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Using electron holography and coherent beam Lorentz microscopy, Akira Tonomura investigated the physics of magnetic flux lines, or vortices, in metal and high-temperature superconductors for more than 20 years. The methodology he developed for doing this made use of coherent electron waves from cold-emission (field-emission) sources and their quantum-mechanical phase shifts. Using 300-kV and 1-MV electron microscopes, Tonomura and his team clarified the dynamic behavior of magnetic flux lines in Pb and Nb superconductors and in high-temperature YBa2Cu3O7-δ and Bi2Sr2CaCu2O8 + δ superconductors. This memorial paper reviews the static and dynamic flux-line behaviors in superconductors as revealed by their results.

A synthesis process for the ionic liquid (IL) 1-butyl-3-methylimidazolium chloride (BMIM.Cl) was developed using a flow microreactor (FMR) in this study. After the reaction rate analysis, the FMR was efficiently designed using computational fluid dynamics simulation, which can shorten the process development time by reducing trial-and-error experimentation. The designed FMR is composed of a V-shaped mixer and a tubular reactor having a millimeter-scale inner diameter. From the viewpoint of process operation and control, reactors with larger inner diameter are basically preferable. The influence of three inner diameters, 0.75, 2.16, and 4.35 mm, that satisfy temperature constraints on product quality, product yield, and production volume was investigated. It was found that as the inner diameter becomes large, the product yield becomes low because the mass transfer approaches the rate-limiting step of the reaction process. To avoid this problem, it was proposed that static mixers should be built in the reac...

Aβ-afferents in maxillary or V2 trigeminal ganglion (TG) neurons are somatosensory neurons that may be involved in both non-nociceptive and nociceptive functions in orofacial regions. However, electrophysiological properties of these V2 trigeminal Aβ-afferent neurons have not been well characterized so far. Here, we used rat ex vivo trigeminal nerve preparations and applied patch-clamp recordings to large-sized V2 TG neurons to characterize their electrophysiological properties. All the cells recorded had afferent conduction velocities in the range of Aβ-afferent conduction speeds. However, these V2 trigeminal Aβ-afferent neurons displayed different action potential (AP) properties. APs showed fast kinetics in some cells but slow kinetics with shoulders in repolarization phases in other cells. Based on the derivatives of voltages in AP repolarization with time (dV/dt), we classified V2 trigeminal Aβ-afferent neurons into four types: type I, type II, type IIIa and type IIIb. Type I V2 trigeminal Aβ-afferent neurons had the largest dV/dt of repolarization, the fastest AP conduction velocities, the shortest AP and afterhyperpolarization (AHP) durations, and the highest AP success rates. In contrast, type IIIb V2 trigeminal Aβ-afferent neurons had the smallest dV/dt of AP repolarization, the slowest AP conduction velocities, the longest AP and AHP durations, and the lowest AP success rates. The type IIIb cells also had significantly lower voltage-activated K+ currents. For type II and type IIIa V2 trigeminal Aβ-afferent neurons, AP parameters were in the range between those of type I and type IIIb V2 trigeminal Aβ-afferent neurons. Our electrophysiological classification of V2 trigeminal Aβ-afferent neurons may be useful in future to study their non-nociceptive and nociceptive functions in orofacial regions.

BACE1 is an attractive target for disease-modifying treatment of Alzheimer's disease. BACE2, having high homology around the catalytic site, poses a critical challenge to identifying selective BACE1 inhibitors. Recent evidence indicated that BACE2 has various roles in peripheral tissues and the brain, and therefore, the chronic use of nonselective inhibitors may cause side effects derived from BACE2 inhibition. Crystallographic analysis of the nonselective inhibitor verubecestat identified explicit water molecules with different levels of free energy in the S2' pocket. Structure-based design targeting them enabled the identification of propynyl oxazine 3 with improved selectivity. Further optimization efforts led to the discovery of compound 6 with high selectivity. The cocrystal structures of 7, a close analogue of 6, bound to BACE1 and BACE2 confirmed that one of the explicit water molecules is displaced by the propynyl group, suggesting that the difference in the relative water displacement cost may contribute to the improved selectivity.

Anaeromyxobacter sp. strain PSR-1, a dissimilatory arsenate [As(V)]-reducing bacterium, can utilize As(V) as a terminal electron acceptor for anaerobic respiration. A previous draft genome analysis revealed that strain PSR-1 lacks typical respiratory As(V) reductase genes (arrAB), which suggested the involvement of another protein in As(V) respiration. Dissimilatory As(V) reductase activity of strain PSR-1 was induced under As(V)-respiring conditions and was localized predominantly in the periplasmic fraction. The activity was visualized by partially denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis identified proteins involved in the active band. Among these proteins, a protein annotated as molybdopterin-dependent oxidoreductase (PSR1_00330) exhibited the highest sequence coverage, 76%. Phylogenetic analysis revealed that this protein was a homolog of tetrathionate reductase catalytic subunit TtrA. However, the crude extract of strain PSR-1 did not show significant tetrathionate reductase enzyme activity. Comparative proteomic analysis revealed that the protein PSR1_00330 and a homolog of tetrathionate reductase electron transfer subunit TtrB (PSR1_00329) were expressed abundantly and specifically under As(V)-respiring conditions, respectively. The genes encoding PSR1_00330 and PSR1_00329 formed an operon-like structure along with a gene encoding a c-type cytochrome (cyt c), and their transcription was upregulated under As(V)-respiring conditions. These results suggest that the protein PSR1_00330, which lacks tetrathionate reductase activity, functions as a dissimilatory As(V) reductase in strain PSR-1. Considering the wide distribution of TtrA homologs among bacteria and archaea, they may play a hitherto unknown role along with conventional respiratory As(V) reductase (Arr) in the biogeochemical cycling of arsenic in nature. IMPORTANCE Dissimilatory As(V)-reducing prokaryotes play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. Generally, such prokaryotes reduce As(V) by means of a respiratory As(V) reductase designated Arr. However, some dissimilatory As(V)-reducing prokaryotes such as Anaeromyxobacter sp. strain PSR-1 lack genes encoding Arr, suggesting the involvement of other protein in As(V) reduction. In this study, using multiple proteomic and transcriptional analyses, it was found that the dissimilatory As(V) reductase of strain PSR-1 was a protein closely related to the tetrathionate reductase catalytic subunit (TtrA). Tetrathionate reductase is known to play a role in anaerobic respiration of Salmonella on tetrathionate, but strain PSR-1 showed neither growth on tetrathionate nor significant tetrathionate reductase enzyme activity. These results suggest the possibility that TtrA homologs encoded in a wide variety of archaeal and bacterial genomes might function as dissimilatory As(V) reductases.

