技术资料

H1N1 virus-induced excessive inflammatory response contributes to severe disease and high mortality rates. There is currently no effective strategy against virus infection in lung. The present study evaluated the protective roles of a natural compound,lapiferin,in H1N1 virus-induced pulmonary inflammation in mice and in cultured human bronchial epithelial cells. Initially,Balb/C mice were grouped as Control,H1N1 infection (intranasally infected with 500 plaque-forming units of H1N1 virus),lapiferin (10 mg/kg),and H1N1+lapiferin (n=10/group). Lung histology,expression of inflammatory factors,and survival rates were assessed after 14 days of exposure. Administration of lapiferin significantly alleviated the virus-induced inflammatory infiltrate in lung tissues. Major pro-inflammatory cytokines,such as interleukin (IL)-1$\beta$,IL-6,and tumor necrosis factor (TNF)-$\alpha$,were decreased at both mRNA and protein levels by lapiferin administration in the lung homogenate. Lapiferin also reduced inflammatory cell numbers in bronchoalveolar fluid. Mechanistically,lapiferin suppressed the transcriptional activity and protein expression of NF-$\kappa$B p65,causing inhibition on NF-$\kappa$B signaling. Pre-incubation of human bronchial epithelial cells with an NF-$\kappa$B signaling specific activator,ceruletide,significantly blunted lapiferin-mediated inhibition of pro-inflammatory cytokines secretion in an air-liquid-interface cell culture experiment. Activation of NF-$\kappa$B signaling also blunted lapiferin-ameliorated inflammatory infiltrate in lungs. These results suggested that lapiferin was a potent natural compound that served as a therapeutic agent for virus infection in the lung. View Publication

Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Here,we evaluated the ability to expand primary NHBE cells in different culture conditions while maintaining their 3D culture characteristics such as ciliated and goblet cells,and ion channel function. Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). Primary cells passaged up to four times maintained airway epithelial characteristics as evidenced by ciliated pseudostratified columnar epithelium with goblet cells,trans-epithelial electrical resistance (TEER) ({\textgreater}400 Ohms.cm2),and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents ({\textgreater}3 µA/cm2). No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. For the first time,this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. Furthermore,this study highlights the criticality of evaluating expansion and differentiation conditions for achieving optimal phenotypic and functional endpoints (CBF,ASL,ion channel function,presence of differentiated cells,TEER) when developing in vitro lung models. View Publication

Mutations in the cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDH1) occur in several types of cancer,and altered cellular metabolism associated with IDH1 mutations presents unique therapeutic opportunities. By altering IDH1,these mutations target a critical step in reductive glutamine metabolism,the metabolic pathway that converts glutamine ultimately to acetyl-CoA for biosynthetic processes. While IDH1-mutated cells are sensitive to therapies that target glutamine metabolism,the effect of IDH1 mutations on reductive glutamine metabolism remains poorly understood. To explore this issue,we investigated the effect of a knock-in,single-codon IDH1-R132H mutation on the metabolism of the HCT116 colorectal adenocarcinoma cell line. Here we report the R132H-isobolome by using targeted (13)C isotopomer tracer fate analysis to trace the metabolic fate of glucose and glutamine in this system. We show that introduction of the R132H mutation into IDH1 up-regulates the contribution of glutamine to lipogenesis in hypoxia,but not in normoxia. Treatment of cells with a d-2-hydroxyglutarate (d-2HG) ester recapitulated these changes,indicating that the alterations observed in the knocked-in cells were mediated by d-2HG produced by the IDH1 mutant. These studies provide a dynamic mechanistic basis for metabolic alterations observed in IDH1-mutated tumors and uncover potential therapeutic targets in IDH1-mutated cancers. View Publication

The upper airway epithelium,which is mainly composed of multiciliated,goblet,club and basal cells,ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases,defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference,we have unraveled trajectories from basal to luminal cells,providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells,which we have entitled deuterosomal cells,is defined by specific markers,such as DEUP1,FOXN4,YPEL1,HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells,thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process,such as keratins or components of the Notch,Wnt or BMP/TGF$\beta$ pathways,can be identified. Confirmation of our results on fresh human and pig airway samples,and on mouse tracheal cells,extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation. View Publication

Human regulatory T (Treg) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains Treg cell identity,yet the complete set of key transcription factors that control Treg cell gene expression remains unknown. Here,we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human Treg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from Treg cells subjected to genetic perturbations and cytokine stimulation,which revealed distinct gene networks individually regulated by FOXP3 and PRDM1,in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2,to our knowledge not previously implicated in Treg cell function,coregulates another gene network with SATB1 and is important for Treg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling,we have uncovered transcriptional regulators and downstream gene networks in human Treg cells that could be targeted for immunotherapies. View Publication

