Identifying ribosome heterogeneity using ribosome profiling

Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the ‘waste’ rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF.     

Origins of Allium ampeloprasum horticultural groups and a molecular phylogeny of the section Allium (Allium: Alliaceae)

The subgenus Allium section Allium includes economically important species, such as garlic and leek, as well as other polyploid minor crops. Phylogenetic studies within this section, with a focus on horticultural groups within A. ampeloprasum, were performed on 31 accessions of 17 species using the nuclear ribosomal DNA internal transcribed spacer (ITS) region and the chloroplast trnL-F and trnD-T regions. The results confirmed the monophyly of section Allium. Four main clades were identified on all ITS analyses but the relationships among those and the remaining species studied within section Allium remained unresolved. Trees based on cpDNA recovered two major clades and a topology only partly congruent with that of the ITS tree. Intra-individual polymorphism of the ITS region proved useful in tracking putative parent species of polyploid taxa. The allopolyploid origin of great headed garlic (GHG), A. iranicum and A. polyanthum was confirmed. No signs of hybridization in leek or kurrat were detected but possible introgression events were identified in pearl onion and bulbous leek. Although GHG is often used as a garlic substitute, molecular analysis revealed only a distant relationship with garlic. We also clarified the previous incorrect classification of cultivated forms within A. ampeloprasum, by showing that leek, kurrat, pearl onion, and bulbous leek should be considered separately from GHG.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 October 2009-30 November 2009

This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.     

The first phospholipase inhibitor from the serum of Vipera ammodytes ammodytes

Ammodytoxins are neurotoxic secretory phospholipase A(2) molecules, some of the most toxic components of the long-nosed viper (Vipera ammodytes ammodytes) venom. Envenomation by this and by closely related vipers is quite frequent in southern parts of Europe and serotherapy is used in the most severe cases. Because of occasional complications, alternative medical treatment of envenomation is needed. In the present study, ammodytoxin inhibitor was purified from the serum of V. a. ammodytes using two affinity procedures and a gel exclusion chromatography step. The ammodytoxin inhibitor from V. a. ammodytes serum consists of 23- and 25-kDa glycoproteins that form an oligomer, probably a tetramer, of about 100 kDa. N-terminal sequencing and immunological analysis revealed that both types of subunit are very similar to gamma-type secretory phospholipase A(2) inhibitors. The ammodytoxin inhibitor from V. a. ammodytes serum is a potent inhibitor of phospholipase activity and hence probably also the neurotoxicity of ammodytoxins. Discovery of the novel natural inhibitor of these potent secretory phospholipase A(2) toxins opens up prospects for the development of new types of small peptide inhibitors for use in regulating the physiological and pathological activities of secretory phospholipases A(2).     

Secreted Phospholipases A2 - not just Enzymes: Revisited

Secreted phospholipases A₂ (sPLA₂s) participate in a very broad spectrum of biological processes through their enzymatic activity and as ligands for membrane and soluble receptors. The physiological roles of sPLA₂s as enzymes have been very well described, while their functions as ligands are still poorly known. Since the last overview of sPLA₂-binding proteins (sPLA₂-BPs) 10 years ago, several important discoveries have occurred in this area. New and more sensitive analytical tools have enabled the discovery of additional sPLA₂-BPs, which are presented and critically discussed here. The structural diversity of sPLA₂-BPs reveals sPLA₂s as very promiscuous proteins, and we offer some structural explanations for this nature that makes these proteins evolutionarily highly advantageous. Three areas of physiological engagement of sPLA₂-BPs have appeared most clearly: cellular transport and signalling, and regulation of the enzymatic activity of sPLA₂s. Due to the multifunctionality of sPLA₂s, they appear to be exceptional pharmacological targets. We reveal the potential to exploit interactions of sPLA₂s with other proteins in medical terms, for the development of original diagnostic and therapeutic procedures. We conclude this survey by suggesting the priority questions that need to be answered.     

Life and death in aluminium-exposed cultures of rat lactotrophs studied by flow cytometry

Prolonged exposure to aluminium may impact health. Aluminium’s deleterious effects are mostly attributed to its selective accumulation in particular organs and cell types. Occupational exposure to aluminium is allied with a reduced level of serum prolactin, a stress peptide hormone mainly synthesised and secreted by the anterior pituitary lactotrophs. Our aim was to study the effect of aluminium on the viability of rat lactotrophs in primary suspension cultures where multicellular aggregates tend to form, comprising approximately two thirds of the total cell population as confirmed by confocal microscopy. Flow cytometric light scattering of calcein acetoxymethyl ester and ethidium homodimer-1 labelled cells was used to define subpopulations of live and dead cells in heterogeneous suspensions comprised of single cells and multicellular aggregates of distinct size. Concentration-dependent effects of AlCl₃ were observed on aggregate size and cell survival. After 24-h exposure to 3 mM AlCl₃, viability of single cells declined from 5% to 3%, while in multicellular aggregates, viability declined from 23% to 20%. The proportion of single cells increased from 30% to 42% within the same concentration range, while in large aggregates, the proportion remained approximately constant representing 35% of the cell suspension. In large aggregates, cell viability (75%) remained unaltered after exposure to AlCl₃ concentrations up to 300 μM, while in single cells, viability was halved at 30 μM. In conclusion, our finding indicates that prolonged exposure to aluminium may lead to significant loss of pituitary cells.     

Expanding the host cell ubiquitylation machinery targeting cytosolic Salmonella

Ubiquitylation is one of the cardinal post‐translational modifications in the cell, balancing several distinct biological processes and acting as a pathogen recognition receptor during bacterial pathogen invasion. A dense layer of polyubiquitin chains marks invading bacteria that gain access to the host cytosol for their selective clearance via xenophagy. However, the enzymes that mediate recognition of cytosolic bacteria and generate this ubiquitin (Ub) coat remain largely elusive. To address this, we employed an image‐based RNAi screening approach to monitor the loss of Ub on Salmonella upon depletion of human Ub E3 ligases in cells. Using this approach, we identified ARIH1 as one of the ligases involved in the formation of Ub coat on cytosolic bacteria. In addition, we provide evidence that the RING‐between‐RING ligase ARIH1, together with LRSAM1 and HOIP, forms part of a network of ligases that orchestrates recognition of intracellular Salmonella and participates in the activation of the host cell immune response.