Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1–D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p?>?0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p?>?0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.     

从猕猴肝脏组织扩增黄嘌呤脱氢酶／氧化酶基因片段

RT-PCR扩增猕猴黄嘌呤脱氢酶／氧化酶（XDH／XO）基因片段,为进一步开展相关研究提供实验资料。方法提取猕猴新鲜肝脏组织总RNA,用RT-PCR二步法进行XDH／XO基因片段扩增,对获得的目的片段进行序列测定,与GenBank上发表的人类（Homosapiens）、小鼠（Musmusculus）、家鼠（Rattusnorvegicus）、野猪（Susscrofa）等物种XDH／XO基因进行该序列同源性比对分析,DNAMAN软件预测该段核苷酸的氨基酸序列,Inter-ProScan及SWISS-MODEL工具进行XDH／XO的编码蛋白结构域及功能预测及三维结构构建。结果RT-PCR产物电泳检测得到了与设计大小相一致的目的条带,序列测定共测到683个核苷酸,DNAMAN软件预测该段核苷酸的氨基酸序列包括了1个编码53个氨基酸的开放阅读框（ORF）,通过该软件包中Multiplealignment对目的基因片段的核苷酸序列与NCBI报道的人类、小鼠、家鼠、野猪XDH／XO基因mRNA互补的cDNA核苷酸序列同源性进行同源性比较分析,结果显示所扩增得到的目的片段与人类同源性最高,为95．6％,与小鼠、家鼠、野猪的同源性分别为85．2％、84．3％、86．1％,说明获得的基因片段是猕猴的XDH／XO基因片段,且该基因在物种间具有较高的相似性。生物信息学预测该段XDH／XO编码蛋白含有醛氧化／脱氢酶的钼喋呤结合点结构域及黄嘌呤脱氢酶结构域。结论在体外成功扩增出猕猴XDH／XO基因片段,为进一步开展高尿酸血症致病机理研究,抗高尿酸血症新药研发奠定工作基础。     

Chromosomal Instability Triggered by Rrm2b Loss Leads to IL-6 Secretion and Plasmacytic Neoplasms

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Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein–protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems‐level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14‐protein core network critical to the viability of multiple EGFR‐mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR‐mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.     

低氧诱导大鼠内脏脂肪组织炎症状态对SePP1的影响

目的：既往研究显示SePP1具有一定的抗氧化作用,而随着年龄的增加机体逐步出现一个慢性低氧、炎症状态,我们通过4％O2浓度体外培养大鼠脂肪前体细胞模拟其体内的低氧状态,进而观察常氧（21％O2）及低氧（4％02）状态下大鼠脂肪前体细胞中炎症因子（IL-6,MCP-1,SePP1）水平的变化及不同状态下硒蛋白SePP1水平的变化。方法：取6—8周SD大鼠肾周脂肪前体细胞,分别于常氧（21％O2）及低氧（4％O2）状态下进行体外培养,诱导分化后采用油红0染色进行鉴定,至第三代后,分别采用PCR及Westem Blot技术检测两种状态下脂肪前体细胞中1L-6,MCP-1,SePPl基因及蛋白表达的不同变化,同时观察不同氧浓度对脂肪前体细胞增殖的影响。结果：4％氧浓度状态下培养的脂肪前体细胞中IL-6,MCP-1的基因及蛋白表达均明显高于正常氧浓度下的脂肪前体细胞,而SePP1的基因及蛋白表达均下降,且低氧状态下脂肪前体细胞增殖较常氧状态下加快。结论：低氧培养可进一步使机体内脏脂肪组织堆积加重,造成脂肪前体细胞的炎症状态,并且可导致SePP1的表达下降,而SePP1具有一定的抗氧化作用,与机体动脉粥样硬化等心血管疾病的发生、发展有一定的关联,本实验结论为通过干预体内SePP1的水平为靶点治疗动脉粥样硬化提供了一定的实验依据,为进一步研究SePP1在低氧状态下对动脉粥样硬化的影响及作用机制提供了一定的试验基础。     

Berberine Targets AP-2/hTERT,NF-κB/COX-2, HIF-1α/VEGF and Cytochrome-c/Caspase Signaling to Suppress Human Cancer Cell Growth

Berberine (BBR), an isoquinoline derivative alkaloid isolated from Chinese herbs, has a long history of uses for the treatment of multiple diseases, including cancers. However, the precise mechanisms of actions of BBR in human lung cancer cells remain unclear. In this study, we investigated the molecular mechanisms by which BBR inhibits cell growth in human non-small-cell lung cancer (NSCLC) cells. Treatment with BBR promoted cell morphology change, inhibited cell migration, proliferation and colony formation, and induced cell apoptosis. Further molecular mechanism study showed that BBR simultaneously targeted multiple cell signaling pathways to inhibit NSCLC cell growth. Treatment with BBR inhibited AP-2α and AP-2β expression and abrogated their binding on hTERT promoters, thereby inhibiting hTERT expression. Knockdown of AP-2α and AP-2β by siRNA considerably augmented the BBR-mediated inhibition of cell growth. BBR also suppressed the nuclear translocation of p50/p65 NF-κB proteins and their binding to COX-2 promoter, causing inhibition of COX-2. BBR also downregulated HIF-1α and VEGF expression and inhibited Akt and ERK phosphorylation. Knockdown of HIF-1α by siRNA considerably augmented the BBR-mediated inhibition of cell growth. Moreover, BBR treatment triggered cytochrome-c release from mitochondrial inter-membrane space into cytosol, promoted cleavage of caspase and PARP, and affected expression of BAX and Bcl-2, thereby activating apoptotic pathway. Taken together, these results demonstrated that BBR inhibited NSCLC cell growth by simultaneously targeting AP-2/hTERT, NF-κB/COX-2, HIF-1α/VEGF, PI3K/AKT, Raf/MEK/ERK and cytochrome-c/caspase signaling pathways. Our findings provide new insights into understanding the anticancer mechanisms of BBR in human lung cancer therapy.     

Historical Biogeography and Diversification of Truffles in the Tuberaceae and Their Newly Identified Southern Hemisphere Sister Lineage

Truffles have evolved from epigeous (aboveground) ancestors in nearly every major lineage of fleshy fungi. Because accelerated rates of morphological evolution accompany the transition to the truffle form, closely related epigeous ancestors remain unknown for most truffle lineages. This is the case for the quintessential truffle genus Tuber, which includes species with socio-economic importance and esteemed culinary attributes. Ecologically, Tuber spp. form obligate mycorrhizal symbioses with diverse species of plant hosts including pines, oaks, poplars, orchids, and commercially important trees such as hazelnut and pecan. Unfortunately, limited geographic sampling and inconclusive phylogenetic relationships have obscured our understanding of their origin, biogeography, and diversification. To address this problem, we present a global sampling of Tuberaceae based on DNA sequence data from four loci for phylogenetic inference and molecular dating. Our well-resolved Tuberaceae phylogeny shows high levels of regional and continental endemism. We also identify a previously unknown epigeous member of the Tuberaceae – the South American cup-fungus Nothojafnea thaxteri (E.K. Cash) Gamundí. Phylogenetic resolution was further improved through the inclusion of a previously unrecognized Southern hemisphere sister group of the Tuberaceae. This morphologically diverse assemblage of species includes truffle (e.g. Gymnohydnotrya spp.) and non-truffle forms that are endemic to Australia and South America. Southern hemisphere taxa appear to have diverged more recently than the Northern hemisphere lineages. Our analysis of the Tuberaceae suggests that Tuber evolved from an epigeous ancestor. Molecular dating estimates Tuberaceae divergence in the late Jurassic (∼156 million years ago), with subsequent radiations in the Cretaceous and Paleogene. Intra-continental diversification, limited long-distance dispersal, and ecological adaptations help to explain patterns of truffle evolution and biodiversity.