TGFß1 signaling protects chondrocytes against oxidative stress via FOXO1-autophagy axis.

OBJECTIVE: The forkhead box O1 (FOXO1) transcription factor is a key regulator of autophagy. In chondrocytes, reduced FOXO1 expression with aging causes osteoarthritis due to dysfunction of autophagy, but the mechanisms underlying regulation of FOXO1 expression and the reduction in expression with aging remain unclear. We investigated the mechanism by which transforming growth factor ß1 (TGFß1) signaling regulates the FOXO1-autophagy axis. METHODS: Expression of FOXO1 was measured in chondrocytes after TGFß1 treatment. Immunohistochemistry was performed to estimate the levels of activin receptor-like kinase 5 (ALK5) and FOXO1 in the knee joints of young, middle-aged and old mice. The effects of the ALK5 inhibitor and SMAD3 or SMAD2 knockdown on FOXO1 expression were evaluated. The role of TGFß1 in autophagy after hydrogen peroxide (H2O2) treatment was analyzed. The protective effect of TGFß1 against H2O2 treatment was assessed by cell viability assay and TUNEL assay. RESULTS: TGFß1 promoted the expression of FOXO1 mRNA and protein. Both ALK5 and FOXO1 expression decreased with aging. ALK5 inhibition and SMAD3 knockdown suppressed induction of FOXO1 expression by TGFß1, whereas SMAD2 knockdown increased it. TGFß1 promoted the expression of microtubule-associated proteins 1A/1B light chain 3B (LC3)-I protein via the SMAD3-FOXO1 pathway. Furthermore, under H2O2 treatment, TGFß1 promoted expression of LC3-II. TGFß1 pretreatment suppressed cell death of chondrocytes following H2O2 treatment, but this protective effect was abolished by FOXO1 knockdown. CONCLUSIONS: TGFß1 protects chondrocytes against oxidative stress via the FOXO1-autophagy axis, and a reduction in ALK5 expression might cause reduced FOXO1 expression with aging.

Smectic Liquid-Crystalline Structure of Skyrmions in Chiral Magnet Co_{8.5}Zn_{7.5}Mn_{4}(110) Thin Film.

The organizing of magnetic skyrmions shows several forms similar to atomic arrays of solid states. Using Lorentz transmission electron microscopy, we report the first direct observation of a stable liquid-crystalline structure of skyrmions in chiral magnet Co_{8.5}Zn_{7.5}Mn_{4}(110) thin film, caused by magnetic anisotropy and chiral surface twist. Elongated skyrmions are oriented and periodically arranged only in the ⟨110⟩ directions, whereas they exhibit short-range order along the ⟨001⟩ directions, indicating a smectic skyrmion state. In addition, skyrmions possess anisotropic interaction with an opposite sign depending on the crystal orientation, in contrast to existing isotropic interaction.

Schizophrenia-like phenotypes in mice with NMDA receptor ablation in intralaminar thalamic nucleus cells and gene therapy-based reversal in adults.

In understanding the mechanism of schizophrenia pathogenesis, a significant finding is that drug abuse of phencyclidine or its analog ketamine causes symptoms similar to schizophrenia. Such drug effects are triggered even by administration at post-adolescent stages. Both drugs are N-methyl-d-aspartate receptor (NMDAR) antagonists, leading to a major hypothesis that glutamate hypofunction underlies schizophrenia pathogenesis. The precise region that depends on NMDAR function, however, is unclear. Here, we developed a mouse strain in which NMDARs in the intralaminar thalamic nuclei (ILN) were selectively disrupted. The mutant mice exhibited various schizophrenia-like phenotypes, including deficits in working memory, long-term spatial memory, and attention, as well as impulsivity, impaired prepulse inhibition, hyperlocomotion and hyperarousal. The electroencephalography analysis revealed that the mutant mice had a significantly reduced power in a wide range of frequencies including the alpha, beta and gamma bands, both during wake and rapid eye movement (REM) sleep, and a modest decrease of gamma power during non-REM sleep. Notably, restoring NMDARs in the adult ILN rescued some of the behavioral abnormalities. These findings suggest that NMDAR dysfunction in the ILN contributes to the pathophysiology of schizophrenia-related disorders. Furthermore, the reversal of inherent schizophrenia-like phenotypes in the adult mutant mice supports that ILN is a potential target site for a therapeutic strategy.

Treatment of acute bleeding in acquired haemophilia A with recombinant activated factor VII: analysis of 10-year Japanese postmarketing surveillance data.

INTRODUCTION: Patients with acquired haemophilia A (AHA) have autoantibodies against factor VIII (FVIII), and may develop spontaneous bleeding that requires treatment with FVIII inhibitor bypassing agents such as recombinant activated FVII (rFVIIa, NovoSeven® ). However, data regarding the use of rFVIIa are limited. AIM: To investigate the use, efficacy and safety of rFVIIa for the treatment of AHA by analysis of 10-year multicentre Japanese postmarketing surveillance data. METHODS: Treatment regimens, haemostatic efficacy and adverse events were recorded for rFVIIa therapy of AHA patients with bleeding episodes. Treatment was evaluated as markedly effective, effective, moderate or ineffective. RESULTS: Data were collected for 371 bleeding episodes in 132 patients. Bleeding improved after rFVIIa therapy in 92% of episodes (markedly effective in 41%, effective in 10%, moderate in 41%). The response rate was significantly better in patients who received an initial dose of ≥90 µg kg-1 than in those who received an initial dose of <90 µg kg-1 . The response rate was also significantly better when rFVIIa was administered earlier after the onset of bleeding. Twelve serious adverse events were recorded in six patients, including five serious thromboembolic events in three patients who were all elderly with significant comorbidities. CONCLUSION: This is the largest, single-country study of rFVIIa therapy in AHA patients reported to date. The Japanese surveillance data show comparable efficacy and safety to prior multinational studies. Doses of 90-120 µg kg-1 and prompt initiation of treatment may be important to achieve good bleeding control.

A high efficient protocol for soybean root transformation by Agrobacterium rhizogenes and most stable reference genes for RT-qPCR analysis.

KEY MESSAGE: A 55% transformation efficiency was obtained by our optimized protocol; and we showed that GmELF1 - ß and GmELF1 - α are the most stable reference genes for expression analyses under this specific condition. Gene functional analyses are essential to the validation of results obtained from in silico and/or gene-prospecting studies. Genetic transformation methods that yield tissues of transient expression quickly have been of considerable interest to researchers. Agrobacterium rhizogenes-mediated transformation methods, which are employed to generate plants with transformed roots, have proven useful for the study of stress caused by root phytopathogens via gene overexpression and/or silencing. While some protocols have been adapted to soybean plants, transformation efficiencies remain limited; thus, few viable plants are available for performing bioassays. Furthermore, mRNA analyses that employ reverse transcription quantitative polymerase chain reactions (RT-qPCR) require the use of reference genes with stable expression levels across different organs, development steps and treatments. In the present study, an A. rhizogenes-mediated soybean root transformation approach was optimized. The method delivers significantly higher transformation efficiency levels and rates of transformed plant recovery, thus enhancing studies of soybean abiotic conditions or interactions between phytopathogens, such as nematodes. A 55% transformation efficiency was obtained following the addition of an acclimation step that involves hydroponics and different selection processes. The present study also validated the reference genes GmELF1-ß and GmELF1-α as the most stable to be used in RT-qPCR analysis in composite plants, mainly under nematode infection.

Ten-year experience of recombinant activated factor VII use in surgical patients with congenital haemophilia with inhibitors or acquired haemophilia in Japan.

