The pressure sensitivity of wrinkled B-doped nanocrystalline diamond membranes.

Nanocrystalline diamond (NCD) membranes are promising candidates for use as sensitive pressure sensors. NCD membranes are able to withstand harsh conditions and are easily fabricated on glass. In this study the sensitivity of heavily boron doped NCD (B:NCD) pressure sensors is evaluated with respect to different types of supporting glass substrates, doping levels and membrane sizes. Higher pressure sensing sensitivities are obtained for membranes on Corning Eagle 2000 glass, which have a better match in thermal expansion coefficient with diamond compared to those on Schott AF45 glass. In addition, it is shown that larger and more heavily doped membranes are more sensitive. After fabrication of the membranes, the stress in the B:NCD films is released by the emergence of wrinkles. A better match between the thermal expansion coefficient of the NCD layer and the underlying substrate results in less stress and a smaller amount of wrinkles as confirmed by Raman spectroscopy and 3D surface imaging.

Detecting and polarizing nuclear spins with double resonance on a single electron spin.

We report the detection and polarization of nuclear spins in diamond at room temperature by using a single nitrogen-vacancy (NV) center. We use Hartmann-Hahn double resonance to coherently enhance the signal from a single nuclear spin while decoupling from the noisy spin bath, which otherwise limits the detection sensitivity. As a proof of principle, we (i) observe coherent oscillations between the NV center and a weakly coupled nuclear spin and (ii) demonstrate nuclear-bath cooling, which prolongs the coherence time of the NV sensor by more than a factor of 5. Our results provide a route to nanometer scale magnetic resonance imaging and novel quantum information processing protocols.

Arterial spin-labeled perfusion imaging reflects vascular density in nonfunctioning pituitary macroadenomas.

BACKGROUND AND PURPOSE: Angiogenesis is very important in clinical features of pituitary adenomas. We investigated the relationship between the blood flow of nonfunctioning pituitary macroadenomas measured by arterial spin-labeled perfusion imaging and the microvessel attenuation of the tissue. MATERIALS AND METHODS: Conventional MR imaging with contrast-enhanced T1WI and arterial spin-labeled perfusion imaging were performed before surgery in 11 consecutive patients with nonfunctioning pituitary macroadenomas. ROIs were drawn on the tumors, and the degrees of enhancement were calculated by dividing the signal intensity on the contrast-enhanced T1WI by that on the nonenhanced TIWI. As an index of tumor perfusion, a quantitative analysis was performed by using normalized tumor blood flow values calculated by dividing the mean value of the tumor region of interest by the mean region of interest values in the 2 cerebellar hemispheres. The relative microvessel attenuation was determined as the total microvessel wall area divided by the entire tissue area on CD-31-stained specimens. The degree of enhancement and the normalized tumor blood flow values were compared with relative microvessel attenuation. Additionally, intra- and postoperative tumor hemorrhages were visually graded. RESULTS: The degree of enhancement was not correlated with relative microvessel attenuation. Statistically significant correlations were observed between normalized tumor blood flow values and relative microvessel attenuation (P < .05). At surgery, 3 cases were visually determined to be hypervascular tumors, and 1 of these cases had symptomatic postoperative hemorrhage. A statistically significant difference in normalized tumor blood flow values was observed visually between the intraoperative hypovascular and hypervascular groups (P < .05). CONCLUSIONS: Arterial spin-labeled perfusion imaging reflects the vascular density of nonfunctioning pituitary macroadenomas, which may be useful in the preoperative prediction of intra- and postoperative tumor hemorrhage.

Visualization of ATP with luciferin-luciferase reaction in mouse skeletal muscles using an "in vivo cryotechnique".

Adenosine triphosphate (ATP) is a well-known energy source for muscle contraction. In this study, to visualize localization of ATP, a luciferin-luciferase reaction (LLR) was performed in mouse skeletal muscle with an "in vivo cryotechnique" (IVCT). First, to confirm if ATP molecules could be trapped and detected after glutaraldehyde (GA) treatment, ATP was directly attached to glass slides with GA, and LLR was performed. The LLR was clearly detected as an intentional design of the ATP attachment. The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. Next, LLR was evaluated in mouse skeletal muscles with IVCT followed by freeze-substitution fixation (FS) in acetone-containing GA. In such tissue sections the histological structure was well maintained, and the intensity of LLR in areas between muscle fibers and connective tissues was different. Moreover, differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine (DAB) reactions were clearly detected in type I muscle fibers and erythrocytes in capillaries, which demonstrated flow shape. Thus, it became possible to perform microscopic evaluation of the numbers of ATP molecules in the mouse skeletal muscles with IVCT, which mostly reflect living states.

