Deep Sequencing of Small RNA Repertoires in Mice Reveals Metabolic Disorders-Associated Hepatic miRNAs

Obesity and associated metabolic disorders contribute importantly to the metabolic syndrome. On the other hand, microRNAs (miRNAs) are a class of small non-coding RNAs that repress target gene expression by inducing mRNA degradation and/or translation repression. Dysregulation of specific miRNAs in obesity may influence energy metabolism and cause insulin resistance, which leads to dyslipidemia, steatosis hepatis and type 2 diabetes. In the present study, we comprehensively analyzed and validated dysregulated miRNAs in ob/ob mouse liver, as well as miRNA groups based on miRNA gene cluster and gene family by using deep sequencing miRNA datasets. We found that over 13.8% of the total analyzed miRNAs were dysregulated, of which 37 miRNA species showed significantly differential expression. Further RT-qPCR analysis in some selected miRNAs validated the similar expression patterns observed in deep sequencing. Interestingly, we found that miRNA gene cluster and family always showed consistent dysregulation patterns in ob/ob mouse liver, although they had various enrichment levels. Functional enrichment analysis revealed the versatile physiological roles (over six signal pathways and five human diseases) of these miRNAs. Biological studies indicated that overexpression of miR-126 or inhibition of miR-24 in AML-12 cells attenuated free fatty acids-induced fat accumulation. Taken together, our data strongly suggest that obesity and metabolic disturbance are tightly associated with functional miRNAs. We also identified hepatic miRNA candidates serving as potential biomarkers for the diagnose of the metabolic syndrome.     

Identification of Volatile Metabolites from Five Fungal Species Cultivated on Two Media

Five fungal species, Aspergillus versicolor, Penicillium commune, Cladosporium cladosporioides, Paecilomyces variotii, and Phialophora fastigiata, were cultivated on two media, malt extract agar and dichloran glycerol agar. Culture flasks provided with inlet and outlet tubes were used and purified, and humidified air was constantly led through the flasks. Air samples from the cultures were sorbed on Tenax GR and analyzed by thermal desorption-gas chromatography. The produced volatile metabolites were analyzed by mass spectrometry. Various hydrocarbons, alcohols, ketones, ethers, esters, sulfur-containing compounds, and terpenes were identified. The most commonly produced substances were 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 3-methylfuran, and dimethyl disulfide. The production was highly dependent on both medium and species.     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.     

Deep Sequencing of Subseafloor Eukaryotic rRNA Reveals Active Fungi across Marine Subsurface Provinces

The deep marine subsurface is a vast habitat for microbial life where cells may live on geologic timescales. Because DNA in sediments may be preserved on long timescales, ribosomal RNA (rRNA) is suggested to be a proxy for the active fraction of a microbial community in the subsurface. During an investigation of eukaryotic 18S rRNA by amplicon pyrosequencing, unique profiles of Fungi were found across a range of marine subsurface provinces including ridge flanks, continental margins, and abyssal plains. Subseafloor fungal populations exhibit statistically significant correlations with total organic carbon (TOC), nitrate, sulfide, and dissolved inorganic carbon (DIC). These correlations are supported by terminal restriction length polymorphism (TRFLP) analyses of fungal rRNA. Geochemical correlations with fungal pyrosequencing and TRFLP data from this geographically broad sample set suggests environmental selection of active Fungi in the marine subsurface. Within the same dataset, ancient rRNA signatures were recovered from plants and diatoms in marine sediments ranging from 0.03 to 2.7 million years old, suggesting that rRNA from some eukaryotic taxa may be much more stable than previously considered in the marine subsurface.     

Complete Plastid Genome Sequencing of Trochodendraceae Reveals a Significant Expansion of the Inverted Repeat and Suggests a Paleogene Divergence between the Two Extant Species

The early-diverging eudicot order Trochodendrales contains only two monospecific genera, Tetracentron and Trochodendron. Although an extensive fossil record indicates that the clade is perhaps 100 million years old and was widespread throughout the Northern Hemisphere during the Paleogene and Neogene, the two extant genera are both narrowly distributed in eastern Asia. Recent phylogenetic analyses strongly support a clade of Trochodendrales, Buxales, and Gunneridae (core eudicots), but complete plastome analyses do not resolve the relationships among these groups with strong support. However, plastid phylogenomic analyses have not included data for Tetracentron. To better resolve basal eudicot relationships and to clarify when the two extant genera of Trochodendrales diverged, we sequenced the complete plastid genome of Tetracentron sinense using Illumina technology. The Tetracentron and Trochodendron plastomes possess the typical gene content and arrangement that characterize most angiosperm plastid genomes, but both genomes have the same unusual ∼4 kb expansion of the inverted repeat region to include five genes (rpl22, rps3, rpl16, rpl14, and rps8) that are normally found in the large single-copy region. Maximum likelihood analyses of an 83-gene, 88 taxon angiosperm data set yield an identical tree topology as previous plastid-based trees, and moderately support the sister relationship between Buxaceae and Gunneridae. Molecular dating analyses suggest that Tetracentron and Trochodendron diverged between 44-30 million years ago, which is congruent with the fossil record of Trochodendrales and with previous estimates of the divergence time of these two taxa. We also characterize 154 simple sequence repeat loci from the Tetracentron sinense and Trochodendron aralioides plastomes that will be useful in future studies of population genetic structure for these relict species, both of which are of conservation concern.     

