技术资料

Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling,infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites,including current University Hospital Southampton COVID-19 risk mitigation measures,and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9{\%}) were ciliated. Fifty-four of 83 (63.9{\%}) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated,scanning electron microscopy demonstrated excellent ciliation,and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary,our ALI culture protocol provides high ciliation rates across three centres,minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful,facilitating PCD research. View Publication

The mechanism of Fas antigen-mediated apoptosis is at present unclear. We show here that the 100,000 x g supernatant from cell lysates prepared from anti-Fas-stimulated JUR-KAT T cells,induces chromatin fragmentation in isolated nuclei with concomitant morphological changes typically seen in apoptosis. The formation of this apoptotic nuclei promoting activity (ANPA) in JURKAT T cells after Fas antigen ligation was blocked by the serine protease inhibitors,TPCK and DCI,and by the interleukin 1-beta-converting enzyme inhibitor,VAD-FMK. In addition,chromatin degradation and morphological changes mediated by the ANPA in isolated nuclei were inhibited by TPCK,but not by DCI or VAD-FMK. These results suggest that Fas-mediated apoptosis in T cells involves the activation of a cascade of proteases. View Publication

Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However,CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR,suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein,we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production,obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures,the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium. View Publication

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions,wherein the regulation of Wnt/$\beta$-catenin pathway is often desirable. Here,we examined the effect of trolox,a vitamin E analog,on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/$\beta$-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/$\beta$-catenin regulator. View Publication

S-glutathionylation of reactive protein cysteines is a post-translational event that plays a critical role in transducing signals from oxidants into biological responses. S-glutathionylation can be reversed by the deglutathionylating enzyme glutaredoxin (GLRX). We have previously demonstrated that ablation of Glrx sensitizes mice to the development of parenchymal lung fibrosis(1). It remains unclear whether GLRX also controls airway fibrosis,a clinical feature relevant to asthma and chronic obstructive pulmonary disease,and whether GLRX controls the biology of airway epithelial cells,which have been implicated in the pathophysiology of these diseases. In the present study we utilized a house dust mite (HDM) model of allergic airway disease in wild type (WT) and Glrx-/- mice on a C57BL/6 background prone to develop airway fibrosis,and tracheal basal stem cells derived from WT mice,global Glrx-/- mice,or bi-transgenic mice allowing conditional ablation of the Glrx gene. Herein we show that absence of Glrx led to enhanced HDM-induced collagen deposition,elevated levels of transforming growth factor beta 1 (TGFB1) in the bronchoalveolar lavage,and resulted in increases in airway hyperresponsiveness. Airway epithelial cells isolated from Glrx-/- mice or following conditional ablation of Glrx showed spontaneous increases in secretion of TGFB1. Glrx-/- basal cells also showed spontaneous TGFB pathway activation,in association with increased expression of mesenchymal genes,including collagen 1a1 and fibronectin. Overall,these findings suggest that GLRX regulates airway fibrosis via a mechanism(s) that involve the plasticity of basal cells,the stem cells of the airways. View Publication

Murine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However,little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here,we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly,their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact-dependent manner through inhibition of cytoplasmic $\beta$-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30{\%} lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore,patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7{\%} whereas patients with higher osteocalcin levels reached a survival rate of 66.8{\%}. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients. View Publication

BACKGROUND Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract,and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1,the roles of other mucins are still poorly understood,especially in viral infections. METHODS To further identify mucins significant in influenza infection,we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus. RESULTS We found that the expression of MUC15 was significantly upregulated upon infection,and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly,positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines,chemokines,EGFR and phosphorylated ERK) started to peak and plateau,MUC15 may potentially serve an immunomodulatory function later during influenza viral infection. CONCLUSIONS Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus,we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection. View Publication

