Metal-Halide Covalency, Exchange Coupling, and Slow Magnetic Relaxation in Triangular (CpiPr5)3U3X6 (X = Cl, Br, I) Clusters.

The actinide elements are attractive alternatives to transition metals or lanthanides for the design of exchange-coupled multinuclear single-molecule magnets. However, the synthesis of such compounds is challenging, as is unraveling any contributions from exchange coupling to the overall magnetism. To date, only a few actinide compounds have been shown to exhibit exchange coupling and single-molecule magnetism. Here, we report triangular uranium(III) clusters of the type (CpiPr5)3U3X (1-X; X = Cl, Br, I; CpiPr5 = pentaisopropylcyclopentadienyl), which are synthesized via reaction of the aryloxide-bridged precursor (CpiPr5)2U2(OPhtBu)4 with excess Me3SiX. Spectroscopic analysis suggests the presence of covalency in the uranium-halide interactions arising from 5f orbital participation in bonding. The dc magnetic susceptibility data reveal the presence of antiferromagnetic exchange coupling between the uranium(III) centers in these compounds, with the strength of the exchange decreasing down the halide series. Ac magnetic susceptibility data further reveal all compounds to exhibit slow magnetic relaxation under zero dc field. In 1-I, which exhibits particularly weak exchange, magnetic relaxation occurs via a Raman mechanism associated with the individual uranium(III) centers. In contrast, for 1-Br and 1-Cl, magnetic relaxation occurs via an Orbach mechanism, likely involving relaxation between ground and excited exchange-coupled states. Significantly, in the case of 1-Cl, magnetic relaxation is sufficiently slow such that open magnetic hysteresis is observed up to 2.75 K, and the compound exhibits a 100-s blocking temperature of 2.4 K. This compound provides the first example of magnetic blocking in a compound containing only actinide-based ions, as well as the first example involving the uranium(III) oxidation state.

Development of Ribityllumazine Analogue as Mucosal-Associated Invariant T Cell Ligands.

Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T cells abundant in human tissues that play a significant role in defense against bacterial and viral infections and in tissue repair. MAIT cells are activated by recognizing microbial-derived small-molecule ligands presented by the MHC class I related-1 protein. Although several MAIT cell modulators have been identified in the past decade, potent and chemically stable ligands remain limited. Herein, we carried out a structure-activity relationship study of ribityllumazine derivatives and found a chemically stable MAIT cell ligand with a pteridine core and a 2-oxopropyl group as the Lys-reactive group. The ligand showed high potency in a cocultivation assay using model cell lines of antigen-presenting cells and MAIT cells. The X-ray crystallographic analysis revealed the binding mode of the ligand to MR1 and the T cell receptor, indicating that it forms a covalent bond with MR1 via Schiff base formation. Furthermore, we found that the ligand stimulated proliferation of human MAIT cells in human peripheral blood mononuclear cells and showed an adjuvant effect in mice. Our developed ligand is one of the most potent among chemically stable MAIT cell ligands, contributing to accelerating therapeutic applications of MAIT cells.

Archaeal Glycerolipids Are Recognized by C-Type Lectin Receptor Mincle.

Recently, various metabolites derived from host microbes have been reported to modulate the immune system, with potential involvement in health or diseases. Archaea, prokaryotic organisms, are present in the human body, but their connection with the host is largely unknown when compared to other microorganisms such as bacteria. This study focused on unique glycerolipids from symbiotic methanogenic archaea and evaluated their activities toward an innate immune receptor. The results revealed that archaeal lipids were recognized by the C-type lectin receptor Mincle and induced immune responses. A concurrent structure-activity relationship study identified the key structural features of archaeal lipids required for recognition by Mincle. Subsequent gene expression profiling suggested qualitative differences between the symbiotic archaeal lipid and the pathogenic bacteria-derived lipid. These findings have broad implications for understanding the function of symbiotic archaea in host health and diseases.

RAB GTPases and SNAREs at the trans-Golgi network in plants.

