技术资料

The competitive advantage of mutant hematopoietic stem and progenitor cells (HSPCs) underlies clonal hematopoiesis (CH). Drivers of CH include aging and inflammation; however,how CH-mutant cells gain a selective advantage in these contexts is an unresolved question. Using a murine model of CH ( Dnmt3a R878H/+ ),we discover that mutant HSPCs sustain elevated mitochondrial respiration which is associated with their resistance to aging-related changes in the bone marrow microenvironment. Mutant HSPCs have DNA hypomethylation and increased expression of oxidative phosphorylation gene signatures,increased functional oxidative phosphorylation capacity,high mitochondrial membrane potential (Δψm),and greater dependence on mitochondrial respiration compared to wild-type HSPCs. Exploiting the elevated Δψm of mutant HSPCs,long-chain alkyl-TPP molecules (MitoQ,d-TPP) selectively accumulate in the mitochondria and cause reduced mitochondrial respiration,mitochondrial-driven apoptosis and ablate the competitive advantage of HSPCs ex vivo and in vivo in aged recipient mice. Further,MitoQ targets elevated mitochondrial respiration and the selective advantage of human DNMT3A -knockdown HSPCs,supporting species conservation. These data suggest that mitochondrial activity is a targetable mechanism by which CH-mutant HSPCs gain a selective advantage over wild-type HSPCs. Subject terms: Ageing,Haematopoietic stem cells,Mitochondria 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

Germline activating and deactivating mutations of STAT5b,part of the JAK-STAT signaling pathway,push the immune system and hematopoiesis in opposing directions,tuning systems either up or down. View Publication

The complete array of genes required for terminal erythroid differentiation remains unknown. To address this knowledge gap,we perform a genome-scale CRISPR knock-out screen in the human erythroid progenitor cell line HUDEP-2 and validate candidate regulators of erythroid differentiation in a custom secondary screen. Comparison of sgRNA abundance in the CRISPR library,proerythroblasts,and orthochromatic erythroblasts,resulted in the identification of genes that are essential for proerythroblast survival and genes that are required for terminal erythroid differentiation. Among the top genes identified are known regulators of erythropoiesis,underscoring the validity of this screen. Notably,using a Log2 fold change of <−1 and false discovery rate of <0.01,the screen identified 277 genes that are required for terminal erythroid differentiation,including multiple genes not previously nominated through GWAS. NHLRC2,which was previously implicated in hemolytic anemia,was a highly ranked gene. We suggest that anemia due to NHLRC2 mutation results at least in part from a defect in erythroid differentiation. Another highly ranked gene in the screen is VAC14,which we validated for its requirement in erythropoiesis in vitro and in vivo. Thus,data from this CRISPR screen may help classify the underlying mechanisms that contribute to erythroid disorders. Subject terms: Erythropoiesis,CRISPR-Cas9 genome editing,Haematopoietic stem cells 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

Osteosarcoma is the most prevalent primary malignant bone tumor affecting pediatric and adolescent individuals. However,despite the passage of three decades,there has been no notable enhancement in the overall survival rate of patients with osteosarcoma. In recent years,CD155 has been reported to exhibit abnormal amplification in a range of tumors,yet the precise underlying mechanism remains elusive. The objective of this study is to investigate the role of CD155 in osteosarcoma,and to identify drugs that specifically target this molecule,thereby offering a novel direction for the treatment of osteosarcoma. The prognosis of patients with osteosarcoma with high and low expression of CD155 was verified by immunohistochemistry. CCK-8 and colony formation assays were used to detect cell proliferation and drug resistance. Transwell experiments were used to detect cell migration and invasion. The sphere formation experiment was used to evaluate the stemness of tumor cells. Additionally,in vivo animal models were utilized to assess the functional role of CD155 in a biological context. RNA-seq and co-immunoprecipitation methods were used to search for downstream target molecules and signaling pathways of CD155. Finally,virtual screening was used to find drugs targeting CD155. In this study,we have established the significant amplification of CD155 in osteosarcoma. Utilizing a comprehensive array of experimental methods,including CCK-8 assay,colony formation assay,Transwell assay,and in vivo animal models,we unequivocally demonstrate that CD155 significantly potentiates the malignancy of osteosarcoma both in vitro and in vivo. Additionally,our findings reveal that CD155 promotes osteosarcoma stemness by modulating the Wnt/β-catenin signaling pathway. Advanced molecular techniques,such as RNA sequencing and co-immunoprecipitation,have been instrumental in elucidating the mechanism of CD155 in activating the Wnt/β-catenin pathway via the SRC/AKT/GSK3β signaling axis,thereby enhancing the stem-cell-like properties of osteosarcoma cells. To explore targeted therapeutic options,we conducted virtual screening and identified troxerutin as a promising CD155 inhibitor. Our findings reveal that troxerutin effectively inhibits CD155,attenuates the SRC/AKT/GSK3β signaling cascade,diminishes the nuclear localization of β-catenin,and consequently mitigates osteosarcoma stemness. These discoveries position troxerutin as a promising candidate for targeted osteosarcoma therapy. The online version contains supplementary material available at 10.1186/s11658-025-00724-8. View Publication

