Copper Complexes with Protein-Based N-Donor Ligands as cis-Selective Nascent Cyclopropanases.

In this study, we aimed to develop protein-based metal ligands to catalyze cis-selective cyclopropanation using the TM1459 cupin protein superfamily. Copper complexes with TM1459 mutants containing the 3-His metal-binding site exhibited excellent diastereoselectivity in cyclopropanation reactions with styrene and ethyl diazoacetate. Further mutations in the secondary coordination sphere increased the cis-preference with t-butyl diazoacetate as the substrate with up to 80 : 20 (cis:trans ratio) and high enantioselectivity (90 % ee).

A thiopyridine-bound mirror-image copper center in an artificial non-heme metalloenzyme.

Artificial metalloenzymes, in which a metal complex and protein matrix are combined, have been synthesized to catalyze stereoselective reactions using the chiral environment provided by the protein cavity. Artificial metalloenzymes can be engineered by the chemical modification and mutagenesis of the protein matrix. We developed artificial non-heme metalloenzymes using a cupin superfamily protein (TM1459) with a 4-His tetrad-metal-binding motif.

Asymmetric Michael addition catalysed by copper-amyloid complexes.

We developed self-assembled peptides containing a partial amyloid β protein sequence and a metal-coordination site. The amyloid fibril-copper complexes exhibited excellent reactivity and moderate enantioselectivity in Michael addition reactions with 2-azachalcone and dimethylmalonate. The catalytic amyloids were characterized using various measurements to confirm their amyloid-like nanofibre structures.

An artificial metallolyase with pliable 2-His-1-carboxylate facial triad for stereoselective Michael addition.

We repurposed the metal-binding site of a cupin superfamily protein into the 2-His-1-carboxylate facial triad, which is one of the common motifs in natural non-heme enzymes, to construct artificial metalloenzymes that can catalyze new-to-nature reactions. The Cu-H52A/H58E variant catalyzed the stereoselective Michael addition reaction and was found to bear a flexible metal-binding site in the high-resolution crystal structure. Furthermore, the H52A/H58E/F104W mutant accommodated a water molecule, which was supported by Glu58 and Trp104 residues hydrogen bonding, presumably leading to high stereoselectivity.

Association between effector-type regulatory T cells and immune checkpoint expression on CD8 T cells in malignant ascites from epithelial ovarian cancer.

Background: Regulatory T cells (Tregs) play an important role in the antitumor immune response in epithelial ovarian cancer (EOC). To understand the immune-inhibitory networks of EOC, we addressed the association between Tregs and immune checkpoint expression on T cells in the tumor microenvironment of EOC.

Methods: A total of 41 patients with stage IIIC and IV EOC were included in the analysis.

Identification of serum cytokine clusters associated with outcomes in ovarian clear cell carcinoma.

Serum cytokine and chemokine networks may reflect the complex systemic immunological interactions in cancer patients. Studying groups of cytokines and their networks may help to understand their clinical biology. A total of 178 cases of ovarian cancer were analyzed in this study, including 73 high-grade serous (HGSC), 66 clear cell (CCC) and 39 endometrioid carcinomas.

Copper-Oxygen Dynamics in the Tyrosinase Mechanism.

The dinuclear copper enzyme, tyrosinase, activates O to form a (μ-η :η -peroxido)dicopper(II) species, which hydroxylates phenols to catechols. However, the exact mechanism of phenolase reaction in the catalytic site of tyrosinase is still under debate. We herein report the near atomic resolution X-ray crystal structures of the active tyrosinases with substrate l-tyrosine.

Cupin Variants as a Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes.

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded β-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantioselective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, calculated substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

His-Cys and Trp-Cys cross-links generated by post-translational chemical modification.

Galactose oxidase and amine oxidase contain a cofactor which is generated by post-translational chemical modification to the corresponding amino acid side chains near the copper active center. Such cofactors provide proteins unusual catalytic ability that canonical amino acids cannot exert as well as their structural stability, and thereby are called as protein-derived cofactors. These cofactors and modifications are mostly derived from aromatic amino acid residues, especially Tyr, Trp, and His.

Irreversible Electroporation versus Radiofrequency Ablation: Comparison of Systemic Immune Responses in Patients with Hepatocellular Carcinoma.

