ABSTRACT Ferroptosis, a regulated form of cell death driven by iron‐dependent lipid peroxidation, emerged as a critical process in the pathogenesis of various diseases, including neurological, cardiovascular, hepatic, pulmonary, and renal disorders. Simultaneously, N6‐methyladenosine (m 6 A), the most abundant reversible RNA modification in eukaryotes, plays a pivotal role in gene regulation by modulating RNA stability, translation, and degradation. Evidence increasingly links m 6 A modifications to the transcriptional and post‐transcriptional regulation of ferroptosis, highlighting their importance in disease mechanisms. This review explores the molecular mechanisms underpinning m 6 A modifications and ferroptosis, detailing their interactions in disease progression. Additionally, it evaluates the therapeutic promise of targeting m 6 A regulators to modulate ferroptosis, offering novel approaches for managing diseases with diverse etiologies. By clarifying the role of m 6 A in ferroptosis, this review underscores the promise of m 6 A‐focused therapeutic strategies in advancing treatment for diseases where ferroptosis plays a key pathological role.
Residual PSC detection follows ultra‑trace‑analysis principles. LOD supports qualitative assessment. Assays employ PSC‑specific markers and proper references, with adaptation to differentiated cell populations from diverse PSC sources.
This document specifies the technical elements for bacteria and fungi detection by PCR assay which enables rapid, broad-spectrum detection of bacteria and fungi with high sensitivity. Qualitative judgement is based on LOD. Key detection points include efficient nucleic acid extraction, broad-spectrum primer-probe design, high amplification efficiency and minimised background interference.
Polyploidy is normally found in numerous tissues and cell types in the generally diploid human body and is crucial for proper development. However, polyploidy, and especially triploidy, can also be found in malignant tumours and is often associated with chemoresistance, stemness and metastatic capabilities. Here, we utilise our isogenic haploid, diploid and triploid human embryonic stem cell (hESC) lines to study the effect of ploidy on the response to four different anticancer drugs. Surprisingly, we show that triploid cells are more sensitive to chemotherapy-induced apoptosis and cell cycle arrest than diploid and haploid cells, correlated with higher levels of DNA damage. This phenotype is regulated by p53, as it was reversed in TP53-KO cells, where triploid mutant cells display higher resistance to the chemotherapies we applied compared to mutant haploid and diploid cells. The reversal from sensitivity to resistance, driven by TP53-KO of the triploid cells, was accompanied by a reversal in the enrichment of DNA repair, replication and cell division related genes, compared to their diploid counterparts. Conversely, triploidy triggered DNA damage-induced differentiation in both WT and TP53-KO treated cells, pointing to a ploidy-dependent response not mediated by p53. In addition, we show that cancer cell lines also display similar ploidy-dependent anticancer drug responses to our TP53-KO hESCs. These findings uncover the interplay between ploidy and the p53 pathway in determining the outcome of anticancer therapeutics, and display the potential use of isogenic hESCs, differing only in their ploidy level, in studying the impact of ploidy on chemotherapy response.
Persistent inflammation, insufficient vascularization and impaired osteogenesis limit the repair of critical-sized femoral defects. Although microRNA-210-3p (miR-210-3p) can regulate these processes, its therapeutic use is constrained by poor extracellular stability and inefficient cellular entry. Here, miR-210-3p was incorporated into a tetrahedral framework nucleic acid (tFNA) to create tFNAs-miR-210-3p. Dynamic light scattering, zeta-potential analysis, atomic force microscopy (AFM) and capillary electrophoresis supported formation of the negatively charged nanosystem. In RAW264.7 macrophages, tFNA delivery increased Cy5-labelled miR-210-3p-positive cells from 1.24% for free miR-210-3p to 99.93%. Under lipopolysaccharide (LPS)-induced inflammatory conditions, tFNAs-miR-210-3p reduced CD86 and inducible nitric oxide synthase, increased CD206 and arginase-1, enhanced endothelial tube formation and improved osteogenic differentiation. It suppressed M1 marker CD86/iNOS and elevated M2 marker CD206/Arg-1 to resolve the inflammatory microenvironment. Mechanistically, miR-210-3p directly binds the 3'UTR of EFNA3 mRNA to inhibit its expression, thereby activating AKT/STAT3 signalling to switch macrophages toward reparative phenotype. In a rat critical-sized femoral defect model, local tFNAs-miR-210-3p treatment improved histological repair, macrophage polarisation, microcomputed-tomography indices, vascularization and osteocalcin expression at 4 and 8 weeks. These findings identify tFNA-mediated miR-210-3p delivery as a promising immunomodulatory strategy for vascularised bone regeneration.
