技术资料

Osteoporosis diagnoses are increasing in the ageing population,and although some treatments exist,these have several disadvantages,highlighting the need to identify new drug targets. G protein-coupled receptors (GPCRs) are transmembrane proteins whose surface expression and extracellular activation make them desirable drug targets. Our previous studies have identified 144 GPCR genes to be expressed in primary human osteoclasts,which could provide novel drug targets. The development of high-throughput assays to assess osteoclast activity would improve the efficiency at which we could assess the effect of GPCR activation on human bone cells and could be utilised for future compound screening. Here,we assessed the utility of a high-content imaging (HCI) assay that measured cytoplasmic-to-nuclear translocation of the nuclear factor of activated T cells-1 (NFATc1),a transcription factor that is essential for osteoclast differentiation,and resorptive activity. We first demonstrated that the HCI assay detected changes in NFATc1 nuclear translocation in human primary osteoclasts using GIPR as a positive control,and then developed an automated analysis platform to assess NFATc1 in nuclei in an efficient and unbiased manner. We assessed six GPCRs simultaneously and identified four receptors (FFAR2,FFAR4,FPR1 and GPR35) that reduced osteoclast activity. Bone resorption assays and measurements of TRAP activity verified that activation of these GPCRs reduced osteoclast activity,and that receptor-specific antagonists prevented these effects. These studies demonstrate that HCI of NFATc1 can accurately assess osteoclast activity in human cells,reducing observer bias and increasing efficiency of target detection for future osteoclast-targeted osteoporosis therapies. View Publication

The CEBPA transcription factor is frequently mutated in acute myeloid leukemia (AML). Mutations in the CEBPA gene,which are typically biallelic,result in the production of a shorter isoform known as p30. Both the canonical 42-kDa isoform (p42) and the AML-associated p30 isoform bind chromatin and activate transcription,but the specific transcriptional programs controlled by each protein and how they are linked to a selective advantage in AML is not well understood. Here,we show that cells expressing the AML-associated p30 have reduced baseline inflammatory gene expression and display altered dynamics of transcriptional induction in response to LPS,consequently impacting cytokine secretion. This confers p30-expressing cells an increased resistance to the adverse effects of prolonged exposure to inflammatory signals. Mechanistically,we show that these differences primarily arise from the differential regulation of AP-1 family proteins. In addition,we find that the impaired function of the AP-1 member ATF4 in p30-expressing cells alters their response to ER stress. Collectively,these findings uncover a link between mutant CEBPA,inflammation and the stress response,potentially revealing a vulnerability in AML. Subject terms: Gene regulation,Acute myeloid leukaemia,Transcriptional regulatory elements,Epigenetics in immune cells View Publication

This study aimed to assess the efficacy of Quality and Quantity media-cultured mononuclear cells (QQ-MNCs) for promoting nerve regeneration in a mouse sciatic nerve transection model. Human peripheral blood mononuclear cells (PB-MNCs) and QQ-MNCs derived from healthy volunteers were used/compared. The left sciatic nerve was surgically transected in 27 mice. After complete nerve transection was confirmed,end-to-end direct epineurial nerve repair was performed using 9–0 nylon. Fibrin glue was applied to the tissue around the injury site to limit diffusion of the study treatment followed by application of 0.5 ml phosphate buffered saline (PBS) or PB-MNCs (2x10 6 cells) or QQ-MNCs (2x10 6 cells) to the injury site. The skin was then closed using 6–0 nylon. Histomorphology,immunohistochemistry,electrophysiologic examination,and functional assessment were evaluated at 12-weeks followed by euthanasia and subsequent harvesting of the left sciatic nerves and the left and right gastrocnemius muscles for examination. QQ-MNCs mice exhibited significant improvement in all histomorphologic parameters (axon fiber diameter,myelin thickness,percentage of nerve density) and immunohistochemistry assays (S100,SOX10,GFAP,neurofilament,IL-1β,VEGF,anti-HNA,TNF-α,vWF) compared to PBS mice (all p < 0.05). QQ-MNCs mice also had a significantly higher Basso Mouse Scale score compared to PBS mice ( p = 0.018). The percentage of nerve density adjacent to the injury site was significantly higher in QQ-MNCs mice than in PB-MNCs mice ( p = 0.049). IL-1β expression was significantly lower in QQ-MNCs mice than in PB-MNCs mice ( p = 0.01). QQ-MNCs mice demonstrated significantly better functional and histomorphologic outcomes of nerve regeneration compared to PB-MNCs mice and PBS mice. View Publication

