Long-term replication of Epstein-Barr virus-derived episomal vectors in the rodent cells

AbstractPlasmids containing the origin of replication, oriP, of the Epstein-Barr virus (EBV) and EBV nuclear antigen-1 genes replicate extrachromosomally in primate cells. However, these plasmids have been believed not to replicate in rodent cells. We demonstrate here that these plasmids can replicate in some types of rodent cells over a long period. This result should offer not only the new insight into the mechanisms of species-specific replication of EBV, but also the possibility that an EBV-based vector can be used for gene transfer experiments in non-primate cells and an animal experiment regarding human gene therapy

CONSTRUCTION PLACEMENT, HARDENED PROPERTIES AND DURABILITY OF SHOTCRETE WITH HIGHLY FUNCTIONAL FLY ASH

Shikoku Electric Power Co., Inc. has developed the technology to manufacture a brand name “Finash” about 12 years ago, by sorting and classifying coal ash generated in coal fired power plants. “Finash” is highly functional fly ash (HFA) is produced by removing irregular coarse particles. It is important for the production of HFA to minimize the variation in quality of coal ash with sophisticated classification technique and extracting good-quality spherical fine particles. The specific surface of HFA is more than 5000 cm2/g. It is now widely utilized as concrete admixture for general civil engineering structures and buildings in Japan. When highly functional fly ash (HFA) is used as shotcrete admixture to substitute for fine aggregate of 100kg/m3, the shotcrete has the advantages of decreasing the amount of dust and rebound during spraying operation, improving the hardened properties and durability of concrete, etc. Therefore, it has been applied in many tunnels by NATM. In order to verify the high performance of shotcrete with HFA, firstly it was carried out the spray tests at the model tunnel using the shotcrete with HFA having the specific surface of 5530cm2/g compared with normal shotcrete without fly ash and shotcrete with the lower fly ash (class 4th-FA) having the specific surface of 1770cm2/g. Secondly it was carried out the spray tests at an actual road tunnel using the shotcrete with HFA having the specific surface of 5450cm2/g compared with normal shotcrete without fly ash and shotcrete with the conventional dust reducing agent of 0.1% mass of the cement. This paper discusses about the various characteristics such as construction placement (dust concentration and rebound rate), hardened properties and durability (compressive strength, accelerating neutralization depth and dry shrinkage) on theses several sorts of shotcrete

Betuletol, a Propolis Component, Suppresses IL-33 Gene Expression and Effective against Eosinophilia

Propolis, a resinous substance produced by honeybees, has been used in folk medicine since ancient times due to its many biological benefits such as antitumor, antioxidant, antimicrobial, anti-inflammatory, and immunomodulatory effects. Propolis contains flavonoids, terpenoids, aromatic aldehydes, and alcohols, which vary with different climate and environmental conditions. In our study, we examined the antiallergic activity of Brazilian green propolis (BGP) and isolated the active compound that can suppress an allergy-sensitive gene, IL-33, expression and eosinophilia. Ethanolic extract of BGP freeze-dried powder was fractionated with several solvent systems, and the active fractions were collected based on activity measurement. The single active compound was found by thin-layer chromatography. Using column chromatography and NMR, the active compound was isolated and identified as 3,5,7-trihydroxy-6,4’-dimethoxyflavone, also known as betuletol. Further, the antiallergic activity of that has been examined in PMA-induced up-regulation of IL-33 gene expression in Swiss 3T3 cells. Our data showed the IL-33 gene suppression both by BGP and the isolated active compound, betuletol. We also found that betuletol suppressed ERK phosphorylation, suggesting it could be effective in suppressing IL-33 mediated eosinophilic chronic inflammation and will provide new insights to develop potent therapeutics against allergic inflammations

Fabrication of the iron-based superconducting wire using Fe(Se, Te)

We have fabricated the Fe(Se, Te) superconducting wire by a special process based on a powder-in-tube method. The pure Fe tube plays the role of not only the sheath but also the raw material for synthesizing the superconducting phases. We succeeded in observing zero resistivity current on the current-voltage measurements for the Fe(Se, Te) wire. Introduction of the pinning centers and fabricating a multi-core wire will enhance the critical current density for the next step.Comment: 16 pages, 6 figures, to appear in Applied Physics Express (APEX

Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.Tsukamoto T., Sakai E., Nishimae F., et al. Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells. Journal of Biotechnology 304, 1 (2019); https://doi.org/10.1016/j.jbiotec.2019.08.004

A trial of somatic gene targeting in vivo with an adenovirus vector

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ(+ )gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro. METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (~1/10000). Our further restriction analysis did not detect any designed recombinant. CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means

Pyrogallol inhibits NFAT signal

As the expression level of allergic disease sensitive genes are correlated with the severity of allergic symptoms, suppression of these gene expressions could be promising therapeutics. We demonstrated that protein kinase Cδ / heat shock protein 90-mediated H1R gene expression signaling and nuclear factor of activated T-cells (NFAT)-mediated IL-9 gene expression signaling are responsible for the pathogenesis of pollinosis. Treatment with Awa-tea combined with wild grape hot water extract suppressed these signaling and alleviated nasal symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats. However, the underlying mechanism of its anti-allergic activity is not elucidated yet. Here, we sought to identify an anti-allergic compound from Awa-tea and pyrogallol was identified as an active compound. Pyrogallol strongly suppressed ionomycin-induced up-regulation of IL-9 gene expression in RBL-2H3 cells. Treatment with pyrogallol in combination with epinastine alleviated nasal symptoms and suppressed up-regulation of IL-9 gene expression in TDI-sensitized rats. Pyrogallol itself did not inhibit calcineurin phosphatase activity. However, pyrogallol suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFAT. These data suggest pyrogallol is an anti-allergic compound in Awa-tea and it suppressed NFAT-mediated IL-9 gene expression through the inhibition of dephosphorylation of NFAT. This might be the underlying mechanism of the therapeutic effects of combined therapy of pyrogallol with antihistamine