技术资料

Evasion of programmed cell death represents a critical form of oncogene addiction in cancer cells. Understanding the molecular mechanisms underpinning cancer cell survival despite the oncogenic stress could provide a molecular basis for potential therapeutic interventions. Here we explore the role of pro-survival genes in cancer cell integrity during clonal evolution in non-small cell lung cancer (NSCLC). We identify gains of MCL-1 at high frequency in multiple independent NSCLC cohorts,occurring both clonally and subclonally. Clonal loss of functional TP53 is significantly associated with subclonal gains of MCL-1. In mice,tumour progression is delayed upon pharmacologic or genetic inhibition of MCL-1. These findings reveal that MCL-1 gains occur with high frequency in lung adenocarcinoma and can be targeted therapeutically. View Publication

BACKGROUND Chronic rhinosinusitis patients (CRS) suffer from chronic inflammation of the sinus mucosa associated with chronic relapsing infections. Mucosal biofilms,associated with Staphylococcus aureus,have been implicated as a cause. We compared the effect of exoproteins secreted from clinical isolates of S aureus from CRS patients in planktonic and biofilm form on the nasal epithelial barrier. METHODS Clinical S aureus isolates from 39 CRS patients were grown in planktonic and biofilm forms and their exoproteins concentrated. These were applied to primary human nasal epithelial cells grown at the air-liquid interface. Transepithelial electrical resistance,permeability of flourescein isothiocyanate-dextrans,and cytotoxicity were measured. Structure and expression of tight junctions zona occludens-1,and claudin-1 proteins were assessed by electron microscopy and immunofluorescence. The Wilcoxon signed rank test was used for statistical analyses. RESULTS S aureus biofilm exoproteins showed dose- and time-dependent reduction of transepithelial electrical resistance,increased cell toxicity,and increased permeability (p {\textless} 0.001) compared with equal concentrations of planktonic cultures. Discontinuity in zona occludens-1 and claudin-1 immunofluorescence was confirmed as disrupted tight junctions on electron microscopy. CONCLUSION S aureus biofilm exoproteins disrupt the mucosal barrier structure in a time- and dose-dependent manner and are toxic. Damage to the mucosal barrier by S aureus biofilm exoproteins may play a major role in CRS etiopathogenesis. View Publication

Uridine,a pyrimidine nucleoside essential for the synthesis of RNA and bio-membranes,is a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardio-circulatory system,at the reproduction level,with both peripheral and central nervous system modulation and with the functionality of the respiratory system. Furthermore,uridine plays a role at the clinical level in modulating the cytotoxic effects of fluoropyrimidines in both normal and neoplastic tissues. The concentration of uridine in plasma and tissues is tightly regulated by cellular transport mechanisms and by the activity of uridine phosphorylase (UPase),responsible for the reversible phosphorolysis of uridine to uracil. We have recently completed several studies designed to define the mechanisms regulating UPase expression and better characterize the multiple biological effects of uridine. Immunohistochemical analysis and co-purification studies have revealed the association of UPase with the cytoskeleton and the cellular membrane. The characterization of the promoter region of UPase has indicated a direct regulation of its expression by the tumor suppressor gene p53. The evaluation of human surgical specimens has shown elevated UPase activity in tumor tissue compared to paired normal tissue. 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

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

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

Drug-induced resistance,or tolerance,is an emerging yet poorly understood failure of anticancer therapy. The interplay between drug-tolerant cancer cells and innate immunity within the tumor,the consequence on tumor growth,and therapeutic strategies to address these challenges remain undescribed. Here we elucidate the role of taxane-induced resistance on natural killer (NK) cell tumor immunity in triple-negative breast cancer (TNBC) and the design of spatio-temporally controlled nanomedicines,which boost therapeutic efficacy and invigorate 'disabled' NK. Drug tolerance limited NK cell immune surveillance via drug-induced depletion of the NK-activating ligand receptor axis,NKG2D and MHC class I polypeptide-related sequence A,B (MICA/B). Systems biology supported by empirical evidence revealed the heat shock protein 90 (Hsp90) simultaneously controls immune surveillance and persistence of drug-treated tumor cells. Based on this evidence,we engineered a 'chimeric' nano-therapeutic tool comprising taxanes and a cholesterol-tethered Hsp90 inhibitor,radicicol,which targets the tumor,reduces tolerance,and optimally re-primes NK cells via prolonged induction of NK-activating ligand receptors via temporal control of drug release in vitro and in vivo. A human ex-vivo TNBC model confirmed the importance of NK cells in drug-induced death under pressure of clinically-approved agents. These findings highlight a convergence between drug-induced resistance,the tumor-immune contexture,and engineered approaches that considers the tumor and microenvironment to improve the success of combinatorial therapy. View Publication