SOT201 and its murine surrogate mSOT201 are novel cis-acting immunocytokines consisting of a humanized/murinized/,Fc-silenced anti-programmed cell death protein 1 (PD-1) monoclonal antibody (mAb) fused to an attenuated human interleukin (IL)-15 and the IL-15Rα sushi+ domain. Murine mPD1-IL2v is a conjugate of a murinized,Fc silenced anti-PD-1 mAb bearing human IL-2 with abolished IL-2Rα binding. These immunocytokines spatiotemporally reinvigorate PD-1 + CD8 + tumor-infiltrating lymphocytes (TILs) via cis-activation and concomitantly activate the innate immunity via IL-2/15Rβγ signaling. Human peripheral blood mononuclear cell and cell lines were used to evaluate cis/trans activity of SOT201. Anti-PD-1 mAb responsive (MC38,CT26) and resistant (B16F10,CT26 STK11 KO) mouse tumor models were used to determine the anticancer efficacy,and the underlying immune cell activity was analyzed via single-cell RNA sequencing and flow cytometry. The expansion of tumor antigen-specific CD8 + T cells by mSOT201 or mPD1-IL2v and memory CD8 + T-cell generation in vivo was determined by flow cytometry. SOT201 delivers attenuated IL-15 to PD-1 + T cells via cis-presentation,reinvigorates exhausted human T cells and induces higher interferon-γ production than pembrolizumab in vitro. mSOT201 administered as a single dose exhibits strong antitumor efficacy with several complete responses in all tested mouse tumor models. While mPD1-IL2v activates CD8 + T cells with a 50-fold higher potency than mSOT201 in vitro,mSOT201 more effectively reactivates effector exhausted CD8 + T cells (Tex),which demonstrate higher cytotoxicity,lower exhaustion and lower immune checkpoint transcriptional signatures in comparison to mPD1-IL2v in MC38 tumors in vivo. This can be correlated with a higher rate of complete responses in the MC38 tumor model following mSOT201 treatment when compared with mPD1-IL2v. mSOT201 increased the relative number of tumor antigen-specific CD8 + T cells,and unlike mPD1-IL2v stimulated greater expansion of adoptively transferred ovalbumin-primed CD8 + T cells simultaneously limiting the peripheral CD8 + T-cell sink,leading to the development of memory CD8 + T cells in vivo. SOT201 represents a promising therapeutic candidate that preferentially targets PD-1 + TILs,delivering balanced cytokine activity for reviving CD8 + Tex cells in tumors. SOT201 is currently being evaluated in the Phase I clinical study VICTORIA-01 ( NCT06163391 ) in patients with advanced metastatic cancer. View Publication

Adeno-associated viral (AAV) vector-based gene therapy is gaining foothold as treatment for genetic neurological diseases with encouraging clinical results. Nonetheless,dose-dependent adverse events have emerged in recent clinical trials through mechanisms that remain unclear. We have modelled here the impact of AAV transduction in cell models of the human central nervous system (CNS),taking advantage of induced pluripotent stem cells. Our work uncovers vector-induced innate immune mechanisms that contribute to cell death. While empty AAV capsids were well tolerated,the AAV genome triggered p53-dependent DNA damage responses across CNS cell types followed by the induction of inflammatory responses. In addition,transgene expression led to MAVS-dependent activation of type I interferon responses. Formation of DNA damage foci in neurons and gliosis were confirmed in murine striatum upon intraparenchymal AAV injection. Transduction-induced cell death and gliosis could be prevented by inhibiting p53 or by acting downstream on STING- or IL-1R-mediated responses. Together,our work identifies innate immune mechanisms of vector sensing in the CNS that can potentially contribute to AAV-associated neurotoxicity. Subject terms: Neuroimmunology,Innate immunity,Neural stem cells View Publication

Ceramides are bioactive sphingolipids that have physiological effects on inflammation,apoptosis,and mitochondrial dysfunction. They may play a critical role in the harm of ischemia/reperfusion (IR). Ceramides and IR injury are not well-studied,and there is a lack of human data. Current studies aimed to investigate the role of ceramide buildup in cardiomyocytes (CMs) death using CMs derived from human induced pluripotent stem cell (hiPSC) as a model for simulating IR injury in vitro. In our model,serum- and glucose-free media was used to expose hiPSC-derived CMs to hypoxia (3% O 2 ) for 6 h (hrs),followed by reoxygenation (20% O 2 ) for 16 h. In contrast to normoxia (control) or hypoxia (ischemia),our data showed that following IR,there was an increase in the formation of mitochondrial superoxide and the mRNA levels of genes regulating ceramide synthesis,such as CerS2 and CerS4 in CMs. Further,there was a considerable rise in the levels of total ceramide,long-chain (C16:0,C18:0,and C18:1),and very long-chain (C22:0 and C24:1) ceramide species in CMs following reperfusion in comparison to control or ischemic CMs. Interestingly,compared to CMs exposed to IR without inhibitor,our data showed that inhibition of ceramide formation with fumonisin B1 (FB1) significantly lowered ceramide levels,reduced apoptosis,improved mitochondrial function,and enhanced survival of CMs exposed to IR. Furthermore,we used a transgenic mouse model,in which the CerS2 gene was overexpressed in the CMs of α-MHC-CerS2 mice,to validate the basic idea that ceramide contributes to heart disease in vivo. Our results showed that the heart tissues of α-MHC-CerS2 mice had significant levels of long-chain and very long-chain ceramides,which causes increased apoptosis,proinflammatory cytokines,interstitial inflammatory cell infiltration,and collagen deposition. Results from both in vitro and in vivo experiments show that ceramides have a significant role in either mediating or inducing damage to CMs. Additionally,in vitro findings show that ceramide reduction improves the outcome of IR injury by lowering intracellular Ca 2+ [Ca 2+ ] i concentration and improves mitochondrial function changes during IR. The online version contains supplementary material available at 10.1186/s13287-025-04340-3. View Publication

Development and lineage choice are driven by interconnected transcriptional,epigenetic and metabolic changes. Specific metabolites,such as α-ketoglutarate (αKG),function as signalling molecules affecting the activity of chromatin-modifying enzymes. However,how metabolism coordinates cell-state changes,especially in human pre-implantation development,remains unclear. Here we uncover that inducing naive human embryonic stem cells towards the trophectoderm lineage results in considerable metabolic rewiring,characterized by αKG accumulation. Elevated αKG levels potentiate the capacity of naive embryonic stem cells to specify towards the trophectoderm lineage. Moreover,increased αKG levels promote blastoid polarization and trophectoderm maturation. αKG supplementation does not affect global histone methylation levels; rather,it decreases acetyl-CoA availability,reduces histone acetyltransferase activity and weakens the pluripotency network. We propose that metabolism functions as a positive feedback loop aiding in trophectoderm fate induction and maturation,highlighting that global metabolic rewiring can promote specificity in cell fate decisions through intricate regulation of signalling and chromatin. Subject terms: Embryonic stem cells,Embryology View Publication

Toll-interacting protein (Tollip) suppresses excessive pro-inflammatory signaling,but its function in airway epithelial responses to IL-13,a key mediator in allergic diseases,remains unclear. This study investigates Tollip knockdown (TKD) effects in primary human airway epithelial cells using single-cell RNA sequencing,providing the first single-cell analysis of TKD and the first exploring its interaction with IL-13. IL-13 treatment upregulated key genes,including SPDEF,MUC5AC,POSTN,ALOX15,and CCL26,confirming IL-13’s effects and validating our methods. IL-13 reduced TNF-α signaling and epithelial-mesenchymal transition in certain cell types,suggesting a dual role in promoting type 2 inflammation while suppressing Th1-driven inflammation. Tollip deficiency alone significantly amplified TNF-α signaling and inflammatory pathways in goblet,club,and suprabasal cells. Comparisons between TKDIL13 vs IL13 and TKD vs CTR revealed that IL-13 does not substantially alter Tollip deficiency response in most cell types,reinforcing findings in TKD vs CTR. Tollip deficiency alters the response to IL-13 in a cell-type-specific manner,strongly downregulating TNF-α signaling in goblet cells but only weakly in basal and club cells. Tollip deficiency enhances IL-13’s suppression of Th1 inflammatory responses in goblet cells. These novel insights in Tollip-IL-13 interactions offer potential therapeutic targets for asthma and related diseases. The online version contains supplementary material available at 10.1186/s13104-025-07255-7. View Publication

Ovarian cancer (OV) has the highest mortality rate among gynecological cancers. As OV progresses,tumor cells spread outside the ovaries to the peritoneal and abdominal cavities,forming cell clusters that float in the ascitic fluid caused by peritonitis carcinomatosa,leading to further dissemination and metastasis. These cell clusters are enriched with cancer stem cells (CSCs) which are responsible for treatment resistance,recurrence,and metastasis. Therefore,targeting CSCs is a potentially effective approach for treating OV. However,understanding how CSCs acquire treatment resistance and identifying targets against CSCs remains challenging. In this study,we demonstrate that 3D-spheroids of OV cell lines exhibit higher stemness than conventional adherent cells. Metabolomics profiling studies have revealed that 3D-spheroids maintain a high-energy state through increased glucose utilization in the citric acid cycle (TCA),efficient nucleotide phosphorylation,and elevated phosphocreatine as an energy buffer. We also found that nicotinamide phosphoribosyltransferase (NAMPT),the rate-limiting enzyme for NAD + production,is highly expressed in OV. Furthermore,the approach based on NAMPT dependence rather than histology found NAMPT to be a potential therapeutic target against CSCs,while also serving as a prognostic indicator in OV. Moreover,we identified a previously unrecognized anti-tumor mechanism whereby disulfiram,an aldehyde dehydrogenase (ALDH) inhibitor,synergistically inhibited mitochondrial function when combined with NAMPT inhibitors - leading to cell cycle arrest in G2/M. Finally,the combination of a NAMPT inhibitor and disulfiram showed significant anti-tumor effects and extended survival in an animal model. Our findings demonstrate the potential of spheroids as a preclinical model for targeting OV CSCs and also indicate that the combination of NAMPT inhibitors and disulfiram is a promising therapeutic strategy to overcome recurrent OV. Subject terms: Ovarian cancer,Metabolomics,Apoptosis,Cancer stem cells View Publication

Low dose methotrexate (LD‐MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti‐inflammatory effects of LD‐MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD‐MTX. This is the first study to characterize the effects of LD‐MTX on HSPC. CD34 + HSPC were isolated from healthy donors' non‐mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD‐MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability,cell cycle,surface abundance of reduced folate carrier 1 (RFC1),proliferation,reactive oxygen species (ROS) levels,DNA double‐strand breaks,p38 activation,and CD34 + subpopulations. HSPC clonogenicity was tested in colony‐forming cell assays. Our results indicate that in cycling HSPC,membrane RFC1 is upregulated and,following LD‐MTX treatment,they accumulate more intracellular MTX than resting HSPC. In cycling HSPC,LD‐MTX inhibits HSPC expansion by promoting S‐phase cell‐cycle arrest,increases intracellular HSPC ROS levels and DNA damage,and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD‐MTX are more evident in CD34 + CD38High progenitors. In non‐cycling HSPC,LD‐MTX also reduces the proliferative response while preserving their clonogenicity. In summary,HSPC uptake LD‐MTX differentially according to their cycling state. In turn,LD‐MTX results in reduced proliferation and the preservation of HSPC clonogenicity. View Publication

The interplay between 3D genomic structure and transposable elements (TE) in regulating cell state-specific gene expression program is largely unknown. Here,we explore the utilization of TE-derived enhancers in naïve and expanded pluripotent states by integrative analysis of genome-wide Hi-C-defined enhancer interactions,H3K27ac HiChIP profiling and CRISPR-guided TE proteomics landscape. We find that short interspersed nuclear elements (SINEs) are the more involved TEs in the active chromatin and 3D genome architecture. In particular,mammalian-wide interspersed repeat (MIR),a SINE family member,is highly associated with naïve-specific genomic interactions compared to the expanded state. Primarily,in the naïve pluripotent state,MIR enhancer is co-opted by ESRRB for naïve-specific gene expression program. This ESRRB and MIR enhancer interaction is crucial for the formation of loops that build a network of enhancers and super-enhancers regulating pluripotency genes. We demonstrate that loss of a ESRRB-bound MIR enhancer impairs self-renewal. We also find that MIR is co-bound by structural protein complex,ESRRB-YY1,in the naïve pluripotent state. Altogether,our study highlights the topological regulation of ESRRB on MIR in the naïve potency state. The online version contains supplementary material available at 10.1186/s13059-025-03577-8. View Publication

Cancer stem cells (CSCs) play crucial roles in tumor metastasis,therapy resistance,and immune evasion. Identifying and understanding the factors that regulate the stemness of tumor cells presents promising opportunities for developing effective therapeutic strategies. In this study on oral squamous cell carcinoma (OSCC),we confirmed the key role of KLF7 in maintaining the stemness of OSCC. Using chromatin immunoprecipitation sequencing and dual-luciferase assays,we identified ITGA2,a membrane receptor,as a key downstream gene regulated by KLF7 in the maintenance of stemness. Tumor sphere formation assays,flow cytometry analyses,and in vivo limiting dilution tumorigenicity evaluations demonstrated that knocking down ITGA2 significantly impaired stemness. Upon binding to its extracellular matrix (ECM) ligand,type I collagen,ITGA2 activates stemness-associated signaling pathways,including PI3K-AKT,MAPK,and Hippo. TC-I 15,a small-molecule inhibitor of the ITGA2-collagen interaction,significantly sensitizes oral squamous cell carcinoma (OSCC) to cisplatin in xenograft models. In summary,we reveal that the KLF7/ITGA2 axis is a crucial modulator of stemness in OSCC. Our findings suggest that ITGA2 is a promising therapeutic target,offering a novel anti-CSC strategy. Subject terms: Cancer stem cells,Cancer therapeutic resistance View Publication