Computational Methods Reveal a Series of Cyclic and Linear Lichenysins and Surfactins from the Vietnamese Marine Sediment-Derived Streptomyces Strain G222.

The Streptomyces strain G222, isolated from a Vietnamese marine sediment, was confidently identified by 16S rRNA gene sequencing. Its AcOEt crude extract was successfully analyzed using non-targeted LC-MS/MS analysis, and molecular networking, leading to a putative annotation of its chemical diversity thanks to spectral libraries from GNPS and in silico metabolite structure prediction obtained from SIRIUS combined with the bioinformatics tool conCISE (Consensus Annotation Propagation of in silico Elucidations). This dereplication strategy allowed the identification of an interesting cluster of a series of putative cyclic and linear lipopeptides of the lichenysin and surfactin families. Lichenysins (3-7) were isolated from the sub-fraction, which showed significant anti-biofilm activity against Pseudomonas aeruginosa MUC-N1. Their structures were confirmed by detailed 1D and 2D NMR spectroscopy (COSY, HSQC, HMBC, TOCSY, ROESY) recorded in CD3OH, and their absolute configurations were determined using the modified Marfey's method. The isolated lichenysins showed anti-biofilm activity at a minimum concentration of 100 µM. When evaluated for antibacterial activity against a panel of Gram-positive and Gram-negative strains, two isolated lichenysins exhibited selective activity against the MRSA strain without affecting its growth curve and without membranotropic activity. This study highlights the power of the MS/MS spectral similarity strategy using computational methods to obtain a cross-validation of the annotated molecules from the complex metabolic profile of a marine sediment-derived Streptomyces extract. This work provides the first report from a Streptomyces strain of combined cyclic and linear lichenysins and surfactins, known to be characteristic compounds of the genus Bacillus.

Iridoid- and flavonoid-enriched fractions of Cornus sanguinea and Cornus mas exert antioxidant and anti-inflammatory effects and inhibit key enzymes in the treatment of metabolic disorders.

Background: Berry fruits are recognized as a "superfood" due to their high content of bioactive compounds and health benefits. Scope and approach: Herein, extracts of Cornus sanguinea and Cornus mas fresh and dried fruits obtained by different extraction procedures (ethanolic and hydroalcoholic maceration, ultrasound-assisted extraction, and Soxhlet apparatus) were analysed using liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-QTOF-MS) and compared to identify the main healthy compounds and their impact on the inhibition of key enzymes (pancreatic lipase, α-glucosidase, and α-amylase) associated with metabolic disorders. The antioxidant activity and inhibition of nitric oxide (NO) and NF-κB pathway were also investigated. Key findings and conclusions: Flavonoids, iridoids, and phenolic acids were the main classes of identified compounds. Herein, kaempferol 3-O-galactoside, kaempferol 3-O-glucoside, quercetin, quercetin 3-O-xyloside, and myricetin 3-O-galactoside were detected for the first time in C. sanguinea. Remarkable antioxidant effects and promising α-glucosidase and lipase inhibitory activity were observed with extracts obtained by hydroalcoholic maceration of both Cornus dried fruits. Consequently, these extracts were subjected to fractionation using Amberlite XAD-16 resin. The most promising biological activities, which are attributed to the presence of some flavonoids and iridoids, were detected with the C. sanguinea fractions, in particular SD2(II). The results of this study offer new insights into the potential development of functional foods, nutraceuticals, and food supplements using the Cornus species.

Molecular acclimation of Halobacterium salinarum to halite brine inclusions.

