技术资料

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

A novel fluorescent derivative of glucose was synthesized by reacting D-glucosamine and NBD-Cl. The TLC analysis of the reaction mixture showed the generation of a single spot with intense fluorescence (lambda Ex = 475 nm,lambda Em = 550 nm). The obtained novel fluorescent product,which was identified as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) by 1H-NMR and FAB-MS spectrometries,was applied to the assessment of the glucose uptake activity of Escherichia coli B. 2-NBDG accumulated in living cells and not in dead cells. The uptake of 2-NBDG was competitively inhibited by D-glucose and not by L-glucose,which suggested the involvement of the glucose transporting system in the uptake of 2-NBDG. 2-NBDG taken into the cytoplasma of E. coli cells was supposedly converted into another derivative in the glucose metabolic pathway. View Publication

Respiratory syncytial virus (RSV) is increasingly recognized for causing severe morbidity and mortality in older adults,but there are few studies on the RSV-induced immune response in this population. Information on the immunological processes at play during RSV infection in specific risk groups is essential for the rational and targeted design of novel vaccines and therapeutics. Here,we assessed the antibody and local cytokine response to RSV infection in community-dwelling older adults (≥60 years of age). During three winters,serum and nasopharyngeal swab samples were collected from study participants during acute respiratory infection and recovery. RSV IgG enzyme-linked immunosorbent assays (ELISA) and virus neutralization assays were performed on serum samples from RSV-infected individuals (n = 41) and controls (n = 563 and n = 197,respectively). Nasal RSV IgA and cytokine concentrations were determined using multiplex immunoassays in a subset of participants. An in vitro model of differentiated primary bronchial epithelial cells was used to assess RSV-induced cytokine responses over time. A statistically significant increase in serum neutralization titers and IgG concentrations was observed in RSV-infected participants compared to controls. During acute RSV infection,a statistically significant local upregulation of beta interferon (IFN-$\beta$),IFN-$\lambda$1,IFN-$\gamma$,interleukin 1$\beta$ (IL-1$\beta$),tumor necrosis factor alpha (TNF-$\alpha$),IL-6,IL-10,CXCL8,and CXCL10 was found. IFN-$\beta$,IFN-$\lambda$1,CXCL8,and CXCL10 were also upregulated in the epithelial model upon RSV infection. In conclusion,this study provides novel insights into the basic immune response to RSV infection in an important and understudied risk population,providing leads for future studies that are essential for the prevention and treatment of severe RSV disease in older adults.IMPORTANCE Respiratory syncytial virus (RSV) can cause severe morbidity and mortality in certain risk groups,especially infants and older adults. Currently no (prophylactic) treatment is available,except for a partially effective yet highly expensive monoclonal antibody. RSV therefore remains a major public health concern. To allow targeted development of novel vaccines and therapeutics,it is of great importance to understand the immunological mechanisms that underlie (protection from) severe disease in specific risk populations. Since most RSV-related studies focus on infants,there are only very limited data available concerning the response to RSV in the elderly population. Therefore,in this study,RSV-induced antibody responses and local cytokine secretion were assessed in community-dwelling older adults. These data provide novel insights that will benefit ongoing efforts to design safe and effective prevention and treatment strategies for RSV in an understudied risk group. View Publication

Dendritic cells (DC) play an essential role in innate immunity and radiation-elicited immune responses. LGP2 is a RIG-I like receptor (RLR) involved in cytoplasmic RNA recognition and anti-viral responses. Although LGP2 has also been linked to cell survival of both tumor cells and T cells,the role of LGP2 in mediating DC function and anti-tumor immunity elicited by radiotherapy remains unclear. Here we report that tumor DC are linked to the clinical outcome of breast cancer patients who received radiotherapy (RT) and the presence of DC correlates with gene expression of LGP2 in the tumor microenvironment. In preclinical models,host LGP2 was essential for optimal anti-tumor control by ionizing radiation (IR). The absence of LGP2 in DC dampened type I interferon production and the priming capacity of DC. In the absence of LGP2,MDA5-mediated activation of type I IFN signaling was abrogated. The MDA5/LGP2 agonist high molecular weight poly I: C improved the anti-tumor effect of IR. This study reveals a previously undefined role of LGP2 in host immunity and provides a new strategy to improve the efficacy of radiotherapy. View Publication

SARS-CoV-2,a $\beta$-coronavirus,has rapidly spread across the world,highlighting its high transmissibility,but the underlying morphogenesis and pathogenesis remain poorly understood. Here,we characterize the replication dynamics,cell tropism and morphogenesis of SARS-CoV-2 in organotypic human airway epithelial (HAE) cultures. SARS-CoV-2 replicates efficiently and infects both ciliated and secretory cells in HAE cultures. In comparison,HCoV-NL63 replicates to lower titers and is only detected in ciliated cells. SARS-CoV-2 shows a similar morphogenetic process as other coronaviruses but causes plaque-like cytopathic effects in HAE cultures. Cell fusion,apoptosis,destruction of epithelium integrity,cilium shrinking and beaded changes are observed in the plaque regions. Taken together,our results provide important insights into SARS-CoV-2 cell tropism,replication and morphogenesis. View Publication

Background: Immunotherapy efficacy in esophageal squamous cell carcinoma (ESCC) is often limited by an immunosuppressive tumor microenvironment (TME). NLRC5,a key regulator of MHC-I antigen presentation,exhibits context-dependent roles in tumor immunity; however,its function in ESCC remains unclear. This study aimed to systematically investigate the expression pattern,prognostic value,and immunological role of NLRC5 in ESCC. Methods: An integrated analysis of bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) data was performed using multiple cohorts,including The Cancer Genome Atlas,Gene Expression Omnibus,and an in-house ESCC cohort. Differential expression,survival analysis,immune infiltration estimation,and functional enrichment analyses were conducted to elucidate the role of NLRC5 in the tumor microenvironment. Results: NLRC5 was significantly upregulated in ESCC and its high expression independently predicted poor patient survival. Although NLRC5 expression was associated with increased CD8+ T cell infiltration,it was paradoxically accompanied by features of T-cell exhaustion and elevated expression of multiple immune checkpoints. Moreover,NLRC5-high tumors were enriched in transcriptional programs related to PANoptosis,indicating an additional immunosuppressive mechanism within the TME. Conclusions: NLRC5 is not only a prognostic biomarker but also a key modulator of an immune-active yet functionally suppressed tumor microenvironment in ESCC. These findings highlight NLRC5 as a potential therapeutic target for restoring effective antitumor immunity. View Publication