Vascularised lung organoids (vLOs) that faithfully mimic human lung tissue architecture and disease pathology are critical for advancing pulmonary research but remain challenging to generate. Here, we developed a robust self-organisation protocol to produce vLOs with cellular heterogeneity and functional vasculature. The engineered blood vessels within vLOs exhibit lung-specific characteristics. As proof of concept, we applied this platform to model chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH). Using patient-derived vLOs, we demonstrated that cigarette smoke extract (CSE) induces pathological features resembling clinical COPD, including epithelial disruption and inflammatory responses. Furthermore, vLOs generated from a PH patient recapitulated disease-associated vascular remodelling, with RNA-seq revealing dysregulated pathways in endothelial dysfunction. Notably, we identified sodium hydrosulfide (NaHS) as a potential therapeutic candidate, as it attenuated aberrant EndoMT in PH-vLOs. In summary, our study establishes a physiologically relevant vLO system that enables modelling of cell-type-specific disease mechanisms and underscores the broad utility of this platform for mechanistic investigations and precision medicine approaches in respiratory disorders.
CKIP-1, as a scaffold protein with special structural domains, treats related diseases through multiple emerging targeted technologies.
Cystic fibrosis (CF) has shifted from a fatal paediatric lung disease to a multi-organ, lifespan-spanning disorder in which gastrointestinal (GI) complications and malignancies are increasingly prominent. With improved survival into mid- and late adulthood, aided by newborn screening, optimised nutrition and the advent of highly effective CF transmembrane conductance regulator (CFTR) modulators, implementation of colonoscopic surveillance has highlighted a several-fold increase in early-onset colorectal cancer (CRC) and a broader spectrum of intestinal pathology. In parallel, work in mouse models, human tissue and patient-derived intestinal organoids now places the CFTR at the centre of a complex epithelial compartment that integrates ion and pH homeostasis, mucus biology, microbiota, redox balance and immune/stromal function. In this review, we advance the hypothesis that CF may be conceptualised as a niche-centric hereditary CRC predisposition syndrome, in which germline CFTR dysfunction chronically destabilises epithelial identity in addition to increasing mutational burden. We synthesise evidence that CFTR loss remodels stem cell regulation, promotes hypoxia and oxidative stress, perturbs microbial ecosystems, drives chronic immune activation and stromal remodelling, and induces epithelial-mesenchymal plasticity (EMP) and DNA-damage vulnerability, collectively creating a pre-neoplastic intestinal ecosystem. We situate this model within contemporary CRC frameworks that emphasise cell-state transitions, hybrid epithelial-mesenchymal (E/M) states and specialised stromal niches, and we distinguish it from oncofoetal reprogramming, an APC-driven programme that CF does not clearly recapitulate. We propose that CF may provide a naturally occurring human context in which chronic epithelial stress sustains EMP-like pressure, a concept that requires direct validation in human CF intestinal tissue. Finally, we consider how CFTR modulators, microbiome-directed therapies and redox-targeted interventions might re-programme the CF intestinal niche and outline experimental and clinical strategies needed to determine whether early, ecosystem-level correction can prevent GI cancers in this high-risk population.