Halophilic microorganisms have long been known to survive within the brine inclusions of salt crystals, as evidenced by the change in color for salt crystals containing pigmented halophiles. However, the molecular mechanisms allowing this survival has remained an open question for decades. While protocols for the surface sterilization of halite (NaCl) have enabled isolation of cells and DNA from within halite brine inclusions, "-omics" based approaches have faced two main technical challenges: (1) removal of all contaminating organic biomolecules (including proteins) from halite surfaces, and (2) performing selective biomolecule extractions directly from cells contained within halite brine inclusions with sufficient speed to avoid modifications in gene expression during extraction. In this study, we tested different methods to resolve these two technical challenges. Following this method development, we then applied the optimized methods to perform the first examination of the early acclimation of a model haloarchaeon (Halobacterium salinarum NRC-1) to halite brine inclusions. Examinations of the proteome of Halobacterium cells two months post-evaporation revealed a high degree of similarity with stationary phase liquid cultures, but with a sharp down-regulation of ribosomal proteins. While proteins for central metabolism were part of the shared proteome between liquid cultures and halite brine inclusions, proteins involved in cell mobility (archaellum, gas vesicles) were either absent or less abundant in halite samples. Proteins unique to cells within brine inclusions included transporters, suggesting modified interactions between cells and the surrounding brine inclusion microenvironment. The methods and hypotheses presented here enable future studies of the survival of halophiles in both culture model and natural halite systems.

Optoproteomics elucidates the interactome of L-type amino acid transporter 3 (LAT3).

Membrane protein interactions are crucial for diverse biological processes. We report the application of genetic code expansion in combination with photo-crosslinking chemistry, as we termed "optoproteomics", to identify proteins interacting with the human L-type membrane amino acid transporter 3 (LAT3, also known as SLC43A1). The site-specifically incorporated photo-cross-linker p-azido-L-phenylalanine (AzF), which reacts with proteins in their proximity, enabled the capture of weak and transient partners of LAT3 in living cells. We identify 11 unique interacting proteins which are light-sensitive and 19 unique proteins that are site-specific, validating the approach and providing insights into the LAT3 protein-protein interaction network currently unavailable.

Untangling the fibre ball: Proteomic characterization of South American camelid hair fibres by untargeted multivariate analysis and molecular networking.

The proteomic analysis of hairs, yarns or textiles has emerged as a powerful method to determine species of origin, mainly used in archaeozoological research and fraud control. Differentiation between the South American camelid (SAC) species (the wild guanaco and vicuña and their respective domesticates the llama and alpaca) is particularly challenging due to poor database information and significant hybridization between species. In this study, we analysed 41 modern and 4 archaeological samples from the four SACs species. Despite strong similarities with Old World Camelidae, we identified 7 peptides specific to SACs assigned to keratin K86 and the keratin-associated proteins KAP13-1 and KAP11-1. Untargeted multivariate analysis of the LC-MS data permitted to distinguish SAC species and propose discriminant features. MS/MS-based molecular networking combined with database-assisted de novo sequencing permitted to identify 5 new taxonomic peptides assigned to K33a, K81 and/or K83 keratins and KAP19-1. These peptides differentiate the two wild species, guanaco and vicuña. These results show the value of combining database search and untargeted metabolomic approaches for paleoproteomics, and reveal for the first time the potential of molecular networks to highlight deamidation related to diagenesis and cluster highly similar peptides related to interchain homologies or intra- or inter-specific polymorphism. SIGNIFICANCE: This study used an innovative approach combining multivariate analysis of LC-MS data together with molecular networking and database-assisted de novo sequencing to identify taxonomic peptides in palaeoproteomics. It constitutes the first attempt to differentiate between hair fibres from the four South American camelids (SACs) based on proteomic analysis of modern and archaeological samples. It provides different proteomic signatures for each of the four SAC species and proposes new SAC taxonomic peptides of interest in archaeozoology and fraud control. SACs have been extensively exploited since human colonization of South America but have not been studied to the extent of their economic, cultural and heritage importance. Applied to the analysis of ancient Andean textiles, our results should permit a better understanding of cultural and pastoral practices in South America. The wild SACs are endangered by poaching and black-market sale of their fibre. For the first time, our results provide discriminant features for the determination of species of origin of contraband fibre.

Deciphering mollusc shell production: the roles of genetic mechanisms through to ecology, aquaculture and biomimetics.

Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing (RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are still debated, with CaCO3 precipitation estimates ranging from 1-2 J/mg to 17-55 J/mg depending on experimental and environmental conditions. However, organic components are more expensive (~29 J/mg) and recent data indicate transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi) and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1 gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs. Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.

Deciphering shell proteome within different Baltic populations of mytilid mussels illustrates important local variability and potential consequences in the context of changing marine conditions.

