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A girl aged 2 years 10 months suddenly went into a deep coma and demonstrated left hemiplegia. At birth, she had exhibited a left-sided facial port-wine stain typical of Sturge-Weber syndrome (SWS) and involving the V1 and V2 distributions of the trigeminal nerve. Computed tomography showed a right thalamic hemorrhage with acute hydrocephalus. Magnetic resonance imaging with Gd enhancement 8 months before the hemorrhage had shown a patent superior sagittal sinus (SSS) and deep venous system. Magnetic resonance imaging and MR angiography studies 2 months before the hemorrhage had revealed obstruction of the SSS and right internal cerebral vein (ICV). Given that a digital subtraction angiography study obtained after the hemorrhage did not show the SSS or right ICV, the authors assumed that impaired drainage was present in the deep venous system at that stage. The authors speculated that the patient's venous drainage pattern underwent compensatory changes because of the occluded SSS and deep venous collectors, shifting outflow through other cortical venous channels to nonoccluded dural sinuses. Sudden congestion (nearly total to total obstruction) of the ICV may have caused the thalamic hemorrhage in this case, which is the first reported instance of pediatric SWS with intracerebral hemorrhage and no other vascular lesion. Findings suggested that the appearance of major venous sinus occlusion in a child with SWS could be a warning sign of hemorrhage.

Background Highly integrated anatomic and functional interactions between the cerebrum and the cerebellum during development have been reported. In our previous study, we conducted a proteome analysis to identify the proteins present in the congenital noncommunicating hydrocephalus in the cerebellum. We found higher expression of high-mobility group box-1 protein (HMGB-1) in hydrocephalic H-Tx rats. Objective We studied the expression pattern of HMGB-1 in the cerebellum. Methods We studied congenital hydrocephalic H-Tx rats aged 1 day and 7 days along with age-matched nonhydrocephalic H-Tx and Sprague-Dawley rats as controls. Gene and protein expressions of HMGB-1 in the cerebellum were assayed by real-time polymerase chain reaction and Western blotting, respectively; furthermore, immunohistochemical analyses were performed by using HMGB-1 (indicator of apoptosis), single-stranded DNA; adhesion factor related to cell migration, HNK-1; and the Purkinje cell-specific antibody, calbindin. Results Cytoplasmic HMGB-1 expression observed in Purkinje cells in the 1-day-old hydrocephalic group was stronger than that in the nonhydrocephalic and Sprague-Dawley groups. Double fluorescent staining with single-stranded DNA confirmed that Purkinje cells were undergoing apoptosis. HNK-1 expression was lower in the Purkinje cell layer in the 7-day-old rats in the hydrocephalic group, and Purkinje cells were disrupted in comparison with the control groups. Morphological changes in the cerebellum were observed in the 7-day-old rats in the hydrocephalic group in comparison with the control groups. Conclusion Our results suggest that cerebellar neuronal cell damage in the early postnatal period may be related to the higher expression of HMGB-1 in the Purkinje cells.

Toxicogenomics is a rapidly developing discipline focused on the elucidation of the molecular and cellular effects of chemicals on biological systems. As a collaborative study group of Toxicogenomics/JEMS·MMS, we conducted studies on hepatocarcinogens in rodent liver in which 100 candidate marker genes were selected to discriminate genotoxic hepatocarcinogens from non-genotoxic hepatocarcinogens. Differential gene expression induced by 13 chemicals were examined using DNA microarray and quantitative real-time PCR (qPCR), including eight genotoxic hepatocarcinogens [o-aminoazotoluene, chrysene, dibenzo[a,l]pyrene, diethylnitrosamine (DEN), 7,12-dimethylbenz[a]anthracene, dimethylnitrosamine, dipropylnitrosamine and ethylnitrosourea (ENU)], four non-genotoxic hepatocarcinogens [carbon tetrachloride, di(2-ethylhexyl)phthalate (DEHP), phenobarbital and trichloroethylene] and a non-genotoxic non-hepatocarcinogen [ethanol]. Using qPCR, 30 key genes were extracted from mouse livers at 4 h and 28 days following dose-dependent gene expression alteration induced by DEN and ENU: the most significant changes in gene expression were observed at 4 h. Next, we selected key point times at 4 and 48 h from changes in time-dependent gene expression during the acute phase following administration of chrysene by qPCR. We successfully showed discrimination of eight genotoxic hepatocarcinogens [2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitsosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane] from four non-genotoxic hepatocarcinogens [1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, DEHP and furan] using qPCR and principal component analysis. Additionally, we successfully identified two rat genotoxic hepatocarcinogens [DEN and 2,6-dinitrotoluene] from a nongenotoxic-hepatocarcinogen [DEHP] and a non-genotoxic non-hepatocarcinogen [phenacetin] at 4 and 48 h. The subsequent gene pathway analysis by Ingenuity Pathway Analysis extracted the DNA damage response, resulting from the signal transduction of a p53-class mediator leading to the induction of apoptosis. The present review of these studies suggests that application of principal component analysis on the gene expression profile in rodent liver during the acute phase is useful to predict genotoxic hepatocarcinogens in comparison to non-genotoxic hepatocarcinogens and/or non-carcinogenic hepatotoxins.

There is a long history of surgical treatment for Parkinson disease (PD). After pioneering trials and errors, the current primary surgical treatment for PD is deep brain stimulation (DBS). DBS is a promising treatment option for patients with medically refractory PD. However, there are still many problems and controversies associated with DBS. In this review, we discuss current issues in DBS for PD, including patient selection, clinical outcomes, complications, target selection, long-term outcomes, management of axial symptoms, timing of surgery, surgical procedures, cost-effectiveness, and new technology.

