Protocol: a simple gel-free method for SNP genotyping using allele-specific primers in rice and other plant species

<p>Abstract</p> <p>Background</p> <p>Genotype analysis using multiple single nucleotide polymorphisms (SNPs) is a useful but labor-intensive or high-cost procedure in plant research. Here we describe an alternative genotyping method that is suited to multi-sample or multi-locus SNP genotyping and does not require electrophoresis or specialized equipment.</p> <p>Results</p> <p>We have developed a simple method for multi-sample or multi-locus SNP genotyping using allele-specific primers (ASP). More specifically, we (1) improved the design of allele-specific primers, (2) established a method to detect PCR products optically without electrophoresis, and (3) standardized PCR conditions for parallel genomic assay using various allele-specific primers. As an illustration of multi-sample SNP genotyping using ASP, we mapped the locus for lodging resistance in a typhoon (<it>lrt5</it>). Additionally, we successfully tested multi-locus ASP-PCR analysis using 96 SNPs located throughout the genomes of rice (<it>Oryza sativa</it>) cultivars 'Koshihikari' and 'Kasalath', and demonstrated its applicability to other diverse cultivars/subspecies, including wild rice (<it>O. rufipogon</it>).</p> <p>Conclusion</p> <p>Our ASP methodology allows characterization of SNPs genotypes without electrophoresis, expensive probes or specialized equipment, and is highly versatile due to the flexibility in the design of primers. The method could be established easily in any molecular biology laboratory, and is applicable to diverse organisms.</p

Reconstruction of radiocesium levels in sediment off Fukushima: Simulation analysis of bioavailability using parameters derived from observed 137Cs concentrations

Radiocesium was released to the North Pacific coastal waters by the accident at the Fukushima Dai-ichi Nuclear Power Plant (1FNPP) of the Tokyo Electric Power Company (TEPCO) in March 2011. Since the radiocesium in the sediment off Fukushima was suggested as a possible source for the transfer of this radionuclide through the benthic food chain, we conducted numerical simulations of 137Cs in sediments off the Fukushima coast by using a model which incorporates dynamic transfer processes between seawater and the labile and refractory fractions in sediment particles. This model reproduced the measured temporal changes of 137Cs concentration in seabed surface sediment off Fukusima coasts, by normalizing the radiocsium transfer between seawater and sediment according to the particle diameter sizes. We found that the 137Cs level in sediment decreased by desorption during the first several months after the accident, followed by a reduction in the labile fraction until the end of 2012. The apparent decrease of the total radiocesium level in surface sediment was estimated to occur at rates of approximately 0.2 y−1 within a 20 km distance from the 1FNPP. The comparison of 137Cs level decreases in the demersal fish and the simulated temporal labile fraction in fine sediment demonstrated that the consideration of radiocesium transfer via sediment is important for determining the 137Cs depuration mechanism in some demersal fish

マクロファージにおけるスフィンゴシン1リン酸2型受容体 (S1P2) は肺線維症を増悪させる

修士課程優秀論

Effect of drought stress on flowering characteristics in rice (Oryza sativa L.): A study using genotypes contrasting in drought tolerance and flower opening time

Rice is most susceptible to heat and drought stress at flowering stage, but flowering characteristics under drought stress are not well characterized. This study investigated flowering characteristics of rice genotypes contrasting in their flower opening time (FOT) and level of drought tolerance. Near-isogenic lines for the early-morning flowering trait (IR64+ qEMF3) and for drought tolerance (IR87707-445-B-B-B), and their recurrent parent cultivar (IR64) were used. IR64+ qEMF3 had stable earlier FOT than IR64 and IR87707-445-B-B-B under drought stress conditions. Drought stress occasionally affects FOT depending on genotype. The number of open spikelets was higher in IR87707-445-B-B-B than in IR64 and IR64+ qEMF3, and the difference among genotypes increased as the rice plants were subjected to more severe stress levels. Panicle temperature increased under drought stress conditions and was similar among genotypes when it was measured at the same time of day, demonstrating that earlier FOT in IR64+ qEMF3 must be beneficial to avoid heat stress at flowering under drought stress conditions. However, IR64+ qEMF3 did not exhibit drought avoidance, as evidenced by the root mass at depth. To assess the potential for the EMF trait to complement ongoing drought breeding efforts, heat tolerance among 13 advanced drought breeding lines and released cultivars was tested. Wide variation in heat tolerance at flowering was observed and, notably, none of the 13 lines possessed the EMF trait. This study therefore proposes that a breeding strategy that transfers the EMF trait into drought tolerant lines could enhance the resilience of rice spikelets to the combined stresses of heat and drought at flowering

The class II phosphoinositide 3-kinases PI3K-C2α and PI3K-C2β differentially regulate clathrin-dependent pinocytosis in human vascular endothelial cells

