Citations

AbstractBackgroundAlzheimer’s disease (AD) risk and progression are significantly influenced by ApoE genotypes, with ApoE4 increasing and ApoE2 decreasing the susceptibility compared to ApoE3. Understanding metabolic pathways affected by ApoE genotypes will help decipher disease development and identify new therapeutic targets.MethodThis study investigates the impact of ApoE genotypes on aging brain metabolic trajectories using human ApoE‐targeted replacement mice. Applying Biocrates P180 targeted metabolomics platform, we analyzed the metabolic impact of ApoE2/2, ApoE3/3, and ApoE4/4 on fatty acid β‐oxidation, amino acids, and phospholipids, which are known to be altered in AD. Furthermore, we compared our rodent model results with human dorsolateral prefrontal cortex data from the Religious Orders Study/Memory and Aging Project (ROS‐MAP).ResultWe found aging mice carrying ApoE2/2 had altered branch‐chain amino acid metabolism and increased C5 acylcarnitine and its ratio to precursor isoleucine, pointing towards increased β‐oxidation and branched‐chain amino acid (BCAA) utilization. Furthermore, ROS‐MAP data revealed the ApoE2 genotype affects similar areas of metabolism in humans. Additionally, our data provide comprehensive insight into age‐related metabolic changes of the current mice model, independent of ApoE genotype, among phospholipids, sphingomyelins, amino acids, and biogenic amines. Many of the observed differences are also known aging markers in humans, with a connection to cognition and Alzheimer’s disease.ConclusionTogether, these results suggest a potential involvement of ApoE2/2 genotype in energy metabolism and characterize the current mice model for further study of ApoE in AD, brain aging, and brain BCAA utilization for energy.

AbstractBackgroundAlzheimer’s disease (AD) is neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often quantified by Thal phase and Braak stage, respectively. Aβ also frequently deposits in the cerebrovasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. These and related measures often show high variability within AD suggesting distinct underlying mechanisms. We hypothesize that, within the AD brain, neuropathology and levels of core AD‐related proteins are influenced by variations in DNA methylation (DNAm). To test this, we performed epigenome‐wide association studies (EWAS) using DNAm measures from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathologic measures (Braak, Thal, CAA) and brain biochemical levels of five proteins (apoE, Aβ40, Aβ42, tau, p‐tau).MethodsDNAm from neuropathologically‐confirmed AD cases was measured by reduced representation bisulfite sequencing (RRBS) from 471 TCX samples, 200 of which also had CER RRBS. TCX levels of five AD‐related proteins from three tissue fractions (buffer‐, detergent‐, and in‐soluble) were measured previously by ELISA (Liu 2020). CpG methylation (CpGm) was binned by a 15‐state chromatin model (Kundaje 2015) and averaged into CpGm clusters (rCpGm). rCpGms were tested for association with each AD endophenotype and expression levels of nearby genes through multi‐variable linear regression. Replication of significant rCpGms was performed in two independent datasets with AD‐related endophenotypes (Shireby 2022, De Jager 2014).ResultsOur innovative binning method demonstrated biologically relevant CpGm patterns. We found epigenome‐wide significant associations primarily with tau‐related endophenotypes including 93 that had significant and concordant associations in the replication datasets. These rCpGms also significantly associated with expression of nearby genes showing enrichment in oligodendrocyte marker genes including those related to myelination. In vitro validation of tau effects on oligodendrocyte gene expression through DNAm is ongoing.ConclusionsAlthough all endophenotypes tested are core to AD pathophysiology, our results suggest each has a distinct epigenetic architecture underlying their variability in the AD brain. In particular, we found evidence of DNAm variability associating with brain oligodendrocyte gene expression and TCX tau levels. By discovering AD brain endophenotype‐specific DNAm changes, we can identify core components of complex mechanisms revealing important biological insights into AD pathophysiology.

AbstractBackgroundThe efficacy of Calorie Restriction (CR) in enhancing cognition, promoting healthy aging, and extending lifespan is well‐established. Yet, it remains unclear whether the apolipoprotein E (APOE) genotype, a known modifier for aging and age‐related disorders, influences the beneficial effects of CR in countering aging.MethodsTo investigate this question, we utilized humanized APOE mouse models, which express APOE2, APOE3, or APOE4 alleles systematically (refer to as E2, E3, and E4 mice). These mice were subjected to either ad libitum (AL) feeding or 30% CR feeding, starting from 12 months and continuing up to 20 months of age (N = 11‐20/ genotype /group, mixed sex). We monitored energy expenditure using the Comprehensive Laboratory Animal Monitoring System (CLAMS). Additionally, we evaluated anxiety levels and memory capabilities through various behavioral tests. Finally, we collected brain, blood, and feces samples for transcriptomic, lipidomic, and microbiomic analyses.ResultsCLAMS measurements showed that CR significantly decreased the metabolic rate in E3 and E4 mice, but not E2 mice, during the non‐feeding period of the CR group, compared to the AL group. Furthermore, CR reduced anxiety and enhanced associative memory in E3 and E4 mice relative to the AL group. Brain transcriptomics revealed that CR upregulated the cholesterol synthesis pathway, potentially improving myelination‐related pathways in E3 and E4 mice. This finding was further validated by the increased staining intensity of myelin basic protein (MBP) observed in E3 and E4 mice from an independent cohort. In contrast, these effects were not pronounced in E2 mice. Additionally, while CR’s impact on the brain lipid profile was minor across all APOE genotypes, it significantly altered blood lipid profile of E2 mice, with minimal changes observed in E3 and E4 mice. Finally, in microbiomics, CR led to notable changes in the abundance of Bifidobacterium species in E3 and E4 mice and Lactobacillus species in E2 mice.ConclusionsOur study highlights the role of APOE genotypes in modulating the diverse effects of CR on cognition, brain transcriptomics, lipid metabolism, and the microbiome, providing a crucial foundation for considering APOE genotypes in developing prevention strategies for aging and age‐related disorders.

Abstract Immune reactions to medical implants often lead to encapsulation by fibrotic tissue and impaired device function. This process is thought to initiate by protein adsorption, which enables immune cells to attach and mount an inflammatory response. Previously, several antifibrotic materials have been either designed to reduce protein adsorption or discovered via high‐throughput screens (HTS) to favorably regulate inflammation. The present work introduces antifouling immunomodulatory (AIM) copolymer coatings, which combine both strategies to effectively enhance implant protection. AIM copolymers synergistically integrate zwitterionic moieties to resist protein fouling, HTS‐derived antifibrotics for immunomodulation, and silane monomers for grafting to diverse substrates including elastomers, ceramics, and metals. Interestingly, simply combining these monomers into conventional random or block copolymer architectures yielded no significant advantage over homopolymers. By contrast, an unusual polymer chain architecture — a zwitterionic block flanked by a mixed zwitterionic immunomodulatory segment — showed superior fibrosis resistance in both peritoneal and subcutaneous sites over one month in immunocompetent mice. This architecture also improved the performance of two different HTS‐derived antifibrotic monomers, suggesting that tailoring AIM architectures may broadly complement immunomodulatory chemistries and provide a versatile approach to improving implant longevity.

Abstract Background The majority of patients with prostate cancer (PCa) exhibit intrinsic resistance to immune checkpoint blockade (ICB) following radiotherapy (RT). This resistance is generally attributed to the limited antigen presentation of heterogeneous cells within tumors. Here, we aimed to isolate and characterize these diverse subgroups of tumor post‐RT to understand the molecular mechanisms of their resistance to ICB. Methods Single‐cell RNA‐sequencing (scRNA‐seq) was used to profile senescent cancer cell clusters induced by RT in LNCaP cells. The expression and phosphorylation levels of ataxia telangiectasia and Rad3‐related protein (ATR) were assessed by immunohistochemistry in clinical samples from patients with or without RT. Co‐immunoprecipitation, mutagenesis, and Western blotting were used to measure the interactions between proteins. Xenograft experiments were performed to assess the tumor immune response in the mice. Results We identified a subset of PCa cells that exhibited resistance to RT, characterized by a reduced antigen presentation capability, which enhanced their ability to evade immune detection and resist cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) blockade. scRNA‐seq revealed that the senescent state was a transient phase of PCa cells post‐RT, particularly in CTLA‐4 blockade treatment‐resistant cells. This state was marked by increased cytosolic ATR level. Cytosolic ATR phosphorylated CD86 in its cytosolic domain and enhanced the interaction between CD86 and its E3 ligase MARCH1 through electrostatic attraction. Depletion or inhibition of Atr increased the sensitivity to immune attack and improved responses to anti‐Ctla‐4 antibody treatment in a mouse model. Conclusions Our findings indicate that the activation of cytosolic ATR, which is associated with cellular senescence, impedes the effectiveness of combined RT and ICB treatments. This discovery may provide valuable insights for improving the efficacy of combined RT and ICB therapies in PCa.

Abstract Chemotherapy regimens for triple-negative breast cancer (TNBC) combine agents without knowing which agents drive response. Consequently, predictors derived from multi-agent regimens cannot be assumed to generalize to individual drugs or other regimens, motivating development of treatment-matched predictors. Here, we used TNBC patient-derived xenografts (PDXs) treated with carboplatin, docetaxel, or the combination to deconvolute drug-specific responses and identify associated molecular features. Combination treatment rarely improved upon the best single agent, with enhanced responses in only 13% of PDXs and antagonism in a comparable fraction. Proteogenomic analyses identified high cytokeratin-5 (KRT5) as a general marker of chemotherapy responsiveness and KRT5 immunohistochemistry discriminated responsive PDXs (AUROC, 0.83). To train treatment-specific predictors, we integrated these data with independent PDX and clinical cohorts with responses assessed after anthracycline-free platinum, taxane, or platinum-taxane therapy, ensuring response corresponded to the modeled treatment. Four feature selection strategies yielded largely nonoverlapping biomarker panels converging on treatment-relevant pathways. On independent test data, RNA-based predictors of complete response (CR) to platinum-based (carboplatin or cisplatin) and taxane-based (docetaxel or paclitaxel) chemotherapy achieved AUROCs of 0.80 and 0.86, respectively. For platinum-taxane regimens (carboplatin plus docetaxel or paclitaxel), proteomic-guided feature selection generated a 10-biomarker, protein- informed RNA predictor of pathologic complete response (pCR) that outperformed RNA-only feature selection and achieved an AUROC of 0.85 in an independent clinical cohort, while retaining practicality as an RNA-based assay. Treatment-matched integration of multi-omic PDX and clinical datasets provides a framework for chemotherapy-specific predictors with clinically relevant performance, supporting biomarker-guided precision selection and treatment optimization for patients with TNBC. Statement of significance Integration of multi-omic data from patient-derived xenografts with treatment-matched clinical cohorts yielded three retrospectively validated predictors of platinum, taxane, and platinum+taxane response, enabling biomarker-guided chemotherapy selection for patients with triple-negative breast cancer.

Reaching is a common daily activity requiring a range of humeral elevation that contributes to rotator cuff compression. The purpose of this study was to estimate supraspinatus and infraspinatus tendon compression risk relative to the acromion and coracoacromial ligament during reaching by manual wheelchair users with spinal cord injury. A cross-sectional design was used to evaluate 8 participants (7 males, median [range] age 36 y [23–61]). Electromagnetic motion capture recorded shoulder kinematics while participants reached for a can at 2 heights: low (0.91 m) and high (1.37 m). Using 1 set of computed tomographic-based bone models and individual glenohumeral kinematics, compression risk was evaluated as the percentage of the reach activity and number of seconds that tendon insertions were less than 5 mm from scapular landmarks. Reach conditions were compared using a Wilcoxon signed-rank test (α = .05). Although not statistically significant, the supraspinatus and infraspinatus relative to the acromion had approximately 40% of the activity duration or 0.8 seconds more time under 5 mm in low versus high reach, indicating increased risk of compression. Compression risk estimates were consistent with prior publications on planar arm movements. Estimating the risk of tendon compression using absolute time may help with understanding cumulative exposure during day-to-day activity.

Abstract Introduction Microdissection testicular sperm extraction (microTESE) is performed in men with nonobstructive azoospermia who desire sperm retrieval for use in IVF. This procedure can be performed in the operating room or in the clinic with local analgesic. Objective To report office-based microTESE sperm retrieval, fertilization, and livebirth rates. Methods A review was conducted of all patients who underwent office-based microTESE for nonobstructive azoospermia with a partner that who underwent concurrent oocyte retrieval for an intended in-vitro fertilization (IVF) intracytoplasmic injection (ICSI) cycle 2022-2024. All male partners underwent Y chromosome microdeletion testing, karyotype analysis, and hormonal testing preoperatively. Patients with a diagnosis of hypogonadism were offered medical therapy and those diagnosed with a varicocele were offered varicocelectomy prior to microTESE. Office-based microTESE was performed using a surgical operating microscope, spermatic cord block, and oral anxiolytic. Female partners underwent oocyte retrieval with oocyte cryopreservation prior to microTESE. Oocyte warming and ICSI was performed only following the collection of motile sperm from the fresh microTESE samples. Relevant demographic and clinical data were collected for patients and their partners. Descriptive data analysis was performed and reported as mean ± standard deviation. Results Ten patients underwent 11 office-based microTESE procedures. One patient had repeat microTESE following an unsuccessful IVF/ICSI cycle. Overall sperm retrieval rate was 70% for first time microTESE with mean search time 197.2 ± 96.5 min. Amongst couples with successful sperm retrieval, normal fertilization rate was 42%. Early cleavage arrest was seen in 84% of normally fertilized oocytes with a 16% blastocyst rate. Embryo development arrested entirely in two couples, while the remaining five couples had at least one blastocyst on day 5 and underwent embryo transfer. Of these, four resulted in a clinical pregnancy (36%). Three couples had a successful live birth and one has an ongoing pregnancy with ultrasound-confirmed fetal heartbeat. Conclusions Our small series demonstrates high sperm retrieval rates from office-based microTESE. Fertilization rates, clinical pregnancies and live birth rates are comparable with those reported in the literature. Given the financial cost associated with microTESE and IVF/ICSI for nonobstructive azoospermia patients, office-based microTESE may be a solution to make the process more financially accessible for couples. Disclosure No.

Abstract Purpose: NFE2L2/KEAP1/CUL3 mutations have been validated for radioresistance in cell-based assays and animal models. However, clinical validation of these biomarkers has been challenging because of multimodality treatment regimens. This study aims to investigate the association between NFE2L2/KEAP1/CUL3 mutations and patient outcomes, including local failure, locoregional failure, disease-free survival (DFS), and overall survival, using samples from a phase III trial in which patients were treated with radiation monotherapy at two controlled doses. Patients and Methods: We investigated NFE2L2/KEAP1/CUL3 mutations in 250 randomized patients with T2N0 glottic squamous cell carcinoma receiving definitive radiotherapy in the NRG/RTOG 9512 trial. A total of 119 patients had available biospecimens that were subjected to amplicon-based next-generation sequencing to assess for the presence of NFE2L2/KEAP1/CUL3 mutations without regard to outcomes. Mutations in NFE2L2/KEAP1/CUL3 were assessed blinded to clinical outcomes. Cox models (two-sided α = 0.05) were used to evaluate the association with clinical outcomes, performed by an independent statistical team. Results: Nineteen of 119 patients (16.0%) had NFE2L2/KEAP1/CUL3 mutations. Patient, treatment, and tumor characteristics were similar between those with and without mutations. Patients with mutation compared with those without had significantly more local failure [HR = 3.50; 95% confidence interval (CI), 1.56–7.89; P = 0.0025] and locoregional failure (HR = 3.80; 95% CI, 1.80–8.03; P = 0.0005). DFS was significantly worse for the mutated compared with the nonmutated group in the first 2 years (HR = 2.88; 95% CI, 1.46–5.66; P = 0.0022). The median DFS was shorter in the mutation group (10.3 months) versus those with intact NFE2L2/KEAP1/CUL3 (4.2 years). Conclusions: NFE2L2/KEAP1/CUL3 mutations may predict radiation treatment failure in T2N0 glottic cancer. See related commentary by Rao, p. 1563

Muscle stem cells (MuSCs) are essential for skeletal muscle regeneration, influenced by a complex interplay of mechanical, biochemical, and molecular cues. Properties of the extracellular matrix (ECM) such as stiffness and alignment guide stem cell fate through mechanosensitive pathways, where forces like shear stress translate into biochemical signals, affecting cell behavior. Aging introduces senescence which disrupts the MuSC niche, leading to reduced regenerative capacity via epigenetic alterations and metabolic shifts. Transplantation further challenges MuSC viability, often resulting in fibrosis driven by dysregulated fibro-adipogenic progenitors (FAPs). Addressing these issues, scaffold designs integrated with pharmacotherapy emulate ECM environments, providing cues that enhance graft functionality and endurance. These scaffolds facilitate the synergy between mechanotransduction and intracellular signaling, optimizing MuSC proliferation and differentiation. Innovations utilizing human pluripotent stem cell-derived myogenic progenitors and exosome-mediated delivery exploit bioactive properties for targeted repair. Additionally, 3D-printed and electrospun scaffolds with adjustable biomechanical traits tackle scalability in treating volumetric muscle loss. Advanced techniques like single-cell RNA sequencing and high-resolution imaging unravel muscle repair mechanisms, offering precise mapping of cellular interactions. Collectively, this interdisciplinary approach fortifies tissue graft durability and MuSC maintenance, propelling therapeutic strategies for muscle injuries and degenerative diseases.