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Background: An association between a high intake of marine-derived n-3 polyunsaturated fatty acids (n-3 PUFAs) with a lower risk of coronary heart disease was previously reported. However, the association between n-3 PUFAs and cerebrovascular lesions remains unclear. We evaluated this association in a general-population-based sample of Japanese men. Methods: Participants were community-dwelling men (40–79 years old) living in Kusatsu City, Shiga, Japan. Serum concentrations of n-3 PUFAs, defined as the sum of eicosapentaenoic and docosahexaenoic acids, were measured via gas-liquid chromatography between 2006 and 2008. Magnetic resonance imaging was used to assess cerebrovascular lesions (including intracerebral large-artery stenosis, lacunar infarction, and microbleeds) and white matter lesions between 2012 and 2015. Logistic regression adjusting for conventional cardiovascular risk factors was used to estimate the odds ratio of prevalent cerebrovascular lesions per 1 standard deviation higher serum concentration of n-3 PUFAs. Results: Of a total of 739 men, the numbers (crude prevalence in %) of prevalent cerebral large-artery stenoses, lacunar infarctions, microbleeds, and white matter lesions were 222 (30.0), 162 (21.9), 103 (13.9), and 164 (22.2), respectively. A 1 standard deviation higher concentration of n-3 PUFAs (30.5 μmol/L) was independently associated with lower odds of cerebral large-artery stenosis (multivariable-adjusted odds ratio, 0.80; 95% confidential interval, 0.67–0.97). There were no significant associations of n-3 PUFAs with the other types of lesions. Conclusions: n-3 PUFAs may have protective effects against large-artery stenosis, but not small vessel lesions, in the brain. [ABSTRACT FROM AUTHOR]

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N -acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-β- d -GlcA-(1→4)-β- d -Glc-(1→3)-β- d -GalNAc-(1→4)-α- d -GalNAc-(1→4)-α- d -GalN-(1→] n (β- d -Glc and α- d -GalNAc are stoichiometrically and substoichiometrically 3- O -acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α- d -GalN and β- d -GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath. [ABSTRACT FROM AUTHOR]

The depolymerization of lignocellulosic biomass is facilitated by feruloyl esterases (FAEs), which hydrolyze ester bonds between lignin and polysaccharides. Fungal FAEs belonging to subfamily (SF) 6 release precursors such as ferulic acid derivatives, attractive for biochemical production. Among these, Aspergillus sydowii FAE (As FaeE), an SF6 FAE, exhibits remarkable activity across various substrates. In this study, we conducted X-ray crystallography and kinetic analysis to unravel the molecular mechanisms governing substrate recognition and catalysis by As FaeE. As FaeE exhibits a typical α/β -hydrolase fold, characterized by a catalytic triad of serine, aspartate, and histidine. Comparative analysis of substrate-free, ferulic acid-bound, and sinapic acid-bound forms of As FaeE suggests a conformational change in the loop covering the substrate-binding pocket upon binding. Notably, Pro158 and Phe159 within this loop cover the phenolic part of the substrate, forming three layers of planar rings. Our structure-based functional mutagenesis clarifies the roles of the residues involved in substrate binding and catalytic activity. Furthermore, distinct substrate-binding mechanisms between As FaeE and other studied FAEs are identified. This investigation offers the initial structural insights into substrate recognition by SF6 FAEs, equipping us with structural knowledge that might facilitate the design of FAE variants capable of efficiently processing a wider range of substrate sizes. • 3D structures of As FaeE in substrate-free and substrate-bound forms were revealed. • The loop covering the substrate-binding pocket plays a pivotal role in binding. • Functional roles of the residues in the substrate-binding pocket are unveiled. • Mutants with different substrate preferences were found. • SF6 FAE's preference towards monomeric hydroxycinnamates is rationally understood. [ABSTRACT FROM AUTHOR]

β-1,4-glucosaminoglucan (GG) was prepared from the sheath of a sulfur-oxidizing bacterium Thiothrix nivea. Recently, GG was found to be adsorbed by cellulose (paper) and is therefore potentially applicable as an aminating agent for cellulose. We attempted to increase the yield of GG using a fed-batch cultivation method. Furthermore, the behavior of GG molecules in water was theoretically and experimentally investigated. NMR analysis in combination with molecular dynamics calculation suggested that GG molecules tend to form soluble aggregates in water. It was experimentally revealed that the self-aggregation is enhanced by the addition of NaCl and reduced temperature. Adsorption of GG onto cellulose via hydrogen bonding was confirmed by molecular dynamics simulation. Adsorption was also promoted in the presence of NaCl but was inhibited by a reduction in temperature. Only 11% of the amino groups in the GG-treated paper was reactive, suggesting that GG molecules adsorbed by the paper were forming aggregates. Attachment of glucosaminoglucan chains and their self-aggregates to cellulose crystalline assemblage via hydrogen bond during molecular dynamics simulation. [ABSTRACT FROM AUTHOR]

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium commonly found in activated sludge. The filament of this bacterium is covered with a sheath. The sheath is an assemblage of macromolecular glucosaminoglucan (GG), [4)-β- d -GlcN-(1 → 4)-β- d -Glc-(1 → ] n , modified with an unidentified deoxy-sugar at position 3 of Glc. GG was obtained by dialysis after the partial hydrolysis of the sheath. The GG hydrogel was prepared by drying a GG solution. Then, the hydrogel was N -acetylated to prepare a stable hydrogel of N -acetylglucosaminoglucan (NGG), [4)-β- d -GlcNAc-(1 → 4)-β- d -Glc-(1 → ] n . The NGG hydrogel was stable in phosphate buffer but was disrupted by lysozyme addition, suggesting that NGG is susceptible to lysozyme degradation and has potential for medical use. The GG solution was N -acetylated to prepare a NGG suspension to confirm enzymatic degradation. The turbidity of the NGG suspension was decreased by lysozyme addition. Sugars released in the reaction mixture were derivatized with 4-aminobenzoic acid ethyl ester (ABEE) followed by HPLC analysis. Two major derivatives were detected, and their concentration was increased in reverse proportion to the turbidity of the reaction mixture. The derivatives were identified as GlcNAc-Glc-GlcNAc-Glc-ABEE and GlcNAc-Glc-ABEE by mass spectrometry. Consequently, NGG was found to be degraded by lysozyme via a mechanism similar to that of chitin degradation. [ABSTRACT FROM AUTHOR]

Background & Aims: A fiber-rich diet has a cardioprotective effect, but the mechanism for this remains unclear. We hypothesized that a fiber-rich diet with brown rice improves endothelial function in patients with type 2 diabetes mellitus. Methods: Twenty-eight patients with type 2 diabetes mellitus at a single general hospital in Japan were randomly assigned to a brown rice (n = 14) or white rice (n = 14) diet and were followed for 8 weeks. The primary outcome was changes in endothelial function determined from flow debt repayment by reactive hyperemia using strain-gauge plethysmography in the fasting state. Secondary outcomes were changes in HbA1c, postprandial glucose excursions, and markers of oxidative stress and inflammation. The area under the curve for glucose after ingesting 250 kcal of assigned rice was compared between baseline (T0) and at the end of the intervention (T1) to estimate glucose excursions in each group. Results: Improvement in endothelial function, assessed by fasting flow debt repayment (20.4% vs. −5.8%, p = 0.004), was significantly greater in the brown rice diet group than the white rice diet group, although the between-group difference in change of fiber intake was small (5.6 g/day vs. −1.2 g/day, p<0.0001). Changes in total, HDL-, and LDL-cholesterol, and urine 8-isoprostane levels did not differ between the two groups. The high-sensitivity C-reactive protein level tended to improve in the brown rice diet group compared with the white rice diet group (0.01 μg/L vs. −0.04 μg/L, p = 0.063). The area under the curve for glucose was subtly but consistently lower in the brown rice diet group (T0: 21.4 mmol/L*h vs. 24.0 mmol/L*h, p = 0.043, T1: 20.4 mmol/L*h vs. 23.3 mmol/L*h, p = 0.046) without changes in HbA1c. Conclusions: Intervention with a fiber-rich diet with brown rice effectively improved endothelial function, without changes in HbA1c levels, possibly through reducing glucose excursions. [ABSTRACT FROM AUTHOR]

Thiothrix fructosivorans forms a microtube (sheath) that encloses a line of cells. This sheath is an assemblage of [→4)-GlcN-(1 → 4)-Glc-(1→] n with side chains of Rha4N-(1 → 3)-Fuc(1→ at position 3 of Glc. The sheath-forming polysaccharide (SFP) may have some substitutions but this is not yet confirmed. To investigate the possible substitutions, the sheath was prepared by mild treatments. Solid-state NMR analysis suggested the presence of N -substitution. The sheath was hydrolyzed with concentrated HCl at 0 °C, followed by derivatization with 4-aminobenzoic acid ethyl ester (ABEE). The presence of N -lactyl-Rha4N-Fuc-ABEE was suggested by NMR spectroscopy. Lactic acid was determined to be the l -isomer by chiral HPLC analysis. To estimate the N -lactylation degree, the sheath was N -acetylated. N -Acetyl-Rha4N-Fuc-ABEE and N -lactyl-Rha4N-Fuc-ABEE were then collectively recovered, and their abundance ratio was determined to be 1:4 by NMR analysis. When hydrolysis was performed at 40 °C, GlcNAc-ABEE was obtained. For estimation of the N -acetylation degree, the sheath was N -acetylated with deuterated acetic anhydride and then N -acetyl-GlcN-ABEE was prepared. The content of deuterated N -acetyl-GlcN-ABEE was determined to be 50% based on the relative intensity of the acetyl proton signal in the 1D- 1 H NMR spectrum. It was concluded that Rha4N is mostly N - l -lactylated and GlcN is substoichiometrically N -acetylated. [ABSTRACT FROM AUTHOR]

An RNA aptamer that strongly binds to a target molecule has the potential to be a nucleic acid drug inside living human cells. To investigate and improve this potential, it is critical to elucidate the structure and interaction of RNA aptamers inside living cells. We examined an RNA aptamer for HIV-1 Tat (TA), which had been found to trap Tat and repress its function in living human cells. We first used in vitro NMR to examine the interaction between TA and a part of Tat containing the binding site for trans-activation response element (TAR). It was revealed that two U-A∗U base triples are formed in TA upon binding of Tat. This was assumed to be critical for strong binding. Then, TA in complex with a part of Tat was incorporated into living human cells. The presence of two U-A∗U base triples was also revealed for the complex in living human cells by in-cell NMR. Thus, the activity of TA in living human cells was rationally elucidated by in-cell NMR. [ABSTRACT FROM AUTHOR]

Abstract: A sheath-forming and sulfur-oxidizing bacterium, Thiothrix fructosivorans, was heterotrophically cultured. The sheath, which is an extracellular microtube, was prepared by selectively removing the cells using lysozyme, sodium dodecyl sulfate, and sodium hydroxide. Solid-state 13C-nuclear magnetic resonance (NMR) spectrum revealed that the sheath is assembled from a glycan possessing acetyl and methyl groups. When the sheath was deacetylated, the original microtube structure was lost and the sheath became soluble under acidic conditions, revealing the importance of acetyl groups in maintaining the sheath structure. Equimolar d-glucose, d-glucosamine, and l-fucose were detected in the acid hydrolysate of the sheath by gas liquid chromatography. In addition to these sugars, β-GlcN-(1→4)-Glc and unidentified sugar were detected by analyzing the hydrolysate using high-performance liquid chromatography analysis. 1H and 13C NMR spectroscopy was used to identify a disaccharide composed of 4-deoxy-4-aminorhamnose (perosamine, Rha4N) and fucose. N-Acetyl-perosamine prepared from the disaccharide was polarimetric and exhibited a d-configuration. The previously unidentified disaccharide was found to be α-d-Rhap4N-(1→3)-d-Fuc. According to 1H and 13C NMR analyses, the deacetylated sheath-forming polysaccharide was found to h have a main chain of [→4)-β-d-GlcpN-(1→4)-β-d-Glcp-(1→] n , to which disaccharide side chains of α-d-Rhap4N-(1→3)-α-l-Fucp-(1→ were attached at position 3 of Glc. [Copyright &y& Elsevier]

Abstract: Sphaerotilus natans is a filamentous sheath-forming bacterium, commonly found in bulking activated sludge. The bulky nature of this bacterium is caused by an extracellular polysaccharide (EPS). EPS is a linear acidic polysaccharide with the following chemical structure: [→4)-α-d-Glcp-(1→2)-β-d-GlcpA-(1→2)-α-l-Rhap-(1→3)-β-l-Rhap-(1→]n. 1H–1H and 1H–13C correlation nuclear magnetic resonance (NMR) experiments were performed to acquire nuclear Overhauser effect signals, which were used for conformational elucidation. Molecular mechanics calculations were performed on each disaccharide unit of the EPS building blocks. On the basis of the results of the calculations, the conformation of a pentasaccharide fragment was estimated. After confirmation of the coincidence between the NMR data and the predicted conformation of the pentasaccharide fragment, the conformation of a heptadecasaccharide fragment was estimated using the same procedure. The heptadecasaccharide was found to form a (12/1) helix and take locally folded chain, which is attributed to a triangular arrangement formed by a series of residues, α-d-Glcp-(1→2)-β-d-GlcpA-(1→2)-α-l-Rhap. This arrangement is caused by the peculiar consecutive (1→2) linkages and reinforced by a hydrogen bond between the α-glucosyl and α-rhamnosyl residues. Considering the steric hindrance due to this triangular arrangement, EPS molecules are not supposed to form double helix in an aqueous environment. We propose the name ‘sphaeran’ to refer to this unique chemical structure and properties of EPS. [Copyright &y& Elsevier]

We introduced an isotopically labeled RNA aptamer for HIV-1 Tat prepared by E. coli transcription into HeLa cells. We successfully recorded the first heteronuclear 2D in-cell NMR spectra, which makes it possible to study the interaction of the RNA aptamer with argininamide in living human cells with higher resolution. [ABSTRACT FROM AUTHOR]

Abstract: A sheath-forming sulfa oxidizer, Thiothrix nivea, was mixotrophically cultured in a medium supplemented with acetic acid and sodium disulfide. Its sheath, a microtube-like extracellular supermolecule, was prepared by selectively removing the cells with lysozyme, sodium dodecyl sulfate, and sodium hydroxide. The sheath was not visibly affected by hydrazine treatment, suggesting that it is not a proteinous supermolecule. From the acid hydrolysate of the sheath, glucose and glucosamine were detected in an approximate molar ratio of 1:1. Three other saccharic compounds were detected and recovered by HPLC as fluorescent derivatives prepared by reaction with 4-aminobenzoic acid ethyl ester. Nuclear magnetic resonance (NMR) analysis suggested that one of the derivatives was derived from an unidentified deoxypentose. NMR analysis for the other 2 derivatives showed that they were derived from β-1,4-linked disaccharides and tetrasaccharides, which were composed of glucose and glucosamine. The sheath was readily broken down by weak HCl treatment, releasing an unidentified deoxypentose and polymer. Chemical analysis showed the presence of β-1,4-linked d-Glcp and d-GlcNp in the polymer. NMR analysis revealed that the polymer had a repeating unit of →4)-d-Glcp-(β1→4)-d-GlcNp-(β1→. The solid-state 1D-13C NMR spectrum of the polymer in N-acetylated form supported this result. The molecular weight of the polymer was estimated to be 8.2×104 by size exclusion chromatography. Based on these results, the sheath of T. nivea is hypothesized to be assembled from alternately β-1,4-linked glucosaminoglucan grafted with unidentified deoxypentose. [Copyright &y& Elsevier]

