The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories. The purpose of this validation study was to assess transferability of the EpiSensA to new laboratories along with its within- (WLR) and between-laboratory reproducibility (BLR). The validation study was organized into two independent stages. As demonstrated during the first stage, where three sensitizers and one non-sensitizer were correctly predicted by all participating laboratories, the EpiSensA was successfully transferred to all three participating laboratories. For Phase I of the second stage, each participating laboratory performed three experiments with an identical set of 15 coded test chemicals resulting in WLR of 93.3%, 93.3%, and 86.7%, respectively. Furthermore, when the results from the 15 test chemicals were combined with those of the additional 12 chemicals tested in Phase II of the second stage, the BLR for 27 test chemicals was 88.9%. Moreover, the predictive capacity among the three laboratories showed 92.6% sensitivity, 63.0% specificity, 82.7% accuracy, and 77.8% balanced accuracy based on murine local lymph node assay (LLNA) results. Overall, this validation study concluded that EpiSensA is easily transferable and sufficiently robust for assessing the skin sensitization potential of chemicals.

Prostanoids Regulate Angiogenesis and Lymphangiogenesis in Pathological Conditions.

Angiogenesis, the formation of new blood vessels from the preexistent microvasculature, is an essential component of wound repair and tumor growth. Nonsteroidal anti-inflammatory drugs that suppress prostanoid biosynthesis are known to suppress the incidence and progression of malignancies including colorectal cancers, and also to delay the wound healing. However, the precise mechanisms are not fully elucidated. Accumulated results obtained from prostanoid receptor knockout mice indicate that a prostaglandin E-type receptor signaling EP3 in the host microenvironment is critical in tumor angiogenesis inducing vascular endothelial growth factor A (VEGF-A). Further, lymphangiogenesis was also enhanced by EP signaling via VEGF-C/D inductions in pathological settings. These indicate the importance of EP receptor to facilitate angiogenesis and lymphangiogenesis in vivo. Prostanoids act beyond their commonly understood activities in smooth muscle contraction and vasoactivity, both of which are quick responses elicited within several seconds on stimulations. Prostanoid receptor signaling will be a potential therapeutic target for disease conditions related to angiogenesis and lymphangiogenesis.

A biologically active lipid, thromboxane, as a regulator of angiogenesis and lymphangiogenesis.

Thromboxane (TX) and prostaglandins are metabolites of arachidonic acid, a twenty-carbon unsaturated fatty acid, and have a variety of actions that are exerted via specific receptors. Angiogenesis is defined as the formation of new blood vessels from pre-existing vascular beds and is a critical component of pathological conditions, including inflammation and cancer. Lymphatic vessels play crucial roles in the regulation of interstitial fluid, immune surveillance, and the absorption of dietary fat from the intestine; and they are also involved in the pathogenesis of various diseases. Similar to angiogenesis, lymphangiogenesis, the formation of new lymphatic vessels, is a critical component of pathological conditions. The TP-dependent accumulation of platelets in microvessels has been reported to enhance angiogenesis under pathological conditions. Although the roles of some growth factors and cytokines in angiogenesis and lymphangiogenesis have been well characterized, accumulating evidence suggests that TX induces the production of proangiogenic and prolymphangiogenic factors through the activation of adenylate cyclase, and upregulates angiogenesis and lymphangiogenesis under disease conditions. In this review, we discuss the role of TX as a regulator of angiogenesis and lymphangiogenesis, and its emerging importance as a therapeutic target.

Pathological significance and predictive value for biochemical recurrence of c-Fes expression in prostate cancer.

