Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo.

Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements-slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.

The effect of RNA base lesions on mRNA translation

The biological effect of oxidatively damaged RNA, unlike oxidatively damaged DNA, has rarely been investigated, although it poses a threat to any living cell. Here we report on the effect of the commonly known RNA base-lesions 8-oxo-rG, 8-oxo-rA, ε-rC, ε-rA, 5-HO-rC, 5-HO-rU and the RNA abasic site (rAS) on ribosomal translation. To this end we have developed an in vitro translation assay based on the mRNA display methodology. A short synthetic mRNA construct containing the base lesion in a predefined position of the open reading frame was 32P-labeled at the 5′-end and equipped with a puromycin unit at the 3′-end. Upon in vitro translation in rabbit reticulocyte lysates, the encoded peptide chain is transferred to the puromycin unit and the products analyzed by gel electrophoresis. Alternatively, the unlabeled mRNA construct was used and incubated with 35S-methionine to prove peptide elongation of the message. We find that all base-lesions interfere substantially with ribosomal translation. We identified two classes, the first containing modifications at the base coding edge (ε-rC, ε-rA and rAS) which completely abolish peptide synthesis at the site of modification, and the second consisting of 8-oxo-rG, 8-oxo-rA, 5-HO-rC and 5-HO-rU that significantly retard full-length peptide synthesis, leading to some abortive peptides at the site of modification.

Translation of ERC resuscitation guidelines into clinical practice by emergency physicians

PURPOSE Austrian out-of-hospital emergency physicians (OOHEP) undergo mandatory biannual emergency physician refresher courses to maintain their licence. The purpose of this study was to compare different reported emergency skills and knowledge, recommended by the European Resuscitation Council (ERC) guidelines, between OOHEP who work regularly at an out-of-hospital emergency service and those who do not currently work as OOHEP but are licenced. METHODS We obtained data from 854 participants from 19 refresher courses. Demographics, questions about their practice and multiple-choice questions about ALS-knowledge were answered and analysed. We particularly explored the application of therapeutic hypothermia, intraosseous access, pocket guide use and knowledge about the participants' defibrillator in use. A multivariate logistic regression analysed differences between both groups of OOHEP. Age, gender, years of clinical experience, ERC-ALS provider course attendance and the self-reported number of resuscitations were control variables. RESULTS Licenced OOHEP who are currently employed in emergency service are significantly more likely to initiate intraosseous access (OR = 4.013, p < 0.01), they initiate mild-therapeutic hypothermia after successful resuscitation (OR = 2.550, p < 0.01) more often, and knowledge about the used defibrillator was higher (OR = 2.292, p < 0.01). No difference was found for the use of pocket guides.OOHEP who have attended an ERC-ALS provider course since 2005 have initiated more mild therapeutic hypothermia after successful resuscitation (OR = 1.670, p <0.05) as well as participants who resuscitated within the last year (OR = 2.324, p < 0.01), while older OOHEP initiated mild therapeutic hypothermia less often, measured per year of age (OR = 0.913, p <0.01). CONCLUSION Licenced and employed OOHEP implement ERC guidelines better into clinical practice, but more training on life-saving rescue techniques needs to be done to improve knowledge and to raise these rates of application.

Cooperative VET in Training Networks: Analysing the Free-Rider Problem in a Sociology-of-Conventions Perspective

In training networks, particularly small and medium-sized enterprises pool their resources to train apprentices within the framework of the dual VET system, while an intermediary organisation is tasked with managing operations. Over the course of their apprenticeship, the apprentices switch from one training company to another on a (half-) yearly basis. Drawing on a case study of four training networks in Switzerland and the theoretical framework of the sociology of conventions, this paper aims to understand the reasons for the slow dissemination and reluctant adoption of this promising form of organising VET in Switzerland. The results of the study show that the system of moving from one company to another creates a variety of free-rider constellations in the distribution of the collectively generated corporative benefits. This explains why companies are reluctant to participate in this model. For the network to be sustainable, the intermediary organisation has to address discontent arising from free-rider problems while taking into account that the solutions found are always tentative and will often result in new free-rider problems.