Cells recognize collagen fibrils as the first step in the process of adherence. Fibrils of chicken skin actinidain-hydrolyzed collagen (low adhesive scaffold collagen, LASCol), in which the telopeptide domains are almost completely removed, cause adhering cells to form spheroids instead of adopting a monolayer morphology. Our goal was to elucidate the ultrastructure of the LASCol fibrils compared with pepsin-hydrolyzed collagen (PepCol) fibrils. At low concentration of 0.2 mg/mL, the time to reach the maximum increasing rate of turbidity for LASCol was all slower than that for PepCol. Differential scanning calorimetry showed that the thermal stability of collagen self-assembly changed significantly between pH 5.5 and pH 6.6 with and without a small number of telopeptides. However, the calorimetric enthalpy change did not vary much in that pH range. The melting temperature of LASCol fibrils at pH 7.3 was 55.1 °C, whereas PepCol fibrils exhibited a peak around 56.9 °C. The D-periodicity of each fibril was the same at 67 nm. Nevertheless, the looseness of molecular packing in LASCol fibrils was demonstrated by circular dichroism measurements and immuno-scanning electron microscopy with a polyclonal antibody against type I collagen. As there is a close relationship between function and structure, loosely packed collagen fibrils would be one factor that promotes cell spheroid formation.

Intervertebral discs are important for maintaining mobility and offer support to the body trunk. If these discs lose their biomechanical features, lower back pain can occur. We previously reported that hepatocyte growth factor (HGF) promotes cell proliferation and suppresses apoptosis, inflammation, and matrix degradation in nucleus pulposus (NP) cells. In the present study, we investigated the molecular mechanisms of how HGF promotes the proliferation of NP cells in hypoxic conditions. Hypoxic stimulation promoted modest cell proliferation, which was further upregulated by HGF. Expression of hypoxia-inducible factor (HIF-1α) protein, which contributes to the maintenance of homeostasis in NP cells, was also upregulated in hypoxia-treated cell groups; HGF further increased HIF-1α expression in NP cells. Additionally, knockdown of HIF-1α expression significantly reduced the proliferation of NP cells. An MAPK inhibitor inhibited the expression of HIF-1α and pERK, as well as cell proliferation in a dose-dependent manner. Similarly, inhibiting the PI3K/Akt and STAT3 pathways also decreased the expression of HIF-1α and cell proliferation. These results show that under hypoxic conditions, HGF promotes NP cell proliferation via HIF-1α-, MAPK-, PI3K/Akt-, and STAT3-mediated signaling which is involved in this pathway. The control of these signaling pathways may be a target for potential therapeutic strategies for the treatment of disc degeneration in hypoxic conditions.

Neuropeptide Y (NPY) Y5 receptor plays a key role in the effects of NPY, an important neurotransmitter in the control of energy homeostasis including stimulation of food intake and inhibition of energy expenditure. The NPY-Y5 receptor system has been an attractive drug target for potential use in treating obesity. Here we report the discovery and characterization of two novel Y5 receptor antagonists, S-2367 and S-234462. Both compounds displayed high affinity for the Y5 receptor in the radio-ligand binding assay, while in the cell-based functional assay, S-2367 and S-234462 showed, respectively, surmountable and insurmountable antagonism. In cell-based washout experiments, S-234462 dissociated from the Y5 receptor more slowly than S-2367. In vivo study showed that S-234462 effectively suppressed food intake induced by acute central injection of a selective Y5 receptor agonist. Furthermore, high-fat diet-induced obese (DIO) mice treated with S-234462 for 5 weeks showed a significant decrease in body weight gain and food intake compared to those treated with S-2367. In conclusion, S-234462 exhibits insurmountable antagonism of NPY Y5 receptor in vitro and superior anti-obesity effects to the surmountable NPY Y5 antagonist S-2367 in DIO mice.

The objective of the present study was to investigate the effect of platelet-rich plasma (PRP) combined with gelatin β-tricalcium phosphate (β-TCP) sponge on bone generation in a lumbar vertebral body defect of ovariectomized rat. After creating critical-size defects in the center of the anterior vertebral body, the defects were filled with the following materials: (1) no material (control group), (2) gelatin β-TCP sponge with PRP (PRP sponge group), and (3) gelatin β-TCP sponge with phosphate-buffered saline (PBS sponge group). Microcomputed tomography and histological evaluation were performed immediately after surgery and at 4, 8, and 12 weeks to assess bone regeneration. Biomechanical test was also performed at postoperative week 12. In the PRP sponge group, both imaging and histological examination showed that visible osteogenesis was first induced and additional growth of bone tissue was observed in the transplanted sponge, compared with the PBS sponge group. There was no negative effect of either PRP sponge or PBS sponge transplantation on bone tissue generation around the periphery of the defect. Biomechanical test showed increased stiffness of the affected vertebral bodies in the PRP sponge group. These results indicate that PRP-impregnated gelatin β-TCP sponge is effective for facilitating bone regeneration in lumbar vertebral bone defect under osteoporotic condition. PRP combined with gelatin β-TCP sponges could be potentially useful for developing a new approach to vertebroplasty for osteoporotic vertebral fracture.