Cystic fibrosis (CF) cells display a more cancer-like phenotype vs. non-CF cells. KLF4 overexpression has been described in CF and this transcriptional factor acts as a negative regulator of wt-CFTR. KLF4 is described as exerting its effects in a cell-context-dependent fashion,but it is generally considered a major regulator of proliferation,differentiation,and wound healing,all the processes that are also altered in CF. Therefore,it is relevant to characterize the differential role of KLF4 in these processes in CF vs. non-CF cells. To this end,we used wt- and F508del-CFTR CFBE cells and their respective KLF4 knockout (KO) counterparts to evaluate processes like cell proliferation,polarization,and wound healing,as well as to compare the expression of several epithelial differentiation markers. Our data indicate no major impact of KLF4 KO in proliferation and a differential impact of KLF4 KO in transepithelial electrical resistance (TEER) acquisition and wound healing in wt- vs. F508del-CFTR cells. In parallel,we also observed a differential impact on the levels of some differentiation markers and epithelial-mesencymal transition (EMT)-associated transcription factors. In conclusion,KLF4 impacts TEER acquisition,wound healing,and the expression of differentiation markers in a way that is partially dependent on the CFTR-status of the cell. View Publication

There is increasing evidence that coronavirus disease 2019 (COVID-19) produces more severe symptoms and higher mortality among men than among women1-5. However,whether immune responses against severe acute respiratory syndrome coronavirus (SARS-CoV-2) differ between sexes,and whether such differences correlate with the sex difference in the disease course of COVID-19,is currently unknown. Here we examined sex differences in viral loads,SARS-CoV-2-specific antibody titres,plasma cytokines and blood-cell phenotyping in patients with moderate COVID-19 who had not received immunomodulatory medications. Male patients had higher plasma levels of innate immune cytokines such as IL-8 and IL-18 along with more robust induction of non-classical monocytes. By contrast,female patients had more robust T cell activation than male patients during SARS-CoV-2 infection. Notably,we found that a poor T cell response negatively correlated with patients' age and was associated with worse disease outcome in male patients,but not in female patients. By contrast,higher levels of innate immune cytokines were associated with worse disease progression in female patients,but not in male patients. These findings provide a possible explanation for the observed sex biases in COVID-19,and provide an important basis for the development of a sex-based approach to the treatment and care of male and female patients with COVID-19. View Publication

The pulmonary system is comprised of two main compartments,airways and alveolar space. Their tissue and cellular complexity ensure lung function and protection from external agents,for example,virus. Two-dimensional (2D) in vitro systems and animal models have been largely employed to elucidate the molecular mechanisms underlying human lung development,physiology,and pathogenesis. However,neither of these models accurately recapitulate the human lung environment and cellular crosstalk. More recently,human-derived three-dimensional (3D) models have been generated allowing for a deeper understanding of cell-to-cell communication. However,the availability and accessibility of primary human cell sources from which generate the 2D and 3D models may be limited. In the past few years,protocols have been developed to successfully employ human pluripotent stem cells (hPSCs) and differentiate them toward pulmonary fate in vitro. In the present review,we discuss the advantages and pitfalls of hPSC-derived lung 2D and 3D models,including the main characteristics and potentials for these models and their current and future applications for modeling development and diseases. Lung organoids currently represent the closest model to the human pulmonary system. We further focus on the applications of lung organoids for the study of human diseases such as pulmonary fibrosis,infectious diseases,and lung cancer. Finally,we discuss the present limitations and potential future applications of 3D lung organoids. This article is categorized under: Adult Stem Cells,Tissue Renewal,and Regeneration {\textgreater} Stem Cells and Disease Adult Stem Cells,Tissue Renewal,and Regeneration {\textgreater} Stem Cell Differentiation and Reversion. View Publication

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin,trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I),consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+,inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential,with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA,and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA {\textless} trilobolide {\textless} thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I,and that binding constants are very similar for E1Ca2,E1 and E2. View Publication

Expression of BAX,without another death stimulus,proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1 beta-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family),as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However,the fall in mitochondrial membrane potential,production of reactive oxygen species,cytoplasmic vacuolation,and plasma membrane permeability that are downstream of BAX still occurred. Thus,BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases. View Publication