A general overview of the multifactorial adaptation to cold: biochemical mechanisms and strategies.

Cold environments are more frequent than people think. They include deep oceans, cold lakes, snow, permafrost, sea ice, glaciers, cold soils, cold deserts, caves, areas at elevations greater than 3000 m, and also artificial refrigeration systems. These environments are inhabited by a diversity of eukaryotic and prokaryotic organisms that must adapt to the hard conditions imposed by cold. This adaptation is multifactorial and includes (i) sensing the cold, mainly through the modification of the liquid-crystalline membrane state, leading to the activation of a two-component system that transduce the signal; (ii) adapting the composition of membranes for proper functions mainly due to the production of double bonds in lipids, changes in hopanoid composition, and the inclusion of pigments; (iii) producing cold-adapted proteins, some of which show modifications in the composition of amino acids involved in stabilizing interactions and structural adaptations, e.g., enzymes with high catalytic efficiency; and (iv) producing ice-binding proteins and anti-freeze proteins, extracellular polysaccharides and compatible solutes that protect cells from intracellular and extracellular ice. However, organisms also respond by reprogramming their metabolism and specifically inducing cold-shock and cold-adaptation genes through strategies such as DNA supercoiling, distinctive signatures in promoter regions and/or the action of CSPs on mRNAs, among others. In this review, we describe the main findings about how organisms adapt to cold, with a focus in prokaryotes and linking the information with findings in eukaryotes.

Embryonic developmental arrest in the annual killifish Austrolebias charrua: A proteomic approach to diapause III.

Diapause is a reversible developmental arrest faced by many organisms in harsh environments. Annual killifish present this mechanism in three possible stages of development. Killifish are freshwater teleosts from Africa and America that live in ephemeral ponds, which dry up in the dry season. The juvenile and adult populations die, and the embryos remain buried in the bottom mud until the next rainy season. Thus, species survival is entirely embryo-dependent, and they are perhaps the most remarkable extremophile organisms among vertebrates. The aim of the present study was to gather information about embryonic diapauses with the use of a "shotgun" proteomics approach in diapause III and prehatching Austrolebias charrua embryos. Our results provide insight into the molecular mechanisms of diapause III. Data are available via ProteomeXchange with identifier PXD025196. We detected a diapause-dependent change in a large group of proteins involved in different functions, such as metabolic pathways and stress tolerance, as well as proteins related to DNA repair and epigenetic modifications. Furthermore, we observed a diapause-associated switch in cytoskeletal proteins. This first glance into global protein expression differences between prehatching and diapause III could provide clues regarding the induction/maintenance of this developmental arrest in A. charrua embryos. There appears to be no single mechanism underlying diapause and the present data expand our knowledge of the molecular basis of diapause regulation. This information will be useful for future comparative approaches among different diapauses in annual killifish and/or other organisms that experience developmental arrest.

On the Track of the Missing tRNA Genes: A Source of Non-Canonical Functions?

Cellular tRNAs appear today as a diverse population of informative macromolecules with conserved general elements ensuring essential common functions and different and distinctive features securing specific interactions and activities. Their differential expression and the variety of post-transcriptional modifications they are subject to, lead to the existence of complex repertoires of tRNA populations adjusted to defined cellular states. Despite the tRNA-coding genes redundancy in prokaryote and eukaryote genomes, it is surprising to note the absence of genes coding specific translational-active isoacceptors throughout the phylogeny. Through the analysis of different releases of tRNA databases, this review aims to provide a general summary about those "missing tRNA genes." This absence refers to both tRNAs that are not encoded in the genome, as well as others that show critical sequence variations that would prevent their activity as canonical translation adaptor molecules. Notably, while a group of genes are universally missing, others are absent in particular kingdoms. Functional information available allows to hypothesize that the exclusion of isodecoding molecules would be linked to: 1) reduce ambiguities of signals that define the specificity of the interactions in which the tRNAs are involved; 2) ensure the adaptation of the translational apparatus to the cellular state; 3) divert particular tRNA variants from ribosomal protein synthesis to other cellular functions. This leads to consider the "missing tRNA genes" as a source of putative non-canonical tRNA functions and to broaden the concept of adapter molecules in ribosomal-dependent protein synthesis.

Fine-tuning the metabolic rewiring and adaptation of translational machinery during an epithelial-mesenchymal transition in breast cancer cells.