Temperature-sensitive two-pore domain potassium channels (thermal K2P) are recently shown to cluster at nodes of Ranvier (NRs) and play a key role in action potential (AP) regeneration and conduction on Aβ-afferent nerves. Cooling temperatures affect AP regeneration and conduction on Aβ-afferent nerves but the underlying mechanisms are not completely understood. Here, we have performed patch-clamp recordings directly at the NR in anex vivotrigeminal nerve preparation. We have characterized the effects of cooling temperatures on intrinsic electrophysiological properties and AP regeneration at the NR on rat Aβ-afferent nerves, and determined whether and how thermal K2P channels may be involved in the effects of cooling temperatures. We show that cooling temperatures from 35°C to 15°C decrease outward leak currents, increase input resistance, depolarize resting membrane potential (RMP), broaden AP width and increase latency of AP threshold at the NR. We further demonstrate that cooling temperatures impair regeneration of high-frequency AP trains at the NR. The effects of cooling temperatures on the intrinsic electrophysiological properties and regeneration of high-frequency AP trains at the NR can be partially reversed by BL-1249 (BL), arachidonic acid (AA), and intra-axonal protons, three thermal K2P activators, indicating the involvement of thermal K2P channels. Moreover, we show that at cooling temperatures there are interplays among thermal K2P channels, RMPs, and voltage-gated Na+channels, which together limit regeneration of high-frequency AP trains at the NR. Our findings demonstrate a new role of thermal K2P channels in temperature-dependent conduction of high-frequency sensory signals.

Excess intramyocellular lipid (IMCL) deposition in skeletal muscle is closely associated with insulin resistance. Pharmacological inhibition of acetyl-CoA carboxylase (ACC) 2 offers a promising approach to treat insulin resistance through stimulation of mitochondrial fatty acid oxidation (FAO) and reduction of IMCL deposition. Previously reported experimental ACC2 inhibitors exhibited plasma glucose-lowering effects in diabetic rodents. However, their antidiabetic action may be potentially biased by off-target effects on triglyceride metabolism or by neurologic side effects. In this study, we investigated a safety profile, target dependency of its action, and antidiabetic efficacy of compound 2e, a novel olefin derivative potent ACC2 selective inhibitor. Four-day administration of suprapharmacological dose of compound 2e did not exhibit any obvious side effects in Sprague-Dawley rats. In db/db mice, single administration of compound 2e led to significantly elevated FAO and reduced IMCL deposition in skeletal muscle. In ACC2 knockout mice, treatment with pharmacological doses of compound 2e did not reduce plasma triglyceride levels, whereas A-908292, a previously reported ACC2 inhibitor, caused a significant triglyceride reduction, showing that compound 2e was devoid of off-target triglyceride-lowering activity. Chronic treatment of db/db mice with compound 2e improved hyperglycemia but did not decrease plasma triglyceride levels. Additionally, compound 2e showed significant improvements of whole-body insulin resistance in the clamp study and insulin tolerance test. Collectively, compound 2e demonstrated a good safety profile and significant antidiabetic effects through inhibition of ACC2-dependent pathways. These findings provide further evidence that selective inhibition of ACC2 is an attractive strategy against insulin resistance and type 2 diabetes. SIGNIFICANCE STATEMENT: This study shows that pharmacological inhibition of acetyl-CoA carboxylase (ACC) 2 leads to significant improvements in whole-body glucose homeostasis, independently of off-target metabolic pathways and toxicity, which were observed in previously reported ACC2 inhibitors. These findings support the concept that ACC2-selective inhibitors will be a novel remedy for treatment of type 2 diabetes.

In the near future, electrification will be introduced to heavy-duty vehicles and passenger cars. However, the wireless power transfer (WPT) requires high energy levels, and the suitability of various types of WPT systems must be assessed. This paper describes a method for solving technical and safety issues associated with this technology. We exposed human corneal epithelial (HCE-T) cells derived from the human eye to 5.8-GHz electromagnetic fields for 24 h. We observed no statistically significant increase in micronucleus (MN) frequency in cells exposed to a 5.8-GHz field at 1 mW/cm2 (the general public level in ICNIRP) relative to sham-exposed or incubator controls. Similarly, the DNA strand breaks, and the expression of heat shock protein (Hsp) Hsp27, Hsp70, and Hsp $90\alpha $ exhibited no statistically significant effects as a result of exposure. These results indicate that the exposure to 5.8-GHz electromagnetic fields at 1 mW/cm2 for 24 h has little or no effect on micronucleus formation, DNA strand breaks, and Hsp expression in human eye cells.

COURSE50 (ultimate reduction of CO2 in the steelmaking process through innovative technology for Cool Earth 50) aims to capture, separate, and recover CO2 from blast furnace gas. From a practical realization viewpoint, three points are important. The first is energy consumption to regenerate the absorbent, second is the energy cost of the heat for regeneration, and third is the facility cost. The advantage afforded by the COURSE50 approach in relation to the CO2 capture process is the utilization of unused waste heat from the steel mills. Energy consumption to regenerate the absorbent is determined mainly by three factors: the regeneration reaction determined primarily by the structure of the chemical absorbent, the energy required to heat that volume of absorption liquid, which is affected by the absorption rate of the agent, and the heat loss from the processes. The most influential factor is the energy required for the regeneration reaction. We discovered high-performance absorbents with the advantages of high absorption rates, high cyclic capacities, and low heats of reaction, and we then compared these with monoethanolamine (MEA) and N-methyldiethanolamine (MDEA). The newly discovered absorbents performed well in terms of absorption rates and cyclic capacities. Among these absorbents, some showed lower heats of reaction than MDEA. These results provide a basic guideline for the discovery of potential amine-based absorbents that may lead to the development of new absorbent systems for CO2 capture.

COURSE50 (CO2 Ultimate Reduction in Steelmaking process by innovative technology for cool Earth 50) aims to increase the proportion of hydrogen reduction in the blast furnace. This objective raises the key issue of heat balance changes in individual regions as well as in the overall blast furnace. In order to compensate for the endothermic reactions of hydrogen, a decrease in direct reduction by carbon, a huge endothermic reaction, is being executed. Among the various hydrogen sources available in the industry, coke oven gas (COG) was chosen because of its availability and stability. However, COG requires reforming for it to be injected into the shaft of the blast furnace because this zone cannot combust the hydrocarbon components of COG. COURSE50 has carried out successful COG and reformed COG injection trials at LKAB’s experimental blast furnace in Lulea, Sweden, in cooperation with LKAB and Swerea MEFOS. Carbon consumption in both the COG and reformed COG injection periods decreased compared with the base period because of the planned increase in hydrogen reduction instead of direct reduction by carbon. These results indicate the possibility of increasing the amount of hydrogen reduction in the blast furnace.

