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Balgach, Switzerland

McCullough K.C.,Institute of Virology and Immunology | Milona P.,Institute of Virology and Immunology | Thomann-Harwood L.,Institute of Virology and Immunology | Demoulins T.,Institute of Virology and Immunology | And 3 more authors.

Dendritic cells (DC) play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA) carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future. © 2014, Vaccines. All rights reserved. Source

Gross A.,ETH Zurich | Zaffarano P.L.,ETH Zurich | Duo A.,ETH Zurich | Grunig C.R.,Microsynth AG
Fungal Genetics and Biology

Ash dieback caused by the fungal pathogen Hymenoscyphus pseudoalbidus is currently ravaging in Europe, killing Fraxinus excelsior and Fraxinus angustifolia trees of all age classes. The aim of this work was to elucidate aspects of the reproduction biology of this fungal pathogen and its cryptic, non-pathogenic sister species Hymenoscyphus albidus. The mating type (MAT) locus of both species was identified, partly sequenced and characterized. Whereas a heterothallic MAT organization was detected in H. pseudoalbidus, H. albidus was shown to be structurally homothallic. The molecular MAT determination of H. pseudoalbidus was confirmed by crossing experiments on sterile ash petioles. Crossings of strains exhibiting alternate MAT idiomorphs produced fertile apothecia whereas crosses of strains with identical MAT idiomorphs were never successful. Offspring genotyping with microsatellites (MSs) and the MAT marker confirmed that both parental strains were involved in apothecia formation. In addition, polymorphic MS were shown to follow Mendelian inheritance. However, for yet unknown reasons the MAT ratio of progenies of one successful cross revealed a significant segregation distortion. Based on the MAT sequences of H. pseudoalbidus a multiplex PCR was developed, allowing for a quick and reliable MAT determination. The PCR was applied to screen the MAT ratio of two H. pseudoalbidus populations derived from the country of the disease outbreak in Poland and two populations from the disease periphery in Switzerland. None of the screened populations showed a significant deviation from the 1:1 ratio, expected under random mating. Therefore, an initial clonal distribution through asexually produced conidiospores as observed for other fungal pathogens holds not true for H. pseudoalbidus. Instead, our data is highly supportive for a distribution through ascospores. Leaf petioles collected in the field were thoroughly analyzed for the number of different colonizing strains and their mating behavior. Up to eight different H. pseudoalbidus genotypes were found on a single petiole. Cross-fertilizations of strains on the same petiole and fertilizations of unknown strains from outside were found, indicating that fertilization is mediated by spermatia. The presented study complements our understanding of the life cycle of this highly destructive pathogen. The possibility to perform sexual crosses in the lab provides ample opportunities for further genetic studies of H. pseudoalbidus and related species in the future. © 2012 Elsevier Inc. Source

Eberhard R.,University of Zurich | Stergiou L.,University of Zurich | Stergiou L.,Redbiotec | Hofmann E.R.,University of Zurich | And 8 more authors.
PLoS Genetics

Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. © 2013 Eberhard et al. Source

Ghodgaonkar M.M.,ETH Zurich | Ghodgaonkar M.M.,Ludwig Maximilians University of Munich | Kehl P.,ETH Zurich | Kehl P.,Microsynth AG | And 4 more authors.
Nature Communications

Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant. Here we studied the phenotype of variants of the essential replicative polymerase-δ carrying missense mutations in its active site, similar to those recently identified in familial colon cancer patients. We show that expression of the mutants but not the wild-type protein endows the engineered cells with a mutator phenotype and that the mutations affect the fidelity and/or the exonuclease activity of the isolated enzyme in vitro. This proof-of-principle study demonstrates the general applicability of this experimental approach in the study of genotype-phenotype correlations. © 2014 Macmillan Publishers Limited. Source

Tellenbach C.,ETH Zurich | Tellenbach C.,Eawag - Swiss Federal Institute of Aquatic Science and Technology | Sumarah M.W.,Agriculture and Agri Food Canada | Sumarah M.W.,Carleton University | And 3 more authors.
Fungal Ecology

Dark septate fungal root endophytes of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are widely distributed throughout the temperate and subtropical regions of the Northern Hemisphere. Previous studies have shown that some PAC members are pathogenic, others suppress oomycete root pathogens and some have no obvious effect on their Norway spruce (Picea abies) host. The activity of 85 PAC isolates against Phytophthora citricola s.l. was investigated by co-culture on plates. We identified a strain of Phialocephala europaea that significantly reduced the growth of P. citricola in vitro. Characterization of its extracellular metabolites resulted in the identification of four major compounds, sclerin, sclerolide, sclerotinin A, and sclerotinin B. These compounds are known for their positive as well as negative effects on plant growth. We found that sclerin and sclerotinin inhibited the growth of P. citricola in vitro at 150 μg ml-1 (∼1 mM). This is the first report of their production by Phialocephala and of activity of these compounds against an oomycete. Therefore, our data suggest that some PAC might reduce disease resulting from P. citricola by the production of antibiotics and plant growth promoting metabolites. © 2012 Elsevier Ltd and The British Mycological Society. Source

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