技术资料

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor,CDC20,preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However,the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression,we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for,gastrointestinal atrophy is detrimental. Remarkably,deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome,while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect,identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast,only overexpression of anti-apoptotic BCL2,but none of the BH3-only protein deficiencies mentioned above,can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo. View Publication

HIV-1 relies on the host-cell machinery to accomplish its replication cycle,and characterization of these helper factors contributes to a better understanding of HIV-host interactions and can identify potential novel antiviral targets. Here we explored the contribution of CIB2,previously identified by RNAi screening as a potential helper factor,and its homolog,CIB1. Knockdown of either CIB1 or CIB2 strongly impaired viral replication in Jurkat cells and in primary CD4+ T-lymphocytes,identifying these proteins as non-redundant helper factors. Knockdown of CIB1 and CIB2 impaired envelope-mediated viral entry for both for X4- and R5-tropic HIV-1,and both cell-free and cell-associated entry pathways were affected. In contrast,the level of CIB1 and CIB2 expression did not influence cell viability,cell proliferation,receptor-independent viral binding to the cell surface,or later steps in the viral replication cycle. CIB1 and CIB2 knockdown was found to reduce the expression of surface molecules implicated in HIV-1 infection,including CXCR4,CCR5 and integrin α4β7,suggesting at least one mechanism through which these proteins promote viral infection. Thus,this study identifies CIB1 and CIB2 as host helper factors for HIV-1 replication that are required for optimal receptor-mediated viral entry. View Publication

The airway epithelium constitutes an essential immunological and cytoprotective barrier to inhaled insults,such as cigarette smoke,environmental particles,or viruses. Although bronchial epithelial integrity is crucial for airway homeostasis,defective epithelial barrier function contributes to chronic obstructive pulmonary disease (COPD). Tight junctions at the apical side of epithelial cell-cell contacts determine epithelial permeability. Cigarette smoke exposure,the major risk factor for COPD,is suggested to impair tight junction integrity; however,detailed mechanisms thereof remain elusive. We investigated whether cigarette smoke extract (CSE) and transforming growth factor (TGF)-$$1 affected tight junction integrity. Exposure of human bronchial epithelial cells (16HBE14o(-)) and differentiated primary human bronchial epithelial cells (pHBECs) to CSE significantly disrupted tight junction integrity and barrier function. Specifically,CSE decreased transepithelial electrical resistance (TEER) and tight junction-associated protein levels. Zonula occludens (ZO)-1 and ZO-2 protein levels were significantly reduced and dislocated from the cell membrane,as observed by fractionation and immunofluorescence analysis. These findings were reproduced in isolated bronchi exposed to CSE ex vivo,as detected by real-time quantitative reverse-transcriptase PCR and immunohistochemistry. Combined treatment of 16HBE14o(-) cells or pHBECs with CSE and TGF-$$1 restored ZO-1 and ZO-2 levels. TGF-$$1 cotreatment restored membrane localization of ZO-1 and ZO-2 protein and prevented CSE-mediated TEER decrease. In conclusion,CSE led to the disruption of tight junctions of human bronchial epithelial cells,and TGF-$$1 counteracted this CSE-induced effect. Thus,TGF-$$1 may serve as a protective factor for bronchial epithelial cell homeostasis in diseases such as COPD. View Publication

ABSTRACTAutism spectrum disorder (ASD) and congenital heart disease (CHD) frequently co-occur,yet the underlying molecular mechanisms of this comorbidity remain unknown. Given that children with CHD are identified as newborns,understanding which CHD variants are associated with autism could help select individuals for early intervention. Autism gene perturbations commonly dysregulate neural progenitor cell (NPC) biology,so we hypothesized that CHD genes disrupting neurogenesis are more likely to increase ASD risk. Therefore,we performed an in vitro pooled CRISPR interference screen to identify CHD genes disrupting NPC biology and identified 45 CHD genes. A cluster of ASD and CHD genes are enriched for ciliary biology,and perturbing any one of seven such genes (CEP290,CHD4,KMT2E,NSD1,OFD1,RFX3 and TAOK1) impairs primary cilia formation in vitro. In vivo investigation of TAOK1 in Xenopus tropicalis reveals a role in motile cilia formation and heart development,supporting its prediction as a CHD gene. Together,our findings highlight a set of CHD genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology. Highlighted Article: The increased likelihood of autism in individuals with congenital heart disease may stem from shared genetic mechanisms centered on cilia biology. View Publication

A rapid and efficient method to stimulate bone regeneration would be useful in orthopaedic stem cell therapies. Rolipram is an inhibitor of phosphodiesterase 4 (PDE4),which mediates cyclic adenosine monophosphate (cAMP) degradation. Systemic injection of rolipram enhances osteogenesis induced by bone morphogenetic protein 2 (BMP-2) in mice. However,there is little data on the precise mechanism,by which the PDE4 inhibitor regulates osteoblast gene expression. In this study,we investigated the combined ability of BMP-2 and cilomilast,a second-generation PDE4 inhibitor,to enhance the osteoblastic differentiation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity of MSCs treated with PDE4 inhibitor (cilomilast or rolipram),BMP-2,and/or H89 was compared with the ALP activity of MSCs differentiated only by osteogenic medium (OM). Moreover,expression of Runx2,osterix,and osteocalcin was quantified using real-time polymerase chain reaction (RT-PCR). It was found that cilomilast enhances the osteoblastic differentiation of MSCs equally well as rolipram in primary cultured MSCs. Moreover,according to the H89 inhibition experiments,Smad pathway was found to be an important signal transduction pathway in mediating the osteogenic effect of BMP-2,and this effect is intensified by an increase in cAMP levels induced by PDE4 inhibitor. View Publication

BACKGROUND Circular RNA of vimentin (circ-VIM) is a predictor for poor prognosis of acute myeloid leukemia,but we had little information on its function in esophageal cancer (EC). Here we examined the effects of circ-VIM together with sevoflurane on immune escape and multiple oncogenic activities of EC. METHODS Bioinformatic tools,luciferase assay,and RNA immunoprecipitation were used to examine regulations between circ-VIM,miR-124-3p (miR-124),and PD-L1. CCK-8,wound healing,and Transwell assays were used to measure cell proliferation,migration,and invasion,respectively. The impacts of EC cells on cytotoxicity,proliferation,and apoptosis of CD8+ T cells were examined using LDH assay,CFSE staining,and Annexin V/PI staining,respectively. The in vivo tumorigenesis and lung metastases were assessed using xenograft model and tail vein injection of EC cells. RESULTS Significant upregulation of circ-VIM and PD-L1 and downregulation of miR-124 were detected in EC tissues or cells. Circ-VIM sponged miR-124 and released its suppression on the downstream target PD-L1. Sevoflurane,independent of circ-VIM,also upregulated miR-124 to lower PD-L1 expression. By modulating miR-124/PD-L1 axis,silencing circ-VIM and applying sevoflurane both inhibited immune escape and multiple oncogenic activities of EC in vitro,and suppressed xenograft growth and lung metastases in vivo. The inactivation of Ras/ERK signaling pathway was involved in suppression of malignant phenotypes by silencing circ-VIM and sevoflurane treatment. CONCLUSIONS Silencing circ-VIM and applying sevoflurane,by separately regulating miR-124/PD-L1 axis,presented synergistic effects in inhibiting immune escape and multiple malignant phenotypes of EC cells. View Publication