Immunosensing is a bioanalytical technique capable of selective detections of pathogens by utilizing highly specific and strong intermolecular interactions between recognition probes and antigens. Here, we exploited the molecular mechanism in artificial nanopores for selective single-virus identifications. We designed hemagglutinin antibody mimicking oligopeptides with a weak affinity to influenza A virus. By functionalizing the pore wall surface with the synthetic peptides, we rendered specificity to virion-nanopore interactions. The ligand binding thereof was found to perturb translocation dynamics of specific viruses in the nanochannel, which facilitated digital typing of influenza by the resistive pulse bluntness. As amino acid sequence degrees of freedom can potentially offer variety of recognition ability to the molecular probes, this peptide nanopore approach can be used as a versatile immunosensor with single-particle sensitivity that promises wide applications in bioanalysis including bacterial and viral screening to infectious disease diagnosis.

Novel acetyl-CoA carboxylase 2 (ACC2) selective inhibitors were identified by the conversion of the alkyne unit of A-908292 to the olefin linker. Modification of the center and left part of the lead compound 1b improved the ACC2 inhibitory activity and CYP450 inhibition profile, and afforded a highly selective ACC2 inhibitor 2e which showed in vivo efficacy in C57BL/6 mice.

Bioinspired pore sensing for selective detection of flagellated bacteria was investigated. The Au micropore wall surface was modified with a synthetic peptide designed from toll-like receptor 5 (TLR5) to mimic the pathogen-recognition capability. We found that intermolecular interactions between the TLR5-derived recognition peptides and flagella induce ligand-specific perturbations in the translocation dynamics of Escherichia coli, which facilitated the discrimination between the wild-type and flagellin-deletion mutant (ΔfliC) by the resistive pulse patterns thereby demonstrating the sensing of bacteria at a single-cell level. These results provide a novel concept of utilizing weak intermolecular interactions as a recognition probes for single-cell microbial identification.

The etiology of intervertebral disc (IVD) degeneration is closely related to apoptosis and extracellular matrix degradation in nucleus pulposus (NP) cells. These defects in NP cells are induced by excessive external stressors such as reactive oxygen species (ROS) and inflammatory cytokines. Recently, hepatocyte growth factor (HGF) has been shown to repair damage in various diseases through anti-apoptotic and anti-inflammatory activity. In this study, we investigated the effects of HGF on NP cell abnormality caused by ROS and inflammatory cytokines by using primary NP cells isolated from rabbit IVD. HGF significantly enhanced the proliferation of NP cells. Apoptosis of NP cells induced by H2 O2 or TNF-α was significantly inhibited by HGF. Induction of mRNA expression of the inflammation mediators cyclooxygenase-2 and matrix metalloproteinase-3 and -9 by TNF-α was significantly suppressed by HGF treatment. Expression of c-Met, a specific receptor for HGF, was confirmed in NP cells and was increased by TNF-α, suggesting that inflammatory cytokines increase sensitivity to HGF. These findings demonstrate that activation of HGF/c-Met signaling suppresses damage caused by ROS and inflammation in NP cells through multiple pathways. We further suggest the clinical potential of HGF for counteracting IVD degradation involved in NP cell abnormalities.

Microreactors with gas–liquid slug flow have an advantage over the conventional batch reactors in mass transfer between mutually immiscible fluids because of the internal circulation flow within the liquid slug. To realize stable long-term operation of microreactors with gas–liquid slug flow, the slug lengths in the microreactors have to be monitored and controlled, because they influence mass transfer performance. In this study, an experimental investigation is carried out to analyze the formation of gas–liquid slug flow in a T-shaped microreactor. The experimental result shows that the pressures in gas and liquid feeding tubes connected to the microreactor oscillate periodically along the formation of a pair of gas and liquid slugs. Based on this result, a method for estimating slug lengths from feed pressure measurements is developed. The method is non-invasive and does not affect slug formation or the manner of gas–liquid slug flow. Its effectiveness was assessed through experimental case studies using T-shaped microreactors having different gas–liquid confluence angles and different gas and liquid flow rates. The relative root mean square errors of estimated slug lengths were within 2% for one minute operation. The results clearly show that the proposed method can be applied to the real time monitoring of slug lengths.

Glutamine synthetase (GS) plays an important role in glutamate neurotransmission or neurological disorder in the brain. [(13) N]Ammonia blood flow tracer has been reported to be metabolically trapped in the brain via the glutamate-glutamine pathway. The present study investigated the effect of an inhibitor of GS on [(13) N]ammonia uptake in order to clarify the feasibility of measuring GS activity in the living brain. l-Methionine sulfoximine (MSO), a selective GS inhibitor was microinjected into the ipsilateral striatum in rats. [(13) N]Ammonia uptake was quantified by autoradiography method as well as small animal positron emission tomography (PET) scans. The GS activity of the brain homogenate was assayed from the γ-glutamyl transferase reaction. Autoradiograms showed a decrease of [(13) N]ammonia radioactivity on the MSO-injected side compared with the saline-injected side of the striatum. This reduction could be detected with a small animal PET scanner. MSO had no effect on cerebral blood flow measured by uptake of [(15) O]H2 O. The reduction of [(13) N]ammonia uptake was closely related to the results of GS activity assay. These results indicated that [(13) N]ammonia may enable measurement of GS activity in the living brain.