Objective Almost two-thirds of patients with Sturge-Weber syndrome (SWS) have epilepsy, and half of them require surgery for it. However, it is well known that scalp electroencephalography (EEG) does not demonstrate unequivocal epileptic discharges in patients with SWS. Therefore, we analyzed interictal and ictal discharges from intracranial subdural EEG recordings in patients treated surgically for SWS to elucidate epileptogenicity in this disorder. Methods Five intractable epileptic patients with SWS who were implanted with subdural electrodes for presurgical evaluation were enrolled in this study. We examined the following seizure parameters: seizure onset zone (SOZ), propagation speed of seizure discharges, and seizure duration by visual inspection. Additionally, power spectrogram analysis on some frequency bands at SOZ was performed from 60 s before the visually detected seizure onset using the EEG Complex Demodulation Method (CDM). Results We obtained 21 seizures from five patients for evaluation, and all seizures initiated from the cortex under the leptomeningeal angioma. Most of the patients presented with motionless staring and respiratory distress as seizure symptoms. The average seizure propagation speed and duration were 3.1 ± 3.6 cm/min and 19.4 ± 33.6 min, respectively. Significant power spectrogram changes at the SOZ were detected at 10–30 Hz from 15 s before seizure onset, and at 30–80 Hz from 5 s before seizure onset. Significance In patients with SWS, seizures initiate from the cortex under the leptomeningeal angioma, and seizure propagation is slow and persists for a longer period. CDM indicated beta to low gamma-ranged seizure discharges starting from shortly before the visually detected seizure onset. Our ECoG findings indicate that ischemia is a principal mechanism underlying ictogenesis and epileptogenesis in SWS.

A large-scale disaster may damage ground communication infrastructure, interfering with the delivery of disaster information. In such cases, satellites will remain unaffected by damage and can be used as a means of providing information. The augmentation signal of a global navigation satellite system (GNSS) can be received using a GNSS receiver, such as that used in cellular mobile phones, which are far more widely used than satellite mobile phones. Research on receiving information with the augmentation signal via a mobile phone is being conducted; however, a method of delivering disaster information to people who do not have a mobile phone requires attention. The purpose of the present research is to develop a system that delivers disaster information via a GNSS to people who do not have a GNSS receiver. To realize this, we aim to design the Machine-to-Machine (M2M) Early Warning System. In our system, various machines of public infrastructure receive information from a GNSS and deliver it immediately. As a first step, we consider the use of public vehicles such as busses and garbage tracks. There are areas where residents cannot hear a message broadcast from distant loudspeakers in disaster administration wireless communication. To deliver disaster information to these areas, public vehicles can receive information from the GNSS and deliver it audibly through speakers attached to the vehicles. Buses in a city would be an ideal means of delivering information employing our system. Through simulation using a geographic information system, we evaluate the area and time zone that information broadcast by public buses will reach. An M2M system is then constructed and tested in the coast area in suburbs of Tokyo, where many buses travel. The loudspeakers automatically broadcast tsunami information received from the Japanese Quasi Zenith Satellite System. The information reaches remote areas where fixed loudspeakers cannot be heard.

Purpose: Diffusional kurtosis imaging (DKI) is an emerging technique that describes diffusion of water molecules in terms of deviation from Gaussian distribution. This study investigated correlations between DKI metrics and cognitive function in patients with idiopathic normal pressure hydrocephalus (iNPH). Materials and Methods: DKI was performed in 29 iNPH patients and 14 age-matched controls. Mini-mental state examination (MMSE), frontal assessment battery (FAB), and trail making test A (TMT-A) were used as cognitive measures. Tract-based spatial statistics (TBSS) analyses were performed to investigate the between-group differences and correlations with the cognitive measures of the diffusion metrics, including mean kurtosis (MK), fractional anisotropy (FA), apparent diffusion coefficient (ADC), axial diffusivity (AD), and radial diffusivity (RD). Results: In iNPH patients, FA and MK identified positive correlations with cognitive function in similar regions, predominantly in the frontal lobes (P < 0.05, corrected for multiple comparisons). The frontoparietal subcortical white matter showed significant correlations with FAB and TMT-A across more extensive areas in MK analyses than in FA. ADC, AD, and RD analyses showed no significant correlations with MMSE and FAB, while negative correlation with TMT-A was observed in the limited portion of the frontal deep white matter. Conclusion: Both FA and MK correlated well with cognitive impairment in iNPH. The observed differences between FA and MK results suggest DKI may play a complementary role to conventional FA and ADC analyses, especially for evaluation of the subcortical white matter.

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.

The genotoxicity of multi-walled carbon nanotubes (MWCNTs) was evaluated in vivo with comet assays using the lung cells of rats given MWCNTs. The MWCNTs were intratracheally instilled as a single dose at 0.2 or 1.0 mg kg–1 or a repeated dose at 0.04 or 0.2 mg kg–1, once a week for 5 weeks, to male rats. The rats were sacrificed 3 or 24 h after the single instillation and were sacrificed 3 h after the last instillation in the repeated instillation groups. Histopathological examinations of the lungs revealed that MWCNTs caused inflammatory changes including the infiltration of macrophages and neutrophils after a single instillation and repeated instillation at both doses. In comet assays using rat lung cells, no changes in % Tail DNA were found in any group given MWCNTs. These findings indicate that MWCNTs do not have the potential to cause DNA damage in comet assays using the lung cells of rats given MWCNTs at doses causing inflammatory responses. Copyright © 2012 John Wiley & Sons, Ltd.

Background Cognitive impairment is difficult to improve after shunt operation in patients with idiopathic normal pressure hydrocephalus (iNPH). This study aims to identify cerebrospinal fluid (CSF) biomarkers predictive of improvement in cognitive function. Methods This study was conducted between January 2008 and December 2010 on consecutive, unselected admissions to our program for the treatment of patients with clinically suspected iNPH. Lumbar CSF concentrations of total tau (Tau), tau phosphorylated at threonine 181 (p-tau), soluble amyloid precursor protein (sAPP), sAPPα, sAPPβ, and β-amyloid1-42 (Aβ42) were analyzed by ELISA. Results Concentrations of p-tau, sAPP, sAPPα, and sAPPβ were strong diagnostic biomarkers for distinguishing between iNPH and Alzheimer's disease (AD). sAPPα exhibited the highest accuracy in differentiating iNPH from patients with AD and normal controls, with an area under the curve value of 0.994. We examined the prognostic value of p-tau and sAPPα for cognition function after surgery. With a cutoff value of 198 ng/ml or less for sAPPα, sensitivity and specificity are 66.7% and 82.9%, respectively, whilst the Mini-Mental State Examination score at 6 months after surgery is expected to be 25 or more. Conclusion Our results show that sAPPα is a suitable biomarker for the diagnosis and prognosis of iNPH.

