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Phospholipase C gamma 2, proline 522 to arginine (PLCγ2-P522R) is a protective variant that reduces the risk of Alzheimer’s disease (AD). Recently, it was shown to mitigate β-amyloid pathology in a 5XFAD mouse model of AD. Here, we investigated the protective functions of the PLCγ2-P522R variant in a less aggressive APP/PS1 mouse model of AD and assessed the underlying cellular mechanisms using mouse and human microglial models. The effects of the protective PLCγ2-P522R variant on microglial activation, AD-associated β-amyloid and neuronal pathologies, and behavioral changes were investigated in PLCγ2-P522R knock-in variant mice crossbred with APP/PS1 mice. Transcriptomic, proteomic, and functional studies were carried out using microglia isolated from mice carrying the PLCγ2-P522R variant. Finally, microglia-like cell models generated from human blood and skin biopsy samples of PLCγ2-P522R variant carriers were employed. The PLCγ2-P522R variant decreased β-amyloid plaque count and coverage in female APP/PS1 mice. Moreover, the PLCγ2-P522R variant promoted anxiety in these mice. The area of the microglia around β-amyloid plaques was also increased in mice carrying the PLCγ2-P522R variant, while β-amyloid plaque-associated neuronal dystrophy and the levels of certain cytokines, including IL-6 and IL-1β, were reduced. These alterations were revealed through [18F]FEPPA PET imaging and behavioral studies, as well as various cytokine immunoassays, transcriptomic and proteomic analyses, and immunohistochemical analyses using mouse brain tissues. In cultured mouse primary microglia, the PLCγ2-P522R variant reduced the size of lipid droplets. Furthermore, transcriptomic and proteomic analyses revealed that the PLCγ2-P522R variant regulated key targets and pathways involved in lipid metabolism, mitochondrial fatty acid oxidation, and inflammatory/interferon signaling in acutely isolated adult mouse microglia and human monocyte-derived microglia-like cells. Finally, the PLCγ2-P522R variant also increased mitochondrial respiration in human iPSC-derived microglia. These findings suggest that the PLCγ2-P522R variant exerts protective effects against β-amyloid and neuronal pathologies by increasing microglial responsiveness to β-amyloid plaques in APP/PS1 mice. The changes observed in lipid/fatty acid and mitochondrial metabolism revealed by the omics and metabolic assessments of mouse and human microglial models suggest that the protective effects of the PLCγ2-P522R variant are potentially associated with increased metabolic capacity of microglia. The online version contains supplementary material available at 10.1186/s12974-025-03387-6.

Spaceflight and microgravity environments have been shown to cause significant health impairments, including bone loss, immune dysfunction, and hematopoietic disorders. Hematopoietic stem cells (HSCs), as progenitors of the hematopoietic system, are critical for the continuous renewal and regulation of immune cells. Therefore, elucidating the regulatory mechanisms governing HSC fate and differentiation in microgravity environments is of paramount importance. In this study, hindlimb unloading (HU) was employed in mice to simulate microgravity conditions. After 28 days of HU, cells were isolated for analysis. Flow cytometry and colony-forming assays were utilized to assess changes in HSC proliferation and differentiation. Additionally, transcriptomic and untargeted metabolomic sequencing were performed to elucidate alterations in the metabolic pathways of the bone marrow microenvironment and their molecular regulatory effects on HSCs fate. Our findings revealed that 28 days of HU impaired hematopoietic function, leading to multi-organ damage and hematological disorders. The simulated microgravity environment significantly increased the HSCs population in the bone marrow, particularly within the long-term and short-term subtypes, while severely compromising the differentiation capacity of hematopoietic stem/progenitor cells. Transcriptomic analysis of HSCs, combined with metabolomic profiling of bone marrow supernatants, identified 1,631 differentially expressed genes and 58 metabolites with altered abundance. Gene set enrichment analysis indicated that HU suppressed key pathways, including hematopoietic cell lineage and MAPK signaling. Furthermore, integrated analyses revealed that metabolites affected by HU, particularly hypoxanthine enriched in the purine metabolism pathway, were closely associated with hematopoietic cell lineage and MAPK signaling pathways. Molecular docking simulations and in vitro experiments confirmed that hypoxanthine interacts directly with core molecules within these pathways, influencing their expression. These findings demonstrate that hypoxanthine in the bone marrow supernatant acts as a signaling mediator under microgravity, influencing HSCs fate by modulating hematopoietic cell lineage and MAPK signaling pathways. This study offers novel insights into the impact of microgravity on HSC fate and gene expression, underscoring the pivotal role of bone marrow microenvironmental metabolic changes in regulating key signaling pathways that determine hematopoietic destiny. The online version contains supplementary material available at 10.1186/s13287-025-04213-9.

The targeting of cancer stem cells (CSCs) has proven to be an effective approach for limiting tumor progression, thus necessitating the identification of new drugs with anti-CSC activity. Through a high-throughput drug repositioning screen, we identify the antibiotic Nifuroxazide (NIF) as a potent anti-CSC compound. Utilizing a click chemistry strategy, we demonstrate that NIF is a prodrug that is specifically bioactivated in breast CSCs. Mechanistically, NIF-induced CSC death is a result of a synergistic action that combines the generation of DNA interstrand crosslinks with the inhibition of the Fanconi anemia (FA) pathway activity. NIF treatment mimics FA-deficiency through the inhibition of STAT3, which we identify as a non-canonical transcription factor of FA-related genes. NIF induces a chemical HRDness (Homologous Recombination Deficiency) in CSCs that (re)sensitizes breast cancers with innate or acquired resistance to PARP inhibitor (PARPi) in patient-derived xenograft models. Our results suggest that NIF may be useful in combination with PARPi for the treatment of breast tumors, regardless of their HRD status. Subject terms: Breast cancer, Mechanisms of disease, Target identification, Cancer stem cells

Psychological stress causes gut microbial dysbiosis and cancer progression, yet how gut microbiota determines psychological stress-induced tumor development remains unclear. Here we showed that psychological stress promotes breast tumor growth and cancer stemness, an outcome that depends on gut microbiota in germ-free and antibiotic-treated mice. Metagenomic and metabolomic analyses revealed that psychological stress markedly alters the composition and abundance of gut microbiota, especially Akkermansia muciniphila ( A. muciniphila ), and decreases short-chain fatty acid butyrate. Supplement of active A. muciniphila , butyrate or a butyrate-producing high fiber diet dramatically reversed the oncogenic property and anxiety-like behavior of psychological stress in a murine spontaneous tumor model or an orthotopic tumor model. Mechanistically, RNA sequencing analysis screened out that butyrate decreases LRP5 expression to block the activation of Wnt/β-catenin signaling pathway, dampening breast cancer stemness. Moreover, butyrate as a HDAC inhibitor elevated histone H3K9 acetylation level to transcriptionally activate ZFP36, which further accelerates LRP5 mRNA decay by binding adenine uridine-rich (AU-rich) elements of LRP5 transcript. Clinically, fecal A. muciniphila and serum butyrate were inversely correlated with tumoral LRP5/β-catenin expression, poor prognosis and negative mood in breast cancer patients. Altogether, our findings uncover a microbiota-dependent mechanism of psychological stress-triggered cancer stemness, and provide both clinical biomarkers and potential therapeutic avenues for cancer patients undergoing psychological stress. Subject terms: Cancer metabolism, Cancer stem cells

Lung transplantation is the primary treatment for end-stage lung diseases. However, ischemia-reperfusion injury (IRI) significantly impacts transplant outcomes. 4-Octyl itaconate (4-OI) has shown potential in mitigating organ IRI, although its effects in lung transplantation require further exploration. BEAS-2B cells were used to model transplantation, assessing the effects of 4-OI through viability, apoptosis, and ROS assays. qRT-PCR analyzed cytokine transcription post-cold ischemia/reperfusion (CI/R). RNA sequencing and Gene Ontology analysis elucidated 4-OI’s mechanisms of action, confirmed by Western blotting. ALI-airway and lung transplantation organoid models evaluated improvements in bronchial epithelial morphology and function due to 4-OI. ELISA measured IL-6 and IL-8 levels. Rat models of extended cold preservation and non-heart-beating transplantation assessed 4-OI’s impact on lung function, injury, and inflammation. Our findings indicate that 4-OI (100 µM) during cold preservation effectively maintained cell viability, decreased apoptosis, and reduced ROS production in BEAS-2B cells under CI/R conditions. It also downregulated pro-inflammatory cytokine transcription, including IL1B, IL6, and TNF. Inhibition of Nrf2 partially reversed these protective effects. In cold preservation solutions, 4-OI upregulated Nrf2 target genes such as NQO1, HMOX1, and SLC7A11. In ALI airway models, 4-OI enhanced bronchial epithelial barrier integrity and ciliary beat function after CI/R. In rat models, 4-OI administration improved lung function and reduced pulmonary edema, tissue injury, apoptosis, and systemic inflammation following extended cold preservation or non-heart-beating lung transplantation. Incorporating 4-OI into cold preservation solutions appears promising for alleviating CI/R-induced bronchial epithelial injury and enhancing lung transplant outcomes via Nrf2 pathway activation. The online version contains supplementary material available at 10.1186/s12931-025-03151-7.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs that function in ribosome and spliceosome biogenesis, primarily by guiding modifying enzymes to specific sites on ribosomal RNA (rRNA) and spliceosomal RNA (snRNA). However, many orphan snoRNAs remain uncharacterized, with unidentified or unvalidated targets, and studies on additional snoRNA-associated proteins are limited. We adapted an enhanced chimeric eCLIP approach to comprehensively profile snoRNA-target RNA interactions using both core and accessory snoRNA-binding proteins as baits. Using core snoRNA-binding proteins, we confirmed most annotated snoRNA-rRNA and snoRNA-snRNA interactions in mouse and human cell lines and called novel, high-confidence interactions for orphan snoRNAs. While some of these interactions result in chemical modification, others may have modification-independent functions. We showed that snoRNA ribonucleoprotein complexes containing certain accessory proteins, like WDR43 and NOLC1, enriched for specific subsets of snoRNA-target RNA interactions with distinct roles in ribosome and spliceosome biogenesis. Notably, we discovered that SNORD89 guides 2′-O-methylation at two neighboring sites in U2 snRNA that fine-tune splice site recognition. Chimeric eCLIP of snoRNA-associating proteins enables a comprehensive framework for studying snoRNA-target interactions in an RNA-binding protein-dependent manner, revealing novel interactions and regulatory roles in RNA biogenesis. The online version contains supplementary material available at 10.1186/s13059-025-03508-7.

Induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) have greater potential for generating chondrocytes without hypertrophic and fibrotic phenotypes compared to bone marrow-derived mesenchymal stem/stromal cells (BMSCs). However, there is a lack of research demonstrating the use of autologous iMSCs for repairing articular chondral lesions in large animal models. In this study, we aimed to evaluate the effectiveness of autologous miniature pig (minipig) iMSC-chondrocyte (iMSC-Ch)-laden implants in comparison to autologous BMSC-chondrocyte (BMSC-Ch)-laden implants for cartilage repair in porcine femoral condyles. iMSCs and BMSCs were seeded into fibrin glue/nanofiber constructs and cultured with chondrogenic induction media for 7 days before implantation. To assess the regenerative capacity of the cells, 19 skeletally mature Yucatan minipigs were randomly divided into microfracture control, acellular scaffold, iMSC, and BMSC subgroups. A cylindrical defect measuring 7 mm in diameter and 0.6 mm in depth was created on the articular cartilage surface without violating the subchondral bone. The defects were then left untreated or treated with acellular or cellular implants. Both cellular implant-treated groups exhibited enhanced joint repair compared to the microfracture and acellular control groups. Immunofluorescence analysis yielded significant findings, showing that cartilage treated with iMSC-Ch implants exhibited higher expression of COL2A1 and minimal to no expression of COL1A1 and COL10A1, in contrast to the BMSC-Ch-treated group. This indicates that the iMSC-Ch implants generated more hyaline cartilage-like tissue compared to the BMSC-Ch implants. Our findings contribute to filling the knowledge gap regarding the use of autologous iPSC derivatives for cartilage repair in a translational animal model. Moreover, these results highlight their potential as a safe and effective therapeutic strategy. The online version contains supplementary material available at 10.1186/s13287-025-04215-7.

Persistence of drug-resistant breast cancer stem cells (brCSCs) after a chemotherapeutic regime correlates with disease recurrence and elevated mortality. Therefore, deciphering mechanisms that dictate their drug-resistant phenotype is imperative for designing targeted and more effective therapeutic strategies. The transcription factor SOX2 has been recognized as a protagonist in brCSC maintenance, and previous studies have confirmed that inhibition of SOX2 purportedly eliminated these brCSCs. However, pharmacological targeting of transcription factors like SOX2 is challenging due to their structural incongruities and intrinsic disorders in their binding interfaces. Therefore, transcriptional co-activators may serve as a feasible alternative for effectively targeting the brCSCs. Incidentally, transcriptional co-activators YAP/TAZ were found to be upregulated in CD44 + /CD24 - /ALDH + cells isolated from patient breast tumors and CSC-enriched mammospheres. Interestingly, it was observed that YAP/TAZ exhibited direct physical interaction with SOX2 and silencing YAP/TAZ attenuated SOX2 expression in mammospheres, leading to significantly reduced sphere forming efficiency and cell viability. YAP/TAZ additionally manipulated redox homeostasis and regulated mitochondrial dynamics by restraining the expression of the mitochondrial fission marker, DRP1. Furthermore, YAP/TAZ inhibition induced DRP1 expression and impaired OXPHOS, consequently inducing apoptosis in mammospheres. In order to enhance clinical relevance of the study, an FDA-approved drug verteporfin (VP), was used for pharmacological inhibition of YAP/TAZ. Surprisingly, VP administration was found to reduce tumor-initiating capacity of the mammospheres, concomitant with disrupted mitochondrial homeostasis and significantly reduced brCSC population. Therefore, VP holds immense potential for repurposing and decisively eliminating the chemoresistant brCSCs, offering a potent strategy for managing tumor recurrence effectively. Subject terms: Cancer stem cells, Cancer stem cells