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Tenascin-C is a member of the matricellular protein family, and its expression level is correlated to poor prognosis in cancer, including glioblastoma, whereas its substantial role in tumor formation and malignant progression remains controversial. We reported previously that peptide TNIIIA2 derived from the cancer-associated alternative splicing domain of tenascin-C molecule has an ability to activate β1-integrin strongly and to maintain it for a long time. Here, we demonstrate that β1-integrin activation by TNIIIA2 causes acquisition of aggressive behavior, dysregulated proliferation, and migration, characteristic of glioblastoma cells. TNIIIA2 hyperstimulated the platelet-derived growth factor–dependent cell survival and proliferation in an anchorage-independent as well as -dependent manner in glioblastoma cells. TNIIIA2 also strongly promoted glioblastoma multiforme cell migration, which was accompanied by an epithelial–mesenchymal transition–like morphologic change on the fibronectin substrate. Notably, acquisition of these aggressive properties by TNIIIA2 in glioblastoma cells was abrogated by peptide FNIII14 that is capable of inducing inactivation in β1-integrin activation. Moreover, FNIII14 significantly inhibited tumor growth in a mouse xenograft glioblastoma model. More importantly, FNIII14 sensitized glioblastoma cells to temozolomide via downregulation of O6-methylguanine-DNA methyltransferase expression. Consequently, FNIII14 augmented the antitumor activity of temozolomide in a mouse xenograft glioblastoma model. Taken altogether, the present study provides not only an interpretation for the critical role of tenascin-C/TNIIIA2 in aggressive behavior of glioblastoma cells, but also an important strategy for glioblastoma chemotherapy. Inhibition of the tenascin-C/β1-integrin axis may be a therapeutic target for glioblastoma, and peptide FNIII14 may represent a new approach for glioblastoma chemotherapy.Significance:These findings provide a proposal of new strategy for glioblastoma chemotherapy based on integrin inactivation.

This file shows the supplementary figure (S1-S8).

This file shows the materials and methods in the results of 'Supplementary Figure'.

Time lapse evaluation of cell scattering (TNIIIA2 + FNIII14)

This file shows the legends of Supplementary Movie S1-S3.

Tenascin-C is a member of the matricellular protein family, and its expression level is correlated to poor prognosis in cancer, including glioblastoma, whereas its substantial role in tumor formation and malignant progression remains controversial. We reported previously that peptide TNIIIA2 derived from the cancer-associated alternative splicing domain of tenascin-C molecule has an ability to activate β1-integrin strongly and to maintain it for a long time. Here, we demonstrate that β1-integrin activation by TNIIIA2 causes acquisition of aggressive behavior, dysregulated proliferation, and migration, characteristic of glioblastoma cells. TNIIIA2 hyperstimulated the platelet-derived growth factor–dependent cell survival and proliferation in an anchorage-independent as well as -dependent manner in glioblastoma cells. TNIIIA2 also strongly promoted glioblastoma multiforme cell migration, which was accompanied by an epithelial–mesenchymal transition–like morphologic change on the fibronectin substrate. Notably, acquisition of these aggressive properties by TNIIIA2 in glioblastoma cells was abrogated by peptide FNIII14 that is capable of inducing inactivation in β1-integrin activation. Moreover, FNIII14 significantly inhibited tumor growth in a mouse xenograft glioblastoma model. More importantly, FNIII14 sensitized glioblastoma cells to temozolomide via downregulation of O6-methylguanine-DNA methyltransferase expression. Consequently, FNIII14 augmented the antitumor activity of temozolomide in a mouse xenograft glioblastoma model. Taken altogether, the present study provides not only an interpretation for the critical role of tenascin-C/TNIIIA2 in aggressive behavior of glioblastoma cells, but also an important strategy for glioblastoma chemotherapy. Inhibition of the tenascin-C/β1-integrin axis may be a therapeutic target for glioblastoma, and peptide FNIII14 may represent a new approach for glioblastoma chemotherapy. Significance: These findings provide a proposal of new strategy for glioblastoma chemotherapy based on integrin inactivation.

