技术资料

Polo-like kinase 1 (PLK1),a key regulator of the G2/M phase in mitosis,is frequently overexpressed in numerous tumors. Although PLK1 inhibitors have emerged as promising therapeutic agents for cancer,their use has been linked to significant anemia in a subset of patients,yet the underlying mechanisms remain poorly understood. In this study,we utilized an in vitro human umbilical cord blood-derived CD34 + cell-based erythroid differentiation system,alongside a murine model,to investigate the impact of PLK1 inhibitors on erythropoiesis. Our results indicate that PLK1 inhibitors,specifically GSK461364 and BI6727,significantly suppress the proliferation of erythroid cells,resulting in G2/M phase cell cycle arrest,increased apoptosis in erythroid cells,and the formation of abnormally nucleated late-stage erythroblasts. In vivo,administration of PLK1 inhibitors in mice induced severe anemia,as evidenced by a marked reduction in red blood cells and hemoglobin levels. More specifically,PLK1 inhibition impaired the differentiation and erythroid commitment of hematopoietic stem cells in the bone marrow,resulting in abnormal accumulation of BFU-E cells and reduced proliferation and differentiation of CFU-E,and a decrease in the number of terminal erythrocytes. Mechanistically,PLK1 inhibitors primarily induce apoptosis in erythroid cells by reducing Mitochondrial membrane potential and arresting the cell cycle at the G2/M phase. Overall,our findings underscore the critical role of PLK1 in erythropoiesis and shed light on the mechanisms underlying PLK1 inhibitor-induced anemia,providing essential guidance for developing strategies to prevent and manage anemia in clinical applications of PLK1-targeted therapies. View Publication

Human norovirus (HuNoV) is a major cause of enteric infectious gastroenteritis and is classified into several genotypes based on its capsid protein amino acid sequence and nucleotide sequence of the polymerase gene. Among these,GII.4 is the major genotype worldwide. Epidemiological studies have highlighted the prevalence of GII.2. Although recent advances using human tissue– and induced pluripotent stem cell (iPSC)–derived intestinal epithelial cells (IECs) have enabled in vitro replication of multiple HuNoV genotypes,GII.2 HuNoV could replicate only in tissue-derived IECs and not in iPSC-derived IECs. We investigated the factors influencing GII.2 HuNoV replication in IECs,focusing on histo-blood group antigens. We also assessed the immunogenicity of GII.2 virus-like particles (VLPs) and their ability to induce neutralizing antibodies. Antibody cross-reactivity was tested to determine whether GII.2 VLPs could neutralize other HuNoV genotypes,including GII.4,GII.3,GII.6,and GII.17. Our findings indicated that GII.2 HuNoV replication in vitro requires the presence of both H and B antigens. Moreover,GII.2 VLPs generated neutralizing antibodies effective against both GII.2 and GII.4 but not against GII.3,GII.6,or GII.17. Comparatively,GII.2 and GII.17 VLPs induced broader neutralizing responses than GII.4 VLPs. The findings of this study suggests that GII.2 and GII.17 VLPs may be advantageous as HuNoV vaccine candidates because they elicit neutralizing antibodies against the predominant GII.4 genotype,which could be particularly beneficial for infants without prior HuNoV exposure. These insights will contribute to the development of effective HuNoV vaccines. View Publication