Molluscs defend themselves against predation and environmental stressors through the possession of mineralized shells. Mussels are widely used to predict the effects of abiotic factors such as salinity and pH on marine calcifiers in the context of changing ocean conditions. Shell matrix proteins are part of the molecular control regulating the biomineralization processes underpinning shell production. Under changing environmental conditions, differential expression of these proteins leads to the phenotypic plasticity of shells seen in many mollusc species. Low salinity decreases the availability of calcium and inorganic carbon in seawater and consequently energetic constraints often lead to thin, small and fragile shells in Mytilid mussels inhabiting Baltic Sea. To understand how the modulation of shell matrix proteins alters biomineralization, we compared the shell proteomes of mussels living under full marine conditions in the North Sea to those living in the low saline Baltic Sea. Modulation of proteins comprising the Mytilus biomineralization tool kit is observed. These data showed a relative increase in chitin related proteins, decrease in SD-rich, GA-rich shell matrix proteins indicating that altered protein scaffolding and mineral nucleation lead to impaired shell microstructures influencing shell resistance in Baltic Mytilid mussels. Interestingly, proteins with immunity domains in the shell matrix are also found to be modulated. Shell traits such as periostracum thickness, organic content and fracture resistance qualitatively correlates with the modulation of SMPs in Mytilid mussels providing key insights into control of biomineralization at molecular level in the context of changing marine conditions.

Global Metabolomic Characterizations of Microcystis spp. Highlights Clonal Diversity in Natural Bloom-Forming Populations and Expands Metabolite Structural Diversity.

Cyanobacteria are photosynthetic prokaryotes capable of synthesizing a large variety of secondary metabolites that exhibit significant bioactivity or toxicity. Microcystis constitutes one of the most common cyanobacterial genera, forming the intensive blooms that nowadays arise in freshwater ecosystems worldwide. Species in this genus can produce numerous cyanotoxins (i.e., toxic cyanobacterial metabolites), which can be harmful to human health and aquatic organisms. To better understand variations in cyanotoxin production between clones of Microcystis species, we investigated the diversity of 24 strains isolated from the same blooms or from different populations in various geographical areas. Strains were compared by genotyping with 16S-ITS fragment sequencing and metabolite chemotyping using LC ESI-qTOF mass spectrometry. While genotyping can help to discriminate among different species, the global metabolome analysis revealed clearly discriminating molecular profiles among strains. These profiles could be clustered primarily according to their global metabolite content, then according to their genotype, and finally according to their sampling location. A global molecular network of all metabolites produced by Microcystis species highlights the production of a wide set of chemically diverse metabolites, including a few microcystins, many aeruginosins, microginins, cyanopeptolins, and anabaenopeptins, together with a large set of unknown molecules. These components, which constitute the molecular biodiversity of Microcystis species, still need to be investigated in terms of their structure and potential bioactivites (e.g., toxicity).

Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver.

Cyanobacterial blooms have become a common phenomenon in eutrophic freshwater ecosystems worldwide. Microcystis is an important bloom-forming and toxin-producing genus in continental aquatic ecosystems, which poses a potential risk to Human populations as well as on aquatic organisms. Microcystis is known to produce along with various bioactive peptides, the microcystins (MCs) that have attracted more attention notably due to their high hepatotoxicity. To better understand the effects of cyanobacterial blooms on fish, medaka fish (Oryzias latipes) were sub-chronically exposed to either non-MC-producing or MC-producing living strains and, for this latter, to its subsequent MC-extract of Microcystis aeruginosa. Toxicological effects on liver have been evaluated through the combined approach of histopathology and 'omics' (i.e. proteomics and metabolomics). All treatments induce sex-dependent effects at both cellular and molecular levels. Moreover, the modalities of exposure appear to induce differential responses as the direct exposure to the cyanobacterial strains induce more acute effects than the MC-extract treatment. Our histopathological observations indicate that both non-MC-producing and MC-producing strains induce cellular impairments. Both proteomic and metabolomic analyses exhibit various biological disruptions in the liver of females and males exposed to strain and extract treatments. These results support the hypothesis that M. aeruginosa is able to produce bioactive peptides, other than MCs, which can induce toxicological effects in fish liver. Moreover, they highlight the importance of considering cyanobacterial cells as a whole to assess the realistic environmental risk of cyanobacteria on fish.

Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa.

Odorant-binding proteins (OBP) are secreted in the nasal mucus at the vicinity of olfactory receptors (ORs). They act, at least, as an interface between hydrophobic and volatile odorant molecules and the hydrophilic medium bathing the ORs. They have also been hypothesized to be part of the molecular coding of odors and pheromones, by forming specific complexes with odorant molecules that could ultimately stimulate ORs to trigger the olfactory transduction cascade. In a previous study, we have evidenced that pig olfactory secretome was composed of numerous olfactory binding protein isoforms, generated by O-GlcNAcylation and phosphorylation. In addition, we have shown that recombinant OBP (stricto sensu) produced in yeast is made up of a mixture of isoforms that differ in their phosphorylation pattern, which in turn determines binding specificity. Taking advantage of the high amount of OBP secreted by a single animal, we performed a similar study, under exactly the same experimental conditions, on native isoforms isolated from pig, Sus scrofa, nasal tissue. Four fractions were obtained by using strong anion exchange HPLC. Mapping of phosphorylation and O-GlcNAcylation sites by CID-nanoLC-MS/MS allowed unambiguous localization of phosphosites at S13 and T122 and HexNAc sites at S13 and S19. T112 or T115 could also be phosphorylated. BEMAD analysis suggested extra phosphosites located at S23, S24, S41, S49, S57, S67, and T71. Due to the very low stoichiometry of GlcNAc-peptides and phosphopeptides, these sites were identified on total mixture of OBP isoforms instead of HPLC-purified OBP isoforms. Nevertheless, binding properties of native OBP isoforms to specific ligands in S. scrofa were monitored by fluorescence spectroscopy. Recombinant phosphorylated OBP-Pichia isoforms bind steroids and fatty acids with slight differences. Native isoforms, that are phosphorylated but also O-GlcNAcylated show radically different binding affinities for the same compounds, which strongly suggests that O-GlcNAcylation increases the binding specificity of OBP isoforms. These findings extend the role of O-GlcNAc in regulating the function of proteins involved in many mechanisms of metabolic homeostasis, including extracellular signaling in olfaction. Data is available via ProteomeXChange with identifier PXD011371.

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods).

First proteomic analyses of the dorsal and ventral parts of the Sepia officinalis cuttlebone.

Protein compounds constituting mollusk shells are known for their major roles in the biomineralization processes. These last years, a great diversity of shell proteins have been described in bivalves and gastropods allowing a better understanding of the calcification control by organic compounds and given promising applications in biotechnology. Here, we analyzed for the first time the organic matrix of the aragonitic Sepia officinalis shell, with an emphasis on protein composition of two different structures: the dorsal shield and the chambered part. Our results highlight an organic matrix mainly composed of polysaccharide, glycoprotein and protein compounds as previously described in other mollusk shells, with quantitative and qualitative differences between the dorsal shield and the chamber part. Proteomic analysis resulted in identification of only a few protein compounds underlining the lack of reference databases for Sepiidae. However, most of them contain domains previously characterized in matrix proteins of aragonitic shell-builder mollusks, suggesting ancient and conserved mechanisms of the aragonite biomineralization processes within mollusks. BIOLOGICAL SIGNIFICANCE: The cuttlefish's inner shell, better known under the name "cuttlebone", is a complex mineral structure unique in mollusks and involved in tissue support and buoyancy regulation. Although it combines useful properties as high compressive strength, high porosity and high permeability, knowledge about organic compounds involved in its building remains limited. Moreover, several cuttlebone organic matrix studies reported data very different from each other or from other mollusk shells. Thus, this study provides 1) an overview of the organization of the main mineral structures found in the S. officinalis shell, 2) a reliable baseline about its organic composition, and 3) a first descriptive proteomic approach of organic matrices found in the two main parts of this shell. These data will contribute to the general knowledge about mollusk biomineralization as well as in the identification of protein compounds involved in the Sepiidae shell calcification.