Titanium dioxide (TiO₂) is widely used as a white pigment in paints, plastics, inks, paper, creams, cosmetics, drugs and foods. In the present study, the genotoxicity of anatase TiO₂ nanoparticles was evaluated in vivo using the comet assay after a single or repeated intratracheal instillation in rats. The nanoparticles were instilled intratracheally at a dosage of 1.0 or 5.0 mg/kg body weight (single instillation group) and 0.2 or 1.0 mg/kg body weight once a week for 5 weeks (repeated instillation group) into male Sprague-Dawley rats. A positive control, ethyl methanesulfonate (EMS) at 500 mg/kg, was administered orally 3 h prior to dissection. Histopathologically, macrophages and neutrophils were detected in the alveolus of the lung in the 1.0 and 5.0 mg/kg TiO₂ groups. In the comet assay, there was no increase in % tail DNA in any of the TiO₂ groups. In the EMS group, there was a significant increase in % tail DNA compared with the negative control group. TiO₂ nanoparticles in the anatase crystal phase are not genotoxic following intratracheal instillation in rats.

The Japanese Environmental Mutagen Society/the Mammalian Mutagenicity Study group conducted a collaborative study to investigate whether cell nuclei or whole cells might be more suitably used to correctly detect genotoxic chemicals in the in vivo rodent alkaline Comet assay. Four participating laboratories applied four sample processing methods, i.e., three homogenization methods using the usual Potter-type shaft, a customized (loose) Potter-type shaft, or a Downs-loose-type shaft, for preparing cell nuclei, and the mesh membrane method for preparing whole cells, to the male rat liver. Homogenization with the usual Potter-type shaft clearly produced damage of the cell nuclei and DNA, while the other three methods seemed to provide similar conditions of the tissue samples. The proportion of cell nuclei: whole cells was 80-90%: 10-20% in all laboratories when the samples were prepared by homogenization using a Downs-loose-type shaft or by the mesh membrane method. The %DNA in tail were comparable in both samples among the negative control groups (single oral administration with physiological saline) of all laboratories, and showed an equal degree of increase in both samples of the ethyl methanesulfonate groups (single oral administration at 250 mg/kg) in all laboratories. In conclusion, the homogenization method using a loosely customized Potter-type shaft or a Downs-loose-type shaft, and the mesh membrane method would be equally acceptable for the in vivo rodent alkaline Comet assay.

The transgenic rodent (TGR) assay has been widely used to study in vivo gene mutations by chemicals or radiation; however, an optimal protocol has not yet been established to assess unknown genotoxic potential. The International Workshop on Genotoxicity Testing (IWGT) strongly recommends a repeated-dose regimen for the TGR assay protocol for regulatory safety assessment as follows: a treatment period of 28 days and a sampling time of 3 days following the final treatment. In this study, TGR assays using F344 gpt delta transgenic rats were conducted at three laboratories to evaluate the validity of the IWGT protocol, as part of a collaborative study of the transgenic rat mutation assay. Male F344 gpt delta transgenic rats were orally treated with 2,4-diaminotoluene (2,4-DAT; hepatic carcinogen in rodents; 10 and 30 mg/kg/day) or 2,6-diaminotoluene (2,6-DAT; non-carcinogen in rodents; 60 mg/kg/day) once daily for 28 days. Rats were euthanized 3 days after the last dosing, and then mutant frequencies (MFs) of the gpt gene in the livers were studied. As a result, a significant increase in the MF was observed at 30 mg/kg in the 2,4-DAT-treated group, but not in the 2,6-DAT-treated group. These results were commonly observed among the three laboratories. In addition, the overall results from the three laboratories were in general agreement. These results indicate that 2,4-DAT induces gene mutation in the liver of gpt delta rats, but 2,6-DAT does not. These results also indicate that the F344 gpt delta transgenic rat mutation assay can distinguish differences in the in vivo mutagenic potential between a hepatic carcinogen and a non-carcinogen. Results from one laboratory showed more variability than those from the other two laboratories, and this appearance was due to the smaller number of colonies scored. Thus, these results demonstrate that the IWGT protocol for the TGR assays is valid, and show that consistent results are obtained among different laboratories.

Mammals, including human beings, have a circadian clock system to regulate behavioral and physiological processes. In this study, we investigated the effect of dosing time on micronucleus induction in the bone marrow by evaluating the frequencies of micronucleated peripheral reticulocytes (MNRETs) in mice exposed to N-ethyl-N-nitrosourea (ENU) to assess any difference in genotoxic sensitivity to chemicals between light and dark periods (inactive phase for rodents and active phase for rodents). Male C3H/He mice were treated intraperitoneally with ENU (12.5 or 25 mg/kg body weight) at zeitgeber time (ZT) 3 in the light period or ZT15 in the dark period, and then the time courses of the frequencies of the MNRETs were determined. The frequencies of the MNRETs induced by ENU increased time-dependently and peaked at 48 hr after treatment for ZT3 and ZT15, and were obviously higher in the ZT15 treatment group than the ZT3 treatment group. The MNRETs were measured at 48 hr after treatment with ENU (25 mg/kg body weight) at various dosing times (ZT0, 3, 6, 12, 15 and 18). The frequencies of the MNRETs in mice treated at ZT0, 15 and 18 were significantly higher than those in mice treated at ZT3, 6 and 12. These results suggest that genotoxic sensitivity to chemicals in mouse bone marrow is different between light and dark periods maybe due to different biological responses (detoxification, cell cycle, DNA repair, etc.) related to circadian rhythms.

