Macrophages participate in the immune system by recognizing and engulfing foreign bodies inside phagosomes, which fuse with lysosomes in their cytoplasm to form mature phagolysosomes. Lysosomes have an acidic interior and generate and release reactive oxygen and nitrogen species (ROS/RNS) to destroy the endocytosed entities. It has been previously reported that intra-lysosomal pH plays an essential role in the regulation of ROS/RNS. However, the exact regulatory mechanism remains to be elucidated. Taking advantage of the large number of active lysosomes distributed along the phagocytic lumen during frustrated phagocytosis of glass fibers by macrophages, the intensity of 4 primary ROS/RNS released fluxes (ONOO⁻, H₂O₂, NO, and NO₂⁻) was monitored with platinum nanoelectrochemical sensors, thereby revealing the important role of intra-lysosomal pH on ROS/RNS fluxes after pharmacological modulations. Acidification (pH <5.0) does not alter the rate of production of ROS/RNS precursors (superoxide ions, O₂^•−, and parent NO) but promotes O₂^•− protonation, leading to an increase of H₂O₂ release. In contrast, the initial production of NO, which subsequently increased the release of ONOO⁻ and NO₂⁻, was enhanced by alkalinization (pH >6.0). The resulting increased oxidative stress was associated with massive proinflammatory cytokine release. Taken together, these results provide important information about the impact of lysosomal pH on ROS/RNS regulation.

Achieving high maturity and functionality in in vitro skeletal muscle models is essential for advancing our understanding of muscle biology, disease mechanisms, and drug discovery. However, current models struggle to fully recapitulate key features such as sarcomere structure, muscle fiber composition, and contractile function while also ensuring consistency and rapid production. Adult stem cells residing in muscle tissue are known for their powerful regenerative potential, yet tissue-derived skeletal muscle organoids have not been established. In this study, we introduce droplet-engineered skeletal muscle organoids derived from primary tissue using cascade-tubing microfluidics. These droplet-engineered organoids (DEOs) exhibit high maturity, including well-developed striated sarcomeres, spontaneous and stimulated contractions, and recapitulation of parental muscle fiber types. Notably, DEOs are produced in just 8 d without the need for primary cell culture—substantially accelerating the 50- to 60-d process required by classical organoid models. Additionally, the cascade-tubing microfluidics platform enables high-throughput production of hundreds of uniform DEO replicates from a small tissue sample, providing a scalable and reproducible solution for skeletal muscle research and drug screening.

Background: Osteosarcoma (OS) is a primary malignant bone tumor predominantly affecting adolescents. Chemotherapeutic agents, such as cisplatin, are commonly used in OS treatment; however, drug resistance markedly undermines treatment efficacy and contributes to reduced patient survival. The mechanisms underlying cisplatin resistance remain poorly understood. Recently, palmitoyl-protein thioesterase 1 (PPT1), a depalmitoylation enzyme, has attracted attention for its role in tumorigenesis and drug resistance. Investigating the mechanisms of PPT1 may offer new strategies to overcome resistance. Methods: This study analyzed multiple Gene Expression Omnibus datasets and utilized the OncoPredict tool to demonstrate the elevated expression of PPT1 in OS and its critical role in cisplatin resistance. By combining single-cell analysis with in vitro and in vivo experiments, we explored how PPT1 influences OS development through depalmitoylation and assessed the antitumor effects of the PPT1 inhibitor Ezurpimtrostat (GNS561), as well as its synergistic effects when combined with cisplatin. Results: We demonstrated that Sprouty 4 (SPRY4) undergoes a dynamic palmitoylation cycle regulated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7) and PPT1, which modulates mitogen-activated protein kinase (MAPK) signaling and subsequently affects tumor cell proliferation, migration, apoptosis, and drug resistance. Further validation confirmed the effectiveness of the PPT1 inhibitor GNS561 in overcoming cisplatin resistance. Notably, GNS561 exhibited a significant synergistic effect when used in combination with cisplatin, greatly enhancing the sensitivity of cisplatin-resistant cells. Conclusion: This study highlights the pivotal role of PPT1 in OS resistance mechanisms. PPT1 and ZDHHC7 regulate SPRY4 through a dynamic palmitoylation–depalmitoylation cycle that modulates MAPK signaling activation and contributes to OS cell proliferation, migration, and drug resistance. As a PPT1 inhibitor, GNS561 not only inhibits OS cell proliferation but also demonstrates synergistic effects with cisplatin, significantly enhancing cisplatin sensitivity in resistant cells and promoting apoptosis. Our findings offer a novel approach for targeting PPT1 in therapeutic strategies. GNS561 holds promise as an adjunctive therapy when combined with cisplatin, potentially overcoming resistance and improving efficacy, thereby enhancing the prognosis for OS patients. Future studies should further investigate the clinical potential of GNS561 and optimize OS treatment strategies.

Arsenic trioxide (ATO) is able to selectively target and degrade the disease-causing PML::RARα (P/R) oncoprotein in acute promyelocytic leukemia (APL) for curing the disease. However, some relapsed patients develop resistance to ATO due to mutations in the promyelocytic leukemia (PML) part of the PML::RARα fusion gene. A relapsed APL patient had shown resistance to ATO and chemotherapy and was identified to harbor a point mutation (A216V) in the unrearranged PML allele rather than the PML::RARα fusion gene. Here, we report that mutations in the unrearranged PML allele impede the ATO-induced destabilization and degradation of the wild-type P/R oncoprotein. Deletion of the coiled-coil domain in a PML mutant completely reversed wild-type P/R protein resistance to ATO by abolishing the interaction between PML and P/R proteins. Collectively, our findings reveal that a point mutation in the unrearranged PML allele can confer ATO resistance through a protein–protein interaction. Therefore, the unrearranged PML allele should also be screened for drug-resistant mutations in relapsed APL patients.

Malignant and premalignant ocular surface tumors (OSTs) can be sight-threatening or even life-threatening if not diagnosed and treated promptly. Artificial intelligence holds great promise for the early detection of these diseases. However, training traditional convolutional neural networks (CNNs) for this task presents challenges due to the lack of large, well-annotated datasets containing OST images labeled according to histopathological results. Here, we introduce the ocular surface pretrained model (OSPM), a domain-specific pretrained model designed to address the scarcity of labeled data. OSPM is constructed utilizing self-supervised learning on approximately 0.76 million unlabeled ocular surface images from 10 clinical centers across China and can be readily adapted to the OST classification task. We then develop and evaluate an OSPM-enhanced classification model (OECM) using 1,455 OST images labeled with histopathological diagnoses to differentiate between malignant, premalignant, and benign OSTs. OECM achieves excellent performance with AUROCs ranging from 0.891 to 0.993 on internal, external, and prospective test datasets, significantly outperforming the traditional CNN models. OECM demonstrated performance comparable to that of senior ophthalmologists and increased the diagnostic accuracy of junior ophthalmologists. Greater label efficiency was observed in OECM compared to CNN models. Our proposed model has high potential to enhance the early detection and treatment of malignant and premalignant OSTs, thereby reducing cancer-related mortality and optimizing functional outcomes.

Background: Ischemic heart disease is a leading cause of mortality and disability worldwide among cardiovascular conditions. Myocardial ischemia–reperfusion injury (MIRI) occurs following percutaneous coronary intervention, during which neutrophils generate neutrophil extracellular traps (NETs) in response to injury. This study aims to elucidate the mechanisms underlying NET activation and its impact on MIRI. Methods: Sham and MIRI rat models were established. Various techniques, including enzyme-linked immunosorbent assay, hematoxylin and eosin staining, Masson staining, and transmission electron microscopy, were used to assess endothelial cell injury and myocardial tissue inflammation. Immunofluorescence was employed to evaluate NET activation in tissues, peripheral blood neutrophils, and protein colocalization. MitoTracker and ER-Tracker staining were conducted to assess the formation of mitochondria-associated membranes (MAMs). Extracted NETs were applied to conduct microvascular endothelial cell tube formation assay and flow cytometry. RNA-sequencing and immunoprecipitation–mass spectrometry were applied to determine the key regulators. Flow cytometry and Western blot were used to assess Ca²⁺ and mitophagy levels in neutrophils. Deoxyribonuclease I, NET inhibitor, was injected into MIRI rats to evaluate the in vivo effects of NET modulation on MIRI severity. Results: MIRI was often accompanied by cardiac microvascular endothelial cell (CMEC) injury and inflammation. Lactate mediated H3K18 lactylation at the MICU3 promoter in neutrophils, enhancing its transcription and leading to elevated MICU3 levels. Besides, lactate also promoted the interaction between MICU3 and AASR1, stabilizing MICU3 through lactylation. Up-regulated MICU3 interacted with VDAC1, facilitating MAM formation, excessive Ca²⁺ uptake, mitochondrial dysfunction, mitophagy activation, and NET activation. Elevated NET level exacerbated CMEC dysfunction, further aggravating MIRI. Conclusion: Lactate-driven MICU3 transcriptional activation and stabilization facilitates NET formation, contributing to MIRI development.