Expression level of tenascin-C is closely correlated to poor prognosis in glioblastoma patients, while the substantial role of tenascin-C responsible for aggressive progression in glioblastoma cells has not been clarified. We previously found that peptide TNIIIA2, which is derived from the tumor-associated tenascin-C variants, has the ability to promote cell adhesion by activating &beta, 1-integrins. Our recent study demonstrated that potentiated activation of integrin &alpha, 5&beta, 1 by TNIIIA2 causes not only a dysregulated proliferation in a platelet-derived growth factor (PDGF)-dependent manner, but also disseminative migration in glioblastoma cells. Here, we show that TNIIIA2 enhances the proliferation in glioblastoma cells expressing PDGF-receptor&beta, even without exogenous PDGF. Mechanistically, TNIIIA2 induced upregulated expression of PDGF, which in turn stimulated the expression of tenascin-C, a parental molecule of TNIIIA2. Moreover, in glioblastoma cells and rat brain-derived fibroblasts, tenascin-C upregulated matrix metalloproteinase-2, which has the potential to release TNIIIA2 from tenascin-C. Thus, it was shown that autocrine production of PDGF triggered by TNIIIA2 functions to continuously generate a functional amount of PDGF through a positive spiral loop, which might contribute to hyper-proliferation in glioblastoma cells. TNIIIA2 also enhanced in vitro disseminative migration of glioblastoma cells via the PKC&alpha, signaling. Collectively, the tenascin-C/TNIIIA2 could be a potential therapeutic target for glioblastoma.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Eur J Clin Invest 2011; 41 (3): 241–255 Abstract Background Urea and creatinine are widely used as biomarkers for disease. However, these parameters have been criticized as markers for several reasons. Thus, we conducted this study to identify novel biomarkers that can be used as alternatives to urea and creatinine to estimate the adequate dialysis dose by metabolomic analyses of plasma samples from patients undergoing haemodialysis. Material and methods Liquid chromatography–electrospray ionization (ESI)–time-of-flight mass spectrometry (MS) was used to analyse low molecular weight molecules present in the plasma samples of 10 patients with end-stage renal disease (ESRD) who were being treated with haemodialysis, and in 16 healthy subjects. Results In plasma samples obtained after haemodialysis, the relative quantities of 54 peaks were significantly (P

A ceiling-mounted robotic C-arm cone beam CT (CBCT) system was developed for use with a 190° proton gantry system and a 6-degree-of-freedom robotic patient positioner. We report on the mechanical design, system accuracy, image quality, image guidance accuracy, imaging dose, workflow, safety and collision-avoidance.The robotic CBCT system couples a rotating C-ring to the C-arm concentrically with a kV X-ray tube and a flat-panel imager mounted to the C-ring. CBCT images are acquired with flex correction and maximally 360° rotation for a 53 cm field of view. The system was designed for clinical use with three imaging locations. Anthropomorphic phantoms were imaged to evaluate the image guidance accuracy.The position accuracy and repeatability of the robotic C-arm was high (0.5 mm), as measured with a high-accuracy laser tracker. The isocentric accuracy of the C-ring rotation was within 0.7 mm. The coincidence of CBCT imaging and radiation isocentre was better than 1 mm. The average image guidance accuracy was within 1 mm and 1° for the anthropomorphic phantoms tested. Daily volumetric imaging for proton patient positioning was specified for routine clinical practice.Our novel gantry-independent robotic CBCT system provides high-accuracy volumetric image guidance for proton therapy. Advances in knowledge: Ceiling-mounted robotic CBCT provides a viable option than CT on-rails for partial gantry and fixed-beam proton systems with the added advantage of acquiring images at the treatment isocentre.

Following inhalation of Mycobacterium tuberculosis, including bacillus Calmette–Guérin (BCG), pathogens enter and grow inside macrophages by taking advantage of their phagocytic mechanisms. Macrophages often fail to eliminate intracellular M. tuberculosis, leading to the induction of host macrophage death. Despite accumulating evidence, the molecular mechanisms underlying M. tuberculosis infection–induced cell death remain controversial. In this study, we show the involvement of two distinct pathways triggered by TLR2 and β2 integrin in BCG infection–induced macrophage apoptosis. First, BCG infection induced activation of ERK1/2, which in turn caused phosphorylation/activation of the proapoptotic protein Bim in mouse macrophage-like Raw 264.7 cells. BCG-infected Raw cells treated with U0126, an MEK/ERK inhibitor, led to the suppression of Bim phosphorylation alongside a remarkable increase in the number of viable macrophages. Small interfering RNA–mediated knockdown of Bim rescued the macrophages from the apoptotic cell death induced by BCG infection. Stimulation with Pam3CSK, a TLR2 agonist, induced macrophage apoptosis with a concomitant increase in the phosphorylation/activation of MEK/ERK and Bim. These observations indicate the important role of the TLR2/MEK/ERK/Bim pathway in BCG infection–induced macrophage apoptosis. Second, we used the β2 integrin agonists C3bi and fibronectin to show that the β2 integrin–derived signal was involved in BCG infection–induced apoptosis, independent of MEK/ERK activation. Interestingly, latex beads coated with Pam3CSK and C3bi were able to induce apoptosis in macrophages to the same extent and specificity as that induced by BCG. Taken together, two distinct pattern-recognition membrane receptors, TLR2 and β2 integrin, acted as triggers in BCG infection–induced macrophage apoptosis, in which MEK/ERK activation played a crucial role following the engagement of TLR2.

