Magnetically Hidden State on the Ground Floor of the Magnetic Devil's Staircase.

We investigated the low-temperature and high-field thermodynamic and ultrasonic properties of SrCu_{2}(BO_{3})_{2}, which exhibits various plateaux in its magnetization curve above 27 T, called a magnetic Devil's staircase. The results of the present study confirm that magnetic crystallization, the first step of the staircase, occurs above 27 T as a first-order transition accompanied by a sharp singularity in heat capacity C_{p} and a kink in the elastic constant. In addition, we observe a thermodynamic anomaly at lower fields around 26 T, which has not been previously detected by any magnetic probes. At low temperatures, this magnetically hidden state has a large entropy and does not exhibit Schottky-type gapped behavior, which suggests the existence of low-energy collective excitations. Based on our observations and theoretical predictions, we propose that magnetic quadrupoles form a spin-nematic state around 26 T as a hidden state on the ground floor of the magnetic Devil's staircase.

Randomly Hopping Majorana Fermions in the Diluted Kitaev System α-Ru_{0.8}Ir_{0.2}Cl_{3}.

dc and ac magnetic susceptibility, magnetization, specific heat, and Raman scattering measurements are combined to probe low-lying spin excitations in α-Ru_{1-x}Ir_{x}Cl_{3} (x≈0.2), which realizes a disordered spin liquid. At intermediate energies (ℏω>3 meV), Raman spectroscopy evidences linearly ω-dependent Majorana-like excitations, obeying Fermi statistics. This points to robustness of a Kitaev paramagnetic state under spin vacancies. At low energies below 3 meV, we observe power-law dependences and quantum-critical-like scalings of the thermodynamic quantities, implying the presence of a weakly divergent low-energy density of states. This scaling phenomenology is interpreted in terms of the random hoppings of Majorana fermions. Our results demonstrate an emergent hierarchy of spin excitations in a diluted Kitaev honeycomb system subject to spin vacancies and bond randomness.

Megabenthic assemblages at the southern Central Indian Ridge - Spatial segregation of inactive hydrothermal vents from active-, periphery- and non-vent sites.

Active hydrothermal vents are small-scale habitats hosting endemic fauna in a well-defined zonation around fluid effluents. The fauna of inactive hydrothermal vents and its relation to active vents and non-vent area is poorly known. Characterizing inactive areas is prerequisite to establish protected areas, especially in the context of potential seafloor massive sulfide mining, which targets inactive sites. Hierarchical clustering and Distance-based Redundancy Analysis revealed five assemblages, with significantly associated substrate types: I) active hydrothermal vent, II) periphery, III) inactive hydrothermal vent and IV) soft- and V) hard-substrate within the non-vent area. For the first time, a unique inactive faunal assemblage could be identified within the hydrothermally extinct inactive Gauss field and on adjacent hard substrates. The spatial separation from the active Edmond field and periphery and the non-vent area indicates the existence of an inactive assemblage.

Three-dimensional longitudinal changes of maxilla and mandible morphology during the predental period.

AIM: The purpose of this study was to longitudinally analyse the morphology of maxilla and mandible over time in infants using a three-dimensional (3D) surface scanner. MATERIALS AND METHODS: Seventeen Japanese full-term infants participated in the study. Dental plaster models were fabricated every 3 months from 1 month of age to 12 months. The plaster models were scanned using the 3D surface scanner to create 3D models. The arch width, arch length, arch angle, palatal depth and palatal area of the 3D models were analysed. RESULTS: The arch width and length of maxilla and mandible increased as the arch angle decreased. The arch width and length of the maxilla were greater than those of the mandible. The total alveolar ridge morphology increased in size in the occlusal view, with marked growth in the sagittal direction. The palatal depth remained virtually unchanged although the palatal area increased as a result of buccal growth of the alveolar ridge. CONCLUSIONS: The morphological growth pattern of the maxilla and mandible in infants can be evaluated quantitatively using 3D analysis. Knowledge about the healthy development of children and their orofacial growth patterns during the predental period can be applied as an index for diagnostic criteria.

A 3D imaging and visualization workflow, using confocal microscopy and advanced image processing for brachyuran crab larvae.

Confocal laser scanning microscopy is an excellent tool for nondestructive imaging of arthropods and can provide detailed information on morphology including fine surface detail. A methodology is presented here for the visualization by confocal microscopy of arthropods, using brachyuran crab zoeal stages as examples and postprocessing techniques derived from micro-CT protocols to improve the final images. This protocol is divided into description of the preprocessing steps (cleaning, staining, digesting and mounting), confocal laser scanning microscopy and data visualization using open-source, freeware programs ImageJ and Drishti. The advantages of using ImageJ to standardize stack data and Drishti for surface rendering are discussed. The methodology has been comprehensively tested using data acquired from all four brands of confocal microscope (Leica, Nikon, Olympus and Zeiss).

Application of three-dimensional digital models for the morphometric analysis of predentition plasters: accuracy and precision.

AIM: This study aimed to test the accuracy and precision of measurements of three-dimensional (3D) digital models from the pre-dentition period using a noncontact 3D measurement system (3D scanner) versus the gold standard method of direct measurements using a digital caliper on plaster models. MATERIALS AND METHODS: Ten pairs of plaster models were obtained from children during the predentition period. Linear measurements were performed using both methods. Three operators were trained in the use of both methods for this study. Measurements were performed with a minimum 2-week interval between measurements in a randomly chosen order. RESULTS: The mean difference between the measured values using the two methods was <0.2 mm for each measurement. There was no linearity in the measurements using pre-dentition digital models. An ANOVA Gage R&R analysis revealed that there was no significant operator difference (P < 0.307). The rate of variation of the 3D scanner over the total variation was 2.8%. The ICC was 0.982 (P< 0.001), suggesting excellent interoperator agreement. CONCLUSION: The results suggest that measurements of digital 3D pre-dentition models are highly accurate and precise, and also comparable to measurements using the gold standard method.

Adiabatic measurements of magneto-caloric effects in pulsed high magnetic fields up to 55 T.

Magneto-caloric effects (MCEs) measurement system in adiabatic condition is proposed to investigate the thermodynamic properties in pulsed magnetic fields up to 55 T. With taking the advantage of the fast field-sweep rate in pulsed field, adiabatic measurements of MCEs were carried out at various temperatures. To obtain the prompt response of the thermometer in the pulsed field, a thin film thermometer is grown directly on the sample surfaces. The validity of the present setup was demonstrated in the wide temperature range through the measurements on Gd at about room temperature and on Gd3Ga5O12 at low temperatures. The both results show reasonable agreement with the data reported earlier. By comparing the MCE data with the specific heat data, we could estimate the entropy as functions of magnetic field and temperature. The results demonstrate the possibility that our approach can trace the change in transition temperature caused by the external field.

Effect of glycogen synthase kinase 3 ß-mediated presenilin 1 phosphorylation on amyloid ß production is negatively regulated by insulin receptor cleavage.

Presenilin 1 (PS1), a causative molecule of familial Alzheimer's disease (AD), is known to be an unprimed substrate of glycogen synthase kinase 3 ß (GSK3ß) [Twomey and McCarthy (2006) FEBS Lett 580:4015-4020] and is phosphorylated at serine 353, 357 residues in its cytoplasmic loop region [Kirschenbaum et al. (2001) J Biol Chem 276:7366-7375]. In this report, we investigated the effect of PS1 phosphorylation on AD pathophysiology and obtained two important results--PS1 phosphorylation increased amyloid ß (Aß) 42/40 ratio, and PS1 phosphorylation was enhanced in the human AD brains. Interestingly, we demonstrated that PS1 phosphorylation promoted insulin receptor (IR) cleavage and the IR intracellular domain (IR ICD) generated by γ-secretase led to a marked transactivation of Akt (PKB), which down-regulated GSK3ß activity. Thus, the cleavage of IR by γ-secretase can inhibit PS1 phosphorylation in the long run. Taken together, our findings indicate that PS1 phosphorylation at serine 353, 357 residues can play a pivotal role in the pathology of AD and that the dysregulation of this mechanism may be causally associated with its pathology.

Angiotensin receptor type 1 antagonists protect against neuronal injury induced by oxygen-glucose depletion.

BACKGROUND AND PURPOSE: Several clinical trials and in vivo animal experiments have suggested that blockade of angiotensin receptor type 1 (AT(1)) improves ischaemic outcomes. However, the mechanism(s) underlying these effects has not been elucidated. Here, we have investigated the protective effects of pretreatment with AT(1) receptor antagonists, losartan or telmisartan, against ischaemic insult to neurons in vitro. EXPERIMENTAL APPROACH: Primary rat neuron-astrocyte co-cultures and astrocyte-defined medium (ADM)-cultured pure astrocyte cultures were prepared. Ischaemic injury was modelled by oxygen-glucose depletion (OGD) and lactate dehydrogenase release after OGD was measured with or without AT(1) receptor antagonists or agonists (L162313), AT(2) receptor antagonist (PD123319) or agonist (CGP-42112A) pretreatment, for 48 h. Activity of glutamate transporter 1 (GLT-1) was evaluated by [(3)H]-glutamate uptake assays, after AT(1) receptor agonists or antagonists. Immunoblot and real-time PCR were used for analysis of protein and mRNA levels of GLT-1. KEY RESULTS: AT(1) receptor agonists augmented OGD-induced cellular damage, which was attenuated by AT(1) receptor antagonists. AT(1) receptor antagonists also suppressed OGD-induced extracellular glutamate release, reactive oxygen species production and nitric oxide generation. GLT-1 expression and glutamate uptake activity were significantly enhanced by AT(1) receptor antagonists and impaired by AT(1) receptor agonists. AT(1) receptor stimulation suppressed both ADM-induced GLT-1 protein expression and mRNA levels. AT(1)b receptor knock-down with siRNA enhanced GLT-1 expression. In postnatal (P1-P21) rat brains, protein levels of GLT-1 and AT(1) receptors were inversely correlated. CONCLUSIONS AND IMPLICATIONS: Suppression of AT(1) receptor stimulation induced GLT-1 up-regulation, which ameliorated effects of ischaemic injury.

Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of alpha7 nicotinic receptors and internalization of NMDA receptors.

BACKGROUND AND PURPOSE: Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer's disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil. EXPERIMENTAL APPROACH: Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca(2+) concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD. KEY RESULTS: Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepezil. In addition, glutamate-mediated Ca(2+) entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of alpha7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca(2+) entry. LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca(2+) entry induced by donepezil. CONCLUSIONS AND IMPLICATIONS: Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of alpha7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.

Protective effect of H2O2 against subsequent H2O2-induced cytotoxicity involves activation of the PI3K-Akt signaling pathway.

Preconditioning of sublethal ischemia implies a cytoprotective mechanism against subsequent ischemia­induced cell death; however, the precise mechanism by which preconditioning protects against ischemic injury is not known. In the present study, we clarified whether pretreatment with a sublethal concentration of H2O2 could counter subsequent H2O2-induced cytotoxicity and also investigated the mechanisms of the cytoprotective effect of a sublethal concentration of H2O2. Using the MTT reduction assay and Calcein-AM staining assay, we showed that pretreatment with H2O2 (10 µM, 24 hr) of COS7 cells partially protected cells against subsequent H2O2 (6 mM, 1 hr) - induced cytotoxicity. The phosphorylation of Akt/PKB, a downstream target of phosphatydylinositol-3 kinase (PI3K), at Ser473 was augmented by H2O2 (10 µM) administration. This augmentation peaked at 10 minutes after H2O2 (10 µM) treatment and fell to the basal level at 24 hr. A blocker of PI3K, LY294002, significantly attenuated H2O2 (10 µM, 24 hr) - induced cytoprotection. In addition, pretreatment with LY294002 reduced H2O2 (10 µM, 10 min)-induced phosphorylation of Akt at Ser473. These findings suggest that a sublethal concentration of H2O2 exerts a cytoprotective effect against subsequent H2O2-induced cell death and that this cytoprotective effect of H2O2 is mediated by activation of the PI3K-Akt signaling pathway.

Practice patterns of Japanese physicians in urologic surveillance and management of spinal cord injury patients.

STUDY DESIGN: Analysis of answers to a new questionnaire. OBJECTIVE: To examine current practice patterns of physicians in the urological surveillance and management of spinal cord injury (SCI) patients in Japan. SETTING: Nationwide questionnaire survey to physicians in Japan. METHODS: A Japanese version of the 14-item questionnaire survey carried out in US was mailed to 770 members of the Japanese Neurogenic Bladder Society (JNBS). RESULTS: We received answers to our questionnaire from 333 (43.2%) members of JNBS. The responders were all urologists. For surveillance of the upper urinary tract (UUT), 239 (71.8%) respondents preferred abdominal ultrasound. Cystometry was performed routinely by 174 (52.3%) respondents for the evaluation of vesicourethral function. Cystoscopy was carried out in cases of hematuria (88.0%) and bladder stone (55.3%). Surveillance of the urinary tract was performed every year in 154 (46.2%). For detection of bladder cancer, which 119 (37.9%) respondents have experienced, 94.9% physicians perform cystoscopy, 76.3% urinary cytology, and 60.4% ultrasound. For initial treatment of detrusor-sphincter dyssynergia (DSD), 225 (69.2%) respondents chose alpha-blocker, and 94 (28.9%) chose clean intermittent catheterization (CIC) with/without anticholinergic agent(s). For initial treatment of overactive bladder, 245 (74.7%) chose anticholinergic agent(s) only and 63 (19.2%) chose anticholinergic agent(s) with CIC. For initial treatment of areflexic bladder, 233 (73.7%) chose CIC and 63 (19.9%) chose Credé maneuver or tapping. CONCLUSIONS: This survey shows that there are some differences in urological surveillance and management of SCI patients between Japan and the US. Reasons for the discrepancy should be examined.

In-situ visualization and quantification of mineralization of cultured osteogenetic cells.

An osteoblastic cell line (HOS cells) produces a prominent osteoid matrix with mineralization. Fibroblasts, on the other hand, do not exhibit this mineralization. To evaluate the degree of mineralization, we added calcein to the culture medium and then observed the culture wells by using an image analyzer. The calcein uptake into the cell/matrix layer was detected in the HOS cells but not in the fibroblasts. The calcein uptake was also quantified in situ by using an image analyzer, which revealed high levels in the HOS cells, which correlated well with the calcium content of the mineralized matrix. Rat marrow cells were also cultured in media containing calcein, fetal bovine serum, beta-glycerophosphate, L-ascorbic acid 2-phosphate, and with or without dexamethasone. With the dexamethasone, the cells exhibited osteogenic differentiation that resulted in mineralized matrix formation after about 10 days. The matrix formation coincided with the appearance of calcein uptake into the cell/matrix layer, with the amount of calcein uptake increasing with time. By contrast, the culture without the dexamethasone did not exhibit matrix formation and the calcein uptake was negligible. In the case of both HOS cell and rat marrow cell cultures in vitro, calcein did not affect expressions of their alkaline phosphatase activity or osteocalcin production. Furthermore, histologic observation revealed that rat marrow cells subcultured with calcein could show osteogenic ability after in vivo implantation. These results suggest that the current method of detecting calcein uptake in a culture allows the monitoring of the osteogenic capacity of cultured cells, as well as the measurement of the amount of mineralization produced by the osteogenic cells. Given that osteogenic cultured cells/mineralized matrices are used in bone reconstruction surgery, the in situ monitoring method is invaluable in that it allows us to evaluate the osteogenic capacity of in vitro constructs.

Extraction of total RNA from leaves of Eucalyptus and other woody and herbaceous plants using sodium isoascorbate.

Rapid extraction of total RNA from Eucalyptus leaves is difficult due to the high content of polyphenolics and polysaccharides. A rapid and simple method was developed by using an extraction buffer containing sodium isoascorbate at a concentration of 500 mM. This method consisted of one or two chloroform extractions, one acid guanidium-phenol-chloroform extraction, and isopropanol precipitation alone. The yields of the RNA fractions were 246-1750 micrograms/g fresh weight when leaves of Eucalyptus, five other woody plants, and four herbaceous plants were used as samples. The contamination of the RNA fractions by proteins and polysaccharides was very limited as judged spectrophotometrically. When the RNA fractions were subjected to agarose gel electrophoresis, intact rRNA bands were detected. The RNA fractions could be used for RT-PCR. These results indicate that our new method achieves a simple and rapid preparation of high-quality RNA from leaves of Eucalyptus and other plant species.

Localization of bradykinin B(2) receptor in the follicles of porcine ovary and increased expression of matrix metalloproteinase-3 and -20 in cultured granulosa cells by bradykinin treatment.

We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B(2)R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B(2)R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B(2)R mRNA. The B(2)R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B(2)R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.

Repeated thermal therapy improves impaired vascular endothelial function in patients with coronary risk factors.

