Accumulated lines of evidence have revealed that a large number of circular RNAs are produced in transcriptomes from fruit fly to mouse and human. Unlike linear RNAs shaped with 5\' cap and 3\' tail, circular RNAs are characterized by covalently closed loop structures without open terminals, thus required specific treatments for their identification and validation. Here, we describe a detailed pipeline for the characterization of circular RNAs. It has been successfully applied to the study of circular intronic RNAs (ciRNAs) derived from intron lariats and circular RNAs (circRNAs) produced from back spliced exons in human.

Creating DNA constructs is a basic and fundamental step in molecular and synthetic biology. While prices for gene synthesis are decreasing, it is still more economical in most cases to assemble constructs from a library of components (Parts). Many methods for DNA assembly are available, but most require either a fixed and inflexible format for the construct, with all Parts first being cloned in specific donor plasmids, or remaking Parts with new homology ends for each specific assembly reaction, requiring large numbers of single-use oligonucleotides. PaperClip assembly allows Parts stored in any format (linear PCR products or synthetic DNA, or cloned in any plasmid) to be used in totally flexible assembly reactions; up to 11 parts can be assembled in a single reaction, in any order, to give a linear or circular construct, and the oligonucleotides required in the assembly process can be reused in any subsequent assembly. In addition to constructing plasmids for bacterial transformation, PaperClip is also well suited to generate linear products for direct transfection of yeast, mammalian, or cyanobacterial cell lines. Thus, PaperClip offers a simple, flexible, and economical route to multipart assembly of constructs for a wide variety of purposes.

Free-air O3 enrichment was used to investigate the responses of different antioxidant mechanisms in different rice (Oryza sativa L.) cultivars - O3-sensitive hybrid indica (O3-S) cultivars and O3-tolerant conventional japonica (O3-T) cultivars across all growth stages. Elevated [O3] induced increases in reactive oxygen species (ROS) production in O3-S cultivars, which were more pronounced in the later growing stages. In O3-S cultivars, continuous O3 stress decreased catalase (CAT), peroxidase (POD) and glutathione peroxidase (GPX) activities, while in O3-T cultivars, short-term O3 stress decreased superoxide dismutase (SOD), CAT, POD and GPX activities. The same POD isozyme patterns were observed in both O3-S and O3-T cultivars, while SOD and APX isozymes varied by cultivar. The results suggest that O3 tolerance might be improved at different rice development stages through regulating the responses of antioxidant mechanisms to O3 stress.

Most traditional post-electrophoretic processes need several hours to several days to finish the whole staining process and traditional staining solutions all contain methanol, acetic acid, or phosphoric acid, which not only produce the unpleasant smell but also cause environmental pollution. Here a fixation-free, fast protein staining method in sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Coomassie blue is described. The protocol includes only staining and quick washing steps, can be completed in 0.5 h. It has a sensitivity of 10 ng. In addition, the dye stain does not contain any acid or methanol.

With the rapid development of sequence specific nucleases (SSNs) for genome targeting, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is now considered the most promising method for functional genetic researches, as well as genetic improvement in crop plants. However, the gene redundancy feature within the allotetraploid rapeseed genome is one of the major obstacles for simultaneous modification of different homologs in the first generation. In addition, large scale screening to identify mutated transgenic plants is very time-and labor-consuming using the conventional restriction enzyme-based approaches. In this study, a streamlined rapeseed CRISPR-Cas9 genome editing platform was developed through synthesizing a premade U6-26 driven sgRNA expression cassette and optimizing polyacrylamide gel electrophoresis (PAGE)-based screening approach. In our experiment, a sgRNA was constructed to target five rapeseed SPL3 homologous gene copies, BnSPL3-A5/BnSPL3-A4/BnSPL3-C3/BnSPL3-C4/BnSPL3-Cnn. High-throughput sequencing analysis demonstrated that the editing frequency of CRISPR/Cas9-induced mutagenesis ranged from 96.8 to 100.0% in plants with obvious heteroduplexed PAGE bands, otherwise this proportion was only 0.00-60.8%. Consistent with those molecular analyses, Bnspl3 mutants exhibited developmental delay phenotype in the first generation. In summary, our data suggest that this set of CRISPR/Cas9 platform is qualified for rapidly generating and identifying simultaneous mutagenesis of multiple gene homologs in allotetraploid rapeseed.

MicroRNA-21 (miR-21) has been regarded as a kind of potential biomarker in several types of cancers. Herein, in this study, a simple, sensitive and cost-effective miR-21 approach was developed utilizing the isothermal target-recycled enzyme-free amplification strategy and polyacrylamide gel electrophoresis (PAGE). In the target-recycled enzyme-free amplification strategy, two rational designed hairpin probes (HPs, HP1 and HP2) can form into HP1-HP2 duplex in the presence of miR-21 under the isothermal condition, producing target signal in PAGE. The sensitivity and the linear range of miR-21 approach were demonstrated by the in vitro detection of miR-21, with the detection limit of 10 pM and the linear range of 50 pM to 8 nM. In particular, the contents of miR-21 in cell extractions of different cell lines were successfully detected through miR-21 approach and the relative expression was highly coincident with the results of stem-loop RT-PCR approach. In summary, the developed approach can detect miR-21 sensitively and easily.

RNA nanoparticles are designed and self-assembled according to noncanonical interactions of naturally conserved RNA motifs and/or canonical Watson-Crick base-pairing interactions, which have potential applications in gene therapy and nanomedicine. These artificially engineered nanoparticles are mainly synthesized from in vitro transcribed RNAs, purified by denaturing and native polyacrylamide gel electrophoresis (PAGE), and characterized with native PAGE, AFM, and TEM technologies. The protocols of in vitro transcription, denaturing and native PAGE, and RNA nanoparticle self-assembly are described in detail.

BACKGROUND: Ferulic acid (FA), ligustrazine (LZ) and tetrahydropalmatine (THP) are separately isolated from Chinese Angelica, Szechwan Lovage Rhizome and Rhizoma in the Jiawei-Foshou-San formula, a popular traditional Chinese medicine for irregular menses. It has been reported that the combination use of FA+LZ+THP has similar effect on endometriosis, but the underlying mechanisms are unclear. This study was to investigate the combination effects and mechanisms of FA+LZ+THP on endometriosis rats.
METHODS: Fifty endometriosis rats were intragastricly treated with FA+LZ+THP for 4 wk. The volume of ectopic endometrial tissue was measured to evaluate the effects. Then the changes in hypothalamic-pituitary-ovarian axis and ERE pathway were indicated by the levels of E2, GnRH, FSH and LH, and the expressions of ER, HSP90 and COX-2, respectively. In addition, peritoneal macrophages of each rat were cultured in vitro and treated with (FA+LZ+THP)-medicated serum for 24h. The proliferation and phagocytosis abilities, the levels of IL-1β and TNF-α, and the expression of IκBα were then measured for the changes of peritoneal macrophage activities.
RESULTS: Combination use of FA+LZ+THP diminished the volume of the ectopic endometrial tissues (P<0.05 or P<0.01). It also decreased the E2 level, suppressed the expression of GnRH, FSH and LH, and decreased the protein expression of ER, HSP90 and COX-2 (all P<0.05 or P<0.01). The phagocytosis ability of peritoneal macrophage was enhanced by (FA+LZ+THP)-medicated serum (P<0.05) with no change of proliferation (P>0.05). Moreover, IL-1β and TNF-α were downregulated (both P<0.05 or P<0.01) and IκBα was upregulated by the (FA+LZ+THP)-medicated serum (P<0.01).
CONCLUSIONS: The combination use of FA, LZ and THP could inhibit the growth of ectopic endometrial tissue in endometriosis rats. It might be related to the down-regulation of hypothalamic-pituitary-ovarian axis, the amelioration in ERE pathway and the improvement of peritoneal macrophage activities.

The WUSCHEL (WUS)-related homeobox (WOX) gene family plays an important role in coordinating gene transcription in the early phases of embryogenesis. In this study, we isolated and characterized WOX5 from common wheat and its relatives Triticum monococcum, Triticum urartu, Aegilops speltoides, Aegilops searsii, Aegilops sharonensis, Aegilops longissima, Aegilops bicornis, Aegilops tauschii, and Triticum turgidum. The size of the characterized WOX5 alleles ranged from 1029 to 1038 bp and encompassed the complete open reading frame (ORF) as well as 5\' upstream and 3\' downstream sequences. Domain prediction analysis showed that the putative primary structures of wheat WOX5 protein include the highly conserved homeodomain besides the WUS-box domain and the EAR-like domain, which is/are present in some members of the WOX protein family. The full-length ORF was subcloned into a prokaryotic expression vector pET30a, and an approximate 26-kDa protein was successfully expressed in Escherichia coli BL21 (DE3) cells with IPTG induction. The WOX5 genes from wheat-related species exhibit a similar structure to and high sequence similarity with WOX5 genes from common wheat. The degree of divergence and phylogenetic tree analysis among WOX5 alleles suggested the existence of three homoeologous copies in the A, B, or D genome of common wheat. Quantitative PCR results showed that TaWOX5 was primarily expressed in the root and calli induced by auxin and cytokinin, indicating that TaWOX5 may play a role related to root formation or development and is associated with hormone regulation in somatic embryogenesis.

Insect chemosensory proteins (CSPs) are proposed to capture and transport hydrophobic chemicals across the sensillum lymph to olfactory receptors (ORs), but this has not been clarified in moths. In this study, we built on our previously reported segment sequence work and cloned the full length CSP19 gene (SinfCSP19) from the antennae of Sesamia inferens by using rapid amplification of cDNA ends. Quantitative real time-PCR (qPCR) assays indicated that the gene was expressed in a unique profile, i.e. predominant in antennae and significantly higher in male than in female. To explore the function, recombinant SinfCSP19 was expressed in Escherichia coli cells and purified by Ni-ion affinity chromatography. Binding affinities of the recombinant SinfCSP19 with 39 plant volatiles, 3 sex pheromone components and 10 pheromone analogs were measured using fluorescent competitive binding assays. The results showed that 6 plant volatiles displayed high binding affinities to SinfCSP19 (Ki = 2.12-8.75 μM), and more interesting, the 3 sex pheromone components and analogs showed even higher binding to SinfCSP19 (Ki = 0.49-1.78 μM). Those results suggest that SinfCSP19 plays a role in reception of female sex pheromones of S. inferens and host plant volatiles.