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: 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: 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: 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: 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: 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.

Objectives/Goals: Neuroendocrine malignancies are heterogeneous cancers with varied clinical outcomes, yet the molecular landscape driving this heterogeneity has not been fully characterized. Here, we investigate the gene expression and mutational profiles of neuroendocrine malignancies to better understand the underlying biology and therapeutic targets. Methods/Study Population: Patients with neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) treated at Cleveland Clinic (2000–2022) with molecular profiling (n = 66) were identified. Mutational and gene expression profiles were abstracted from electronic health records (EHR). Clinico-pathological characteristics and overall survival (OS) were obtained from EHR. Statistical analyses were performed by R v.4.0.5 and R package Limma for differential gene expression, as well as Chi-square, Fisher’s exact, and Wilcoxon rank sum tests. Results/Anticipated Results: The cohort consisted of 38 cases with NEC, 18 NET g3, and 10 NET g1/2. EZH2 and cyclin E1 were differentially over-expressed in NEC vs. NET (p < 0.05), while PTEN and MSLN were differentially under-expressed in NEC vs. NET (p < 0.005). Several recurrent alterations co-segregated with aggressive histology (NEC vs. NET): TP53 (p 60. Also, there was no difference in gene expression profiles between the two age groups among NETs or NECs. Discussion/Significance of Impact: This study explores the molecular landscape of NETs and NECs, revealing distinct gene expression and mutation profiles related to clinical outcomes. High expressions of cyclin D1 and EGFR were significantly associated with improved 2-year OS in NECs, highlighting potential therapeutic targets. Future studies are needed to validate these findings.

Objectives/Goals: Cognitive decline is a known sequalae of intracranial radiation in the treatment of brain metastases. In this study, we investigate global structural changes in the brain akin to accelerated aging and compare aging kinetics between patients treated with whole-brain radiation therapy (WBRT) and stereotactic radiosurgery (SRS). Methods/Study Population: This retrospective study consists of patients with brain metastases treated with WBRT and SRS at our institution. Brain MRI images collected prior to radiation therapy and at approximately three and six months following radiation will be analyzed, excluding patients with evidence of worsening disease burden in the brain. Surface morphology of the cerebral cortex and sub-cortical structures will be extracted using Freesurfer and converted to graphs. Data will then be input into a validated graph convolutional neural network model to estimate brain age at each time point. A generalized linear model will be used to estimate the aging pace between baseline and follow-up for each subject within the whole brain as well as the sub-cortical structures, which will be compared between WBRT and SRS treatment groups. Results/Anticipated Results: We anticipate that intracranial radiation will accelerate brain aging to a greater extent following WBRT compared to SRS. Additionally, this accelerated aging will occur globally in the whole brain as well as within individual substructures, including the cerebral cortex, nucleus accumbens, amygdala, caudate, hippocampus, pallidum, putamen, and thalamus. Discussion/Significance of Impact: This study will demonstrate structural changes in the brain analogous to accelerated aging, supporting its potential use as an imaging biomarker to monitor cognitive decline after radiation therapy. Future work will explore the relationship between structural brain aging and assessments of neurocognitive function.

