Plasmid Construction Using Recombination Activity in the Fission Yeast Schizosaccharomyces pombe

BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC), is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp). No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different markers constructed in this research are available from NBRP-yeast (http://yeast.lab.nig.ac.jp/)

Contributing Processes to Arctic Temperature Amplification for a Range of Forcing in MIROC GCM

第3回極域科学シンポジウム/第35回極域気水圏シンポジウム 11月30日（金） 国立国語研究所 ２階多目的

ハーブティーの摂取がヘアレスマウスの皮膚機能に及ぼす影響

Ultraviolet(UV)rays are suggested to be one of the factors responsible for skin photoaging induced by active oxygen species. In recent years, it has been reported that the intake of some compounds such as carotenoids prevented UV-induced photoaging. This study aims to investigate the effect of herb tea intake on the skin function of hairless mice irradiated with UV rays. The mice were given the following types of herb tea: Lavandula angustifolia, Malva sylvestris, Calluna vulgaris, Rosa laevigata, and Emblica officinalis. The skin condition of the Malva sylvestris-given mice was the best among all the experimental mice groups. Malva sylvestris had the lowest antioxidant activity and total polyphenolic content. While we could not identify the underlying mechanism, we infer that the long-term intake of herb tea such as Malva sylvestris increases the barrier function of the corneum and the water content of the epidermis

Isolation and molecular characterization of multidrug-resistant Gram-negative bacteria from imported flamingos in Japan

Imported animals, especially those from developing countries, may constitute a potential hazard to native animals and to public health. In this study, a new flock of lesser flamingos imported from Tanzania to Hiroshima Zoological Park were screened for multidrug-resistant Gram-negative bacteria, integrons and antimicrobial resistance genes. Thirty-seven Gram-negative bacterial isolates were obtained from the flamingos. Seven isolates (18.9%) showed multidrug resistance phenotypes, the most common being against: ampicillin, streptomycin, tetracycline, trimethoprim/sulfamethoxazole and nalidixic acid. Molecular analyses identified class 1 and class 2 integrons, β-lactamase-encoding genes, blaTEM-1 and blaCTX-M-2 and the plasmid-mediated quinolone resistance genes, qnrS and qnrB. This study highlights the role of animal importation in the dissemination of multidrug-resistant bacteria, integrons and antimicrobial resistance genes from one country to another

A Japanese version of the stressors in nursing students (SINS) scale

© 2018 Chinese Nursing Association Objectives: To translate and study the factor structure of a Japanese version of the Stressors in Nursing Students scale. Methods: The Stressors in Nursing Students scale was translated into Japanese and administered to a large cohort (N = 1298) of female Japanese nursing students across five universities and across all four years of the nursing programme. The data were analysed using exploratory factor analysis. Results: Exploratory factor analysis revealed four factors: ‘Clinical’; ‘Conflict and confidence’; ‘Education’; and ‘Free time’. The relationship between the total score on the Stressors in Nursing Students scale and the demographic aspects of the sample was weak but there was a significant increase in the perception of stressors between first and second year students. Conclusions: There were similarities and differences between the perception of stressors by nursing students in Japan compared with, for example, the United Kingdom and China mainland. While a four-factor structure was found here, the distribution of items in the Stressors in Nursing Students scale differed from previous studies in the United Kingdom and China with the ‘Free time’ factor being unique to this study. Stress is an issue for nursing students and is related to the specific stressors they encounter in the process of their work and study. Findings can inform the development of strategies to reduce such stressors among nursing students in multiple, global contexts

UNC93B1 Physically Associates with Human TLR8 and Regulates TLR8-Mediated Signaling

Toll-like receptors (TLRs) 3, 7, 8, and 9 are localized to intracellular compartments where they encounter foreign or self nucleic acids and activate innate and adaptive immune responses. The endoplasmic reticulum (ER)-resident membrane protein, UNC93B1, is essential for intracellular trafficking and endolysosomal targeting of TLR7 and TLR9. TLR8 is phylogenetically and structurally related to TLR7 and TLR9, but little is known about its localization or function. In this study, we demonstrate that TLR8 localized to the early endosome and the ER but not to the late endosome or lysosome in human monocytes and HeLa transfectants. UNC93B1 physically associated with human TLR8, similar to TLRs 3, 7, and 9, and played a critical role in TLR8-mediated signaling. Localization analyses of TLR8 tail-truncated mutants revealed that the transmembrane domain and the Toll/interleukin-1 receptor domain were required for proper targeting of TLR8 to the early endosome. Hence, although UNC93B1 participates in intracellular trafficking and signaling for all nucleotide-sensing TLRs, the mode of regulation of TLR localization differs for each TLR

Disruption of a Novel NADH-Glutamate Synthase2 Gene Caused Marked Reduction in Spikelet Number of Rice

Inorganic ammonium ions are assimilated by a coupled reaction of glutamine synthetase and glutamate synthase (GOGAT). In rice, three genes encoding either ferredoxin (Fd)-GOGAT, NADH-GOGAT1, or NADH-GOGAT2, have been identified. OsNADH-GOGAT2, a newly identified gene, was expressed mainly in fully expanded leaf blades and leaf sheaths. Although the distinct expression profile to OsNADH-GOGAT1, which is mainly detected in root tips, developing leaf blades, and grains, was shown in our previous studies, physiological role of NADH-GOGAT2 is not yet known. Here, we isolated retrotransposon mediated-knockout mutants lacking OsNADH-GOGAT2. In rice grown under paddy field conditions, disruption of the OsNADH-GOGAT2 gene caused a remarkable decrease in spikelet number per panicle associated with a reductions in yield and whole plant biomass, when compared with wild-type (WT) plants. The total nitrogen contents in the senescing leaf blade of the mutants were approximately a half of the WT plants. Expression of this gene was mainly detected in phloem companion cells and phloem parenchyma cells associated with large vascular bundles in fully expanded leaf blades, when the promoter region fused with a β-glucuronidase gene was introduced into the WT rice. These results suggest that the NADH-GOGAT2 is important in the process of glutamine generation in senescing leaves for the remobilization of leaf nitrogen through phloem to the panicle during natural senescence. These results also indicate that other GOGATs, i.e., NADH-GOGAT1 and ferredoxin-GOGAT are not able to compensate the function of NADH-GOGAT2

Essential Role of the Zinc Transporter ZIP9/SLC39A9 in Regulating the Activations of Akt and Erk in B-Cell Receptor Signaling Pathway in DT40 Cells

The essential trace element zinc is important for all living organisms. Zinc functions not only as a nutritional factor, but also as a second messenger. However, the effects of intracellular zinc on the B cell-receptor (BCR) signaling pathway remain poorly understood. Here, we present data indicating that the increase in intracellular zinc level induced by ZIP9/SLC39A9 (a ZIP Zrt-/Irt-like protein) plays an important role in the activation of Akt and Erk in response to BCR activation. In DT40 cells, the enhancement of Akt and Erk phosphorylation following BCR activation requires intracellular zinc. To clarify this event, we used chicken ZnT5/6/7-gene-triple-knockout DT40 (TKO) cells and chicken Zip9-knockout DT40 (cZip9KO) cells. The levels of Akt and ERK phosphorylation significantly decreased in cZip9KO cells. In addition, the enzymatic activity of protein tyrosine phosphatase (PTPase) increased in cZip9KO cells. These biochemical events were restored by overexpressing the human Zip9 (hZip9) gene. Moreover, we found that the increase in intracellular zinc level depends on the expression of ZIP9. This observation is in agreement with the increased levels of Akt and Erk phosphorylation and the inhibition of total PTPase activity. We concluded that ZIP9 regulates cytosolic zinc level, resulting in the enhancement of Akt and Erk phosphorylation. Our observations provide new mechanistic insights into the BCR signaling pathway underlying the regulation of intracellular zinc level by ZIP9 in response to the BCR activation