Abstract: A typical filamentous bacterium, Sphaerotilus natans, secretes a thiolic glycoconjugate which is assembled into a microtube, so called sheath. The glycoconjugate is known to consist of a pentasaccharide-dipeptide repeating unit, but its chemical structure has not been completely elucidated. In order to determine its chemical structure, the sheath was broken down by performic acid oxidation. The released sulfonated derivative was water soluble which was suitable for detailed NMR analysis. The data exhibited the presence of two stoichiometric and one substoichiometric (relative abundance was about 0.5) acetylations, suggesting that the glycoconjugate is composed of two equimolar pentasaccharide-dipeptide repeating units each having either two or three acetyl groups. However, the position of substoichiometric acetylation could not be defined. To determine the position, the sheath was derivatized with a thiol selective fluorescent reagent followed by digestion with a specific polysaccharide lyase prepared from a sheath-degrading bacterium, Paenibacillus koleovorans. As expected, two fluorescent digests were recovered by reverse-phase HPLC and were subjected to NMR analysis. The data revealed that both digests are pentasaccharide-dipeptides which have unsaturated glucuronic acid and galactosamine residues at their reducing and non-reducing ends, respectively. It was also confirmed that one digest has 3-O-acetylated glucose residue while the other has non-derivatized glucose residue. The substoichiometric acetylation was thus identified with the 3-O-acetylation, and structural determination of the thiolic glycoconjugate was completed. By virtue of the clarification of the two digests’ structures, the cleavage site was specified as (1→4)-α-galactosaminic bond to glucuronic acid. Based on the present and earlier findings, we propose a novel glycoconjugate category named thiopeptidoglycan and a novel polysaccharide lyase named thiopeptidoglycan lyase. [Copyright &y& Elsevier]

Abstract: The sheath of Leptothrix cholodnii is constructed from a structural glycoconjugate, a straight-chained amphoteric heteropolysaccharide modified with glycine and cysteine. Though the structure of the glycan core is already determined, its modifications with amino acids and other molecules are not fully resolved. In this study, we aimed to determine the chemical structure of the glycoconjugate as a whole. Enantiomeric determination of cysteine in the sheath was performed and as a result, l-cysteine was detected. NMR spectroscopy was endeavored to determine overall structure of the glycoconjugate. Prior to NMR analysis, solubilization of the glycoconjugate was attempted by adding denaturing reagents or by derivatization. As far as tested, sulfonation by performic acid oxidation was suitable for solubilization, but further improvement was achieved by N-acetylation. The approximate molecular weight of the derivative was estimated to be 4.5×104 by size-exclusion chromatography. The NMR studies for the sulfonated glycoconjugate and its N-acetylated derivative revealed that the sheath glycoconjugate is a glycosaminoglycan consisting of a pentasaccharide repeating unit which is substoichiometrically esterified with 3-hydroxypropionic acid and stoichiometrically amidated with acetic acid and glycyl-l-cysteine. [Copyright &y& Elsevier]

The Shiga Epidemiological Study of Subclinical Atherosclerosis was conducted in Kusatsu City, Shiga, Japan, from 2006 to 2008. Participants were measured for LDL-p through nuclear magnetic resonance technology. 740 men participated in follow-up and underwent 1.5 T brain magnetic resonance angiography from 2012 to 2015. Participants were categorized as no-ICAS, and ICAS consisted of mild-ICAS (1 to < 50%) and severe-ICAS (≥ 50%) in any of the arteries examined. After exclusion criteria, 711 men left for analysis, we used multiple logistic regression to examine the association between lipid profiles and ICAS prevalence. Among the study participants, 205 individuals (28.8%) had ICAS, while 144 individuals (20.3%) demonstrated discordance between LDL-c and LDL-p levels. The discordance "low LDL-c–high LDL-p" group had the highest ICAS risk with an adjusted OR (95% CI) of 2.78 (1.55–5.00) in the reference of the concordance "low LDL-c–low LDL-p" group. This was followed by the concordance "high LDL-c–high LDL-p" group of 2.56 (1.69–3.85) and the discordance "high LDL-c–low LDL-p" group of 2.40 (1.29–4.46). These findings suggest that evaluating LDL-p levels alongside LDL-c may aid in identifying adults at a higher risk for ICAS. [ABSTRACT FROM AUTHOR]

Objective: SGLT2 inhibitors reduce body weight (BW) due to negative energy balance. Although previous reports suggested that SGLT2 inhibitor improved liver dysfunction and steatosis, the efficacy of SGLT2 inhibitor on intrahepatic triglyceride (IHTG) were merely quantified. Methods: The study was an open label randomized controlled trial. Fifty-two overweight patients (BMI > 23) who had inadequate glycemic control with oral antidiabetic agents were randomly assigned to dapagliflozin (Dapa) or standard treatment (Con) and followed for 24 weeks. The Primary endpoint was the change in BW from baseline to week 24 between the two groups. The secondary endpoint was IHTG which was assessed by magnetic resonance spectroscopy (MRS). Results: The change in BW was significantly larger in Dapa group compared to Con. The mean difference in changes between two groups was -1.72 kg (95% CI: -2.85, -0.59, p = 0.004) which was comparable with the previous studies of Caucasians. IHTG measured with MRS was significantly reduced in Dapa group (p = 0.033). Conclusion: Dapagliflozin treatment for 24 weeks resulted in reduction of BW and IHTG. These findings suggest the therapeutic potential of dapagliflozin for patients with NAFLD. Clinical study registration number: UMIN000020239. Disclosure: K. Morino: Research Support; Self; Astellas Pharma Inc., AstraZeneca, CMIC Pharma Science, Miki Corp., Ono Pharmaceutical Co., Ltd., Sunstar Inc. K. Kondo: None. S. Tanaka: None. I. Miyazawa: None. K. Fuse: None. S. Ida: None. H. Arima: Consultant; Self; Kyowa Hakko Kirin Co., Ltd. Speaker's Bureau; Self; Bayer Yakuhin, Ltd., Daiichi Sankyo Company, Limited, Takeda Pharmaceutical Company Limited, Teijin Pharma Limited. K. Miura: None. N. Ohashi: None. S. Ugi: None. H. Maegawa: Research Support; Self; Antares Pharma, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Takeda Pharmaceutical Company Limited. Funding: AstraZeneca; Ono Pharmaceutical Co., Ltd. [ABSTRACT FROM AUTHOR]

The conformation of lignin in solvents is major key factors governing the physicochemical properties of aromatic polymers. However, the conformational changes of lignin in good and poor solvents is not clearly understood at the molecular level. In this study, short- (DP 2.77) and long-chain (DP 4.49) lignin oligomer model compounds composed solely of interunit β–O–4 bonds with a narrow polydispersity were synthesized, and their NMR spectra were recorded to evaluate the molecular structural changes induced by addition of water to an organic solvent. The spectral signals were completely assigned in DMSO-d6 and D2O by applying 2D 1H–13C HSQC, HMBC, and long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC). The conformation of the long- and short-chain lignin models were analyzed by 2D 1H–1H ROESY. In all the solvent systems, consisting of DMSO-d6 and containing 0–90% volume of acetic acid-d4 buffer in D2O (pD 5.0), the lignin models were found to have folded conformations, but more compact structures were observed in D2O compared with DMSO-d6. [ABSTRACT FROM AUTHOR]

Aims Enzymatic degradation of β-1,4-linked glucose and glucosamine (glucosaminoglucan, GG), which is prepared from Thiothrix nivea and can act as a cellulose-aminating agent with a strong affinity to cellulose, was attempted. Methods and results A chitosanase-secreting fungal strain was isolated as a GG-degrading microbe. GG was found to be degraded by not only chitosanases but also cellulases. Based on nuclear magnetic resonance spectroscopy, both enzymes were found to produce GlcN-Glc from GG. The cellulases also produced GlcN-Glc-GlcN-Glc as an additional final digest. Furthermore, aminated (GG-coated) cellulose nanofibers exhibited cellulase resistance. The flexibility of GG adsorbed onto a cellulose crystal was almost identical to that of cellulose, as estimated via the molecular dynamics calculations. Conclusions The chitosanase and cellulase hydrolyzed the β-1,4-linkage from Glc to GlcN and were expected to recognize the tetramer and hexamer units of GG depending on their final products. The cellulose nanofibers acquired cellulase resistance via amination with GG, probably because of the lower activity of cellulase to GG than cellulose. [ABSTRACT FROM AUTHOR]