BACKGROUND: During breast cancer progression, the epithelial to mesenchymal transition has been associated with metastasis and endocrine therapy resistance; however, the underlying mechanisms remain elusive. To gain insight into this process, we studied the transition undergone by MCF7-derived cells, which is driven by the constitutive nuclear expression of a MKL1 variant devoid of the actin-binding domain (MKL1 ΔN200). We characterized the adaptive changes that occur during the MKL1-induced cellular model and focused on regulation of translation machinery and metabolic adaptation. METHODS: We performed a genome-wide analysis at the transcriptional and translational level using ribosome profiling complemented with RNA-Seq and analyzed the expression of components of the translation machinery and enzymes involved in energy metabolism. NGS data were correlated with metabolomic measurements and quantification of specific mRNAs extracted from polysomes and western blots. RESULTS: Our results reveal the expression profiles of a luminal to basal-like state in accordance with an epithelial to mesenchymal transition. During the transition, the synthesis of ribosomal proteins and that of many translational factors was upregulated. This overexpression of the translational machinery appears to be regulated at the translational level. Our results indicate an increase of ribosome biogenesis and translation activity. We detected an extensive metabolic rewiring occurring in an already "Warburg-like" context, in which enzyme isoform switches and metabolic shunts indicate a crucial role of HIF-1α along with other master regulatory factors. Furthermore, we detected a decrease in the expression of enzymes involved in ribonucleotide synthesis from the pentose phosphate pathway. During this transition, cells increase in size, downregulate genes associated with proliferation, and strongly upregulate expression of cytoskeletal and extracellular matrix genes. CONCLUSIONS: Our study reveals multiple regulatory events associated with metabolic and translational machinery adaptation during an epithelial mesenchymal-like transition process. During this major cellular transition, cells achieve a new homeostatic state ensuring their survival. This work shows that ribosome profiling complemented with RNA-Seq is a powerful approach to unveil in-depth global adaptive cellular responses and the interconnection among regulatory circuits, which will be helpful for identification of new therapeutic targets.

Correction to: Neuropilin-1 receptor in the rapid and selective estrogen-induced neurovascular remodeling of rat uterus.

The article title of the original publication contains error for the term "estrogen" was captured twice.

Neuropilin-1 receptor in the rapid and selective estrogen-induced neurovascular remodeling of rat uterus.

Sympathetic nerves innervate most organs and regulate organ blood flow. Specifically, in the uterus, estradiol (E2) elicits rapid degeneration of sympathetic axons and stimulates the growth of blood vessels. Both physiological remodeling processes, critical for reproduction, have been extensively studied but as independent events and are still not fully understood. Here, we examine the neuropilin-1 (NRP1), a shared receptor for axon guidance and angiogenic factors. Systemic estradiol or vehicle were chronically injected to prepubertal rats and uterine and sympathetic chain sections immunostained for NRP1. Uterine semaphorin-3A mRNA was evaluated by in situ hybridization. Control sympathetic uterine-projecting neurons (1-month-old) expressed NRP1 in their somas but not in their intrauterine terminal axons. Estradiol did not affect NRP1 in the distal ganglia. However, at the entrance of the organ, some sympathetic NRP1-positive nerves were recognized. Vascular NRP1 was confined to intrauterine small-diameter vessels in both hormonal conditions. Although the overall pattern of NRP1-IR was not affected by E2 treatment, a subpopulation of infiltrated eosinophil leukocytes showed immunoreactivity for NRP1. Sema3A transcripts were detected in this cellular type as well. No NRP1-immunoreactive axons nor infiltrated eosinophils were visualized in other estrogenized pelvic organs. Together, these data suggest the involvement of NRP1/Sema3A signaling in the selective E2-induced uterine neurovascular remodeling. Our data support a model whereby NRP1 could coordinate E2-induced uterine neurovascular remodeling, acting as a positive regulator of growth when expressed in vessels and as a negative regulator of growth when expressed on axons.

Echinococcus granulosus: Insights into the protoscolex F-actin cytoskeleton.

Echinococcus granulosus is a cestode parasite whose cytoskeleton plasticity allows it to enter and develop inside its hosts, completing thus its life cycle. We focused our attention on F-actin organization and distribution in E. granulosus protoscoleces (PSC) in order to contribute to the knowledge of the parasite cytoskeleton. In particular, we addressed some aspects of F-actin rearrangements in PSC at different stages of the evagination/invagination process. The use of light microscopy allowed us to identify different PSC structures and phalloidin staining displayed a parasite's highly organized F-actin cytoskeleton. Suckers exhibit an important musculature composed of a set of radial fibers. At the rostellum, the F-actin filaments are arranged in a bulbar shape with perforations that appear to be the attachment places for the hooks. Also, "circular" structures of F-actin were identified, which remind the flame cells. Furthermore, parasite F-actin filaments, unevenly distributed, seem to have remained substantially unchanged during the evagination/invagination process. Finally, we showed that the scolex of an evaginated E. granulosus PSC reinvaginates in vitro without any treatment.

A high resolution analysis of the structure and chemical composition of the calcareous corpuscles from Mesocestoides corti.

Mesocestoides corti (syn. vogae), similar to many other cestode platyhelminthes, contains abundant calcium carbonate structures called calcareous corpuscles. These concretions that may constitute as much as 40% of the dry weight of the body, and were proposed to form intracellularly in certain parenchymal cells. As an approach to elucidate the biological role of calcareous corpuscles in cestodes, our aim was to characterize more precisely the structure and topological composition of the corpuscles from M. corti. Employing a variety of high resolution technical approaches, we found that the calcareous corpuscles are spheroid or ovoid layered concretions. They are formed by topographically homogeneous but compositionally heterogeneous layers, suggesting a cyclic process of biomineralization. The layers are composite structures, with granules of tens of nanometers, each surrounded by a cortex of about eight nanometers.

Estrogen up-regulation of semaphorin 3F correlates with sympathetic denervation of the rat uterus.

Current evidence indicates that rises in systemic levels of estrogen create in the uterus an inhibitory environment for sympathetic nerves. However, molecular insights of these changes are far from complete. We evaluated if semaphorin 3F mRNA, a sympathetic nerve repellent, was produced by the rat uterus and if its expression was modulated by estrogen. We also analyzed whether uterine nerves express the semaphorin 3F binding receptor, neuropilin-2. Uterine levels of semaphorin 3F mRNA were measured using real time reverse transcriptase-polymerase chain reaction in prepubertal rat controls and following chronic estrogen treatment. Localization of semaphorin 3F transcripts was determined by in situ hybridization and the expression of neuropilin-2 was assessed by immunohistochemistry. These studies showed that: (1) chronic estrogen treatment led to a 5-fold induction of semaphorin 3F mRNA in the immature uterus; (2) estrogen provoked a tissue-specific induction of semaphorin 3F which was particularly localized in the connective tissue that borders muscle bundles and surrounds intrauterine blood vessels; (3) two major cell-types were recognized in the areas where transcripts were concentrated, fibroblast-like cells and infiltrating eosinophil leukocytes; and (4) some delicate nerve terminal profiles present in the estrogenized uterus were immunoreactive for neuropilin-2. Temporal and spatial expression patterns of semaphorin 3F/neuropilin-2 are consistent with a possible role of this guidance cue in the remodeling of uterine sympathetic innervation by estrogen. Though correlative in its nature, these data support a model whereby semaphorin 3F, in combination with other inhibitory molecules, converts the estrogenized myometrium to an inhospitable environment for sympathetic nerves.

Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus.

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 micromol min(-1)mg(-1)), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

Monohydrocalcite in calcareous corpuscles of Mesocestoides corti.

Mesocestoides corti (syn. vogae), as many other cestode platyhelminthes, contains abundant mineralized structures called calcareous corpuscles. These concretions may constitute as much as 40% of the dry weight of the organisms, but their function remains poorly understood. In this work, we reviewed the mineral composition of the calcareous corpuscles of M. corti. X-ray diffraction pattern showed that the major mineral component of the corpuscles is a hydrated form of calcium carbonate, monohydrocalcite, also confirmed by infrared spectrometry. The baseline shift of the X-ray diffraction spectra suggested the presence of amorphous calcium carbonate, accordingly to previous reports, and an organic matrix was confirmed by FTIR. Monohydrocalcite is a rare mineral unusually found in biominerals. Although the significance of monohydrocalcite in biominerals has not been determined, the knowledge of corpuscles composition is of relevance to establish their function and for the elucidation of the mechanisms involved in mineralization processes.

Characterization of hypoxia induced gene 1: expression during rat central nervous system maturation and evidence of antisense RNA expression.

Although recent studies have provided a detailed understanding of cellular interactions occurring during the development of the CNS, little is known about the molecular signals which during the peri and postnatal periods ensure its maturation and functionality. Using the mammalian spinal cord as a model, we have designed experiments to examine the main changes in gene expression occurring during this critical transition. In this paper we describe the cloning and characterization of the rat hypoxia induced gene-1 (Hig-1), its expression pattern during spinal cord maturation and in situ localization of its mRNA. We show an increase in Hig-1 expression between P1 and P15 in the spinal cord and a differential spatial pattern. In the P1 spinal cord we observed preferential expression in regions of dorsal laminae II and III and laminae IX ventrally; while in P8, the distribution was more widespread and overall expression was increased. Hig-1 is also widely expressed in the brain. Results of in situ hybridization experiments, as well as particular features concerning ESTs, led us to propose the expression of an antisense mRNA. Primer-specific RTPCR demonstrates the presence of this aHig-1 transcript whose structure has not yet been characterized. The high homology between putative rHig-1 protein and human- and murine-predicted sequences, as well as its characteristic expression in the Central Nervous System, are indicative of a specific role which could be related to apoptosis signaling during postnatal maturation.

Linked thioredoxin-glutathione systems in platyhelminths.

The thioredoxin and glutathione systems play a central role in thiol-disulfide redox homeostasis in many organisms by providing electrons to essential enzymes, and defence against oxidative stress. These systems have recently been characterized in platyhelminth parasites, and the emerging biochemical scenario is the existence of linked processes with the enzyme thioredoxin glutathione reductase supplying reducing equivalents to both pathways. In contrast to their hosts, conventional thioredoxin reductase and glutathione reductase enzymes appear to be absent. Analysis of published data and expressed-sequence tag databases indicates the presence of linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of these parasites.

Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus.

As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 degrees C, in the range 6.5-7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.

Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase.

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.