Identification of the blast resistance gene Pit in rice cultivars using functional markers.

DNA markers that allow for identification of resistance genes in rice germplasm have a great advantage in resistance breeding because they can assess the existence of the genes without laborious inoculation tests. Functional markers (FMs), which are designed from functional polymorphisms within the sequence of genes, are unaffected by nonfunctional allelic variation and make it possible to identify an individual gene. We previously showed that the resistance function of the rice blast resistance gene Pit in a resistant cultivar, K59, was mainly acquired by up-regulated promoter activity through the insertion of a long terminal repeat (LTR) retrotransposon upstream of Pit. Here, we developed PCR-based DNA markers derived from the LTR-retrotransposon sequence and used these markers to screen worldwide accessions of rice germplasm. We identified 5 cultivars with the LTR-retrotransposon insertion out of 68 rice accessions. The sequence and expression pattern of Pit in the five cultivars were the same as those in K59 and all showed Pit-mediated blast resistance. The results suggest that the functional Pit identified using the markers was derived from a common progenitor. Additionally, comparison of the Pit coding sequences between K59 and susceptible cultivars revealed that one nucleotide polymorphism, which caused an amino acid substitution, offered another target for a FM. These results indicate that our DNA markers should enhance prediction of Pit function and be applicable to a range of rice varieties/landraces cultivated in various regions worldwide and belonging to the temperate japonica, tropical japonica, and indica groups.

IL-8 in cerebrospinal fluid from children with acute encephalopathy is higher than in that from children with febrile seizure.

We identify possible differences in the cytokine/chemokine profiles in cerebrospinal fluid (CSF) from children with encephalopathy and febrile seizure. Interleukin (IL)-1beta, 2, 4, 5, 6, 7, 8, 10, 12, 13, 17, interferon-gamma, tumour necrosis factor-alpha, granulocyte colony-stimulating factor, granulocyte monocyte colony-stimulating factor, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1beta were measured simultaneously in CSF supernatants from children with encephalopathy (n = 8), febrile seizure (n = 16) and fever without neurological complications (n = 8). IL-8 in CSF from children with encephalopathy was significantly elevated compared to that in CSF from children with febrile seizure and fever without neurological complications. IL-8 in CSF was also higher than serum IL-8, suggesting that increased IL-8 was generated from glia cells or astrocytes, not by leakage from serum. Increased IL-8 in CSF in encephalopathy may protect against severe brain damage.

Imaging mass spectrometry revealed the production of lyso-phosphatidylcholine in the injured ischemic rat brain.

To develop an effective neuroprotective strategy against ischemic injury, it is important to identify the key molecules involved in the progression of injury. Direct molecular analysis of tissue using mass spectrometry (MS) is a subject of much interest in the field of metabolomics. Most notably, imaging mass spectrometry (IMS) allows visualization of molecular distributions on the tissue surface. To understand lipid dynamics during ischemic injury, we performed IMS analysis on rat brain tissue sections with focal cerebral ischemia. Sprague-Dawley rats were sacrificed at 24 h after middle cerebral artery occlusion, and brain sections were prepared. IMS analyses were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) in positive ion mode. To determine the molecular structures, the detected ions were subjected to tandem MS. The intensity counts of the ion signals of m/z 798.5 and m/z 760.5 that are revealed to be a phosphatidylcholine, PC (16:0/18:1) are reduced in the area of focal cerebral ischemia as compared to the normal cerebral area. In contrast, the signal of m/z 496.3, identified as a lyso-phosphatidylcholine, LPC (16:0), was clearly increased in the area of focal cerebral ischemia. In IMS analyses, changes of PC (16:0/18:1) and LPC (16:0) are observed beyond the border of the injured area. Together with previous reports--that PCs are hydrolyzed by phospholipase A(2) (PLA(2)) and produce LPCs,--our present results suggest that LPC (16:0) is generated during the injury process after cerebral ischemia, presumably via PLA(2) activation, and that PC (16:0/18:1) is one of its precursor molecules.

Long-term results of the Japanese Childhood Cancer and Leukemia Study Group studies 811, 841, 874 and 911 on childhood acute lymphoblastic leukemia.

We analyzed the long-term outcomes of 1021 patients with acute lymphoblastic leukemia (ALL), enrolled in four successive clinical trials (ALL811, ALL841, ALL874 and ALL911) between 1981 and 1993. All patients received risk-adopted therapy according to leukocyte count and age at the time of diagnosis. The median follow-up durations of the four studies were 17.8 years in ALL811, 15.5 years in ALL841, 11.9 years in ALL874 and 15.8 years in ALL911. Patients' event-free survival (EFS) and overall survival (OS) rates at 12 years were 41.0 and 54.3% in ALL811, 50.2 and 60.2% in ALL841, 57.3 and 64.7% in ALL874, and 63.4 and 71.7% in ALL911, respectively. Thus, cure can become a reality for about 70% of children with ALL. There is, however, still a significant difference in survival outcomes according to risk group. Late effects were observed in 70 patients out of 834 (8.4%); hepatitis and short stature were most commonly reported. Reduction of late adverse effects for all patients and development of new treatment strategies for very-high-risk patients are major issues for upcoming trials to address.

Dynamic nuclear polarization study of UV-irradiated butanol for hyperpolarized liquid NMR.

We have carried out a dynamic nuclear polarization (DNP) study of proton spins of UV-irradiated solid butanol containing phenol at 0.1-1%. By the aid of butyl free radicals produced by the photolysis, the butanol sample was dynamically polarized up to the polarization of 1.4+/-0.1% at 1.5 K and 1.2 T, which is about 4000 times larger than that at the thermal equilibrium at an ambient temperature. Unlike persistent free radicals such as nitroxy and trityl free radicals commonly used for DNP, the butyl free radicals immediately decay by melting. We propose that free radicals produced by UV-photolysis are applicable to the DNP-enhanced hyperpolarized liquid-state NMR (Ardenkjaer-Larsen et al. [6]). The hyperpolarization of the UV-irradiated samples should remain longer than that of the samples having persistent free radicals, because of the absence of the free radicals after the melting in the UV-irradiated samples.

Life-threatening airway obstruction due to retropharyngeal and cervicomediastinal hematomas following stellate ganglion block.

Airway obstruction by retropharyngeal or cervicomediastinal hematomas following stellate ganglion block is life-threatening. The onset of the initial symptoms of retropharyngeal or cervicomediastinal hematoma usually occurs 2 hours or more after stellate ganglion block. We report the rare complication of airway obstruction leading to respiratory arrest caused by retropharyngeal and cervicomediastinal hematomas due to rebleeding of an ascending cervical artery 3 days after stellate ganglion block.

Expression of periostin in patients with non-small cell lung cancer: correlation with angiogenesis and lymphangiogenesis.

The aim of this study was to evaluate periostin expression measured immunohistochemically in patients with non-small cell lung cancer (NSCLC) and to determine its association with clinical features, prognosis, angiogenesis, and lymphangiogenesis. We investigated periostin expression in a series of 88 patients with NSCLC. We also determined whether expression of periostin correlated with microvessel density and lymphatic microvessel density. Periostin was expressed in 42% of 88 patients. Its expression was significantly correlated with tumor size, lymph node metastasis, disease stage, and lymphatic invasion (p=0.0128, 0.0015, 0.0310 and 0.0273, respectively). There also was a significant relation between periostin expression and microvessel density and lymphatic microvessel density (all p<0.0001). Five-year survival rates were better in patients with negative periostin expression than in those with positive periostin expression (p=0.0044). Periostin expression was not significant in a multivariate additive model. Our findings show that periostin correlates with increased tumor progression and a worse prognosis in NSCLC, as well as with angiogenesis and lymphangiogenesis.

Molecular characterization of the Fusarium graminearum species complex in Japan.

Members of the Fusarium graminearum species complex are important cereal pathogens worldwide and belong to one of at least nine phylogenetically distinct species. We examined 298 strains of the F. graminearum species complex collected from wheat or barley in Japan to determine the species and trichothecene chemotype. Phylogenetic analyses and species-diagnostic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLPs) revealed the presence and differential distribution of F. graminearum sensu stricto (s. str.) and F. asiaticum in Japan. F. graminearum s. str. is predominant in the north, especially in the Hokkaido area, while F. asiaticum is predominant in southern regions. In the Tohoku area, these species co-occurred. Trichothecene chemotyping of all strains by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 (70%) out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by use of 15 PCR-RFLP markers developed in this study. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

[Resected lung cancer with production of CA19-9].

