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Background/Objectives: Muscle aging is a complex, dynamic process that impairs overall metabolism and physiological function. The molecular mechanisms underlying declines in muscle performance and metabolic efficiency remain poorly understood, largely due to the time and resource demands of traditional model organisms. The hawk moth Manduca sexta offers a promising alternative, with a short adult lifespan (~10 days) and notable similarities to vertebrate muscle systems, making it well-suited for time-course molecular dissection of muscle aging. Methods: In this study, we performed high-resolution temporal analysis of muscle tissues from aging M. sexta, spanning the physiomuscular aging process from middle age to advanced age. Results: We observed decreased expression of genes involved in fatty acid β-oxidation, ATP synthase subunits, superoxide dismutase, glutathione S-transferases, and heat shock proteins. In contrast, genes associated with proteolysis, catabolic processes, insulin signaling, akirin, titin, high-affinity choline transporters, and vesicular acetylcholine transporters were increased in expression. Conclusions: These changes suggest a shift toward increased proteolysis and protein catabolism with age. Our findings support the use of M. sexta as a complementary model for muscle aging research. However, it remains unclear whether the observed gene expression changes are driven by intrinsic, sex-specific age-related muscle aging or confounded by potential starvation effects in older males.

Introduction: Higher mtDNA-CN reflects better mitochondrial function, while lower levels indicate mtDNA release and increased inflammation. Although mtDNA-CN serves as a marker of cardiovascular health, the importance and mechanism of risk associated with mtDNA in high-risk individuals with chronic coronary artery disease (CAD) remains poorly understood. To address this gap, we leveraged the ISCHEMIA Biorepository to: (1) evaluate the association between mtDNA-CN and all-cause death (ACD); and (2) define a transcriptomic profile associated with high mtDNA-CN and ACD risk for high-risk patients with CAD. Hypothesis: Higher mtDNA-CN is associated with reduced ACD risk and inflammation in high-risk patients enrolled in the ISCHEMIA Biorepository. Methods: Low pass whole genome sequencing and whole blood RNA-Seq were performed on 594 ISCHEMIA and ISCHEMIA-CKD participants with moderate-severe ischemia. Data were processed using the NYU deciphEHR pipeline and mtDNA was quantified using mitoAnalyzer. mtDNA-CN was median split, with samples having values ≤ median classified as the 'low' group and those > median as the 'high' group. Unadjusted group differences were assessed using Chi-squared tests. Survival analysis was performed using Cox proportional hazard models, adjusted for age, sex, diabetes, eGFR, dialysis status, ischemia severity, and left ventricular ejection fraction. Adjusted differential gene expression (DGE) and gene set enrichment analysis (GSEA) were performed to explore molecular mechanisms underlying mtDNA-CN. Results: Median age was 67 (IQR: 57–78); 19% were female, 84% white, and 6% Hispanic. Hypertension (84%), diabetes (43%), and obesity (45%) were common; 26% had eGFR <60 mL/min/1.73 m 2 . Median mtDNA-CN was 153 counts per sample. After multivariate adjustment , mtDNA-CN > median was associated with a 48% reduction in ACD (aHR = 0.52, 95% CI: 0.30–0.90; P = 0.020) (Figure 1A) . DGE revealed downregulation of inflammatory genes in the mtDNA-CN group, including S100A9 (FC=-.41, p=3.32e-10) and S100A12 (FC=-.56, p=3.32e-10). Inflammatory pathways such as the inflammasome complex (NES=-2.17, p=9.32e-03) and interleukin-1 production (NES= -2.02, p=6.60e-03) were downregulated among participants in the high mtDNA-CN ( Figure 1B) . Conclusion: Higher mtDNA-CN is associated with lower risk of ACD and reduced inflammasome activity, suggesting that enhanced mitochondrial health may mitigate inflammasome-related pathology linked to ACD in patients with CAD.

Importance Contemporary guidelines recommend sentinel lymph node biopsy (SLNB) for patients with melanoma with predicted risk of SLN metastasis greater than 10% and consideration of SLNB when the risk is 5% to 10%. A gene expression profile (GEP)–based test that can accurately identify patients with a low risk of SLN metastasis would refine selection for SLNB. Objective To establish the predictive capability of the combined clinicopathological factors and GEP (CP-GEP) test to identify patients with primary cutaneous melanoma who can safely forgo SLNB and to investigate the prognostic value of CP-GEP regarding the outcome in patients with cutaneous melanoma after a negative SLNB (not reported herein). Design, Setting, and Participants This prognostic study was conducted from September 2021 to June 2024 at 9 academic medical centers with experienced melanoma surgeons. Included were patients with biopsy-proven invasive cutaneous melanoma with T1 to T3 tumors and clinically negative regional LNs. All patients were deemed candidates for SLNB using standard clinical criteria. GEP was performed on formalin-fixed, paraffin-embedded tissue from the primary melanoma biopsy. Analyses were performed from December 2024 to August 2025. Intervention CP-GEP testing to determine risk of having a positive SLNB in patients with T1 to T3 cutaneous melanoma who were considered appropriate candidates for the procedure based on standard clinical parameters. Main Outcomes and Measures CP-GEP results were reported as low risk or high risk; the primary outcome measure was negative predictive value (NPV) in low-risk cases. Analyses included NPV assessment by tumor (T) subcategory, primary site, and age. Results A total of 1761 patients (median [IQR] age, 64 [53-72] years; 997 male [56.6%]) underwent SLNB (310 [17.6%] SLN positive) and had a successful CP-GEP test; GEP was successful in 97.7% of samples. A total of 651 patients (37.0%) were considered low risk by CP-GEP. Among low-risk cases, 46 (7.1%) were SLN positive, and NPV was 92.9% (95% CI, 90.7%-94.8%). High-risk cases had a 23.8% (264 of 1110) SLN-positive rate. The percentage of cases with low-risk CP-GEP declined with increasing T category (T1 = 346 of 507 [68.2%]; T2 = 295 of 897 [32.9%]; T3 = 10 of 357 [2.8%]). CP-GEP results were consistent in discriminating SLN-positive rates across primary sites, histologic subtypes, and mitotic count categories. In 2 large subsets, clinical stage IB (n = 1187) and patients 65 years and older (n = 832), 6.5% (95% CI, 4.6%-8.8%) of low-risk clinical stage IB cases and 6.6% (95% CI, 4.2%-9.7%) of all low-risk cases in patients 65 years and older were SLN positive vs 18.3% (95% CI, 15.3%-21.6%) and 20.3% (95% CI, 16.8%-24.2%) for high-risk cases, respectively. Conclusions and Relevance In this multicenter, prospective, blinded, prognostic study, the CP-GEP test reliably identified patients with melanoma with less than 10% SLN metastasis risk. SLN metastasis rates were approximately 3-fold greater for high-risk vs low-risk CP-GEP cases. In appropriately selected patients, the CP-GEP test may add value for shared patient-surgeon decision-making.

Abstract Background and Aims Patients with chronic kidney disease (CKD) have a higher risk of developing heart failure with preserved ejection fraction (HFpEF). Growing evidence supports that changes in cardiac microcirculation are a major pathophysiological driver of HFpEF. We created a model of CKD stage 3 that subsequently developed a cardiac phenotype with numerous traits of HFpEF (CKD-HFpEF), including microvascular (MV) rarefaction and decreased availability of the pro-angiogenic growth factor vascular endothelial growth factor (VEGF) in the heart. We hypothesize that cardiac VEGF therapy will attenuate MV injury and improve cardiac function in CKD-HFpEF. Method CKD and normal pigs (n = 6 each, 3 males/3 females) were studied for 14 weeks. In addition, CKD-HFpEF pigs (n = 6, 3 males/3 females) were treated after 6 weeks of disease with a single intracoronary (IC) administration of VEGF attached to a drug-delivery vector (ELP-VEGF) that increases cardiac targeting, and effects quantified 8 weeks later. Renal (multi-detector CT) and cardiac (echocardiography, PV loops) morphology and function were quantified in vivo, microvascular (MV) density (3D micro-CT) ex vivo, and cardiac expression of VEGF was quantified by qPCR and western blot, as VEGF-related genes by bulk mRNA-sequencing (n = 3/group). Results A single IC administration of ELP-VEGF improved MV density and rescued cardiac function compared to placebo similarly in male and female pigs and independent of hypertension (Fig. 1A–C). These improvements were accompanied by improved mRNA and protein expression of VEGF and restored expression of VEGF-related pro- and anti-angiogenic genes (Fig. 1D and E). Conclusion Our study supports the prominent role of cardiac MV disease in the development of HFpEF in CKD, supports the critical need for healthy VEGF signaling to preserve the heart in CKD, and shows the feasibility of a potential new therapeutic strategy via a targeted mechanistic intervention.

Abstract Background Sickle cell disease (SCD) is a hemoglobinopathy with limited treatment options. Genetic modification of the patient's own hematopoietic stem cells (HSCs) offers an avenue to curative therapy using the patient's own cells. Many challenges exist in collecting sufficient numbers of starting material for manufacturing, beginning with the pre‐mobilization optimization of the patient to the mobilization, collection, and post‐collection phases. Study Design and Methods A working group within the Cellular Therapy Subcommittee of the American Society for Apheresis Clinical Applications Committee describes the challenges associated with apheresis collection of the starting material for gene therapy (GT) product manufacturing for patients with SCD and discusses potential strategies for overcoming these challenges. Results and Discussion Patients must undergo automated red blood cell exchange (RCE) to lower hemoglobin S in a relatively short window of time before apheresis collection of HSCs. The risks and benefits of transfusion as well as the logistics of providing adequate and appropriate blood products must be considered. Limited agents are available for mobilization of HSCs into the peripheral blood for collection and the timing of administration is short. Each collection cycle consists of RCE followed by at least three mobilization and apheresis collection events in addition to collecting backup cells. More than one cycle is typically needed to collect enough cells for GT drug product manufacturing. Optimization of patient factors and apheresis instrument parameters leads to better collection yields. Early and frequent ongoing coordination with all stakeholders involved is critical to developing personalized treatment plans for patients.

Abstract Objectives Granulomatous myositis (GM) is defined by focal collections of activated macrophages that fuse to form multinucleated cells that aggregate into granulomas within skeletal muscle. This study aimed to elucidate the pathophysiology of GM by defining its specific transcriptomic profile. Methods Bulk RNA sequencing was performed on 722 muscle biopsies, including 38 from patients with GM, other myopathies and healthy comparators. Spatial transcriptomics and immunohistochemistry assays were used to identify the location of specific transcripts and proteins within the tissue. Results We conducted a systematic analysis of the inflammatory pathways implicated in GM. This analysis revealed that GM muscle biopsies are characterized by elevated levels of IFNγ, IFNγ-inducible genes and proinflammatory cytokine genes including IL1B, TNF and TGFB1. Additionally, we identified a specific gene signature in GM, comprising 1293 genes that were significantly upregulated and 256 significantly downregulated. These genes were strongly correlated with transcriptomic markers of disease activity. Using this gene signature, support vector machine models accurately identified GM, achieving an area under the curve of 99.6% (99.0–99.9%) and an accuracy of 98.6% (98.2–98.9%). Among the identified genes, CHIT1 emerged as the most significantly upregulated gene, with its expression strongly correlating with disease severity. Furthermore, CHIT1 was detected at both the RNA and protein levels in granulomas and giant cells. Conclusion GM is transcriptomically characterized by a strong IFNγ signature and overexpression of proinflammatory cytokines, including IL1B, TNF and TGFB1. Additionally, it exhibits a unique transcriptomic profile, including CHIT1, which correlates with disease activity.