Objectives/Goals: Cerebral amyloid angiopathy (CAA) characterized by the accumulation of amyloid-beta in the cerebrovasculature, affects blood vessel integrity leading to brain hemorrhages and an accelerated cognitive decline in Alzheimer’s disease patients. In this study, we are conducting a genome-wide association study to identify genetic risk factors for CAA. Methods/Study Population: We genotyped 1253 additional AD cases using and curated existing genome-wide genotype data from 110 AD and 502 non-AD donors from the Mayo Clinic Brain Bank. We performed QC and imputation of all datasets. We conducted GWAS in AD only (N =  1,363), non-AD only, as well as the combined cohort (N = 1,865) by testing imputed variant dosages for association with square root transformed CAA using linear regression, adjusting for relevant covariates. To assess associations in the context of major CAA risk factors, we performed interaction analysis with APOEe4 presence and sex; and pursued stratified analyses. We collected peripheral gene expression measures using RNA isolated from 188 PAXgene tube samples of 95 donors collected across multiple time points. More than 1/3 of these participants have MRI measures collected. Results/Anticipated Results: Variants at the APOE locus were identified as the most significant in our study. In addition, several other variants with suggestive association were found under the main model adjusting for AD neuropathology (Braak and Thal). LINC-PINT splice variant remained associated with lower CAA scores in AD cases without the APOEe4 risk allele. To enhance the robustness of our findings, we are pursuing further expansion of our study cohort. In the periphery, we expect to identify expression changes associated with neuroimaging indicators of CAA and determine if variants and genes discovered via GWAS are implicated in these changes. Discussion/Significance of Impact: We expect this study will provide further insight into the genetic architecture underlying risk for CAA both in the context of significant AD pathology and without. Characterization of genetic variants and functional outcomes in the context of neuropathology may lead to new avenues of research aimed at identifying biomarkers and therapies to treat CAA

Objectives/Goals: Baseline lung allograft dysfunction (BLAD) is defined as the failure to attain normal lung function after transplant and has been associated with impaired survival. BLAD has no consensus definition and assessment of varying thresholds of abnormality may identify an impact on survival or development of chronic lung allograft dysfunction (CLAD). Methods/Study Population: This is a retrospective cohort analysis of bilateral lung transplant recipients who were transplanted between 1/1/2012 and 12/31/2022 who have complete pulmonary function data posttransplant. Thresholds of BLAD including percent predicted levels of FEV1 and FVC at 80%, 75%, 70%, 65%, and 60% were assessed. Outcomes evaluated include survival, development of CLAD, and association of key risk factors with the development of BLAD including donor, recipient, operative, and postoperative characteristics. Results/Anticipated Results: Totally, 680 bilateral lung transplant recipients were identified. Prevalence of BLAD ranged from 41.9% to 9.7% at specified thresholds. We anticipate performing survival analyses and evaluating development of CLAD in patients with BLAD at varying thresholds. We are assessing key donor, recipient, operative, and postoperative variables for association with BLAD. Preliminary analyses demonstrate significant associations of BLAD with recipient-donor height mismatch, prolonged mechanical ventilation time posttransplant, increased length of hospitalization posttransplant, the use of cardiopulmonary bypass intraoperatively and surgical allograft downsizing. Discussion/Significance of Impact: A threshold of BLAD at 70% predicted FEV1 and FVC or lower suggests importance for developing CLAD. Key characteristics associated with BLAD suggest importance of height mismatch, operative complexity, frailty, and severity of disease at time of transplant and immediately postoperatively.

Objectives/Goals: Our study’s overarching goal is to characterize the relationship between water transfer rate (Kw) across the blood–brain barrier (BBB) as measured by diffusion prepared (DP) and multi-echo (ME) ASL in two cohorts that have been shown to have regionally different water transfer rates due to underlying changes in BBB physiology. Methods/Study Population: Ten young, healthy participants (aged 21–30 years, 4f) and 12 elderly participants (aged 66–84 years, 8f) underwent MRI scans on a 3T Siemens Prisma scanner. Structural scans, along with DP and ME ASL, were acquired from each of the participants. The order of the DP and ME ASL sequences was reversed in half the participants to account for ordinal bias. FreeSurfer was used to segment the structural image into respective gray matter, white matter, and deep cortical gray regions to perform region of interest (ROI) analysis. Results/Anticipated Results: We are still in the project’s analysis phase. The anticipated result is that we will see different water transfer rate (Kw) patterns between the old and young groups and between the two sequence groups. Discussion/Significance of Impact: The significance of the results is that we can answer two questions: 1) if there are any differences between water transfer rates in the two age groups and 2) whether there are any variations in performance differences between the sequences.

