Diversity of phototrophic phytoplankton in Northern South China Sea indicated by <Emphasis Type="Italic">rbcL</Emphasis> analysis

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) as an important enzyme in photosynthetic process exists in various marine phytoplankton. To investigate the photosynthetic phytoplankton communities, conservative encoding gene of RubisCO large subunit (rbcL) was chosen as a target gene in this study. We constructed 33 clone libraries from samples collected in the north of South China Sea (NSCS) and retained 3173 sequences for further analysis. Results of BLASTp showed Stramenopiles and Haptophyta were predominant taxonomic groups in this area, while only five harmful species of Dinophyta were observed. According to the estimators of biodiversity, the photosynthetic community of N709 had very low genetic diversity and richness, which could be explained by the influence of brackish estuarine environment. Beta diversity showed that all samples could be clustered into three groups and those samples with approximately the same distance to land clustered together. Temperature, depth, and latitude of stations as biogeographic factors were indicated to have a significantly positive or negative relation with biodiversity estimators of the phytoplankton community. We concluded that biogeographic factors could be linked with difference in diversity and population of natural phytoplankton assemblages in horizontal surface of NSCS in summer 2007.     

两种近缘鸟种麻雀和山麻雀的咬合力比较

鸟类的咬合力受食性、种内竞争和捕食压力等多种生态因素的影响,可作为其生态适应特征的重要指标。但目前关于鸟类的咬合力及其影响因素却鲜有研究,为此,我们使用咬合力传感器,对同属的两个近缘鸟种,麻雀(Passer montanus)和山麻雀(P.cinnamomeus)的咬合力进行了比较研究。结果表明,山麻雀(n=12)的咬合力显著大于麻雀(n=59)(t=3.754,P0.01),但山麻雀(t=0.449,P0.05)和麻雀(Z=﹣1.198,P0.05)的雌雄个体间咬合力均无差异,同时,山麻雀的头宽(t=﹣3.713,P0.01)、头高(t=﹣5.405,P0.01)和喙宽(t=﹣6.201,P0.01)均显著大于麻雀。尽管个体的咬合力与其身体各参数指标无显著相关性,但在种间,头和喙的大小可能是影响两者咬合力的重要因素,由于两者的一些生态适应特征可通过头大小和喙型体现,推测两者生境和食性的差异可能是影响其咬合力大小的主要原因。     

Identifying the antiasthmatic target of doxofylline using immobilized β2‐adrenoceptor based high‐performance affinity chromatography and site‐directed molecular docking

As a xanthine derivative, doxofylline is believed to be dominant for fighting against asthma in practice. Unlike other xanthines, the antiasthmatic effects of doxofylline lack any definite proof of target and mediating mechanism according to previous reports. In this work, the interaction between doxofylline and β₂‐AR was investigated by high performance affinity chromatography using frontal analysis and nonlinear model. The methodology involved the immobilization of β₂‐AR on the silica gel by a random linking method, the determination of the binding parameters by frontal analysis and nonlinear chromatography and the exploration of the binding mechanism by site‐directed molecular docking. The association constant for doxofylline binding to immobilized β₂‐AR was determined to be 7.70 × 10⁴ M^?1 by nonlinear chromatography and 5.91 × 10⁴ M^?1 by frontal analysis. Ser¹⁶⁹ and Ser¹⁷³ were the binding sites for the receptor–drug interaction on which hydrogen bond was believed to be the main driven force during the interaction. These results indicated that the antiasthmatic effects of doxofylline may be behind the mediating mechanism of β₂‐AR. High performance affinity chromatography based on immobilized receptor has potential to become an alternative for drug target confirmation and drug–receptor interaction analysis. Copyright © 2016 John Wiley & Sons, Ltd.     

Harnessing Type I and Type III CRISPR-Cas systems for genome editing

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are widespread in archaea and bacteria, and research on their molecular mechanisms has led to the development of genome-editing techniques based on a few Type II systems. However, there has not been any report on harnessing a Type I or Type III system for genome editing. Here, a method was developed to repurpose both CRISPR-Cas systems for genetic manipulation in Sulfolobus islandicus, a thermophilic archaeon. A novel type of genome-editing plasmid (pGE) was constructed, carrying an artificial mini-CRISPR array and a donor DNA containing a non-target sequence. Transformation of a pGE plasmid would yield two alternative fates to transformed cells: wild-type cells are to be targeted for chromosomal DNA degradation, leading to cell death, whereas those carrying the mutant gene would survive the cell killing and selectively retained as transformants. Using this strategy, different types of mutation were generated, including deletion, insertion and point mutations. We envision this method is readily applicable to different bacteria and archaea that carry an active CRISPR-Cas system of DNA interference provided the protospacer adjacent motif (PAM) of an uncharacterized PAM-dependent CRISPR-Cas system can be predicted by bioinformatic analysis.     

Fully Automated RNAscope In Situ Hybridization Assays for Formalin‐Fixed Paraffin‐Embedded Cells and Tissues

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal‐to‐noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA‐box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201–2208, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.