Unique Rhizobial Communities Dominated by Bradyrhizobium liaoningense and Bradyrhizobium ottawaense were Found in Vegetable Soybean Nodules in Osaka Prefecture, Japan.

Vegetable soybean (Glycine max [L.]) is mainly consumed in Asian countries, but has recently attracted attention worldwide due to its high nutritional value. We aimed to identify the indigenous rhizobia of vegetable soybean in Yao City, Osaka Prefecture, Japan, and to clarify the relationships between the rhizobial community and soil environmental factors. Soil samples were collected from 12 vegetable soybean cultivation fields under two different conditions (six greenhouses and six open fields) in Yao City with different varieties of vegetable soybean. A total of 217 isolates were obtained from the nodules and clustered into nine operational taxonomic units (OTUs) with 97% homology based on the 16S-23S rRNA internal transcribed spacer (ITS) region. A phylogenetic ana-lysis showed that OTUs were closely related to Bradyrhizobium liaoningense, B. ottawaense, B. elkanii, and other Bradyrhizobium species and were dominant in this order. B. liaoningense was widely found in sampled sites and accounted for 50.7% of all isolates, while B. ottawaense was mostly limited to open fields. This rhizobial community differed from Japanese soybean rhizobia, in which B. diazoefficiens, B. japonicum, and B. elkanii were dominant. These results imply the characteristic differences among host plants or regional specialties. A non-metric multidimensional scaling (NMDS) ana-lysis revealed the significant impact of soil pH and the contents of Ca, Mg, Mn, total nitrogen (TN), and total carbon (TC) on the distribution of rhizobia. B. liaoningense was detected in soils with a neutral pH, and high TN and low Mn contents increased its abundance. The present study provides novel insights into Japanese rhizobia and potentially novel resources for sustainable agriculture.

TAIM: Tool for Analyzing Root Images to Calculate the Infection Rate of Arbuscular Mycorrhizal Fungi.

Arbuscular mycorrhizal fungi (AMF) infect plant roots and are hypothesized to improve plant growth. Recently, AMF is now available for axenic culture. Therefore, AMF is expected to be used as a microbial fertilizer. To evaluate the usefulness of AMF as a microbial fertilizer, we need to investigate the relationship between the degree of root colonization of AMF and plant growth. The method popularly used for calculation of the degree of root colonization, termed the magnified intersections method, is performed manually and is too labor-intensive to enable an extensive survey to be undertaken. Therefore, we automated the magnified intersections method by developing an application named "Tool for Analyzing root images to calculate the Infection rate of arbuscular Mycorrhizal fungi: TAIM." TAIM is a web-based application that calculates the degree of AMF colonization from images using automated computer vision and pattern recognition techniques. Experimental results showed that TAIM correctly detected sampling areas for calculation of the degree of infection and classified the sampling areas with 87.4% accuracy. TAIM is publicly accessible at http://taim.imlab.jp/.

Multivariate analysis of causal factors influencing accuracy of guided implant surgery for partial edentulism: a retrospective clinical study.

BACKGROUND: In dental implant treatment, the placement position of the implant body is important. The hypothesis is that there are factors that have a greater impact than the factors that have been studied so far. MATERIAL AND METHODS: The deviation between planned and actually placed implants was measured three-dimensionally by modified treatment evaluation method in 110 patients who underwent implant placement with guided surgery for partial edentulism. Ten factors that seemed to affect errors in placement were selected: the type of tooth, type of edentulism, distance from the remaining teeth, the type of implant, implant length, number of implants, method of guidance, the number of teeth supporting the surgical guide, number of anchor pins, and presence or absence of a reinforcement structure. The effect of each factor that corrected each confounding was calculated using multivariate analysis. RESULTS: In this study, 188 implant bodies were set to target, and the errors measurement data of the implant position were as follows: average Angle, 2.5 ± 1.6° (95% CI 2.25-2.69); Base, 0.67 ± 0.37 mm (95% CI 0.62-0.72); and Apex, 0.92 ± 0.47 mm (95% CI 0.86-0.98). As the result of multivariate analysis, larger errors were present in the partially guided group than the fully guided group. The number of teeth supporting the surgical guide significantly influenced the error in placement position. The error caused by the number of anchor pins was significantly different for the Angle. Similarly, the presence of the reinforcement structure influenced the error significantly for the Angle. CONCLUSIONS: It was suggested that the smaller errors could be present by performing guided surgery with full guidance and devising the design of the guide such as the number of teeth supporting the surgical guide, the setting of the anchor pin, and the reinforcement structure.

