Cytotoxic CD8+ T lymphocytes (CTLs) efficiently eliminate infected and cancerous cells throughout the body. T cell receptor (TCR)-induced Hedgehog signalling contributes to CTL-mediated killing, but how the pathway is activated downstream of the TCR is unknown. Here, we show that extracellular calcium (Ca2+) flux through L-type voltage-gated Ca2+ (Cav1) channels at the plasma membrane downstream of the TCR drives induction of the Hedgehog transcription factor Gli1, which is important for CTL killing in vitro and in vivo. This previously unknown non-canonical Hedgehog pathway is independent of canonical signalling and represents a primary mechanism of Gli1 induction in naive CD8+ T cells, whereas CTLs can also activate Gli1 via MAPK. We further show that Cav1 channel-controlled Gli1 induction is functionally important for CTL killing in mice and humans and other cytotoxic lymphocytes. Notably, killing capacity can be amplified using a small molecule Cav1 agonist or by overexpressing a gain-of-function Cav1 subunit. These findings suggest a strategy to improve cytotoxic lymphocyte function in the clinic, including in CAR T cell therapy.

Selective removal of endoplasmic reticulum (ER) is important for cell health. Macroautophagy is the primary mechanism for the removal of the ER, but the ER can be cleared in a macroautophagy-independent manner. However, the physiological relevance and mechanisms underlying macroautophagy-independent ER clearance remain largely unknown. Here we show that ER is cleared by lysosomes in a macroautophagy Atg gene-independent manner during development. This developmentally programmed Atg-independent ER clearance by lysosomes requires the ER protein Vap33 that promotes ER and lysosome contact. Oxysterol-binding protein (Osbp) is known to associate with Vap33, and Osbp lysosomal localization is required for ER clearance in cells lacking macroautophagy. Significantly, the cholesterol transport-associated protein Start1 regulates ER and lysosome contact, macroautophagy-independent ER clearance, and cholesterol transport from ER to the lysosome. These studies reveal that Vap33, Osbp, and Start1 promote ER clearance by lysosomes that is associated with cholesterol trafficking.

Background Apocrine carcinoma (AC) is a rare cancer that arises either from cutaneous apocrine sweat glands (cutaneous AC) or as breast carcinoma with apocrine differentiation (breast AC). Differentiation between them is clinically critical because their management and outcomes differ significantly: breast AC is treated according to established breast cancer therapies, whereas cutaneous AC lacks standardized systemic treatment and often carries a poorer prognosis. A diagnostic challenge arises when AC occurs in the axilla, where both entities may arise, given their highly similar histology. Methods In this study, we performed a retrospective multi-cohort transcriptomic analysis integrating institutional, multi-institutional, and publicly available datasets to characterize the molecular differences between breast and cutaneous AC, and developed a transcriptomic classifier using differentially expressed genes and a LASSO logistic regression model for tissue-of-origin classification. Results Transcriptomic profiling revealed that breast ACs exhibited higher steroid hormone-related signaling and lower proliferative activity compared with cutaneous ACs, while immune-related features were largely comparable between the two tumor types. These findings suggest that, despite shared apocrine morphology, breast and cutaneous ACs retain distinct biological characteristics reflective of their tissue of origin. Building on this, we further developed a transcriptomic classifier, the Breast Apocrine Carcinoma (BAC) score, which accurately distinguished breast and cutaneous ACs, suggesting that these tumors retain distinct tissue-of-origin transcriptional signatures despite shared apocrine differentiation. Furthermore, application of the BAC score to tumors with unknown primary origin in the axillary region enabled estimation of tissue origin, highlighting its potential clinical utility in diagnostically challenging cases. Conclusion Breast and cutaneous ACs exhibit distinct biological features despite shared apocrine morphology. The BAC score accurately distinguished tumors of breast and cutaneous origin, including within AC cohorts, highlighting the potential utility of transcriptomic profiling for improving their classification and diagnostic evaluation.

Despite advances in molecular oncology, head and neck cancer diagnosis still relies on anatomical site and histopathology. In up to 5% of metastatic cases, no identifiable primary tumor is found, defining carcinoma of unknown primary (CUP) and posing significant diagnostic and therapeutic challenges. We conducted a narrative review of the literature focusing on current diagnostic and treatment strategies for head and neck CUP, including imaging, viral biomarkers (HPV, EBV), minimally invasive procedures, and transoral robotic surgery (TORS). While TORS plays a dual role in diagnosis and treatment, a multidisciplinary approach of neck dissection, radiotherapy, and chemotherapy in high-risk patients, remains the cornerstone of management. Personalization is increasingly crucial in this comprehensive context, mainly involving treatment tailoring and radiotherapy planning, where limited evidence on field definition is available. Ongoing trials, such as UK FIND study, highlight the potential of targeted surgery to tailor and possibly reduce radiotherapy fields for better patient outcomes and improved quality of life.

Recent work on double descent has challenged classical bias-variance tradeoffs, showing that test error can decrease, increase sharply near the interpolation threshold, and then decrease again as model capacity grows. This phenomenon has been documented in regression and classification, but its relevance to survival analysis remains unclear. Survival data are subject to censoring, which obscures true event times, and widely used models such as the Cox proportional hazards model are optimized via partial likelihoods that emphasize ranking rather than calibrated risk estimation. It is therefore unknown whether double descent occurs in this setting, how censoring influences its manifestation, or how it interacts with standard performance metrics.We investigate these questions using synthetic survival data generated from Weibull hazards with controlled censoring, allowing systematic variation of model capacity from under to over parameterized regimes.While we verified double descent occurs in survival models, calibration plateaus and decouples from discrimination, even under strong [Formula: see text] regularization. This decoupling arises because the Cox partial likelihood optimizes rankings rather than magnitudes, producing extreme risk scores that break the Breslow estimator used to estimate survival probabilities. Validation on two real-world clinical datasets, the METABRIC breast cancer cohort and the SUPPORT study of seriously ill hospitalized adults, confirms the calibration-discrimination decoupling: IBS saturates at a constant value as network width grows while concordance varies, reproducing the primary synthetic finding across distinct clinical domains and sample sizes. These results highlight limitations of discrimination-based model selection in survival analysis and underscore the need for calibration-aware evaluation in high-capacity prognostic models.

Myocardial ischemia-reperfusion injury (MIRI) is the primary cause of death for acute myocardial infarction. LncRNA NNT-AS1 was upregulated in the H9c2 hypoxia-reoxygenation (H/R) model. However, the interaction between sevoflurane and NNT-AS1 is unknown in MIRI. The specific aim of this study was to explore the mechanism by which sevoflurane regulates cardiomyocyte injury via the NNT-AS1/miR-23a-3p/USP24 axis. The Caspase-3 level and apoptosis rate were used to assess the cell apoptosis level. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), GSH/GSSG ratio, the total iron content, and Glutathione Peroxidase 4 (GPX4) protein content were measured to evaluate the level of ferroptosis. The targeting relationship was detected by the dual-luciferase reporter gene assay. In the H9c2 H/R model, the NNT-AS1 and USP24 levels were increased, but miR-23a-3p was decreased. The cell damage was aggravated, and apoptosis and ferroptosis were promoted. However, the NNT-AS1 and USP24 expression was downregulated, and miR-23a-3p expression was upregulated by sevoflurane pre-treatment. Cell damage, apoptosis, and ferroptosis were also improved. NNT-AS1 functions as a molecular sponge for miR-23a-3p. The NNT-AS1 overexpression further aggravated cell damage, apoptosis, and ferroptosis, while miR-23a-3p overexpression reversed the damaging effect of NNT-AS1. In summary, sevoflurane pretreatment alleviates apoptosis and ferroptosis in H/R-stimulated cardiomyocytes by regulating the NNT-AS1/miR-23a-3p/USP24 signaling axis, which may provide a potential target for MIRI treatment.