Patients with congenital haemophilia with inhibitors or acquired haemophilia are at risk of bleeding complications during surgery. In these patients, replacement therapy for the missing coagulation factor is ineffective, and a bypassing agent such as recombinant activated factor VII (rFVIIa) is required to manage bleeding. To evaluate the safety and haemostatic efficacy of rFVIIa treatment in Japanese patients with congenital haemophilia with inhibitors to FVIII/FIX or acquired haemophilia undergoing surgery. Postmarketing surveillance data from May 2000 to March 2010 were analysed to assess the haemostatic efficacy of 38 procedures in 22 patients with congenital haemophilia A, 13 procedures in seven patients with congenital haemophilia B, and five procedures in five patients with acquired haemophilia. Postoperative bleeding control was judged to be effective (bleeding was stopped completely or reduced considerably) for 34/38 procedures (89%) in patients with congenital haemophilia A, 10/13 procedures (77%) in patients with congenital haemophilia B, and 4/5 procedures (80%) in patients with acquired haemophilia. Tranexamic acid was used concomitantly for 36/56 procedures (64%). Safety was analysed for 66 procedures in 37 patients. Adverse effects potentially related to rFVIIa treatment included mild superficial thrombophlebitis, mild decrease in platelet count, and mild elevation of the serum alanine transaminase level in one patient each. All adverse effects resolved without treatment. Administration of rFVIIa provided adequate haemostasis without serious adverse effects in the majority of cases. The efficacy and safety data in Japanese patients were similar to previously published data from other countries.

Plasma membrane-associated sialidase (NEU3) regulates progression of prostate cancer to androgen-independent growth through modulation of androgen receptor signaling.

Prostate cancers generally become androgen-independent and resistant to hormone therapy with progression. To understand the underlying mechanisms and facilitate the development of novel treatments for androgen-independent prostate cancer, we have investigated plasma membrane-associated sialidase (NEU3), the key enzyme for ganglioside hydrolysis participating in transmembrane signaling. We have discovered NEU3 to be upregulated in human prostate cancer compared with non-cancerous tissue, correlating with the Gleason score. NEU3 silencing with siRNA in prostate cancer PC-3 and LNCaP cells resulted in increased expression of differentiation markers and in cell apoptosis, but decrease in Bcl-2 as well as a progression-related transcription factor, early growth response gene (EGR-1). In androgen-sensitive LNCaP cells, forced overexpression of NEU3 significantly induced expression of EGR-1, androgen receptor (AR) and PSA both with and without androgen, the cells becoming sensitive to androgen. The NEU3-mediated induction was abrogated by inhibitors for PI-3 kinase and MAP kinase and more specifically by their silencing in the absence of androgen, being confirmed by increased phosphorylation of AKT and ERK1/2 in NEU3 overexpressing cells. NEU3 siRNA introduction caused reduction of cell growth of an androgen-independent PC-3 cells in culture and of transplanted tumors in nude mice. These data suggest that NEU3 regulates tumor progression through AR signaling, and thus be a potential tool for diagnosis and therapy of androgen-independent prostate cancer.

Diffuse dermal angiomatosis.

Diffuse dermal angiomatosis (DDA) is characterized clinically by painful erythematous lesions with ulcers and histologically by a benign, diffuse, and self-limited proliferation of tiny blood vessels in the superficial layers of the reticular dermis. Here we describe a case of DDA with leg ulcer. Erythematous lesions presented around the ulcer and angiogram revealed an occlusion of the superficial femoral artery. The erythematous lesions disappeared after revascularization. Although DDA is extremely rare, early correction of the ischemia in the peripheral artery should be taken into consideration.

Increased fat:carbohydrate oxidation ratio in Il1ra (-/-) mice on a high-fat diet is associated with increased sympathetic tone.

