Kitasatospora setae NBRC 14216T (=KM-6054T) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K. setae NBRC 14216T as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8 783 278 bp with terminal inverted repeats of 127 148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.
Liver fibrosis is final stage of chronic liver diseases which may cause liver failure and liver cancer. While various diagnostic methods, including serum marker have been established, no standard therapy has developed. The objective of this study is to assess the approach of overexpressing matrix metalloproteinase-13 gene (MMP13) in rat liver to block liver fibrosis. The rat liver fibrosis model was developed by the bile duct ligation (BDL). And the liver-targeted hydrodynamic gene delivery procedure involves catheter insertion via the inferior vena cava (IVC) to the junction of the IVC and the hepatic veins followed by the hydrodynamic injection of MMP13 expression vector, containing CAG promoter-MMP13-IRES-tdTomato-polyA cassette with temporal blood flow occlusions at the supra- and infra-hepatic IVC. Three groups (n=5 in each group) of animals, BDL alone (non-treated), BDL simultaneously followed by hydrodynamic delivery of pBGI-MMP13 (treated group), and hydrodynamic delivery of pBGI-MMP13 (control) were analyzed for: 1) MMP13 expression in serum and cells in the liver by ELISA, western blotting, immunohistochemical staining, and fluorescent detection; and 2) therapeutic effect on liver fibrosis by analysis of serum fibrotic markers of hyaluronic acid and collagen type IV, and histological analysis including silver staining. Serum concentration of MMP13 in pBGI-MMP13 treated group reached 71.±5.1 pg/ml (p<0.001) compared to ≈5 pg/ml in non-treated group in 2 weeks after the procedure which sustained for following 8 weeks and slowly declined to 37.±5.9 pg/ml in 20 weeks. The protein expression in the liver was also confirmed by western blotting and immunohistochemical staining of the liver samples. The expression of MMP13 contributed on the therapeutic effect which was evidenced by the statistically lower level of the hyaluronic acids of 50.±7.0 ng/ml in treated group than that of 20 ± 19 ng/ml in non-treated group (p<0.5) 2 weeks after the BDL, which sustained for 8 weeks. The serum level of collagen type IV showed similar pattern although no statistical difference was marked. Silver staining showed a significantly lower volume of the fibrotic tissue in treated group than that of non-treated. These results suggest that the liver-targeted hydrodynamic injection of pBGI-MMP13 could achieve long-term gene expression in rat liver and prevent the fibrotic development in the liver.
We report a 53-year-old female autopsy case of multiple sclerosis with bilateral continuous cystic lesions along the lateral ventricles and caudate-callosal angles (Wetterwinkel). The pathophysiological mechanisms underlying these peculiar huge cystic lesions can be explained by the appearance of necrotic tissue during the recurrent relapsing stages of the disease, and then, by the absorption and scavenging of activated microglias. Poor astrocytic gliosis, which might be an effect of frequent use of corticosteroids during the clinical course makes the cavities bigger.
Abstract Background: EMT (Epitherial-Mesenchymal transition) is a significant event in tumor metastasis and malignancy. Cancer cells are stimulated by EMT-inducible agonists from the surrounding stromal cells, and cause transformation such as weakening of cell-cell adhesion and the accompanying acceleration of cell migration and invasion. Inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we developed a high-throughput assay system of EMT inhibitor screen in 3D cell culture. Method: We adopted 3D cell culture method on NanoCulture® Plate (NCP), a scaffold type spheroid culture plate, as a culture method which can confirm spheroid morphologies. TGF-β and its inhibitor, SB431542, were used as an EMT inducer and as a positive control for EMT inhibitor, respectively. Expressions of E-cadherin, N-cadherin, vimentin and zeb-1, represent EMT maker genes, were evaluated by qRT-PCR. As hypoxia inside the spheroids reflected the spheroid size and cell-cell adhesion strength, we visualized the intra-spheroid hypoxia, and used it as an indicator for EMT induction and inhibition. Result: A549 cell spheroids gradually collapsed, by the weakening of cell-cell adhesion, and cells migrating out from the spheroids, by treating with TGF-β. This collapse of the spheroids was inhibited by SB431542 treatment. SB431542 treatment also accompanied inhibition of E-cadherin down-regulation, and N-cadherin, vimentin and zeb-1 up-regulation. These results indicate that this method is clearly reproducing EMT and its inhibition. And we elucidated that intra-spheroid hypoxicity was closely correlated with spheroid morphology. From this, it is demonstrated that this method is simple but high-precision assay. Conclusion: We developed a high-throughput assay system for EMT inhibitor screen, by evaluating intra-spheroid hypoxicity as an indicator for alternation of the spheroid morphology. This method is an unique method which enables to evaluate EMT condition, by monitoring the collapse of spheroids directly. We are now running a screen for EMT inhibitor candidate compounds, by using this assay system. Citation Format: Kazuya Arai, Ruriko Sakamoto, Manabu Itoh, Hiromi Miura, Manami Shimomura, Tetsuya Nakatsura. Development of novel high-throughput screening assay system for exploring EMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5525. doi:10.1158/1538-7445.AM2013-5525
Hepatocellular carcinoma (HCC) is a major global malignancy, responsible for >90% of primary liver cancers. Currently available therapeutic options have poor performances due to the highly heterogeneous nature of the tumor cells; recurrence is highly probable, and some patients develop resistances to the therapies. Accordingly, the development of a novel therapy is essential. We assessed gene therapy for HCC using a diphtheria toxin fragment A (DTA) gene-expressing plasmid, utilizing a non-viral hydrodynamics-based procedure. The antitumor effect of DTA expression in HCC cell lines (and alpha-fetoprotein (AFP) promoter selectivity) is assessed in vitro by examining HCC cell growth. Moreover, the effect and safety of the AFP promoter-selective DTA expression was examined in vivo using an HCC mice model established by the hydrodynamic gene delivery of the yes-associated protein (YAP)-expressing plasmid. The protein synthesis in DTA transfected cells is inhibited by the disappearance of tdTomato and GFP expression co-transfected upon the delivery of the DTA plasmid; the HCC cell growth is inhibited by the expression of DTA in HCC cells in an AFP promoter-selective manner. A significant inhibition of HCC occurrence and the suppression of the tumor marker of AFP and des-gamma-carboxy prothrombin can be seen in mice groups treated with hydrodynamic gene delivery of DTA, both 0 and 2 months after the YAP gene delivery. These results suggest that DTA gene therapy is effective for HCC.
CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.
