Discovery of 17 conserved structural RNAs in fungi

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.     

miR-106a-5p调控 PTEN对鼻咽癌细胞SUNE2增殖和迁移的抑制作用研究

目的研究miR-106a-5p对鼻咽癌细胞SUNE2增殖和迁移的影响。 方法将体外培养的鼻咽癌细胞SUNE2分成对照组（细胞未做任何处理）、Anti-NC组（转染阴性对照抑制剂）、Anti-miR-106a-5p组（转染miR-106a-5p抑制剂）、后期实验另设Anti-miR-106a-5p-inhibitor+si-NC组（转染miR-106a-5p抑制剂、siRNA阴性对照）、Anti-miR-106a-5p-inhibitor+si-PTEN组（转染miR-106a-5p抑制剂、PTEN siRNA）,采用Realtime PCR测定miR-106a-5p表达,MTT法检测增殖,Transwell小室检测细胞侵袭和迁移能力变化,用Western blot方法测定vimentin、E-cadherin蛋白表达。在线靶基因预测软件预测miR-106a-5p的靶基因可能为PTEN,双荧光素酶报告系统鉴定miR-106a-5p和PTEN的靶向关系。两组间比较用独立样本t检验,多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与正常鼻咽上皮细胞NP69比较,鼻咽癌细胞CNE-2、HK1、SUNE2中miR-106a-5p水平（1.00±0.11比1.84±0.13、2.19±0.14、2.87±0.25）升高,差异具有统计学意义（P < 0.05）。与对照组、Anti-NC组比较,Anti-miR-106a-5p组鼻咽癌细胞miR-106a-5p水平（1.00±0.10、0.99±0.12比0.76±0.08）降低,OD值（0.56±0.05、0.57±0.04比0.32±0.02）,细胞侵袭数[（128.47±11.65）个、（129.84±10.93）个比（85.12±6.75）个],迁移数[（182.51±14.81）个、（180.68±17.64）个比（122.01±11.62）个],vimentin蛋白表达水平（0.84±0.09、0.82±0.07比0.30±0.05）降低,E-cadherin蛋白表达水平（0.29±0.04、0.28±0.05比0.76±0.08）升高,差异具有统计学意义（P均< 0.05）。与Anti-miR-106a-inhibitor+si-NC组比较,Anti-miR-106a-inhibitor+si-PTEN组细胞OD值（0.33±0.03比0.52±0.05）、侵袭数[（84.16±5.91）个比（105.79±8.63）个]、迁移数[（118.42±10.25）个比（164.28±12.05）个]、vimentin蛋白表达水平（0.34±0.06比0.68±0.05）均升高,E-cadherin蛋白表达水平（0.72±0.06比0.29±0.05）降低,差异具有统计学意义（P均< 0.05）。 结论miR-106a-5p可通过靶向调控PTEN抑制鼻咽癌细胞SUNE2增殖和迁移潜能。     

Ohmefentanyl stereoisomers induce changes of CREB phosphorylation in hippocampus of mice in conditioned place preference paradigm

The present study was designed to determine the changes of phosphorylation of cAMP-response element binding protein(CREB)in hippocampus induced by ohmefentanyl stereoisomers(F9202 and F9204) in conditioned place preference(CPP)paradigm.The results showed that mice receiving F9202 and F9204 displayed obvious CPP.They could all significantly stimulate CREB phosphorylation and maintained for a long time without affecting total CREB protein levels.The effect of F9204 was similar to morphine which effect was more potent and longer than F9202.We also examined the effects of ketamine,a noncompetitive N-mthyl-D-asartate receptor(NR)antagonist,on morphine-,F9202-and F9204-induced CPP and phosphorylation of CREB in hippocampus.Ketamine could suppress not only the place preference but also the phosphorylation of CREB produced by morphine,F9202 and F9204.These findings suggest that alterations in the phosphorylation of CREB be relevant to opiates signaling and the development of opiates dependence.NR antagonists may interfere with opiates dependence and may have potential therapeutic implications.     

The protective effect of cycloastragenol on aging mouse circadian rhythmic disorder induced by d-galactose

Aging process in mammals is associated with a decline in amplitude and a long period of circadian behaviors which are regulated by a central circadian regulator in the suprachiasmatic nucleus (SCN) and local oscillators in peripheral tissues. It is unclear whether enhancing clock function can retard aging. Using fibroblasts expressing per2::lucSV and senescent cells, we revealed cycloastragenol (CAG), a natural aglycone derivative from astragaloside IV, as a clock amplitude enhancing small molecule. CAG could activate telomerase to antiaging, but no reports focused on its effects on circadian rhythm disorders in aging mice. Here we analyze the potential effects of CAG on d -galactose-induced aging mice on the circadian behavior and expression of clock genes. For this purpose, CAG (20 mg/kg orally), was administered daily to d -galactose (150 mg/kg, subcutaneous) mice model of aging for 6 weeks. An actogram analysis of free-running activity of these mice showed that CAG significantly enhances the locomotor activity. We further found that CAG increase expressions of per2 and bmal1 genes in liver and kidney of aging mouse. Furthermore, CAG enhanced clock protein BMAL1 and PER2 levels in aging mouse liver and SCN. Our results indicated that the CAG could restore the behavior of circadian rhythm in aging mice induced by d -galactose. These data of present study suggested that CAG could be used as a novel therapeutic strategy for the treatment of age-related circadian rhythm disruption.     

