Comparison of mucosa-associated microbiota in Crohn's disease patients with and without anti-tumor necrosis factor-α therapy.

Most studies on the gut microbiome of Crohn's disease have been conducted using feces, instead of intestinal mucus to analyze the mucosa-associated microbiota. To investigate the characteristics of mucosa-associated microbiota in Crohn's disease patients and the effect of anti-tumor necrosis factor (TNF)-α therapy on mucosa-associated microbiota, we analyzed microbiota in Crohn's disease patients using brushing samples taken from terminal ileum. The recruited subjects were 18 Crohn's disease patients and 13 controls.

Distribution of Antimicrobial Resistance and Virulence Genes within the Prophage-Associated Regions in Nosocomial Pathogens.

Prophages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage.

Development of multiplexing gene silencing system using conditionally induced polycistronic synthetic antisense RNAs in Escherichia coli.

Although efficient methods of gene silencing have been established in eukaryotes, many different techniques are still used in bacteria due to the lack of a standardized tool. Here, we developed a convenient and efficient method to downregulate the expression of a specific gene using ∼140 nucleotide RNA with a 24-nucleotide antisense region from an arabinose-inducible expression plasmid by taking Escherichia coli lacZ and phoA genes encoding β-galactosidase and alkaline phosphatase, respectively, as target genes to evaluate the model. We examined the antisense RNA (asRNA) design, including targeting position, uORF stability elements at the 5'-end, and Hfq-binding module at the 3'-end, and inducer amount required to obtain effective experimental conditions for gene silencing.

Comprehensive analysis of liver and blood miRNA in precancerous conditions.

Streptozotocin administration to mice (STZ-mice) induces type I diabetes and hepatocellular carcinoma (HCC). We attempted to elucidate the carcinogenic mechanism and the miRNA expression status in the liver and blood during the precancerous state. Serum and liver tissues were collected from STZ-mice and non-treated mice (CTL-mice) at 6, 10, and 12 W.

Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach.

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium.

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis.

Functional characterization of the type I toxin Lpt from Lactobacillus rhamnosus by fluorescence and atomic force microscopy.

Lpt is a 29 amino acid long type I toxin identified in the plasmid DNA of wild Lactobacillus rhamnosus strains isolated from food. We previously reported that transcription of the encoding gene was upregulated under nutritional starvation conditions mimicking cheese ripening environment. The heterologous expression of the Lpt peptide in E.

Circulating osteocyte-derived exosomes contain miRNAs which are enriched in exosomes from MLO-Y4 cells.

Signaling molecules produced by osteocytes have been proposed to serve as soluble factors that contribute to bone remodeling, as well as to homeostasis of other organs. However, to the best of our knowledge, there are currently no studies investigating the role of osteocyte-secreted exosomes. In the present study, ablation of osteocytes in mice [osteocyte-less (OL)] was used to examine the microRNA (miRNA) levels of plasma-circulating exosomes.

Involvement of an intracellular vesicular transport process in naked-sgRNA-mediated TRUE gene silencing.

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology with therapeutic potential. Recently, our group demonstrated that a heptamer, mh1 (Bcl‑2), targeting human Bcl-2 mRNA, can be taken up by cells without the use of any transfection reagents and can induce the apoptosis of leukemia cells. However, little is known regarding the mechanism of naked small guide (sg)RNA uptake by cultured cells.

Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.

Several pieces of evidence suggest that small RNA degradation products together with tRNase ZL appear to form another layer of the whole gene regulatory network. The degraded RNA such as a 5'-half-tRNA and an rRNA fragment function as small guide RNA (sgRNA) to guide the enzyme to target RNA. We were curious whether there exist RNAs in plasma that can function as sgRNAs for tRNase ZL, whether these RNAs are working as signaling molecules between cells to fulfill physiological roles, and whether there are any differences in plasma sgRNA species and levels between normal and pathological conditions.

Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.

tRNase-Z(L)-utilizing efficacious (TRUE) gene silencing is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the property of tRNase Z(L) that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA). To search for novel potential therapeutic sgRNAs for hematological malignancies, we screened a library composed of 156 sgRNAs, and found that 20 sgRNAs can efficiently induce apoptosis in leukemia and/or myeloma cells.

Comparison of CAGE and RNA-seq transcriptome profiling using clonally amplified and single-molecule next-generation sequencing.

