Biosensing Techniques in Yeast: G-Protein Signaling and Protein-Protein Interaction Assays for Monitoring Ligand Stimulation and Oligomer Formation of Heterologous GPCRs

Guanine nucleotide-binding proteins (G-proteins) act as transducers of external stimuli for intracellular signaling, and control various cellular processes in cooperation with seven transmembrane G-protein-coupled receptors (GPCRs). Because GPCRs constitute the largest family of eukaryotic membrane proteins and enable the selective recognition of a diverse range of molecules (ligands), they are the major molecular targets in pharmaceutical and medicinal fields. In addition, GPCRs have been known to form heteromers as well as homomers, which may result in vast physiological diversity and provide opportunities for drug discovery. G-proteins and their signal transduction machinery are universally conserved in eukaryotes; thereby, the yeast Saccharomyces cerevisiae has been used to construct artificial in vivo GPCR biosensors. In this chapter, we focus on the yeast-based GPCR biosensors that can detect ligand stimulation and oligomer formation, and summarize their techniques using the G-protein signaling and protein-protein interaction assays

Sodium/Lithium-Ion Transfer Reaction at the Interface between Low-Crystallized Carbon Nanosphere Electrodes and Organic Electrolytes

Carbon nanosphere (CNS) electrodes are the candidate of sodium-ion battery (SIB) negative electrodes with small internal resistances due to their small particle sizes. Electrochemical properties of low-crystallized CNS electrodes in dilute and concentrated sodium bis(trifluoromethanesulfonyl) amide/ethylene carbonate + dimethyl carbonate (NaTFSA/EC + DMC) were first investigated. From the cyclic voltammograms, both lithium ion and sodium ion can reversibly insert into/from CNSs in all of the electrolytes used here. The cycling stability of CNSs in concentrated electrolytes was better than that in dilute electrolytes for the SIB system. The interfacial charge-transfer resistances at the interface between CNSs and organic electrolytes were evaluated using electrochemical impedance spectroscopy. In the Nyquist plots, the semicircles at the middle-frequency region were assigned to the parallel circuits of charge-transfer resistances and capacitances. The interfacial sodium-ion transfer resistances in concentrated organic electrolytes were much smaller than those in dilute electrolytes, and the rate capability of CNS electrodes in sodium salt-concentrated electrolytes might be better than in dilute electrolytes, suggesting that CNSs with concentrated electrolytes are the candidate of SIB negative electrode materials with high rate capability. The calculated activation energies of interfacial sodium-ion transfer were dependent on electrolyte compositions and similar to those of interfacial lithium-ion transfer

Kinetic properties of sodium-ion transfer at the interface between graphitic materials and organic electrolyte solutions

Graphitic materials cannot be applied for the negative electrode of sodium-ion battery because the reversible capacities of graphite are anomalously small. To promote electrochemical sodium-ion intercalation into graphitic materials, the interfacial sodium-ion transfer reaction at the interface between graphitized carbon nanosphere (GCNS) electrode and organic electrolyte solutions was investigated. The interfacial lithium-ion transfer reaction was also evaluated for the comparison to the sodium-ion transfer. From the cyclic voltammograms, both lithium-ion and sodium-ion can reversibly intercalate into/from GCNS in all of the electrolytes used here. In the Nyquist plots, the semi-circles at the high frequency region derived from the Solid Electrolyte Interphase (SEI) resistance and the semi-circles at the middle frequency region owing to the charge-transfer resistance appeared. The activation energies of both lithium-ion and sodium-ion transfer resistances were measured. The values of activation energies of the interfacial lithium-ion transfer suggested that the interfacial lithium-ion transfer was influenced by the interaction between lithium-ion and solvents, anions or SEI. The activation energies of the interfacial sodium-ion transfer were larger than the expected values of interfacial sodium-ion transfer based on the week Lewis acidity of sodium-ion. In addition, the activation energies of interfacial sodium-ion transfer in dilute FEC-based electrolytes were smaller than those in concentrated electrolytes. The activation energies of the interfacial lithium/sodium-ion transfer of CNS-1100 in FEC-based electrolyte solutions were almost the same as those of CNS-2900, indicating that the mechanism of interfacial charge-transfer reaction seemed to be the same for highly graphitized materials and low-graphitized materials each other

Operando analysis of graphite intercalation compounds with fluoride-containing polyatomic anions in aqueous solutions

The formation of graphite intercalation compounds (GICs) in aqueous solutions has attracted much attention, but reversibility in the formation/deformation of GICs is a challenging issue to construct highly safe rechargeable batteries. In this study, we used an operando analysis (X-ray diffraction and Raman spectroscopy) to discuss the feasibility of using fluoride-containing polyatomic anions in the formation of GICs in aqueous highly concentrated solutions. We found that the intercalation of anions containing a C₂F₅ moiety (such as [N(SO₂CF₃)(SO₂CF₂CF₃)]⁻ or [N(SO₂CF₂CF₃)₂]⁻) does not occur in the bulk of graphite, but only in the surface region. In addition, anions containing a CF₃ moiety show different behaviors: SO₃CF₃⁻ shows greater reversibility and larger stage-number than N(SO₂CF₃)₂⁻ in the formation of GICs. These results provide design guidelines for the reversible intercalation and de-intercalation of anions and their application as a cathode material in aqueous rechargeable batteries