BACKGROUND: c-Fes is a proto-oncogene encoded non-receptor protein-tyrosine kinase (PTK). However, genetic studies have indicated that it has anti-tumorigenic effects in certain cancers. The pathological and clinical significance of c-Fes in prostate cancer are unknown. METHODS: Expression of c-Fes was evaluated in normal glands, prostatic intraepithelial neoplasia (PIN), cancer cells in tissues of knock-in mouse adenocarcinoma prostate (KIMAP) model, and prostate cancer patients free of metastasis. Expression of c-Fes was analyzed by immunohistochemistry, and quantified by using the immunoreactivity score (IRS) (staining intensity × percentage of positive cells). Relationships between c-Fes expression and pT stage, Gleason's score (GS), and biochemical recurrence in patients who underwent radical surgery were also investigated. RESULTS: In KIMAP, the percentage in normal glands, PIN and cancer cells positive for c-Fes expression were 0 (0/7), 25.0 (2/8), and 100% (7/7), respectively. In human tissues, c-Fes expression was also significantly higher in cancer cells than in normal cells and PIN, and it correlated with pT stage (P < 0.001) and GS (P = 0.047). Multivariate analysis showed that c-Fes expression was an independent predictor of poor outcome poor prognosis (hazard ratio = 3.21, 95% confidence interval = 1.11-9.37, P = 0.032). CONCLUSION: The results suggested that c-Fes expression is a useful predictor of biochemical recurrence after radical surgery. The results also suggested that c-Fes is a potentially useful therapeutic target in prostate cancer and a predictor of biochemical recurrence after radical prostatectomy.

Thrombospondin-1-derived 4N1K peptide expression is negatively associated with malignant aggressiveness and prognosis in urothelial carcinoma of the upper urinary tract.

BACKGROUND: Thrombospondin (TSP) is a multi-functional protein that appears to have dual roles in cancer, that is, either as a promoter or a suppressor. 4N1K is a TSP-derived peptide that has been reported to be associated with neovascularity, cell survival, and invasion. There is a little information regarding its pathological roles in human cancer tissues. Our aim was to clarify clinical significance and prognostic value of 4N1K expression in patients with urothelial carcinoma of the upper urinary tract (UC-UUT). METHODS: We investigated 4N1K expression in 97 surgically excised, non-metastasized UC-UUT specimens and five normal tissues via immunohistochemistry. Microvessel density (MVD), lymph vessel density (LVD), cancer cell proliferation (PI), apoptotic index (AI), and matrix metalloproteinase (MMP)-9 expression was also determined. The relationships 4N1K expression and pT stage, grade, and prognosis were analysed. In addition, correlations with these cancer-related and TSP-related factors were also investigated. RESULTS: Strong and moderate 4N1K expression was found in normal urothelial tissues. Of the 97 specimens, 45 patients were positive for 4N1K expression, which was primarily located in the interstitial areas of the cancer tissue. 4N1K expression was negatively associated with pT stage (p = 0.003) and grade (p = 0.002). Survival analyses revealed that 4N1K is a predictor of metastasis-free (p = 0.036) and cause-specific survival (p = 0.009). 4N1K expression was closely associated with malignant behaviour, specifically MVD (p = 0.001), AI (p = 0.013), and MMP-9 expression (p = 0.036), but not PI and LVD, as determined via multivariate analysis models. CONCLUSIONS: 4N1K expression appears to be associated with cancer cell progression and survival in UC-UUT patients via the regulation of angiogenesis, apoptosis, and MMP-9 expression. There is a possibility that the 4N1K-peptide may be a useful marker and novel therapeutic target in patients with UC-UUT.

Discovery of a new pyrimidine synthesis inhibitor eradicating glioblastoma-initiating cells.

BACKGROUND: Glioblastoma-initiating cells (GICs) comprise a tumorigenic subpopulation of cells that are resistant to radio- and chemotherapies and are responsible for cancer recurrence. The aim of this study was to identify novel compounds that specifically eradicate GICs using a high throughput drug screening approach. METHODS: We performed a cell proliferation/death-based drug screening using 10 560 independent compounds. We identified dihydroorotate dehydrogenase (DHODH) as a target protein of hit compound 10580 using ligand-fishing and mass spectrometry analysis. The medical efficacy of 10580 was investigated by in vitro cell proliferation/death and differentiation and in vivo tumorigenic assays. RESULTS: Among the effective compounds, we identified 10580, which induced cell cycle arrest, decreased the expression of stem cell factors in GICs, and prevented tumorigenesis upon oral administration without any visible side effects. Mechanistic studies revealed that 10580 decreased pyrimidine nucleotide levels and enhanced sex determining region Y-box 2 nuclear export by antagonizing the enzyme activity of DHODH, an essential enzyme for the de novo pyrimidine synthesis. CONCLUSION: In this study, we identified 10580 as a promising new drug against GICs. Given that normal tissue cells, in particular brain cells, tend to use the alternative salvage pathway for pyrimidine synthesis, our findings suggest that 10580 can be used for glioblastoma therapy without side effects.Key Points1. Chemical screening identified 10580 as a novel GIC-eliminating drug that targets DHODH, an essential enzyme for the de novo pyrimidine synthesis pathway. 2. Compound 10580 induced cell cycle arrest, apoptosis, and differentiation in GICs.

Pathological significance and prognostic significance of FES expression in bladder cancer vary according to tumor grade.

PURPOSE: The feline sarcoma oncogene protein (FES) is a non-receptor tyrosine kinase implicated in both oncogenesis and tumor suppression. Here, cancer cell lines and human tissues were employed to clarify the pathological and prognostic significance of FES in bladder cancer. METHODS: The relationship between FES expression and cancer aggressiveness was investigated using 3 cell lines (T24: corresponding to grade 3, 5637: corresponding to grade 2, and RT4: corresponding to grade 1) and 203 tissues derived from human bladder malignancies. Proliferation, invasion, and migration of cancer cells were assessed following the knockdown (KD) of FES expression by the siRNA method. Relationships between FES expression and pathological features, aggressiveness, and outcome were investigated. RESULTS: FES-KD inhibited the proliferation, migration, and invasion of T24 cells but not of RT4 cells and 5637 cells. Considering all patients, FES expression demonstrated a negative relationship with grade but no association with muscle invasion or cancer cell proliferation. However, it was positively correlated with pT stage and cell proliferation in high-grade tumors (p = 0.002); no such association was found for low-grade tumors. In addition, elevated FES expression was a negative prognostic indicator of metastasis after radical surgery for patients with high-grade tumors (p = 0.021) but not for those with low-grade malignancies. CONCLUSIONS: FES appeared to act as a suppressor of carcinogenesis, being associated with low tumor grade in the overall patient group. However, its expression correlated with cancer aggressiveness and poor outcome in high-grade bladder cancer. FES, therefore, represents a potential therapeutic target and useful prognostic factor for such patients.

Human antigen R as a predictive marker for response to gemcitabine-based chemotherapy in advanced cisplatin-resistant urothelial cancer.

In patients with advanced urothelial cancer (UC), a combination of cisplatin (CDDP) and gemcitabine (GEM) is the most commonly used first-line systematic chemotherapy regimen. Although no standard regime for the treatment of CDDP-resistant UC has been established, GEM-based regimens are frequently used in these patients. In other types of cancer, human antigen R (HuR) status in cancer cells is closely associated with patient response to GEM. The aim of the present study was to establish the predictive potential of HuR expression for disease progression and survival in patients with UC who were treated with GEM-based regimens as a first or second-line chemotherapy. A total of 50 patients with advanced UC were enrolled in the current study. As first-line chemotherapy, methotrexate, vinblastine, epirubicin and CDDP (MVEC) combination therapy and GEM and CDDP combination therapy were administered in 34 (68.0%) and 16 patients (32.0%), respectively. Following progression, 45 patients (90.0%) were treated with combined GEM and paclitaxel therapy, and 5 patients (10.0%) were treated with GEM monotherapy. Cytoplasmic and nuclear HuR expression was evaluated using immunohistochemical techniques. The associations between HuR expression levels and local tumor response and treatment outcomes were analyzed. In first-line chemotherapy, no anticancer effects were observed to be significantly associated with nuclear or cytoplasmic HuR expression. In second-line chemotherapy nuclear HuR expression also exhibited no significant association with anticancer effects; however, the local tumor response was significantly improved if positive cytoplasmic HuR expression was present (P=0.002). Multivariate analyses revealed that cytoplasmic HuR expression levels were a significant predictive marker for longer OS (hazard ratio, 0.22; 95% confidence interval, 0.09-0.56; P=0.001). No significant association was observed between nuclear HuR expression levels and the overall survival. Therefore, cytoplasmic HuR expression is a significant predictive marker of response to GEM-based chemotherapy in patients with CDDP-resistant UC. Despite the limitations of a small and retrospective study, the results of the present study may facilitate the development of novel treatment strategies and provide a focus for additional basic and clinical studies.