Purpose: Irreversible electroporation (IRE) differs from thermal radiofrequency (RF) ablation, especially in terms of the reparative process in the ablation zone induced. To elucidate this, the systemic immune responses after 2 mechanistically different types of ablation (IRE and RF ablation) were evaluated in patients with hepatocellular carcinoma (HCC).

Materials And Methods: Twenty-one patients with HCC who underwent either RF ablation (n = 11) or IRE (n = 10) were studied.

Expression of multiple immune checkpoint molecules on T cells in malignant ascites from epithelial ovarian carcinoma.

Expression of immune checkpoint molecules, including programmed cell death protein-1 (PD-1), has been reported on T cells in various types of cancer. However, the expression status of these molecules in the tumor microenvironment of epithelial ovarian cancer (EOC) has not yet been studied. A total of 54 cases of malignant ascites from patients with EOC were analyzed in the present study.

NK cell and IFN signatures are positive prognostic biomarkers for resectable pancreatic cancer.

To establish prognostic biomarkers and to identify potential novel therapeutic targets, we performed integrative immunomonitoring of blood and tumor in patients with resectable pancreatic cancer. Flow cytometry (FC) was employed for phenotyping immune cells, multiplex bead assays for plasma cytokine and chemokine determination, and RNA-Seq for the analysis of gene expression in the tumor. Nineteen pancreatic cancer patients were stratified into those with longer or shorter than median recurrence-free survival after surgery (median, 426 days).

Geometric effects on OO bond scission of copper(II)-alkylperoxide complexes.

Copper(II) complexes supported by N-tridentate ligands, consisting of a rigid cyclic diamine (8-membered cyclic-diamine; L8 or 7-membered cyclic-diamine; L7) and a 2-(2-pyridyl)ethyl (-CHCHPy) group, were synthesized and structurally characterized. Reaction of the copper(II) complexes and cumene hydroperoxide (CmOOH) in the presence of triethylamine in CHCN gave the corresponding cumylperoxide complexes L8CuOOCm and L7CuOOCm. The UV-vis and EPR spectra suggested that L8CuOOCm takes a tetrahedrally distorted structure, whereas L7CuOOCm has a planar geometry in solution.

Tetrahedral Copper(II) Complexes with a Labile Coordination Site Supported by a Tris-tetramethylguanidinato Ligand.

A new tridentate N ligand (TMGtach) consisting of cis,cis-1,3,5-triaminocyclohexane (tach) and three N,N,N',N'-tetramethylguanidino (TMG) groups has been developed to prepare copper complexes with a tetrahedral geometry and a labile coordination site. Treatment of the ligand with CuX (X = Cl and Br) gave copper(II)-halide complexes, [Cu(TMGtach)Cl] (2) and [Cu(TMGtach)Br] (2), the structures of which have been determined by X-ray crystallographic analysis. The complexes exhibit a four-coordinate structure with C symmetry, where the labile halide ligand (X) occupies a position on the trigonal axis.

Generation and characterisation of a stable nickel(ii)-aminoxyl radical complex.

A stable nickel(ii)-aminoxyl radical complex was generated by the reaction of a nickel(ii) complex supported by a tren ligand (tris(2-aminoethyl)amine) having bulky m-terphenyl substituents (TIPT: 3,5-bis(2,6-diisopropylphenyl)phenyl) and m-CPBA (m-chloroperoxybenzoic acid). The formation mechanism of the nickel(ii)-aminoxyl radical complex was examined.

A Well-Defined Osmium-Cupin Complex: Hyperstable Artificial Osmium Peroxygenase.

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (T ≈ 120 °C).

CD27(-)CD45(+) γδ T cells can be divided into two populations, CD27(-)CD45(int) and CD27(-)CD45(hi) with little proliferation potential.

In addition to the majority of T cells which carry the αβ T cell receptor (TCR) for antigen, a distinct subset of about 1-5% of human peripheral blood T cells expressing the γδ TCR contributes to immune responses to infection, tissue damage and cancer. T cells with the Vδ2(+) TCR, usually paired with Vγ9, constitute the majority of these γδ T cells. Analogous to αβ T cells, they can be sorted into naive (CD27(+)CD45RA(+)), central memory (CD27(+)CD45RA(-)), effector memory (CD27(-)CD45RA(-)), and terminally-differentiated effector memory (CD27(-)CD45RA(+)) phenotypes.

