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.

Objectives/Goals: Objectives/Goals (300 characters): Sarcopenia is a progressive skeletal muscle disorder associated with adverse outcomes. There is a gab of having objective measures upon performing interventions in patients with muscular dystrophies. The object of the present study is to describe the severity of sarcopenia in DMD patients in Puerto Rico. Methods/Study Population: Methods/Study Population (700 characters): Forty to 30 patients with DMD who are followed in MDA Care Center in the “Instituto De Rehabilitacion del Caribe.” Diagnosis will be confirmed with genetic testing and/or muscle biopsy. Lean muscle mass will be measured with a Whole Body Dexa (WBD) in a Nuclear Medicine Lab. Hand grip, elbow flexor, and knee extensor muscles strength will be measures with an isometric dynamometer. Patients’ functionality will done using the North Star Ambulatory Assessment scale and Brook and Vignos scales, which have been validated for patients with DMD and neuromuscular disease respectively. Correlations will be made with lean body mass (independent variable) and muscle strength and functionality (dependent variable). Results/Anticipated Results: Results/Anticipated Results (700 characters): We expect to find severe sarcopenia in patients with DMD in PR and that it will be more severe with older age. There will be a direct correlation between lean muscle mass and muscle strength, and functionality in DMD patients. Discussion/Significance of Impact: Discussion/Significance of Impact (300 characters): The findings of our study can help us to explore the possibility that Whole Body DEXA can serve as a potential biomarker for future studies since there is a need to develop noninvasive biomarkers that correlate with disease progression and interventions in DMD patients.

Objectives/Goals: To determine the heterogeneity in CD4:CD8 ratio in a well-characterized cohort of PWH and to investigate the predictors of intestinal CD4:CD8 ratio reconstitution (CD4:CD8>1) and its impact on systemic inflammation. Methods/Study Population: We enrolled 52 PWH on ART and with peripheral HIV-RNA Results/Anticipated Results: PWH had a lower CD4:CD8 ratio both in the peripheral blood [p1. This subset of PWH was more likely female (62% vs. 38%, p = 0.0158), diagnosed with HIV for a longer time [p = 0.0347] have longer duration of most recent viral suppression [p = 0.0365] higher CD4+ T cells at enrollment [p =  0.0262] and higher CD4+ T cell nadir. Multiple logistic regression showed that duration of HIV infection [OR 1.13 (95% C.I. 1.02–1.3)] and CD4 =  T cell nadir[OR 1.01 (95% C.I. 1.001–1.016)] were associated with colonic CD4:CD8 >1. Colonic CD4:CD8 ratio partially correlated with the peripheral blood CD4:CD8 ratio (r = 0.274, p = 0.068) and with the pro-inflammatory cytokines IL-20 (r =  -0.413, p = 0.036) and SLAMF-1 (r =  -0.329, p = 0.074). Discussion/Significance of Impact: In PWH, CD4:CD8 ratio

Objectives/Goals: We have shown that parathyroid hormone-related protein (PTHrP) is enriched at the LIFR promoter in breast cancer cells and inhibits the expression of dormancy-associated genes including LIFR. The objective of this study is to define where all PTHrP binds DNA and identify pathways that are regulated by PTHrP that promote breast cancer colonization of the bone. Methods/Study Population: In this study, we use human estrogen receptor-positive MCF7 breast cancer cells which we and others have reported lie dormant in the bone. MCF7 cells were engineered to express either PTHrP with an HA-tag (MCF7P), or a vector control (MCF7V). We use Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a method of mapping protein-DNA interactions, to define where PTHrP binds DNA. Here, an HA-specific antibody identifies regions of DNA that are bound to PTHrP in MCF7P cells compared to MCFV cells. Next, we perform DNA sequencing and gene set enrichment analysis (GSEA) on genes identified by CUT&RUN to identify pathways that are regulated by PTHrP. These experiments will determine how PTHrP regulates dormancy and breast cancer colonization in the bone. Results/Anticipated Results: We completed IgG (-control), H3K4me3 (+ control), and HA (PTHrP) CUT&RUN on MCF7V and MCF7P cells, and submitted DNA for sequencing. This study will define where PTHrP binds the genome and identify pathways regulated by PTHrP. Previously, through ChIP-qPCR we showed that PTHrP binds the LIFR promoter to repress LIFR expression. Given this result, we expect that PTHrP binds to the promoters of dormancy-associated genes including LIFR in MCF7P cells compared to MCF7V cells. PTHrP may be involved in regulating other processes besides dormancy to induce expansion of breast cancer cells in the bone, so we will use GSEA to identify pathways that are altered in MCF7P cells when PTHrP is over-expressed compared to MCF7V cells. Together, this will define how PTHrP regulates gene expression of bone metastatic breast cancer cells. Discussion/Significance of Impact: This study will unveil mechanisms of metastatic breast cancer expansion in the bone by defining where PTHrP binds the genome to regulate gene expression. These findings will reveal therapeutic vulnerabilities that will be used to target bone-disseminated tumor cells to prevent lethal recurrence.