Quantitative evaluation of connective tissue grafts on peri-implant tissue morphology in the esthetic zone: A 1-year prospective clinical study.

BACKGROUND: In the treatment of anterior implants, few studies have quantitatively evaluated the effects of connective tissue grafts on labial bone resorption and soft tissue recession. PURPOSE: To evaluate the influence of connective tissue grafting (CTG) on the peri-implant tissue morphology by quantitatively measuring change over time the tissue surrounding the implant in the anterior esthetic zone. MATERIAL AND METHODS: Twenty-six patients who received implants with platform shifting in the anterior esthetic region were included in this follow-up study. Patients were classified as those who received CTG [CTG (+) group] and those who did not [CTG (-) group]. The vertical and horizontal dimensions of the buccal alveolar bone of the implant and its surrounding soft tissues were evaluated using cone-beam computed tomography. RESULTS: At 1 year after connection of the superstructure, labial soft tissue recession was on average 0.64 mm in the CTG (-) group and 0.09 mm in the CTG (+) group, and this difference was significant (P < .001). Furthermore, mean labial bone resorption was 0.65 mm in the CTG (-) group and 0.13 mm in the CTG (+) group, and also this difference was significant (P = .003). CONCLUSIONS: Within the limitations of this study, these findings suggest that CTG may be effective in both reducing labial bone resorption around the implant and reducing the recession of the soft tissue.

Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis.

In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay under ER stress, whereas the mutant does not exhibit severe developmental defects under normal conditions. In this study, we focused on the Arabidopsis IRE1C gene, whose product lacks a sensor domain. We found that the ire1a/b/c triple mutant is lethal, and heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate regulated IRE1-dependent decay but not bZIP60 splicing. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain.

[A Case of Solid Pseudopapillary Neoplasm of the Pancreas in a Male Patient].

The patient was a 50-year-old man. Abdominal ultrasound performed as part of a regular check-up suggested the presence of a tumor in the left kidney, and he was referred to our hospital for further examination. Computed tomography(CT) and magnetic resonance imaging(MRI)revealed the presence of a cystic neoplasm of the pancreas with partial calcification. The tumor had a maximum diameter of approximately 12 cm. Based on these findings, the patient was diagnosed with solid pseudopapillary neoplasm(SPN)and underwent distal pancreatectomy and splenectomy. Pathological findings indicated that a large proportion of the tumor was composed of cells with clear cytoplasm and small, oval nuclei, while some parts of the tumor were composed of atypical cells. Immunohistochemistry further demonstrated that the tumor expressed vimentin, nuclear b-catenin, and CD10. These findings confirmed the diagnosis of SPN. Atypical cells in the tumor were p53-positive, and some were exposed with damaged membranes. The patient has been followed up due to the possibility of tumor recurrence. The tumor has not recurred for approximately 32 months since surgery. Previous studies demonstrated that SPN is more common in women. We report the case of SPN in a male patient and discuss the literature.

[A Case of Stage IV Breast Cancer with Extensive Metastasis Successfully Treated with S-1 plus Anastrozole Therapy].

