Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 μm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'.

Morphomics via next-generation electron microscopy.

The living body is composed of innumerable fine and complex structures. Although these structures have been studied in the past, a vast amount of information pertaining to them still remains unknown. When attempting to observe these ultra-structures, the use of electron microscopy (EM) has become indispensable.

Compact laboratory-based X-ray microscope enabling nondestructive 3D structure acquisition of mouse nephron with high speed and better user accessibility.

X-ray microscopes adopting computed tomography enable nondestructive 3D visualization of biological specimens at micron-level resolution without conventional 2D serial sectioning that is a destructive/laborious method and is routinely used for analyzing renal biopsy in clinical diagnosis of kidney diseases. Here we applied a compact commercial system of laboratory-based X-ray microscope to observe a resin-embedded osmium-stained 1-mm strip of a mouse kidney piece as a model of renal biopsy, toward a more efficient diagnosis of kidney diseases. A reconstructed computed tomography image from several hours of data collection using CCD detector allowed us to unambiguously segment a single nephron connected to a renal corpuscle, which was consistent with previous reports using serial sectioning.

Changes in TCA cycle and TCA cycle-related metabolites in plasma upon citric acid administration in rats.

Recent studies have reported that plasma levels of tricarboxylic acid (TCA) cycle metabolites and TCA cycle-related metabolite change in patients with chronic fatigue syndrome (CFS) and in healthy humans after exercise. Exogenous dietary citric acid has been reported to alleviate fatigue during daily activities and after exercise. However, it is unknown whether dietary citric acid affects the plasma levels of these metabolites.

Comprehensive Evaluation of the Binding of Lipocalin-Type Prostaglandin D Synthase to Poorly Water-Soluble Drugs.

Low water solubility of candidate drug compounds is a major problem in pharmaceutical research and development. We developed a novel drug delivery system (DDS) for poorly water-soluble drugs using lipocalin-type prostaglandin D synthase (L-PGDS), which belongs to the lipocalin superfamily and binds a large variety of hydrophobic molecules. In this study, we comprehensively evaluated the capability of L-PGDS to bind and solubilize various poorly water-soluble drugs using structure-based docking.

Capillary-Inserted Rotor Design for HRµMASNMR-Based Metabolomics on Mass-Limited Neurospheres.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique and has been widely used in metabolomics. However, the intrinsic low sensitivity of NMR prevents its applications to systems with limited sample availabilities. In this study, a new experimental approach is presented to analyze mass-scarce samples in limited volumes of less than 300 nL with simple handling.

Music Improves Subjective Feelings Leading to Cardiac Autonomic Nervous Modulation: A Pilot Study.

It is widely accepted that listening to music improves subjective feelings and reduces fatigue sensations, and different kinds of music lead to different activations of these feelings. Recently, cardiac autonomic nervous modulation has been proposed as a useful objective indicator of fatigue. However, scientific considerations of the relation between feelings of fatigue and cardiac autonomic nervous modulation while listening to music are still lacking.

NG2 glial cells regulate neuroimmunological responses to maintain neuronal function and survival.

NG2-expressing neural progenitor cells (i.e., NG2 glial cells) maintain their proliferative and migratory activities even in the adult mammalian central nervous system (CNS) and produce myelinating oligodendrocytes and astrocytes.

Energetic basis on interactions between ferredoxin and ferredoxin NADP reductase at varying physiological conditions.

In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.

Black hairy tongue after chemotherapy for malignant brain tumors.

Black hairy tongue (BHT) developed in five patients (2.6%) among 192 patients undergoing chemotherapy for malignant brain tumors. Three patients with a history of diabetes mellitus developed BHT within 10 days after the initiation of chemotherapy.

Non-covalent forces tune the electron transfer complex between ferredoxin and sulfite reductase to optimize enzymatic activity.

Although electrostatic interactions between negatively charged ferredoxin (Fd) and positively charged sulfite reductase (SiR) have been predominantly highlighted to characterize complex formation, the detailed nature of intermolecular forces remains to be fully elucidated. We investigated interprotein forces for the formation of an electron transfer complex between Fd and SiR and their relationship to SiR activity using various approaches over NaCl concentrations between 0 and 400 mM. Fd-dependent SiR activity assays revealed a bell-shaped activity curve with a maximum ∼40-70 mM NaCl and a reverse bell-shaped dependence of interprotein affinity.

Index markers of chronic fatigue syndrome with dysfunction of TCA and urea cycles.

Chronic fatigue syndrome (CFS) is a persistent and unexplained pathological state characterized by exertional and severely debilitating fatigue, with/without infectious or neuropsychiatric symptoms, lasting at least 6 consecutive months. Its pathogenesis remains incompletely understood. Here, we performed comprehensive metabolomic analyses of 133 plasma samples obtained from CFS patients and healthy controls to establish an objective diagnosis of CFS.

Noninvasive Evaluation of Cellular Proliferative Activity in Brain Neurogenic Regions in Rats under Depression and Treatment by Enhanced [18F]FLT-PET Imaging.

