CAIcal: A combined set of tools to assess codon usage adaptation

 

A recent systematic survey suggested that the YRG (or YawG/YlqF) family with the G4-G5-G1-G2-G3 order of the conserved GTPase motifs represents the only possible circularly permuted variation of the canonical GTPase structure. Here we show that a different circularly permuted GTPase domain actually does exist, conforming to the pattern G3-G4-G5-G1-G2. The domain, dubbed cpRAS, is a variant of RAS family GTPases and occurs in two types of larger proteins, either inserted into a region homologous to a bacterial group of proteins classified as COG2373 and potentially related to the alpha-2-macroglobulin family (so far a single protein in Dictyostelium) or in combination with a von Willebrand factor type A (VWA) domain. For the latter protein type, which was found in a few metazoans and several distantly related protists, existence in the common ancestor of opisthokonts, Amoebozoa and excavates followed by at least eight independent losses may be inferred. Our findings thus bring further evidence for the importance of parallel reduction of ancestral complexity in the eukaryotic evolution.

Reviewers

This article was reviewed by Lakshminarayan Iyer and Fyodor Kondrashov. For the full reviews, please go to the Reviewers' comments section.     

Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

Dysregulation of intracellular Ca²⁺ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer''s Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ_1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca²⁺ in neurons and promote mitochondrial Ca²⁺ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca²⁺ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca²⁺ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca²⁺ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca²⁺ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.     

A new antigen retrieval technique for human brain tissue

Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times.     

A QM/MM study on the enzymatic inactivation of cefotaxime

The reaction between the antibiotic cefotaxime and the CTX-M-14 class A serine hydrolase is addressed from a theoretical point of view, by means of hybrid quantum mechanics/molecular mechanical (QM/MM) calculations, adopting a new approach that postulates that the residue Ser70 itself should play the role of the acid-base species required for the cefotaxime acylation. The proposed mechanism differs from earlier proposals existing in literature for other class A β-lactamases. The results confirm the hypothesis, and show that the reaction should occur via a concerted mechanism in which the acylation of the lactam carbonyl carbon, protonation of the N₇ lactam atom, and opening of the β-lactam ring occurs simultaneously. Exploration of the potential energy surface shows three critical points, associated with reactants, transition state and product. The transition state is characterized by frequency, intrinsic reaction coordinate, atomic charge, and bond orders calculations. The calculated activation barrier is 20 kcal mol^?1, and the reaction appears to be slightly endothermic by about 12 kcal mol^?1. We conclude that this approach is feasible, and should be considered as an alternative mechanism or may exist in competition with others already published in the literature. This information should be useful for the design of novel antibiotics and β-lactamase inhibitors.

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Graphical abstract Three-dimensional view of the potential energy surface of cefotaxime

     

Mapping the Spatial Proteome of Metastatic Cells in Colorectal Cancer

Colorectal cancer (CRC) is the second deadliest cancer worldwide. Here, we aimed to study metastasis mechanisms using spatial proteomics in the KM12 cell model. Cells were SILAC‐labeled and fractionated into five subcellular fractions corresponding to: cytoplasm, plasma, mitochondria and ER/golgi membranes, nuclear, chromatin‐bound and cytoskeletal proteins and analyzed with high resolution mass spectrometry. We provide localization data of 4863 quantified proteins in the different subcellular fractions. A total of 1318 proteins with at least 1.5‐fold change were deregulated in highly metastatic KM12SM cells respect to KM12C cells. The protein network organization, protein complexes and functional pathways associated to CRC metastasis was revealed with spatial resolution. Although 92% of the differentially expressed proteins showed the same deregulation in all subcellular compartments, a subset of 117 proteins (8%) showed opposite changes in different subcellular localizations. The chaperonin CCT, the Eif2 and Eif3 initiation of translation and the oxidative phosphorylation complexes together with an important number of guanine nucleotide‐binding proteins, were deregulated in abundance and localization within the metastatic cells. Particularly relevant was the relationship of deregulated protein complexes with exosome secretion. The knowledge of the spatial proteome alterations at subcellular level contributes to clarify the molecular mechanisms underlying colorectal cancer metastasis and to identify potential targets of therapeutic intervention.     

Exploring growth‐defence trade‐offs in Arabidopsis: phytochrome B inactivation requires JAZ10 to suppress plant immunity but not to trigger shade‐avoidance responses

Under conditions that involve a high risk of competition for light among neighbouring plants, shade‐intolerant species often display increased shoot elongation and greater susceptibility to pathogens and herbivores. The functional links between morphological and defence responses to crowding are not well understood. In Arabidopsis, the protein JAZ10 is thought to play a key role connecting the inactivation of the photoreceptor phytochrome B (phyB), which takes place under competition for light, with the repression of jasmonate‐mediated plant defences. Here, we show that a null mutation of the JAZ10 gene in Arabidopsis did not affect plant growth nor did it suppress the shade‐avoidance responses elicited by phyB inactivation. However, the jaz10 mutation restored many of the defence traits that are missing in the phyB mutant, including the ability to express robust responses to jasmonate and to accumulate indolic glucosinolates. Furthermore, the jaz10phyB double mutant showed a significantly increased resistance to the pathogenic fungus Botrytis cinerea compared with the phyB parental line. Our results demonstrate that, by inactivating JAZ10, it is possible to partially uncouple shade avoidance from defence suppression in Arabidopsis. These findings may provide clues to improve plant resistance to pathogens in crops that are planted at high density.     

Phylogeny and taxonomy of the genus Ilyodon Eigenmann, 1907 (Teleostei: Goodeidae), based on mitochondrial and nuclear DNA sequences

Taxonomy of the live‐bearing fish of the genus Ilyodon Eigenmann, 1907 (Goodeidae), in Mexico, is controversial, with morphology and mitochondrial genetic analyses in disagreement about the number of valid species. The present study accumulated a comprehensive DNA sequences dataset of 98 individuals of all Ilyodon species and mitochondrial and nuclear loci to reconstruct the evolutionary history of the genus. Phylogenetic inference produced five clades, one with two sub‐clades, and one clade including three recognized species. Genetic distances in mitochondrial genes (cytb: 0.5%–2.1%; coxI: 0.5%–1.1% and d‐loop: 2.3%–10.2%) were relatively high among main clades, while, as expected, nuclear genes showed low variation (0.0%–0.2%), with geographic concordance and few shared haplotypes among river basins. High genetic structure was observed among clades and within basins. Our genetic analyses, applying the priority principle, suggest the recognition only of Ilyodon whitei and Ilyodon furcidens, with I. cortesae relegated to an invalid species, the populations of which belong to I. whitei.     

Genetic favouring of pheomelanin‐based pigmentation limits physiological benefits of coloniality in lesser kestrels Falco naumanni

Pheomelanin contributes to the pigmentation phenotype of animals by producing orange and light brown colours in the integument. However, pheomelanin synthesis in melanocytes requires consumption of glutathione (GSH), the most important intracellular antioxidant. Therefore, a genetic control favouring the production of large amounts of pheomelanin for pigmentation may lead to physiological costs under environmental conditions that promote oxidative stress. We investigated this possibility in the context of breeding coloniality, a reproductive strategy that may affect oxidative stress. We found in lesser kestrel Falco naumanni nestlings that the GSH:GSSG ratio, which decreases with systemic oxidative stress, increased with the size of the colony where they were reared, but the expression in feather melanocytes of five genes involved in pheomelanin synthesis (Slc7a11, Slc45a2, CTNS, MC1R and AGRP) did not vary with colony size. The antioxidant capacity (TEAC) of lesser kestrel nestlings also increased with colony size, but in a manner that depended on Slc7a11 expression and not on the expression of the other genes. Thus, antioxidant capacity increased with colony size only in nestlings least expressing Slc7a11, a gene with a known role in mediating cysteine (a constituent amino acid of GSH) consumption for pheomelanin production. The main predictor of the intensity of pheomelanin‐based feather colour was Slc45a2 expression followed in importance by Slc7a11 expression, hence suggesting that the genetic regulation of the pigmentation phenotype mediated by Slc7a11 and a lack of epigenetic lability in this gene limits birds from benefiting from the physiological benefits of coloniality.     

No evidence for costs of being large in females of Orgyia spp. (Lepidoptera, Lymantriidae): larger is always better

Strong correlation between female body size and potential fecundity is often observed in insects. Directional selection favouring increased body sizes is thus predicted in the absence of opposing selection pressure. The evolutionary forces capable of counterbalancing such a 'fecundity advantage' are poorly documented. This study focuses on revealing the costs of large body size in the wingless females of Orgyia antiqua and O. leucostigma, two related species of lymantriid moths. Extreme behavioural simplicity of these animals allows systematic assessment of various fitness components in conditions that are close to natural. A linear relationship between pupal weight and potential fecundity was observed. This association was found to be independent of particular rearing conditions. There was no evidence that the relationship between fecundity and body mass becomes asymptotic when body sizes increases. No component of fitness showed a negative phenotypic correlation with body size; some displayed a weakly positive one. In particular, pupal mortality, adult longevity, mating and oviposition success, as well as egg hatching rate and egg size, were established as independent of body size in a series of field and laboratory experiments. There was a very high overall efficiency of converting resources accumulated during the larval stage to egg masses. With no costs of large adult size, selective forces balancing the fecundity advantage should operate in the course of immature development. The strong dependence of realized fecundity on body size is considered characteristic of the capital breeding strategy.