OBJECT The authors’ goal in this paper is to provide the first clinical, radiological, and genetic studies of panventriculomegaly (PaVM) defined by a wide foramen of Magendie and large cisterna magna. METHODS Clinical and brain imaging data from 28 PaVM patients (including 10 patients from 5 families) were retrospectively studied. Five children were included. In adult patients, the age at onset was 56.0 ± 16.7 years. Tetraventricular dilation, aqueductal opening with flow void on T2-weighted images, and a wide foramen of Magendie and large cisterna magna (wide cerebrospinal fluid space at the fourth ventricle outlet) were essential MRI findings for PaVM diagnosis. 3D fast asymmetrical spin echo sequences were used for visualization of cistern membranes. Time-spatial labeling inversion pulse examination was performed to analyze cerebrospinal fluid movement. Copy number variations were determined using high-resolution microarray and were validated by quantitative polymerase chain reaction with breakpoint sequencing. RESULTS Adult patients showed gait disturbance, urinary dysfunction, and cognitive dysfunction. Five infant patients exhibited macrocranium. Patients were divided into 2 subcategories, those with or without downward bulging third ventricular floors and membranous structures in the prepontine cistern. Patients with bulging floors were successfully treated with endoscopic third ventriculostomy. Genetic analysis revealed a deletion in DNAH14 that encodes a dynein heavy chain protein associated with motile cilia function, and which co-segregated with patients in a family without a downward bulging third ventricular floor. CONCLUSIONS Panventriculomegaly with a wide foramen of Magendie and a large cisterna magna may belong to a subtype of congenital hydrocephalus with familial accumulation, younger age at onset, and symptoms of normal pressure hydrocephalus. In addition, a family with PaVM has a gene mutation associated with dysfunction of motile cilia.

A 49-year-old man with Parkinson's disease developed the wearing-off and levodopa-induced dyskinesia with a dystonic component in the left upper limb. He decided to undergo deep brain stimulation. Asymmetric targets were chosen. For right side, globus pallidus interna was selected to improve the dystonia in his left upper extremity, and for left side, the subthalamic nucleus, which had the expected benefit of reducing the patient's medications, was selected. After the surgery, the levodopa-induced dystonia in his left arm was markedly improved, and the levodopa equivalent dose was reduced by 21.4%. Our case suggests that deep brain stimulation with a different target in the left and right hemisphere may be an effective therapeutic option for patients with asymmetric symptoms. This article is protected by copyright. All rights reserved.

A 64-year-old woman with Parkinson's disease who developed motor fluctuations, and both levodopa-induced and stimulation-induced dyskinesia, after long-term treatment of bilateral subthalamic nucleus (STN) deep brain stimulation (DBS), underwent implantation of additional globus pallidus internus (GPi) DBS leads. Although an appropriate DBS target should be chosen, “rescue” GPi DBS can synergistically work with pre-existing STN DBS for the treatment of dyskinesia, which might provide a “third honeymoon.”

Background and aims The mechanisms of cancer cell growth and metastasis are still not entirely understood, especially from the viewpoint of chemical reactions in tumours. Glycolytic metabolism is markedly accelerated in cancer cells, causing the accumulation of glucose (a reducing sugar) and methionine (an amino acid), which can non-enzymatically react and form carcinogenic substances. There is speculation that this reaction produces gaseous sulfur-containing compounds in tumour tissue. The aims of this study were to clarify the products in tumour and to investigate their effect on tumour proliferation. Methods Products formed in the reaction between glucose and methionine or its metabolites were analysed in vitro using gas chromatography. Flatus samples from patients with colon cancer and exhaled air samples from patients with lung cancer were analysed using near-edge x-ray fine adsorption structure spectroscopy and compared with those from healthy individuals. The tumour proliferation rates of mice into which HT29 human colon cancer cells had been implanted were compared with those of mice in which the cancer cells were surrounded by sodium hyaluronate gel to prevent diffusion of gaseous material into the healthy cells. Results Gaseous sulfur-containing compounds such as methanethiol and hydrogen sulfide were produced when glucose was allowed to react with methionine or its metabolites homocysteine or cysteine. Near-edge x-ray fine adsorption structure spectroscopy showed that the concentrations of sulfur-containing compounds in the samples of flatus from patients with colon cancer and in the samples of exhaled air from patients with lung cancer were significantly higher than in those from healthy individuals. Animal experiments showed that preventing the diffusion of sulfur-containing compounds had a pronounced antitumour effect. Conclusions Gaseous sulfur-containing compounds are the main products in tumours and preventing the diffusion of these compounds reduces the tumour proliferation rate, which suggests the possibility of a new approach to cancer treatment.

Background Pathological gambling can develop in Parkinson’s disease (PD), and impairment of decision-making may play an important role in the mechanism. To assess acute effects of bilateral subthalamic nucleus deep brain stimulation (STN-DBS) on decision-making, patients with PD who were eligible for STN-DBS were evaluated using the Iowa Gambling Task (IGT). Methods The study participants were 16 patients with PD who underwent bilateral STN-DBS, and 16 age-matched control patients with PD. The participants performed the IGT pre-operatively and 2–4 weeks post-operatively with on- and off-stimulation. Participants’ one hundred card selections were divided into five blocks of 20 cards each. Results The total IGT score was not significantly different before surgery, on-stimulation or off-stimulation, but DBS patients tended to perform worse in the on-DBS session compared to off-DBS session ( P = 0.019) only in the last block of the task. The IGT score did not correlate with levodopa equivalent dose or performance on the measures of executive function, but did correlate with self-reported depression symptoms, and active contact of stimulation. Conclusion Bilateral STN-DBS may affect decision-making in acute post-operative stage.

A collaborative trial was conducted to evaluate the possibility of integrating the rat-liver Comet assay into repeat-dose toxicity studies. Fourteen laboratories from Europe, Japan and the USA tested fifteen chemicals. Two chemicals had been previously shown to induce micronuclei in an acute protocol, but were found negative in a 4-week Micronucleus (MN) Assay (benzo[a]pyrene and 1,2-dimethylhydrazine; Hamada et al., 2001); four genotoxic rat-liver carcinogens that were negative in the MN assay in bone marrow or blood (2,6-dinitrotoluene, dimethylnitrosamine, 1,2-dibromomethane, and 2-amino-3-methylimidazo[4,5-f]quinoline); three compounds used in the ongoing JaCVAM (Japanese Center for the Validation of Alternative Methods) validation study of the acute liver Comet assay (2,4-diaminotoluene, 2,6-diaminotoluene and acrylamide); three pharmaceutical-like compounds (chlordiazepoxide, pyrimethamine and gemifloxacin), and three non-genotoxic rodent liver carcinogens (methapyrilene, clofibrate and phenobarbital). Male rats received oral administrations of the test compounds, daily for two or four weeks. The top dose was meant to be the highest dose producing clinical signs or histopathological effects without causing mortality, i.e. the 28-day maximum tolerated dose. The liver Comet assay was performed according to published recommendations and following the protocol for the ongoing JaCVAM validation trial. Laboratories provided liver Comet assay data obtained at the end of the long-term (2- or 4-week) studies together with an evaluation of liver histology. Most of the test compounds were also investigated in the liver Comet assay after short-term (1-3 daily) administration to compare the sensitivity of the two study designs. MN analyses were conducted in bone marrow or peripheral blood for most of the compounds to determine whether the liver Comet assay could complement the MN assay for the detection of genotoxins after long-term treatment. Most of the liver genotoxins were positive and the three non-genotoxic carcinogens gave negative result in the liver Comet assay after long-term administration. There was a high concordance between short- and long-term Comet assay results. Most compounds when tested up to the maximum tolerated dose were correctly detected in both short- and long-term studies. Discrepant results were obtained with 2,6 diaminotoluene (negative in the short-term, but positive in the long-term study), phenobarbital (positive in the short-term, but negative in the long-term study) and gemifloxacin (positive in the short-term, but negative in the long-term study). The overall results indicate that the liver Comet assay can be integrated within repeat-dose toxicity studies and efficiently complements the MN assay in detecting genotoxins. Practical aspects of integrating genotoxicity endpoints into repeat-dose studies were evaluated, e.g. by investigating the effect of blood sampling, as typically performed during toxicity studies, on the Comet and MN assays. The bleeding protocols used here did not affect the conclusions of the Comet assay or of the MN assays in blood and bone marrow. Although bleeding generally increased reticulocyte frequencies, the sensitivity of the response in the MN assay was not altered. These findings indicate that all animals in a toxicity study (main-study animals as well as toxicokinetic (TK) satellite animals) could be used for evaluating genotoxicity. However, possible logistical issues with scheduling of the necropsies and the need to conduct electrophoresis promptly after tissue sampling suggest that the use of TK animals could be simpler. The data so far do not indicate that liver proliferation or toxicity confound the results of the liver Comet assay. As was also true for other genotoxicity assays, criteria for evaluation of Comet assay results and statistical analyses differed among laboratories. Whereas comprehensive advice on statistical analysis is available in the literature, agreement is needed on applying consistent criteria.

Hydrogen-dissolved water has been suggested to be effective for alleviating the oxidative stress. In the present study, neutral-pH hydrogen-enriched electrolyzed water (NHE-water; dissolved hydrogen: 0.90—1.14 parts per million [ppm]; oxido-reduced potential: —150 ∼ —80 mV), which was prepared with a water-electrolysis apparatus equipped with a non-diaphragm cell and a highly compressed activated-carbon block, was evaluated for the mutagenic and genotoxic potentials, at concentrations up to 100% dose/plate, and for the subchronic toxicity. NHE-water did not induce reverse mutations in Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537, and Escherichia coli strain WP2uvrA, in either the absence or presence of rat liver S9 for exogenous metabolic activation. Similarly, NHE-water did not induce chromosome aberrations in Chinese hamster lung fibroblast cells (CHL/IU), in short-term (6-hour) tests, with or without rat liver S9, or in a continuous treatment (24-hour) test. To evaluate the subchronic toxicity, Crj:CD(SD) specific pathogen free (SPF)-rats were administered with NHE-water at a dose of 20 mL/kg/day for 28 days via intragastric infusion. NHE-water-related toxic changes were not seen in terms of any items such as clinical symptoms, body weight, food consumption, urinalysis, hematology, blood chemistry, necropsy, each organ weight and histopathology. Thus, the no-observable-adverse-effect level (NOAEL) for NHE-water was estimated to be greater than 20 mL/kg/day under the conditions examined, demonstrating the consistency with the expected safety for a human with a body weight of 60 kg to drink the NHE-water up to at least 1.2 L/day.

Object: Cerebrospinal fluid (CSF) shunting can improve symptoms of elderly patients idiopathic normal pressure hydrocephalus (iNPH). However, adjunctive means for confirming the diagnosis remain unavailable. We have previously reported specific increase of leucine-rich alpha-2-glycoprotein (LRG) in iNPH CSF, and present study investigates its potential clinical applications. Methods: We performed CSF tap test (TT) on 90 patients and shunting in 52 patients (mean age 73.5 years), evaluating symptom improvement and higher cerebral functions-MMSE and Frontal Assessment Battery (FAB) before and twelve months after shunting. LRG and tau protein concentrations in TT CSF were simultaneously measured using ELIZA. Then we compared the predictive value of these concentrations with TT results regarding successful shunting outcomes. Findings: Positive combinations of TT and LRG concentrations of 67ng/mL or higher, gave 81.6% sensitivity and 78.6% specificity. Therefore we used LRG (67ng/mL) and tau (200pg/mL) cutoff values, LRG≥67ng/ml and tau

