参考文献

The cell cycle (CC) underpins diverse cell processes like cell differentiation,cell expansion,and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases,rely on transcriptomic signatures,use imaging modalities limited to adherent cells,or lack high-throughput multiplexing. To solve this,we develop an expanded,Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight,we quantify scCC states across suspension and adherent cell lines,and stimulated primary human T cells. Our approach captures the diversity of scCC states,including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably,primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems,Proteomics,Cell biology,Immunology,Systems biology View Publication

Cost-effective,practical,and reproducible culture of human pluripotent stem cells (hPSCs) is required for basic and translational research. Basal 8 (B8) has emerged as a cost-effective solution for weekend-free and chemically-defined hPSC culture. However,the requirement to home-produce some recombinant growth factors for B8 can hinder access and reproducibility. Moreover,we found the published B8 formulation suboptimal in widely-used normoxic hPSC culture. Lastly,the performance of B8 in functional applications such as genome editing or organoid differentiation required systematic evaluation. We formulated B8 with commercially available,growth factors and adjusted its composition to support normoxic culture of WTC11 human induced pluripotent stem cell line. We compared this formulation (B8+) with commercial Essential 8 (cE8) and a home-made,weekend-free E8 formulation (hE8). We measured pluripotency marker expression and cell cycle by flow cytometry,and investigated the transcriptional profiles by bulk and single-cell RNA sequencing. We further assessed genomic stability,genome editing efficiency,single-cell cloning,and differentiation in both monolayer and organoids. Finally,we validated key findings using male (H1) and female (H9) human embryonic stem cells. hE8 performed comparably to cE8 across most functional assays and cell lines. In contrast,cells in B8+ displayed higher NANOG expression and improved genome editing efficiency. At the same time,B8+ led to gene expression changes indicative of marked lineage priming,reflected in altered morphology and differential response to some differentiation protocols. Both weekend-free media resulted in a modest transcriptional shift towards a less metabolically active state,consistent with intermittent media starvation. Homemade weekend-free media can provide a cost-effective alternative to commercial formulations. hE8,integrating some features of B8 while resembling cE8,emerges as a robust and practical option with limited compromises. B8+,though advantageous in some contexts,warrants caution due to lineage priming effects that may impact differentiation outcomes. View Publication

Cancer drug resistance poses a significant challenge in oncology,often driven by intricate cross-talk among membrane-bound receptors that compromise mono-targeted therapies. We develop a dual membrane receptor degradation strategy leveraging Folate Receptor α (FRα) to address this issue. Folate Receptor α Targeting Chimeras-dual (FolTAC-dual) are engineered degraders designed to selectively and simultaneously degrade distinct receptor pairs: (1) EGFR/HER2 and (2) PD-L1/VISTA. Through modular optimization of modality configurations and geometries,we identify the “string” format as the most effective construct. Mechanistic studies demonstrate an ~85% increase in EGFR-binding affinity compared to the conventional knob-into-hole design,likely contributing to the improved efficiency of dual-target degradation. Proof-of-concept studies reveal that EGFR and HER2 FolTAC-dual effectively counteracts resistance in Trastuzumab/Lapatinib-resistant HER2-positive breast cancer models,while PD-L1 and VISTA FolTAC-dual rejuvenates immune responses in PD-L1 antibody-resistant syngeneic mouse models. These findings establish FolTAC-dual as a promising dual-degradation platform for clinical translation. Subject terms: Cancer immunotherapy,Targeted therapies,Protein design,Drug discovery and development View Publication

Proliferating cell nuclear antigen (PCNA),a well-documented anticancer target,is critical for DNA synthesis,replication,and repair. AOH1996,a small-molecule PCNA inhibitor,is currently undergoing clinical trials for the treatment of advanced solid tumors. However,the therapeutic effect of AOH1996 on head and neck squamous cell carcinoma (HNSCC) remains unclear. The effects of AOH1996 on HNSCC biological behaviors and cancer stemness were tested in HNSCC cells and nude mice. The combination treatment of AOH1996 and anti-PD1 was performed in a 4-nitroquinoline N-oxide (4NQO)-induced HNSCC mouse model. RNA sequencing,Western Blotting,immunofluorescence staining,comet assays,and qRT‒PCR were conducted for mechanistic studies. Our results showed that AOH1996 effectively inhibited HNSCC proliferation and invasion both in vitro and in vivo. AOH1996 suppressed HNSCC stemness,development,and metastasis. Moreover,AOH1996 altered the tumor immune microenvironment into an inflamed state with increased CD8 + T-cell infiltration,rendering it a favorable partner for combination therapy with immune checkpoint inhibitors. Mechanistically,AOH1996 induced cellular DNA damage,suppressed cancer stemness through the upregulation of p-TBK1,and promoted the secretion of CD8 + T-cell-recruiting chemokines by stimulating IRF3-mediated transcription. Taken together,our results demonstrated that AOH1996 suppressed tumor growth,eliminated cancer stem cells (CSCs),and synergistically enhanced the efficacy of anti-PD1 immunotherapy in HNSCC. The online version contains supplementary material available at 10.1186/s13287-025-04607-9. View Publication