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death worldwide. Although immune checkpoint inhibitors (ICIs) have shown remarkable clinical efficacy,they can also induce a paradoxical cancer acceleration,known as hyperprogressive disease (HPD),whose causative mechanisms are still unclear. This study investigated the mechanisms of ICI resistance in an HPD-NSCLC model. Two primary cell cultures were established from samples of a NSCLC patient,before ICI initiation (“baseline”,NSCLC-B) and during HPD (“hyperprogression”,NSCLC-H). The cell lines were phenotypically and molecularly characterized through immunofluorescence,Western Blotting and RNA-Seq analysis. To assess cell plasticity and aggressiveness,cellular growth patterns were evaluated both in vitro and in vivo through 2D and 3D cell growth assays and patient-derived xenografts establishment. In vitro investigations,including the evaluation of cell sensitivity to interferon-gamma (IFN-γ) and cell response to PD-L1 modulation,were conducted to explore the influence of these factors on cell plasticity regulation. NSCLC-H exhibited increased expression of specific CD44 isoforms and a more aggressive phenotype,including organoid formation ability,compared to NSCLC-B. Plastic changes in NSCLC-H were well described by a deep transcriptome shift,that also affected IFN-γ–related genes,including PD-L1. IFN-γ–mediated cell growth inhibition was compromised in both 2D-cultured NSCLC-B and NSCLC-H cells. Further,the cytokine induced a partial activation of both type I and type II IFN-pathway mediators,together with a striking increase in NSCLC-B growth in 3D cell culture systems. Finally,low IFN-γ doses and PD-L1 modulation both promoted plastic changes in NSCLC-B,increasing CD44 expression and its ability to produce spheres. Our findings identified plasticity as a relevant hallmark of ICI-mediated HPD by demonstrating that ICIs can modulate the IFN-γ and PD-L1 pathways,driving tumor cell plasticity and fueling HPD development. The online version contains supplementary material available at 10.1186/s12967-024-06023-8. View Publication

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However,cellular and humoral immunogenicity of genome editing tools,which originate from bacteria,complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases,SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis,we identify putative immunogenic epitopes on each nuclease. Using computational modeling,we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components,including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses,yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9,but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection. Subject terms: Protein design,Immunogenetics,CRISPR-Cas9 genome editing View Publication

Our study highlights the role of epigenetic machinery in transformation of normal pluripotent stem cells to variant pluripotent state. We demonstrate the importance of non-histone protein methylation,which underlie the EMT and abnormal differentiation behaviour of variant hESCs. View Publication

Th2 inflammation and epithelial-mesenchymal transition (EMT) play crucial roles in the pathophysiology of chronic rhinosinusitis with nasal polyps (CRSwNP). This study aimed to investigate the hypothesis that MMP-12,produced by M2 macrophages,induces EMT in nasal epithelial cells,thereby contributing to airway inflammation and remodeling in CRSwNP. The expression levels of MMP-12 were measured by RT-PCR in CRS nasal mucosa and THP-1 cells. mRNA and protein levels of E-cadherin,vimentin,α-SMA,and fibronectin were determined using RT-PCR,western blotting,and immunofluorescence staining in primary nasal epithelial cells and air-liquid interface culture. The expression of MMP-12 was significantly increased in CRSwNP and M2-like THP-1 cells. In co-culture with primary nasal epithelial cells and M2-like THP-1 cells,E-cadherin expression was inhibited,and fibronectin,vimentin,and α-SMA expression were increased. MMP-12 decreased E-cadherin but induced fibronectin,vimentin,and α-SMA mRNA and protein expression in primary nasal epithelial cells and air-liquid interface culture. MMP408,an MMP-12 inhibitor,inhibited EMT-related factors. These findings suggest that MMP-12 expression in M2 macrophages induces EMT in nasal epithelial cells and may contribute to the pathogenesis of CRSwNP. View Publication

Micropapillary adenocarcinoma (MPC) is an aggressive histological subtype of lung adenocarcinoma (LUAD). MPC is composed of small clusters of cancer cells exhibiting inverted polarity. However,the mechanism underlying its formation is poorly understood. Here we show that hypoxia is involved in MPC formation. Hypoxia induced the formation of MPC-like structures (MLSs) in a three-dimensional culture system using A549 human LUAD cells,and HIF-1α was indispensable for MLS formation. RNA sequencing analysis demonstrated that A549 cells forming MLSs exhibited a gene expression signature similar to that of lung MPC. Moreover,MLS formation enhanced the resistance of A549 cells to natural killer cell cytotoxicity. Our findings suggest that hypoxia drives lung MPC formation through HIF-1α and that immune escape from natural killer cells might underlie the aggressiveness of MPC. View Publication

