In vivo testing of gold nanoparticles using the Caenorhabditis elegans model organism

Gold nanoparticles (AuNPs) are present in many man-made products and cosmetics, and are also used by the food and medical industries. Tight regulations regarding the use of mammalian animals for product testing can hamper the study of the specific interactions between engineered nanoparticles and biological systems. Invertebrate models, such as the nematode Caenorhabditis elegans (C. elegans), can offer alternative approaches during the early phases of nanoparticle discovery. Here, we thoroughly evaluated the biodistribution of 11-nm and 150-nm citrate-capped AuNPs in the model organism C. elegans at multiple scales, moving from micrometric to nanometric resolution and from the organismal to cellular level. We confirmed that the nanoparticles were not able to cross the intestinal and dermal barriers. We investigated the effect of AuNPs on the survival and reproductive performance of C. elegans, and correlated these effects with the uptake of AuNPs in terms of their number, surface area, and metal mass. In general, exposure to 11-nm AuNPs resulted in a higher toxicity than the larger 150-nm AuNPs. NP aggregation inside C. elegans was determined using absorbance microspectroscopy, which allowed the plasmonic properties of AuNPs to be correlated with their confinement inside the intestinal lumen, where anatomical traits, acidic pH and the presence of biomolecules play an essential role on NP aggregation. Finally, quantitative PCR of selected molecular markers indicated that exposure to AuNPs did not significantly affect endocytosis and intestinal barrier integrity. Statement of significance This work highlights how the simple, yet information-rich, animal model C. elegans is ideally suited for preliminary screening of nanoparticles or chemicals mitigating most of the difficulties associated with mammalian animal models, namely the ethical issues, the high cost, and time constraints. This is of particular relevance to the cosmetic, food, and pharmaceutical industries, which all have to justify the use of animals, especially during the discovery, development and initial screening phases. This work provides a detailed and thorough analysis of 11-nm and 150-nm AuNPs at multiple levels of organization (the whole organism, organs, tissues, cells and molecules).Peer ReviewedPostprint (author's final draft

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

SummaryNuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation

Evolution of the nucleus

Under a Creative Commons license.The nucleus represents a major evolutionary transition. As a consequence of separating translation from transcription many new functions arose, which likely contributed to the remarkable success of eukaryotic cells. Here we will consider what has recently emerged on the evolutionary histories of several key aspects of nuclear biology; the nuclear pore complex, the lamina, centrosomes and evidence for prokaryotic origins of relevant players.Work in our laboratories was supported by the following agencies, and which is gratefully acknowledged; MRC and Wellcome Trust (MR/K008749/1 and 090007/Z/09/Z respectively, to MCF), C2A Junta de Andalucia to DPD and DFG GR1642/4-1 to RG.Open Access funded by Wellcome Trust.Peer Reviewe

Building bridges between cellular and molecular structural biology

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.Peer reviewe

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Chromatin-remodeling factor ISWI is a microtubule-associated protein that contributes to chromosome segregation by stabilizing microtubules during anaphase

Genome Analysis of Planctomycetes Inhabiting Blades of the Red Alga

Porphyra is a macrophytic red alga of the Bangiales that is important ecologically and economically. We describe the genomes of three bacteria in the phylum Planctomycetes (designated P1, P2 and P3) that were isolated from blades of Porphyra umbilicalis (P.um.1). These three Operational Taxonomic Units (OTUs) belong to distinct genera; P2 belongs to the genus Rhodopirellula, while P1 and P3 represent undescribed genera within the Planctomycetes. Comparative analyses of the P1, P2 and P3 genomes show large expansions of distinct gene families, which can be widespread throughout the Planctomycetes (e.g., protein kinases, sensors/response regulators) and may relate to specific habitat (e.g., sulfatase gene expansions in marine Planctomycetes) or phylogenetic position. Notably, there are major differences among the Planctomycetes in the numbers and sub-functional diversity of enzymes (e.g., sulfatases, glycoside hydrolases, polysaccharide lyases) that allow these bacteria to access a range of sulfated polysaccharides in macroalgal cell walls. These differences suggest that the microbes have varied capacities for feeding on fixed carbon in the cell walls of P.um.1 and other macrophytic algae, although the activities among the various bacteria might be functionally complementary in situ. Additionally, phylogenetic analyses indicate augmentation of gene functions through expansions arising from gene duplications and horizontal gene transfers; examples include genes involved in cell wall degradation (e.g., κ-carrageenase, alginate lyase, fucosidase) and stress responses (e.g., efflux pump, amino acid transporter). Finally P1 and P2 contain various genes encoding selenoproteins, many of which are enzymes that ameliorate the impact of environmental stresses that occur in the intertidal habitat

Genome-Wide Influence of Indel Substitutions on Evolution of Bacteria of the PVC Superphylum, Revealed Using a Novel Computational Method

Whole-genome scans for positive Darwinian selection are widely used to detect evolution of genome novelty. Most approaches are based on evaluation of nonsynonymous to synonymous substitution rate ratio across evolutionary lineages. These methods are sensitive to saturation of synonymous sites and thus cannot be used to study evolution of distantly related organisms. In contrast, indels occur less frequently than amino acid replacements, accumulate more slowly, and can be employed to characterize evolution of diverged organisms. As indels are also subject to the forces of natural selection, they can generate functional changes through positive selection. Here, we present a new computational approach to detect selective constraints on indel substitutions at the whole-genome level for distantly related organisms. Our method is based on ancestral sequence reconstruction, takes into account the varying susceptibility of different types of secondary structure to indels, and according to simulation studies is conservative. We applied this newly developed framework to characterize the evolution of organisms of the Planctomycetes, Verrucomicrobia, Chlamydiae (PVC) bacterial superphylum. The superphylum contains organisms with unique cell biology, physiology, and diverse lifestyles. It includes bacteria with simple cell organization and more complex eukaryote-like compartmentalization. Lifestyles range from free-living organisms to obligate pathogens. In this study, we conduct a whole-genome level analysis of indel substitutions specific to evolutionary lineages of the PVC superphylum and found that indels evolved under positive selection on up to 12% of gene tree branches. We also analyzed possible functional consequences for several case studies of predicted indel events