BackgroundAcute respiratory distress syndrome (ARDS) is a life-threatening condition associated with the inflammatory activation of alveolar macrophages. Here,we examined the role of circVAPA in regulating inflammasome activation and macrophage inflammatory polarization in an ARDS model.MethodscircVAPA expression levels were analyzed in macrophages isolated from healthy controls and patients with ARDS. In vitro cell models of mouse alveolar macrophages and an in vivo mouse ARDS model were established through Lipopolysaccharide (LPS) stimulation. The effects of circVAPA knockdown on macrophage inflammatory polarization,inflammasome activation,and pulmonary tissue damage were investigated in both cell and animal models. The interaction between circVAPA and downstream factors was verified through a luciferase reporter assay and by silencing circVAPA.ResultscircVAPA upregulation in alveolar macrophages was associated with the inflammation in ARDS patients. circVAPA was also upregulated in LPS-stimulated mouse alveolar macrophages (MH-S cells). Additionally,circVAPA knockdown attenuated the inflammatory activation of MH-S cells and reduced the expression of pyroptosis-related proteins. circVAPA silencing also mitigated the inflammatory effects of LPS-stimulated MH-S cells on lung epithelial cells (MLE-12),and alleviated the inflammatory damage in the pulmonary tissue of ARDS mouse model. We further showed that miR-212-3p/Sirt1 axis mediated the functional role of circVAPA in the inflammatory polarization of MH-S cells.ConclusionOur data suggest that circVAPA promotes inflammasome activity and macrophage inflammation by modulating miR-212-3p/Sirt1 axis in ARDS. Targeting circVAPA may be employed to suppress the inflammatory activation of alveolar macrophages in ARDS.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10735-024-10312-3. View Publication

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here,we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to an atlas of cell-type-specific DNA methylation patterns derived from 476 methylomes of purified cells. For liver cell types,DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and functionally important regulatory regions. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However,sustained elevation of hepatocyte and biliary epithelial cfDNA within the first month indicates early-onset allograft injury. Further,cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and intervention. Current approaches to detect allograft damages non-invasively are limited and do not differentiate between cellular mechanisms. Here,the authors show that the composition of cell-free DNA in blood samples can reveal cellular causes of allograft injury after liver transplant. View Publication

Although Hodgkin and Reed-Sternberg (HRS) cells are B lymphoid cells,they are unlike any normal cells of that lineage. Moreover,the limited proliferative potential of HRS cells belies the clinical aggressiveness of Hodgkin lymphoma (HL). More than 20 years ago,the L428 HL cell line was reported to contain a small population of phenotypic B cells that appeared responsible for the continued generation of HRS cells. This observation,however,has never been corroborated,and such clonotypic B cells have never been documented in HL patients. We found that both the L428 and KM-H2 HL cell lines contained rare B-cell subpopulations responsible for the generation and maintenance of the predominant HRS cell population. The B cells within the HL cell lines expressed immunoglobulin light chain,the memory B-cell antigen CD27,and the stem cell marker aldehyde dehydrogenase (ALDH). Clonal CD27(+)ALDH(high) B cells,sharing immunoglobulin gene rearrangements with lymph node HRS cells,were also detected in the blood of most newly diagnosed HL patients regardless of stage. Although the clinical significance of circulating clonotypic B cells in HL remains unclear,these data suggest they may be the initiating cells for HL. View Publication