The Globally Harmonized System of Classification and Labeling of Chemicals (GHS) requires classification of chemicals on germ cell mutagenicity. The Japanese government has conducted GHS classification on about 1400 chemicals in a 2-year project (J-GHS) for implementing GHS domestically. Prior to the classification work, the technical guidance for classification of germ cell mutagens was prepared. This guidance introduces the concept of heritable mutagenicity, and presents detailed criteria for germ cell mutagens, test data to be used, and a practical decision tree for classification. These practical guidance and supporting explanations are useful for non-expert Classifiers (scientists applying the classification criteria). Several issues, however, were identified during the course of J-GHS and in re-evaluating the classification results. These include: (1) the information sources when available data are limited; (2) lack of understanding GHS classification criteria or insufficient review of the information by Classifiers; (3) varying opinions of experts on data quality and weight of evidence, and; (4) decision tree approaches, e.g., inadequacy for use in overall evaluation in some cases. Ideally, classification should be performed by Classifiers with high expertise using high quality information sources. Genetic toxicologists as experts should consider data quality and reliability, and give a critical review of all available information for support of classification. A weight of evidence approach is also required to assess mutagenic potential of chemicals. Critical points for suitable classification for GHS are discussed.

The authors have developed a minimally invasive lumboperitoneal shunt placement procedure conducted after administration of a local anesthetic. The procedure involves placing a guide wire and a peel-away sheath under fluoroscopic and CT guidance. Between June 2004 and August 2006, 40 patients (21 men and 19 women; mean age 72.5 years [range 33–86 years]) underwent surgery. A Codman Hakim programmable valve system (82–3844, Codman & Shurtleff, Inc.) was used for the procedure. The mean operating time was 53 minutes, and 7 patients (17.5%) developed shunt dysfunction complications. These complications comprised an infected shunt valve in 2 patients, postoperative lower-limb pain in 1 patient, and shunt obstruction (caused by debris and hemorrhage) at the ventral and lumbar ends in 2 patients each. This procedure is less invasive than conventional lumboperitoneal shunt insertion and could be performed as an outpatient surgery for treatment of idiopathic normal-pressure hydrocephalus.

A single-dose oral toxicity study was conducted to examine the qualitative and quantitative toxicity of a commercial-grade batch of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS No. 205687-03-2). Dihydrocapsiate was administered once by gavage to ICR mice at dose levels of 0 (vehicle) or 5000 mg/kg/day. No mortality was observed during the 14 day observation period following test article administration. During the 2 h immediately following dosing, mice of both sexes treated with dihydrocapsiate were observed to exhibit one or more of the following: staggered gait, decreased spontaneous movement, increased time in the prone position, tremors, gasping, or red-brownish urine. All mice had completely recovered by the 6 h observation interval. No effects on body weights or necropsy findings were observed as a result of dihydrocapsiate administration. These results suggested that the lethal dose of dihydrocapsiate was >5000 mg/kg. In an in vivo micronucleus test using BDF1 male mice, a commercial grade of dihydrocapsiate neither increased the incidence of micronucleated polychromatic erythrocytes (MNPCEs) nor decreased the ratio of polychromatic erythrocytes (PCEs) in any of the treatment groups. The results suggest that commercial-grade dihydrocapsiate is unlikely to be an in vivo clastogen.

A series of studies was performed to evaluate the safety of dihydrocapsiate (4-hydroxy-3-methoxybenzyl 8-methylnonanoate; CAS no. 205687-03-2). This study evaluated the potential genotoxicity of this compound using a variety of in vitro and in vivo test systems, including bacterial reverse mutation test, chromosomal aberration test, micronucleus test, gene mutation assay with transgenic rats, and single-cell gel (SCG) assay (Comet assay). In vitro tests (bacterial reverse mutation test and chromosomal aberration test) produced positive results in the absence of metabolic activation, but negative results in the presence of metabolic activation. The in vivo gene mutation assay (with transgenic rats) produced negative results, as did the in vivo mouse micronucleus assay, which failed to induce micronucleated polychromatic erythrocytes. Although the rat SCG assay produced statistically significant increases in the Olive tail moment and % tail DNA of the liver and intestine in the 2000 mg/kg group (compared with the negative-control group), a number of factors caused the authors to question the validity of these findings. Taken together, these results suggest that dihydrocapsiate has a low or extremely low likelihood of inducing genotoxicity.

Object Some cases of compensatory hydrocephalus have been reported in which cognitive deficiency progresses despite the absence of progressive ventricular dilation. In this study, the differentially expressed genes in compensated hydrocephalic H-Tx rat cortices were determined. A molecular mechanism that induces neuronal death in the cerebral cortex of compensated hydrocephalus is proposed. Methods The cerebral cortices of 8-week-old H-Tx rats with spontaneously arrested hydrocephalus (hH-Tx) and nonhydrocephalic H-Tx (nH-Tx) control rats were subjected to cDNA microarray analysis followed by canonical pathway analysis. Results In the hH-Tx rats, many genes in the amyloidal processing pathway showed altered expression, including Akt3 and p38 MAPK. These latter genes are involved in tau protein phosphorylation, and their increased expression in hydrocephalus was confirmed by real-time polymerase chain reaction analysis. Immunohistological and immunoblot analysis revealed elevated phosphorylated tau expression in the cerebral cortex neurons of the hH-Tx rats. Conclusions The accumulation of phosphorylated tau protein in the cerebral cortex may be one of the mechanisms by which later cognitive dysfunction develops in patients with compensated hydrocephalus. More work needs to be done to determine if the accumulation of phosphorylated tau in the cortex can help predict which patients may decompensate thus requiring more aggressive treatment for compensated hydrocephalus.

At the Plymouth Third International Workshop on Genotoxicity Testing in June 2002, a new expert group started a working process to provide guidance on a common strategy for genotoxicity testing beyond the current standard battery. The group identified amongst others "Follow-up testing of tumorigenic agents not positive in the standard genotoxicity test battery" as one subject for further consideration [L. Müller, D. Blakey, K.L. Dearfield, S. Galloway, P. Guzzie, M. Hayashi, P. Kasper, D. Kirkland, J.T. MacGregor, J.M. Parry, L. Schechtman, A. Smith, N. Tanaka, D. Tweats, H. Yamasaki, Strategy for genotoxicity testing and stratification of genotoxicity test results-report on initial activities of the IWGT Expert Group, Mutat. Res. 540 (2003) 177-181]. A workgroup devoted to this topic was formed and met on September 9-10, 2005, in San Francisco. This workgroup was devoted to the discussion of when it would be appropriate to conduct additional genetic toxicology studies, as well as what type of studies, if the initial standard battery of tests was negative, but tumor formation was observed in the rodent carcinogenicity assessment. The important role of the standard genetic toxicology testing to determine the mode of action (MOA) for carcinogenesis (genotoxic versus non-genotoxic) was discussed, but the limitations of the standard testing were also reviewed. The workgroup also acknowledged that the entire toxicological profile (e.g. structure-activity relationships, the nature of the tumor finding and metabolic profiles) of a compound needed to be taken into consideration before the conduct of any additional testing. As part of the meeting, case studies were discussed to understand the practical application of additional testing as well as to form a decision tree. Finally, suitable additional genetic toxicology assays to help determine the carcinogenic MOA or establish a weight of evidence (WOE) argument were discussed and formulated into a decision tree.

As part of the Fourth International Workshop on Genotoxicity Testing (IWGT), held 9-10 September 2005 in San Francisco, California, an expert working group on the Comet assay was convened to review and discuss some of the procedures and methods recommended in previous documents. Particular attention was directed at the in vivo rodent, alkaline (pH >13) version of the assay. The aim was to review those protocol areas which were unclear or which required more detail in order to produce a standardized protocol with maximum acceptability by international regulatory agencies. The areas covered were: number of dose levels required, cell isolation techniques, measures of cytotoxicity, scoring of comets (i.e., manually or by image analysis), and the need for historical negative/positive control data. It was decided that a single limit dose was not sufficient although the required number of dose levels was not stipulated. The method of isolating cells was thought not to have a qualitative effect on the assay but more data were needed before a conclusion could be drawn. Concurrent measures of cytotoxicity were required with histopathological examination of tissues for necrosis or apoptosis as the "Gold Standard". As for analysing the comets, the consensus was that image analysis was preferred but not required. Finally, the minimal number of studies required to generate a historical positive or negative control database was not defined; rather the emphasis was placed on demonstrating the stability of the negative/positive control data. It was also agreed that a minimum reporting standard would be developed which would be consistent with OECD in vivo genotoxicity test method guidelines.

A new specimen preparation method named “cell suspension method” for in vivo micronucleus test is proposed. Generally, a specimen of the micronucleus test is prepared by smearing the sample cells on a slide glass before staining with Giemsa or acridine orange (A.O.). In this new method, the sample cells are suspended with formalin, and stained with A.O., then put on a slide glass and examined under a microscope without smearing. This method is more convenient than the conventional method in consideration of preparation and observation of specimens. Furthermore, preparation is repeatable after the observation in this method, the specimen is suitable for a long storage, and this method is applicable to specimen preparation using peripheral blood.

A 56-year-old man complained of gait disturbance and confused thinking. Magnetic resonance imaging(MRI)revealed an arteriovenous malformation(AVM)of the cerebellar vermis(Spetzler-Martin grade IV)causing hydrocephalus. One dilated precentral cerebellar vein was compressing the aqueduct. After feeder embolization over 3 sessions using N-butyl cyanoacrylate(NBCA), the nidus was reduced to one-third in size. However, symptoms remained unimproved, and endoscopic third ventriculostomy(ETV)was performed. The third ventricle showed thinning of the floor, with a fenestration in part of the floor. Radiological findings and clinical symptoms improved, and the patient returned home after rehabilitation. The condition of the patient remained stable as of six months later. On angiography, the draining vein showed a pressure of 20 mmHg with no change in the residual AVM. Embolization alone achieved a reduction in nidus volume, but could not reduce venous pressure, and combination therapy including ETV proved necessary. Cases with hydrocephalus due to aqueductal stenosis by AVM are extremely rare. This pathology is discussed with reference to the literature.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this validation effort was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The purpose of the pre-validation studies (i.e., Phase 1 through 3), conducted in four or five laboratories with extensive comet assay experience, was to optimize the protocol to be used during the definitive validation study.

The in vivo rodent alkaline comet assay (comet assay) is used internationally to investigate the in vivo genotoxic potential of test chemicals. This assay, however, has not previously been formally validated. The Japanese Center for the Validation of Alternative Methods (JaCVAM), with the cooperation of the U.S. NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)/the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), the European Centre for the Validation of Alternative Methods (ECVAM), and the Japanese Environmental Mutagen Society/Mammalian Mutagenesis Study Group (JEMS/MMS), organized an international validation study to evaluate the reliability and relevance of the assay for identifying genotoxic carcinogens, using liver and stomach as target organs. The ultimate goal of this exercise was to establish an Organisation for Economic Co-operation and Development (OECD) test guideline. The study protocol was optimized in the pre-validation studies, and then the definitive (4th phase) validation study was conducted in two steps. In the 1st step, assay reproducibility was confirmed among laboratories using four coded reference chemicals and the positive control ethyl methanesulfonate. In the 2nd step, the predictive capability was investigated using 40 coded chemicals with known genotoxic and carcinogenic activity (i.e., genotoxic carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and non-genotoxic non-carcinogens). Based on the results obtained, the in vivo comet assay is concluded to be highly capable of identifying genotoxic chemicals and therefore can serve as a reliable predictor of rodent carcinogenicity.