Extracellular vesicles (EVs) are nano-sized lipid vesicles released into the extracellular space. We investigated the role of mouse brain-derived EVs in α-synuclein (α-syn) degradation and pathology transmission. Using sucrose gradient isolation and biochemical characterization,we found that EVs harbor active proteases that cleave both monomeric α-syn and pre-formed fibrils (PFFs). Protease activity and inhibitor profiling identified cathepsins B and S as key enzymes mediating this cleavage. EV-mediated proteolysis reduced the seeding capacity of α-syn PFFs in vitro and in vivo,whereas protease inhibition enhanced aggregation. Proteomic analysis revealed a restricted protease repertoire within EV cargo. Our findings suggest that EVs regulate extracellular α-syn levels via proteolysis,thereby modulating its prion-like spreading potential. We suggest that EVs represent a novel post-translational mechanism to regulate the levels of extracellular α-syn and may thus affect the spreading of α-syn pathology. Targeting this proteolytic capacity may offer new therapeutic interventions for mitigating synucleinopathies. Subject terms: Biochemistry,Cell biology,Neuroscience,Pathogenesis View Publication

Background: Lipoprotein(a) [Lp(a)]-induced inflammation contributes to coronary artery spasm (CAS) by the contraction of vascular smooth muscle cells. However,the interaction between Lp(a) and soluble CD36 (sCD36)/interleukin (IL)-6/RAS Homolog Family Member A (RhoA)-GTP signaling pathway has not been evaluated. Methods: We investigated the relevance of Lp(a)/CD36 signaling in CAS patient monocyte-derived macrophages (PMDMs) and a human coronary artery smooth muscle cell (HCASMC) line using expression profile correlation analyses,molecular docking,RNA sequencing,flow cytometry,immunoblotting,and quantitative reverse transcription polymerase chain reaction. Results: Plasma Lp(a) and sCD36 levels in 41 CAS patients were significantly higher ( p = 0.001) and positively correlated (r 2 = 0.3145,p < 0.001),a trend not observed in 36 non-CAS controls. RNA sequencing indicated a significant co-overexpression of CD36 and RhoA in Lp(a)-treated CAS PMDMs and HCASMCs,of which the mRNA and protein expression of CD36 and RhoA were significantly enhanced ( p < 0.001) dose-dependently. Lp(a) rather than LDL preferentially induced CD80+ PMDM (M1) polarization. In HCASMCs,the CD36 knockdown using either short hairpin RNA or natural biflavonoid amentoflavone suppressed Lp(a)-upregulated protein expression of CD36,RhoA-GTP,IL-6,tumor necrosis factor (TNF)-α,nuclear factor (NF)-κB,and CD80; however,overexpressed CD36 increased their levels. Lp(a) decreased and amentoflavone increased the epigenetic expression of CD36 inhibitors,miR-335-5p,and miR-448,respectively. Reciprocally,an miRNA inhibitor or mimic could magnify or diminish Lp(a)-induced CD36,TNF-α,NF-κB and IL-6 expressions in HCASMCs,respectively. Conclusions: Elevated Lp(a) levels upregulate the CD36-dependent TNF-α/NF-κB/IL-6/RhoA-GTP signaling pathway in CAS PMDMs and HCASMCs,indicating that Lp(a)/CD36 inflammatory signaling,HCASMC activation,and macrophage M1 polarization mediate CAS development. View Publication