Chronic graft-versus-host disease (cGVHD) is a common late complication of allogeneic stem cell transplantation (allo-SCT). Some cGVHD patients develop skin lesions,and the skin lesions in sclerodermatous cGVHD (s-cGVHD) patients resemble those in progressive systemic sclerosis (PSS),which is characterized by impaired production of circulating endothelial progenitor cells (EPCs). We investigated,retrospectively,whether low EPC production may promote the development of sclerodermatous lesions in cGVHD. Peripheral blood (PB) was obtained from 14 healthy volunteers and 27 allo-SCT patients. Five patients developed s-cGVHD. CD34(+) cells were purified by using the magnetic cell-sorting separation system,and the CD34(+)/CD133(+)/vascular endothelial growth factor (VEGF) receptor-2(+) EPCs were quantified. The endothelial cell colony-formation potential was evaluated. Serum VEGF and basic fibroblast growth factor (b-FGF) concentrations were measured by ELISA. The s-cGVHD patients had significantly lower median circulating EPCs frequencies than non-s-cGVHD patients or control (145 of 20 mL [interquartial range-IQR 107-193] versus 1083.5 [IQR 669.3-2151]; P = .0023,and versus 1530.5 [IQR 961.3-2158]; P = .0012,respectively). They also had impaired median endothelial-forming ability compared to non-s-cGVHD patients or controls (3.8 [IQR 1.0-4.3] versus 12.8 [IQR 8.8-28.8],and versus 26.4 [IQR 23.6-30.6],respectively; P = .0012). Their VEGF and b-FGF serum levels were also higher than in controls. In conclusion,s-cGVHD patients show findings consistent with those seen in PSS with impaired vasculogenesis that may limit blood perfusion and may contribute to the development of sclerodermatous lesions. View Publication

Circulating CD34+ cells are haemopoietic progenitors that may play a role in tissue repair. No data are available on circulating progenitors in chronic obstructive pulmonary disease (COPD). Circulating CD34+ cells were studied in 18 patients with moderate-to-severe COPD (age: mean+/-sd 68+/-8 yrs; forced expiratory volume in one second: 48+/-12% predicted) and 12 controls,at rest and after endurance exercise. Plasma concentrations of haematopoietic growth factors (FMS-like tyrosine kinase 3 (Flt3) ligand,kit ligand),markers of hypoxia (vascular endothelial growth factor (VEGF)) and stimulators of angiogenesis (VEGF,hepatocyte growth factor (HGF)) and markers of systemic inflammation (tumour necrosis factor (TNF)-alpha,interleukin (IL)-6,IL-8) were measured. Compared with the controls,the COPD patients showed a three-fold reduction in CD34+ cell counts (3.3+/-2.5 versus 10.3+/-4.2 cells.microL-1),and a 50% decrease in AC133+ cells. In the COPD patients,progenitor-derived haemopoietic and endothelial cell colonies were reduced by 30-50%. However,four COPD patients showed progenitor counts in the normal range associated with lower TNF-alpha levels. In the entire sample,CD34+ cell counts correlated with exercise capacity and severity of airflow obstruction. After endurance exercise,progenitor counts were unchanged,while plasma Flt3 ligand and VEGF only increased in the COPD patients. Plasma HGF levels were higher in the COPD patients compared with the controls and correlated inversely with the number of progenitor-derived colonies. In conclusion,circulating CD34+ cells and endothelial progenitors were decreased in chronic obstructive pulmonary disease patients and could be correlated with disease severity. View Publication

BACKGROUND: Although current evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood,we hypothesized that such cells circulate but that their concentration has been vastly underestimated owing to the use of assays that required adherence to plastic. We further reasoned that the concentration of these cells is elevated during times of increased bone formation,such as during pubertal growth. METHODS: We used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [+/-SD] age,14.5+/-0.7 vs. 37.7+/-7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. RESULTS: Cells positive for osteocalcin and cells positive for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1 to 2 percent of mononuclear cells). There were more than five times as many cells positive for osteocalcin in the circulation of adolescent boys (whose markers of bone formation were clearly increased as a result of pubertal growth) as compared with adult subjects (Ptextless0.001). The percentage of cells positive for osteocalcin correlated with markers of bone formation. Sorted osteocalcin-positive cells expressed osteoblastic genes,formed mineralized nodules in vitro,and formed bone in an in vivo transplantation assay. Increased values were also found in three adults with recent fractures. CONCLUSIONS: Osteoblast-lineage cells circulate in physiologically significant numbers,correlate with markers of bone formation,and are markedly higher during pubertal growth; therefore,they may represent a previously unrecognized circulatory component to the process of bone formation. View Publication