Stromal expression of Fer suppresses tumor progression in renal cell carcinoma and is a predictor of survival.

Fps/Fes related (Fer) is a non-receptor tyrosine kinase that is expressed in fibroblasts, immune cells and endothelial cells. Fer serves an important pathological role in cell survival, angiogenesis and the immune system. However, the pathological role of Fer expression in the stromal cells surrounding renal cell carcinoma (RCC) has not been previously investigated. In the present study, immunohistochemical analysis of Fer was performed using the formalin-fixed tissue samples of 152 patients with RCC. The proliferative and apoptotic indices were used to represent the percentage of proliferation marker protein Ki-67- and cleaved caspase-3-positive cells, respectively. The microvessel density was defined as the number of cluster of differentiation (CD) 31-positively stained vessels/mm2. In addition, CD57+ and CD68+ cells were counted using semi-quantification of natural killer (NK) cells and macrophages. Fer expression in stromal cells was negatively associated with Fuhrman grade, pathological tumor stage and metastasis (P<0.001). Fer expression in stromal cells was negatively associated with CD68+ macrophage density, whereas it was positively associated with CD57+ NK cell density. Kaplan-Meier estimators indicated that decreased stromal Fer expression was a predictive marker of decreased cause-specific survival rate (P<0.001). Furthermore, low expression of Fer was identified as being an independent marker of decreased cause-specific survival using multivariate analysis (hazard ratio, 7.4; 95% confidence interval, 1.7-33.0; P<0.001). The results of the present study suggested that low Fer expression in stromal cells is associated with increased malignant aggressiveness and decreased survival in patients with RCC. CD57+ NK cell and CD68+ macrophage regulation in cancer-stromal tissue is considered to affect RCC pathology.

Design, construction, and analysis of a novel class of self-folding RNA.

RNA can play multiple biological roles through use of its three-dimensional (3-D) structures. Recent advances in RNA structural biology have revealed that complex RNA 3D structures are assemblages of double-stranded helices with a variety of tertiary structural motifs. By employing RNA tertiary structural motifs together with the helices, we designed a novel class of self-folding RNA. In RNA composed of three helices (P1, P2, and P3), P1 interacts with P3 via a tetraloop-receptor interaction and P2 forms consecutive base-triples. Two designed RNAs of this class were prepared and their folding properties indicate that they form defined tertiary structures as designed. These RNAs may be used as modular units for constructing artificial ribozymes or nanometer-scale materials.

Infestivity of Demodex canis to hamster skin engrafted onto SCID mice.

We demonstrated that Demodex canis was transferred to skin xenografts of a dog and a hamster onto severe combined immunodeficiency mice. After the transfer of mites, the number of eggs, larvae, nymphs and adult mites per gram of canine and hamster xenografts increased, whereas no live mites were detected on murine allograft. These results indicate that D. canis proliferates in hair follicles of dog and hamster skins but not in murine allograft. Therefore, D. canis may have host preference but not strict host-specificity.

Smoking-induced changes in cancer-related factors in patients with upper tract urothelial cancer.

Cigarette smoking is a major risk factor for urothelial cancer (UC) development. However, the associations between smoking and changes in the pathological characteristics and molecular expression of cancer-related molecules in upper tract (UT) UC have not been fully elucidated. We investigated the associations between smoking status and cancer-related factors, including cancer cell proliferation, apoptosis, angiogenesis, lymphangiogenesis and expression of vascular endothelial growth factor-A and -C, matrix metalloproteinase (MMP)-2 and -9, cyclooxygenase (COX)-2 and urokinase-type plasminogen activator, in patients with UTUC. A total of 134 patients who underwent nephroureterectomy were retrospectively investigated. Proliferation index (PI), microvessel density and lymphatic vessel density (LVD) were measured using anti-Ki-67, anti-CD105 and anti-D2-40 antibodies in formalin-fixed specimens. The apoptotic index was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Other cancer-related molecules were investigated by immunohistochemistry in similar specimens. The patients were divided into three groups; non-smoker (n=54, 40.3%), former smoker (n=46, 34.3%) and current smoker (n=34, 25.4%). The PI and the apoptotic index were not found to be correlated with smoking status; however, the mean/standard deviation level of LVD in current smokers (40.9/12.9) was significantly higher (P=0.034) compared to that in patients who had never smoked (34.4/10.6). In addition, smoking status was positively correlated with the presence of intratumoral lymphatic vessels (iLV) (P=0.010) and the expression of COX-2 and MMP-9 (P=0.032). The multivariate analysis demonstrated that current smoking was independently associated with all the abovementioned smoking-related factors. However, former smoking was correlated with LVD and the presence of iLV. In the survival analysis, LVD, the presence of iLV and the expression of COX-2 and MMP-9 were identified as predictive factors for metastasis following surgery. In conclusion, lymphangiogenesis and the expression levels of COX-2 and MMP-9 were found to be associated with the smoking status of UTUC patients. Our results may provide important insights into the pathological changes precipitated by smoking in these patients.

Development of novel diastereoselective alkenylation of enolates using alkenylselenonium salts.

[reaction: see text] A novel alkenylation of enolates using alkenylselenonium salts is described. A reaction of lithium enolates, which were prepared in situ by the reaction of LiHMDS and carbonyl compounds, with alkenylselenonium salts gave the ethenylation products of carbonyl compounds in high yield. Diastereoselective alkenylation was also accomplished by the reaction of the enolates derived from N-acyl-1,3-oxazolidin-2-ones with the alkenylselenonium salt to afford good results (up to 92% yield and up to 95% de).

One-pot asymmetric synthesis of beta-cyanohydroxymethyl alpha-amino acid derivatives: formation of three contiguous stereogenic centers.

[reaction: see text] One-pot asymmetric Mannich-hydrocyanation reactions are described. Reaction of unmodified aldehydes with N-PMP-protected alpha-imino ethyl glyoxylate in the presence of catalytic amounts of L-proline followed by the addition of Et(2)AlCN provided highly enantiomerically pure beta-cyanohydroxymethyl alpha-amino acid derivatives possessing three contiguous stereogenic centers as single diastereomers (93-99% ee). Control of reaction temperature during the cyanation step directed whether cyclization of the products to lactones occurred.

TNFalpha induces acetylation of p53 but attenuates its transcriptional activation in rheumatoid synoviocytes.

Synovial hyperplasia is an important feature of rheumatoid arthritis (RA) and we have reported that several transcription factors were highly activated in rheumatoid synoviocytes. The purpose of this study was to examine nuclear acetylation in synoviocytes as an activation marker and determine its role in cell activation. Autonomous acetylation of approximately 53 and 62 kDa nuclear proteins was detected in rheumatoid synoviocytes by anti-acetylated lysine specific antibody. Furthermore, tumor necrosis factor alpha (TNFalpha), a potent mitogen for synoviocytes, dose-dependently increased their state of acetylation. Immunoprecipitation analysis revealed that 53 kDa acetylated protein (ap53) was identical with p53, a tumor suppressor gene product. Since enhanced p53 binding to the promoter by TNFalpha treatment was detected by gel shift assay, we analyzed p53 promoter activity by reporter assay system. Contrary to enhanced binding activity, the transcriptional activity was attenuated in a TNFalpha concentration-dependent manner. Since p53 activation requires recruitment of CREB binding protein (CBP) as a coactivator, we also examined the effect of CBP on TNFalpha-induced attenuation of p53 promoter activation. Overexpression of CBP induced p53 transcriptional activity and recovery of TNFalpha-induced inhibition. Our results clearly indicate that autonomous nuclear acetylation is characteristically enhanced in rheumatoid synoviocytes and that p53 is one of acetylated protein. Our results also demonstrate that TNFalpha-induced acetylation of p53 attenuated its transcriptional activation via CBP depletion, and that overexpression of CBP enhanced TNFalpha-induced cell death in rheumatoid synoviocytes, suggesting that regulation of transcriptional coactivator become a novel strategy for RA therapy.

Successful treatment for retroperitoneal cavernous hemangioma adjacent to the renal hilum via the laparoscopic approach: a case report.

INTRODUCTION: Cavernous hemangiomas are common benign tumors of the skin or liver but can also rarely originate from the retroperitoneal space, especially adjacent to the renal hilum. Qualitative characterization of these retroperitoneal tumors using available imaging modalities is relatively difficult. CASE PRESENTATION: A 40-year-old Japanese woman was incidentally noted to have a round homogenous tumor adjacent to the left renal hilum on computed tomography. The preoperative diagnosis was paraganglioma according to hormonal and clinical findings. The tumor was successfully resected via a laparoscopic approach, and histopathological examination of the tumor revealed cavernous hemangioma. CONCLUSIONS: Cavernous hemangioma is a rare but relatively benign disease when considering the different types of retroperitoneal tumors. We were able to effectively treat the retroperitoneal cavernous hemangioma via laparoscopy.

In vitro analysis of the effect of alkyl-chain length of anionic surfactants on the skin by using a reconstructed human epidermal model.

We investigated the effect of the alkyl-chain length of anionic surfactants on the skin using an in vitro model. The evaluated anionic surfactants were sodium alkyl sulfate (AS) and sodium fatty acid methyl ester sulfonate (MES), which had different alkyl-chain lengths (C8-C14). Skin tissue damage and permeability were examined using a reconstructed human epidermal model, LabCyte EPI-MODEL24. Skin tissue damage was examined by measuring cytotoxicity with an MTT assay. Liquid chromatography/tandem mass spectrometry (LC/MS-MS) and liquid chromatography/mass spectrometry (LC/MS) were used to detect surfactants that permeated into the assay medium through an epidermal model. To assess the permeation mechanism and cell damage caused by the surfactants through the epidermis, we evaluated the structural changes of Bovine Serum Albumin (BSA), used as a simple model protein, and the fluidity of 1,2-dipalmitoyl-sn-glycero-3-phosphpcholine (DPPC) liposome, which serves as one of the most abundant phospholipid models of living cell membranes in the epidermis. The effects of the surfactants on the proteins were measured using Circular Dichroism (CD) spectroscopy, while the effects on membrane fluidity were investigated by electron spin resonance (ESR) spectroscopy. ET50 (the 50% median effective time) increased as follows: C10 < C12 < C8 < C14 in AS and C8, C10 < C12 < C14 in MES. The order of permeation through the LabCyte EPI-MODEL24 was C10 > C12 > C14, for both AS and MES. For both AS and MES, the order parameter, which is the criteria for the microscopic viscosity of lipid bilayers, increased as follows: C10 < C12 < C14, which means the membrane fluidity is C10 > C12 > C14. It was determined that the difference in skin tissue damage in the LabCyte EPI-MODEL24 with C10 to C14 AS and MES was caused by the difference in permeation and cell membrane fluidity through the lipid bilayer path in the epidermis.

Plasma spectroscopy of metal ions for hyper-electron cyclotron resonance ion source.

In this research, the optical line spectra of metal ions from ECR plasma were observed using a grating monochromator with a photomultiplier. The light intensity of line spectrum from the ECR plasma had a strong correlation with ion beam intensity measured by a magnetic mass analyzer. This correlation is a significant information for the beam tuning process, because it allows to conduct the extraction of the desired metal ion species from the ECR plasma. Separation of ion species of the same charge to mass ratio with an electromagnetic mass analyzer is known to be an exceptionally complex process, but this research provides a new approach for its simplification. In this paper the grating monochromator method for metal ion beam tuning such as (40)Ca(12+), (56)Fe(15+), and (85)Rb(20+) of hyper-ECR ion source as an injector for RIKEN Azimuthal Varying Field cyclotron is described.

Canola and hydrogenated soybean oils accelerate ectopic bone formation induced by implantation of bone morphogenetic protein in mice.

Canola oil (Can) and hydrogenated soybean oil (H2-Soy) are commonly used edible oils. However, in contrast to soybean oil (Soy), they shorten the survival of stroke-prone spontaneously hypertensive (SHRSP) rats. It has been proposed that the adverse effects of these oils on the kidney and testis are caused at least in part by dihydro-vitamin K (VK) 1 in H2-Soy and unidentified component(s) in Can. Increased intake of dihydro-VK1 is associated with decreased tissue VK2 levels and bone mineral density in rats and humans, respectively. The aim of the present study was to determine the effects of these oils on bone morphogenetic protein (BMP)-induced ectopic bone formation, which is promoted by VK2 deficiency, in relation to the role of VK in the γ-carboxylation of osteocalcin and matrix Gla protein. A crude extract of BMPs was implanted into a gap in the fascia of the femoral muscle in 5-week-old mice maintained on a Soy, Can, or H2-Soy diet. Newly formed bone volume, assessed by three-dimensional X-ray micro-computed tomography and three-dimensional reconstruction imaging for bone, was 4-fold greater in the Can and H2-Soy groups than in the Soy group. The plasma carboxylated osteocalcin (Gla-OC) and total OC (Gla-OC plus undercarboxylated osteocalcin [Glu-OC]) levels were significantly lower in the Can group than in the Soy group (p < 0.05). However, these levels did not significantly differ between the H2-Soy and Soy groups. The plasma Gla-OC/Glu-OC ratio in the Can and H2-Soy groups was significantly lower (in Can; p = 0.044) or was almost significantly lower (in H2-Soy; p = 0.053) than that in the Soy group. In conclusion, Can and H2-Soy accelerated BMP-induced bone formation in mice to a greater extent than Soy. Further research is required to evaluate whether the difference in accelerated ectopic bone formation is associated with altered levels of VK2 and VK-dependent protein(s) among the three dietary groups.

Note: 6Li III light intensity observation for 6Li3+ ion beam operation at Hyper-Electron Cyclotron Resonance ion source.

The light intensity of (6)Li III line spectrum at λ = 516.7 nm was observed during (6)Li(3+) beam tuning at the Hyper-Electron Cyclotron Resonance (ECR) ion source. Separation of ion species of the same charge to mass ratio with an electromagnetic mass analyzer is known to be an exceptionally complex process. However, (6)Li III line intensity observation conducted in this study gives new insights into its simplification of this process. The light intensity of (6)Li III line spectrum from the ECR plasma was found to have a strong correlation with the extracted (6)Li(3+) beam intensity from the RIKEN Azimuthal Varying Field cyclotron.