Membrane traffic is a fundamental cellular system to exchange proteins and membrane lipids among single membrane-bound organelles or between an organelle and the plasma membrane in order to keep integrity of the endomembrane system. RAB GTPases and SNARE proteins, the key regulators of membrane traffic, are conserved broadly among eukaryotic species. However, genome-wide analyses showed that organization of RABs and SNAREs that regulate the post-Golgi transport pathways is greatly diversified in plants compared to other model eukaryotes. Furthermore, some organelles acquired unique properties in plant lineages. Like in other eukaryotic systems, the trans-Golgi network of plants coordinates secretion and vacuolar transport; however, uniquely in plants, it also acts as a platform for endocytic transport and recycling. In this review, we focus on RAB GTPases and SNAREs that function at the TGN, and summarize how these regulators perform to control different transport pathways at the plant TGN. We also highlight the current knowledge of RABs and SNAREs' role in regulation of plant development and plant responses to environmental stimuli.

Establishment and characterization of highly osteolytic luminal breast cancer cell lines by intracaudal arterial injection.

Bone is one of the most common metastatic sites of breast cancer, and bone metastasis profoundly affects the quality of life of breast cancer patients. Bone metastasis is commonly observed among all the subtypes of breast cancer; however, its molecular mechanism has been analyzed only in triple-negative subtype of breast cancer (TNBC). To characterize the molecular mechanisms of bone metastasis of luminal breast cancer, we established a bone-metastatic model of the MCF7, luminal breast cancer cell line, with enhanced osteolytic activity by intracaudal arterial injection (CAI). Pathological analysis of the established cell lines revealed that they exhibited fierce osteolytic ability by promoting osteoclast differentiation and activity. The signature genes extracted from highly osteolytic MCF7 cell lines were differed from those of bone-metastatic TNBC cell lines. Our results suggest that unique mechanisms of osteolysis in bone-metastatic lesions of luminal breast cancer. In addition, several up-regulated genes in MCF7-BM (Bone Metastasis) 02 cell lines correlated with poor prognosis with luminal breast cancer patients. Our findings support further study on the bone-metastatic mechanisms of luminal breast cancer.

Distinct sets of tethering complexes, SNARE complexes, and Rab GTPases mediate membrane fusion at the vacuole in Arabidopsis.

Membrane trafficking plays pivotal roles in various cellular activities and higher-order functions of eukaryotes and requires tethering factors to mediate contact between transport intermediates and target membranes. Two evolutionarily conserved tethering complexes, homotypic fusion and protein sorting (HOPS) and class C core vacuole/endosome tethering (CORVET), are known to act in endosomal/vacuolar transport in yeast and animals. Both complexes share a core subcomplex consisting of Vps11, Vps18, Vps16, and Vps33, and in addition to this core, HOPS contains Vps39 and Vps41, whereas CORVET contains Vps3 and Vps8. HOPS and CORVET subunits are also conserved in the model plant Arabidopsis. However, vacuolar trafficking in plants occurs through multiple unique transport pathways, and how these conserved tethering complexes mediate endosomal/vacuolar transport in plants has remained elusive. In this study, we investigated the functions of VPS18, VPS3, and VPS39, which are core complex, CORVET-specific, and HOPS-specific subunits, respectively. Impairment of these tethering proteins resulted in embryonic lethality, distinctly altering vacuolar morphology and perturbing transport of a vacuolar membrane protein. CORVET interacted with canonical RAB5 and a plant-specific R-soluble NSF attachment protein receptor (SNARE), VAMP727, which mediates fusion between endosomes and the vacuole, whereas HOPS interacted with RAB7 and another R-SNARE, VAMP713, which likely mediates homotypic vacuolar fusion. These results indicate that CORVET and HOPS act in distinct vacuolar trafficking pathways in plant cells, unlike those of nonplant systems that involve sequential action of these tethering complexes during vacuolar/lysosomal trafficking. These results highlight a unique diversification of vacuolar/lysosomal transport that arose during plant evolution, using evolutionarily conserved tethering components.

Peptide Sequence Mapping around Bisecting GlcNAc-Bearing N-Glycans in Mouse Brain.

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.

Controlled Dimerization and Bonding Scheme of Icosahedral M@Au12 (M=Pd, Pt) Superatoms.

Targeted syntheses of MM'Au36 (PET)24 (M, M'=Pd, Pt; PET=SC2 H4 Ph) were achieved by hydride-mediated fusion reactions between [MAu8 (PPh3 )8 ]2+ and [M'Au24 (PET)18 ]- . Single-crystal X-ray diffraction analysis indicated that the products have bi-icosahedral MM'Au21 cores composed of M@Au12 and M'@Au12 superatoms. Although the MM'Au21 superatomic molecules correspond to O2 in terms of the number of valence electrons (12 e), the distances between the icosahedrons were larger than that in the bi-icosahedral Au23 core of Au38 (PET)24 corresponding to F2 and the spin state was singlet. These counterintuitive results were explained by a "bent bonding model" based on tilted (non-orthogonal) bonding interaction between the 1P superatomic orbitals of M@Au12 and M'@Au12 superatoms.

Glucoselysine is derived from fructose and accumulates in the eye lens of diabetic rats.

Prolonged hyperglycemia generates advanced glycation end-products (AGEs), which are believed to be involved in the pathogenesis of diabetic complications. In the present study, we developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae. Although the molecular weight of GL was identical to that of fructoselysine (FL), GL was distinguishable from FL because GL was resistant to acid hydrolysis, which converted all of the FLs to furosine. We also detected GL in vitro when reduced BSA was incubated with fructose for 1 day. However, when we incubated reduced BSA with glucose, galactose, or mannose for 14 days, we did not detect GL, suggesting that GL is dominantly generated from fructose. LC-ESI-MS/MS experiments with synthesized [13C6]GL indicated that the GL levels in the rat eye lens time-dependently increase after streptozotocin-induced diabetes. We observed a 31.3-fold increase in GL 8 weeks after the induction compared with nondiabetic rats, and Nϵ-(carboxymethyl)lysine and furosine increased by 1.7- and 21.5-fold, respectively, under the same condition. In contrast, sorbitol in the lens levelled off at 2 weeks after diabetes induction. We conclude that GL may be a useful biological marker to monitor and elucidate the mechanism of protein degeneration during progression of diabetes.

Cholesteryl 6-O-acyl-α-glucosides from diverse Helicobacter spp. signal through the C-type lectin receptor Mincle.

Helicobacter spp. are Gram-negative bacteria that cause a spectrum of disease in the gut, biliary tree and liver. Many Helicobacter spp. produce a range of cholesteryl α-glucosides that have the potential to act as pathogen associated molecular patterns. We report a highly stereoselective α-glucosylation of cholesterol using 3,4,6-tri-O-acetyl-2-O-benzyl-d-glucopyranosyl N-phenyl-2,2,2-trifluoroacetimidate, which allowed the synthesis of cholesteryl α-glucoside (αCG) and representative Helicobacter spp. cholesteryl 6-O-acyl-α-glucosides (αCAGs; acyl = C12:0, 14:0, C16:0, C18:0, C18:1). All αCAGs, irrespective of the nature of their acyl chain composition, strongly agonised signalling through the C-type lectin receptor Mincle from human and mouse to similar degrees. By contrast, αCG only weakly signalled through human Mincle, and did not signal through mouse Mincle. These results provide a molecular basis for understanding of the immunobiology of non-pylori Helicobacter infections in humans and other animals.

Autocrine and Paracrine Interactions between Multiple Myeloma Cells and Bone Marrow Stromal Cells by Growth Arrest-specific Gene 6 Cross-talk with Interleukin-6.

The pathogenesis of multiple myeloma (MM) has not yet been fully elucidated. Our microarray analysis and immunohistochemistry revealed significant up-regulation of growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein with a structural homology with protein S, in bone marrow (BM) cells of MM patients. ELISA showed that the serum levels of soluble Gas6 were significantly increased in the MM patients when compared with healthy controls. Gas6 was overexpressed in the human CD138-positive MM cell line RPMI-8226. Exogenous Gas6 suppressed apoptosis induced by serum deprivation and enhanced cell proliferation of the MM cells. The conditional medium from the human BM stromal cell line HS-5 induced cell proliferation and anti-apoptosis of the MM cells with extracellular signal-regulated kinase, Akt, and nuclear factor-κB phosphorylation, which were reversed by the neutralizing antibody to Gas6 or IL-6. The TAM family receptor Mer, which has been identified as a Gas6 receptor, was overexpressed in BM cells of MM patients. The knockdown of Mer by siRNA inhibited cell proliferation, anti-apoptosis, and up-regulation of intercellular cell adhesion molecule-1 (ICAM-1) in MM cells stimulated by an HS-5 cell-conditioned medium. Furthermore, the Gas6-neutralizing antibody reduced the up-regulation of IL-6 and ICAM-1 induced by a HS-5 cell-conditioned medium in MM cells. The present study provides new evidence that autocrine and paracrine stimulation of Gas6 in concert with IL-6 contributes to the pathogenesis of MM, suggesting that Gas6-Mer-related signaling pathways may be a promising novel target for treating MM.

