参考文献

We examined the potential role of myosin and actin in the release of human immunodeficiency virus type 1 (HIV-1) from infected cells. Wortmannin (100 nM to 5 microM),an effective inhibitor of myosin light chain kinase,blocked the release of HIV-1 from infected T-lymphoblastoid and monocytoid cells in a concentration-dependent manner. Cytochalasin D,a reagent that disrupts the equilibrium between monomeric and polymeric actin,also partially inhibited the release of HIV-1 from the infected cells. At the budding stage,myosin and HIV-1 protein were detected in the same areas on the plasma membrane by using dual-label immunofluorescence microscopy and immunoelectron microscopy. In the presence of 5 microM wortmannin,viral components were observed on the plasma membrane by using immunofluorescence microscopy and electron microscopy,implying that wortmannin did not disturb the transport of viral proteins to the plasma membrane but rather inhibited budding. View Publication

UNLABELLED The effects of oleuropein on the processes of osteoblastogenesis and adipogenesis in mesenchymal stem cells (MSCs) from human bone marrow have been studied. We report that oleuropein,a polyphenol abundant in olive tree products,reduces the expression of peroxisome proliferator-activated receptor gamma (PPAR$\gamma$),inhibits adipocyte differentiation,and enhances differentiation into osteoblast. INTRODUCTION Age-related bone loss is associated with osteoblast insufficiency during continuous bone remodeling. It has been suggested that the formation of osteoblasts in bone marrow is closely associated with adipogenesis,and age-related changes in this relationship could be responsible for the progressive adiposity of bone marrow which occurs with osteoporosis. In addition,the consumption of oleuropein,a major polyphenol in olive leaves and olive oil,has been associated with a reduction in bone loss. METHODS We have analyzed the effects of oleuropein-at concentrations between 10(-6) and 10(-4) M-on the processes of osteoblastogenesis and adipogenesis in MSCs from human bone marrow. RESULTS The results show an increase in osteoblast differentiation and a decrease in adipocyte differentiation when there is oleuropein in the culture media. The gene expression of osteoblastogenesis markers,RUNXII,osterix,collagen type I,osteocalcin,or alkaline phosphatase (ALP),was higher in osteoblast-induced oleuropein-treated cells. Also,the ALP activity and extracellular matrix mineralization were higher when oleuropein was present in the media. Oleuropein in MSCs induced adipocytes to produce a decrease in the expression of the genes involved in adipogenesis,the PPAR$\gamma$,lipoprotein lipase,or fatty acid-binding protein 4,and minor fat accumulation. CONCLUSION Our data suggest that oleuropein,highly abundant in olive tree products included in the traditional Mediterranean diet,could prevent age-related bone loss and osteoporosis. View Publication

Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs alpha and beta) that bind triiodothyronine (T(3),3,5,3'-triiodo-l-thyronine) with high affinity,and its precursor thyroxine (T(4),3,5,3',5'-tetraiodo-l-thyronine) with lower affinity. T(4) contains a bulky 5' iodine group absent from T(3). Because T(3) is buried in the core of the ligand binding domain (LBD),we have predicted that TH analogues with 5' substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T(4) affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics,migration on hydrophobic interaction columns,and non-denaturing gels) that TR-T(4) complexes adopt a conformation that differs from TR-T(3) complexes in solution. Nonetheless,T(4) behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells,when conversion to T(3) does not contribute to agonist activity. We determined x-ray crystal structures of the TRbeta LBD in complex with T(3) and T(4) at 2.5-A and 3.1-A resolution. Comparison of the structures reveals that TRbeta accommodates T(4) through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket,which,together,enlarge a niche that permits helix 12 to pack over the 5' iodine and complete the coactivator binding surface. While T(3) is the major active TH,our results suggest that T(4) could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5' extension should be considered in TR ligand design. View Publication

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2,an emerging virus that utilizes host proteins ACE2 and TMPRSS2 as entry factors. Understanding the factors affecting the pattern and levels of expression of these genes is important for deeper understanding of SARS-CoV-2 tropism and pathogenesis. Here we explore the role of genetics and co-expression networks in regulating these genes in the airway,through the analysis of nasal airway transcriptome data from 695 children. We identify expression quantitative trait loci for both ACE2 and TMPRSS2,that vary in frequency across world populations. We find TMPRSS2 is part of a mucus secretory network,highly upregulated by type 2 (T2) inflammation through the action of interleukin-13,and that the interferon response to respiratory viruses highly upregulates ACE2 expression. IL-13 and virus infection mediated effects on ACE2 expression were also observed at the protein level in the airway epithelium. Finally,we define airway responses to common coronavirus infections in children,finding that these infections generate host responses similar to other viral species,including upregulation of IL6 and ACE2. Our results reveal possible mechanisms influencing SARS-CoV-2 infectivity and COVID-19 clinical outcomes. View Publication

In vitro cultures of primary human airway epithelial cells (hAECs) grown at air-liquid interface have become a valuable tool to study airway biology under normal and pathologic conditions,and for drug discovery in lung diseases such as cystic fibrosis (CF). An increasing number of different differentiation media,are now available,making comparison of data between studies difficult. Here,we investigated the impact of two common differentiation media on phenotypic,transcriptomic,and physiological features of CF and non-CF epithelia. Cellular architecture and density were strongly impacted by the choice of medium. RNA-sequencing revealed a shift in airway cell lineage; one medium promoting differentiation into club and goblet cells whilst the other enriched the growth of ionocytes and multiciliated cells. Pathway analysis identified differential expression of genes involved in ion and fluid transport. Physiological assays (intracellular/extracellular pH,Ussing chamber) specifically showed that ATP12A and CFTR function were altered,impacting pH and transepithelial ion transport in CF hAECs. Importantly,the two media differentially affected functional responses to CFTR modulators. We argue that the effect of growth conditions should be appropriately determined depending on the scientific question and that our study can act as a guide for choosing the optimal growth medium for specific applications. View Publication

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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

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by defects in the NADPH oxidase complex. Mutations in NCF2 encoding the cytosolic factor p67phox result in autosomal recessive CGD. We describe three patients with a novel c.855G{\textgreater}C NCF2 mutation presenting with diverse clinical phenotype. Two siblings were heterozygous for the novel mutation and for a previously described exon 8-9 duplication,while a third unrelated patient was homozygous for the novel mutation. Mutation pathogenicity was confirmed by abnormal DHR123 assay and absent p67phox production and by sequencing of cDNA which showed abnormal RNA splicing. Clinically,the homozygous patient presented with suspected early onset interstitial lung disease and NCF2 mutation was found on genetic testing performed in search for surfactant-related defects. The two siblings also had variable presentation with one having history of severe pneumonia,lymphadenitis,and recurrent skin abscesses and the other presenting in his 30s with discoid lupus erythematosus and without significant infectious history. We therefore identified a novel pathogenic NCF2 mutation causing diverse and unusual clinical phenotype. View Publication

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development,cell growth,and migration,as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers,and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here,we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands,[(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)-phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist),was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1),an antagonist,did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay,SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists SANT-1 and SANT-2 bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway." 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