CAGE (cap analysis gene expression) and RNA-seq are two major technologies used to identify transcript abundances as well as structures. They measure expression by sequencing from either the 5' end of capped molecules (CAGE) or tags randomly distributed along the length of a transcript (RNA-seq). Library protocols for clonally amplified (Illumina, SOLiD, 454 Life Sciences [Roche], Ion Torrent), second-generation sequencing platforms typically employ PCR preamplification prior to clonal amplification, while third-generation, single-molecule sequencers can sequence unamplified libraries.

Temporal dynamics and transcriptional control using single-cell gene expression analysis.

Background: Changes in environmental conditions lead to expression variation that manifest at the level of gene regulatory networks. Despite a strong understanding of the role noise plays in synthetic biological systems, it remains unclear how propagation of expression heterogeneity in an endogenous regulatory network is distributed and utilized by cells transitioning through a key developmental event.

Results: Here we investigate the temporal dynamics of a single-cell transcriptional network of 45 transcription factors in THP-1 human myeloid monocytic leukemia cells undergoing differentiation to macrophages.

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells.

Novel small noncoding RNAs in mouse spermatozoa, zygotes and early embryos.

The recent discovery of a significant amount of RNA in spermatozoa contradicted the previously held belief that paternal contribution was limited to one copy of the genome. Furthermore, detection of RNA in sperm raised the intriguing question of its possible role in embryonic development. The possibility that RNAs may serve as epigenetic determinants was supported by experiments showing inheritance of epigenetic traits in mice mediated by RNA.

pre-miRNA profiles obtained through application of locked nucleic acids and deep sequencing reveals complex 5'/3' arm variation including concomitant cleavage and polyuridylation patterns.

Recent research hints at an underappreciated complexity in pre-miRNA processing and regulation. Global profiling of pre-miRNA and its potential to increase understanding of the pre-miRNA landscape is impeded by overlap with highly expressed classes of other non coding (nc) RNA. Here, we present a data set excluding these RNA before sequencing through locked nucleic acids (LNA), greatly increasing pre-miRNA sequence counts with no discernable effect on pre-miRNA or mature miRNA sequencing.

miRNA profiling of bilateral rat hippocampal CA3 by deep sequencing.

MicroRNAs (miRNAs) have been demonstrated to be potent post-trascriptional modulators of protein expression. miRNA expression was profiled in the left and right dorsal hippocampal CA3 of mature rats by high-throughput deep sequencing. Among the sequenced and cross-mapped small RNAs, 88% belonged to the miRNAs annotated in the miRBase 15 database.

Reduction of non-insert sequence reads by dimer eliminator LNA oligonucleotide for small RNA deep sequencing.

Here we describe a method for constructing small RNA libraries for high-throughput sequencing in which we have made a significant improvement to commonly available standard protocols. We added a locked nucleic acid (LNA) oligonucleotide--named dimer eliminator--that is complementary to the adapter-dimer ligation products during the reverse transcription reaction. It reduces adapter-dimers, which often contaminate standard libraries and increase the number of non-insert sequence reads.

Cross-mapping and the identification of editing sites in mature microRNAs in high-throughput sequencing libraries.

MicroRNAs (miRNAs) are short (20-23 nt) RNAs that are sequence-specific mediators of transcriptional and post-transcriptional regulation of gene expression. Modern high-throughput technologies enable deep sequencing of such RNA species on an unprecedented scale. We find that the analysis of small RNA deep-sequencing libraries can be affected by cross-mapping, in which RNA sequences originating from one locus are inadvertently mapped to another.

Repression of small toxic protein synthesis by the Sib and OhsC small RNAs.

The sequences encoding the QUAD1 RNAs were initially identified as four repeats in Escherichia coli. These repeats, herein renamed SIB, are conserved in closely related bacteria, although the number of repeats varies. All five Sib RNAs in E.

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli.

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations.

An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin.

Only few small, regulatory RNAs encoded opposite another gene have been identified in bacteria. Here, we report the characterization of a locus where a small RNA (SymR) is encoded in cis to an SOS-induced gene whose product shows homology to the antitoxin MazE (SymE). Synthesis of the SymE protein is tightly repressed at multiple levels by the LexA repressor, the SymR RNA and the Lon protease.

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli.

The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid.

Detection of 5'- and 3'-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli.

Evidence is accumulating that small, noncoding RNAs are important regulatory molecules. Computational and experimental searches have led to the identification of approximately 60 small RNA genes in Escherichia coli. However, most of these studies focused on the intergenic regions and assumed that small RNAs were >50 nt.