AIMS/HYPOTHESIS: Proinflammatory cytokines, including IL-1, exert pleiotropic effects on the neuro-immuno-endocrine system. Previously, we showed that mice with knockout of the gene encoding IL-1 receptor antagonist (Il1ra (-/-), also known as Il1rn (-/-)) have a lean phenotype. The present study was designed to analyse the mechanisms leading to this lean phenotype. METHODS: Il1ra (-/-) mice were fed a high-fat diet following weaning. Energy expenditure, body temperature, heart rate, blood parameters, urinary catecholamines and adipose tissue were analysed. RESULTS: Il1ra (-/-) mice exhibited resistance to obesity induced by a high-fat diet; this resistance was associated with increased energy expenditure and a decreased respiratory quotient, indicating that the ratio of fat:carbohydrate metabolism in Il1ra (-/-) mice is greater than in controls. Activity level in Il1ra (-/-) mice was significantly decreased and body temperature was significantly increased, compared with wild-type (WT) mice. Inguinal white adipose tissues in Il1ra (-/-) mice express increased levels of Ucp1 and mitochondrial respiratory chain genes compared with WT mice. Histological analysis of adipose tissue in Il1ra (-/-) mice revealed that brown adipose tissue is hyperactive and inguinal white adipose tissue contains smaller cells, which exhibit the distinctive multilocular appearance of brown adipocytes. Urinary epinephrine and norepinephrine excretion in Il1ra (-/-) mice was significantly increased compared with WT mice, suggesting that Il1ra (-/-) mice have increased sympathetic tone. Consistent with this, heart rate in Il1ra (-/-) mice was also significantly increased. CONCLUSIONS/INTERPRETATION: Our results show that Il1ra (-/-) mice have increased energy expenditure, fat:carbohydrate oxidation ratio, body temperature, heart rate and catecholamine production. All of these observations are consistent with an enhanced sympathetic tone.

Expression and function of the Delta-1/Notch-2/Hes-1 pathway during experimental acute kidney injury.

The Notch signaling pathway consists of several receptors and their ligands Delta and Jagged and is important for embryogenesis, cellular differentiation and proliferation. Activation of Notch receptors causes their cleavage yielding cytoplastic domains that translocate into the nucleus to induce target proteins such as the basic-loop-helix proteins Hes and Hey. Here we sought to clarify the significance of the Notch signaling pathway in acute kidney injury using a rat ischemia-reperfusion injury model and cultured NRK-52E cells. Analysis of the whole kidney after injury showed increased expression of Delta-1 and Hes-1 mRNA and protein along with processed Notch-2. Confocal microscopy, using specific antibodies, showed that Delta-1, cleaved Notch-2 and Hes-1 colocalized in the same segments of the injured renal proximal tubules. Recombinant Delta-1 significantly stimulated NRK-52E cell proliferation. Our study suggests that the Delta-1/Notch-2/Hes-1 signaling pathway may regulate the regeneration and proliferation of renal tubules during acute kidney injury.

Bromide removal by hydrotalcite-like compounds in a continuous system.

Bromide ion removal from a real water matrix by hydrortalcite-like compounds (HTCs) was attempted in a column reactor to control the formation of brominated disinfection by-products in drinking water treatment process. The performance of HTCs was found to be comparable to a commercially available ion exchange resin for relatively low alkalinity water. Also, it was deduced that HTCs are better than ion exchange resins for high sulfate water because of their unique ion selectivity. In addition, the ion exchange reactions by HTCs were faster than a commercially available resin. Thus, HTCs are expected to provide similar performance to organic resins without the concern about secondary contamination (i.e., elution of organic compounds from resins).

Association between PADI4 and rheumatoid arthritis: a meta-analysis.

