技术资料

Protein disulfide isomerase (PDI,PDIA1) is an emerging therapeutic target in oncology. PDI inhibitors have demonstrated a unique propensity to selectively induce apoptosis in cancer cells and overcome resistance to existing therapies,although drug candidates have not yet progressed to the stage of clinical development. We recently reported the discovery of lead indene compound E64FC26 as a potent pan-PDI inhibitor that enhances the cytotoxic effects of proteasome inhibitors in panels of Multiple Myeloma (MM) cells and MM mouse models. An extensive medicinal chemistry program has led to the generation of a diverse library of indene-containing molecules with varying degrees of proteasome inhibitor potentiating activity. These compounds were generated by a novel nucleophilic aromatic ring cyclization and dehydration reaction from the precursor ketones. The results provide detailed structure activity relationships (SAR) around this indene pharmacophore and show a high degree of correlation between potency of PDI inhibition and bortezomib (Btz) potentiation in MM cells. Inhibition of PDI leads to ER and oxidative stress characterized by the accumulation of misfolded poly-ubiquitinated proteins and the induction of UPR biomarkers ATF4,CHOP,and Nrf2. This work characterizes the synthesis and SAR of a new chemical class and further validates PDI as a therapeutic target in MM as a single agent and in combination with proteasome inhibitors. View Publication

Although a link between the gut microbiota and alcohol-related liver diseases (ALDs) has previously been suggested,the causative effects of specific taxa and their functions have not been fully investigated to date. Here,we analyze the gut microbiota of 410 fecal samples from 212 Korean twins by using the Alcohol Use Disorders Identification Test (AUDIT) scales to adjust for host genetics. This analysis revealed a strong association between low AUDIT scores and the abundance of the butyrate-producing genus Roseburia. When Roseburia spp. are administered to ALD murine models,both hepatic steatosis and inflammation significantly improve regardless of bacterial viability. Specifically,the flagellin of R. intestinalis,possibly through Toll-like receptor 5 (TLR5) recognition,recovers gut barrier integrity through upregulation of the tight junction protein Occludin and helps to restore the gut microbiota through elevated expression of IL-22 and REG3$\gamma$. Our study demonstrates that Roseburia spp. improve the gut ecosystem and prevent leaky gut,leading to ameliorated ALDs. View Publication

INTRODUCTION Complexins (CPLXs),initially identified in neuronal presynaptic terminals,are cytoplasmic proteins that interact with the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) complex to regulate the fusion of vesicles to the plasma membrane. Although much is known about CPLX function in neuronal synaptic vesicle exocytosis,their distribution and role in immune cells are still unclear. In this study,we investigated CPLX2 knockout (KO) mice to reveal the role of CPLXs in exocytosis of lymphocytes. METHODS We examined the expression of CPLXs and SNAREs in lymphocytes. To study the effect of CPLXs on the immune system in vivo,we analyzed the immune phenotype of CPLX2 KO mice. Furthermore,antibodies secretion from the peritoneal cavity,spleen,and bone marrow cells of wild-type (WT) and CPLX2 KO mice were determined. RESULTS CPLX2 was detected in B cells but not in T cells,while other CPLXs and SNAREs were expressed at a similar level in both B and T cells. To clarify the function of CPLX2 in B lymphocytes,serum concentrations of immunoglobulin G (IgG),IgA,IgM,and IgE were measured in WT and CPLX2 KO mice using enzyme-linked immunosorbent assay. The level of IgM,which mainly consists of natural antibodies,was higher in KO mice than that in WT mice,while the levels of other antibodies were similar in both types of mice. Additionally,we found that spontaneous secretion of IgM and IgG1 was enhanced from the splenic antibody-secreting cells (ASCs) of CPLX2 KO mice. CONCLUSION Our data suggest that CPLX2 inhibits spontaneous secretion of IgM and IgG1 from splenic ASCs. This study provides new insight into the mechanism of antibody secretion of ASCs. View Publication