We report the case of a patient in which S-1 plus anastrozole was administered as first-line chemotherapy for Stage IV breast cancer with skin invasion, multiple lymph node metastasis, and lymphangitis carcinomatosis. A 77-year-old woman had a mass destroyed immediately outside the axilla with dry coughs. An 11mm unpalpable mass in the right breast and an axillary mass were confirmed to be scirrhous carcinoma(Luminal type B), respectively, by core needle biopsy. In one course, S- 1(100mg/day)therapy involves taking 2 courses of 14 days of administration and 7 days off the drug. Anastrozole(1mg/ day)was administered daily. After completion of one course, marked shrinkage of the axillary tumor and supraclavicular lymph node, and lightness of coughing was observed. The metastatic lymph nodes and pulmonary metastatic lesions reduced in size by over 30%, as revealed using CT. The adverse event was only grade 1 pigmentation and lacrimation. Ten months later, the self-destructed skin was completely scarred, and metastatic lesions had maintained their reduction in size. According to the results of the SELECT BC study, S-1 as primary chemotherapy for breast cancer is an evidence-based drug that can reduce the decrease in QOL, such as hair loss, and it can be positively selected.

[Long-Term Disease-Free Survival by Curative Resection in a Case of Remnant Gastric Cancer with Peritoneal Recurrence].

The patient was 69-year-old man. For the remnant gastric cancer, partial resection of the remnant stomach with combined resection of mesentery of transverse colon was performed. Pathological diagnosis was adenocarcinoma(tub2>tub1), M, B- 50-AJ, type 3, pT4b(mesentery of transverse colon), pN0, CY0. A CT scan of 6 months after the surgery showed a tumor on the left side abdomen and diagnosed as peritoneal recurrence. Chemotherapy consisted of 1 course of TS-1(100mg/body) plus cisplatin(70mg/body), 4 courses(2 weeks administration and 1 week break)of TS-1(100mg/body), 8 courses of docetaxel(80mg/body). Tumor shrinkage and internal necrosis were observed. Peritoneal tumor was resected 19 months after the first surgery, and partial resection of the invaded transverse colon and jejunum was performed. Pathological diagnosis was metastasis of remnant gastric cancer. After that, it is 61 months since the first surgery and 42 months from the recurrence surgery without relapse.

[A Case of Perforation Peritonitis with Multiple Duodenal and Intestinal Malignant Lymphomas].

Malignant lymphomas of the duodenum and small intestine are relatively rare, but are clinically important, as they may result in perforation peritonitis. Here, we report a case of perforation peritonitis caused by multiple duodenal and small intestinal malignant lymphomas. An 84-year-old man was diagnosed with malignant lymphoma of the duodenum. Although chemotherapy was planned, emergency surgery was performed to treat perforation peritonitis. Laparoscopic observation revealed that the perforation was not in the duodenum, but in the ileum. In addition, numerous lymphoma lesions were revealed throughout the small intestine. Partial resection of the small intestine including the perforation was performed. Primary gastrointestinal malignant lymphoma may exist over multiple digestive tracts and it is necessary to carefully diagnose and treat, even in emergency surgery.

[Two Cases of Colorectal Cancer with Tumor Thrombus in the Inferior Mesenteric Vein].

In colorectal cancer, progression with an intravenous tumor thrombus is very rare. Here, we report 2 cases of colorectal cancer which showed a tumor thrombus in the inferior mesenteric vein (IMV). Case 1: A 69-year-old woman was admitted for the treatment of advanced rectal cancer, and underwent a low anterior resection. Six months of post-operative therapy was carried out with mFOLFOX6, but a metachronous lung metastasis was detected and a lung partial resection was performed. Case 2: A 67-year-old man was admitted for the treatment of advanced sigmoid colon cancer with simultaneous liver metastasis, and underwent a laparoscopic high anterior resection. Four courses of mFOLFOX6+bevacizumab chemotherapy were carried out after surgery, and subsequently he underwent a partial hepatectomy. In both cases IMV tumor thrombus was suspected from abdominal contrast-enhanced computed tomography (CT). Tumor thrombus filling the lumen of the IMV was confirmed on histopathological examination. Colorectal cancer with IMV tumor thrombus is a form of advanced cancer with advanced vascular invasion, and there is a high risk of simultaneous or metachronous hematogenous metastasis. Combined modality therapy should therefore be given to improve the prognosis.