Fungal glucuronoyl esterases (FGEs) catalyze cleavage of the ester bond connecting a lignin alcohol to the xylan-bound 4-O-methyl-D-glucuronic acid of glucuronoxylans. Thus, FGEs are capable of degrading lignin-carbohydrate complexes and have potential for biotechnological applications toward woody biomass utilization. Therefore, identification and characterization of new FGEs are of critical importance. Firstly, in this study, we built a phylogenetic tree from almost 400 putative FGEs obtained on BLAST analysis and defined six main clades. In the phylogenetic tree, all the putative FGEs of ascomycetes cluster in clades I to IV, and most of the putative FGEs of basidiomycetes (B-FGEs) cluster in clades V to VI. Interestingly, several B-FGEs were found to cluster in clade II; most FGEs of clade II were found to have higher theoretical isoelectric points than those in the other five clades. To gain an insight into the putative FGEs in the clades that have not been characterized yet, we chose the FGEs of Ceriporiopsis subvermispora (CsGE) and Pleurotus eryngii (PeGE), which belong to clades V and II, respectively. The catalytic domains of both CsGE and PeGE were successfully expressed using Pichia pastoris, and then purified. Benzyl glucuronic acid was used as a substrate to confirm the activities of the CsGE and PeGE, and the hydrolyzed product, glucuronic acid, was quantified spectrophotometrically. Both CsGE and PeGE clearly exhibited the esterase activity. Additionally, we demonstrated that PeGE exhibits high tolerance toward several denaturing agents, which may make it a potentially more applicable enzyme. [ABSTRACT FROM AUTHOR]

Background and purpose: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a new target for reducing low‐density lipoprotein cholesterol and incident cardiovascular disease, including stroke. However, the clinical relevance of circulating PCSK9 levels has been poorly elucidated in the general population, particularly in association with subclinical cerebrovascular disease including cerebral small vessel disease (CSVD) and intracranial artery stenosis (ICAS). Methods: In community‐dwelling Japanese men (n = 526) aged 46–82 years without a history of cardiovascular disease, the associations of serum PCSK9 levels with the prevalence of CSVD and ICAS were assessed using magnetic resonance imaging. CSVD included lacunar infarction, deep and subcortical white matter hyperintensity, periventricular hyperintensity and cerebral microbleeds. Results: The median (interquartile range) age at baseline and serum PCSK9 levels were 69 (63–74) years and 240 (205–291) ng/ml, respectively. After adjusting for traditional cardiovascular risk factors including low‐density lipoprotein cholesterol, multivariable Poisson regression with robust error variance revealed a significant association between PCSK9 levels (per 1 SD) and ICAS (relative risks 1.18, 95% confidence interval 1.02–1.37). Multivariable ordinal logistic regression for ICAS, with stenosis graded as mild (<50%) or moderate–severe (≥50%), revealed a similar association (common odds ratio 1.31, 95% confidence interval 1.04–1.64). However, no significant association was observed between serum PCSK9 levels and CSVD. Conclusions: Higher circulating PCSK9 levels were independently associated with an ICAS prevalence but not with CSVD prevalence. The quantification of circulating PCSK9 levels may help to identify individuals at high risk for cerebrovascular disease in the general population. [ABSTRACT FROM AUTHOR]

Glutathione S-transferases (GSTs) of wood-degrading fungi play essential roles in cellular detoxification processes and endogenous metabolism. Fungal GSTs of GSTFuA class are suggested to be involved in lignin degradation. Ceriporiopsis subvermispora is one of the important model fungi of the selective lignin degraders, we found it interesting to study its GSTs. Here, we characterized the activities of two GSTs of the GSTFuA class of C . subvermispora (CsGST63524 and CsGST83044). A high-yield expression systems involving Escherichia coli was developed for each of these enzymes. Both enzymes were found to exhibit GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene, and GSH-peroxidase activity toward cumene hydroperoxide. Both enzymes showed high GSH-conjugation activity under basic conditions (pH 8.0 to 9.0), and the optimum temperature for their activity was 40 °C. In addition, three fluorescent compounds were used i . e ., methylumbelliferyl acetovanillone was used to monitor etherase activity, and 5-chloromethylfluorescein diacetate and 4-methylumbelliferyl acetate to monitor esterase activity. CsGST83044 exhibited both etherase and esterase activities, while CsGST63524 displayed only esterase activity, which was much higher than that of CsGST83044. These findings imply the functional diversity of the GSTFuA class GSTs of C . subvermispora , suggesting that each protein plays distinctive roles in both the fungal detoxification system and wood compound metabolism. [ABSTRACT FROM AUTHOR]

Aims/Introduction: To examine the association between diabetes and prediabetes at baseline, and disability, mortality over a 22‐year period among middle‐aged Japanese adults. Materials and Methods: Participants consisted of 1,788 adults aged 45–64 years at baseline from the cohort study National Integrated Project for Prospective Observation of Non‐communicable Disease and its Trends in the Aged 1990 (NIPPON DATA90). Disability, defined as having a decline in activities of daily living (ADL), was assessed by a modified Katz questionnaire at four time points. Disability and death without disability for 22‐year follow up were used as outcomes to test the association with a diagnosis of diabetes or prediabetes at baseline, using multinomial logistic regression. Adjusted odds ratios (ORs) were obtained from four models that contained appropriate adjustment factors, such as age, sex, smoking status, drinking status, body mass index and cardiovascular risk factors (hypertension, hypercholesterolemia, triglycerides, low serum high‐density lipoprotein), at baseline. Results: In the present study, 334 participants (18.7%) reported at least one disability, and 350 (19.6%) were reported dead without observation of disability during follow up. Adjusting sex and other risk factors, participants with diabetes and prediabetes had a higher risk for disability (OR 1.43, 95% confidence interval [CI] 1.07–1.91 and OR 1.66, 95% CI 1.10–2.50, respectively) and for mortality (OR 1.56, 95% CI 1.16–2.08 and OR 1.77, 95% CI 1.18–2.65, respectively) than individuals with normal glucose tolerance. Conclusions: In middle‐aged Japanese adults, individuals with diabetes and prediabetes were more likely to be associated with disability and mortality. Our findings suggest that prediabetes and diabetes in middle‐aged adults should be paid more attention, and requires more intervention to prevent disability and mortality in later life. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to investigate the incidence rates and predictors of lower limb fractures in a general Japanese population. Methods: NIPPON DATA is a nationwide, long-term, prospective cohort study of individuals who participated in the National Cardiovascular Survey Japan and the National Nutrition Survey in 1990. Overall, 3,134 individuals (1,827 women, 1,307 men) who participated in follow-up assessments in 1995, 2000, and/or 2006 were included in the present analysis. The outcomes of this study were lower limb fractures (including proximal femur fractures). Results: The mean age at baseline was 63.8 years in women and 63.1 years in men. The average body mass index (BMI) was 23.3 kg/m2 in women and 22.9 kg/m2 in men. During a mean follow-up of 12.1 years, 271 total lower limb fractures were observed. In women, older age, lower BMI, and less intake of vegetables were associated with increased risks of proximal femur fractures. With regard to the outcome of total lower limb fractures, less intake of vegetables and regular exercise were significant predictors in women. Calcium intake was not significantly associated with proximal femur or total lower limb fractures. There were no significant predictors of proximal femur or total lower limb fractures in men, except for age. Conclusions: Aging was a significant risk factor for proximal femur and total lower limb fractures in both men and women. With regard to modifiable risk factors, low BMI and low intake of vegetables were associated with increased risks of proximal femur and/or total lower limb fractures in the general population of Japanese women. [ABSTRACT FROM AUTHOR]

Metaplastic breast cancers (MBCs) [spindle cell carcinoma (SpCC), squamous cell carcinoma (SCC), and matrix-producing carcinoma (MPC)] and invasive carcinomas with central acellular zones (CACs) were analyzed with respect to biological potential by immunohistochemical analyses. Specimens from 40 patients [20 with MBCs (7 with SCC, 6 with SpCC, 5 with MPC, and 2 with mixed type)] and 20 with CACs were analyzed using antibodies to cytokeratin (CK) 8, 5/6, 14, AE1/AE3, 34αE12, involucrin, c-kit, vimentin (VIM), alpha-smooth muscle actin, p63, epidermal growth factor receptor, epithelial cell adhesion molecule, and estrogen receptor (ER)/progesterone receptor (PR)/HER2. Expression of CK5/6, 34βE12, VIM, nuclear p63, and cytoplasmic p63 was significantly higher with MBCs than CACs (38%/13%, 70%/43%, 85%/33%, 68%/40%, and 48%/18%, respectively). Other markers were expressed at various levels in these tumors, but the difference between them was not significant. Eighteen MBC and 8 CAC cases were triple (ER/PR/HER2) negative; 17 MBCs and 7 CACs were basal-like tumors. Several differences were seen in MBCs and CACs, but they were heterogeneous, differentiating multipotentially into mesenchymal, myoepithelial, basal-like phenotypes with 'stem cell-like' features. Thus, CACs are related to MBCs by immunohistochemical analyses as well as according to morphological findings. [ABSTRACT FROM AUTHOR]