Although acute myeloid leukemia (AML) affects hematopoietic stem cell (HSC)-supportive microenvironment,it is largely unknown whether leukemia-modified bone marrow (BM) microenvironment can be remodeled to support normal hematopoiesis after complete remission (CR). As a key element of BM microenvironment,endothelial progenitor cells (EPCs) provide a feasible way to investigate BM microenvironment remodeling. Here,we find reduced and dysfunctional BM EPCs in AML patients,characterized by impaired angiogenesis and high ROS levels,could be partially remodeled after CR and improved by N-acetyl-L-cysteine (NAC). Importantly,HSC-supporting ability of BM EPCs is partially recovered,whereas leukemia-supporting ability is decreased in CR patients. Mechanistically,the transcriptome characteristics of leukemia-modified BM EPCs return to near-normal after CR. In a classic AML mouse and chemotherapy model,BM vasculature and normal hematopoiesis are reversed after CR. In summary,we provide further insights into how leukemia-modified BM microenvironment can be remodeled to support normal hematopoiesis after CR,which can be further improved by NAC. Subject terms: Translational research,Acute myeloid leukaemia View Publication

The hippocampus is associated with mood disorders,and the activation of quiescent neurogenesis has been linked to anxiolytic effects. Near-infrared (NIR) light has shown potential to improve learning and memory in human and animal models. Despite the vast amount of information regarding the effect of visible light,there is a significant gap in our understanding regarding the response of neural stem cells (NSCs) to NIR stimulation,particularly in anxiety-like behavior. The present study aimed to develop a new optical manipulation approach to stimulate hippocampal neurogenesis and understand the mechanisms underlying its anxiolytic effects. We used 940 nm NIR (40 Hz) light exposure to stimulate hippocampal stem cells in C57BL/6 mice. The enhanced proliferation and astrocyte differentiation of NIR-treated NSCs were assessed using 5-ethynyl-2’-deoxyuridine (EdU) incorporation and immunofluorescence assays. Additionally,we evaluated calcium activity of NIR light-treated astrocytes using GCaMP6f recording through fluorescence fiber photometry. The effects of NIR illumination of the hippocampus on anxiety-like behaviors were evaluated using elevated plus maze and open-field test. NIR light effectively promoted NSC proliferation and astrocyte differentiation via the OPN4 photoreceptor. Furthermore,NIR stimulation significantly enhanced neurogenesis and calcium-dependent astrocytic activity. Moreover,activating hippocampal astrocytes with 40-Hz NIR light substantially improved anxiety-like behaviors in mice. We found that flickering NIR (940 nm/40Hz) light illumination improved neurogenesis in the hippocampus with anxiolytic effects. This innovative approach holds promise as a novel preventive treatment for depression. The online version contains supplementary material available at 10.1186/s13287-024-04114-3. View Publication

Mild therapeutic hypothermia showed potential neuroprotective properties during and after cerebral hypoxia or ischemia in experimental animal studies. However,in clinical trials,where hypothermia is mainly applied after reperfusion,results were divergent and neurophysiological effects unclear. In our current study,we employed human-derived neuronal networks to investigate how treatment with hypothermia during hypoxia influences neuronal functionality and whether it improves post-hypoxic recovery. We differentiated neuronal networks from human induced pluripotent stem cells on micro-electrode arrays (MEAs). We studied the effect of hypothermia (34°C)–as well hyperthermia (39°C) ‐ on neuronal functionality during and after hypoxia using MEAs. We also studied the effects on the number of synaptic puncta and cell viability by immunocytochemistry. In comparison to neuronal networks under normothermia,we found that hypothermia during hypoxia improved functional neuronal network recovery,expressed as enhanced neuronal network activity. This was associated with prevention of synaptic loss during and after the hypoxic phase. Furthermore,hypothermia improved cell viability after the hypoxic phase. Instead,hyperthermia during hypoxia had detrimental effects,with an irreversible loss of neuronal network function,loss of synaptic puncta and decreased cell viability. Our results show potential neuroprotective properties of hypothermia occurring during hypoxia,indicating that administering hypothermia to bridge the time to reperfusion may be beneficial in clinical settings. View Publication