Liver organoids are three-dimensional miniature liver models that recapitulate the complex architecture and key functions of the human liver in vitro, offering powerful platforms for both fundamental research and translational applications. This review systematically summarises current fabrication strategies, disease-modelling utilities and regenerative potentials of liver organoids, alongside the major challenges and future directions. In recent years, the field has witnessed several breakthroughs. Through endothelial co-culture approaches, vascularised and metabolically zonated liver organoids have been successfully generated, achieving endothelial coverage exceeding 85%. Prime editing enables precise correction of pathogenic mutations in patient-derived organoids, with no off-target effects detected at the genome-wide level. In disease modelling, iPSC-derived liver organoids faithfully recapitulate the pathological progression of metabolic dysfunction-associated steatotic liver disease (MASLD) and verify the lipid-lowering efficacy of semaglutide. Macrophage-integrated organoid models support the full life cycles of HEV, SARS-CoV-2 and dengue virus, providing new tools for antiviral drug screening. Large-scale patient-derived tumour organoid biobanks successfully preserve the heterogeneity and clinical drug-resistance signatures of liver cancers. In regenerative medicine, encapsulated hepatocyte organoids and the UTOpiA bioartificial liver system have effectively rescued acute liver failure in animal models, while gene-edited autologous organoids offer potential curative strategies for genetic disorders such as Wilson disease. Nevertheless, insufficient hepatocyte functional maturity, difficulties in constructing vascular networks, and the lack of standardised culture protocols remain major obstacles to clinical translation. By bridging fundamental liver biology and clinical practice, liver organoid technology lays a solid foundation for precision hepatology and regenerative therapies. Continued interdisciplinary efforts are still required to overcome current limitations and facilitate its clinical adoption.
Dentinogenesis, a process essential for tooth function, relies on the precise differentiation of dental papilla cells into odontoblasts. This lineage commitment is governed by complex transcription factor networks. RUNX2 and KLF4, known key TFs co-expressed in this process, were proven to operate synergistically, but the specific cofactors coordinating their activity at the chromatin level remain unknown. Here, we identify the chromatin remodeller BRD9 as a novel interactor of both RUNX2 and KLF4 during odontoblastic differentiation. To probe their shared function, we generated neural crest-specific conditional knockout mice. Wnt1-Cre; Brd9fl/fl mice exhibited a disordered odontoblast layer with reduced secretion of extracellular matrix proteins (DMP1 and DSPP), strikingly phenocopying the compound Wnt1-Cre; Runx2fl/wt; Klf4fl/fl mutants. In vitro, chemical BRD9 degradation suppressed odontoblastic differentiation and mineral deposition. Integrative analyses of RNA-seq and ATAC-seq revealed that BRD9 maintained chromatin accessibility at odontogenesis-associated regions enriched with RUNX2 and KLF4 motifs, specifically at the Fam20c enhancer, thereby facilitating RUNX2 and KLF4 binding for transcriptional activation. Crucially, exogenous supplementation of FAM20C protein partially rescued the odontoblastic differentiation defects upon BRD9 loss. These findings establish BRD9 as a critical epigenetic orchestrator that coordinates RUNX2-KLF4 synergy to activate key odontogenic genes like Fam20c, thus affecting odontoblastic differentiation and dentinogenesis.