Programmed cell death 1‐expressing CD56‐negative natural killer (NK) cell expansion is a hallmark of chronic NK cell activation during dasatinib treatment

Dasatinib treatment markedly increases the number of large granular lymphocytes including natural killer (NK) cells in a proportion of Ph+ leukemia patients, which associates with a better prognosis. In-depth immune profiling of NK cells can predict therapeutic response in these patients. In the present study, we showed that CD56-negative (CD56neg) NK cells increased exclusively in cytomegalovirus-seropositive (CMV+) patients treated with dasatinib. The increase longitudinally paralleled with progressive differentiation of CD56dim NK cells during dasatinib therapy driven by CMV reactivation as shown by principal component analysis on 19 NK cell markers. The CD56neg NK cells showed downregulation of NK-activating receptors, upregulation of PD-1, and lower cytotoxicity and cytokine production, indicating that these cells are anergic and dysfunctional as seen in chronic infections with HIV-1 or hepatitis C virus. Moreover, cytolytic activity of CD56dim and CD56neg NK cells against leukemia cells was partially restored by nivolumab in proportion to the frequency of PD-1+ NK cells. The proportion of patients who achieved deep molecular responses at 2 years was significantly higher in dasatinib-treated patients with ≥3% CD56neg NK cells than in those with fewer CD56neg NK cells (54.5% vs 15.8%, P = .0419). These findings suggest that CD56neg NK cells may be an exhausted population induced by chronic activation through CMV reactivation during dasatinib therapy. Expansion of CD56neg NK cells is a hallmark of chronic NK cell activation in patients treated with dasatinib and may predict a better clinical outcome. Furthermore, PD-1 blockade may enhance anti-leukemia responses of such NK cells

The EZH2 inhibitor tazemetostat upregulates the expression of CCL17/TARC in B‐cell lymphoma and enhances T‐cell recruitment

An inhibitor of the histone methyltransferase enhancer of zeste homologue 2 (EZH2), tazemetostat, has been developed for the treatment of B-cell lymphoma, but its mechanisms of action are not fully elucidated. We screened for genes targeted by tazemetostat in eleven B-cell lymphoma cell lines and found that tazemetostat significantly increased the expression of chemokine (C-C motif) ligand 17 (CCL17)/thymus- and activation-regulated chemokine (TARC) in all, which codes for a chemokine that is a hallmark of Hodgkin/Reed-Sternberg (H/RS) cells in Hodgkin lymphoma. Notably, gene set enrichment analysis demonstrated a positive correlation between the genes upregulated by tazemetostat in five follicular lymphoma (FL) cell lines and those reported to be overexpressed in H/RS cells. The CCL17 promoter region was enriched in repressive histone modification H3K27me3, and tazemetostat induced H3K27 demethylation and activated gene transcription. CCL17 protein secretion was also induced by EZH2 inhibition, which was further enhanced by concurrent CpG stimulation. In vitro transwell migration assay demonstrated that CCL17 produced by tazemetostat-treated B cells enhanced the recruitment of T cells, which had the potential to exert antilymphoma response. Analysis of publicly available human lymphoma databases showed that CCL17 gene expression was inversely correlated with the EZH2 activation signature and significantly paralleled the CD4⁺ and CD8⁺ T-cell–rich signature in FL and germinal center B-cell–like diffuse large B-cell lymphoma. Our findings indicate that tazemetostat can potentially activate antilymphoma response by upregulating CCL17 expression in B-cell lymphoma cells and promote T-cell recruitment, which provides a rationale for its combination with immunotherapy

Proteomic and meta-transcriptomic study on lymph node metastasis in gastric cancer

AbstractTo examine the proteomic background of lymph node metastasis (LNM) in gastric cancer, we performed protein expression profiling of paired non-tumor, primary tumor, and LNM tissues. Using a label-free proteomic approach, we generated protein expression profiles of 3894 unique proteins and identified 109 differentially expressed proteins. Functional pathway analysis of the differentially expressed proteins showed that members of the beta-3 integrin (ITGB3) pathway were significantly enriched. Aberrations of ITGB3 were reported in various malignancies; however, ITGB3 in LNM tissues has not been examined to date. Different level of ITGB3 expression was confirmed in 20 gastric cancer cases by Western blotting. We analyzed the mRNA levels of the differentially expressed proteins by using a public mRNA expression database; 38.8% of the proteins examined, including those involved in oxidation and reduction, showed correlation between protein and mRNA levels. Proteins without such correlation included factors related to cell adhesion. Our study suggests a novel role for the integrin pathway in the development of LNM in gastric cancer and indicated possible benefits of observational transcriptomic analysis for proteomic studies