棉蚜2个乙酰胆碱酯酶cDNA片段的基因克隆与序列分析

采用RT－PCR技术,利用简并引物从棉蚜（Aphis gossypii Glover)中克隆出2个乙酰胆碱酯酶基因的cDNA片段,Ag,ace 1l和Ag.ace2.Ag.ace1基因的cDNA片段为282bp,编码94个氨基酸;Ag.ace2基因的cDNA片段为264bp,编码88个氨基酸。扩增获得的2个乙酰胆碱酯酶基因cDNA片段所编码的氨基酸序列均与其他昆虫的乙酰胆碱酯酶基因有很高的同源性。首次从一种昆虫中克隆出2个乙酰胆碱酯酶基因片段,为同一种昆虫中存在多个乙酰胆碱酯酶基因的假设提供了直接的分子生物学证据。     

Transcriptome Sequencing of Two Phenotypic Mosaic Eucalyptus Trees Reveals Large Scale Transcriptome Re-Modelling

Phenotypic mosaic trees offer an ideal system for studying differential gene expression. We have investigated two mosaic eucalypt trees from two closely related species (Eucalyptus melliodora and E. sideroxylon), which each support two types of leaves: one part of the canopy is resistant to insect herbivory and the remaining leaves are susceptible. Driving this ecological distinction are differences in plant secondary metabolites. We used these phenotypic mosaics to investigate genome wide patterns of foliar gene expression with the aim of identifying patterns of differential gene expression and the somatic mutation(s) that lead to this phenotypic mosaicism. We sequenced the mRNA pool from leaves of the resistant and susceptible ecotypes from both mosaic eucalypts using the Illumina HiSeq 2000 platform. We found large differences in pathway regulation and gene expression between the ecotypes of each mosaic. The expression of the genes in the MVA and MEP pathways is reflected by variation in leaf chemistry, however this is not the case for the terpene synthases. Apart from the terpene biosynthetic pathway, there are several other metabolic pathways that are differentially regulated between the two ecotypes, suggesting there is much more phenotypic diversity than has been described. Despite the close relationship between the two species, they show large differences in the global patterns of gene and pathway regulation.     

Deep Sequencing of the Scutellaria baicalensis Georgi Transcriptome Reveals Flavonoid Biosynthetic Profiling and Organ-Specific Gene Expression

Scutellaria baicalensis Georgi has long been used in traditional medicine to treat various such widely varying diseases and has been listed in the Chinese Pharmacopeia, the Japanese Pharmacopeia, the Korean Pharmacopoeia and the European Pharmacopoeia. Flavonoids, especially wogonin, wogonoside, baicalin, and baicalein, are its main functional ingredients with various pharmacological activities. Although pharmaological studies for these flavonoid components have been well conducted, the molecular mechanism of their biosynthesis remains unclear in S. baicalensis. In this study, Illumina/Solexa deep sequencing generated more than 91 million paired-end reads and 49,507 unigenes from S. baicalensis roots, stems, leaves and flowers. More than 70% unigenes were annotated in at least one of the five public databases and 13,627 unigenes were assigned to 3,810 KEGG genes involved in 579 different pathways. 54 unigenes that encode 12 key enzymes involved in the pathway of flavonoid biosynthesis were discovered. One baicalinase and three baicalein 7-O-glucuronosyltransferases genes potentially involved in the transformation between baicalin/wogonoside and baicalein/wogonin were identified. Four candidate 6-hydroxylase genes for the formation of baicalin/baicalein and one candidate 8-O-methyltransferase gene for the biosynthesis of wogonoside/wogonin were also recognized. Our results further support the conclusion that, in S. baicalensis, 3,5,7-trihydroxyflavone was the precursor of the four above compounds. Then, the differential expression models and simple sequence repeats associated with these genes were carefully analyzed. All of these results not only enrich the gene resource but also benefit research into the molecular genetics and functional genomics in S. baicalensis.     

Deep Sequencing Reveals Low Incidence of Endogenous LINE-1 Retrotransposition in Human Induced Pluripotent Stem Cells

Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3′ end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.     

Some Differences in the Responses of Stomata of the Two Leaf Surfaces in Cotton

Some differences in the responses of the upper and lower stomatain cotton (Gossypium hirsutuni) are presented. These differenceswere observed in the course of some studies in which the transpirationof the two leaf surfaces was measured under controlled environmentconditions and the transpiration data used as an estimate ofstomatal response. In darkness the upper stomata were more or less effectivelyclosed while the lower stomata were partially open. Upon illuminationof the leaf with non-saturation or saturation radiation theupper stomata were slower to open than the lower stomata. Thereductions in stomatal aperture which occurs with the increasein age of leaves commenced earlier in the upper stomata andproceeded at a faster rate than the lower stomata. Sudden exposureto saturation radiation caused the stomata of the two leaf surfacesto oscillate. These oscillations were not observed in youngleaves but in older leaves. During ageing of leaves oscillationsof the upper stomata commenced earlier than oscillations ofthe lower stomata. When the petiole was excised in darknessor light the upper stomata showed a transient increase but notthe lower stomata. Gossypium hirsutum, stomatal responses, transpiration