The Role of HSPA12B in Regulating Neuronal Apoptosis

Heat shock protein A12B (HSPA12B) is the newest member of a recently defined subfamily of proteins distantly related to the 70-kDa family of heat shock proteins (HSP70) family. HSP70s play a crucial role in protecting cells, tissues, organs and animals from various noxious conditions. Here we studied the dynamic expression changes and localization of HSPA12B after middle cerebral artery occlusion (MCAO) with reperfusion induced ischemic insult processes in adult rats. Apoptosis, as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, was also increased in the peri-ischemic cortex compared to non-ischemic hemisphere. The expression of HSPA12B was strongly induced in the ischemic hemisphere of MCAO reperfusion rats in vivo. In vitro studies indicated that the up-regulation of HSPA12B may be involved in oxygen-glucose deprivation-induced PC12 cell death. And knockdown of HSPA12B in cultured differentiated PC12 cells by siRNA showed that HSPA12B inhibited the expression of active caspase-3. Collectively, these results suggested that HSPA12B may be required for protecting neurons from ischemic insults.     

Insights into Avian Influenza Virus Pathogenicity: the Hemagglutinin Precursor HA0 of Subtype H16 Has an Alpha-Helix Structure in Its Cleavage Site with Inefficient HA1/HA2 Cleavage

With a new serotype (H17) of hemagglutinin (HA) recently being discovered, there are now 17 serotypes (H1 to H17) of influenza A viruses in total. It is believed that HA is initially expressed as a precursor of HA0 and then cleaved into HA1 and HA2, forming a disulfide bond-linked complex, for its full function. Structural data show that a loop structure exists in the cleavage site between HA1 and HA2, and this flexible loop is crucial for the efficient cleavage of HA0. Here, the crystal structures of H16 (a low-pathogenicity avian influenza virus) in their HA0 form (H16HA0) have been solved at 1.7-Å and 2.0-Å resolutions. To our surprise, an α-helix element in the cleavage site which inserts into the negatively charged cavity with the key residue R329 hidden behind the helix was observed. In vitro trypsin cleavage experiments demonstrated inefficient cleavage of H16HA0 under both neutral and low-pH conditions. The results provide new insights into influenza A virus pathogenicity; both the relatively stable α-helix structure in the flexible cleavage loop and inaccessibility of the cleavage site likely contribute to the low pathogenicity of avian influenza A virus. Furthermore, compared to all of the HAs whose structures have been solved, H16 is a good reference for assigning the HA subtypes into two groups on the basis of the three-dimensional structure, which is consistent with the phylogenetic grouping. We conclude that in light of the current H16HA0 structure, the natural α-helix element might provide a new opportunity for influenza virus inhibitor design.     

Three RNA Binding Proteins Form a Complex to Promote Differentiation of Germline Stem Cell Lineage in Drosophila

In regenerative tissues, one of the strategies to protect stem cells from genetic aberrations, potentially caused by frequent cell division, is to transiently expand the stem cell daughters before further differentiation. However, failure to exit the transit amplification may lead to overgrowth, and the molecular mechanism governing this regulation remains vague. In a Drosophila mutagenesis screen for factors involved in the regulation of germline stem cell (GSC) lineage, we isolated a mutation in the gene CG32364, which encodes a putative RNA-binding protein (RBP) and is designated as tumorous testis (tut). In tut mutant, spermatogonia fail to differentiate and over-amplify, a phenotype similar to that in mei-P26 mutant. Mei-P26 is a TRIM-NHL tumor suppressor homolog required for the differentiation of GSC lineage. We found that Tut binds preferentially a long isoform of mei-P26 3′UTR, and is essential for the translational repression of mei-P26 reporter. Bam and Bgcn are both RBPs that have also been shown to repress mei-P26 expression. Our genetic analyses indicate that tut, bam, or bgcn is required to repress mei-P26 and to promote the differentiation of GSCs. Biochemically, we demonstrate that Tut, Bam, and Bgcn can form a physical complex in which Bam holds Tut on its N-terminus and Bgcn on its C-terminus. Our in vivo and in vitro evidence illustrate that Tut acts with Bam, Bgcn to accurately coordinate proliferation and differentiation in Drosophila germline stem cell lineage.     

Temporal and spatial distribution of ammonia-oxidizing organisms of two types of wetlands in Northeast China

Applied Microbiology and Biotechnology - Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) contribute significantly to the nitrogen cycle. The community structure of AOA and AOB...