Currently, no targeted therapy exists for idiopathic pulmonary fibrosis (IPF). The hallmark pathological feature of excessive extracellular matrix (ECM) deposition severely undermines the efficacy of mesenchymal stem cell (MSC)-based treatments. While existing MSC therapeutic strategies primarily focus on modulating inflammation in early stages, they have not yet established precise interventions addressing the core pathological mechanism-ECM dysregulation. Previous studies demonstrated the therapeutic potential of human embryonic stem cell (hESCs)-derived immunity-and-matrix-regulatory cells (IMRCs) in lung injury and fibrosis models. However, the critical biomarkers and underlying mechanisms mediating IMRCs' efficacy in IPF remain poorly understood. In this study, we generated MMP1 knockout IMRCs (IMRCs-MMP1 KO) using CRISPR-based gene editing. We then characterized whether MMP1 ablation affected key properties of IMRCs, including cell morphology, proliferation, migration, marker protein expression, transcriptomic profile, and cytokine secretion. Subsequently, the ability of IMRCs-MMP1 KO to degrade collagen was tested using in vivo and in vitro pulmonary fibrosis models. MMP1 knockout was successfully achieved and did not compromise typical IMRC characteristics or impair their immunomodulatory capacity. However, MMP1 deficiency significantly attenuated the ability of IMRCs to degrade TGF-β1-induced collagen I deposition in A549 cells. Importantly, wild-type IMRCs demonstrated superior therapeutic efficacy in ameliorating bleomycin-induced lung injury and fibrosis in mice compared with IMRCs-MMP1 KO. Furthermore, IMRCs exhibited significantly greater capability to directly degrade the pericellular collagen I and modulate fibroblasts' activation progression within fibrotic lung tissues in a MMP1-dependent manner. In summary, our data establish that MMP1 plays an essential functional role in IMRC-mediated attenuation of PF. MMP1 thus represents a key therapeutic biomarker for IMRC-based treatment. This work provides a foundation for developing stem cell therapies tailored to the pathological features of IPF, potentially enabling adaptive treatment strategies.
Autoimmune diseases, including psoriasis (Ps), rheumatoid arthritis (RA) and ulcerative colitis (UC), pose significant health burdens worldwide. A more refined classification of these diseases is essential for enabling targeted therapeutic strategies. Traditional Chinese Medicine (TCM) is gaining increasing recognition globally, offering potential insights into disease heterogeneity. In this study, we performed comprehensive T cell receptor (TCR) and B cell receptor (BCR) repertoire sequencing in 59 participants, including patients with Ps, RA, UC and healthy controls. Patients were further stratified into Dampness and non-Dampness groups based on standardized TCM diagnostic criteria. Repertoire composition, clonotype richness, diversity metrics, V gene usage and shared CDR3 patterns were analysed. Machine learning approaches (LASSO, GLM and OPLS-DA) were applied to identify diagnostic biomarkers, followed by validation in an independent cohort. Compared with healthy controls, Ps, RA and UC patients exhibited reduced clonotype richness and diminished TCR/BCR diversity, indicating adaptive immune contraction. Dampness ZHENG-specific alterations were observed, including selective IgK and IgL clonotype richness reduction in Ps with dampness, increased TRA/TRB diversity in RA with dampness, and elevated TRG clonotype counts/read proportions in UC with dampness. Stratification by Dampness ZHENG revealed distinct immune repertoire architectures characterized by increased D50 index, reduced proportions of large clones and preferential V gene usage, including TRAV20, TRAV38-2DV8 and TRAV8-3. Unsupervised dimensionality reduction demonstrated significant separation between Dampness and non-Dampness groups across diseases. A three-gene V-segment model achieved an AUC of 0.804 and 74.7% accuracy in an independent validation cohort, supporting its potential utility as an objective biomarker for Dampness ZHENG. Our findings suggest that TCM-based phenotyping may offer a meaningful approach for subgrouping autoimmune diseases, thereby providing a foundation for more syndrome differentiation-based treatment strategies.
Chimeric antigen receptor (CAR) T-cell therapy has achieved durable efficacy in hematologic malignancies but encounters persistent obstacles in solid tumours, including antigen heterogeneity, a suppressive tumour microenvironment (TME), and intrinsic T-cell dysfunction. This review examines the transition from single-axis engineering to an integrated framework that addresses these hurdles in sequence. We delineate how next-generation CAR-T cells are designed for precise spatiotemporal activation through logic-gated and pharmacologically regulatable receptors, while being reinforced by metabolic and epigenetic reprogramming to resist TME-driven exhaustion. We also assess strategies that actively reshape the immunosuppressive TME, including depletion of regulatory cell populations, blockade of 'don't eat me' signals, and the use of biomaterial scaffolds for locoregional delivery. The synthesis of controllable activation, intrinsic resilience, and extrinsic TME modulation is defining a class of adaptive therapeutic systems. Clinical implementation of this approach requires careful management of toxicities, notably cytokine release syndrome (CRS), and support from advanced monitoring technologies. Progress will depend on rational combinations that move beyond isolated optimisations, enabling cellular therapies to dynamically respond to evolving tumour ecosystems and narrowing the efficacy gap between hematologic and solid cancers.