The prevalence of hepatitis C virus (HCV) infection among the Patients of 22 renal dialysis units in Wakayama Prefecture; part of Sakai City, Osaka Prefecture; and Kasai City, Hyogo Prefecture was examined by three kinds of second generation HCV antibody detection kits (Particle agglutination, PA, Ortho; Passive hemagglutination, PHA, Dainabot; Enzyme immunoassay, UBI, Kyokuto). A total of 1, 170 patients received hemodialysis (HD) and 23 received continuous ambulatory peritoneal dialysis (CAPD).Anti-HCV was detected in 360 (30.2%) of the 1, 193 dialysis patients, with good agreement seen among the three different methods. Anti-HCV positive rate of HD patients (30.3%) was higher than that of the CAPD patients (21.7%).The frequency of HCV infection correlated with the duration of dialysis treatment, HD treatment and the amount of blood transfusion. Transfusion history of anti-HCV positive patients was 37.0% of HD and 36.4% of CAPD patients. CAPD patients without blood transfusion, however, had a higher anti-HCV rate (8.3%) than the general Japanese HCV career rate (1.2%).These results suggest that HCV transmission may occur in any renal dialysis unit.

Superconducting Bi,Pb–Sr–Ca–Cu–O material has been prepared by an improved coprecipitation method; mixed metal oxalates as the precursor were precipitated from a homogeneous acetic acid solution of the constituent metal acetates and dimethyl oxalate. This method permits easier fabrication of the superconducting material which is almost free from low-Tc phase.

An advanced photomask dry etching system (NLDE-9035) has been evaluated. The NLD plasma has an advantage to have a capability to control the plasma distribution and density. In our previous work, it has been confirmed to obtain excellent CD uniformity, CD linearity and good pattern fidelity. To improve the CD uniformity further, the neutral loop modulation etching technique has been evaluated. As a result, a further improvement of CD uniformity has been confirmed by using neutral loop (NL) diameter modulation etching technique.

The dry etching process by using NLD (Neutral Loop Discharge Plasma) has been evaluated. The loading effect was measured applying the CAR (Chemically Amplified Resist) negative resist process in the low pressure etching condition, where an excellent CD (Critical Dimension) uniformity was obtained.

As the LSI pattern density increases, the minimum feature size on reticle decreases and the required dimensional accuracy becomes more severe. To write patterns for 130nm- node device, the proximity effect correction is essential for electron beam mask writing system to obtain enough CD accuracy. We optimized the proximity effect correction parameters in EB mask writer, and the evaluated results are presented.

We reported the high effectiveness of electrolyzed strong acid aqueous solution (ESAAS) in cleaning hemodialysis lines. Although ESAAS has a strong bactericidal action, one concern is its strong acidity. It has a pH of 2.3-2.7, more than 1,000 mV in oxidation-reduction potential (ORP), and 10-50 ppm of available chlorine. The possibility of metal corrosion, degradation of synthetic resins, chorine gas emission, or dissolving calcium carbonate (CaCO3) deposits due to ESAAS's acidity was tested using in vitro experiments. The bactericidal and antiviral effects of various ESAAS's were also tested. Metal corrosion and synthetic resin degradation, although they occurred, were not serious. There were no problems with chlorine gas emission and dissolving of CaCO3 deposits. Each type of ESAAS showed almost the same bactericidal and antiviral effect, but in some cases differences were observed.

For the next generation reticle fabrication, one of the key technologies is a high accelerating voltage EB writing system in combination with CAR (Chemically Amplified Resist) process. So we have evaluated an advanced electron beam mask-writing system (EBM-210VS/Toshiba Machine, Ltd) using EP002 resist (Tokyo Ohka, Ltd) and RE514OP (Hitachi Chemical, Ltd). The system adopts accelerating voltage of 50 kV, variable shaped beam, vector scanning, continuous moving stage and 230 mm mask capability. In the results of the exposure evaluations, using 4 pass writing strategy, global positioning accuracy of 23 nm (3 (sigma) ), local positioning accuracy of 16 nm (3 (sigma) ), global CD accuracy of 11 nm (3 (sigma) ), local CD accuracy of 14.5 nm (3 (sigma) ), L/S CD linearity of 23 nm and stitching accuracy of 20 nm were obtained. These results are satisfactory for our first target.

This work demonstrated the ability of 230 mm reticle manufacture by a current technology. The CD accuracy of 17 nm (3 (sigma) ) and image placement accuracy of 30 nm (3 (sigma) ) were obtained in the 230 mm reticle. It was able to be confirmed that there was no big problem though the improvement was still necessary for manufacturing equipment of 230 mm reticle.

An advanced photomask dry etching system (NLDE-9035 Prototype) has been evaluated. This system adopts new plasma source NLDE, and has a 230 mm mask capability. In this experiment, etching uniformity, selectivity and etching pattern profile were mainly evaluated. Etching uniformity of 20 nm (range) was obtained and good pattern fidelity was confirmed.

Superconducting Bi,Pb–Sr–Ca–Cu–O material has been prepared by an improved coprecipitation method; mixed metal oxalates as the precursor were precipitated from a homogeneous acetic acid solution of the constituent metal acetates and dimethyl oxalate. This method permits easier fabrication of the superconducting material which is almost free from low-Tc phase.