We report 2 cases of resected CA19-9 producing adenocarcinoma of the lung. Immunohistochemical studies of the specimens in 2 cases showed positive staining for CA19-9 in most of tumor cells. The serum CA19-9 levels decreased after operations and effective chemotherapy in these cases. But the serum CA19-9 levels increased with regrowth of tumors or with no therapeutic gain by chemotherapy. The serum CA19-9 level was useful to evaluate the therapeutic effect of surgery and chemotherapy, and may be effective in detecting the recurrence of lung cancer.

Bacterium organizes hierarchical amorphous structure in microbial cellulose.

A pellicle, a gel film of microbial cellulose, is a supermolecular system containing 99% of water by weight, which is closely related to an amorphous structure in it. Using ultra-small-angle neutron scattering, in order to cover over a wide range of length scales from nm to 10 microm, we examined the hierarchical amorphous structure in the microbial cellulose, which is synthesized by a bacterium (Acetobacter xylinum). The microbial cellulose swollen by water shows small-angle scattering that obeys a power law q -behavior according to q -alpha as a function of the magnitude of the scattering vector q . The power law, determined by scattering, is attributed to a mass fractal due to the distribution of the center of mass for the crystallite (microfibril) in amorphous cellulose swollen by water. As q increases, alpha takes the values of 2.5, 1, and 2.35, corresponding, respectively, to a gel network composed of bundles, a bundle composed of cellulose ribbons, and concentration fluctuations in a bundle. From the mass fractal q -behavior and its length scale limits, we evaluated a volume fraction of crystallite in microbial cellulose. It was found that 90% of the cellulose bundle is occupied by amorphous cellulose containing water.

Modification of the hepatic mitochondrial proteome in response to ischemic preconditioning following ischemia-reperfusion injury of the rat liver.

BACKGROUND/AIM: Ischemic preconditioning (IPC) may reduce hepatic ischemia-reperfusion (IR) injury, but efficacy of IPC on mitochondrial proteome is not demonstrated. We investigated how IPC modifies the mitochondrial proteome after IR injury. METHODS: Rats were subjected to 25 min of portal triad crossclamping (IR group, n = 8). In the IPC group (n = 8), 10 min of temporal portal triad clamping was performed before 25 min of portal clamping. Samples were obtained after 24 h. The mitochondrial inner-membrane potential was measured by the uptake of a lipophilic cationic carbocyanine probe and mitochondrial proteome was also investigated using 2-dimensional differential in-gel electrophoresis and liquid chromatography-tandem mass spectrometry. RESULTS: Mitochondrial inner-membrane potential and glutathione were lower and serum transaminase was higher in the IPC group than in the IR group. The mitochondrial precursor of aldehyde dehydrogenase 2 and alpha-methylacyl-CoA-racemase were upregulated in the IPC group in comparison to the IR group. In contrast, protein disulfide-isomerase A3 precursor, 60S acid ribosomal protein P0, carbonic anhydrase 3 and superoxide dismutase were significantly more downregulated in the IPC group than in the IR group. CONCLUSIONS: A hepatoprotective effect by IPC was not shown; however, IPC caused significant up- or downregulation of several mitochondrial proteins.

Real-time monitoring of nitric oxide (NO) and pO2 levels under ischemic conditions associated with small bowel ischemia/reperfusion injury using selective electrodes for NO and oxygen molecules.

The present study demonstrated the feasibility of monitoring nitric oxide (NO) and pO2 levels under ischemic conditions associated with small bowel ischemia/reperfusion (I/R) injury through the use of selective electrodes for NO and oxygen molecules. NO levels gradually increased during ischemia. When reperfusion was started, the NO level decreased suddenly and returned to pre-ischemia values within 10 minutes. After clamping, pO2 decreased rapidly. When reperfusion was started, pO2 increased suddenly, returning to pre-ischemia values within 10 minutes. We concluded that it is feasible to monitor NO and pO2 levels under ischemic conditions of small bowel I/R injury through the use of electrodes selective for NO and oxygen molecules.

Protective effects of MnM2Py4P and Mn-salen against small bowel ischemia/reperfusion injury in rats using an in vivo and an ex vivo electron paramagnetic resonance technique with a spin probe.