[Protein-losing gastroenteropathy in a case of colon cancer].

A 74-year-old man was admitted with primary complaints of appetite loss, abdominal swelling, and pedal edema. Laboratory data revealed the presence of anemia and hypoalbuminemia. Barium enema and colonofiberscopy demonstrated an advanced cancer in the transverse colon. Albumin scintigraphy revealed high uptake by the tumor in the transverse colon. Preoperative administration of albumin did not improve hypoalbuminemia. Serum protein and albumin levels improved immediately after resection of the tumor. Therefore, in the presence of hypoalbuminemia due to leakage from the tumor, it is important to operate promptly, without waiting to correct the hypoalbuminemia.

[A case of small bowel cancer with positive peritoneal cytology and five-year recurrence-free survival].

Small bowel cancer is frequently detected at an advanced stage and its prognosis is poor. We report on a patient with small bowel cancer with positive peritoneal cytology who survived for 5 years without recurrence after surgery.The case involved a 73-year-old woman who had undergone partial resection of the small intestine and lymphadenectomy for a small bowel tumor with obstruction. Pathological examination confirmed papillary adenocarcinoma with partial serosal invasion. Ascites cytology indicated a class V tumor. Adjuvant chemotherapy with TS-1 was administered for 20 months, and the patient has survived without evidence of disease for over 5 years.In this case, it is possible that TS-1 chemotherapy was effective for prevention against small bowel cancer recurrence.Furthermore , peritoneal cytology in patients with small bowel cancer should be evaluated as a predictor of prognosis.

[Validation study on a multi-residue method for determination of pesticide residues in agricultural products by new automatic pretreatment equipment (FASRAC) and GC-MS/MS].

A validation study was performed on a multiresidue method for determination of pesticide residues in agricultural products according to the method validation guideline of the Ministry of Health, Labour and Welfare of Japan. FASRAC (Food Automatic Analytical Systems for Residual Agricultural Chemicals) automatically performs extraction of pesticide residues from agricultural products with acetonitrile, filtration, constant volume, mixing with the use of air, mixing acetonitrile with buffer solvent, separation, and dehydration with sodium sulfate. The extract was purified with a GC/NH2 column. For wheat flour and soybeans, a purification step with a C18 column was added before a GC/NH2 column. After removal of the solvent, the extract was resolved in n-hexane/acetone solvent for GC-MS/MS analysis. In the case of manual analysis, pesticide residues were analyzed according to official multiresidue methods and purification steps were the same as in FASRAC. Recovery tests were performed with wheat flour, soybeans, spinach and apples, by addition of 302 pesticides at the concentrations 0.01 mg/kg. The results indicate that automatic extraction using FASRAC is superior to manual analysis in trueness, repeatability and within-run reproducibility. Specially, automatic extraction using FASRAC is superior to manual analysis in trueness because it is optimized in various respects, for example reextraction at salting-out.

Interruption of the HGF paracrine loop by NK4, an HGF antagonist, reduces VEGF expression of CT26 cells.

Hepatocyte growth factor (HGF), acting through the c­Met receptor, plays an important role in solid tumors. Various malignant cells utilize the biological actions of the HGF/c­Met pathway for their dissociative, invasive and metastatic behaviors. HGF also binds to the receptor expressed on endothelial cells that stimulates angiogenesis, a process critical to continued growth of solid tumors. It is known that HGF induces in vitro expression of vascular endothelial growth factor (VEGF), a key agonist of tumor angiogenesis. In the present study, we showed using in vitro co-culture system with fibroblasts that VEGF expression of CT26 cells was amplified through tumor-stromal interaction, i.e., the HGF paracrine loop. This action was inhibited by interruption of the HGF paracrine loop by gene transfer of NK4, an HGF antagonist. In in vivo experiments, CT26 tumor growth and angiogenesis were markedly enhanced by fibroblast co-inoculation, while the effect of fibroblasts was not observed in NK4­expressing CT26 cells. These findings suggest that NK4 exerted potent anti­angiogenic action via indirectly inhibiting VEGF expression of tumor cells in addition to direct effects on endothelial cells. Thus, the HGF/c­Met pathway may be a considerable candidate for molecular targeting strategy against tumor angiogenesis.