OBJECTIVE: Polymorphisms and haplotypes of the peptidylarginine deiminase type 4 gene (PADI4) have been reported to be associated with rheumatoid arthritis (RA) in a Japanese population. However, subsequent replication studies showed conflicting results. The aim of this study was to determine whether meta-analysis would prove the existence of the association. METHODS: PubMed was searched using the term 'PADI4' for articles from the publication of the first study to December 2005. Replication studies that tested the association between PADI4 and RA were reviewed for meta-analysis. The Breslow-Day test for homogeneity across the studies was calculated. The Mantel-Haenszel procedure was used to pool odds ratios (OR) with 95% confidence intervals (CI) to evaluate the association. RESULTS: Six replication studies, one from Japan and five from Europe and North America, fulfilled the selection criteria for inclusion in the meta-analysis. Homogeneity was confirmed across the replication studies. The common OR was 1.14 (95% CI = 1.07-1.21) for allelic distribution. The association was confirmed when only five replication studies in the European descent populations were combined (P = 0.0096, common OR = 1.10). CONCLUSIONS: Our meta-analysis showed a positive association between PADI4 and RA not only in the Japanese population but also in populations of European descent.

Localization and expression of R-cadherin in skeletal muscle following nerve injury.

Rats in which the sciatic nerves were cut were divided into two groups: animals with nerve sutured and animals with nerve not sutured. In the unsutured group, the levels of R-cadherin expression increased and then decreased to values lower than those of controls. In the sutured group, the levels of R-cadherin expression increased and then decreased to almost control values. These results suggest that R-cadherin plays some role in cells of normal and regenerating muscles.

Unconstrained cardiorespiratory and body movement monitoring system for home care.

An unconstrained respiratory rate (RR) and heart rate (HR) monitoring system to be used during sleeping is proposed. The system consisted of eight polyvinylidene fluoride cable sensors, charge amplifiers and measuring software, together with an analogue-to-digital converter unit. The cable sensors were horizontally embedded into a textile sheet on a bed surface covering the upper half of the body. The digital infinite impulse response filters were constructed to extract cardiorespiratory signals from displacement of the sensors. The system software automatically searched the optimum sensor(s) based on the power of the respective filter outputs. Then, the system obtained the 5 s average HR and 15 s average RR by measuring the intervals between the peaks of the respective autocorrelation functions of the filtered output. If the subject changed his posture, the system captured the image of the body position as a time stamp using a CCD camera. To show the validity of this method, the HR and RR obtained by this monitor were compared with those simultaneously measured using respiratory flow and an electrocardiogram. The results showed that the mean frame-by-frame difference ranged from -1.2 to 0.2 beats min(-1) for the HR and, for RR, ranged from -0.8 to 1.4 breath min(-1) during the short-term recordings. Similar differences were obtained during the first 2 h of overnight recordings. The proposed system is feasible for the combined long-term monitoring of a person's RR and HR with sleep posture changes and may be helpful for practical use in the home.

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

Production and induction of manganese peroxidase isozymes in a white-rot fungus Pleurotus ostreatus.

The production of MnP by Pleurotus ostreatus in different liquid cultures was investigated. The highest level of activity was observed after 8 days of culture in peptone-glucose-yeast extract medium (PGY), whereas maximal activity was achieved after 30 days in glucose-yeast extract medium (GY). MnP was purified to homogeneity from PGY (designated MnP-PGY) and GY (MnP-GY). The isoelectric points of MnP-PGY and MnP-GY were 3.77 and 4.06, respectively. The molecular mass of both enzymes was 42 kDa. Analysis of the N-terminal amino acid sequence of purified MnPs and nucleotide sequence of cloned mnp indicated that MnP-GY has VTCATGQTTANE at the N-terminus, whereas MnP-PGY has ATCADGRTTANA. A putative exposed tryptophan residue (W170) was found in MnP-GY. Both isozymes oxidized veratryl alcohol, although the K(m) of MnP-GY was lower than that of MnP-PGY. Thus, the presence of peptone in the medium affected the production of MnP isozymes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that the synthesis of MnP isozymes is controlled by culture conditions at the transcriptional level.

Daily use of dentifrice with and without xylitol and fluoride: effect on glucose retention in humans in vivo.