Sensitive Multiplexed Quantitative Analysis of Autoantibodies to Cancer Antigens with Chemically S-Cationized Full-Length and Water-Soluble Denatured Proteins.

Humoral immune responses against tumor-associated antigens (TAAs) or cancer/testis antigens (CTAs) aberrantly expressed in tumor cells are frequently observed in cancer patients. Recent clinical studies have elucidated that anticancer immune responses with increased levels of anti-TAA/CTA antibodies improve cancer survival rates. Thus, these antibody levels are promising biomarkers for diagnosing the efficiency of cancer immunotherapy.

Generation, Characterization, and Reactivity of a Cu(II)-Alkylperoxide/Anilino Radical Complex: Insight into the O-O Bond Cleavage Mechanism.

The reaction of [Cu(I)(TIPT3tren) (CH3CN)]ClO4 (1) and cumene hydroperoxide (C6H5C(CH3)2OOH, ROOH) at -60 °C in CH2Cl2 gave a Cu(II)-alkylperoxide/anilino radical complex 2, the formation of which was confirmed by UV-vis, resonance Raman, EPR, and CSI-mass spectroscopy. The mechanism of formation of 2, as well as its reactivity, has been explored.

Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding.

As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase bearing a putative metal binding site leads to the emergence of peroxidase activity ( 7.8 × 10 s, 1.

Direct hydroxylation of benzene to phenol using hydrogen peroxide catalyzed by nickel complexes supported by pyridylalkylamine ligands.

Selective hydroxylation of benzene to phenol has been achieved using H2O2 in the presence of a catalytic amount of the nickel complex [Ni(II)(tepa)](2+) (2) (tepa = tris[2-(pyridin-2-yl)ethyl]amine) at 60 °C. The maximum yield of phenol was 21% based on benzene without the formation of quinone or diphenol. In an endurance test of the catalyst, complex 2 showed a turnover number (TON) of 749, which is the highest value reported to date for molecular catalysts in benzene hydroxylation with H2O2.

Cytotoxic T lymphocytes block tumor growth both by lytic activity and IFNγ-dependent cell-cycle arrest.

To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors and B16 melanoma cells as targets. In addition to upregulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell-cycle-promoting genes were downregulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing a fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) and performed CTL therapy in mice bearing B16-fucci tumors.

Geometric control of nuclearity in copper(I)/dioxygen chemistry.

Copper(I) complexes supported by a series of N3-tridentate ligands bearing a rigid cyclic diamine framework such as 1,5-diazacyclooctane (L8, eight-membered ring), 1,4-diazacycloheptane (L7, seven-membered ring), or 1,4-diazacyclohexane (L6, six-membered ring) with a common 2-(2-pyridyl)ethyl side arm were synthesized and their reactivity toward O2 were compared. The copper(I) complex of L8 preferentially provided a mononuclear copper(II) end-on superoxide complex S as reported previously [Itoh, S., et al.

Redox chemistry of nickel(II) complexes supported by a series of noninnocent β-diketiminate ligands.

Nickel complexes of a series of β-diketiminate ligands ((R)L(-), deprotonated form of 2-substituted N-[3-(phenylamino)allylidene]aniline derivatives (R)LH, R = Me, H, Br, CN, and NO2) have been synthesized and structurally characterized. One-electron oxidation of the neutral complexes [Ni(II)((R)L(-))2] by AgSbF6 or [Ru(III)(bpy)3](PF6)3 (bpy = 2,2'-bipyridine) gave the corresponding metastable cationic complexes, which exhibit an EPR spectrum due to a doublet species (S = 1/2) and a characteristic absorption band in near IR region ascribable to a ligand-to-ligand intervalence charge-transfer (LLIVCT) transition. DFT calculations have indicated that the divalent oxidation state of nickel ion (Ni(II)) is retained, whereas one of the β-diketiminate ligands is oxidized to give formally a mixed-valence complex, [Ni(II)((R)L(-))((R)L(•))](+).

[A case of hypoglycemia caused by the accidental ingestion of glimepiride in an elderly dementia patient diagnosed based on the serum glimepiride concentration].

A 77-year-old man being treated for Alzheimer-type dementia and an old cerebral infarction was admitted to our hospital due to disturbance of consciousness. The patient's Mini-mental State Examination and Hasegawa Dementia Scale scores were 23 and 17 points, respectively. His blood glucose level was low (18 mg/dl), with a relatively high insulin level (15.