Cloning, characterization and widespread expression analysis of testicular piRNA-like chicken RNAs

Piwi-interacting RNAs (piRNAs) are small RNAs abundant in the germline that have been implicated in germline development and maintenance of genomic integrity across several animal species including human, mouse, rat, zebrafish and drosophila. Tens of thousands of piRNAs have been discovered, yet abundant piRNAs have still not been detected in various eukaryotic organisms. This is a report on the characterization, cloning and expression profiling of piRNA-like chicken RNAs. Here, we identified 19 piRNAs, each 23–39 nucleotides long, from chicken testis using a small RNA cDNA library and T-A cloning methods. Three different pilRNAs were selected according to size, homology and secondary structure for temporal and spatial expression by Q-PCR technology in different tissues at five growth and four development stages of Chinese indigenous Rugao chickens (RG) and introduced recessive white feather chickens (RW). We found that, consistent to other organisms, pilRNA-encoding sequences within the chicken genome were asymmetrically distributed on the chromosomes while displaying a preference for intergenic regions across the genome. Interestingly, unlike miRNAs with unique stem-loop structures (mature miRNAs form stem section and the rest form loop section), distinct secondary structures of pilRNAs were predicted. In addition, chicken pilRNAs were not only abundant in the germline but also existed in somatic tissues, where, expression levels were influenced mainly by different pilRNAs, breed and gender. Taken together, our results suggest that two distinct secondary structures exist between pilRNAs and miRNAs, which may clarify the splicing and processing mechanisms of the two small RNAs are possible different. Moreover, our results suggest that pilRNAs may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. Additionally, different pilRNAs may be involved in the unique regulatory machinery of complex biological processes.     

小鼠Toll样受体3基因的克隆及真核表达

采用RT-PCR方法从小鼠巨噬细胞中克隆小鼠Toll样受体3(TLR3)基因,基因测序表明获得了小鼠全长TLR3cDNA,构建了真核表达质粒p3XFLAG-CMV-7.1-TLR3.重组质粒转染293T细胞,Western blotting检测蛋白表达,表达蛋白质的相对分子量与预计相符.采用TLR3的阳性刺激物poly(I∶C)刺激重组质粒转染的293T细胞,双荧光素酶报告基因系统检测发现能激活下游转录因子NF-κB的转录活性,并能诱导TLR3下游细胞因子IL-6和TNF-α的表达.小鼠TLR3基因的克隆和表达,为研究TLR3介导的信号通路及其在抗病毒免疫中的作用打下基础.     

基于物种运动路径识别的动物通道选址——以武深高速为例

动物通道是缓解高速公路对其周边野生动物生境隔离的有效措施,通道的位置是影响其使用效率的关键因素,然而现有研究对通道的选址却甚少涉及。以武深高速为例,推荐一种基于物种运动路径识别的通道选址方法,选取影响动物生境选择的环境因子构建评价体系,借助GIS手段对公路周边野生动物生境适宜性进行分析,在此基础上借鉴水文分析原理快速准确地刻画出物种在生境中的潜在活动路径,从而确定了5处高速公路上建设动物通道的理想位置。结果表明,该方法能定量地反映出生境的质量格局对于物种运动的影响,准确定位出物种运动受到阻碍的关键区域,在景观层次上,提出的通道位置能有效地缓解栖息地破碎化造成的生态压力;研究不但能弥补目前研究的不足,同时亦为道路网设计、城市生态规划等相关领域研究提供科学参考。     

Plant-derived phosphocholine facilitates cellular uptake of anti-pulmonary fibrotic HJT-sRNA-m7

Pulmonary fibrosis, a progressive chronic disease with a high mortality rate, has limited treatment options. Currently, lung transplantation remains the only effective treatment. Here we report that a small RNA, HJT-sRNA-m7, from a Chinese herbal medicine Hong Jing Tian(HJT, RHODIOHAE CRENULATAE RADIX ET RHIZOMA, Rhodiola crenulata) can effectively reduce the expressions of fibrotic hallmark genes and proteins both in alveolar in vitro and in mouse lung tissues in vivo. We also discovered over one hundred oil-soluble chemicals from HJT decoctions, most of which are found in lipid extracts from other Chinese herbals decoctions, including Pu Gong Ying(PGY, TARAXACI HERBA, Taraxacum mongolicum), Chuan Xin Lian(CXL, changed to "ANDROGRAPHIS HERBA, Andrographis paniculata"), and Jin Yin Hua(JYH, lonicera japonica or Honeysuckle). We identified the active component in these decoctions as two forms of phosphocholines, PC(18:0/18:2) and PC(16:0/18:2). These PCs potentially could form liposomes with small RNAs to enter human alveolar and gastric cells. Our experimental results suggest an unprecendent lipid complex route through which botanic small RNA can enter human bodies.Our results provide an innovative treatment strategy for oral delivery of siRNAs as therapeutic medication.     

