A Generic System for the Expression and Purification of Soluble and Stable Influenza Neuraminidase

The influenza surface glycoprotein neuraminidase (NA) is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir) and Relenza (zanamivir) that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent ‘swine flu’ pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1) H274Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS) that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development

Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection

[EN] Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) engineered to silence endogenous transcripts as well as viral RNAs in plants. Here, we explore the possibility of using amiRNAs and syn-tasiRNAs to specifically interfere with infections by viroids, small (250–400-nucleotide) non-coding circular RNAs with compact secondary structure infecting a wide range of plant species. The combined use of recent highthroughput methods for artificial sRNA construct generation and the Potato spindle tuber viroid (PSTVd)–Nicotiana benthamiana pathosystem allowed for the simple and time-effective screening of multiple artificial sRNAs targeting sites distributed along PSTVd RNAs of (1) or (–) polarity. The majority of amiRNAs were highly active in agroinfiltrated leaves when co-expressed with an infectious PSTVd transcript, as were syn-tasiRNAs derived from a construct including the five most effective amiRNA sequences. A comparative analysis showed that the effects of the most effective amiRNA and of the syn-tasiRNAs were similar in agroinfiltrated leaves, as well as in upper non-agroinfiltrated leaves in which PSTVd accumulation was significantly delayed. These results suggest that amiRNAs and syntasiRNAs can be used effectively to control viroid infections in plants.We thank Veronica Aragones, Teresa Cordero, Arnau Puigvert and Cristina Beceiro for invaluable technical assistance. This study was supported by grants BIO2014-54269-R and AGL2013-49919-EXP from the Ministerio de Economia y Competitividad (MINECO, Spain). Alberto Carbonell was supported by an Individual Fellowship from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No. 655841.Carbonell Olivares, A.; Daros Arnau, JA. (2016). Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection. Molecular Plant Pathology. 18(5):746-753. https://doi.org/10.1111/mpp.12529S74675318

Genome-wide identification of the miRNAome in response to salinity stress in date palm (Phoenix dactylifera L.)

Although date palm is relatively salt-tolerant, little is known about the underlying molecular mechanisms that contribute to its salt tolerance. Only recently, investigators have uncovered microRNA-mediated post-transcriptional gene regulation, which is critical for typical plant development and adaptation to stress conditions such as salinity. To identify conserved and novel miRNAs in date palm and to characterize miRNAs that could play a role in salt tolerance, we have generated sRNA libraries from the leaves and roots of NaCl-treated and untreated seedlings of date palm. Deep sequencing of these four sRNA libraries yielded approximately 251 million reads. The bioinformatics analysis has identified 153 homologs of conserved miRNAs, 89 miRNA variants, and 180 putative novel miRNAs in date palm. Expression profiles under salinity revealed differential regulation of some miRNAs in date palm. In leaves, 54 of the identified miRNAs were significantly affected and the majority (70%) of them were upregulated, whereas in roots, 25 of the identified miRNAs were significantly affected and 76% of them were upregulated by the salinity stress. The salt-responsiveness of some of these miRNAs was further validated using semi-quantitative PCR (qPCR). Some of the predicted targets for the identified miRNA include genes with known functions in plant salt tolerance, such as potassium channel AKT2-like proteins, vacuolar protein sorting-associated protein, calcium-dependent and mitogen-activated proteins. As one of the first cultivated trees in the world that can tolerate a wide range of abiotic stresses, date palm contains a large population of conserved and non-conserved miRNAs that function at the post-transcriptional level. This study provided insights into miRNA-mediated gene expression that are important for adaptation to salinity in date palms.Peer reviewedBiochemistry and Molecular Biolog

A new fast and robust technique for pricing and hedging Asian options.

An Asian option is a financial contract with payoff depending on the average of an asset price over a predetermined period. It is known to be one of the hard computational problems in mathematical finance as the PDE derived for its price is two-dimensional degenerate, and creates many numerical problems. Monte Carlo simulation methods work, but they are slow. We exploit a recently proposed formulation and use the simple form of the derived PDE to custom-design a fast and accurate numerical procedure. The PDE admits an explicit, closed-form solution when we replace the non-smooth terminal condition by a polynomial, but the solution becomes prohibitively complex as the degree of the polynomial increases. We prove the existence of a weak solution to the terminal value problem and use Hormander's Hypoellipticity Theorem to prove that the solution is smooth. Through a priori bounds on the solution and artifacts of the financial model, we show that the solution is given in closed form to the right of the degeneracy curve and we justify the boundary conditions that we use in our numerical solutions. Moreover, through comparison principles and numerical investigation, we examine the sensitivity of the resulting prices as we vary model parameters and contrast the results with those for vanilla options. We demonstrate that our method's results are consistent with those in the literature, but that our method performs better and faster than other PDE pricing methods.Ph.D.FinanceMathematicsPure SciencesSocial SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124042/2/3121914.pd

Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems

Induction of Promoter DNA Methylation Upon High-Pressure Spraying of Double-Stranded RNA in Plants

Exogenous application of RNA molecules is a potent method to trigger RNA interference (RNAi) in plants in a transgene-free manner. So far, all exogenous RNAi (exo-RNAi) applications have aimed to trigger mRNA degradation of a given target. However, the issue of concomitant epigenetic changes was never addressed. Here, we report for the first time that high-pressure spraying of dsRNAs can trigger de novo methylation of promoter sequences in plants

Epigenetic modifications: An unexplored facet of exogenous RNA application in plants

Exogenous RNA interference (exo-RNAi) is a powerful transgene-free tool in modern crop improvement and protection platforms. In exo-RNAi approaches, double-stranded RNAs (dsRNAs) or short-interfering RNAs (siRNAs) are externally applied in plants in order to selectively trigger degradation of target mRNAs. Yet, the applied dsRNAs may also trigger unintended epigenetic alterations and result in epigenetically modified plants, an issue that has not been sufficiently addressed and which merits more careful consideration. © 2020 by the authors. Licensee MDPI, Basel, Switzerland