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). HGF‑induced HIF‑1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF‑induced HIF‑1α 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/c‑Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF/c‑Met signalling may be a promising target for anti-angiogenic strategies.

The Arabidopsis unfolded protein response transducer IRE1 contributes to male gametophyte development using an alternative activation mechanism bypassing the unfolded protein-sensing domain.

A broad-range PCR assay for the detection of bacteria belonging to the Enterobacteriaceae family was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 72 different bacterial species (of 49 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Vibrioaceae or Enterobacteriaceae strain except Proteus mirabilis. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA for PCR was prepared by a simple procedure with the use of Chelex 100 resin from culture after growth in brain heart infusion medium. To test this PCR assay for the monitoring of the Enterobacteriaceae family, either Escherichia coli or Salmonella Enteritidis was inoculated into various foods as an indicator. Prior to the PCR, the inoculation of 10 to 40 CFU of bacteria per g of food was followed by a 5-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. When actual examinations of the contamination of 15 noodle foods with Enterobacteriaceae by this PCR assay were conducted, 33% (5 of 15) of the samples tested positive. These results agreed with those of the Petrifilm Enterobacteriaceae Count Plate assay. Including the enrichment culture step, the entire PCR detection process can be completed within 7 h.

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 periimplant 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.

No abstract

As an initial step for the unfolded protein response (UPR) pathway, the luminal domain of inositol requiring enzyme 1 (IRE1) senses unfolded proteins in the endoplasmic reticulum (ER). Recent findings in yeast and metazoans suggest alternative IRE1 activation without the sensor domain, although its mechanism and physiological significance remain to be elucidated. In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay (RIDD) under ER stress, while the mutant does not exhibit severe developmental defects and is fertile under non-stress conditions. In this study, we focused on a third Arabidopsis IRE1 gene, designated as IRE1C, whose product lacks a sensor domain. We found that even though ire1c and ire1a/c mutants did not exhibit defective bZIP60 splicing and RIDD under ER stress, the ire1a/b/c triple mutant is lethal. 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) in the ire1a/b mutant did not complement defects in ER stress-dependent bZIP60 splicing and RIDD. Nevertheless, expression of Δ LD complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the Δ LD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate RIDD but not bZIP60 splicing. Phenotypes of IRE1B mutants lacking the sensor domain produced by CRISPR/Cas9-mediated gene editing in the ire1a/c mutant background were essentially same as those of Δ LD-expressing ire1a/b mutant. 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. KiM directed the project. KiM and NK design the experiments. KiM, YI, RH and NN produced transgenic plants. TM and KiM performed histological analysis. AM performed fatty acid analysis.KiM performed all other experiments. KiM and YI provided the materials and reagents. KiM, YI, and NK wrote the manuscript. All authors have read and approved the manuscript. Conflict of interestThe authors declare that they have no conflict of interest. ReferencesChen Y, Brandizzi F (2012) AtIRE1A/AtIRE1B and AGB1 independently control two essential unfolded protein response pathways in Arabidopsis. Plant J 69: 266-277 Clough SJ, Bent AF (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 Deng Y, Humbert S, Liu JX, Srivastava R, Rothstein SJ, Howell SH (2011) Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis.

To select Lactobacillus acidophilus group bacteria as a probiotic yogurt starter, we designed a new screening method that measures the binding activity of surface layer protein to rat colonic mucin, which contains sugar chains similar to those in human colonic mucin. The B1 subgroup (Lactobacillus gasseri), which is the dominant strain in the human intestinal tract, showed the highest binding activity to rat colonic mucin among all the subgroups of L. acidophilus. The binding activity of the surface layer protein was also shown to be significantly reduced after periodate oxidation of the rat colonic mucin. This new screening method is useful for rapid selection of L. acidophilus strains that have high adhesion to the human intestinal tract. Lectin-like proteins that were bound to rat colonic mucin were isolated from the surface layer proteins with a rat colonic mucin-coated membrane and were analyzed by SDS-PAGE. A few main bands together with several minor bands were observed on the electrophoretograms obtained from the strains tested. It is possible that those lectin-like proteins contribute to adhesion of the bacterial cell to human colonic mucosa by binding specifically to carbohydrate portions.

A broad-range PCR assay for the detection of bacteria belonging to Bacillus and Staphylococcus genera was developed. Primers targeting the bacterial 16S rRNA gene were newly designed and used in a PCR assay. To determine the specificity of the assay, 81 different bacterial strains (of 50 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Bacillus, Staphylococcus, or Aerococcus strain. In addition, the result for Listeria grayi was positive with lower PCR product. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA was prepared with the use of achromopeptidase and Chelex 100 resin from culture after growth in brain heart infusion medium. To test the sensitivity of this PCR assay for Bacillus or Staphylococcus genus, either Bacillus cereus or Staphylococcus aureus was inoculated into various foods with undetectable levels of endogenous microbial contamination as an indicator. Inoculation of bacteria at 10 to 30 CFU/g of food was followed by a 5-h enrichment culture step after which the PCR assay allowed the detection of bacterial cells. When the inoculation (B. cereus or S. aureus) of 10 to 90 CFU/g into noodle foods containing endogenous microflora (10(3) to 10(5) CFU/g) was followed by a 6-h enrichment culture step, the PCR assay detected the bacteria. Including the enrichment culture step, the entire PCR detection process can be completed within 8.5 h.