Chrysophanol exhibits inhibitory activities against colorectal cancer by targeting decorin

Colorectal cancer (CRC) is a common human malignancy that accounts for 600,000 deaths annually worldwide. Chrysophanol, a naturally occurring anthraquinone compound, exhibits anti-neoplastic effects in various cancer cells. The aim of this study was to explore the biological effects of chrysophanol on CRC cells, and determine the underlying mechanism. Chrysophanol inhibited proliferation of and promoted apoptosis in CRC cells by activating the intrinsic mitochondrial apoptotic pathway. In addition, chrysophanol also suppressed tumor growth in vivo and increased the percentage of apoptotic cells in tumor xenografts, without general toxicity. Proteomic iTRAQ analysis revealed decorin (DCN) as the major target of chrysophanol. DCN was upregulated in the tumor tissues following chrysophanol treatment, and ectopic DCN expression markedly augmented the pro-apoptotic effects of chrysophanol in CRC cells. In contrast, DCN knockdown significantly abrogated chrysophanol-induced apoptosis in CRC cells. Taken together, chrysophanol exerts anti-neoplastic effects in vitro and in vivo in CRC cells by modulating DCN, there by highlighting its therapeutic potential in CRC.     

澳门生态一区和二区水鸟谱系多样性的季节变化

季节变化是鸟类群落的重要特征之一,其引起的环境变化决定着鸟类群落构建过程中不同驱动因子的作用力。因此,了解鸟类群落结构的季节变化,对于全面认识生物群落结构具有重要意义,尤其利用谱系多样性来探讨鸟类的季节性变化和推断群落聚集规律越来越受到关注。2018年1—12月,采用样线法对澳门生态一区和二区2块湿地进行逐月调查,记录水鸟的物种丰富度和多度,并分析水鸟谱系多样性的季节动态。结果显示,1)共记录水鸟37种,隶属于6目9科,目、科、种中数量最多的依次为:鸻形目Charadriiformes物种数为15种,鹭科Ardeidae物种数为11种,白鹭Egretta garzetta多度为540只。2)生态一区和二区水鸟的物种丰富度在冬季最高,其次为秋季;生态一区和二区的物种多度分别在秋季和冬季最高;谱系多样性和平均成对谱系距离的季节变化规律与物种丰富度的相似。3)生态一区和二区的鸟类群落分别在春、秋、冬季和秋季趋向于谱系发散,而在其他时间大多呈现出谱系聚集。谱系发散可能表明了种间竞争在群落构建中作用更大,而谱系聚集则表明了环境过滤对繁殖季水鸟群落构建具有重要作用。结果表明,湿地水鸟群落的驱动因子存在季节变化,考虑这些变化是全面认识群落结构的前提。     

Silver nanocrystals sensitize magnetic-nanoparticle-mediated thermo-induced killing of cancer cells

Magnetic nanoparticles (MNPs) can heat up tumor tissues and induce killing of cancer cells under external AC magnetic field. However, magnetic nanoparticles hyperthermia (MNPH) requires high concentration of MNPs that are injected into the tumor in order to obtain clinically needed thermal dose because of the complicated heat transfer in vivo and the limited heat quality of MNPs. To cut down the dose of MNPs and enhance the effect of this Nanotherapy, we prepared silver nanoparticles (AgNPs) with different sizes and investigated the effects of these AgNPs on cancer cells in MNPH treatment. It was found that AgNPs could enhance thermo-sensitivity of glioma cells and this effect was size dependent. AgNPs could induce cell cycles arrested in G(2)/M phase and enhanced the apoptosis rate of cancer cells after hyperthermia. In glioma bearing rats model, MNPH combined with AgNPs could enhance Bax expression in cancer cells. Our results suggested that AgNPs could be a potential thermo-sensitizer and could be further developed for the design of Ag nanostructure-based thermal seeds for MNPH therapy.     

Rab35 is required for Wnt5a/Dvl2-induced Rac1 activation and cell migration in MCF-7 breast cancer cells

The small GTPases regulate many major biological processes in both tumorigenesis and tumor progression such as cell survival, actin cytoskeleton organization, cell polarity and movement. Wnt5a, a non-canonical Wnt family member, is implicated in the activation of small GTPases in breast cancer. We previously demonstrated that Wnt5a signaling stimulates the migration of breast cancer cells MDA-MB-231 via activating RhoA. However, we found here that RhoA activation was not enhanced by Wnt5a in breast cancer cells MCF-7. The conflicting results prompted us to further probe novel small GTPases in response to Wnt5a and investigate the mechanisms whereby cell migration is regulated. We showed here that Wnt5a dose dependently activated Dvl2, Rab35 and Rac1 and subsequently promoted the migration of MCF-7 cells, which was, however, abolished by knocking down Wnt5a expression via small interfering RNA (siRNA) transfection. Dvl2 siRNA significantly decreased background and Wnt5a-induced Rab35/Rac1 activation and, consequently, cell migration. Rab35 short hairpin RNA (shRNA) remarkably inhibited background and Wnt5a-induced Rac1 activation and cell migration. Additionally, blockade of Rac1 activation with Rac1 siRNA suppressed background and Wnt5a-induced cell migration. Co-immunoprecipitation and immunofluorescence assays showed that Dvl2 bound to Rab35 in mammalian cells. Taken together, we demonstrated that Wnt5a promotes breast cancer cell migration via the Dvl2/Rab35/Rac1 signaling pathway. These findings implicate Wnt5a signaling in regulating small GTPases, which could be targeted for manipulating breast cancer cell migration.