Construction of a genomewide RNAi mutant library in rice

Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification‐mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down‐regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double‐stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross‐silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA‐derived siRNAs were characteristic of Argonaute‐binding small RNAs. Our results indicate that RNAi mutant library is a high‐efficient approach for genomewide gene identification in plants.     

Dahuang Zhechong pill attenuates CCl4-induced rat liver fibrosis via the PI3K-Akt signaling pathway

It is well characterized that activated hepatic stellate cells (HSCs) exert critical functions in accelerating the progression of liver fibrosis. Previous studies have indicated that Dahuang Zhechong pill (DHZCP), a traditional Chinese herbal medicine, is capable of inactivating HSCs and thus attenuate the formation of liver fibrosis in rats. However, pharmacological mechanisms of DHZCP in alleviating liver fibrosis remain unclear. This study aims to investigate the antifibrotic role of DHZCP through inhibiting the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) pathway. DHZCP was found to significantly suppresses extracellular matrix formation and immune cell infiltration, thus alleviating liver fibrosis symptoms in the in vivo model. Moreover, DHZCP reduced serum levels of transforming growth factor β1 and tumor necrosis factor-α in rats with liver fibrosis. DHZCP treatment remarkably downregulated protein levels of PI3K and phosphorylated Akt, as well as fibrosis markers. In vitro experiments further demonstrated that DHZCP markedly suppressed HSCs proliferation by downregulating PI3K/Akt, which exerted a synergistic effect with the PI3K inhibitor LY294002. To sum up, our results confirmed that DHZCP exerted an antifibrotic effect in the animal model through inactivating the PI3K/Akt pathway, thus protecting rats from liver injury.     

长江口及邻近海域富营养化指标响应变量参照状态的确定

对长江口富营养化指标中的响应变量进行了筛选,并在长江口分区的基础上,运用参照点或观测点指标频数分布曲线法,对1992年-2010年的数据进行分析,确定了长江口外海区和舟山海区富营养化指标中响应变量的参照状态.选择叶绿素a和底层溶解氧作为响应指标的必选指标,浮游植物密度和CODMn作为辅助指标.经分析,长江口口外海区叶绿素a、浮游植物密度、CODMn和底层溶解氧的春夏秋3个季节的参照状态分别为0.87mg,/m3,17.44× 103个/L,0.42mg/L,8.36mg/L;1.88mg/m3,25.96×103个/L,0.56mg/L,4.22mg/L;0.84mg/m3,12.10×103个/L,0.46mg/L,6.95mg/L;舟山海区叶绿素a、浮游植物密度、CODMn和溶解氧的春夏秋3个季节的参照状态分别为0.73mg/m3,6.77×103个/L,0.51 mg/L,8.75mg/L;1.00mg/m3,9.72×103个/L,0.37mg/L,5.94mg/L;0.78mg/m3,4.59×103个/L,0.55mg/L,7.40mg/L.本研究确定的参照状态值能较为客观的反映该海域的富营养化参照状态,且不同分区,不同季节间的指标的参照状态亦存在着显著的差异.     

Gene expression profiles of sodium-dependent vitamin C transporters in mice after alcohol consumption

Alcoholic liver disease （ALD） is a serious fiver problem in western countries. Our previous study has demonstrated that vitamin C plays a protective role in ALD. The vitamin C homeostasis is tightly regulated by sodium-dependent vitamin C transporters （SVCTs） 1 and 2. But the role of two SVCTs in ALD is less understood. In this study, we exam- ined the expression patterns of two SVCTs in mice after alcohol consumption. Our results suggested that alcohol con- sumption obviously increased the expression of two SVCTs in liver and SVCT1 in kidney and intestine, which is important for vitamin C absorption. Vitamin C supplement increased the sera vitamin C content and ameliorated the symptom of ALD. Intestinal absorption and renal re-absorption mediated by SVCTI are key factors to increase the sera vitamin C content after alcohol consumption. We proposed that both reactive oxygen species and low vitamin C concentration regulate the expression of SVCTs, and the protective role of vitamin C is mediated by suppressing the stability of hypoxia-inducible factor-loL. Thus, our study is significant for the understanding of vitamin C homeostasis in ALD and for better use of other antioxidants in ALD therapy.     

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.     

A new species, Embellisia astragali sp. nov., causing standing milk-vetch disease in China

A new species, Embellisia astragali sp. nov., is described from necrotic leaves, petioles and stems of Astragalus adsurgens in China. The morphology of E. astragali is compared and contrasted to that of four similar species, E. abundans, E. oxytropis, E. phragmospora and E. telluster. The fungus grew intercellularly in stems and leaf blades and intracellularly in leaves. It was isolated from most sources of seeds at frequencies of 0.1-44.6%. Growth rates of colonies on potato-carrot agar, potato-dextrose agar, wheat hay decoction (WHDA) and V8 at 25 C were 0.48, 0.32, 0.68 and 0.27 mm d(-1), respectively. The optimal temperature for colony growth on WHDA was 20-25 C, and no growth was measured above 30 C. Five week old standing milk-vetch seedlings were inoculated with E. astragali by dipping whole roots and pruned roots in a conidial suspension and pouring the suspension onto the soil surface in which two seedlings had been planted. After 20 wk 66.5%, 62.1% and 85.0% plants were diseased and 24.1%, 20.7% and 17.5% plants were dead, respectively. Symptoms included the development of more side shoots with small, curved, necrotic and yellowed young leaves, plant stunting, reddish brown lesions, stem browning, dieback, shoot blight, crown rot, root black rot and plant death. This is first report of a pathogenic Embellisia on legumes.     

Optimal amounts of fluorescent dye improve expression microarray results in tumor specimens

Expression microarrays have great potential for clinical use but variability of the results represents a challenge for reliable practical application. The amount of fluorescent dye used in microarray experiments is a significant source of variability that has not been systematically studied. Here we demonstrate that the quantity of Cy3 dye affects microarray results performed on tumor specimens. Signal-to-noise ratios and coefficients of variation are significantly improved by increasing Cy3 to 150–180 pmol, but any further increase does not improve the data. In conclusion, optimal amounts of dye reduce variability and improve reliability of expression microarray experiments.