論文

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. Paired-end RNA sequencing (RNA-seq) is usually applied to the quantification of long transcripts such as messenger or long non-coding RNAs, in which case overlapping pairs are discarded. In contrast, RNA-seq on short RNAs (≤ 200 nt) is typically carried out in single-end mode, as the additional cost associated with paired-end would only translate into redundant sequence information. Here, we exploit paired-end sequencing of short RNAs as a strategy to filter out sequencing errors and apply this method to the identification of adenosine-to-inosine (A-to-I) RNA editing events on human precursor microRNA (pre-miRNA) and mature miRNA. Combined with RNA immunoprecipitation sequencing (RIP-seq) of A-to-I RNA editing enzymes, this method takes full advantage of deep sequencing technology to identify RNA editing sites with unprecedented resolution in terms of editing efficiency.

Halomonas hydrothermalis strain Slthf2 is a Gram-negative bacterium isolated from low-temperature hydrothermal fluids in South Pacific Ocean vent fields located at 2,580-m depth. Here, we report the complete genome sequence of this strain, which has a genome size of 4.12 Mb, with a GC content of 53.2%.

Halomonas meridiana strain Eplume2 (ATCC BAA-804) is a Gram-negative bacterium isolated from hydrothermal plume seawater in the Northeast Pacific Ocean at a depth of 2,000 m. Here, we report the complete genome sequence of this strain, which has a total size of 4.12 Mbp and a 56.6% G+C content.

Halomonas meridiana strain Slthf1 (ATCC BAA-801) is a Gram-negative bacterium that was isolated from a thermal vent in 1998. Here, we report the complete genome sequence of this strain, which has a 3.6-Mbp genome, containing 3,400 protein-coding sequences.

Here, we report the complete genome sequence of Halomonas olivaria strain TYRC17, a moderately halophilic, Gram-negative bacterium that was isolated from olive processing effluents. The 5-Mbp genome consists of 7,375 protein-coding sequences, including a variety of genes involved in tyrosol metabolism, nitrate respiration, and the production of polysaccharides.

We report the complete genome sequence of Halomonas sulfidaeris ATCC BAA-803, isolated from a metal sulfide rock at a depth of 2,200 m in the Northeast Pacific Ocean. The assembled genome comprised one circular chromosome of 4.20 Mb and one large plasmid of 273 kb. The chromosome harbors 6,705 protein-coding genes.

Halomonas axialensis is a halophilic bacterial species discovered near a deep-sea hydrothermal vent. Here, we report the first single closed genome sequence of the original strain, Halomonas axialensis strain Althf1. The genome was assembled by Nanopore sequencing and consisted of a single chromosome of 3.6 Mbp with 56.8% G+C content.

Because of its small size, Gram-negative Sphingomonas paucimobilis can pose a risk of nosocomial infection. We report the complete circular genome sequence of S. paucimobilis strain Kira, which was isolated from retinoic acid-supplemented SHSY5Y human cell cultures, to be 3,917,410bp (G1Ccontent,65.7%; 3,672 protein-coding sequences), with two plasmids (79,575bp and 44,333bp).

Correlative microscopy and block-face imaging (CoMBI), a method that we previously developed, is characterized by the ability to correlate between serial block-face images as 3-dimensional (3D) datasets and sections as 2-dimensional (2D) microscopic images. CoMBI has been performed for the morphological analyses of various biological specimens, and its use is expanding. However, the conventional CoMBI system utilizes a cryostat, which limits its compatibility to only frozen blocks and the resolution of the block-face image. We developed a new CoMBI system that can be applied to not only frozen blocks but also paraffin blocks, and it has an improved magnification for block-face imaging. The new system, called CoMBI-S, comprises sliding-type sectioning devices and imaging devices, and it conducts block slicing and block-face imaging automatically. Sections can also be collected and processed for microscopy as required. We also developed sample preparation methods for improving the qualities of the block-face images and 3D rendered volumes. We successfully obtained correlative 3D datasets and 2D microscopic images of zebrafish, mice, and fruit flies, which were paraffin-embedded or frozen. In addition, the 3D datasets at the highest magnification could depict a single neuron and bile canaliculus.

The skin is at the interface of living organisms and their environment, and has evolved interesting structural properties usually over the course of millions of years, providing clues for the design of manmade biomimetic surfaces with favorable fluid mechanics properties. Here, we describe steps to produce silicone rubber films based on microscopy data from shark skin denticles, from data acquisition and manufacturing to attachment to an airfoil for experimental fluid dynamics in large towing tanks. This method is relatively low-cost, may be generalized to other types of patterned micro-structured surfaces, and the manufacturing process may be reproduced by anyone equipped with a 3D printer.

