogs increase the surface calreticulin levels. Treatment of MDA-MB-231 cells with these compounds also resulted in an increase in p62 and LC3II expression, suggesting a disruption of the autophagic process. Discussion/Significance of Impact: These results suggest that monensin and its analogs not only exhibit anti-breast cancer cell activity but also modulate immune-related pathways. By disrupting autophagy and enhancing calreticulin levels, these compounds may potentiate antitumor immune responses, providing a promising avenue for drug repositioning in cancer therapy.

Objectives/Goals: This study investigates the contribution of the stool secretome (the soluble factors secreted by microbes into extracellular space) to in vitro α-synuclein (αSyn) oligomerization using stool cultures from patients with multiple system atrophy (MSA), a rare neurodegenerative disease hallmarked by pathologic αSyn aggregates. Methods/Study Population: Stool samples from MSA patients (n = 20), household controls (n = 20), and healthy controls (n = 20) will be cultured using an adapted dilution-to-extinction approach. The goal is to reduce microbial complexity progressively to produce random secretome combinations that may affect αSyn oligomerization differentially. The original inoculant and dilutions will be cultured anaerobically to collect conditioned media (CM) enriched with microbial secretomes. CM will be used to expose a fluorescence resonance energy transfer (FRET) biosensor assay and a Gaussia luciferase protein complementation assay – both modified to quantify αSyn-αSyn interaction indicating oligomerization. Any CM-altering αSyn oligomerization will undergo multiomic characterization to identify potential causative agent(s). Results/Anticipated Results: Specific microbe-produced molecules from the literature are anticipated to modulate αSyn oligomerization, identified by targeted, reductionist studies that selected and tested separately single microbial factors on αSyn aggregation in vitro and in vivo. From these studies, lipopolysaccharide and bacterial amyloid protein are expected to increase αSyn oligomerization, while short-chain fatty acids, such as butyrate, are expected to interfere with and decrease oligomerization. As a complementary systemic approach, this study’s agnostic methods involving MSA stool culture combined with the proposed dilution-to-extinction method are expected to identify additional MSA stool secretome components modulating αSyn oligomerization that might otherwise be missed in earlier reductionist approaches. Discussion/Significance of Impact: Completion of this reverse-translational work will aid in discovering MSA stool secretome components modulating αSyn oligomerization. Identification of specific factors contributing to pathologic αSyn behavior might set the stage for patient screenings for identified stool markers and could lead to microbiome-based interventions for MSA.

Objectives/Goals: The second highest fear of the aging population is cognitive decline. Diet is associated with brain aging; therefore, the objective is to determine the effects of a Western diet (WD) on cognitive decline and the efficacy of a Mediterranean diet (MeDi) fecal microbiota transplant (FMT) in WD-induced cognitive deficit progression in aged rats. Methods/Study Population: For Study 1, 12-month-old Fischer344 rats (NIA Aging Colony) will be randomly assigned to a WD, MeDi, or control (positive control) for 6 or 12 months. Microbiota composition, blood pressure, and body composition (DXA Scan) will be longitudinally assessed. Groups will undergo a battery of neurobehavioral assessments to measure cognitive performance. At the end of the study, mitochondria bioenergetic assays in isolated cerebral microvessels will be used to determine changes in cerebrovascular function. For Study 2, 18-month-old Fischer344 rats (NIA Aging Colony) will be randomly assigned to a WD, MeDi, or control for 6 months. At month 4, the WD+ MeDi-FMT group will receive once weekly MeDi-FMT for two months. Assessments will be performed as described in Study 1. Results/Anticipated Results: It is anticipated that the WD-related gut dysbiosis will increase blood pressure, fat-free mass, neurovascular dysfunction, and induce cognitive impairment relative to a MeDi. When using a MeDi-FMT as an intervention, it is anticipated that there will be measurable improvements in cognitive function relative to a WD through the regulation of gut dysbiosis, blood pressure, fat-free mass, and neurovascular dysfunction. Discussion/Significance of Impact: These results are expected to have an important positive impact because they will provide insights into the WD-induced gut dysbiosis-associated cognitive impairments, and evaluate the roles and mechanisms of MeDi-FMT in the therapeutic intervention of aged rats.

Objectives/Goals: Bacterial dysbiosis has emerged as an accomplice in the progression of many cancers. The pancreas microbiome changes in pancreatic cancer patients. The mechanisms via which components of the microbiome regulate tumor growth is unclear. We seek to determine if bacterial dysbiosis influences cancer cell behavior thereby promoting tumor progression. Methods/Study Population: We performed immunohistochemistry for lipopolysaccharide and observed bacteria preferentially located in close proximity to cancer cells. We utilized an in vitro cell culture system and in vivo mouse models, in the presence and absence of gut bacteria, to assess the effect of bacteria and bacterial metabolites on pro-tumorigenic signaling and transcriptional changes in the cancer cell. We analyzed cancer cells and epithelial cells using RNA sequencing, flow cytometry, and enzyme-linked immunosorbent assay. We also used targeted metabolomics to identify bacterial and cancer cell produced metabolites. Results/Anticipated Results: We found microbial dysbiosis can induce proliferation, an inflammatory response and an increase in tryptophan metabolism via the kynurenine pathway in the pancreatic cancer cell. Along with upregulated expression of IDO1 in vivo, we observe an increase in nicotinic adenine mononucleotide. Also, we observe an increase in nicotinic acid in vitro and nicotinic adenine dinucleotide within the cancer cell compartment in the presence of bacteria and bacteria conditioned media. Due to the critical role in many vital pathways of cell survival, NAD+ production is thought to play a significant role in cancer progression. Nicotinic acid can stimulate NAD production to protect cells from cell death. Discussion/Significance of Impact: Pancreatic cancer is associated with a distinct tumor microbiome and ablation slows disease progression. Our data delineate mechanisms via which microbes modulate the pancreatic cancer cell and provide insight into therapeutic strategies for gut microbial modulation in treating pancreatic cancer.

Objectives/Goals: The extracellular matrix (ECM) of the sinoatrial node (SAN) is critical for maintaining automaticity in hiPSC-derived pacemaking cardiomyocytes (PCMs) under cyclic strain. We aim to determine the ECM ligands responsible for cell-ECM mediated mechanotransduction and the resulting phenotype in PCMs. Methods/Study Population: HiPSCs are differentiated to PCM and replated on substrate with 5 or 15 kPa PDMS that are coated with 5 or 25 ug/cm of either collagen I or fibronectin at sub-confluent density to restrict junction engagement to only costameres. Then, PCM are subjected to 10% cyclic mechanical strain at 1 Hz for 48 hours, with static culture as control. PCMs from all conditions are subsequently fixed and stained for cardiomyocyte-specific troponin T (TnT), pacemaking HCN4 channel, and pro-pacemaking transcription factors (Shox2, Isl1, Tbx3, Tbx18). Additionally, PCM cell size will also be assessed. Results/Anticipated Results: Considering the amount of hypertrophy and myofilament in CMs correlates with mechanical strain, we expect a reduced degree of mechanotransduction in hiPSC-PCM on collagen I with a stiffness 15 kPa to induce smaller cell size with fewer myofilament and an upregulation of HCN4 and pro-pacemaking transcription factors than those on 5 kPa and those on fibronectin of either 5 or 15 kPa after cyclic strain. This is because COL1 is reported to have a lower signaling threshold but a limited sensitivity to force which contributes to the diminished mechanotransduction signaling. Discussion/Significance of Impact: Effects of the microenvironment on hiPSC-PCMs via costamere mechanotransduction may provide insights for engineering biopacemakers with a suitable ECM, to potentially preserve automaticity in hiPSC-PCMs and sustain long-term pacemaking function, making biopacemakers a step closer to reality.

Objectives/Goals: Optimal hydrocephalus treatment with permanent cerebrospinal fluid (CSF) diversion in myelomeningocele (MMC) patients is not well understood, especially how treatment response varies with time of MMC repair. We evaluate two treatment methods in this population–endoscopic third ventriculostomy (ETV) and ventriculoperitoneal shunting (VPS). Methods/Study Population: We retrospectively identified patients from St. Louis Children’s Hospital who were diagnosed with MMC prenatally and underwent either prenatal or postnatal repair and subsequently underwent permanent treatment for hydrocephalus (VPS or ETV +/- choroid plexus cauterization) between 2018 and 2024. The primary outcome was failure (defined as need for revision) of procedure and time to failure. All revisions were shunt insertions/revisions. Differences in preoperative and 6-month postoperative head circumference (HC) and WHO standard HC z-score were examined. Differences in preoperative and 6-month follow-up fronto-occipital horn ratios (FOHR), a validated age-independent measure of CSF within the brain, on CT and MRI were additionally examined. Results/Anticipated Results: Eighty-three MMC patients were identified. 46 (55%) underwent CSF diversion: 37 (80.4%, 9 pre- and 28 postnatal) VPS and 9 (19.6%, 5 pre- and 4 postnatal) ETV +/- choroid plexus cauterization. Six (16%) VPS patients required revision vs. 3 (33%) ETV patients (∆17%; 95% CI -9 – 50). Mean time to failure was longer after VPS vs. ETV (516, SD 470 vs. 34, SD 7 days) [∆482; 95% CI 163 – 800]. The decrease between pre- and postoperative FOHR was greater after VPS vs. ETV (6 mo: -0.14, SD 0.10 vs. -0.03, SD 0.07) [∆0.11; 95% CI 0.04 – 0.18]. Differences in pre- and postoperative HC were similar (VPS 5.67, SD 2.91 vs. ETV 4.04, SD 1.66 cm) [∆1.63; 95% CI -0.71 – 3.98]. Greater, but not significant, z-score decreases were seen after VPS vs. ETV (-1.04, SD 2.22 vs. -0.13, SD 1.11) [∆-0.91; 95% CI -2.68 – 0.86]. Similar trends were observed in pre- and postnatal MMC repair groups. Discussion/Significance of Impact: Failure rates in MMC-associated hydrocephalus patients were greater in the ETV group, but no definitive conclusion can be made due to imprecision. Those treated with ETV have less time to failure and smaller FOHR decreases than those treated with VPS, indicating less CSF drainage. Due to the need for more subjects, future research should be multicenter.

Objectives/Goals: Mood and anxiety disorders are a risk factor (Ozer et al., 2003) for posttraumatic stress disorder (PTSD) following trauma exposure. As such, a latent internalizing dimension may also be a risk factor. We examine how the shared variance between mood and anxiety symptoms (as in HiTOP; Kotov et al., 2021) impacts development of posttraumatic stress (PTS) symptoms, and symptom clusters. Methods/Study Population: Using data from a prior study of individuals who arrived at emergency rooms and were assessed at later time points (AURORA study; McLean et al., 2020), our sample included 1866 participants (1208 females, Mage  =  38.49 years) with available data for the proposed analyses. A latent factor (INTtotal) was operationalized as the shared variance between mood and anxiety symptoms (PROMIS Anxiety and Depression; Cella et al., 2010) as well as PTS symptoms (PCL-5, Weathers et al., 2013). We computed a second internalizing factor excluding PTS symptoms (INTma) to isolate the contribution of baseline affect and anxiety from PTS at baseline. We examined how baseline PTS symptoms, INTtotal, and INTma compare as prospective predictors for PTS symptoms at later time points and how these variables predict individual PTS symptoms. Results/Anticipated Results: Baseline INTma, INTtotal, and PTS symptoms were significant prospective predictors of PTS symptoms across all time points (all with t > 10, p < .005). When focusing on INTma relative to DSM-5 PTSD criterion (American Psychiatric Association, 2013), INTma significantly predicted later symptoms at six months posttrauma pertaining to Criterion D (t  =  18.88, p < .005), negative alterations in cognition and mood, Criterion E (t  =  15.44, p < .005) arousal and reactivity, and Criterion B, intrusion (t  =  15.44, p < .005). INTma significantly predicted symptoms in Criterion C, avoidance, though to a lesser degree (t  =  12.87, p Discussion/Significance of Impact: These findings bolster the utility of examining PTSD risk factors through a transdiagnostic lens. INTma was predictive of later PTS symptoms, independent of baseline PTS. Our analyses reveal clinical implications for the assessment of PTSD, and the tailoring of treatment for patients high in internalizing following trauma exposure.

Objectives/Goals: The primary objective of this study is to investigate the relationship between human leukocyte antigen (HLA) alleles to COVID-19 clinical severity, specifically: hospitalization, mortality, pneumonia by COVID-19, post-acute sequelae of SARS-CoV-2 infection (PASC), and clinical lab values. Methods/Study Population: We are conducting a retrospective cohort study utilizing the All of Us controlled tier dataset. The base population was defined as any patients with a COVID-19 diagnosis code (ICD-10: U07.1 or SNOMED: 840539006) and genomic sequencing data. PASC definitions were developed by the N3C consortium and refined in house. A total of 15,252 patients (64.5% female; 50.4% self-reported European ancestry; 18.8% self-reported African ancestry; 34.5% > 65 years old) are included in this study. HLA Class I and Class II alleles will be imputed from a global diversity reference panel utilizing the HIBAG “R” package. Results/Anticipated Results: Controlling for age, sex, race, and COVID-19 vaccination status, we anticipate determining the HLA alleles associated with severe clinical outcomes, such as Pneumonia by COVID (n = 1,436) and PASC (ICD-10:U09.9 or SNOMED:119303003 or OMOP:OMOP5160861 [n = 498]). We will assess which HLA alleles are associated with markedly different IgM and IgG COVID-19 serum antibody levels (n = 1,024). Coexisting conditions, i.e., type 2 diabetes, chronic obstructive pulmonary disease, and hypertension, will be controlled for with the Charlson comorbidity index. The accuracy of HLA allelic imputation will be validated in patients with long-read whole genome sequences. Discussion/Significance of Impact: Our findings can help identify patients who may be at risk of severe COVID-19 infection, particularly those undergoing bone marrow or organ transplantation. We hope this study will accelerate personalized care of COVID-19 in vulnerable populations.

Objectives/Goals: This study aims to explore transcriptional adaptation, where mutations in one gene trigger compensatory changes in related genes, and how this affects the variability in clinical manifestations of ALS. Our findings will provide insights for therapeutic strategies, while we also use gene editing to investigate correcting variants in ALS patients. Methods/Study Population: The presence of a premature termination codon triggers transcriptional activation. Therefore, we utilized CRISPR-Cas9 tool to generate a premature termination codon in CHCHD10 gene in multiple types of cells, including induced pluripotent stem cells derived from patient samples with known CHCHD10 mutations causative for Amyotrophic lateral sclerosis. CRISPR-Cas9 tool was delivered via ribonucleoprotein electroporation and transfected cell’s DNA was sequenced to validate gene editing. To confirm transcriptional adaption, changes in levels of protein and gene expression will be measured via immunoblot and quantification of CHCHD10 and CHCHCD2 from whole cell lysates of the edited cells. Results/Anticipated Results: Utilizing CRISPR-Cas9 tools, we anticipate that CHCHD2 gene can functionally compensate for the loss of function in the CHCHD10 locus through transcriptional adaptation. Additionally, employing single-stranded oligodeoxynucleotides (ssODNs) we aim to accurately correct the genetic aberrations in ALS patient cells, and study the pathomechanisms of aberrant CHCHD10. Discussion/Significance of Impact: The significance of this research lies in its potential to uncover transcriptional adaptation in humans, which could explain why patients with the same genetic variant experience different symptoms. By understanding this mechanism, we could pave the way for novel therapies, especially for CHCHD10-associated ALS.

Objectives/Goals: One in 14 individuals have a substance use disorder (SUD). We suggest that a trait of poor impulse control, or high impulsivity, may predict relapse risk. We explore how changes in brain structure linked to decision-making and reward might drive high impulsivity, helping create a “biosignature” to identify those most at risk and guide treatment choices. Methods/Study Population: Male rats were phenotyped as high impulsive (HI) or low impulsive (LI) based on premature responses on the one-choice serial reaction time (1-CSRT) task. Rats then received an intracranial infusion of a retrograde virus (AAVr2) in the nucleus accumbens (NAc) to trace corticoaccumbens neurons back to the medial prefrontal cortex (mPFC). After impulsivity phenotyping (ITI8), another cohort of animals performed cocaine self-administration followed by 30 days of abstinence. Cue reactivity, a measure of relapse-like behaviors, was performed on abstinence day 30. Analyses of microtubule-associated protein 2 (MAP2), a cytoskeletal marker of dendrites, spines, and somas was performed with western blotting and fluorescent images of brain slices after phenotyping and cocaine abstinence. Results/Anticipated Results: HI rats made greater premature responses, a marker of impulsive action vs. LI rats at baseline (p Discussion/Significance of Impact: Poor inherent impulse control and drug cues heighten relapse risk. We found high impulsivity linked to brain structure differences and lower protein markers of synaptic (units supporting signaling) strengthening. Future investigations into brain-behavior links with impulsivity may further identify a SUD relapse vulnerability biosignature.