Objectives/Goals: To use patient-level Center for Medicare and Medicaid Services (CMS) mandated quality metrics for inpatient rehabilitation facilities (IRFs) to develop and validate predictive models of functional recovery and interactions of baseline characteristics with therapy time. Methods/Study Population: Retrospective cohort study of a national US sample of ~40,000 adults with a primary diagnosis of stroke admitted to IRFs in 2023. Records will be randomly allocated to equal training and validation samples. We will use a random forest approach to generate predictive models for self-care and mobility functional outcomes using patient baseline and demographic data from a CMS-mandated assessment for IRFs (Section GG). We will also examine how predictive variables modulate the effects of occupational, physical, and speech-language therapy minutes. The random forest is a machine-learning approach that trains multiple models and combines their predictions to improve their overall performance. Results/Anticipated Results: Predictive models developed from the training sample will be applied to the validation sample to confirm their capacity to support new observations. Preliminary results will be reported, including the F1 score and area under the curve (AUC), with 95% confidence intervals. A unique feature of this study is the large sample, which contrasts with prior research in stroke rehabilitation using machine learning approaches. This study will produce powerful models that will inform the design of a clinical decision-support tool for application into clinical practice in a future study. Discussion/Significance of Impact: By using CMS-mandated quality metrics that are collected as part of standard clinical practice in IRFs, results will support clinical interpretation and application of metrics and inform the development of a clinician-facing intervention to support personalized rehabilitation approaches.

Objectives/Goals: Our aim is to compare scores collected from a health utilities measure (Health Utility Index, HUI) to those collected from a profile measure (Child Health Ratings Inventories, CHRIs- Global) among youth with newly diagnosed, high-risk classic Hodgkin lymphoma. Methods/Study Population: We will analyze existing data collected during the Children’s Oncology Group AHOD 1331 trial, which was a phase 3 clinical trial comparing the efficacy of adding brentuximab vedotin to standard-of-care treatment with multiagent chemotherapy in children and adolescents with high-risk Hodgkin lymphoma. The study also had a prespecified patient-reported outcomes (PRO) secondary aim, which involved recruiting a subset of the initial 309 patients aged 11 years or older enrolled in the trial for serial PRO measures taken over the trial period. Health-related quality of life (HRQoL) was assessed by CHRIs, HUI version 2, and HUI version 3 assessments at six planned points throughout treatment. Results/Anticipated Results: The first step of our analysis will be to ascertain agreement in scoring for parent–child dyads for the HUI2, HUI3, and CHRIs scores by comparing mean scores via two-sample t-testing. Bland–Altman plots will be constructed to compare agreement between the scores for HUI2/3 and the CHRIs. Similarly, Spearman’s correlation coefficients will be calculated for CHRIs with HUI2/3 for both parents and children. We hypothesize the CHRIs and HUI scores should roughly correlate with one another, but there may be divergence of correlation because the HUI has greater emphasis on functionality (e.g., sensation, mobility), and the CHRIs further emphasize social and emotional well-being in addition to physical health. Discussion/Significance of Impact: The composite score of the HUI 2/3 has allowed for direct comparison with other global HRQoL measures, providing greater clarity of its performance in different patient populations and clinical settings. The current study will improve understanding of the HUI 2/3 performance in a pediatric cancer population over time.

Objectives/Goals: Accurately stratifying patients with clinically isolated syndrome by risk of developing multiple sclerosis is of great clinical importance. Though numerous prediction models attempt to achieve this goal, no systematic review exists to independently evaluate these models. We aim to systematically identify and assess the risk of bias in all such models. Methods/Study Population: Studies developing or validating prediction models to assess risk of developing MS in patients with CIS who are not receiving an MS-indicated disease-modifying therapeutic will be identified via a systematic literature search. Studies will be evaluated for overall risk of bias using PROBAST (Prediction model Risk Of Bias Assessment Tool). Briefly, data sources, predictor, and outcome definition and assessment, applicability, and analysis will be assessed for each model in each identified study, and an overall risk of biased judgment will be assigned. Identified studies, predictors incorporated, results, and risk of bias assessment with accompanying rationale will be summarized in the final report. Results/Anticipated Results: Based on an initial exploratory search, we anticipate that most, if not all, identified prediction models will have high risk of bias. We anticipate that many studies will have limited applicability due to the use of outdated diagnostic criteria for definition of outcomes, or high risk of bias concerns originating from their analysis due to insufficient volume of included participants or poor model validation practices. We further anticipate that most, if not all, of the identified prediction models will have limited potential to be translated to use in a clinical setting. Discussion/Significance of Impact: Understanding how to identify patients with high-risk CIS may inform and improve clinician treatment decisions, patient outcomes, and future research study design. This work may also reveal flaws in current prediction models for CIS, opening new avenues of research and prompting development of improved prognostic models for patients with CIS.

Objectives/Goals: The goal of this project was to engineer 3D lung models by embedding human epithelial cells and fibroblasts within hybrid-hydrogels containing human decellularized extracellular matrix (dECM) from healthy and fibrotic lungs. This platform will enable us to study cell–matrix interactions involved lung fibrosis pathogenesis. Methods/Study Population: To incorporate dECM into hybrid-hydrogels it must be digested and functionalized. We determined the best conditions for pepsin digesting dECM from healthy and fibrotic human lung by collecting samples every 12 hours up to 96 hours and measuring total protein (BCA assay), total amine concentration (ninhydrin assay), and protein fragment size (SDS PAGE). Next, several molar excesses of Traut’s reagent were tested and functionalization was verified by comparing amine content (ninhydrin assay) to thiol content (Ellman’s assay). Hydrogel stiffness was measured initially and after stiffening using parallel-plate rheology. Results/Anticipated Results: The dECM was successfully pepsin-digested, with the 48-hour time point yielding the highest free amine levels. A 75-molar excess of Traut’s reagent was best for converting free amines to thiols. Dynamic stiffening allowed the creation of hybrid-hydrogels mimicking both healthy (1–5 kPa) and fibrotic (>10 kPa) lung microenvironments. We anticipate that this model will demonstrate differential fibroblast activation based on hybrid-hydrogel dECM source (healthy or fibrotic), microenvironmental stiffness, and cell source (healthy or fibrotic). Validation of this 3D co-culture system could accelerate drug discovery by providing a more accurate in vitro platform for high-throughput screening. Discussion/Significance of Impact: This work advances pulmonary fibrosis modeling by creating human dECM-based hydrogels that recapitulate the cellular and mechanical microenvironment of healthy and diseased lung, potentially enabling us to uncover novel therapeutic targets and improving drug efficacy testing in vitro.

Objectives/Goals: This study tests how fiber microstructural integrity and myelination levels within the cingulum connectome are associated with information processing speed (IPS) in relapsing-remitting multiple sclerosis (RRMS). We investigate the functional impact of structural coherence, myelin content, and white matter hyperintensities (WMH) load on IPS. Methods/Study Population: Data from 63 RRMS and 25 healthy controls (HC) were used. We hypothesize that the structural integrity of the cingulum bundle and its structural network – or connectome – is distinctly associated with IPS function in people with RRMS (vs. HC) due to myelin-related plasticity across the wiring. Using diffusion spectrum imaging and high-resolution tract segmentation, we constructed individualized white matter connectomes. Diffusion quantitative anisotropy (QA) and myelin fractions (MWF) were used to quantify structural coherence and myelination. WMH load was measured with T2-FLAIR imaging. Bayesian–Pearson correlations, mixed-linear, and moderation models explored how fiber-specific QA, MWF, and WMH load relate to IPS function in RRMS, as measured by Symbol Digit Modalities Test (SDMT). Results/Anticipated Results: We theorize that (1) QA in the cingulum connectome correlates with SDMT performance dimensionally, indicating that structural coherence in the white matter supports IPS function among both groups; (2) increased myelination will strengthen the positive association between QA and SDMT scores, suggesting that connectome-specific myelin content facilitates IPS; (3) conversely, WMH load within the cingulum connectome is expected to inversely correlate with SDMT scores, reflecting the detrimental impact of lesion burden on IPS function; (4) myelination in specialized tracts within the cingulum connectome play a compensatory role to support IPS function in the RRMS group. These investigations can offer a mechanistic clue to potential neuroplastic targets for cognitive interventions in MS. Discussion/Significance of Impact: By linking white matter integrity to cognitive function at the connectome level, this study can support neuroregenerative strategies to mitigate cognitive burden in RRMS. Our findings may advance understanding of how structural coherence, tract myelination, and WMH affect IPS, shaping personalized prognostic and therapeutic interventions.

Objectives/Goals: Obesity has been classified as a global epidemic and origin of numerous health issues. The central hypothesis of this study is that psychological measures, DNA methylation, and gene transcription will predict obesity-related outcomes after lifestyle interventions, and such interventions may alter DNA methylation profiles. Methods/Study Population: This study consisted of a randomized-controlled trial examining the effects of lifestyle +/- stress reduction interventions in 285 highly stressed parents with obesity, followed for 2 years. Full participants received nutrition and activity counseling, and were randomized to either a stress reduction intervention or a contact control. Those who otherwise qualified for the study but unable to fully participate were included in a no intervention control group. The intervention consisted of 12 weeks of nutrition and activity counseling +/- 2-hour weekly stress reduction interventions using MBSR and CBT-based strategies. DNA methylation was assessed using Illumina EPIC arrays. Results/Anticipated Results: Using linear mixed models (LMMs), this study will first examine the hypothesis that baseline psychological measures and pre-existing methylation sites associated with obesity and glycemic control (e.g., ABCG1, ATP10A, TXNIP, SREBF1, RNF39, and SOCS3) predict changes in BMI, HOMA-IR, and HgbA1C post-intervention and at 1 and 2 year follow-ups. Using sites that demonstrate statistical significance, we will develop a polymethylation risk score predictor of change in BMI. Next, we will examine the hypothesis that interventions which reduce obesity may also lead to improvements in epigenetic aging using LMMs to determine if changes in BMI or HOMA-IR predict changes in epigenetic age acceleration over the course of the study. Discussion/Significance of Impact: This work examines whether psychological factors and/or epigenetic markers may be used in patient stratification at initiation of treatment, enabling improved treatment selection, fewer years of obesity and decreased risk of comorbidities. This proposal also asks whether lifestyle interventions impact the aging process itself.

Objectives/Goals: Our team has developed a 24-well donor-derived skeletal muscle microphysiological system (MPS) to study signaling pathways associated with a variety of muscle diseases. 3D muscle will be utilized to evaluate pharmacologic interventions for these muscle conditions to improve both muscle mass and function. Methods/Study Population: In this study, muscle MPS were formed from healthy young female and male subjects. 3D muscle underwent a 21-day differentiation with an electrical stimulation (e-stim) regimen twice daily beginning on Day 14. Functional assessments in permeabilized fibers of both sexes included isometric and isotonic calcium-induced contractions, allowing for the characterization of force-pCa (-log[Ca2+]), force-velocity and force-power relationships. Samples from Day 17 and Day 21 will be assessed for pro-growth protein signaling via western blotting and a subset of samples will be analyzed by histology and microscopy for fiber type and size. Finally, culture media pre- and post-terminal e-stim on Day 21 will be collected for extracellular vesicle (EV) isolation and EVs will be assessed by standard proteomics analysis. Results/Anticipated Results: Permeabilized fibers from both sexes reproduced the well-established sigmoidal force-pCa and the curvilinear force-velocity and force-power relationships reported in native striated muscle. Maximum specific force and force-pCa relationship were not different between sexes. Isotonic contractile measurements revealed that these male and female fibers also exhibit similar force-velocity and force-power relationships. We anticipate that 3D muscles from day 17 compared to day 21 will exhibit higher levels of pro-growth protein signaling due to e-stim application and no differences in fiber type or size between sexes. Additionally, we expect that EV quantity will depend upon 3D muscle maturity and presence of e-stim. Discussion/Significance of Impact: This study demonstrates the similarities of functional characteristics and exercise (or e-stim) adaptation between native human skeletal muscle and 3D bioengineered skeletal muscle. Ultimately, this data further validates the muscle MPS system to study muscle diseases and to enhance the translation of therapeutics to clinical settings.