金沢大学医薬保健研究域医学系Pinocytosis is an important fundamental cellular process that is used by the cell to transport fluid and solutes. Phosphoinositide 3-kinases (PI3Ks) regulate a diverse array of dynamic membrane events. However, it is not well-understood which PI3K isoforms are involved in specific mechanisms of pinocytosis. We performed knockdown studies of endogenous PI3K isoforms and clathrin heavy chain (CHC) mediated by small interfering RNA (siRNA). The results demonstrated that the class II PI3K PI3K-C2α and PI3K-C2β, but not the class I or III PI3K, were required for pinocytosis, based on an evaluation of fluorescein-5-isothiocyanate (FITC)–dextran uptake in endothelial cells. Pinocytosis was partially dependent on both clathrin and dynamin, and both PI3K-C2α and PI3K-C2β were required for clathrin-mediated—but not clathrin-non-mediated—FITC-dextran uptake at the step leading up to its delivery to early endosomes. Both PI3K-C2α and PI3K-C2β were co-localized with clathrin-coated pits and vesicles. However, PI3K-C2β, but not PI3K-C2α, was highly co-localized with actin filament-associated clathrin-coated structures and required for actin filament formation at the clathrin-coated structures. These results indicate that PI3K-C2α and PI3K-C2β play differential, indispensable roles in clathrin-mediated pinocytosis. © 2018, The Physiological Society of Japan and Springer Japan KK, part of Springer Nature.Embargo Period 12 month

Class II PI3Ks α and β Are Required for Rho-Dependent Uterine Smooth Muscle Contraction and Parturition in Mice

金沢大学医薬保健研究域医学系Class II phosphoinositide 3-kinases (PI3Ks), PI3K-C2α and PI3K-C2β, are highly homologous and distinct from class I and class III PI3Ks in catalytic products and domain structures. In contrast to class I and class III PI3Ks, physiological roles of PI3K-C2α and PI3K-C2β are not fully understood. Because we previously demonstrated that PI3K-C2α is involved in vascular smooth muscle contraction, we studied the phenotypes of smooth muscle-specific knockout (KO) mice of PI3K-C2α and PI3K-C2β. The pup numbers born from single PI3K-C2α-KO and single PI3K-C2β-KO mothers were similar to those of control mothers, but those from double KO (DKO) mothers were smaller compared with control mice. However, the number of intrauterine fetuses in pregnant DKO mothers was similar to that in control mice. Both spontaneous and oxytocin-induced contraction of isolated uterine smooth muscle (USM) strips was diminished in DKO mice but not in either of the single KO mice, compared with control mice. Furthermore, contraction of USM of DKO mice was less sensitive to a Rho kinase inhibitor. Mechanistically, the extent of oxytocin-induced myosin light chain phosphorylation was greatly reduced in USM from DKO mice compared with control mice. The oxytocin-induced rise in the intracellular Ca2+ concentration in USM was similar in DKO and control mice. However, Rho activation in the intracellular compartment was substantially attenuated in DKO mice compared with control mice, as evaluated by fluorescence resonance energy transfer imaging technique. These data indicate that both PI3K-C2α and PI3K-C2β are required for normal USM contraction and parturition mainly through their involvement in Rho activation.Embargo Period 12 month

MTMR4, a phosphoinositide-specific 3\u27-phosphatase, regulates TFEB activity and the endocytic and autophagic pathways.

金沢大学医薬保健研究域医学系Phosphatidylinositol 3-phosphate (PI(3)P) is the predominant phosphoinositide species in early endosomes and autophagosomes, in which PI(3)P dictates traffic of these organelles. Phosphoinositide levels are tightly regulated by lipid-kinases and -phosphatases; however, a phosphatase that converts PI(3)P back to phosphatidylinositol in the endosomal and autophagosomal compartments is not fully understood. We investigated the subcellular distribution and functions of myotubularin-related protein-4 (MTMR4), which is distinct among other MTMRs in that it possesses a PI(3)P-binding FYVE domain, in lung alveolar epithelium-derived A549 cells. MTMR4 was localized mainly in late endosomes and autophagosomes. MTMR4 knockdown markedly suppressed the motility, fusion, and fission of PI(3)P-enriched structures, resulting in decreases in late endosomes, autophagosomes, and lysosomes, and enlargement of PI(3)P-enriched early and late endosomes. In amino acid- and serum-starved cells, MTMR4 knockdown decreased both autophagosomes and autolysosomes and markedly increased PI(3)P-containing autophagosomes and late endosomes, suggesting that the fusion with lysosomes of autophagosomes and late endosomes might be impaired. Notably, MTMR4 knockdown inhibited the nuclear translocation of starvation stress responsive transcription factor-EB (TFEB) with reduced expression of lysosome-related genes in starved cells. These findings indicate that MTMR4 is essential for the integrity of endocytic and autophagic pathways. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.Embargo Period 12 month

Sphingosine-1-phosphate receptor-2 facilitates pulmonary fibrosis through potentiating IL-13 pathway in macrophages.

金沢大学医薬保健研究域医学系Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2 LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow–derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin–administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline–administered wild-type mice. Interestingly, in bleomycin–adminis-tered S1pr2 -/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2 +/+ mice alleviated bleomycin–induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis. © 2018 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

大学を核とした総合型地域スポーツクラブの創設・育成・運営の可能性について : 仙南広域スポーツ研究会の活動報告から

In 1995, the (then) Ministry of Education launched the "Project for the General-purpose Local Sports Clubs Development Model," and in 2000 it devised the Basic Plan for the Promotion of Sports, which set the target of "developing at least one general-purpose local sports club in each municipality nationwide by 2010." Meanwhile, revision of the School Education Act made it compulsory for all universities to undergo regular third-party evaluation, and universities themselves were required to make tangible local contributions. As a result, some universities started to establish and operate general-purpose local sports clubs as a project for contributing to the local community. This paper investigates the possibility of establishing, developing and operating general-purpose local sports clubs centering on universities, including the development status of the general-purpose local sports clubs in Miyagi Prefecture. It also reports on the activities of the Support Project for General-purpose Local Sports Clubs