OBJECTIVES: We sought to determine whether sauna therapy, a thermal vasodilation therapy, improves endothelial function in patients with coronary risk factors such as hypercholesterolemia, hypertension, diabetes mellitus and smoking. BACKGROUND: Exposure to heat is widely used as a traditional therapy in many different cultures. We have recently found that repeated sauna therapy improves endothelial and cardiac function in patients with chronic heart failure. METHODS: Twenty-five men with at least one coronary risk factor (risk group: 38 +/- 7 years) and 10 healthy men without coronary risk factors (control group: 35 +/- 8 years) were enrolled. Patients in the risk group were treated with a 60 degrees C far infrared-ray dry sauna bath for 15 min and then kept in a bed covered with blankets for 30 min once a day for two weeks. To assess endothelial function, brachial artery diameter was measured at rest, during reactive hyperemia (flow-mediated endothelium-dependent dilation [%FMD]), again at rest and after sublingual nitroglycerin administration (endothelium-independent vasodilation [%NTG]) using high-resolution ultrasound. RESULTS: The %FMD was significantly impaired in the risk group compared with the control group (4.0 +/- 1.7% vs. 8.2 +/- 2.7%, p < 0.0001), while %NTG was similar (18.7 +/- 4.2% vs. 20.4 +/- 5.1%). Two weeks of sauna therapy significantly improved %FMD in the risk group (4.0 +/- 1.7% to 5.8 +/- 1.3%, p < 0.001). In contrast, %NTG did not change after two weeks of sauna therapy (18.7 +/- 4.2% to 18.1 +/- 4.1%). CONCLUSIONS: Repeated sauna treatment improves impaired vascular endothelial function in the setting of coronary risk factors, suggesting a therapeutic role for sauna treatment in patients with risk factors for atherosclerosis.

Apolipoprotein J/clusterin is induced in vascular smooth muscle cells after vascular injury.

BACKGROUND: Understanding the precise molecular mechanisms underlying the phenomenon of restenosis after PTCA may help us to develop a new strategy for the treatment of restenosis after PTCA. The purpose of this study was to identify the genes involved in vascular restenosis. METHODS AND RESULTS: Applying a differential hybridization method to a model of the balloon-injured rabbit aorta, we identified 6 cDNA clones that were upregulated after injury. Northern blot showed that 5 genes, but not apolipoprotein J (apoJ)/clusterin, were constitutively expressed in noninjured aorta and upregulated after balloon injury. ApoJ mRNA was not detectable in noninjured aorta (control), began to be expressed at 6 hours after injury, showed a peak level at 24 hours (a 48-fold increase), gradually declined, and returned to the control level at 24 weeks. Western blot and immunohistochemistry demonstrated no expression of apoJ protein in noninjured aorta, an expression of apoJ at 2 days after balloon injury, and a peak level (a 55-fold increase) at 2 to 8 weeks. The expression of apoJ protein continued until 24 weeks after injury. In situ hybridization revealed that apoJ mRNA was expressed in smooth muscle cells (SMCs) of media at 2 days after injury and in SMCs of media and neointima at 2 weeks. To analyze the function of apoJ, stably transfected rabbit SMCs were created. The expression of apoJ stimulated proliferation and migration of SMCs. CONCLUSIONS: ApoJ is dramatically induced in media and neointima after vascular injury, suggesting that apoJ contributes to restenosis after angioplasty.

Increased constriction of the ductus arteriosus with combined administration of indomethacin and L-NAME in fetal rats.

We studied age-related changes and the caliber of the ductus arteriosus (DA) after two-pathway inhibition of prostaglandin E(2) and nitric oxide (NO) by the combined administration of indomethacin, a cyclooxygenase inhibitor, and N(G)-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in fetal rats. Pregnant rats from day 18 to 21 of gestation were used. They were administered indomethacin orally (3 mg/kg) 3 h before cesarean section, and then L-NAME (50 mg/kg) was injected intraperitoneally 3 h before the rats were killed. Using rapid-freezing and shaving methods, the caliber of the DA in fetal rats was measured. Compared with the indomethacin alone group, indomethacin plus L-NAME further constricted the DA after indomethacin and L-NAME were simultaneously administered 3 h before the rats were sacrificed. The extent of the final DA constriction was almost equal to the addition of each effect of indomethacin and L-NAME. We concluded that the magnitude of DA constriction following indomethacin plus L-NAME was due to the additive effects of these agents, suggesting a possible method to treat patent DA in premature infants.