The disruption of ferroptosis, an emerging form of programmed cell death, is crucial in the development and aggressiveness of tumors. Meanwhile, the mechanisms and treatments that control ferroptosis in neuroblastoma (NB), a prevalent extracranial cancer in children, are still unknown. In this study, forkhead box C1 (FOXC1) and O-GlcNAc transferase (OGT) are identified as regulators of asparagine- and alanine-mediated ferroptosis repression in NB. Mechanistically, OGT facilitates FOXC1 stabilization via inducing O-GlcNAcylation in liquid condensates to increase the expression of asparagine synthetase (ASNS) and glutamate pyruvate transaminase 2 (GPT2), resulting in asparagine and alanine biogenesis, and subsequent synthesis of cystathionine β-synthase (CBS) or ferritin heavy chain 1 (FTH1). Meanwhile, exonic circular OGT RNA (ecircOGT) is able to encode a novel protein (OGT-570aa) containing domain essential for binding of OGT to FOXC1, which competitively decreases the OGT–FOXC1 interaction. Preclinically, miconazole nitrate facilitates the interaction of OGT-570aa with FOXC1, suppresses ferroptosis resistance of NB cells, and inhibits their growth, invasion, and metastasis. In clinical NB cases, higher OGT, FOXC1, ASNS, GPT2, CBS, or FTH1 levels are correlated with worse survival, while lower ecircOGT or OGT-570aa expression is associated with tumor progression. These results indicate that targeting the ecircOGT/OGT/FOXC1 axis inhibits asparagine- and alanine-mediated ferroptosis repression in NB progression.

Oxalate-induced crystalline kidney injury is a common form of crystal nephropathy. The accumulation of calcium oxalate (CaOx) crystal could lead to renal epithelium injury and inflammation. The underlying cellular events in kidney after CaOx crystal formation are largely unknown. This study was aimed to gain a better understanding of mouse kidney function in the development of renal CaOx formation. The study utilized a mouse CaOx model to analyze the cellular response at 5 time points using single-cell RNA sequencing and investigate the interaction of different cells during renal CaOx crystal formation. Additionally, the study investigated the communication between these cells and macrophages, as well as the role of chemokines in recruiting infiltrating macrophages. RNA velocity analysis uncovered an alternative differentiation pathway for injured and S1 proximal tubule cells, which mainly communicate with macrophages through the SPP1–CD44 pair, along with the expression of proinflammatory factors and stone matrix genes during renal CaOx crystal formation. Furthermore, resident Fn1 macrophages were found to express chemokines, such as CCL2, which recruited infiltrating macrophages. The CCL2 secretion was mediated by the CD44–AKT pathway. Blocking CCL2 decreased the expression of injury markers in the kidney, including CLU, LCN2, and KIM-1, and inhibited CaOx crystal deposition. The study identified potential cell types and target genes involved in renal tubule injury in oxalate-related crystal nephropathy. The findings shed light on the cellular processes that contribute to the formation and damage caused by CaOx crystals within the kidney, which could lead to the development of potential cell types and target genes for treating this condition.

Autoimmune kidney diseases (AIKDs) depict a range of disorders involving immune-mediated damage to the kidneys, where conventional biologic therapies involving monoclonal antibodies often prove insufficient because of persistent autoreactive B cell reservoirs in lymphoid organs and inflammatory tissues. The appearance of chimeric antigen receptor (CAR)-T cell therapies targeting B cells has shown transformative potential, with recent clinical trials showing the remarkable efficacy of anti-CD19 CAR-T cells in achieving profound B cell depletion, reducing immune complex deposition, and ameliorating renal inflammation in AIKDs. While these results highlight the potential of CAR-T cell therapy in facilitating immune reset and overcoming treatment resistance, further clinical investigations are imperative to establish its long-term safety and sustained therapeutic benefits. This review synthesizes current evidence on CAR-T cell applications in AIKDs, discusses critical considerations for clinical translation, identifies existing limitations and challenges, and proposes strategic directions for therapeutic optimization and advancement.

Vascular inflammatory aging is strongly associated with multimorbidity, including immunosenescence. Here, bioinformatic analysis indicated elevated expression of the lysozyme (LYZ) gene in age-dependent vascular diseases. Lyz1 deficiency led to vascular inflammatory aging, including damage to indicators related to oxidative stress, vascular function, and inflammation in the serum and vascular tissues of wild-type (WT) and Lyz1^−/− mice. The 16S ribosomal RNA sequencing of intestinal contents revealed increased Bifidobacterium and its metabolism of acetate, butyrate, omega-muricholic acid, propionate, and valeric acid in Lyz1^−/− mice compared with that in WT mice. Additionally, RNA sequencing of vascular tissues identified differentially expressed genes in Lyz1^−/− mice compared with those in WT mice, as well as enrichment of the common phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway. Vascular inflammatory aging phenotypes were detected in the blood vessels of antibiotic-treated and germ-free mice, and the PI3K–Akt signaling pathway was inhibited. Importantly, intravenous LYZ administration worsened the pathological conditions, whereas oral LYZ administration successfully restored the gut microbial balance and reversed the vascular inflammatory aging phenotypes. Collectively, this study establishes LYZ as a novel biomarker for age-related vascular diseases and the gut microbiota–PI3K–Akt axis as a promising therapeutic target.