A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections

A new multiple fluorescence in situ hybridization method based on hybridization chain reaction was recently reported, enabling simultaneous mapping of multiple target mRNAs within intact zebrafish and mouse embryos. With this approach, DNA probes complementary to target mRNAs trigger chain reactions in which metastable fluorophore-labeled DNA hairpins self-assemble into fluorescent amplification polymers. The formation of the specific polymers enhances greatly the sensitivity of multiple fluorescence in situ hybridization. In this study we describe the optimal parameters (hybridization chain reaction time and temperature, hairpin and salt concentration) for multiple fluorescence in situ hybridization via amplification of hybridization chain reaction for frozen tissue sections. The combined use of fluorescence in situ hybridization and immunofluorescence, together with other control experiments (sense probe, neutralization and competition, RNase treatment, and anti-sense probe without initiator) confirmed the high specificity of the fluorescence in situ hybridization used in this study. Two sets of three different mRNAs for oxytocin, vasopressin and somatostatin or oxytocin, vasopressin and thyrotropin releasing hormone were successfully visualized via this new method. We believe that this modified protocol for multiple fluorescence in situ hybridization via hybridization chain reaction would allow researchers to visualize multiple target nucleic acids in the future

Co-localization of Pirt protein and P2X2 receptors in the mouse enteric nervous system

P2X2 receptors, with other P2X receptor subtypes, have an important role mediating synaptic transmission in regulating the functions of the gastrointestinal tract. Our recent work has found a new regulator of P2X receptor function, called phosphoinositide-interacting regulator of transient receptor potential channels (Pirt). In the present work, we have shown that Pirt immunoreactivity was localized in nerve cell bodies and nerve fibers in the myenteric plexus of the stomach, ileum, proximal, and distal colon and in the submucosal plexus of the jejunum, ileum, proximal, and distal colon. Almost all the Pirt-immunoreactive (ir) neurons were also P2X2-ir, and co-immunoprecipitation experiments have shown that Pirt co-precipitated with the anti-P2X2 antibody. This work provides detailed information about the expression of Pirt in the gut and its co-localization with P2X2, indicating its potential role in influencing P2X2 receptor function

A novel role of dendritic gap junction and mechanisms underlying its interaction with thalamocortical conductance in fast spiking inhibitory neurons

<p>Abstract</p> <p>Background</p> <p>Little is known about the roles of dendritic gap junctions (GJs) of inhibitory interneurons in modulating temporal properties of sensory induced responses in sensory cortices. Electrophysiological dual patch-clamp recording and computational simulation methods were used in combination to examine a novel role of GJs in sensory mediated feed-forward inhibitory responses in barrel cortex layer IV and its underlying mechanisms.</p> <p>Results</p> <p>Under physiological conditions, excitatory post-junctional potentials (EPJPs) interact with thalamocortical (TC) inputs within an unprecedented few milliseconds (i.e. over 200 Hz) to enhance the firing probability and synchrony of coupled fast-spiking (FS) cells. Dendritic GJ coupling allows fourfold increase in synchrony and a significant enhancement in spike transmission efficacy in excitatory spiny stellate cells. The model revealed the following novel mechanisms: <b><it>1) </it></b>rapid capacitive current (I_cap) underlies the activation of voltage-gated sodium channels; <b><it>2) </it></b>there was less than 2 milliseconds in which the I_capunderlying TC input and EPJP was coupled effectively; <b><it>3) </it></b>cells with dendritic GJs had larger input conductance and smaller membrane response to weaker inputs; <b><it>4) </it></b>synchrony in inhibitory networks by GJ coupling leads to reduced sporadic lateral inhibition and increased TC transmission efficacy.</p> <p>Conclusion</p> <p>Dendritic GJs of neocortical inhibitory networks can have very powerful effects in modulating the strength and the temporal properties of sensory induced feed-forward inhibitory and excitatory responses at a very high frequency band (>200 Hz). Rapid capacitive currents are identified as main mechanisms underlying interaction between two transient synaptic conductances.</p

An efficient approach to finding Siraitia grosvenorii triterpene biosynthetic genes by RNA-seq and digital gene expression analysis