The clinical implications of alcohol consumption have been extensively examined; however, its effects on brain structures in apparently healthy community-dwellers remain unclear. Therefore, we investigated the relationship between alcohol consumption and brain gray matter volume (GMV) in community-dwelling Japanese men using voxel-based morphometry (VBM). We recruited cognitively intact Japanese men, aged 40-79 years, from a population-based cohort in Shiga, Japan. Brain magnetic resonance imaging was performed, on average, 2 years after demographic and medical information was obtained in 2010-2014. A multivariable linear regression analysis of 639 men was conducted to elucidate the relationship between the amount of alcohol consumed and GMV. VBM statistics were analyzed by threshold-free cluster enhancement with a family-wise error rate of <0.05. The results obtained demonstrated that the amount of alcohol consumed was associated with lower GMV. The VBM analysis showed lower GMV within the parahippocampal, entorhinal, cingulate, insular, temporal, and frontal cortices and cerebellum in very heavy drinkers (≥42 ethanol g/day) than in non-drinkers. Furthermore, alcohol consumption was associated with a higher white matter lesion volume. These results suggest subclinical structural changes similar to alcohol-related neurological diseases. [ABSTRACT FROM AUTHOR]

Human origin recognition complex (hORC) binds to the DNA replication origin and then initiates DNA replication. However, hORC does not exhibit DNA sequence-specificity and how hORC recognizes the replication origin on genomic DNA remains elusive. Previously, we found that hORC recognizes G-quadruplex structures potentially formed near the replication origin. Then, we showed that hORC subunit 1 (hORC1) preferentially binds to G-quadruplex DNAs using a hORC1 construct comprising residues 413 to 511 (hORC1413−511). Here, we investigate the structural characteristics of hORC1413−511 in its free and complex forms with G-quadruplex DNAs. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopic studies indicated that hORC1413−511 is disordered except for a short α-helical region in both the free and complex forms. NMR chemical shift perturbation (CSP) analysis suggested that basic residues, arginines and lysines, and polar residues, serines and threonines, are involved in the G-quadruplex DNA binding. Then, this was confirmed by mutation analysis. Interestingly, CSP analysis indicated that hORC1413−511 binds to both parallel- and (3 + 1)-type G-quadruplex DNAs using the same residues, and thereby in the same manner. Our study suggests that hORC1 uses its intrinsically disordered G-quadruplex binding region to recognize parallel-type and (3 + 1)-type G-quadruplex structures at replication origin. • hORC1 binds to G-quadruplex (G4) DNAs. • The G4 DNA-binding region of hORC1, hORC1413−511, is intrinsically disordered. • hORC1413−511 remains disordered upon binding to G4 DNAs. • Interaction of basic and polar residues of hORC1413−511 with G4 DNAs is suggested. • hORC1413−511 binds to both parallel and (3 + 1)-type G4s in the same manner. [ABSTRACT FROM AUTHOR]

Dysfunctional high-density lipoprotein (HDL) is a risk factor for cardiovascular disease (CVD) beyond HDL concentrations. Recently, a novel method has been introduced to measure LOX-1 ligand containing apolipoprotein AI (LAA), which is an indicator of various types of modified HDL with binding capacity to LOX-1 and related to impaired anti-atherogenic functions of HDL. This study aimed to examine the relationship between LAA as a novel marker of dysfunctional HDL and coronary artery calcification (CAC). We selected 910 community-dwelling Japanese men aged 40–79 years without a history of CVD. The odds ratios per 1SD of LAA for the presence of CAC (Agatston score >10) were estimated using logistic regression model adjusted for confounders, including HDL-C or HDL particle (HDL-P) concentration. In addition, we performed further analysis stratified by age (<65 and ≥ 65 years). The mean age of the participants was 63.6 years, and the median LAA was 187.0 ng/mL. The prevalent CAC was 46.2%. The multivariable adjusted odds ratio (95% confidence interval) per 1SD of LAA for CAC was 1.14 (0.96–1.36) for all participants. After stratification by age, multivariable adjusted odds ratios per 1SD of LAA were 1.34 (1.02–1.76) and 0.97 (0.77–1.23) in men aged <65 and ≥ 65 years, respectively. The present study showed that LAA was associated with CAC independent of HDL-C or HDL-P in middle-aged Japanese men. This finding suggests that LAA might be an early marker for CVD events. Future longitudinal studies are warranted. Image 1 • We measured LOX-1 ligand containing apolipoprotein AI (LAA) as a novel marker of dysfunctional HDL in Japanese men. • LAA was positively associated with coronary artery calcification independent of HDL-C levels in middle-aged men. • This finding suggests that LAA might be an early marker for cardiovascular disease. [ABSTRACT FROM AUTHOR]

Background: Highly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated. Results: Three types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H–13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π–π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16–4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. Conclusion: The CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process. [ABSTRACT FROM AUTHOR]

To examine whether smoking habits, including smoking amount and cessation duration at baseline, are associated with atherosclerosis progression. At baseline (2006–08, Japan), we obtained smoking status, amount of smoking and time since cessation for quitters in a community-based random sample of Japanese men initially aged 40–79 years and free of cardiovascular disease. Coronary artery calcification (CAC) and aortic artery calcification (AAC) as biomarker of atherosclerosis was quantified using Agatston's method at baseline and after 5 years of follow-up. We defined progression of CAC and AAC (yes/no) using modified criteria by Berry. A total of 781 participants was analyzed. Multivariable adjusted odds ratios (ORs) of CAC and AAC progression for current smokers were 1.73 (95% CI, 1.09–2.73) and 2.47 (1.38–4.44), respectively, as compared to never smokers. In dose-response analyses, we observed a graded positive relationship of smoking amount and CAC progression in current smokers (multivariable adjusted ORs: 1.23, 1.72, and 2.42 from the lowest to the highest tertile of pack-years). Among the former smokers, earlier quitters (≥10.7 years) had similar ORs of the progression of CAC and AAC to that of participants who had never smoked. Compared with never smokers, current smokers especially those with greater pack-years at baseline had higher risk of atherosclerosis progression in community-dwelling Japanese men. Importantly, the residual adverse effect appears to be present for at least ten years after smoking cessation. The findings highlight the importance of early avoidance or minimizing smoking exposure for the prevention of atherosclerotic disease. • Longitudinal study on relationship between smoking habits and atherosclerosis. • Continued smokers had artery calcification progression two times than never smokers. • Residual effect on atherosclerosis appears at least ten years after smoking cessation. [ABSTRACT FROM AUTHOR]

Aims: Ectopic fat accumulation is related to insulin resistance and diabetes mellitus (DM). However, the effect of fatty liver on DM in non-obese individuals has not been clarified. We investigated whether liver fat accumulation assessed by computed tomography (CT) is associated with the incidence of DM.Methods: In a prospective population-based study, 640 Japanese men were followed up for 5 years. The liver to spleen (L/S) ratio of the CT attenuation value was used as the liver fat accumulation index. We calculated the odds ratio (OR) and 95% confidence interval (CI) for the DM incidence of per 1 standard deviation (SD) lower L/S and those of L/S < 1.0 compared with L/S ≥ 1.0, using logistic regression models.Results: Both per 1 SD lower L/S and L/S < 1.0 were significantly associated with a risk for DM incidence (1 SD lower L/S: OR = 1.57, 95%CI = 1.14-2.16; L/S < 1.0: OR = 2.27, 95%CI = 1.00-5.14). The relationship between L/S and incidence of DM was consistent in the obese and non-obese groups, with thresholds of BMI 25 kg/m2, waist circumference 85 cm, or visceral adipose tissue 100 cm2.Conclusions: Liver fat accumulation assessed by CT was associated with the incidence of DM. [ABSTRACT FROM AUTHOR]

Although heavy alcohol consumption has been identified as a risk factor for adverse cognitive functioning, it currently remains unclear whether moderate alcohol consumption exerts similar effects. Observational studies previously reported the potential benefits of moderate alcohol consumption on cognition, particularly in the elderly; however, these effects have not yet been demonstrated in Asian populations. The aim of the present study was to investigate the relationship between alcohol consumption levels and global and domain-specific cognitive functions in cognitively intact elderly Japanese men. Cross-sectional data from the Shiga Epidemiological Study of Subclinical Atherosclerosis (SESSA), an ongoing prospective, population-based study in Shiga, Japan, were used to examine the relationship between alcohol consumption and cognitive function. Men (n = 585) aged ≥65 years provided information on their weekly consumption of alcohol, and the data obtained were used to construct categories of never, ex- (quit before interview), very light (<14 g/day), light (14-23 g/day), moderate (>23-46 g/day), and heavy (>46 g/day) drinkers. Cognitive function was measured using the Cognitive Abilities Screening Instrument (CASI). A fractional logistic regression model adjusted for age, education, body mass index, smoking, exercise, hypertension, diabetes, and dyslipidemia showed that the CASI scores for global and domain-specific cognitive functions were not significantly different between all subgroups of current drinkers and never-drinkers. However, the CASI score of ex-drinkers (multivariable adjusted mean CASI score [SD]) was significantly lower than that of never-drinkers in the global [never vs. ex: 90.16 (2.21) vs. 88.26 (2.58)] and abstraction and judgment domain [never vs. ex: 9.48 (0.46) vs. 8.61 (0.57)]). The present results do not suggest any beneficial or adverse relationship between current alcohol consumption levels and cognitive functioning (both global and domain specific) in elderly Japanese men; however, low cognitive function among ex-drinkers warrants future investigations to identify the factors causing drinkers to quit. [ABSTRACT FROM AUTHOR]

Introduction: Evaluation of total energy expenditure (TEE) and physical activity level (PAL) is important for treatment of patients with type 2 diabetes mellitus (T2DM). However, the validity of accelerometers (ACC) and physical activity questionnaires (PAQ) for estimating TEE and PAL remains unknown in elderly populations with T2DM. We evaluated the accuracy of TEE and PAL results estimated by an ACC (TEEACC and PALACC) and a PAQ (TEEPAQ and PALPAQ) in elderly patients with T2DM. Methods: Fifty-one elderly patients with T2DM (aged 61–79 years) participated in this study. TEEACC was calculated with PALACC using a triaxial ACC (Active style Pro HJA-750c) over 2 weeks and predicted basal metabolic rate (BMR) by Ganpule's equation. TEEPAQ was estimated using predicted BMR and the PALPAQ from the -Japan Public Health Center Study-Long questionnaire. We compared the results to TEEDLW measured with the doubly labeled water (DLW) method and PALDLW calculated with BMR using indirect calorimetry. Results: TEEDLW was 2,165 ± 365 kcal/day, and TEEACC was 2,014 ± 339 kcal/day; TEEACC was strongly correlated with TEEDLW (r = 0.87, p < 0.01) but significantly underestimated (–150 ± 183 kcal/day, p < 0.05). There was no significant difference in TEEPAQ and TEEDLW (–49 ± 284 kcal/day), while the range of difference seemed to be larger than TEEACC. PALDLW, PALACC, and PALPAQ were calculated to be 1.71 ± 0.17, 1.69 ± 0.16, and 1.78 ± 0.24, respectively. -PALACC was strongly correlated with PALDLW (r = 0.71, p < 0.01), and there was no significant difference between the 2 values. PALPAQ was moderately correlated with PALDLW (r = 0.43, p < 0.01) but significantly overestimated. Predicted BMR was significantly lower than the BMR -measured by indirect calorimetry (1,193 ± 186 vs. 1,262 ± 155 kcal/day, p < 0.01). Conclusions: The present ACC and questionnaire showed acceptable correlation of TEE and PAL compared with DLW method in elderly patients with T2DM. Systematic errors in estimating TEE may be improved by the better equation for predicting BMR. [ABSTRACT FROM AUTHOR]

Aims/Introduction: Sodium–glucose cotransporter 2 inhibitors reduce bodyweight (BW) by creating a negative energy balance. Previous reports have suggested that this BW reduction is mainly loss of body fat and that ~20% of the reduction is lean mass. However, the effects of sodium–glucose cotransporter 2 inhibitors on BW and body composition remain unclear. We examined these effects in Japanese patients with type 2 diabetes mellitus treated with insulin. Materials and Methods: In this open‐label, randomized controlled trial, 49 overweight patients (body mass index ≥23 kg/m2) with inadequate glycemic control (hemoglobin A1c >7.0%) receiving insulin treatment were randomly assigned to receive add‐on ipragliflozin or no additional treatment (control group). Patients were followed for 24 weeks. The goal for all patients was to achieve glycated hemoglobin <7.0% without hypoglycemia. The primary end‐point was a change in BW from baseline to week 24. Body composition was assessed with dual‐energy X‐ray absorptiometry and bioelectrical impedance analysis. Results: BW change was significantly larger in the ipragliflozin group than in the control group (−2.78 vs −0.22 kg, P < 0.0001). Total fat mass was reduced evenly in the arms, lower limbs and trunk in the ipragliflozin group. Total muscle mass and bone mineral content were maintained, but muscle mass in the arms might have been affected by ipragliflozin treatment. Conclusions: Ipragliflozin treatment for 24 weeks resulted in reduced BW, mainly from fat mass loss. Muscle mass and bone mineral content were maintained. Further study is necessary to elucidate the long‐term effects of ipragliflozin. [ABSTRACT FROM AUTHOR]

Ceriporiopsis subvermispora (C. subvermispora) is a selective degrader of lignin in the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in cellular detoxification and metabolism. The crystal structures of a GST of C. subvermispora , CsGST83044, in GSH-free and -bound forms were solved at 1.95 and 2.19 Å resolution, respectively. The structure of the GSH-bound form revealed that CsGST83044 can be categorized as an atypical-type of GST. In the GSH-bound form of CsGST83044, Asn22, Asn24, and Tyr46 are located closest to the sulfur atom and form hydrogen bonds with the thiol group. The functional mutagenesis indicated that they are critical for the enzymatic activities of CsGST83044. The critical residues of an atypical-type GST belonging to the GSTFuA class were revealed for the first time. A previous study indicated that CsGST83044 and another GST, CsGST63524, differ in substrate preference; CsGST83044 prefers smaller substrates than CsGST63524 for its esterase activity. The GSH-bound pocket of CsGST83044 turns out to be small, which may explain the preference for smaller substrates. Protein engineering of GSTs of C. subvermispora in the light of the obtained insight may pave a path in the future for utilization of the woody biomass. [ABSTRACT FROM AUTHOR]

Abstract Ceriporiopsis subvermispora (C. subvermispora), one of the white-rot fungi, is known as a selective lignin degrader of the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that are capable of catalyzing the reactions involved in detoxification and metabolic pathways. In this study, a GST of C. subvermispora , named CsGST63524, was overexpressed in E. coli , and then purified by affinity, anion exchange, and size exclusion column chromatography. The crystal structures of the CsGST63524 in ligand-free and complex with GSH were refined at 2.45 and 2.50 Å resolutions, respectively. The sulfur atom of glutathione forms a hydrogen bond with Ser21 of CsGST63524, indicating it is a serine-type GST. Mutagenesis of Ser21 unexpectedly indicated that this serine residue is not essential for the enzymatic activity of CsGST63524. Comparative sequence and structural analyses, together with functional mutagenesis, newly identified the enzymatically important non-canonical amino acid residues, Asn23 and Tyr45, other than the serine residue. Highlights • GSH-free and -bound structures of a C. subvermispora GST, CsGST63524, were solved. • Ser21 is located closest to a sulfur of GSH, indicating CsGST63524 is a serine-type. • S21A mutant unexpectedly indicated Ser21 is not crucial for the enzymatic activity. • Functional mutagenesis revealed Asn23 and Tyr45 are crucial for enzymatic activity. • A putative substrate-binding site was deduced from those of homologous proteins. [ABSTRACT FROM AUTHOR]