Ribosome-associated ncRNAs (rancRNAs) An emerging class of translation regulators

Small non-protein-coding RNA (ncRNA) molecules represent major contributors to regulatory networks in controlling gene expression in a highly efficient manner. Most of the recently discovered regulatory ncRNAs acting on translation target the mRNA rather than the ribosome (e.g.: miRNAs, siRNAs, antisense RNAs). To address the question, whether ncRNA regulators exist that are capable of modulating the rate of protein production by directly interacting with the ribosome, we have analyzed the small ncRNA interactomes of ribosomes. Deep-sequencing analyses revealed thousands of putative rancRNAs in various model organisms (1,2). For a subset of these ncRNA candidates we have gathered experimental evidence that they associate with ribosomes in a stress-dependent manner and fine-tune the rate of protein biosynthesis (3,4). Many of the investigated rancRNAs appear to be processing products of larger functional RNAs, such as tRNAs (2,3), mRNAs (3), or snoRNAs (2). Post-transcriptional cleavage of RNA to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. Our data disclose the ribosome as target for small regulatory RNAs. rancRNAs are found in all domains of life and represent a prevalent but so far largely unexplored class of regulatory molecules (5). Ongoing work in our lab revealed first insight into rancRNA processing and mechanism of this emerging class of translation regulators.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

Oxidized bases in the ribosome's peptidyl transferase center and their effects on translation

The ribosome is central to protein biosynthesis and the focus of extensive research. Recent biochemical and structural studies, especially detailed crystal structures and high resolution Cryo-EM in different functional states have broadened our understanding of the ribosome and its mode of action. However, the exact mechanism of peptide bond formation and how the ribosome catalyzes this reaction is not yet understood. Also, consequences of direct oxidative stress to the ribosome and its effects on translation have not been studied. So far, no conventional replacement or even removal of the peptidyl transferase center's bases has been able to affect in vitro translation. Significant contribution to the catalytic activity seems to stem from the ribose-phosphate backbone, specifically 2'OH of A2451. Using the technique of atomic mutagenesis, novel unnatural bases can be introduced to any desired position in the 23S rRNA, surpassing conventional mutagenesis and effectively enabling to alter single atoms in the ribosome. Reconstituting ribosomes in vitro using this approach, we replaced universally conserved PTC bases with synthetic counterparts carrying the most common oxidations 8-oxorA, 5-HOrU and 5-HOrC. To investigate the consequent effects on translation, the chemically engineered ribosomes were studied the in various functional assays. Incorporation of different oxidized bases into the 70S ribosome affected the ribosomes in different ways. Depending on the nucleobase modified, the reconstituted ribosomes exhibited radical deceleration of peptide bond formation, decrease of synthesis efficiency or even an increase of translation rate. These results may further our understanding of the residues involved in the peptide bond formation mechanism, as well as the disease-relevant effects of oxydative stress on the translation machinery.

tRNA-derived fragments target the small ribosomal subunit to fine-tune translation

Post-transcriptional cleavage of RNA molecules to generate smaller fragments is a widespread mechanism that enlarges the structural and functional complexity of cellular RNomes. In particular, fragments deriving from both precursor and mature tRNAs represent one of the rapidly growing classes of post-transcriptional RNA pieces. Importantly, these tRNA-derived fragments (tRFs) possess distinct expression patterns, abundance, cellular localizations, or biological roles compared with their parental tRNA molecules [1]. Here we present evidence that tRFs from the archaeon Haloferax volcanii directly bind to ribosomes. In a previous genomic screen for ribosome-associated small RNAs we have identified a 26 residue long fragment originating from the 5’ part of valine tRNA (Val-tRF) to be by far the most abundant tRF in H. volcanii [2]. The Val-tRF is processed in a stress- dependent manner and was found to primarily target the small ribosomal subunit in vitro and in vivo. Translational activity was markedly reduced in the presence of Val-tRF, while control RNA fragments of similar length did not show inhibition of protein biosynthesis. Crosslinking experiments and subsequent primer extension analyses revealed the Val-tRF interaction site to surround the mRNA path in the 30S subunit. In support of this, binding experiments demonstrated that Val-tRF does compete with mRNAs for ribosome binding. Therefore this tRF represents a ribosome-bound non-protein-coding RNA (ncRNA) capable of regulating gene expression in H. volcanii under environmental stress conditions probably by fine-tuning the rate of protein production [1].