Biliverdin IXβ reductase (BLVRB) is an NADPH-dependent enzyme previously implicated in a redox-regulated mechanism of thrombopoiesis distinct from the thrombopoietin (TPO)/c-MPL axis. Here,we apply computational modeling to inform molecule design,followed by de novo syntheses and screening of unique small molecules retaining the capacity for selective BLVRB inhibition as a novel platelet-enhancing strategy. Two distinct classes of molecules are identified,and NMR spectroscopy and co-crystallization studies confirm binding modes within the BLVRB active site and ring stacking between the nicotinamide moiety of the NADP + cofactor. A diazabicyclo derivative displaying minimal off-target promiscuity and excellent bioavailability characteristics promotes megakaryocyte speciation in biphenotypic (erythro/megakaryocyte) cellular models and synergizes with TPO-dependent megakaryocyte formation in hematopoietic stem cells. Upon oral delivery into mice,this inhibitor expands platelet recovery in stress thrombopoietic models with no adverse effects. In this work,we identify and validate a cellular redox inhibitor retaining the potential to selectively promote megakaryocytopoiesis and enhance stress-associated platelet formation in vivo distinct from TPO receptor agonists. Subject terms: Target validation,Medicinal chemistry,X-ray crystallography,Computational biophysics View Publication

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

Lipid dyshomeostasis and tau pathology are present in frontotemporal lobar degeneration (FTLD) and Alzheimer’s disease (AD). However,the relationship between lipid dyshomeostasis and tau pathology remains unclear. We report that GRAM Domain Containing 1B (GRAMD1B),a nonvesicular cholesterol transporter,is increased in excitatory neurons of human neural organoids (HNOs) with the MAPT R406W mutation. Human FTLD,AD cases,and PS19 tau mice also have increased GRAMD1B expression. We show that overexpression of GRAMD1B increases levels of free cholesterol,lipid droplets,and impairs autophagy flux. Modulating GRAMD1B in iPSC-derived neurons also alters key autophagy-related components such as PI3K,phospho-AKT,and p62,as well as phosphorylated tau,and CDK5R1. Blocking GRAMD1B function decreases free cholesterol and lipid droplets. Knocking down GRAMD1B additionally reduces phosphorylated tau,and CDK5R1 expression. Our findings elucidate the role of GRAMD1B in the nervous system and highlight its relevance to FTLD and AD. Subject terms: Diseases of the nervous system,Ageing View Publication

Myelodysplastic syndrome (MDS) is a malignant hematologic disorder with limited curative options,primarily reliant on hematopoietic stem cell transplantation. Anemia,a prevalent symptom of MDS,has few effective treatment strategies. Realgar,though known for its therapeutic effects on MDS,remains poorly understood in terms of its mechanism of action. In this study,both in vivo and in vitro experiments were conducted using Realgar and its primary active component,As 2 S 2,to examine their impact on mouse erythroblasts at the single-cell level. Realgar treatment significantly altered the transcriptional profiles and cellular composition of bone marrow in mice,both in vivo and in vitro. Differentially expressed genes in erythroblasts regulated by Realgar were identified,unveiling potential regulatory functions and signaling pathways,such as heme biosynthesis,hemoglobin production,oxygen binding,IL-17 signaling,and MAPK pathways. These findings suggest that Realgar enhances the differentiation of erythroblasts in mouse bone marrow and improves overall blood cell counts. This work offers preliminary insights into Realgar’s mechanisms,expands the understanding of this mineral medicine,and may inform strategies to optimize its therapeutic potential in hematologic diseases. The online version contains supplementary material available at 10.1186/s12935-025-03768-0. View Publication

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex,cerebellum and remaining areas. First,each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second,the cerebral regions were subjected to the decellularization process and their respective characterization using molecular,histological,and ultrastructural techniques. Third,the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth,we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells,as a unidirectional model of differentiation. Finally,in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and,in addition,it possesses a combination of structural and biochemical signals (e.g.,neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro. The online version contains supplementary material available at 10.1038/s41598-025-95656-w. View Publication

Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs,FR-GECO1a and FR-GECO1c,based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm,respectively,display large responses to Ca 2+ in vitro (Δ F / F 0 = 6 for FR-GECO1a,18 for FR-GECO1c),are bright under both one-photon and two-photon illumination,and have high affinities (apparent K d = 29 nM for FR-GECO1a,83 nM for FR-GECO1c) for Ca 2+ . FR-GECOs offer sensitive and fast detection of single action potentials in neurons,and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators. Subject terms: Fluorescent proteins,Optogenetics,Zebrafish,Molecular neuroscience,Calcium signalling View Publication