To assess reactive oxygen species (ROS) production by detecting the fluorescent oxidation product, hydroethidine has been used extensively. The present study was undertaken to evaluate the potential of the hydroethidine derivative as a radiotracer to measure in vivo brain ROS production. [3H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([3H]Hydromethidine) was synthesized, and evaluated using in vitro radical-induced oxidization and in vivo brain ROS production model. In vitro studies have indicated that [3H]Hydromethidine is converted to oxidized products by a superoxide radical (O2•-) and a hydroxyl radical (OH•-) but not hydrogen peroxide (H2O2). In vivo whole-body distribution study showed that [3H]Hydromethidine rapidly penetrated the brain and then was washed out in normal mice. Microinjection of sodium nitroprusside (SNP) into the brain was performed to produce ROS such as OH•- via Fenton reaction. A significant accumulation of radioactivity immediately after [3H]Hydromethidine injection was seen in the side of the brain treated with SNP (5 and 20 nmol) compared with that in the contralateral side. These results indicated that [3H]Hydromethidine freely penetrated into the brain where it was rapidly converted to oxidized forms, which were trapped there in response to the production of ROS. Thus, [3H]Hydromethidine should be useful as a radical trapping radiotracer in the brain.

Traditional biomarkers such as alanine and aspartate aminotransferase (ALT, AST) and total bilirubin (TBIL) have been widely used for detecting drug-induced liver injury (DILI). Although the Food and Drug Administration (FDA) proposed standardized thresholds for human as Hy's law, those for animals have not been determined, and predictability of these biomarkers for future onset of hepatic lesions remains unclear. In this study, we investigated these diagnostic and predictive performance of 10 traditional biomarkers for liver injury by receiver-operating characteristic (ROC) curve, using a free-access database where 142 hepatotoxic or non-hepatotoxic compounds were administrated to male rats (n=5253). Standardization of each biomarker value was achieved by calculating the ratio to control mean value, and the thresholds were determined under the condition of permitting 5% false positive. Of these 10 biomarkers, AST showed the best diagnostic performance. Furthermore, ALT and TBIL also showed high performance under the situation of hepatocellular necrosis and bile duct injury, respectively. Additionally, the availability of the diagnostic thresholds in difference testing facility was confirmed by the application of these thresholds to in-house prepared dataset. Meanwhile, incorrect diagnosis by the thresholds was also observed. Regarding prediction, all 10 biomarkers showed insufficient performance for future onset of hepatic lesions. In conclusion, the standardized diagnostic thresholds enable consistent evaluation of traditional biomarkers among different facilities, whereas it was suggested that novel biomarker is required for more accurate diagnosis and prediction of DILI.

Rapid conduction of nerve impulses is critical in life and relies on action potential (AP) leaps through the nodes of Ranvier (NRs) along myelinated nerves. While NRs are the only sites where APs can be regenerated during nerve conduction on myelinated nerves, ion channel mechanisms underlying the regeneration and conduction of APs at mammalian NRs remain incompletely understood. Here, we show that TREK-1 and TRAAK, the thermosensitive and mechanosensitive two-pore-domain potassium (K2P) channels, are clustered at NRs of rat trigeminal Aβ-afferent nerves with a density over 3,000-fold higher than that on their somas. These K2P channels, but not voltage-gated K+ channels as in other parts of nerves, are required for rapid AP repolarization at the NRs. Furthermore, these channels permit high-speed and high-frequency AP conduction along the myelinated afferent nerves, and loss of function of these channels at NRs retards nerve conduction and impairs sensory behavioral responses in animals.

Nephrotoxicity is a common side effect observed during both nonclinical and clinical drug development investigations. The present study aimed to identify metabolomic biomarkers that could provide early and sensitive indication of nephrotoxicity in rats. Metabolomic analyses were performed using capillary electrophoresis-time-of-flight mass spectrometry on rat plasma collected at 9 and 24 h after a single dose of 2-bromoethylamine or n-phenylanthranilic acid and at 24 h after 7 days of repeated doses of gentamicin, cyclosporine A or cisplatin. Among a total of 169 metabolites identified, 3-methylhistidine (3-MH), 3-indoxyl sulfate (3-IS) and guanidoacetate (GAA) were selected as candidate biomarkers. The biological significance and reproducibility of the observed changes were monitored over time in acute nephrotoxicity model rats treated with a single dose of cisplatin, with the glomerular filtration rate monitored by determination of creatinine clearance. Increased plasma levels of 3-MH and 3-IS were related to a decline in glomerular filtration due to a renal failure. In contrast, the decrease in plasma GAA, which is synthesized from arginine and glycine in the kidneys, was considered to reflect decreased production due to renal malfunction. Although definitive validation studies are required to confirm their usefulness and reliability, 3-MH, 3-IS and GAA may prove to be valuable plasma biomarkers for monitoring nephrotoxicity in rats.

5-Ethyl-3-methyl-2′,4′:3′,5′-di-O-methylenedioxy-riboflavinium perchlorate (DMRFlEt+ClO4 –) is readily derived from commercially available vitamin B2 (riboflavin) and exhibits high catalytic activity for the oxidation of organic sulfides under an oxygen atmosphere (1 atm) with the assistance of hydrazine hydrate as a reductant. This is an inexpensive, convenient, and environmentally benign method for the selective oxidative transformation of sulfides into sulfoxides.

Recent efforts have been made to identify useful urinary biomarkers of nephrotoxicity. Furthermore, the application of urine to the other toxicities as new biomarker source has been recently expanded. Meanwhile, correction of urinary biomarker concentrations according to fluctuations in urine flow rate is required for adequate interpretation of the alteration. The urinary biomarker-to-creatinine ratio (UBCR) is widely used because of the convenience, while the urinary biomarker-excretion rate is regarded as the gold standard corrective method. Because creatinine is a catabolite in energy production in muscles, we hypothesized that altered muscle mass could affect creatinine kinetics, ultimately affecting UBCR. However, no study has examined this hypothesis. In this study, we examined the influence of muscle mass gain on UBCR, using male Sprague-Dawley rats during the growth phase, 6–12-week old. Both plasma creatinine and excretion of urinary creatinine (Ucr excretion) showed increases with muscle mass gain in rats, in which the alterations of UBCR were lowered. The renal mRNA level of the organic cation transporter-2 (Oct2), a creatinine transporter, showed an age-related increase, whereas the mRNA level of multidrug and toxin extrusions-1 (Mate1) remained constant. Multiple regression analysis showed that the increase in creatinine clearance highly contributed to the age-related increase in Ucr excretion compared to the mRNA levels of Oct2 and Mate1. This suggested that the age-related increase in Ucr excretion may be attributable to the increased transglomerular passage of creatinine. In conclusion, the results suggest that muscle mass gain can affect creatinine kinetics, leading to underestimation of UBCR. Therefore, it is important to understand the characteristics of the corrective method when using urinary biomarker, the failure of which can result in an incorrect diagnosis.