R428 (BGB324) is an anti-cancer drug candidate under clinical investigation. It inhibits the receptor tyrosine kinase Axl and induces apoptosis of many types of cancer cells,but the relationship between the two has not been well established. We investigated the molecular mechanisms of the R428-induced apoptosis and found that R428 induced extensive cytoplasmic vacuolization and caspase activation,independent of its inhibitory effects on Axl. Further analyses revealed that R428 blocked lysosomal acidification and recycling,accumulated autophagosomes and lysosomes,and induced cell apoptosis. Inhibition of autophagy by autophagy inhibitors or autophagic gene-knockout alleviated the R428-induced vacuoles formation and cell apoptosis. Our study uncovered a novel function and mechanism of R428 in addition to its ability to inhibit Axl. These data will help to better direct the application of R428 as an anti-cancer reagent. It also adds new knowledge to understand the regulation of autophagy and apoptosis. View Publication

Recent studies suggest that forced activation of AMP-activated protein kinase (AMPK) could inhibit melanoma cell proliferation. In this report,we evaluated the anti-melanoma cell activity by a novel small-molecular AMPK activator,GSK621. Treatment of GSK621 decreased survival and proliferation of human melanoma cells (A375,WM-115 and SK-Mel-2 lines),which was accompanied by activation of caspase-3/-9 and apoptosis. Reversely,caspase inhibitors attenuated GSK621-induced cytotoxicity against melanoma cells. Significantly,GSK621 was more potent than other AMPK activators (A769662,Compound 13 and AICAR) in inhibiting melanoma cells. Intriguingly,same GSK621 treatment was non-cytotoxic or pro-apoptotic against human melanocytes. Molecularly,we showed that activation of AMPK mediated GSK621's activity against melanoma cells. AMPK$\alpha$1 shRNA knockdown or dominant negative mutation (T172A) dramatically attenuated GSK621-induced melanoma cell lethality. Further studies revealed that MEK-ERK activation might be the primary resistance factor of GSK621. MEK-ERK inhibition,either genetically or pharmacologically,significantly sensitized melanoma cells to GSK-621. Remarkably,intraperitoneal (i.p.) injection of GSK621 inhibited A375 tumor growth in SCID mice. Co-administration of MEK-ERK inhibitor MEK162 further sensitized GSK621-induced anti-A375 tumor activity in vivo. Together,the results imply that targeted activation of AMPK by GSK621 inhibits melanoma cell survival and proliferation. MEK-ERK inhibition may further sensitize GSK621's anti-melanoma cell activity in vitro and in vivo. View Publication

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes,but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study,we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs,enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro,as well as heterotopic bone formation in vivo. Similarly,treating hMSC with Phalloidin,which is known to stabilize polymerized actin filaments,increased hMSCs viability and OB differentiation. Conversely,Cytocholasin D,an inhibitor of actin polymerization,reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level,preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover,depolymerizing actin reduced FAK,p38 and JNK activation during OB differentiation of hMSCs,while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation. View Publication

Adenomyosis is also called internal endometriosis and affects about 20{\%} of reproductive-aged women. It seriously reduces life quality of patients because current drug therapies face with numerous challenges. Long-term clinical application of mifepristone exhibits wonderful therapeutic effects with mild side-effects in many disorders since 1982. Since adenomyosis is a refractory disease,we investigate whether mifepristone can be applied in the treatment of adenomyosis. In this study,we investigated the direct effects of mifepristone on human primary eutopic endometrial epithelial cells and stromal cells in adenomyosis. We found that mifepristone causes cell cycle arrest through inhibiting CDK1 and CDK2 expressions and induces cell apoptosis via the mitochondria-dependent signalling pathway in endometrial epithelial cells and stromal cells of adenomyosis. Furthermore,mifepristone inhibits the migration of endometrial epithelial cells and stromal cells through decreasing CXCR4 expression and restricts the invasion of endometrial epithelial cells via suppression of epithelial-mesenchymal transition in adenomyosis. We also found that mifepristone treatment decreases the uterine volume,CA125 concentration and increases the haemoglobin concentration in serum for adenomyosis patients. Therefore,we demonstrate that mifepristone could serve as a novel therapeutic drug in the treatment of adenomyosis,and therefore,the old dog can do a new trick. View Publication

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells,making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis,Nampt,in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells,but not normal donor as well as MM patients PBMCs. Importantly,FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6,IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly,cytotoxicity of FK866 triggered autophagy,but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally,FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model,associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology,providing the basis for a novel targeted therapeutic approach. View Publication