Donor-π-Acceptor Type Unsymmetrical Triarylborane-Based Fluorophores: Synthesis, Fluorescence Properties, and Photostability.

A two-step synthesis to prepare tricoordinate organoboron compounds bearing three different aryl groups has been developed. After the first aryl substitution to an aryl boronic ester took place, the intermediate species, that is, bis(diarylborinate) species, was isolated as an air- and moisture-stable solid, which allowed the second aryl substitution to carry out in a selective manner. Subsequently, a series of unsymmetrical triarylboranes possessing a sterically bulky aryl group, triarylamine moiety, and para-functionalized phenyl ring was synthesized. Not only did these triarylboranes exhibit remarkable solvent-dependent fluorescence as expected for donor-π-acceptor (D-π-A) systems, they were also accompanied by profound persistence against photoirradiation especially for that bearing a 1,3,5-tri- tert-butylphenyl ring. This survey exemplifies that sufficient electronic and steric modification is key to construct photostable D-π-A type triarylborane-based fluorophores.

Visual Search Load Effects on Age-Related Cognitive Decline: Evidence From the Yakumo Longitudinal Study.

The validity of Bucur and Madden's (2010) proposal that an age-related decline is particularly pronounced in executive function measures rather than in elementary perceptual speed measures was examined via the Yakumo Study longitudinal database. Their proposal suggests that cognitive load differentially affects cognitive abilities in older adults. To address their proposal, linear regression coefficients of 104 participants were calculated individually for the digit cancellation task 1 (D-CAT1), where participants search for a given single digit, and the D-CAT3, where they search for 3 digits simultaneously. Therefore, it can be conjectured that the D-CAT1 represents primarily elementary perceptual speed and low-visual search load task. whereas the D-CAT3 represents primarily executive function and high-visual search load task. Regression coefficients from age 65 to 75 for the D-CAT3 showed a significantly steeper decline than that for the D-CAT1, and a large number of participants showed this tendency. These results support the proposal by Brcur and Madden (2010) and suggest that the degree of cognitive load affects age-related cognitive decline.

[Exploring the Possibility of New Biomarkers of Immune Checkpoint Inhibitors for Non-Small Cell Lung Cancer (NSCLC)].

Mutation burden in a tumor, presumably involving neo-antigens in the tumor tissue, is also thought to be one of the better predictors for the efficacy of immune checkpoint inhibitors. However, it is difficult to analyze the mutation burden routinely in the clinic. Here, we describe more convenient factors that can be used as surrogate markers of mutation burden. Ninety-four patients with NSCLC who underwent resection in our institution were recruited for this study. Mutation burden and major gene alterations were analyzed by using next generation sequencing. Several immunological parameters were also assessed using immunohistochemistry. Statistical analysis was performed on mutation burden, major gene alternations, immunohistochemistry, and clinical parameters. The median mutation load was 54 mutations(range, 10-363 mutations). Squamous cell carcinoma, EGFRmutation -negativity, and TP53 alteration-positivity were closely connected with higher mutation burden. Multiple regression analysis showed that mutation burden in the tumor could be associated with EGFRmutation and TP53 alteration status.

Modulation of Plant RAB GTPase-Mediated Membrane Trafficking Pathway at the Interface Between Plants and Obligate Biotrophic Pathogens.

RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.

Mass isotopomer analysis of metabolically labeled nucleotide sugars and N- and O-glycans for tracing nucleotide sugar metabolisms.

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.

Developmental trajectories of verbal and visuospatial abilities in healthy older adults: comparison of the hemisphere asymmetry reduction in older adults model and the right hemi-ageing model.

Two models of cognitive ageing, the hemisphere asymmetry reduction in older adults (HAROLD) model and the right hemi-ageing model, were compared based upon the verbal memory and visuospatial task performance of 338 elderly participants. Comparison of the developmental trajectories for four age groups (50s, 60s, 70s and 80s) supported the HAROLD model, but not the right hemi-ageing model. Performance differences between the verbal memory and visuospatial tasks in the earlier age groups decreased in the later age groups. There was a sex difference in the cognitive-decline trajectories for verbal and visuospatial task performance after the 50s.