BACKGROUND: Reperfusion of ischemic tissues results in the formation of toxic reactive oxygen species (ROS), such as superoxide anion, hydroxyl radicals, hydroperoxide, and peroxynitrite. ROS are potent oxidizing agents, capable of damaging cellular membranes by lipid peroxidation. In the present study, we applied an in vivo electron paramagnetic resonance (EPR)/spin probe and an ex vivo EPR technique to provide direct evidence of ROS following experimentally induced small bowel ischemia/reperfusion (I/R) injury. MATERIALS AND METHODS: We used a rat model of small bowel I/R injury to explore the possibility that MnM2Py4P or Mn-salen can prevent the accumulation of ROS species following experimentally induced I/R injury. We examined the ability of MnM2Py4P and Mn-salen to scavenge radicals in living Sprague-Dawley (SD) rats using an in vivo and an ex vivo EPR technique with a spin probe. RESULTS: The CP decay rates in the MnM2Py4P- and Mn-salen-treated rats were significantly higher than those in the untreated rats and almost equal to those in sham group rats. There were no significant differences between the MnM2Py4P-treated group and the Mn-salen-treated group. Superoxide scavenging activities (SSA) in the MnM2Py4P- and EUK-8-treated group were higher than those in the untreated group and almost equal to the sham group. CONCLUSION: The present study suggested that the protective effects of MnM2Py4P and Mn-salen against small bowel IR injury were mediated by the inhibition of O2, H2O2, and NO production.

Analysis of MPO-ANCA subtypes in a patient with propylthiouracil-induced vasculitis with multiple complications.

BACKGROUND: We report a 16 year-old girl with propylthiouracil (PTU)-induced antineutrophil cytoplasmic antibody (ANCA)-positive glomerulonephritis combined with Henoch-Schönlein purpura nephritis (HSPN) and antiphospholipid syndrome (APS). CASE AND METHODS: The patient had Graves' disease and had been treated with PTU for about 6 years. She complained of arthralgia, epigastralgia, purpura of the lower extremities, anemia, and abnormal urinalysis. Lupus anticoagulant was positive. Additionally, a high level of anti-myeloperoxidase (MPO) antibodies (IgG) and a low level of coagulation factor XIII were recognized. She had several complications including lung bleeding, lacuna infarctions of the right frontal and parietal brain lobes, and deep vein thrombosis of the left lower extremity. We studied tissue histology and carried out MPO-ANCA subtype analysis by immunofluorescence and flow cytometry and MPO-ANCA epitope analysis. RESULTS: Histologically, purpura showed leukocytoclastic vasculitis with perivascular depositions of IgA and complement C3. Renal biopsy showed necrotizing glomerulonephritis with crescents and mesangial IgA deposits. Notably, IgG, IgM, and IgA ANCA were detected in the patient's serum by flow cytometry and immunofluorescence. We diagnosed an overlap syndrome of ANCA-positive vasculitis, HSPN, and APS. A change in the reactivity of MPO-ANCA from recognition of only the Hg epitope in the C-terminal region to recognition of multiple MPO epitopes was accompanied by a remission of symptoms. CONCLUSIONS: This report may provide a very rare description of an overlap syndrome of PTU-induced ANCA vasculitis, HSPN, and APS in which not only IgG ANCA but also IgA and IgM ANCA were found. Epitope analysis may be a useful marker for disease-monitoring of PTU-induced ANCA-positive vasculitis. This case may provide insight into the pathological mechanism underlying each of these diseases.

Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase.

Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.

Clonotypic analysis of T cell reconstitution after haematopoietic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency.

Haematopoietic stem cell transplantation (HSCT) is performed for treatment of a broad spectrum of illnesses. Reconstitution of an intact immune system is crucial after transplantation to avoid infectious complications, and above all, the establishment of T cell receptor (TCR) diversity is the most important goal in the procedure. Until recently, little has been known of the mechanism of T cell reconstitution in the very early period after HSCT. In this study, we analysed TCR repertoires sequentially in four patients with severe combined immunodeficiency (SCID) before and after HSCT. In all patients, the TCR repertoires were extremely abnormal before HSCT, whereas after transplantation there was progressive improvement in TCR diversity, based on analysis of the TCR Vbeta repertoire and CDR3 size distributions. Somewhat unexpectedly, there was a significant but transient expansion of TCR diversity 1 month after transplantation in all cases. Clonotypic analysis of TCRs performed in one case showed that many T cell clones shared identical CDR3 sequences at 1 month and that the shared fraction decreased progressively. These results indicate that early expansion of TCR diversity may reflect transient expansion of pre-existing mature T cells from the donor blood, independent of de novo T cell maturation through the thymus.