Myelodysplastic syndromes (MDSs) are a group of clonal disorders of hematopoietic stem cells,resulting in ineffective hematopoiesis. Previous studies have reported that decitabine (DAC) plays an essential role in cell cycle arrest and cell death induction in multiple cell types. Nevertheless,the effect of decitabine on mesenchymal stromal cells derived from bone marrow of patients with MDSs is not completely clarified. Here,we explored the apoptotic and anti-proliferative effect of DAC on MSCs isolated from patients with MDSs. Treatment with DAC inhibited cell growth in a concentration- and time-dependent manner by inducing apoptosis. We found a positive relationship between cell death triggered by DAC in MSCs and the death receptor family members Fas and FasL mRNA and protein levels (***P {\textless} 0.00085),cleaved caspase (-3,-8,and -9) activity,and mitochondrial membrane potential reduction. Additionally,DAC-induced apoptosis was inhibited by Kp7-6,a FasL/Fas antagonist,indicating a crucial role of FasL/Fas,a cell death receptor,in mediating the apoptotic effect of DAC. DAC also induced reactive oxygen species (ROS) generation in MSCs derived from MDSs patients (*P = 0.038). Furthermore,N-acetyl-L-cysteine (NAC),a widely accepted ROS scavenger,efficiently reversed DAC-induced apoptosis by inhibiting ROS generation (***P {\textless} 0.00051) in mitochondria and restoring mitochondrial membrane potential. Furthermore,ROS production was found to be a consequence of caspase activation via caspases inhibition. Our data imply that DAC triggers ROS production in human MSCs,which serves as a crucial factor for mitochondrial membrane potential reduction,and DAC induces cell death prior to FasL/Fas stimulation. View Publication

Graphene quantum dots (GQDs) have gained significant attention in various biomedical applications. The physicochemical properties of these nanoparticles,including toxic effects,are largely determined by their surface modifications. Previous studies have demonstrated high in vitro cytotoxicity of the hydroxylated GQDs (OH-GQDs). The focus of this study was on the intestinal toxicity of OH-GQDs. Briefly,C57BL/6J mice were given daily oral gavage of 0.05,0.5 or 5 mg/kg OH-GQD for 7 days,and the indices of intestinal damage were evaluated. Higher doses of the OH-GQDs caused significant intestinal injuries,such as enhanced intestinal permeability,shortened villi and crypt loss. The number of Lgr5+ intestinal stem cells also decreased dramatically upon OH-GQDs exposure,which also inhibited the Ki67+ proliferative progenitor cells. In addition,an increased number of crypt cells harboring the oxidized DNA base 8-OHdG and $\gamma$H2AX foci were also detected in the intestines of OH-GQD-treated mice. Mechanistically,the OH-GQDs up-regulated both total and phosphorylated p53. Consistent with this,the average number of TUNEL+ and cleaved caspase-3+ apoptotic intestinal epithelial cells were significantly increased after OH-GQDs treatment. Finally,a 3-dimensional organoid culture was established using isolated crypts,and OH-GQDs treatment significantly reduced the size of the surviving intestinal organoids. Taken together,the intestinal toxicity of the OH-GQDs should be taken into account during biomedical applications. View Publication

Advancements in vaccination and sanitation have significantly reduced the prevalence and burden of infectious diseases; however,these benefits have coincided with a marked rise in autoimmune and allergic disorders. Recent studies have investigated these linked trends through the lens of host-microbiome alterations,proposing these shifts as a potential explanatory mechanism. Previously,we demonstrated that vancomycin-induced depletion of short-chain fatty acid (SCFA)-producing bacteria results in hyperactivation of ILC2s and exacerbated allergic responses. Here we investigate the effects of low-dose streptomycin on innate and adaptive immune cell populations and their activation states. Although streptomycin-treated mice exhibit normal allergic responses,they display heightened susceptibility to Th1/Th17-mediated disease,specifically hypersensitivity pneumonitis (HP). This is characterized by a two-fold increase in ILC3s and Th17 cells in the lungs,alongside activation of antigen-presenting cells (APCs) at steady state-an effect that is further amplified upon exposure to HP-inducing agents. Shotgun metagenomic analysis revealed that streptomycin-induced dysbiosis reduces microbial diversity,depletes bile acid-metabolizing bacteria,and enriches for metabolic pathways involved in branched-chain amino acid biosynthesis,including leucine-a known activator of mTORC1. Strikingly,administration of the secondary bile acid metabolite isolithocholic acid (an inverse agonist of RORγt),or an IL-23 neutralizing antibody,reverses the enhanced susceptibility to HP. Inhibition of mTORC1 significantly reduced Th17/ILC3 responses and histopathology. Our findings underscore microbial equilibrium as a key determinant of susceptibility to HP and uncover a positive feedback loop between IL and 23-producing APCs and ILC3/Th17 cells that mechanistically links dysbiosis to sustained type 3 inflammation,and we identify a simple,actionable means of intervention. View Publication

