J Vis Exp

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged

Regulating Data Exchange in Service Oriented Applications

We define a type system for COWS, a formalism for specifying and combining services, while modelling their dynamic behaviour. Our types permit to express policies constraining data exchanges in terms of sets of service partner names attachable to each single datum. Service programmers explicitly write only the annotations necessary to specify the wanted policies for communicable data, while a type inference system (statically) derives the minimal additional annotations that ensure consistency of services initial configuration. Then, the language dynamic semantics only performs very simple checks to authorize or block communication. We prove that the type system and the operational semantics are sound. As a consequence, we have the following data protection property: services always comply with the policies regulating the exchange of data among interacting services. We illustrate our approach through a simplified but realistic scenario for a service-based electronic marketplace

Enhancing nonclassical bosonic correlations in a quantum walk network through experimental control of disorder

The presence of disorder and inhomogeneities in quantum networks has often been unexpectedly beneficial for both quantum and classical resources. Here we experimentally realize a controllable inhomogenous quantum walk (QW) dynamics, which can be exploited to investigate the effect of coherent disorder on the quantum correlations between two indistinguishable photons. Through the imposition of suitable disorder configurations, we observe two-photon states that exhibit an enhancement in the quantum correlations between two selected modes of the network, compared to the case of an ordered QW. Different configurations of disorder can steer the system toward different realizations of such an enhancement, thus allowing spatial and temporal manipulation of quantum correlations between remote modes of QW networks

Wave-Like Variations and Sudden Density Decreases in the Lower Thermosphere as Measured by the San Marco V Satellite

Neutral density measurements were carried out by the microaccelerometer on board the Italian San Marco V satellite in 1988. During the final week of its existence the satellite's perigee decreased to as low as 130 km. Measured density values were compared to the corresponding CIRA '86 (MSIS '86) or to our dMSIS model values. The residuals reveal a wavy structure of different time scales. Characterising the wave amplitude by the average deviation of the residuals, its dependence on different parameters was studied. These investigations demonstrated that the wave-amplitude varies with height, local solar time and geomagnetic disturbance level. There is a particularly developed wave pattern in the average deviations below 200 km. Case studies indicated that there are sudden density decreases of 20–30 sec duration that might be in connection with plasma bubble crossings by the satellite. Altogether 261 such cases were identified and their distribution as a function of height, LST and longitude have been investigated

Timing Issues in Web Services Composition

Abstract. webπ is a recent process calculus introduced to formally specify Web Services composition. It extends the π-calculus with timed workunits, namely an asynchronous and temporized mechanism for events raising and catching. In this paper we encode Berger-Honda Timed-π in webπ timed workunits and we prove a simulation theorem. The overall perspective of this work is to make webπ comparable with both real composition languages and well established models for dis-tributed components.

Experimental Multi-state Quantum Discrimination in the Frequency Domain with Quantum Dot Light

The quest for the realization of effective quantum state discrimination strategies is of great interest for quantum information technology, as well as for fundamental studies. Therefore, it is crucial to develop new and more efficient methods to implement discrimination protocols for quantum states. Among the others, single photon implementations are more advisable, because of their inherent security advantage in quantum communication scenarios. In this work, we present the experimental realization of a protocol employing a time-multiplexing strategy to optimally discriminate among eight non-orthogonal states, encoded in the four-dimensional Hilbert space spanning both the polarization degree of freedom and photon energy. The experiment, built on a custom-designed bulk optics analyser setup and single photons generated by a nearly deterministic solid-state source, represents a benchmarking example of minimum error discrimination with actual quantum states, requiring only linear optics and two photodetectors to be realized. Our work paves the way for more complex applications and delivers a novel approach towards high-dimensional quantum encoding and decoding operations

Overexpression of miR-128 specifically inhibits the truncated isoform of NTRK3 and upregulates BCL2 in SH-SY5Y neuroblastoma cells

<p>Abstract</p> <p>Background</p> <p>Neurotrophins and their receptors are key molecules in the regulation of neuronal differentiation and survival. They mediate the survival of neurons during development and adulthood and are implicated in synaptic plasticity. The human neurotrophin-3 receptor gene <it>NTRK3 </it>yields two major isoforms, a full-length kinase-active form and a truncated non-catalytic form, which activates a specific pathway affecting membrane remodeling and cytoskeletal reorganization. The two variants present non-overlapping 3'UTRs, indicating that they might be differentially regulated at the post-transcriptional level. Here, we provide evidence that the two isoforms of <it>NTRK3 </it>are targeted by different sets of microRNAs, small non-coding RNAs that play an important regulatory role in the nervous system.</p> <p>Results</p> <p>We identify one microRNA (miR-151-3p) that represses the full-length isoform of <it>NTRK3 </it>and four microRNAs (miR-128, miR-485-3p, miR-765 and miR-768-5p) that repress the truncated isoform. In particular, we show that the overexpression of miR-128 - a brain enriched miRNA - causes morphological changes in SH-SY5Y neuroblastoma cells similar to those observed using an siRNA specifically directed against truncated <it>NTRK3</it>, as well as a significant increase in cell number. Accordingly, transcriptome analysis of cells transfected with miR-128 revealed an alteration of the expression of genes implicated in cytoskeletal organization as well as genes involved in apoptosis, cell survival and proliferation, including the anti-apoptotic factor <it>BCL2</it>.</p> <p>Conclusions</p> <p>Our results show that the regulation of <it>NTRK3 </it>by microRNAs is isoform-specific and suggest that neurotrophin-mediated processes are strongly linked to microRNA-dependent mechanisms. In addition, these findings open new perspectives for the study of the physiological role of miR-128 and its possible involvement in cell death/survival processes.</p

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

Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma

Integration of Expressed Sequence Tag Data Flanking Predicted RNA Secondary Structures Facilitates Novel Non-Coding RNA Discovery

Many computational methods have been used to predict novel non-coding RNAs (ncRNAs), but none, to our knowledge, have explicitly investigated the impact of integrating existing cDNA-based Expressed Sequence Tag (EST) data that flank structural RNA predictions. To determine whether flanking EST data can assist in microRNA (miRNA) prediction, we identified genomic sites encoding putative miRNAs by combining functional RNA predictions with flanking ESTs data in a model consistent with miRNAs undergoing cleavage during maturation. In both human and mouse genomes, we observed that the inclusion of flanking ESTs adjacent to and not overlapping predicted miRNAs significantly improved the performance of various methods of miRNA prediction, including direct high-throughput sequencing of small RNA libraries. We analyzed the expression of hundreds of miRNAs predicted to be expressed during myogenic differentiation using a customized microarray and identified several known and predicted myogenic miRNA hairpins. Our results indicate that integrating ESTs flanking structural RNA predictions improves the quality of cleaved miRNA predictions and suggest that this strategy can be used to predict other non-coding RNAs undergoing cleavage during maturation