Aim: Most patients with dementia frequently encounter various problems in their daily lives. Those troubles embarrass both the patients and their families, and cause problems for society. However, there have been few scientific reports on the difficulties in the daily life of patients with dementia. Therefore, we tried to clarify the frequency and characteristics of troubles experienced by patients with dementia. Methods: Seven medical centers treating dementia patients in Okayama Prefecture, Japan, participated in this survey. A total of 737 patients were placed in one of the three groups: a dementia group (n = 478), a mild cognitive impairment group (n = 199) and a control group (n = 60). The frequency of 13 difficulties was scored for each patient. Results: Among normal participants, no person caused these problems once a year or more frequently. "Massive, recurrent buying" and "acts that risk causing a fire" were reported once a year or more for >10% of mild cognitive impairment patients. "Troubles with wealth management" and "troubles with money management" were the most frequent problems of dementia patients. Conclusions: Several problems are already sometimes encountered in patients with mild cognitive impairment. It would be useful to know which social difficulties are often seen in dementia patients in order to protect the safety of the patients. It is always difficult to balance respecting the autonomy of dementia patients and ensuring their safely. Geriatr Gerontol Int 2019; 19: 113–118. [ABSTRACT FROM AUTHOR]

Thiothrix nivea is a filamentous sulfur-oxidizing bacterium common in activated sludge and its filament is covered with a polysaccharide layer called sheath. In this study, we found that T. nivea aggregates under acidic conditions. A hexagonal lattice pattern, a typical morphological feature of proteinaceous S-layers, was newly observed on the surface of the sheath by transmission electron microscopy. The pattern and the acid-dependent aggregation were not observed in T. fructosivorans, a relative sheath-forming bacterium of T. nivea. The putative S-layer of T. nivea was detached by washing with unbuffered tris(hydroxymethyl)aminomethane base (Tris) solution and a protein of 160 kDa was detected by electrophoresis. Based on partial amino acid sequences of the protein, its structural gene was identified. The gene encodes an acidic protein which has a putative secretion signal and a Ca2+-binding domain. The protein was solubilized with urea followed by dialysis in the presence of calcium. A hexagonal lattice pattern was observed in the aggregates formed during dialysis, revealing that the protein is responsible for S-layer formation. Biosorption ability of copper, zinc, and cadmium onto the T. nivea filament decreased upon pretreatment with Tris, demonstrating that the S-layer was involved in metal adsorption. Moreover, aggregation of Escherichia coli was promoted by acidification in the presence of the S-layer protein, suggesting that the protein is potentially applicable as an acid-driven flocculant for other bacteria. [ABSTRACT FROM AUTHOR]

Background It is widely supposed that there has been no evidence of increased survival in patients with advanced dementia receiving enteral tube feeding. However, more than a few studies have reported no harmful outcome from tube feeding in dementia patients compared to in patients without dementia. Methods This was a retrospective study. Nine psychiatric hospitals in Okayama Prefecture participated in this survey. All inpatients fulfilling the entry criteria were evaluated. All subjects suffered from difficulty with oral intake. Attending physicians thought that the patients could not live without long-term artificial nutrition. The physicians decided whether to make use of long-term artificial nutrition between January 2012 and December 2014. Results We evaluated 185 patients. Their mean age was 76.6 ± 11.4 years. Of all subjects, patients with probable Alzheimer's disease ( n = 78) formed the biggest group, schizophrenia patients ( n = 44) the second, and those with vascular dementia ( n = 30) the third. The median survival times were 711 days for patients with tube feeding and 61 days for patients without tube feeding. In a comparison different types of tube feeding, median survival times were 611 days for patients with a nasogastric tube and more than 1000 days for those with a percutaneous endoscopic gastrostomy tube. Conclusion Patients with tube feeding survived longer than those without tube feeding, even among dementia patients. This study suggests that enteral nutrition for patients with dementia prolongs survival. Additionally, percutaneous endoscopic gastrostomy tube feeding may be safer than nasogastric tube feeding among patients in psychiatric hospitals. [ABSTRACT FROM AUTHOR]

Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans , are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [→4)-β- d -GlcN-(1 → 4)-β- d -Glc-(1→] n modified with unusual deoxy sugars. In an attempt to elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were determined in this study. Prior to analysis, both sheaths were confirmed to be highly de- N -acetylated. Sheaths in viable filaments were N -biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addition, the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chemically fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N -acetylation. [ABSTRACT FROM AUTHOR]

Aim: Artificial nutrition, including tube feeding, continues to be given to dementia patients in numerous geriatric facilities in Japan. However, the clinical characteristics of patients receiving artificial nutrition have not been fully investigated. Therefore, we tried to evaluate the clinical features of those patients in this study. Methods: Various clinical characteristics of all inpatients at 18 of 20 psychiatric hospitals in Okayama Prefecture, Japan, with a percutaneous endoscopic gastrostomy tube, nasogastric tube, or total parenteral nutrition were evaluated. Results: Two hundred twenty ‐ one patients (5.4% of all inpatients) had been receiving artificial nutrition for more than 1 month, and 187 (130 women, 57 men; 84.6% of 221 patients) were fully investigated. The mean age was 78.3 years old, and the mean duration of artificial nutrition was 29.8 months. Eighty ‐ four patients (44.7% of 187 patients) were receiving artificial nutrition for more than 2 years. Patients with Alzheimer's disease (n = 78) formed the biggest group, schizophrenia (n = 37) the second, and vascular dementia (n = 26) the third. Conclusion: About one ‐ fifth of the subjects receiving artificial nutrition were in a vegetative state. More than a few patients with mental disorders, including schizophrenia, also received long ‐ term artificial nutrition. We should pay more attention to chronic dysphasia syndrome in mental disorders. [ABSTRACT FROM AUTHOR]

Efficient solid–liquid phase separation of excess sludge from the mixed-liquor suspended solids in wastewater treatment is difficult to achieve without prior sludge conditioning. Several physical conditioners have been assessed as skeleton builders or filtering aids to reduce sludge compressibility; however, little is known about how the properties of the physical conditioner affect sludge compressibility and dewaterability. In this study, synthetic poly(ethylene terephthalate) (PET), polypropylene, and viscose rayon (VR) fibers were used as physical conditioners for activated sludge, and the effects of the fiber compressibility on the dewaterability of activated sludge collected from a laboratory-scale reactor were assessed. Based on expression experiments using a laboratory-scale apparatus, it was found that: (1) the rate of consolidation of sludge cakes increased after incorporation of synthetic fibers, particularly at the primary stage. (2) The porosity of the sludge cake was maintained only with fibers that required high pressures for compaction (i.e., PET and VR); thus, high dewaterability of the cakes was achieved with these fibers. It is proposed that the compressibility of the physical conditioner may be one of the more important factors for improving sludge dewaterability. [ABSTRACT FROM AUTHOR]

Aim: We aimed to determine whether SGLT2 inhibitor dapagliflozin treatment affects body composition and amino acid (AA) metabolism.Methods: Fifty-two overweight patients treated by oral antidiabetic agents were randomly assigned to dapagliflozin (Dapa) or a standard treatment (Con) and followed for 24 weeks. The primary outcome was the change in body mass (BM) between baseline and week 24. Body composition, intrahepatic triglyceride (IHTG) content, and plasma AA concentrations were examined as secondary outcomes.Results: The change in BM was significantly larger in the Dapa than in the Con group, with a difference in the mean change of -1.72 kg (95 %CI: -2.85, -0.59; P = 0.004) between the groups. Total fat mass was reduced by dapagliflozin treatment, but fat-free mass was maintained. IHTG content was significantly reduced in the Dapa than in the Con (P = 0.033). Changes in AAs showed small differences between the groups, but only serine concentrations were significantly reduced in the Dapa. Intra-group analysis showed that positive associations were observed between changes in branched chain AA concentrations and body composition only in the Dapa.Conclusions: Dapagliflozin treatment causes a reduction in BM mainly by reducing fat mass. AA metabolism shows subtle changes with dapagliflozin treatment. [ABSTRACT FROM AUTHOR]