HGF regulates VEGF expression via the c-Met receptor downstream pathways, PI3K/Akt, MAPK and STAT3, in CT26 murine cells.

In the present study, we assessed the involvement of hepatocyte growth factor (HGF)/c-Met signalling with vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor (HIF)-1α expression in the downstream pathways phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in CT26 cells, to determine the mechanisms of the potent anti-angiogenic effect of NK4. We established genetically modified CT26 cells to produce NK4 (CT26-NK4). VEGF expression in subcutaneous CT26 tumours in vivo and in culture supernatants in vitro was determined by ELISA. HIF-1α expression in nuclear extracts was evaluated by western blot analysis. VEGF and HIF-1α mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The DNA binding activity of HIF-1α was evaluated using an HIF-1α transcription factor assay kit. Our results demonstrated that VEGF expression was reduced in homografts of CT26-NK4 cells, compared to those of the control cells. In vitro, VEGF expression, which was induced by HGF, was inhibited by anti-HGF antibody, NK4 and by kinase inhibitors (PI3K, LY294002; MAPK, PD98059; and STAT3, Stattic). HGF­induced HIF­1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF­induced HIF­1α mRNA expression was not inhibited by LY294002 and PD98059, but was inhibited by Stattic. These data suggest that the PI3K/Akt, MAPK and STAT3 pathways, downstream of HGF/c­Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF/c­Met signalling may be a promising target for anti-angiogenic strategies.

[Two cases of chylous ascites after laparoscopic colorectal cancer surgery].

We report the cases of 2 patients in whom chylous ascites developed after laparoscopic colorectal cancer surgery. Case 1 involved a 64-year-old woman who underwent laparoscopic right hemicolectomy with D3 lymphadenectomy for transverse colon cancer. Chylous ascites occurred immediately after the resumption of oral food intake on postoperative day 3. The patient gradually recovered by undergoing immediate treatment and by consuming a low-fat diet. The drain was removed on postoperative day 8, and the patient experienced no adverse events thereafter. Case 2 involved an 80-year-old man who underwent laparoscopic high anterior resection with D2 lymphadenectomy for multiple sigmoid cancers. Chylous ascites occurred a day after the resumption of oral food intake on postoperative day 3; however, food intake was continued. Because of its small volume, the chylous ascites was easily drained on postoperative day 6. Most cases of chylous ascites after colorectal cancer surgery can be easily resolved. However, if involvement of a major lymph duct is suspected during surgery, it should be ligated or clipped.

[A case of esophageal cancer with a funnel chest].

Esophageal cancer is a disease that is difficult to manage before and after surgery and is associated with a high in-hospital mortality rate despite there being reports of improved outcomes after multidisciplinary treatment. Meanwhile, although funnel chest is generally a subclinical condition, patients with this deformity may sometimes present with cardiac failure and chest pain. We report a case of advanced esophageal cancer with a funnel chest deformity that was very difficult to reconstruct after thoracoscopy-assisted resection.

NK4 gene expression enhances 5-fluorouracil-induced apoptosis of murine colon cancer cells.