3-Chloro-4-(dichloromethyl)−5-hydroxy-2(5H)-furanone (MX), a by-product of water chlorination, is a potent bacterial mutagen and rat carcinogen. In the present study, the in vivo mutagenicity, cell proliferative activity, and carcinogenicity of MX were investigated in gpt delta mice. Groups of 5 male and female 7-week-old gpt delta C57BL/6J transgenic mice were given MX at doses of 0, 10, 30, or 100 ppm in their drinking water for 12 weeks, and then killed to assess in vivo mutagenicity using 6-thioguanine and Spi− selection, and cell proliferative activity using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Further groups of 10 male and female gpt delta mice were given 0 or 100 ppm MX for 78 weeks, and a full necropsy with histopathological examination of all organs was conducted to detect neoplastic lesions. The 12-week MX treatment did not result in mutagenicity in the livers or lungs or cell proliferative activity in several organs of the mice, and the 78-week treatment did not cause carcinogenicity. Additional investigations were conducted to evaluate the potential of MX to inhibit gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape loading/dye transfer method. Inhibition of GJIC was detected within 2 hr with a noncytotoxic dose of MX (4 μg/ml), followed by partial restoration after 5 hr. A second phase of inhibition occurred after 10 hr and then the lowered level persisted for the 24 hr-incubation period. Dose-dependent inhibition was evident at both 2 hr and 24 hr, with much stronger effects at the former time. These findings indicate that MX is not mutagenic, mitogenic or carcinogenic in mice, and suggest that the compound exerts epigenetic actions leading to GJIC inhibition. Environ. Mol. Mutagen., 2006. © 2005 Wiley-Liss, Inc.

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its inter-laboratory reproducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin–transferrin– ethanolamine–sodium selenite and 2% fetal bovine serum (FBS) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12 -O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1μg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In the presence of 0.2μg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.

In this study, we examined how the press formability of A1 alloys sheets and the generation of residual stresses was influenced by pre-coating resins, lubricants, die shoulder radius, the punch shoulder radius, and so on. The use of a pre-coated layer offers advantages by preventing direct contact between sheet metal and the face of the die, and also by enabling the use of volatile oil lubricants that keep the factory environment cleaner. The circumferential residual stress (σC ) of a cup becomes lower when the lubricant is less effective, and when the die shoulder radius is smaller.

A large and extensive body of data on the use of human liver S9 fractions in the Salmonella mutagenicity test (Ames test) is presented; the data were obtained from a collaborative study by JEMS/BMS (Bacterial Mutagenicity Test Study Group) members and the Human and Animal Bridging Research Organization (HAB). In this study, the mutagenicity of 58 chemicals, many of which were judged to be human carcinogens by the IARC, was determined by the Ames test (the pre-incubation method at 37 degrees C for 20 min) in the presence of a selected human liver S9 fraction with a high drug-metabolic activity or a pooled human liver S9 fraction with a moderate drug-metabolic activity. For reference, mutagenicity was also examined in the presence of a phenobarbital/5,6-benzoflavone-pretreated rat liver S9 fraction, which is normally used in mutagenicity testing systems. The bacterial test strains consisted of Salmonella typhimurium TA100, TA98 or YG7108. The data indicated that the mutagenicity of chemicals in the rat and human liver S9 fractions varied considerably, depending on the chemicals in question. In addition, a large inter-individual diversity in the mutagenic response to mutagens, depending on the chemical structures of the mutagens, was also demonstrated using two selected human S9 fractions. Most of the mutagens tested in this study (75%; 36 out of 48 compounds that were judged to be mutagenic in at least one S9 fraction) were less mutagenic in the presence of the two human S9 fractions than in the presence of the rat S9 fraction. On the other hand, the other compounds (25%), including some aromatic amines and nitrosamines, showed a more potent mutagenicity in the presence of either one of the two human S9 fractions than in the presence of the rat S9 fraction. These data strongly suggest that the use of human liver S9 fraction in mutagenicity testing systems may be useful for a better understanding of the mutagenic effects of chemicals on humans.

Numerical results simulating a three-dimensional separated flow and heat transfer around surface-mounted rectangular blocks in a plane channel are presented. Flow configuration treated in the present study is transversal two blocks. The finite difference method is employed to directly solve Navier-Stokes, continuity and energy equations, and resulting finite difference equations are solved with the SMAC method in Reynolds number range from 100 to 500. Present results are compared with available flow visualization ones and it is found that the present numerical method well simulates the observed results such as horseshoe vortices and the recirculating separated flow. Local and mean heat transfer characteristics are clarified and their correlations with the flow structure are investigated.

A novel bioabsorbable bone substitute composed of a β-tricalcium phosphate (β-TCP) and a carboxymethyl-chitin (CM-chitin) sodium has been developed. Rabbit tibia defects (4 mm in diameter) were repaired after 4 weeks more effectively by the composite compared with a sham-operation group. To further investigate the biological safety of the components, genotoxicity and carcinogenicity of an extract prepared from the composite were determined using four different in vitro assays. The main extract component was identified as CM-chitin sodium [average molecular weight (Mw) ∼230 kDa] as determined by Fourier transform infrared spectroscopy and gel permeation chromatography analysis. The concentrations of P and Ca possibly derived from β-TCP were 17.7 and 37.1 μg/g, respectively, as determined by inductively coupled plasma mass spectroscopy. Both the metabolic activation and nonactivation (−S9) systems of the rat microsome S9 fraction were used to perform a genotoxicity evaluation using the Ames test and chromosome aberration assay on Chinese hamster lung fibroblast cells treated with the extract. In these assays, no genotoxicity was detected with doses ≤5 mg/mL (maximum concentration). The cell transformation assay using BALB/c 3T3 cells and the metabolic cooperation assay with V79 cells both showed negative results for any tumor-promoting activity caused by the extract (∼5 mg/mL). These results indicate that the bioabsorbable β-TCP/CM-chitin composite is a highly biocompatible bone substitute. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 635–643, 2004