Monoclonal antibody-based immune checkpoint inhibitors,which have brought breakthrough effects in cancer treatments,are expected to assist in the treatment of viral diseases. However,antibody therapies may cause immune-related side effects,such as inflammation and pneumonia,due to cytokine storms. Small-molecule PD-1/PD-L1 inhibitors are an alternative to monoclonal antibody-based therapeutics. We have identified a novel small-molecule PD-1/PD-L1 inhibitor having a functional group (disulfide group),namely compound 2 (molecular weight: 456.6),from our library of sulfur-containing protein–protein interaction inhibitor compounds. Compound 2 selectively bound to PD-L1 over PD-1,with the dissociation rate constant (K D ) of 77.60 ± 4.44 nM (obtained by affinity analysis) and showed promising T cell activation recovery. A molecular docking simulation study between 2 and PD-L1 suggested that 2 binds to PD-L1 in a binding mode different from those of other small-molecule PD-L1/PD-1 inhibitors. Notably,oral administration of 2 to mice pre-infected with influenza A virus (A/NWS/33,H1N1 subtype) caused a significant increase in the neutralizing antibody titers,as well as recovery from influenza-induced pneumonia. Overall,2 provides insight for the development of therapeutic drugs against early viral infections,with both virus titer-reducing and antibody titer-boosting effects. Moreover,2 is widely used as a rubber peptizing agent in the production process of tires and other rubber products. Our findings may provide useful information for investigating its influence on living organisms. The online version contains supplementary material available at 10.1038/s41598-025-17982-3. Subject terms: Drug discovery and development,Pharmacology,Screening,Structure-based drug design View Publication

Immune checkpoint blockers (ICBs) have demonstrated substantial efficacy across various malignancies,yet the benefits of ICBs are limited to a subset of patients. Therefore,it is essential to identify novel therapeutic targets. By integrating multi-omics data from cohorts of patients with melanoma treated with ICBs,a positive correlation is observed between tumor CD137L expression and the efficacy of PD-1 blockade. Functionally,CD137L induction in cancer cells significantly enhances anti-tumor immunity by promoting CD8 + T cell survival,both in vivo and in vitro. Mechanistically,helicase-like transcription factor (HLTF) is identified as a pivotal transcriptional regulator of CD137L,controlling its expression through phosphorylation of serine at position 398. Therapeutically,the AMPK agonist AICAR (acadesine) as an inducer of CD137L,exhibiting synergistic effects with PD-1 or CTLA-4 blockade. In summary,our findings elucidate a mechanism controlling CD137L expression and highlight a promising combination therapy to enhance the efficacy of ICBs in melanoma. One Sentence Summary: Inducing co-stimulatory immune checkpoint CD137L expression in melanoma cells enhances T cell-mediated anti-tumor immunity. Subject terms: Tumour immunology,Cancer immunotherapy View Publication

Hsp90 is a molecular chaperone that is often overexpressed across multiple cancer types and has a potential value as a prognostic marker as well as a therapeutic target. Given the high interest in Hsp90 therapies,positron emission tomography or PET imaging of Hsp90 can be a valuable tool for patient selection. The limitations of the previously developed Hsp90 tracers prompted us to evaluate the recently developed brain-permeable [ 11 C]HSP990 PET probe to advance the development of Hsp90-targeted therapeutics. Given the brain accumulation of [ 11 C]HSP990 probe,application for glioblastoma imaging of this tracer is of particular interest. In vitro [ 11 C]HSP990 binding was assessed in breast cancer and glioma cell lines including patient-derived cells using Hsp90 inhibitors and RNA interference knockdown of Hsp90 isoforms. Saturation binding studies were conducted on these cells and tumour tissue homogenates,and autoradiography was performed on tissue sections. Ex vivo biodistribution and in vivo dynamic µPET/CT studies were performed in healthy mice and tumour-bearing mice,including immunocompromised subcutaneous human U87 and MDA-MB-231models and immunocompetent intracranial murine NS/CT-2A models at baseline and following a pre-treatment with Hsp90 inhibitors. High Hsp90-specific tracer uptake was observed in breast cancer and glioma cells,with Hsp90β inhibition resulting in the most substantial reduction in uptake. In vivo uptake was high in U87 tumours but low in MDA-MB-231,presumably due to the differences in Hsp90 expression in tumour tissue versus cultured cells. Differences in maximum binding capacity or B max across cell and tissue types support this hypothesis,especially given that the affinity measured as dissociation constant K d remained similar across all tissue types. Despite high NS/CT-2A tumour uptake in vitro,no contrast between the healthy brain tissue and the NS/CT-2A glioma was observed in vivo due to the high uptake by the healthy brain. [ 11 C]HSP990 is a promising tracer for identifying Hsp90-overexpressing tumours and may hold potential for patient stratification,prognosis,and therapy monitoring of novel Hsp90 therapeutics. High healthy brain uptake of this tracer precluded the differentiation of the tumour in the intracranial NS/CT-2A tumour model,therefore [ 11 C]HSP990 might not be a suitable tracer for the glioblastoma imaging. Tracer with a longer half-life might be needed to compare the washout of the tracer from the brain and the tumour tissue over several hours to identify a suitable imaging window. The online version contains supplementary material available at 10.1186/s41181-025-00386-z. View Publication

Antibiotic resistance is a threat to human health,yet recent work highlights how loss of resistance may drive pathogenesis in some bacteria. In two recent studies,we found that β-lactam antibiotics and nutrient stresses faced during infection selected for genetic inactivation of the Pseudomonas aeruginosa antibiotic efflux pump mexEFoprN . Unexpectedly,efflux pump mutations increased P. aeruginosa virulence during infection; however,neither the prevalence of mexEFoprN inactivating mutations in real human infections,nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for virulence enhancing mutations. Using genome sequencing of clinical isolates,we show that mexEFoprN efflux pump inactivating mutations are enriched in P. aeruginosa isolates from cystic fibrosis infections relative to isolates from acute respiratory infections. Combining RNA-seq,metabolomics,genetic approaches,and infection models we show that efflux pump mutants have elevated quorum sensing driven expression of elastase and rhamnolipids which increase P. aeruginosa virulence during acute and chronic infections. Restoration of the efflux pump in a representative respiratory isolate and the notorious cystic fibrosis Liverpool epidemic strain reduced their virulence. These findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity. Subject terms: Antimicrobial resistance,Pathogens,Bacteriology,Molecular evolution View Publication

Suspension bath bioprinting,whereby bioinks are extruded into a yield stress bath with rapid recovery from shearing,has enabled the printing of low viscosity bioinks into constructs with high geometric complexity. Previous studies have often relied upon external stabilisation of the suspension bath (e.g. collagen) in order to culture soft materials without loss of printed structure. Here,we report a systematic investigation of suspension bath properties that support the printing,fusion,and culture of spheroid-based bioinks without added stabilisation. Specifically,agarose fluid gels of varied polymer concentrations and dilutions were produced and characterised morphologically and rheologically. Juvenile bovine chondrocytes or mesenchymal stromal cells (MSCs) were formed into spheroids of ∼150 µ m in diameter and investigated within agarose suspension baths either for their fusion in hanging drop cultures or as jammed bioinks. MSC spheroids were also printed when mixed with hydrogel microparticles to demonstrate additional versatility to the approach. Suspension baths of lower polymer concentrations and increased dilution enabled faster spheroid fusion; however,the most heavily diluted suspension bath was unable to maintain print fidelity. Other formulations supported the printing,fusion,and culture of spheroid-based inks,either as simple lines or more complex patterns. These findings help to inform the design of suspension baths for bioprinting and culture. View Publication

Tissue remodeling is a key feature of allergic rhinitis (AR),but its underlying molecular mechanisms remain unclear. Lysyl oxidase-like 2 (LOXL2),a regulator of tissue remodeling,has not been studied in AR. Proteomic analysis was performed on nasal mucosal tissues from 8 AR patients and 8 healthy controls (HCs) to identify differentially expressed proteins (DEPs). The top three upregulated DEPs and their association with tissue remodeling markers were validated by immunofluorescence,Western blot,and RT-qPCR in an independent cohort of 30 AR patients and 30 HCs. In vitro,human nasal epithelial cells (HNECs) were treated with IL-4,and the effects of candidate protein inhibitors on remodeling were assessed. An AR mouse model was used to evaluate the impact of these inhibitors on nasal inflammation and remodeling. Proteomic analysis revealed a disease-specific protein expression profile in the nasal mucosa of AR patients,with the top three upregulated proteins being LOXL2,TGF-β1,and TIRAP. Tissue validation showed that LOXL2 was significantly upregulated in the nasal mucosa of AR patients compared to HCs and was significantly correlated with EMT markers (TGF-β1,α-SMA,and E-cadherin). In vitro,IL-4 stimulation significantly upregulated LOXL2,TGF-β1,and α-SMA,while downregulating E-cadherin in a dose-dependent manner in human nasal epithelial cells. These effects were reversed by inhibition of LOXL2. Further investigations demonstrated that LOXL2 promotes tissue remodeling through activation of the TGF-β1/Smad signaling pathway. In the AR mouse model,LOXL2 inhibitors significantly reduced nasal mucosal inflammation and tissue remodeling. Our proteomic analysis suggests that LOXL2 may be involved in the pathological remodeling processes of AR,potentially through modulation of the TGF-β1/Smad signaling pathway. These findings provide preliminary evidence that LOXL2 could serve as a candidate biomarker and a possible therapeutic target in AR,warranting further investigation. View Publication