BACKGROUND: The hepatocyte growth factor (HGF)-Met pathway plays a role in progression to malignant characteristics in tumors, including that of resistance to anticancer drugs. The purpose of this study was to elucidate the possibility of the combination therapy of NK4, a competitive antagonist for HGF. MATERIALS AND METHODS: We established a genetically modified murine colon cancer cell, CT26, to produce abundant NK4 (CT26/NK4). Cell proliferation, apoptosis, cell cycle, intracellular signaling, and 5-fluorouracil (5-FU) metabolism of this cell line were examined. RESULTS: There was no difference in thymidylate synthase mRNA level between mock-transfected control CT26 cells and CT26/NK4 cells, suggesting that NK4 expression does not change 5-FU metabolism. NK4 gene expression enhanced 5-FU-induced caspase-3 and -7 activation of CT26. Cell cycle analysis showed that NK4 gene expression and 5-FU treatment caused an increase in the proportion of sub-G(1) cells. On 5-FU treatment, phosphorylation of Akt and Erk1/2 was suppressed in CT26/NK4 less than in mock-transfected cells. 5-FU showed a stronger cytotoxic activity towards CT26/NK4 cells than control CT26 cells. CONCLUSION: 5-FU exerts an additional effect on apoptosis of NK4-expressing CT26 cells by down-regulating intracellular signaling of the HGF/c-Met pathway.

Gene transfer of NK4, an angiogenesis inhibitor, induces CT26 tumor regression via tumor-specific T lymphocyte activation.

Hepatocyte growth factor (HGF) has been shown to be involved in malignant behaviors, such as invasion and metastasis, in different tumors. Hence, HGF could be a target molecule for control of the malignant potential of cancer. NK4 is a competitive antagonist for HGF and exerts an antitumor activity, not only by HGF antagonism but also by antiangiogenesis. Here, we studied the participation of cellular immunity in CT26 tumor regression by NK4 gene transfer. In vivo experiments showed that NK4-induced inhibition of subcutaneous tumor growth (as demonstrated in immunocompetent BALB/c mice) was weakened in T lymphocyte-deficient nude mice. In addition, the immunocompetent BALB/c mice that had shown complete regression of CT26-NK4 tumors generated an immune memory against repeated challenge with the same tumor antigen. Immunohistochemistry of tumor-infiltrating lymphocytes showed that the ratio of CD8/CD4 in CT26-NK4 tumors was significantly higher than that in control tumors. Also, the presence of tumor-specific cytotoxic T lymphocytes (CTL) was demonstrated by cytotoxicity assays. Depletion of CD8+ T lymphocytes markedly abrogated the antitumor activity of NK4. However, NK4 had no direct effect on the in vitro cellular immune system. Taken together, these data indicate that NK4 expression by gene transfer, at the tumor site, triggers tumor-specific CTL activation, resulting in complete CT26 tumor regression in vivo. This action was considered to be due to apoptosis induced by NK4's potent antiangiogenic and HGF antagonistic effects.

NK4, an HGF antagonist, prevents hematogenous pulmonary metastasis by inhibiting adhesion of CT26 cells to endothelial cells.

Hepatocyte growth factor (HGF) plays a definitive role in invasive, angiogenic, and metastatic activities of tumor cells by binding to the c-Met receptor. NK4, a competitive antagonist for HGF and the c-Met receptor, prevents tumor cell growth and metastasis via its bifunctional properties to act as an HGF antagonist and angiogenesis inhibitor. In the present study, we investigated the inhibitory effectiveness of NK4 on hematogenous pulmonary metastasis of the CT26 murine colon cancer cell line, focusing on tumor cell adhesion to endothelial cells. In an in vitro adhesion assay, HGF facilitated adhesion of CT26 cells to a murine endothelial cell line (F-2) in a dose-dependent manner. Furthermore, the enhancing effect of HGF on CT26-F-2 cell interaction was blocked by NK4 as well as by anti-HGF antibody. Similarly, HGF-induced phosphorylation of focal adhesion kinase (FAK), downstream of integrin signaling, was reduced by NK4 and by anti-HGF antibody. However, distinct integrin expression on the surface of CT26 cells was not altered by HGF. In an in vivo experimental pulmonary metastasis assay, stable NK4 expression potently decreased the number of pulmonary metastatic foci. The NK4-induced suppression of pulmonary metastasis was partially reversed when HGF was intraperitoneally administered in an adhesive phase. These results suggest that NK4 could act on tumor cells to inhibit CT26 adhesion to endothelial cells by reducing FAK phosphorylation, which is regulated by inside-out HGF/c-Met signaling, and thereby suppress hematogenous pulmonary metastasis.