The airway epithelium contains ionocytes,a rare cell type with high expression of Forkhead Box I1 (FOXI1) transcription factor and Cystic Fibrosis Transmembrane conductance Regulator (CFTR),a chloride channel that is defective in cystic fibrosis (CF). Our aim was to verify if ionocyte development is altered in CF and to investigate the relationship between ionocytes and CFTR-dependent chloride secretion. We collected nasal cells by brushing to determine ionocyte abundance. Nasal and bronchial cells were also expanded in vitro and reprogrammed to differentiated epithelia for morphological and functional studies. We found a relatively high ({\~{}}3{\%}) ionocyte abundance in ex vivo nasal samples,with no difference between CF and control individuals. In bronchi,ionocytes instead appeared very rarely as previously reported,thus suggesting a possible proximal-distal gradient in human airways. The difference between nasal and bronchial epithelial cells was maintained in culture,which suggests an epigenetic control of ionocyte development. In the differentiation phase of the culture procedure,we used two media that resulted in a different pattern of CFTR expression: confined to ionocytes or more broadly expressed. CFTR function was similar in both conditions,thus indicating that chloride secretion equally occurs irrespective of CFTR expression pattern. View Publication

PURPOSE PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3,a critical player for efficient mitotic progression,is required for the stabilization of the mitotic spindle by regulation of the mitotic components,NuMA and Tankyrase 1. METHODS The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V,a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. RESULTS PARP3 inhibitors sensitize breast cancer cells to vinorelbine,a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold,respectively,potentiating vinorelbine-induced arrest at the G2/M boundary. In addition,PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore,olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition,mitotic arrest,and apoptosis. CONCLUSION Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases. View Publication

Thyromimetics that specifically target TR$\beta$ have been shown to reduce plasma cholesterol levels and avoid atherosclerosis through the promotion of reverse cholesterol transport in an animal model. We designed novel thyromimetics with high receptor (TR$\beta$) and organ (liver) selectivity based on the structure of eprotirome (3) and molecular modeling. We found that indane derivatives are potent and dual-selective thyromimetics expected to avoid hypothyroidism in some tissues as well as heart toxicity. KTA-439 (29),a representative indane derivative,showed the same high human TR$\beta$ selectivity in a binding assay as 3 and higher liver selectivity than 3 in a cholesterol-fed rat model. View Publication

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however,the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely,shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1,verteporfin,significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties,suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population. View Publication

Pulmonary sarcoidosis has unknown etiology,a difficult diagnostic procedure and no curative treatment. Extracellular vesicles including exosomes are nano-sized entities released from all cell types. Previous studies of exosomes from bronchoalveolar lavage fluid (BALF) of sarcoidosis patients have revealed pro-inflammatory components and abilities,but cell sources and mechanisms have not been identified. In the current study,we found that BALF exosomes from sarcoidosis patients,but not from healthy individuals,induced a dose-dependent elevation of intracellular IL-1$\beta$ in monocytes. Analyses of supernatants showed that patient exosomes also induced release of IL-1$\beta$,IL-6 and TNF from both PBMCs and enriched monocytes,suggesting that the observed effect is direct on monocytes. The potently chemotactic chemokine CCL2 was induced by exosomes from a subgroup of patients,and in a blocking assay the exosome-induced CCL2 was reduced for 13 out of 19 patients by the asthma drug Montelukast,a cysteinyl leukotriene receptor antagonist. Further,reactive oxygen species generation by PBMCs was induced to a higher degree by patient exosomes compared to healthy exosomes. These findings add to an emerging picture of exosomes as mediators and disseminators of inflammation,and open for further investigations of the link between CCL2 and exosomal leukotrienes in sarcoidosis. View Publication

Detection and characterization of rare circulating tumor cells (CTCs) in patients' blood is important for the diagnosis and monitoring of cancer. The traditional way of counting CTCs via fluorescent images requires a series of tedious experimental procedures and often impacts the viability of cells. Here we present a method for label-free detection of CTCs from patient blood samples,by taking advantage of data analysis of bright field microscopy images. The approach uses the convolutional neural network,a powerful image classification and machine learning algorithm to perform label-free classification of cells detected in microscopic images of patient blood samples containing white blood cells and CTCs. It requires minimal data pre-processing and has an easy experimental setup. Through our experiments,we show that our method can achieve high accuracy on the identification of rare CTCs without the need for advanced devices or expert users,thus providing a faster and simpler way for counting and identifying CTCs. With more data becoming available in the future,the machine learning model can be further improved and can serve as an accurate and easy-to-use tool for CTC analysis. View Publication

Membrane potential (Vmem) is a key bioelectric property of non-excitable cells that plays important roles in regulating cell proliferation. However,the regulation of Vmem itself remains largely unexplored. We found that,under nutrient starvation,during which cell division is inhibited,MKN45 gastric cancer cells were in a hyperpolarized state associated with a high intracellular chloride concentration. AMP-activated protein kinase (AMPK) activity increased,and expression of cystic fibrosis transmembrane conductance regulator (CFTR) decreased,in nutrient-starved cells. Furthermore,the increase in intracellular chloride concentration level and Vmem hyperpolarization in nutrient-starved cells was suppressed by inhibition of AMPK activity. Intracellular chloride concentrations and hyperpolarization increased after over-activation of AMPK using the specific activator AICAR or suppression of CFTR activity using specific inhibitor GlyH-101. Under these conditions,proliferation of MKN45 cells was inhibited. These results reveal that AMPK controls the dynamic change in Vmem by regulating CFTR and influencing the intracellular chloride concentration,which in turn influences cell-cycle progression. These findings offer new insights into the mechanisms underlying cell-cycle arrest regulated by AMPK and CFTR. View Publication