SENP8 regulates a non-canonical NEDD8-dependent pathway to stabilize STAT1, promote CCL2/CCL5 transcription, and maintain germ cell proliferation and migration.
Excessive mechanical stress is a main cause of intervertebral disc degeneration (IDD). However, the specific mechanism remains unclear. We established in vivo and in vitro models to investigate the role of cytoskeletal proteins in excessive mechanical stress-induced NP cell pyroptosis and IDD. The expression level of Vimentin was decreased in degenerated NP cells induced by excessive mechanical stress. Knockdown of Vimentin promoted NP cell pyroptosis and IDD in rats, whereas Vimentin overexpression significantly alleviated excessive mechanical stress-induced NP cell pyroptosis and degeneration. Further mechanistic studies revealed that Vimentin ameliorated mitochondrial dysfunction triggered by excessive mechanical stress through PINK1-Parkin-dependent mitophagy, thereby attenuating NP cell pyroptosis and degeneration. Co-immunoprecipitation-mass spectrometry analysis suggested an interaction between Itgb1 and Vimentin, which was validated by Co-immunoprecipitation assays. Itgb1 enhanced Vimentin protein stability via the ubiquitin-proteasome pathway and inhibited mechanical stress-mediated Vimentin degradation. Subsequent experiments confirmed that Itgb1 reduced Vimentin ubiquitination and degradation by blocking the binding of MNAT1 to Vimentin. Itgb1 ameliorated excessive mechanical stress-induced NP cell pyroptosis and degeneration via Vimentin. Restoring Vimentin function through gene overexpression effectively inhibited NP cell pyroptosis and delayed the progression of IDD in rats. In summary, this study reveals a mechanotransduction pathway from mechanical stress sensing to cellular functional regulation in IDD, providing novel insights into the pathological mechanisms underlying IDD. Moreover, this study demonstrates that Vimentin exerts a significant protective effect against excessive mechanical stress-induced NP cell pyroptosis and IDD, offering a potential therapeutic target for the clinical management of IDD.
In mammals, Müllerian ducts (MDs) are the precursors of the female reproductive tract which regress in males during embryonic development. Failure of MD regression results in persistent Müllerian duct syndrome (PMDS) in humans. Although several factors essential for MD regression have been identified, the underlying regulatory network remains incompletely understood. In this study, we found that the Wilms tumour gene (Wt1) was highly expressed in the MD mesenchyme of male mice. Mesenchyme-specific inactivation of Wt1 resulted in MD retention and male infertility. The expression of Amh in the testes and its receptor Amhr2 in the MD mesenchyme remained unchanged in Wt1-/flox; Amhr2-cre male mice. Instead, the expression of Wnt inhibitory factor 1 (Wif1) and Osterix (Osx) was significantly reduced, accompanied by nuclear accumulation of β-catenin in the MD mesenchyme of Wt1-deficient mice. Further studies revealed that Wif1 and Osx were direct transcriptional targets of WT1. These findings identify Wt1 as a previously unrecognized mesenchymal regulator of MD regression by inducing Wif1 and Osx expression and provide new insights into the regulatory mechanisms underlying MD regression as well as the aetiology of reproductive tract disorders associated with WT1 mutations.
Colorectal cancer (CRC) exhibits heterogeneity based on tumour laterality, with right-sided tumours demonstrating distinct genetic, immunogenic and clinical behaviours compared to left-sided counterparts. In this study, we conducted single-cell RNA sequencing and spatial transcriptomics to dissect the distinct molecular landscapes of left- and right-sided CRC. We identified enhanced germinal centre B cell infiltration and CXCL13+ T cell enrichment, features linked to adaptive immune priming and better immunotherapy responsiveness. Right-sided CRC epithelial cells exhibited proliferative and immunogenic phenotypes, marked by upregulated immune-related pathways and chemokine-driven interactions with lymphocytes. Spatial analysis revealed organized tertiary lymphoid structure-immune microenvironment crosstalk in right-sided tumours, mediated by CCL19/21-CCR7 signalling. In contrast, left-sided CRC tumours displayed stromal-epithelial interactions favouring angiogenesis and metabolic reprogramming. Our findings established a laterality-specific immune microenvironment in CRC, providing insights for precise therapeutic strategies of left- and right-sided CRC.