The effect of daily use of three different dentifrices on glucose retention after glucose mouth rinsing was tested in this study regarding xylitol and fluoride. Six experimental groups used three different dentifrices produced by two different companies: xylitol- and fluoride-containing dentifrice (XF), non-xylitol- and fluoride-containing dentifrice (F), and non-xylitol- and non-fluoride-containing dentifrice (NonX-NonF). Subjects were divided at random and rinsed their mouths for 15s with 20ml of 0.5M glucose solution. Glucose and lactate retention were determined by collecting samples of saliva from the approximal areas of the maxillary and mandibular anterior teeth and using the enzyme membrane test. Samples were collected 0, 1 and 2 months after the start of regular dentifrice use. There were significant differences in glucose retention in relation to the dentifrice used, month of sampling, site of sampling, and time since start of rinsing. Their contribution ratios were 2.0, 4.4, 11.7 and 7.4%, respectively (P<0.01). There were significant differences observed between the XF and NonX-NonF groups, with the XF group presenting lower glucose retention than the NonX-NonF group. The XF group presented lower glucose retention than the F group. The F group showed lower glucose retention than the NonX-NonF group. There were significant differences in lactate retention in relation to the month and site of sampling, and their contribution ratios were 3.3 and 2.8%, respectively (P<0.01). There were, however, no significant differences in glucose and lactate retention in relation to the dentifrice manufacturer. It was concluded that the XF dentifrice was the most effective, and the F dentifrice was more effective in reducing glucose retention than the NonX-NonF dentifrice.

Impact of respiratory syncytial virus infection as a cause of lower respiratory tract infection in children younger than 3 years of age in Japan.

BACKGROUND: Respiratory syncytial virus (RSV) is the most important viral pathogen for lower respiratory tract infection (LRI) in infants and children. An RSV-specific monoclonal antibody has been developed to provide prophylaxis against RSV associated LRI (RSV-LRI). The objective of this study was to determine the impact of RSV as a cause of LRI in children younger than 3 years of age to provide data to aide in the implementation of forthcoming prophylaxis against RSV. METHODS: We analyzed the viral etiology of LRI in hospitalized Japanese children younger than 3 years of age admitted to Shizuoka Red Cross Hospital from July, 1997 to June, 2000. RESULTS: A total of 535 patients younger than 3 years of age were hospitalized with LRI at Shizuoka Red Cross Hospital from July 1, 1997 to June 30, 2000. Of these, a positive diagnosis of RSV infection was made in 168 patients (31.4%). Most of the patients with RSV infection had been well and had had no underlying disease that was defined as risk factor of RSV infection (94.0%). The peak incidence of LRI was observed in the winter each year and the number of LRI was strongly associated with the epidemic of RSV (r=0.700, P<0.0001). The number of patients with LRI younger than 6 months of age was 116 (21.7%). Of these 116 patients younger than 6 months with LRI, 55 patients (47.4%) were confirmed to have RSV infection. The proportions of RSV infection to total LRI was greatest in early infants younger than 6 months (P<0.0001). The number of patients with which RSV infection was detected in LRI patients younger than 3 years was highest during the first five months of life and there was a dramatic decrease in incidence of RSV infection with increasing age thereafter. CONCLUSIONS: The incidence of LRI hospitalization is highly affected by RSV infection epidemic. The proportion of RSV infections among early infants younger than 6 months is greater than that of older patients. The prophylaxis against RSV will be needed to be toward early infants.

Use of facial artery musculomucosal flap for closure of soft tissue defects of the mandibular vestibule.

We present the use of the inferiorly based facial artery musculomucous (FAMM) flap to repair a defect of the mandibular vestibule, and discuss the advantages and disadvantages of this procedure. The FAMM flap an effectively cover defects of the mandibular vestibule and other regions of the oral cavity, and its softness, toughness, and minimal shrinkage allow for the use of dental prostheses.