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

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

Genetically modified organism-free RNA interference: Exogenous application of RNA molecules in plants1[open]

Since its discovery more than 20 years ago, RNA interference (RNAi) has been extensively used in crop protection platforms. So far, RNAi approaches have been conventionally based on the use of transgenic plants expressing double-stranded RNAs (dsRNAs) against selected targets. However, the use of transgenes and genetically modified organisms (GMOs) has raised considerable scientific and public concerns. Hence emerged the need for alternative approaches that avoid the use of transgenes and resort instead to direct exogenous application of RNA molecules that have the potential to trigger RNAi. Here, we highlight the most important advances in this field, discussing the various methods of RNA delivery in plants against diverse targets such as plant genes, viruses, viroids, fungi, insects, mites, and nematodes. In addition, we examine the possible shortcomings of these methods, underline the critical parameters that have to be met for a desired outcome, and explore feasible possibilities to increase their efficiency and applicability, even against bacterial pathogens. © 2020 American Society of Plant Biologists. All rights reserved

Correction to: The mystery of “missing” visits in an emergency cardiology department, in the era of COVID-19.; a time-series analysis in a tertiary Greek General Hospital (Clinical Research in Cardiology, (2020), 109, 12, (1483-1489), 10.1007/s00392-020-01682-1)

The original version of this article unfortunately contained a mistake. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature

Genome-wide identification of endogenous RNA-directed DNA methylation loci associated with abundant 21-nucleotide siRNAs in Arabidopsis

In Arabidopsis, the 24-nucleotide (nt) small interfering RNAs (siRNAs) mediates RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of transposable elements (TEs). In the present study, we examined genome-wide changes in DNA methylation and siRNA accumulation in Arabidopsis induced by expression of the Cucumber mosaic virus silencing suppressor protein 2b known to directly bind to both the 21/24-nt siRNAs as well as their associated Argonaute proteins. We demonstrated a genome-wide reduction of CHH and CHG methylation in the 2b-transgenic plants. We found that 2b suppressed RdDM not only at the previously annotated loci directed by 24-nt siRNAs but also a new set of loci associated with 21/22-nt siRNAs. Further analysis showed that the reduced methylation of TEs and coding genes targeted by 21/22-nt siRNAs was associated with sequestration of the duplex siRNAs by the 2b protein but not with changes in either siRNA production or transcription. Notably, we detected both the deletion and/or the transposition of multicopy TEs associated with 2b-induced hypomethylation, suggesting potential TE reactivation. We propose that the silencing of many TEs in Arabidopsis is controlled by the 24- and 21-nt endogenous siRNAs analogous to Drosophila TE silencing by PIWI-interacting RNAs and siRNAs