The effect of shark skin on the boundary-layer separation process under dynamic conditions (maneuvers) has been studied experimentally. We use a foil covered with biomimetic shark skin to explore how this type of surface impacts boundary-layer dynamics in both steady and accelerating conditions. The effect of denticles is assessed via particle image velocimetry in the wake. It is shown that dynamic conditions and small-scale disturbances can mitigate boundary-layer separation through instantaneous modification of the local pressure-gradient distribution. For instance, the region of favourable pressure gradient can be extended by accelerating the foil. The acceleration results in a thinner separated shear layer on the foil surface when compared to the steady reference case. This remarkable difference indicates that local roughness (introduced through for instance biomimetic shark skin) may trigger an interaction with relatively large-scale structures in the boundary layer for effective boundary-layer control during unsteady propulsion and maneuvering.

© 2018, © 2018 Taylor & Francis Group, LLC. Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities.

Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding machinery. Genome-wide studies have revealed that the majority of editing events occur in non-coding RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs that regulate the expression of target mRNAs with complementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex globally reassigns their target mRNAs in vivo, and reveal that miRNA containing inosine in the seed region exhibits a different degree of silencing efficiency compared to the corresponding miRNA with guanosine at the same position. The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

RNA editing by deamination of adenosine to inosine is an evolutionarily conserved process involved in many cellular pathways, from alternative splicing to miRNA targeting. In humans, it is carried out by no less than three major adenosine deaminases acting on RNA (ADARs): ADAR1-p150, ADAR1-p110, and ADAR2. However, the first two derive from alternative splicing, so that it is currently impossible to delete ADAR1-p110 without also knocking out ADAR1-p150 expression. Furthermore, the expression levels of ADARs varies wildly among cell types, and no study has systematically explored the effect of each of these isoforms on the cell transcriptome. In this study, RNA immunoprecipitation (RIP)-sequencing on overexpressed ADAR isoforms tagged with green fluorescent protein (GFP) shows that each ADAR is associated with a specific set of differentially expressed genes, and that they each bind to distinct set of RNA targets. Our results show a good overlap with known edited transcripts, establishing RIP-seq as a valid method for the investigation of RNA editing biology.

Pleural fluid accumulation is a frequent clinical observation in diffuse malignant pleural mesothelioma (MPM). The cytological analysis of pleural fluid often reveals the presence of free spheroid aggregates of malignant cells, giving rise to the question of the ability of non-adherent tumor cells to resist the loss of anchorage-induced apoptosis (termed as anoikis), and to develop new tumor foci in the pleural cavity. Here, we show that MPM cells cultured under non-adherent conditions form well-organized aggregates composed of viable cells, which progressively enter in G(0). Although the PI3K/Akt, ERK and SAPK/JNK signaling pathways are activated in adherent MPM cells, loss of anchorage results in the inactivation of these pathways. By comparison, we show that the non-tumoral mesothelial cells MeT-5A enter anoikis in an SAPK/JNK-, Bim- and caspase-9-dependent pathway. The survival of MPM cells can be reversed by activating SAPK/JNK with anisomycin, according to a Bim-dependent mitochondrial pathway. Finally, our findings show that impairment of cell aggregation activates SAPK/JNK and Bim and induces anoikis. Our results underline the importance of intercellular contacts in the anoikis resistance of MPM cells.

Long noncoding RNAs (lncRNAs) play important roles in the regulation of gene expression. In fission yeast, glucose starvation triggers a transcriptional cascade of polyadenylated lncRNAs in the upstream region of the fructose-1,6-bisphosphatase gene (fbp1(+) ), which is correlated with stepwise chromatin remodeling and necessary for the massive induction of fbp1(+) mRNA. Here, we show that these novel metabolic stress-induced lncRNAs (mlonRNAs) are 5'-capped, less stable than fbp1(+) mRNA and sensitive to a certain extent to the nuclear exosome cofactor Rrp6. However, most mlonRNAs seem to escape nuclear degradation and are exported to the cytoplasm, where they localize to polysomes precisely during glucose starvation-induced global translation inhibition. It is likely that ribosomes tend to accumulate in the upstream region of mlonRNAs. Although mlonRNAs contain an unusual amount of upstream AUGs (uAUGs) and small open reading frames (uORFs), they escape Upf1-mediated targeting to the non-sense-mediated decay (NMD) pathway. The deletion of Upf1 had no effect on mlonRNA stability, but considerably destabilized fbp1(+) mRNA, hinting toward a possible novel role of Upf1. Our findings suggest that the stability of mlonRNAs is distinctly regulated from mRNA and previously described noncoding transcripts.