High sensitivity and label-free oligonucleotides detection using photonic bandgap sensing structures biofunctionalized with molecular beacon probes

A label-free sensor, based on the combination of silicon photonic bandgap (PBG) structures with immobilized molecular beacon (MB) probes, is experimentally developed. Complementary target oligonucleotides are specifically recognized through hybridization with the MB probes on the surface of the sensing structure. This combination of PBG sensing structures and MB probes demonstrates an extremely high sensitivity without the need for complex PCR-based amplification or labelling methods

Increased Neural Activity of a Mushroom Body Neuron Subtype in the Brains of Forager Honeybees

Honeybees organize a sophisticated society, and the workers transmit information about the location of food sources using a symbolic dance, known as ‘dance communication’. Recent studies indicate that workers integrate sensory information during foraging flight for dance communication. The neural mechanisms that account for this remarkable ability are, however, unknown. In the present study, we established a novel method to visualize neural activity in the honeybee brain using a novel immediate early gene, kakusei, as a marker of neural activity. The kakusei transcript was localized in the nuclei of brain neurons and did not encode an open reading frame, suggesting that it functions as a non-coding nuclear RNA. Using this method, we show that neural activity of a mushroom body neuron subtype, the small-type Kenyon cells, is prominently increased in the brains of dancer and forager honeybees. In contrast, the neural activity of the two mushroom body neuron subtypes, the small-and large-type Kenyon cells, is increased in the brains of re-orienting workers, which memorize their hive location during re-orienting flights. These findings demonstrate that the small-type Kenyon cell-preferential activity is associated with foraging behavior, suggesting its involvement in information integration during foraging flight, which is an essential basis for dance communication

Beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

The Wnt/beta-catenin/TCF4 pathway plays critical roles in the maintenance of small intestinal epithelium; however, downstream targets of the beta-catenin/TCF4 complex are not extensively characterized. We identified miR-30e as an immediate target activated by the beta-catenin/TCF4 complex. miR-30e was detected in the peri-nuclear region of the intestinal crypt IEC-6 cells. Bioinformatics analysis revealed clustered beta-catenin/TCF4 binding sites within the miR-30e promoter region. This promoter region was cloned into pGL3-control luciferase reporter vector, with the enhancer region removed. Transfection of pCMV-SPORT6-beta-catenin expression vector dose-dependently increased luciferase activity, and co-transfection of pCMV-SPORT6-TCF4 expression vector further enhanced the promoter activity. Dexamethasone-induced IEC-6 cells differentiation caused a 2.5-fold increase in miR-30e expression, and upon beta-catenin siRNA transfection, miR-30e increased 1.3-fold. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay confirmed the binding between beta-catenin/TCF4 complexes from IEC-6 nuclear extracts and the putative sequences in the miR-30e promoter. These results demonstrate that beta-catenin/TCF4 transactivates miR-30e during intestinal cell differentiation

Widespread Regulation of miRNA Biogenesis at the Dicer Step by the Cold-Inducible RNA-Binding Protein, RBM3

MicroRNAs (miRNAs) play critical roles in diverse cellular events through their effects on translation. Emerging data suggest that modulation of miRNA biogenesis at post-transcriptional steps by RNA-binding proteins is a key point of regulatory control over the expression of some miRNAs and the cellular processes they influence. However, the extent and conditions under which the miRNA pathway is amenable to regulation at posttranscriptional steps are poorly understood. Here we show that RBM3, a cold-inducible, developmentally regulated RNA-binding protein and putative protooncogene, is an essential regulator of miRNA biogenesis. Utilizing miRNA array, Northern blot, and PCR methods, we observed that over 60% of miRNAs detectable in a neuronal cell line were significantly downregulated by knockdown of RBM3. Conversely, for select miRNAs assayed by Northern blot, induction of RBM3 by overexpression or mild hypothermia increased their levels. Changes in miRNA expression were accompanied by changes in the levels of their ∼70 nt precursors, whereas primary transcript levels were unaffected. Mechanistic studies revealed that knockdown of RBM3 does not reduce Dicer activity or impede transport of pre-miRNAs into the cytoplasm. Rather, we find that RBM3 binds directly to ∼70 nt pre-miRNA intermediates and promotes / de-represses their ability as larger ribonucleoproteins (pre-miRNPs) to associate with active Dicer complexes. Our findings suggest that the processing of a majority of pre-miRNPs by Dicer is subject to an intrinsic inhibitory influence that is overcome by RBM3 expression. RBM3 may thus orchestrate changes in miRNA expression during hypothermia and other cellular stresses, and in the euthermic contexts of early development, differentiation, and oncogenesis where RBM3 expression is highly elevated. Additionally, our data suggest that temperature-dependent changes in miRNA expression mediated by RBM3 may contribute to the therapeutic effects of hypothermia, and are an important variable to consider in in vitro studies of translation-dependent cellular events

Changes in Brain MicroRNAs Contribute to Cholinergic Stress Reactions

Mental stress modifies both cholinergic neurotransmission and alternative splicing in the brain, via incompletely understood mechanisms. Here, we report that stress changes brain microRNA (miR) expression and that some of these stress-regulated miRs regulate alternative splicing. Acute and chronic immobilization stress differentially altered the expression of numerous miRs in two stress-responsive regions of the rat brain, the hippocampal CA1 region and the central nucleus of the amygdala. miR-134 and miR-183 levels both increased in the amygdala following acute stress, compared to unstressed controls. Chronic stress decreased miR-134 levels, whereas miR-183 remained unchanged in both the amygdala and CA1. Importantly, miR-134 and miR-183 share a common predicted mRNA target, encoding the splicing factor SC35. Stress was previously shown to upregulate SC35, which promotes the alternative splicing of acetylcholinesterase (AChE) from the synapse-associated isoform AChE-S to the, normally rare, soluble AChE-R protein. Knockdown of miR-183 expression increased SC35 protein levels in vitro, whereas overexpression of miR-183 reduced SC35 protein levels, suggesting a physiological role for miR-183 regulation under stress. We show stress-induced changes in miR-183 and miR-134 and suggest that, by regulating splicing factors and their targets, these changes modify both alternative splicing and cholinergic neurotransmission in the stressed brain

Genome-wide microRNA screening in Nile tilapia reveals pervasive isomiRs’ transcription, sex-biased arm switching and increasing complexity of expression throughout development

MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs

Mir-132/212 is required for maturation of binocular matching of orientation preference and depth perception

MicroRNAs (miRNAs) are known to mediate post-transcriptional gene regulation, but their role in postnatal brain development is still poorly explored. We show that the expression of many miRNAs is dramatically regulated during functional maturation of the mouse visual cortex with miR-132/212 family being one of the top upregulated miRNAs. Age-downregulated transcripts are significantly enriched in miR-132/miR-212 putative targets and in genes upregulated in miR-132/212 null mice. At a functional level, miR-132/212 deletion affects development of receptive fields of cortical neurons determining a specific impairment of binocular matching of orientation preference, but leaving orientation and direction selectivity unaltered. This deficit is associated with reduced depth perception in the visual cliff test. Deletion of miR-132/212 from forebrain excitatory neurons replicates the binocular matching deficits. Thus, miR-132/212 family shapes the age-dependent transcriptome of the visual cortex during a specific developmental window resulting in maturation of binocular cortical cells and depth perception

MicroRNA Expression Is Down-Regulated and Reorganized in Prefrontal Cortex of Depressed Suicide Subjects

<div><h3>Background</h3><p>Recent studies suggest that alterations in expression of genes, including those which regulate neural and structural plasticity, may be crucial in the pathogenesis of depression. MicroRNAs (miRNAs) are newly discovered regulators of gene expression that have recently been implicated in a variety of human diseases, including neuropsychiatric diseases.</p> <h3>Methodology/Principal Findings</h3><p>The present study was undertaken to examine whether the miRNA network is altered in the brain of depressed suicide subjects. Expression of miRNAs was measured in prefrontal cortex (Brodmann Area 9) of antidepressant-free depressed suicide (n = 18) and well-matched non-psychiatric control subjects (n = 17) using multiplex RT-PCR plates. We found that overall miRNA expression was significantly and globally down-regulated in prefrontal cortex of depressed suicide subjects. Using individual tests of statistical significance, 21 miRNAs were significantly decreased at p = 0.05 or better. Many of the down-regulated miRNAs were encoded at nearby chromosomal loci, shared motifs within the 5′-seeds, and shared putative mRNA targets, several of which have been implicated in depression. In addition, a set of 29 miRNAs, whose expression was not pairwise correlated in the normal controls, showed a high degree of co-regulation across individuals in the depressed suicide group.</p> <h3>Conclusions/Significance</h3><p>The findings show widespread changes in miRNA expression that are likely to participate in pathogenesis of major depression and/or suicide. Further studies are needed to identify whether the miRNA changes lead to altered expression of prefrontal cortex mRNAs, either directly (by acting as miRNA targets) or indirectly (e.g., by affecting transcription factors).</p> </div

MicroRNA and Target Protein Patterns Reveal Physiopathological Features of Glioma Subtypes

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets