参考文献

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

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

Mammalian target of rapamycin (mTOR) is frequently upregulated in hepatocellular carcinoma (HCC). Blockage of mTOR was found to induce marked reduction in HCC growth in preclinical models. In the present study,we tested a novel mTOR inhibitor,Torin-2,for its antitumor efficacy in HCC cell lines Hep G2,SNU-182 and Hep 3B2.1-7. The HCC cell lines were cultured in vitro. These cells were treated with Torin-2. Cell apoptosis was evaluated by Annexin V staining. Cell proliferation and cell cycle progression were determined by Ki67 staining and propidium iodide staining,respectively. mTOR signaling,autophagy induction and expression of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) were assessed by western blot analysis. The UHRF1 mRNA level was determined by real-time PCR. We found that Torin-2 effectively suppressed the growth and survival of HCC cell lines,demonstrated by reduced proliferation and a high rate of apoptosis. Further study elucidated that in addition to blocking mTOR complex 1 (mTORC1)-associated cell cycle progression and induction of autophagy,Torin-2 downregulated transcription of UHRF1,an essential regulator of DNA methylation that is highly expressed in HCC cell lines. Consistently,the level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) was higher after treatment of the HCC cell lines with Torin-2. The downregulation of UHRF1 by Torin-1 was partially due to a decrease in the UHRF1 mRNA level. Torin-2 effectively inhibited HCC cell proliferation through induction of autophagy. Torin‑2-induced downregulation of UHRF1 expression may also contribute to its antitumor effect. Our research provides new clues regarding the antitumor effects of Torin-2 and sheds light on a novel therapeutic approach for HCC. View Publication

The function of a T cell depends on its subtype and activation state. Here,we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2,CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99{\%} accuracy,and according to activation state (quiescent or activated) and subtype (CD3+CD8+ or CD3+CD4+) with 97{\%} accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function. 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

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

To avoid innate sensing and immune control,human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here,we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein,unleashing end degradation of viral cDNA. Concomitantly,Vpu inhibits RAD52-mediated homologous repair of viral cDNA,preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs. View Publication

Lymphatic endothelial cells (LECs) chemoattract na{\{i}}ve T cells and promote their survival in the lymph nodes and can cross-present antigens to na{\"{i}}ve CD8+ T cells to drive their proliferation despite lacking key costimulatory molecules. However the functional consequence of LEC priming of CD8+ T cells is unknown. Here we show that while many proliferating LEC-educated T cells enter early apoptosis the remainders comprise a long-lived memory subset with transcriptional metabolic and phenotypic features of central memory and stem cell-like memory T cells. In vivo these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8+ T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.""" View Publication

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however,our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here,we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins,including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders. View Publication

Recent studies suggest the effect of radiation is observed not only in irradiated cells but also in adjacent non-irradiated cells (bystander effect),although the mechanism has not yet been fully revealed. This bystander effect may be caused by intercellular communication via a gap junction or by messengers released from irradiated cells,such as reactive oxygen species,nitric oxide,or cytokines. However,an unknown mechanism is also possible in the bystander effect. On the other hand,it is known that extracellular ATP,ADP,uridine 5'-triphosphate (UTP),and uridine 5'-diphosphate (UDP),which are released from cells,act as intercellular signaling molecules by activating purinergic P2X and P2Y receptors (purinergic signaling). Recently,I have suggested these extracellular nucleotides may be novel mediators of a radiation-induced bystander effect,because our recent studies indicated that purinergic signaling is involved in important cellular responses to radiation. Our data indicate that ionizing irradiation causes activation of the transient receptor potential melastatin type 2 (TRPM2) channel,and then ATP is released from cells through the anion channel or connexin43 hemichannel mediated by the activation of a P2X7 receptor. The released nucleotides activate P2Y6 and P2Y12 receptors,which are involved in the DNA damage response after irradiation. Activation of the P2Y6 receptor is also involved in radiation-induced activation of the epithelial growth factor receptor-extracellular signal regulated protein kinase (EGFR-ERK)1/2 pathway and subsequent nuclear translocation of EGFR,which plays a role in DNA repair. Further,the induction of an antioxidant after irradiation is also mediated by the activation of the P2Y receptor. In conclusion,purinergic signaling could play an important role in the protective cellular response to ionizing irradiation. View Publication