<p>Abstract</p> <p>Background</p> <p><it>Siraitia grosvenorii </it>(Luohanguo) is an herbaceous perennial plant native to southern China and most prevalent in Guilin city. Its fruit contains a sweet, fleshy, edible pulp that is widely used in traditional Chinese medicine. The major bioactive constituents in the fruit extract are the cucurbitane-type triterpene saponins known as mogrosides. Among them, mogroside V is nearly 300 times sweeter than sucrose. However, little is known about mogrosides biosynthesis in <it>S. grosvenorii</it>, especially the late steps of the pathway.</p> <p>Results</p> <p>In this study, a cDNA library generated from of equal amount of RNA taken from <it>S. grosvenorii </it>fruit at 50 days after flowering (DAF) and 70 DAF were sequenced using Illumina/Solexa platform. More than 48,755,516 high-quality reads from a cDNA library were generated that was assembled into 43,891 unigenes. De novo assembly and gap-filling generated 43,891 unigenes with an average sequence length of 668 base pairs. A total of 26,308 (59.9%) unique sequences were annotated and 11,476 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. cDNA sequences for all of the known enzymes involved in mogrosides backbone synthesis were identified from our library. Additionally, a total of eighty-five cytochrome P450 (CYP450) and ninety UDP-glucosyltransferase (UDPG) unigenes were identified, some of which appear to encode enzymes responsible for the conversion of the mogroside backbone into the various mogrosides. Digital gene expression profile (DGE) analysis using Solexa sequencing was performed on three important stages of fruit development, and based on their expression pattern, seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as the candidates most likely to be involved in mogrosides biosynthesis.</p> <p>Conclusion</p> <p>A combination of RNA-seq and DGE analysis based on the next generation sequencing technology was shown to be a powerful method for identifying candidate genes encoding enzymes responsible for the biosynthesis of novel secondary metabolites in a non-model plant. Seven <it>CYP450</it>s and five <it>UDPG</it>s were selected as potential candidates involved in mogrosides biosynthesis. The transcriptome data from this study provides an important resource for understanding the formation of major bioactive constituents in the fruit extract from <it>S. grosvenorii</it>.</p

Breast cancer epithelial-to-mesenchymal transition: examining the functional consequences of plasticity

The epithelial-to-mesenchymal transition (EMT) is a critical developmental process that has recently come to the forefront of cancer biology. In breast carcinomas, acquisition of a mesenchymal-like phenotype that is reminiscent of an EMT, termed oncogenic EMT, is associated with pro-metastatic properties, including increased motility, invasion, anoikis resistance, immunosuppression and cancer stem cell characteristics. This oncogenic EMT is a consequence of cellular plasticity, which allows for interconversion between epithelial and mesenchymal-like states, and is thought to enable tumor cells not only to escape from the primary tumor, but also to colonize a secondary site. Indeed, the plasticity of cancer cells may explain the range of pro-metastatic traits conferred by oncogenic EMT, such as the recently described link between EMT and cancer stem cells and/or therapeutic resistance. Continued research into this relationship will be critical in developing drugs that block mechanisms of breast cancer progression, ultimately improving patient outcomes

FOXP3 and FOXP3-regulated microRNAs suppress SATB1 in breast cancer cells

The transcription factor FOXP3 has been identified as a tumour suppressor in the breast and prostate epithelia, but little is known about its specific mechanism of action. We have identified a feed-forward regulatory loop in which FOXP3 suppresses the expression of the oncogene SATB1. In particular, we demonstrate that SATB1 is not only a direct target of FOXP3 repression, but that FOXP3 also induces two miRs, miR-7 and miR-155, which specifically target the 3′-UTR of SATB1 to further regulate its expression. We conclude that FOXP3-regulated miRs form part of the mechanism by which FOXP3 prevents the transformation of the healthy breast epithelium to a cancerous phenotype. Approaches aimed at restoring FOXP3 function and the miRs it regulates could help provide new approaches to target breast cancer.N McInnes, TJ Sadlon, CY Brown, S Pederson, M Beyer, JL Schultze, S McColl, GJ Goodall and SC Barr

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe