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.

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.