This paper reports the recent results of improving the radiation hardness of silicon solar cells, which is SHARP and NASDA's project since 1998 (Tonomura et al., Second World Conference on Photovoltaic Solar Energy, 1998, pp. 3511–3514). Newly developed 2×2 cm2 Si solar cells with ultrathin substrates and both-side junction (BJ) structure showed 72.0 mW (13.3% efficiency) maximum output power at AM0, 28°C after 1 MeV electron irradiation up to 1×1015 e/cm2 and the best cell showed 72.5 mW (13.4%) maximum output power. These solar cells have p–n junctions at both front and rear surfaces and showed less radiation degradation and better remaining factor than previous solar cells.

Cardiotoxicity and musculoskeletal toxicity can be life-threatening, and thus have strong impact on both the development and marketing of drugs. Because the conventional biomarkers such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) have low detection power, there has been increasing interest in developing biomarkers with higher detection power. The current study examined the usefulness of several promising biomarkers, cardiac and skeletal muscle troponins (cTnI, cTnT and sTnI), fatty acid binding protein 3 (FABP3) and myosin light chain 3 (MYL3), and compared the obtained data to AST, LDH and CK in rat models treated with various myotoxic and non-myotoxic compounds (isoproterenol, metaproterenol, doxorubicin, mitoxantrone, allylamine, cyclosporine A, cyclophosphamide, aminoglutethimide, acetaminophen, methapyrilene, allylalcohol and α-naphthylisothiocyanate). These promising biomarkers were found to be superior to the conventional biomarkers, as they had a specific and abundant distribution within the heart and/or skeletal muscles; exhibited a positive correlation between the amplitude of increases and the degree of pathological alterations; had higher diagnostic accuracy for detecting pathological alterations; and had the additive effect of improving the diagnostic accuracy of conventional biomarkers. However, these promising biomarkers have several drawbacks including a rapid clearance, the fact that they are affected by renal dysfunction, and different reactivity to the mode of action of individual myotoxicants. In conclusion, the promising biomarkers cTnI, cTnT, FABP3, MYL3, and sTnI demonstrated sensitivity and specificity for cardiac and skeletal myotoxicity that was superior to those of conventional biomarkers, while we should pay attention to the drawbacks of these biomarkers when used in toxicity studies.

Among 13 kinds of metal oxides, ceria (CeO 2 ) shows the highest catalytic activity for one-pot selective synthesis of the secondary amide from 2-cyanopyridine and n -octylamine as a test reaction. CeO 2 acts as a reusable heterogeneous catalyst, and it is effective for the secondary amide formation from various nitriles and amines in high yields (73% to >99%). Pharmacologically important products such as heteroaromatic N-alky amides and morpholine amide are effectively synthesized, indicating that CeO 2 can be a practically useful catalyst. A proposed mechanism includes (1) hydration of nitrile to the primary amide and (2) transamidation of the primary amide with amine as the rate-limiting step. This reaction mechanism provides a reason why the present catalytic system gives high selectivity.

Recent research has revealed several useful urinary biomarkers of renal dysfunction such as acute kidney injury (AKI). For adequate evaluation of altered urinary biomarkers, it is necessary to consider the influence of varied urine flow rate (UFR). Calculation of the excretion rate of a urinary biomarker (UFR-correction) is the gold standard for the correction of UFR variation. An alternative method that is widely used is to calculate the ratio of the biomarker level to urinary creatinine (Ucr-correction). To date, the equivalence between these two methods has been examined only in a steady state situation such as diabetic nephropathy, and the urinary biomarkers examined have been limited to proteinuria and albuminuria. Therefore, we comprehensively addressed the relationship between Ucr-correction and UFR-correction of ten urinary biomarkers N-acetyl-β-d-glucosaminidase (NAG), lactate dehydrogenase (LDH), total protein, albumin, kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β(2)-microglobulin, cystatin-c and glutathione S-transferase-α in non-steady state situations such as AKI. All ten urinary biomarkers showed larger amplitude increases in AKI by Ucr-correction than by UFR-correction in linear regression analysis. Moreover, receiver operating characteristic curves analysis suggested that, at least for the biomarkers NAG and LDH, Ucr-correction had higher diagnostic power than UFR-correction. We observed a decrease in the Ucr excretion in AKI that was accompanied by a reduction in creatinine clearance and reduced mRNA expression of the renal organic cation transporter-2, which is known to function as a transporter for creatinine. These results may provide a mechanistic explanation for the phenomena obtained in Ucr-correction. In conclusion, while Ucr-correction could overestimate the degree of AKI, it could also provide higher diagnostic power for AKI than UFR-correction. We should take into consideration of these backgrounds when using the Ucr-correction.

The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real-time RT-PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression.

Phosphoric acid alkyl diphenyl esters were prepared by reacting diphenyl phosphoryl chloride with alcohols in the presence of amines, and then subjected to fluorinative hydrolysis with Bu4 NF to give phosphorofluoridic acid ammonium salt monoesters in moderate to high yields. The salts were converted to the corresponding acids by treatment with Amberlyst 15. The NMR properties of a series of organophosphorus compounds are presented. The catalytic activities of the acids for the condensation reaction of α-bromobutanoic acid and 3-hexyne-2-ol were tested. © 2011 Wiley Periodicals, Inc. Heteroatom Chem 22:417–425, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/hc.20700

To screen candidate materials for dynamic random-access memory capacitors, the tunneling probability at a constant equivalent oxide thickness (EOT) of metal-insulator-metal (MIM) capacitors was theoretically maximized according to a tradeoff between permittivity and band offset. As a result, it was found that cubic HfO2 with a TiN electrode is a promising candidate. TiN/Al-doped HfO2/TiN MIM capacitors were fabricated by inserting Al2O3 layers for phase control of HfO2 and for suppression of TiN oxidation. The fabricated capacitors exhibit leakage current of 80 nA/cm2 at 1 V and EOT of 0.7 nm. Moreover, the main leakage current was estimated to originate from oxygen vacancies.

Cardiotoxicity represents one of the most serious side effects of new drugs. It is essential for pharmaceutical companies to detect potential cardiotoxicity of candidate drugs in non-clinical studies during the early stages of drug development. In this study, we aimed to detect potential genomic biomarkers of rat cardiotoxicity using a toxicogenomics approach. In order to achieve this, we induced cardiac lesions in rats following treatment with the three prototypical cardiotoxic compounds isoproterenol, doxorubicin and carbofuran. We then undertook histopathological examination and microarray analysis at 8 or 24 h after single dosing. Using statistical and cluster analysis, we extracted 36 probe sets commonly up-regulated by the three cardiotoxic compounds. GO analysis revealed that these genes were functionally associated with either chemotaxis, tissue regeneration, positive regulation of cell proliferation, cellular organization and morphogenesis events in accordance with the degeneration of myocardium and inflammation observed in the histopathology analysis. Most of selected genes showed transient up-regulation at different time point for each compound. However, among these genes, Spp1, Fhl1, Timp1, Ccl7 and Reg3b revealed a sustained up-regulation with high expression levels at both time points for all three compounds. In conclusion, even though definitive validation studies are required for the establishment of their usefulness and reliability, these identified genes may prove to be the most promising candidate genomic biomarkers of cardiotoxicity in rats.

The effect of scaffold shape on dentin regeneration is not well understood. In this study, porous hydroxyapatite/beta-tricalcium phosphate (HAp/beta-TCP), powdered HAp/beta-TCP, and polyglycolic acid (PGA) fiber mesh were used as scaffolds and transplanted with cultured porcine dental pulp-derived cells into the backs of nude mice. Samples were harvested after 6 weeks. Newly-formed hard tissue was observed in all transplants. When porous HAp/beta-TCP was used, dentin-like hard tissue was observed on the inner wall with minimum cell inclusions and odontoblast-like cells were aligned adjacent to the hard tissue. When HAp/beta-TCP powders or PGA were used, bone-like hard tissues showed cell inclusions and cell alignment was not observed. Hard tissue from the HAp/beta-TCP block group was positive for type I collagen, osteonectin, bone sialoprotein and dentin sialoprotein (DSP), which are markers for dentin. This result was confirmed by in situ hybridization with a dsp probe. Only the aligned cells were positive with an antisense probe. On the other hand, hard tissue from other scaffolds showed incomplete expression of both bone and dentin markers and they were negative for osteonectin and DSP. These results suggest that scaffold shape affects the type of tissue regenerated by dental pulp-derived cells.

The purpose of this study was to investigate the influence of hydrostatic pressure (HP) on the gene expression of cartilage matrix, cytokines, and apoptosis-associated factors in chondrocytes in which the cartilage was in extracellular matrix (ECM)-rich or ECM-poor condition.Chondrocytes were isolated from rabbit joints and cultured in alginate beads. Immediately after embedding (0W group) or after 2 weeks culture (2W group), the amounts of glycosaminoglycan (GAG) in the alginate beads were quantified. Both groups were exposed to continuous HP of 10 or 50 MPa for 12 h. The expression of inflammatory cytokines, proteases, and apoptosis-related factors were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of proteoglycan core protein (PG) and collagen type II were quantified by real-time RT-PCR.All of the GAG components in alginate beads markedly increased in the 2W group. The expression of PG and collagen type II increased after exposure to 10 MPa in both groups. In the 0W group, these levels decreased after exposure to 50 MPa of HP. The expression of interleukins IL-6 and IL-8 increased after exposure to HP in the 0W group. HP at 50 MPa induced mRNA expression of ADAMTS-5 in the 0W group but not in the 2W group. The expression of Fas increased after exposure to HP in the 0W group.These findings suggested that nonphysiological, excessive HP on chondrocytes with the ECM in poor condition reduced matrix gene expression and increased expression of the genes associated with apoptosis and catabolism of the cartilage matrix. These results might therefore be associated with the pathogenesis of osteoarthritis.

Collagen is composed of fibrils that are formed by self-assembly of smaller units, monomers which are triple-helical polypeptide. However, the mechanism of fibril formation at the level of individual molecules has remained to be clarified. We found that the fluorescence of thioflavin T, which has been widely used as a specific dye for amyloid fibrils, also increased by binding with fibrils of atelocollagen prepared from yellowfin tuna skin. There was a linear correlation between the fluorescence increase and the amount of atelocollagen within a collagen concentration range of 0-0.15 mg/ml at pH 6.5 with 50 microM thioflavin T. In contrast, neither actinidain-processed collagen that keeps monomeric nature nor heat-denatured collagen could cause the fluorescence increase of thioflavin T at all. The relationship between the fluorescence increase and thioflavin T concentration was fit to a theoretical binary binding curve. An apparent dissociation constant, K(d), and a maximal fluorescence increase, DeltaF(max), were calculated at various pHs. The values of K(d) and DeltaF(max) were dependent on pH (K(d) was 9.4 microM at pH 6.5). The present finding demonstrates that thioflavin T specifically binds to collagen fibrils and may be used as a sensitive tool for the study of collagen structure.

In this paper, we characterized aluminum/sapphire interface structure by using high-resolution transmission electron microscopy. It was found that step structures of sapphire formed at the aluminum/sapphire interfaces during the liquid-phase bonding. It was discovered that the addition of silicon in aluminum significantly reduces the step growth at the interface. Silicon was found to segregate and precipitate at the interface. These results suggest that the strong preference of silicon at the interface may inhibit the step growth reactions during liquid-phase bonding. [doi:10.2320/matertrans.MC200810]