Chronic kidney disease (CKD) remains a major global health challenge. Angiotensin II (Ang II)-induced lipotoxicity is an important contributor to podocyte injury. Perilipin 5 (PLIN5) is a lipid droplet-associated protein that helps maintain cellular metabolic homeostasis. However, how PLIN5 protects podocytes from lipotoxic stress remains incompletely understood. In this study, we generated podocyte-specific PLIN5 knockout mice using the Cre-loxP system and induced PLIN5 overexpression in vivo and in vitro. We found that Ang II markedly downregulated PLIN5 expression in podocytes both in vivo and in vitro. Podocyte-specific deletion of PLIN5 aggravated Ang II-induced lipid accumulation, mitochondrial dysfunction and apoptosis, whereas PLIN5 overexpression alleviated these abnormalities. Proteomic screening identified FK506-binding protein 8 (FKBP8), an outer mitochondrial membrane protein, as a PLIN5-interacting partner. Co-immunoprecipitation and proximity ligation assays showed that the PLIN5-FKBP8 interaction was reduced under Ang II stimulation. Functionally, FKBP8 knockdown disrupted lipid droplet-mitochondria contact and exacerbated Ang II-induced podocyte lipotoxicity. Domain-mapping and rescue experiments further demonstrated that the 70-200 amino acid region of FKBP8 is required for PLIN5 binding and for preservation of lipid droplet-mitochondria contact under lipotoxic stress. In addition, disruption of the PLIN5-FKBP8 axis was associated with impaired fatty acid utilisation and altered mitochondrial homeostasis. Collectively, these findings support a model in which PLIN5 protects podocytes, at least in part, by interacting with FKBP8 and preserving lipid droplet-mitochondria contact, thereby limiting Ang II-induced lipotoxic injury.
Colorectal cancer (CRC) is the third most common malignancy worldwide. Epidemiological studies have suggested a positive association between periodontitis (PD) and CRC risk; however, the mechanistic basis underlying this relationship remains unclear. Extracellular vesicles (EVs) represent an important mode of systemic communication and may mediate the distal effects between PD and CRC. PD model was established in Apc+/- mice with spontaneous intestinal tumorigenesis. Tumour onset, burden, and progression were evaluated in the colorectum and small intestine. Circulating EVs were isolated from the plasma of PD or sham mice and characterised. The functional contribution of EVs was assessed using pharmacological inhibition of EV release and MC38 syngeneic tumour models. Metabolomic profiling, RNA sequencing, and in vitro functional assays were performed to investigate EV cargo and underlying mechanisms. PD significantly accelerated CRC onset and increased tumour number and size in Apc+/- mice. Inhibition of EV release by GW4869 attenuated PD-driven tumour progression, indicating a critical role of periodontitis-associated EVs (PDEVs). PDEVs promoted tumour growth and induced an immunosuppressive tumour microenvironment in MC38 transplanted tumours. Metabolomic analysis revealed marked enrichment of carnosine in PDEVs. Under acidic conditions, EV-delivered carnosine alleviated intracellular acidosis, preserved lysosomal positioning and acidification, and promoted proliferation, migration, and epithelial-mesenchymal transition of MC38 cells. Collectively, circulating EV-mediated metabolic communication pathway linking PD to CRC progression. By delivering carnosine, PDEVs support malignant phenotypes and facilitate tumour adaptation to acidic stress. Circulating EV-associated carnosine may represent a potential biomarker and a candidate target for modulating CRC progression in high-risk populations.