Highlights: [•] Fish-oil diet increased 4-hydroxy hexenal, a peroxidative lipid, in multiple organs. [•] The expression of HO-1 mRNA was increased with fish-oil diet in most tissues. [•] These increases were paralleled by an increase in the level of docosahexaenoic acid. [•] These phenomena may explain the pleiotropic effect of fish-oil in multiple organs. [ABSTRACT FROM AUTHOR]

Recent studies have proposed that n-3 polyunsaturated fatty acids (n-3 PUFAs) have direct antioxidant and anti-inflammatory effects in vascular tissue, explaining their cardioprotective effects. However, the molecular mechanisms are not yet fully understood. We tested whether n-3 PUFAs showed antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor for antioxidant genes. C57BL/6 or Nrf2−/− mice were fed a fish-oil diet for 3 weeks. Fish-oil diet significantly increased the expression of heme oxygenase-1 (HO-1), and endothelium-dependent vasodilation in the aorta of C57BL/6 mice, but not in the Nrf2−/− mice. Furthermore, we observed that 4-hydroxy hexenal (4-HHE), an end-product of n-3 PUFA peroxidation, was significantly increased in the aorta of C57BL/6 mice, accompanied by intra-aortic predominant increase in docosahexaenoic acid (DHA) rather than that in eicosapentaenoic acid (EPA). Human umbilical vein endothelial cells were incubated with DHA or EPA. We found that DHA, but not EPA, markedly increased intracellular 4-HHE, and nuclear expression and DNA binding of Nrf2. Both DHA and 4-HHE also increased the expressions of Nrf2 target genes including HO-1, and the siRNA of Nrf2 abolished these effects. Furthermore, DHA prevented oxidant-induced cellular damage or reactive oxygen species production, and these effects were disappeared by an HO-1 inhibitor or the siRNA of Nrf2. Thus, we found protective effects of DHA through Nrf2 activation in vascular tissue, accompanied by intra-vascular increases in 4-HHE, which may explain the mechanism of the cardioprotective effects of DHA. [ABSTRACT FROM AUTHOR]

Anti-diabetic agent-related hypoglycemia is a serious complication in type 2 diabetic patients on hemodialysis. Therefore, we assessed the efficacy and tolerability of 24 weeks of monotherapy with vildagliptin, a dipeptidyl peptidase four inhibitor, which is a new class of antidiabetic agent. This open-label, single-arm clinical trial was performed on 26 patients on hemodialysis. The primary assessments were changes in postprandial glucose level and glycated albumin (GA). During the study, three patients dropped out, and data from 23 patients were analyzed. Significant reductions were seen in postprandial glucose (−2.60 ± 3.80 mmol/L, P < 0.001) and GA (−2.59 ± 2.33%, P < 0.001) levels. No serious drug-related adverse events were observed. Vildagliptin monotherapy can be recommended for glycemic control in type 2 diabetic patients on hemodialysis. This trial was registered with the University Hospital Medical Information Network (no. UMIN000003661). (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00169.x, 2011) [ABSTRACT FROM AUTHOR]

Background Little is known about the relationships between higher-level functional capacity (HLFC) and mortality, and its gender difference. Methods NIPPON DATA90 is the cohort study of the National Cardiovascular Survey of Japan in 1990. We followed up 1824 Japanese adults over 65 years who were independent in basic activities of daily living in 1995 until 2010. HLFC was measured in 1995 using the total score and 3 subscales (instrumental activities of daily living [IADL], intellectual activity, social role) by the Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG-IC). The association of HLFC to mortality was evaluated by adjusted hazard ratio (HR) and 95% confidence interval (CI) using the Cox proportional hazard model. Results During average 12.2-year follow-up, 836 participants (45.8%) died. Participants with impaired total TMIG-IC score showed significantly higher risk of mortality (adjusted HR 1.37; 95%CI 1.14-1.66) compared with normal participants, even after adjusting for other variables. The corresponding HR was significant only in women (HR 1.42; 95% CI 1.10-1.83). Among the 3 subscales, only impaired social role showed significantly higher risk of mortality (HR 1.37; 95%CI 1.12-1.67) in men. In contrast, IADL and intellectual activity showed significantly higher risk in women (HR 1.51; 95%CI 1.16-1.96) (HR 1.46; 95%CI 1.19-1.80). Conclusions Impaired HLFC was related to higher risk of long-term mortality in Japanese elderly at baseline. Especially among women, IADL and intellectual activity were important predictors, whereas social role was important in men. Key messages Impaired HLFC was related to long-term mortality in Japanese elderly. [ABSTRACT FROM AUTHOR]

Background Premature atrial contractions (PACs) are known predictor of atrial fibrillation; however very little has been revealed on its factors among Asian general population. We assessed the frequency of higher PAC counts and its association with relevant factors in general Japanese men. Methods We have conducted a population based cross sectional study among 517 healthy men from Kusatsu, Japan, aged between 40 and 79 years. 24 hours Holter electrocardiogram was performed to assess the PAC frequency. We divided participants into quartiles based on number of PACs/hour to compare the characteristics by using Cochran–Mantel–Hanzal test and ANOVA. We defined the highest quartile as event and multivariable logistic regression was performed to assess the significance with relevant factors. Results Median number of PACs was 2.84 PACs/hour. The highest quartile of PAC counts was >7.86PACs/hour. Age, triglycerides, blood pressure, ventricular contractions and heart rates were different among quartiles. In multivariable logistic regression analysis, higher age (OR, 95% CI: 1.35, 1.01-1.67), lower mean heart rates (OR, 95% CI: 0.97, 0.94-0.99), higher heart rate difference in a day (OR, 95% CI: 1.40, 1.12-1.75) and lower triglyceride (OR, 95% CI: 0.53, 0.33-0.82) were independently, significantly associated with highest quartile of PACs. Conclusions Higher PACs evaluated by Holter ECG was associated with higher age, lower heart rate, higher heart rate difference, and lower triglyceride in general Japanese men. Key messages The PAC frequency was high. Age, mean heart rates, difference between maximum and resting heart rates, triglycerides are possible risk factors for frequent PAC counts. [ABSTRACT FROM AUTHOR]

Palladium-catalyzed cyclization-methoxycarbonylation of (2R,3S)-3-methylpent-4-yne-1,2-diol (6) derived from (2R,3S)-epoxybutanoate 5 followed by methylation gave the tetrahydro-2-furylidene acetate (−)-7, which was converted to the left-half aldehyde (+)-3. A Wittig reaction between (+)-3 and the phosphoranylide derived from the bithiazole-type phosphonium iodide 4 using lithium bis(trimethylsilyl)amide afforded the (+)-cystothiazole G (2), whose spectral data were identical with those of the natural product (+)-2. Thus, the stereochemistry of cystothiazole G (2) was proved to be (4R,5S,6(E)). [Copyright &y& Elsevier]

Origin recognition complex (ORC) binds to replication origins in eukaryotic DNAs and plays an important role in replication. Although yeast ORC is known to sequence-specifically bind to a replication origin, how human ORC recognizes a replication origin remains unknown. Previous genome-wide studies revealed that guanine (G)-rich sequences, potentially forming G-quadruplex (G4) structures, are present in most replication origins in human cells. We previously suggested that the region comprising residues 413–511 of human ORC subunit 1, hORC1413–511, binds preferentially to G-rich DNAs, which form a G4 structure in the absence of hORC1413–511. Here, we investigated the interaction of hORC1413-511 with various G-rich DNAs derived from human c-myc promoter and telomere regions. Fluorescence anisotropy revealed that hORC1413–511 binds preferentially to DNAs that have G4 structures over ones having double-stranded structures. Importantly, circular dichroism (CD) and nuclear magnetic resonance (NMR) showed that those G-rich DNAs retain the G4 structures even after binding with hORC1413–511. NMR chemical shift perturbation analyses revealed that the external G-tetrad planes of the G4 structures are the primary binding sites for hORC1413–511. The present study suggests that human ORC1 may recognize replication origins through the G4 structure. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to the development of a method using selective fluorescent labeling of the sheath of bacteria Sphaerotilus natans by conventional reagents. It provides the explanation to the phenomena of sheath formation and ubiquitous cell growth of the bacteria. The result of the study reveals that the method can be used in environmental sampling to detect the presence of Sphaerotilus sheath.