技术资料

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

2-Hydroxyglutarate (2HG) exists as two enantiomers,(R)-2HG and (S)-2HG,and both are implicated in tumor progression via their inhibitory effects on $\alpha$-ketoglutarate ($\alpha$KG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations,whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5'-diphosphate-ribose) polymerase (PARP) inhibitors. This BRCAness" phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme and conversely it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1-dependent PARP inhibitor sensitivity in a range of clinically relevant models including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH rather than attempting to block 2HG production by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore our results uncover an unexpected link between oncometabolites altered DNA repair and genetic instability." View Publication

Type III interferon (interferon lambda [IFN-$\lambda$]) is known to be a potential immune modulator,but the mechanisms behind its immune-modulatory functions and its impact on plasmablast differentiation in humans remain unknown. Human B cells and their subtypes directly respond to IFN-$\lambda$. Using B cell transcriptome profiling,we investigate the immune-modulatory role of IFN-$\lambda$ in B cells. We find that IFN-$\lambda$-induced gene expression in B cells is steady,prolonged,and importantly,cell type specific. Furthermore,IFN-$\lambda$ enhances the mTORC1 (mammalian/mechanistic target of rapamycin complex 1) pathway in B cells activated by the B cell receptor (BCR/anti-IgM). Engagement of mTORC1 by BCR and IFN-$\lambda$ induces cell-cycle progress in B cells. Subsequently,IFN-$\lambda$ boosts the differentiation of naive B cells into plasmablasts upon activation,and the cells gain effector functions such as cytokine release (IL-6 and IL-10) and antibody production. Our study shows how IFN-$\lambda$ systematically boosts the differentiation of naive B cells into plasmablasts by enhancing the mTORC1 pathway and cell-cycle progression in activated B cells. 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 human nasal epithelium is the primary site of exposure to influenza virus,the initiator of host responses to influenza and the resultant pathologies. Influenza virus may cause serious respiratory infection resulting in major complications,as well as severe impairment of the airways. Here,we elucidated the global transcriptomic changes during H3N2 infection of human nasal epithelial cells from multiple individuals. Using RNA sequencing,we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. We used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. Statistical analysis highlighted strong transcriptomic signatures significantly associated with 24 and 48 h after infection,but not at the earlier 8-h time point. In particular,we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling,B-cell signaling,apoptosis,necrosis,smooth muscle proliferation,and metabolic alterations. These molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway,and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management. View Publication

Apoptosis,an evolutionarily conserved form of cell suicide,requires specialized machinery. The central component of this machinery is a proteolytic system involving a family of proteases called caspases. These enzymes participate in a cascade that is triggered in response to proapoptotic signals and culminates in cleavage of a set of proteins,resulting in disassembly of the cell. Understanding caspase regulation is intimately linked to the ability to rationally manipulate apoptosis for therapeutic gain. 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

Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels,but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected,leading to liver-selective pharmacology,when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality,as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441,which reduces glucose levels and improves lipid profiles in an animal model of diabetes. 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