Unlabelled: Neural stem cells in two neurogenic regions, the subventricular zone and the subgranular zone (SGZ) of the hippocampal dentate gyrus, can divide and produce new neurons throughout life. Hippocampal neurogenesis is related to emotions, including depression/anxiety, and the therapeutic effects of antidepressants, as well as learning and memory. The establishment of in vivo imaging for proliferative activity of neural stem cells in the SGZ might be used to diagnose depression and to monitor the therapeutic efficacy of antidepressants.

Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease.

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death.

Physicochemical nature of interfaces controlling ferredoxin NADP(+) reductase activity through its interprotein interactions with ferredoxin.

Although acidic residues of ferredoxin (Fd) are known to be essential for activities of various Fd-dependent enzymes, including ferredoxin NADP(+) reductase (FNR) and sulfite reductase (SiR), through electrostatic interactions with basic residues of partner enzymes, non-electrostatic contributions such as hydrophobic forces remain largely unknown. We herein demonstrated that intermolecular hydrophobic and charge-charge interactions between Fd and enzymes were both critical for enzymatic activity. Systematic site-directed mutagenesis, which altered physicochemical properties of residues on the interfaces of Fd for FNR /SiR, revealed various changes in activities of both enzymes.

Potential biomarkers of fatigue identified by plasma metabolome analysis in rats.

In the present study, prior to the establishment of a method for the clinical diagnosis of chronic fatigue in humans, we validated the utility of plasma metabolomic analysis in a rat model of fatigue using capillary electrophoresis-mass spectrometry (CE-MS). In order to obtain a fatigued animal group, rats were placed in a cage filled with water to a height of 2.2 cm for 5 days.

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9.

Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system.

Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system.

CRISPR/Cas9-mediated genome editing is a next-generation strategy for genetic modifications, not only for single gene targeting, but also for multiple targeted mutagenesis. To make the most of the multiplexity of CRISPR/Cas9, we established a system for constructing all-in-one expression vectors containing multiple guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette. We further demonstrated successful examples of multiple targeting including chromosomal deletions in human cells using the all-in-one CRISPR/Cas9 vectors constructed with our novel system.

Brain interleukin-1β and the intrinsic receptor antagonist control peripheral Toll-like receptor 3-mediated suppression of spontaneous activity in rats.

During acute viral infections such as influenza, humans often experience not only transient fever, but also prolonged fatigue or depressive feelings with a decrease in social activity for days or weeks. These feelings are thought to be due to neuroinflammation in the brain. Recent studies have suggested that chronic neuroinflammation is a precipitating event of various neurological disorders, but the mechanism determining the duration of neuroinflammation has not been elucidated.

Fine-tuned broad binding capability of human lipocalin-type prostaglandin D synthase for various small lipophilic ligands.

The hydrophobic cavity of lipocalin-type prostaglandin D synthase (L-PGDS) has been suggested to accommodate various lipophilic ligands through hydrophobic effects, but its energetic origin remains unknown. We characterized 18 buffer-independent binding systems between human L-PGDS and lipophilic ligands using isothermal titration calorimetry. Although the classical hydrophobic effect was mostly detected, all complex formations were driven by favorable enthalpic gains.

Rhodium-catalyzed asymmetric hydroarylation of 3-pyrrolines giving 3-arylpyrrolidines: protonation as a key step.

A hydroxorhodium complex coordinated with (R)-segphos was found to catalyze the hydroarylation of 3-pyrrolines with arylboroxines under neutral conditions to give 3-arylpyrrolidines with high enantioselectivity in high yields.

Differential gene regulation by V(IV) and V (V) ions in the branchial sac, intestine, and blood cells of a vanadium-rich ascidian, Ciona intestinalis.

Ascidians are hyperaccumulators that have been studied in detail. Proteins and genes involved in the accumulation process have been identified, but regulation of gene expression related to vanadium accumulation remains unknown. To gain insights into the regulation of gene expression by vanadium in a genome-wide manner, we performed a comprehensive study on the effect of excess vanadium ions on a vanadium-rich ascidian, Ciona intestinalis, using a microarray.

Systematic interaction analysis of human lipocalin-type prostaglandin D synthase with small lipophilic ligands.

L-PGDS [lipocalin-type PG (prostaglandin) D synthase] is a multi-functional protein, acting as a PGD₂-producing enzyme and a lipid-transporter. In the present study, we focus on the function of L-PGDS as an extracellular transporter for small lipophilic molecules. We characterize the binding mechanism of human L-PGDS for the molecules, especially binding affinity stoichiometry and driving force, using tryptophan fluorescence quenching, ICD (induced circular dichroism) and ITC (isothermal titration calorimetry).

Drug delivery system for poorly water-soluble compounds using lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily and a secretory lipid-transporter protein, which binds a wide variety of hydrophobic small molecules. Here we show the feasibility of a novel drug delivery system (DDS), utilizing L-PGDS, for poorly water-soluble compounds such as diazepam (DZP), a major benzodiazepine anxiolytic drug, and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and anticonvulsant. Calorimetric experiments revealed for both compounds that each L-PGDS held three molecules with high binding affinities.