Objectives/Goals: To determine the safety and feasibility of single-pulse transcranial magnetic stimulation (spTMS) for assessing corticospinal tract (CST) excitability and integrity in infants with perinatal brain injury, bridging foundational neuroscience to potential early diagnosis and clinical interventions during critical neuroplasticity periods. Methods/Study Population: Nineteen infants with perinatal brain injury underwent 1–3 spTMS sessions at three developmental time points: 3–6 months, 12 ± 1 month, and 18 ± 1 month. spTMS targeted the primary motor cortex to elicit motor-evoked potentials (MEPs), recorded via electromyography (EMG) from bilateral wrist flexor muscles. Safety monitoring included heart rate (HR), respiratory rate (RR), the Modified Behavioral Pain Scale (MBPS), and caregiver feedback. Feasibility was evaluated based on the ability to elicit MEPs, the number of trials that elicited MEPs, and procedure tolerability. Pre- and post-spTMS physiological and behavioral data were analyzed using linear mixed-effects models (LMEM) to account for repeated measures within subjects. Results/Anticipated Results: Thirty-five spTMS sessions were conducted in 19 infants (mean age 8.75 ± 5.12 months) with perinatal brain injury, delivering 1936 pulses with a median inter-pulse interval of 24.7 seconds. Analysis with LMEM found no significant changes in HR (mean difference  =  0.51 bpm, p  =  0.81) or RR (mean difference  =  0.69 breaths/min, p  =  0.66). MBPS scores showed a small statistically significant increase (mean difference  =  0.57, p  =  0.046), but overall remained low (mean score change from 1.94 to 2.51 on 0–10 scale). The median change score was 0, and 18/35 sessions showed no change in MBPS, indicating low discomfort with TMS. No adverse events were reported during or after the sessions. The feasibility of eliciting MEPs in this population was confirmed, with 235 MEPs identified in 17/19 participants. Discussion/Significance of Impact: Understanding neurodevelopment after injury is crucial for early diagnosis and targeted rehabilitation. Our study demonstrates that spTMS is a safe, feasible tool for assessing motor pathways in infants with early brain injury, highlighting its potential for clinical translation in neurodevelopmental disorders, and offering a pathway to improved care.

Objectives/Goals: Second-generation antipsychotics (SGA) are used to treat mental disorders in youth but are linked metabolic syndrome (MetS). Most data on prescribing practices and risk factors are from short-term studies (6–12 months). We aim to characterize prescribing and identify clinical and genetic predictors of MetS using electronic health records (EHR). Methods/Study Population: EHR data were extracted from Cincinnati Children’s Hospital Medical Center (CCHMC) for patients aged ≤21 years prescribed SGAs from 7/1/2009 and 7/1/2024, identifying prescribing prevalence. Next steps are to create an SGA-MetS case–control dataset 8 weeks after an SGA prescription. A case will be defined by meeting 3 of 5 criteria: 1) BMI ≥95th percentile for age/sex; 2) fasting glucose ≥100 mg/dL or use of anti-diabetics; 3) triglycerides ≥110 mg/dL; 4) HDL-C ≤40 mg/dL; 5) systolic/diastolic BP ≥90th percentile for age/sex or use of antihypertensives. The prevalence of SGA-MetS will be calculated by dividing SGA-MetS cases by total SGA users. Logistic regression will identify clinical predictors of MetS, and we will evaluate the association of polygenic risk scores (PRS) of BMI and type 2 diabetes with SGA-MetS risk. Results/Anticipated Results: Our preliminary analysis identified 30,076 patients who were prescribed SGAs (mean age 12 years, SD = 4; 58.8% female; n =  17685). Most self-identified as non-Hispanic (95%, n = 28,595) and of White race (76%; n =  22,935), with 18.5% self-identifying as Black or African American (n = 5,579). The most commonly prescribed SGAs were risperidone (n = 12,382, 41.1%), aripiprazole (n = 9,847, 32.7%), and quetiapine (n = 5,263, 17.5%), with much lower prescribing rates of other SGA known of their low risk of MetS (e.g., ziprasidone 5.5%, lurasidone 1.4%, paliperidone (n = 316, 1.1%), or others cariprazine (n = 72), asenapine (n = 43), brexipiprazole (n = 39), iloperidone (n = 24), and clozapine (n = 20). Discussion/Significance of Impact: Our analyses found that risperidone, quetiapine, and aripiprazole were the most prescribed SGA, with risperidone/quetiapine linked to a higher risk of MetS. We will present ongoing work identifying risk factors for SGA-MetS and examining the association with PRS. Our work has the potential to identify high-risk patients for personalized treatment.

Objectives/Goals: Nucleoside transport by ENT2 facilitates transit of the lupus anti-DNA autoantibody Deoxymab into cells and across the blood–brain barrier into brain tumors. This work examines the Deoxymab-nucleoside interactions that contribute to membrane crossing and apply them in brain tumor therapeutics. Methods/Study Population: Deoxymab interactions with individual nucleosides, nucleobases, and pentose sugars are examined by surface plasma resonance (SPR) and cell penetration assays in a panel of cell lines including glioblastoma, breast cancer, and normal breast epithelial cells. Deoxymab-conjugated gold nanoparticles are generated and tested for binding to normal human astrocytes and glioma cells, and the impact of supplemental nucleosides on this binding is determined. Deoxymab-gold nanoparticles are tested for brain tumor localization by systemic and local administration in mice bearing orthotopic glioblastoma brain tumors and enhancement of laser interstitial thermal therapy (LITT) examined. Results/Anticipated Results: Individual nucleosides significantly increase the efficiency of cell penetration by Deoxymab in all cell lines tested. In contrast, component nucleobases and pentose sugars significantly suppress the uptake of the autoantibody into cells. Deoxymab-conjugated gold nanoparticles bind DNA in vitro and to astrocytes in culture and are anticipated will enhance the efficacy if LITT in vivo by associating with DNA released by necrotic tumors and/or by locally administered nucleosides in brain tumor environments and subsequently act as heat sink to amplify LITT impact. Discussion/Significance of Impact: Deoxymab is a DNA-targeting, cell-penetrating autoantibody. These findings establish nucleosides as the components of DNA that promote autoantibody membrane crossing through ENT2 activity and indicate potential for use of Deoxymab-gold nanoparticles in combination with LITT for brain tumor therapy.

Objectives/Goals: The overarching goal of the GRAMM study is to address racial health disparities by increasing access to genetic counseling and testing among the Black community. Specific objectives are to determine patient acceptability of risk assessment at time of mammography and to evaluate subsequent access to and uptake of genetic counseling and testing. Methods/Study Population: All women presenting for screening mammography at the Ypsilanti Health Center under the University of Michigan were invited to enroll. After providing written informed consent, study participants entered family cancer and personal health information in the InheRET software tool which links to the National Comprehensive Cancer Network genetic testing guidelines. Upon completion, each participant was informed immediately if they did or did not meet the criteria to meet with a genetic counselor. For those who met the criteria, referral to genetic counseling was provided. All enrollees were invited to complete a post-assessment survey on acceptability of the service and genetics knowledge. Patients will be followed over time for completion of genetic counseling and testing. The study was approved by the Umich IRB. Results/Anticipated Results: As of 10/18/24, a total of 53 were enrolled, with 52 eligible for the study and were between the ages of 30–75 years. 51 identified as women and 1 identified as nonbinary. 21 (40.4%) identified as Black, 30 (57.7%) identified as White, 2 (3.8%) identified as Hispanic, and 1 (1.9%) identified as mixed race. Of the total enrolled, 25 (48.1%) met criteria to meet with a genetic counselor. Twelve (23.1%) have been scheduled to with meet with a genetic counselor and 2 (3.8%) of this group completed their appointment, but did not pursue genetic testing. 28 (53.8%) completed the survey and reported that they were satisfied with the service. Of the 16 people who screened positive and completed the survey, all 16 (100%) stated that they intended on proceeding with testing. Our study is still ongoing. Discussion/Significance of Impact: While this model has demonstrated acceptability so far, there are still possible barriers to genetic counseling and testing after the referral has been provided that need to be explored. However, this approach could provide a novel framework for combining risk assessment with screening mammography for all women nationwide.