Objectives/Goals: This review examined if sleep duration is associated with established Alzheimer’s disease (AD) fluid biomarkers, such as amyloid-β peptides (Aβ40 and Aβ42), total-tau (t-tau), phosphorylated tau (p-tau181 and p-tau217), neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP). Methods/Study Population: We searched PubMed, CINAHL, and SCOPUS through September 15, 2024, using keywords and appropriate subject headings related to AD, fluid biomarkers, and sleep. The search was developed and conducted in collaboration with a medical librarian. We also searched Google Scholar and screened the reference lists of relevant reviews. Two independent reviewers screened 1,657 peer-reviewed articles, of which 21 met the inclusion criteria (14 with biomarkers measured in cerebrospinal fluid [CSF] and 7 in blood). Two review authors independently extracted study details from included articles using a standardized data extraction template. Results/Anticipated Results: Sample sizes ranged from 18 to 4,712 participants. Sleep duration was assessed using self-reported measures in 8 studies and objective measures in 13. For the 14 studies using CSF biomarkers, lower Aβ42 (3/14), Aβ40 (1/14), or the ratio (1/14) were associated with either short or long sleep duration; t-tau (3/14) and p-tau181 (4/14) levels were mostly associated with short sleep. For the 7 blood-based biomarker studies, Aβ42 (2/7), Aβ40 (2/7), and the ratio (3/7) had mixed results with either short or long sleep. T-tau (1/7) and p-tau181 (1/7) levels were associated with long sleep; NfL (2/7) was associated with both short and long sleep. Six studies reported nonlinear relationships, with both short and long sleep associated with unfavorable biomarker profiles. None of the studies investigated p-tau 217 or GFAP. Discussion/Significance of Impact: Our results suggest that the relationship between sleep duration and AD fluid biomarkers is very complex, and it highlights the importance of sleep in AD risk assessment and prevention. The inconsistency in findings stresses the need for standardized study design and measurement methods to clarify causality and inform clinical guidelines.

Objectives/Goals: Neuromodulation strategies like transcranial magnetic stimulation (TMS) can target specific neural circuits underlying particular psychiatric symptoms, potentially 1) enhancing understanding of mechanisms of illness and recovery and 2) acting as novel therapeutics. These feasibility studies lay foundation for a study of major depression. Methods/Study Population: Four healthy volunteers completed structural and functional MRI (fMRI). fMRI included a trait-adjective task, a negative self-referential processing task known to activate VMPFC, which is known to be abnormal in major depression. During the task, participants respond on a task pad whether they feel that each of a series of displayed adjectives (positive, negative, or neutral) applies to them. Three participants then participated in a simulated image-guided TMS session using their MRI data to target their VMPFC. Three-dimensional tracking of the participant’s head and the TMS coil was used to position the coil for peak stimulation of the targeted brain region. Results/Anticipated Results: Our team collected quality neural and behavioral data on the fMRI task; participants reported a tolerable experience. Simulated neuronavigated TMS showed feasibility and tolerability of positioning the device to stimulate VMPFC. The fMRI task activated the VMPFC as predicted. The MRI and TMS protocols were replicable and tolerable. These procedures can now be used experimentally by our team with confidence to test our hypothesis that targeting the VMPFC within the brain’s default-mode network may normalize aberrant VMPFC activity seen in major depression, thereby improving excessive negative self-referential processing. Discussion/Significance of Impact: This project lays essential groundwork for my K12 project, “Targeting Negative-Self Referential Processing in Depression with TMS,” a longitudinal neuroimaging and behavioral study using these methods in the study population of people with major depression.