Abstract Our inability to obtain nerve samples from the vast majority of neuropathic pain patients impedes our ability to understand the disease, creates challenges in understanding mechanisms in specific patient populations, and limits our ability to make treatment decisions based on quantifiable molecular data. Fields like oncology have overcome these problems to take advantage of the insight that sequencing offers for understanding mechanisms of disease and have leveraged these molecular insights to dramatically change the treatment landscape in the past decade. Here we tested the hypothesis that skin biopsies could be used to gain insight into neuronal transcriptomic changes in patients with paclitaxel-induced peripheral neuropathy (PIPN). Our analysis reveals that hundreds of differentially expressed genes (DEGs) found through bulk RNA sequencing in these skin biopsies are likely contributed by dorsal root ganglion (DRG) neuronal axons and/or terminals. Up-regulated genes were representative of broad class of nociceptors whereas down-regulated genes were associated with putative injured DRG neurons expressing the PDIA2 gene. DEGs that could be confidently associated with specific subsets of skin cells were mostly expressed by keratinocytes supporting a growing literature tying keratinocyte-neuron communication abnormalities to pain in PIPN. We validated these findings by analyzing additional previously published datasets and through prospectively conducted spatial transcriptomics experiments on human skin biopsies. Our findings warrant further assessment of skin biopsies in additional neuropathic pain populations to gain insight into DRG neuron changes that have previously been thought to be inaccessible in routine clinical or scientific assessments in most patients.

PURPOSE Recent studies reveal that 5%-18% of children with cancer harbor pathogenic variants in known cancer-predisposing genes. However, DNA damage repair (DDR) genes, which are frequently somatically altered in pediatric tumors, have not been systematically examined as a source of novel cancer-predisposing signals. METHODS To address this gap, we interrogated 189 DDR genes for presence of germline predisposing variants (PV) among 5,993 childhood cancer cases and 14,477 adult noncancer controls (discovery cohort). PV were determined using a tiered approach incorporating ClinVar annotations, InterVar classification, and in silico tools (REVEL, CADD, and MetaSVM). Using logistic and firth regression, we identified genes with PV statistically enriched in the germline of children with tumors and replicated findings among 1,497 additional childhood cancer cases across three independent cohorts. RESULTS Analysis across all cases with cancer revealed enrichment of TP53 PV. Cancer-specific analyses confirmed known associations including germline TP53 PV in adrenocortical carcinoma, high-grade glioma (HGG), and medulloblastoma (MB), PMS2 in HGG and non-Hodgkin lymphoma (NHL), MLH1 in HGG, BRCA2 in NHL, and BARD1 in neuroblastoma. In addition, four novel associations were uncovered, including BRCA1 in ependymoma, SPIDR in HGG, SMC5 in MB, and SMARCAL1 in osteosarcoma (OS). Importantly, the SMARCAL1 :OS association was significant in the discovery (6/230, 2.6%, false discovery rate [FDR] logistic = 0.0189) as well as all three replication cohorts (Childhood Cancer Survivor Study: 8/275, 2.9%; P Fisher < .0001; Cancer Predisposition Syndrome-German Childhood Cancer Registry: 4/135, 3%, P Fisher = .002; Individualized Therapy for Relapsed Malignancies in Childhood: 4/217, 1.8%, P Fisher = .012). The remaining wild-type SMARCAL1 allele was deleted in three of four OS tumors with available data. CONCLUSION Our study confirms the relevance DDR genetic variation in pediatric cancer risk and establishes SMARCAL1 as a novel OS predisposing gene, providing insights into tumor biology and creating opportunities to optimize care for patients with this challenging tumor.