Fluorescence microscopy has undergone rapid advancements,offering unprecedented visualization of biological events and shedding light on the intricate mechanisms governing living organisms. However,the exploration of rapid biological dynamics still poses a significant challenge due to the limitations of current digital camera architectures and the inherent compromise between imaging speed and other capabilities. Here,we introduce sHAPR,a high-speed acquisition technique that leverages the operating principles of sCMOS cameras to capture fast cellular and subcellular processes. sHAPR harnesses custom fiber optics to convert microscopy images into one-dimensional recordings,enabling acquisition at the maximum camera readout rate,typically between 25 and 250 kHz. We have demonstrated the utility of sHAPR with a variety of phantom and dynamic systems,including high-throughput flow cytometry,cardiomyocyte contraction,and neuronal calcium waves,using a standard epi-fluorescence microscope. sHAPR is highly adaptable and can be integrated into existing microscopy systems without requiring extensive platform modifications. This method pushes the boundaries of current fluorescence imaging capabilities,opening up new avenues for investigating high-speed biological phenomena. The authors introduce a highspeed acquisition technique,sHAPR,for rapid exploration of biodynamics using fluorescence microscopy. The method leverages sCMOS cameras and custom fibre optics to convert microscopy images into 1D recordings,enabling acquisition at the maximum camera readout rate. View Publication

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia,platelet dysfunction,and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation,the DNA methylation status in FPDMM remains unknown,largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here,using genome editing techniques,we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks),consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs,with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1,an ETS family member,was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation,and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation. View Publication

BackgroundCHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders,including CHARGE (Coloboma,Heart defects,Atresia of the choanae,Retardation of growth and development,Genital hypoplasia,and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus,liver,pancreas,and intestine.MethodsIn this study,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7?/?),heterozygous mutant (CHD7+/?),and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers,mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).ResultsWe found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.ConclusionsCHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04437-9. View Publication

SUMMARYUnderstanding how human gene expression is coordinately regulated by functional units of proteins across the genome remains a major biological goal. Here,we present COMET,a high-throughput screening platform for combinatorial effector targeting for the identification of transcriptional modulators. We generate libraries of combinatorial dCas9-based fusion proteins,containing two to six effector domains,allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription. Importantly,we keep full proteins or domains intact,maintaining catalytic cores and surfaces for protein-protein interactions. We observe more than 5800 significant hits that modulate transcription,we demonstrate cell type specific transcriptional modulation,and we further investigate epistatic relationships between our effector combinations. We validate unexpected combinations as synergistic or buffering,emphasizing COMET as both a method for transcriptional effector discovery,and as a functional genomics tool for identifying novel domain interactions and directing locus-specific biochemistry. View Publication

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract,referred to as mutant ataxin-3. In this study,we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First,we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently,the pluripotency of GE-iPSCs was confirmed,and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons,restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells),and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion,our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8. View Publication

Human-induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) have been shown to be useful for the development of cell-based regenerative strategies and for modelling drug discovery. However,stem cell-derived HLCs are not identical in nature to primary human hepatocytes (PHHs),which could affect the cell phenotype and,potentially,model reliability. Therefore,we employed the in-depth gene expression profiling of HLCs and other important and relevant cell types,which led to the identification of clear similarities and differences between them at the transcriptional level. Through gene set enrichment analysis,we identified that genes that are critical for immune signalling pathways become downregulated upon HLC differentiation. Our analysis also found that TAV.HLCs exhibit a mild gene signature characteristic of acute lymphoblastic leukaemia,but not other selected cancers. Importantly,HLCs present significant similarity to PHHs,making them genuinely valuable for modelling human liver biology in vitro and for the development of prototype cell-based therapies for pre-clinical testing. View Publication