The aim of this study was to clarify the significance of subchondral bone in the pathology of osteoarthritis (OA) by investigating the expression of inflammatory cytokines, proteases, and receptor activator of NF-kappaB ligand (RANKL)/receptor activator of NF-kappaB (RANK)/osteoprotegerin (OPG) involved in cartilage degeneration.Subchondral bone was obtained from 19 patients diagnosed with knee OA and 4 patients diagnosed with femoral neck fracture. Subchondral bone osteoblasts (SBOs) were isolated, and total RNA was extracted. Messenger RNA expression of inflammatory cytokines, proteases, and RANKL/RANK/OPG were analyzed using a real-time reverse transcription-polymerase chain reaction (RT-PCR).Real-time RT-PCR showed that mRNA expressions of interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13), and RANKL were significantly enhanced in OA SBOs compared to SBOs without OA. The expressions of these genes was greater in patients with severe cartilage damage than in those with mild cartilage damage. A high correlation between mRNA expression of IL-6 and that of MMP-13 was found in OA SBOs.The increases in IL-6, MMP-13, and RANKL expression in OA SBOs suggest that in subchondral bone OA progression involves abnormal osseous tissue remodeling, which induces mechanical property changes. Cartilage degeneration in OA may also be due, at least in part, to IL-6 and MMP-13 produced by SBOs. Comprehensive research on these pathological features may lead to the development of more effective therapies for OA by administration of molecules that affect bone remodeling and metabolism.

We present a SAR method that can predict estrogen-like endocrine disrupting chemical (EDC) activity as well as key biodegradation steps for detoxification. This method is based on a recent graph-mining algorithm developed by Kudo et al., which generates a set of descriptors from all potent chemical fragments (including rings). This method is novel in that it achieves chemical diversity in the training data set by sampling another data set of larger diversity. The model achieved an 83% accuracy prediction rate, and identified 1291 EDC candidates from the KEGG database. From this set of candidate compounds, bisphenol A was chosen for assay validation and biodegradation pathway analysis. Results showed that bisphenol A exhibited estrogen-like activity and was degraded in three distinct reactions. The prediction model provided information on the mechanism of the ligand-target binding, such as key functional groups involved. We focused on the enzyme commission number, which is useful for analyses of biodegradation pathways. Results identified oxygenases, ether hydrolases, and carbon-halide lyases as being important in the biodegradation pathway. This combined approach provided new information regarding the biodegradation of EDCs, and can potentially be extended to applications with transcriptomic, proteomic, and metabolomic data to provide a quick screen of biological activity and biodegradation pathway(s).

A robust method for generating odontoblasts from cultured dental pulp cells has not been established. In this study, efficient methods for deriving odontoblasts from cultured human and porcine dental pulp-derived cells were investigated with special attention to species differences. Cultured human cells showed relatively low alkaline phosphatase (ALP) activity in the presence of dexamethasone (Dex) and beta-glycerophosphate (beta-Gly). In contrast, the addition of 1,25-dihydroxyvitaminD(3) (VitD3) significantly increased the ALP activity. In porcine cells, beta-Gly alone or a combination of Dex and beta-Gly significantly increased ALP activity; however, addition of VitD3 reduced this activity. RT-PCR and Western blotting analysis revealed that the combination of three induction reagents on human cells significantly upregulates the expression of osteocalcin mRNA, and dentin sialoprotein. We propose that the combination of Dex, beta-Gly, and VitD3 is critical for differentiation of human dental pulp-derived cells into odontoblasts. In addition, the inducibility of dental pulp-derived cells presented remarkable species differences.

Background Reactive oxygen species (ROS) have been implicated in cisplatin-induced nephrotoxicity. The aim of this study was to investigate the potential of using [3H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([3H]hydromethidine) for ex vivo imaging of regional ROS overproduction in mouse kidney induced by cisplatin. Methods Male C57BL/6 J mice were intraperitoneally administered with a single dose of cisplatin (30 mg/kg). Renal function was assessed by measuring serum creatinine and blood urea nitrogen (BUN) levels and morphology by histological examination. Renal malondialdehyde levels were measured as a lipid peroxidation marker. Autoradiographic studies were performed with kidney sections from mice at 60 min after [3H]hydromethidine injection. Results Radioactivity accumulation after [3H]hydromethidine injection was observed in the renal corticomedullary area of cisplatin-treated mice and was attenuated by pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger. Cisplatin administration significantly elevated serum creatinine and BUN levels, caused renal tissue damage, and promoted renal lipid peroxidation. These changes were significantly suppressed by DMTU pretreatment. Conclusions The present study showed that [3H]hydromethidine was rapidly distributed to the kidney after its injection and trapped there in the presence of ROS such as hydroxyl radicals, suggesting that [3H]hydromethidine is useful for assessment of the renal ROS amount in cisplatin-induced nephrotoxicity.

Background Reactive oxygen species (ROS) have been implicated in the pathophysiology of the brain after ischemic stroke. In this study, we investigate the generation of brain ROS after transient focal ischemia in mice using a radical trapping radiotracer, [3H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([3H]hydromethidine), which we recently reported as a ROS imaging probe. We also examined the effect of dimethylthiourea (DMTU), a hydroxyl radical scavenger, on brain ROS generation and infarct volume after transient focal ischemia in mice. Methods [3H]Hydromethidine was intravenously injected into mice at 1, 2, 5, and 7 h after transient middle cerebral artery occlusion (tMCAO), and then, the brain autoradiogram was acquired at 60 min after tracer injection. Brain infarct volumes at 24 h after tMCAO were assessed by 2,3,5-triphenyltetrazolium chloride staining. Results Accumulation of radioactivity was observed in the ipsilateral striatum and cortex at 1 h after tMCAO. The increase of radioactivity was attenuated at 2 h after tMCAO and then became maximized at 5 h. The high accumulation of radioactivity remained until 7 h after tMCAO. DMTU treatment significantly attenuated the accumulation of radioactivity in the ipsilateral hemisphere at 1, 5, and 7 h after tMCAO. Brain infarct volumes were also significantly reduced in DMTU-treated mice at 24 h after tMCAO. Conclusions These results indicated that [3H]hydromethidine is a useful radiotracer for detecting in vivo brain ROS generation such as hydroxyl radical after ischemic injury.

Here, we report a draft genome sequence of Anaeromyxobacter sp. strain PSR-1, an arsenate-respiring bacterium isolated from arsenic-contaminated soil. It contained three distinct arsenic resistance gene clusters ( ars operons), while no respiratory arsenate reductase gene ( arr ) was identified.