Insights from the Shell Proteome: Biomineralization to Adaptation.

Bivalves have evolved a range of complex shell forming mechanisms that are reflected by their incredible diversity in shell mineralogy and microstructures. A suite of proteins exported to the shell matrix space plays a significant role in controlling these features, in addition to underpinning some of the physical properties of the shell itself. Although, there is a general consensus that a minimum basic protein tool kit is required for shell construction, to date, this remains undefined. In this study, the shell matrix proteins (SMPs) of four highly divergent bivalves (The Pacific oyster, Crassostrea gigas; the blue mussel, Mytilus edulis; the clam, Mya truncata, and the king scallop, Pecten maximus) were analyzed in an identical fashion using proteomics pipeline. This enabled us to identify the critical elements of a "basic tool kit" for calcification processes, which were conserved across the taxa irrespective of the shell morphology and arrangement of the crystal surfaces. In addition, protein domains controlling the crystal layers specific to aragonite and calcite were also identified. Intriguingly, a significant number of the identified SMPs contained domains related to immune functions. These were often are unique to each species implying their involvement not only in immunity, but also environmental adaptation. This suggests that the SMPs are selectively exported in a complex mix to endow the shell with both mechanical protection and biochemical defense.

An Antarctic molluscan biomineralisation tool-kit.

The Antarctic clam Laternula elliptica lives almost permanently below 0 °C and therefore is a valuable and tractable model to study the mechanisms of biomineralisation in cold water. The present study employed a multidisciplinary approach using histology, immunohistochemistry, electron microscopy, proteomics and gene expression to investigate this process. Thirty seven proteins were identified via proteomic extraction of the nacreous shell layer, including two not previously found in nacre; a novel T-rich Mucin-like protein and a Zinc-dependent metalloprotease. In situ hybridisation of seven candidate biomineralisation genes revealed discrete spatial expression patterns within the mantle tissue, hinting at modular organisation, which is also observed in the mantle tissues of other molluscs. All seven of these biomineralisation candidates displayed evidence of multifunctionality and strong association with vesicles, which are potentially involved in shell secretion in this species.

An integrated omic analysis of hepatic alteration in medaka fish chronically exposed to cyanotoxins with possible mechanisms of reproductive toxicity.

Cyanobacterial blooms threaten human health as well as the population of other living organisms in the aquatic environment, particularly due to the production of natural toxic components, the cyanotoxin. So far, the most studied cyanotoxins are microcystins (MCs). In this study, the hepatic alterations at histological, proteome and transcriptome levels were evaluated in female and male medaka fish chronically exposed to 1 and 5 µg L-1 microcystin-LR (MC-LR) and to the extract of MC-producing Microcystis aeruginosa PCC 7820 (5 µg L-1 of equivalent MC-LR) by balneation for 28 days, aiming at enhancing our understanding of the potential reproductive toxicity of cyanotoxins in aquatic vertebrate models. Indeed, both MC and Microcystis extract adversely affect reproductive parameters including fecundity and egg hatchability. The liver of toxin treated female fish present glycogen storage loss and cellular damages. The quantitative proteomics analysis revealed that the quantities of 225 hepatic proteins are dysregulated. In particular, a notable decrease in protein quantities of vitellogenin and choriogenin was observed, which could explain the decrease in reproductive output. Liver transcriptome analysis through Illumina RNA-seq reveals that over 100-400 genes are differentially expressed under 5 µg L-1 MC-LR and Microcystis extract treatments, respectively. Ingenuity pathway analysis of the omic data attests that various metabolic pathways, such as energy production, protein biosynthesis and lipid metabolism, are disturbed by both MC-LR and the Microcystis extract, which could provoke the observed reproductive impairment. The transcriptomics analysis also constitutes the first report of the impairment of circadian rhythm-related gene induced by MCs. This study contributes to a better understanding of the potential consequences of chronic exposure of fish to environmental concentrations of cyanotoxins, suggesting that Microcystis extract could impact a wider range of biological pathways, compared with pure MC-LR, and even 1 µg L-1 MC-LR potentially induces a health risk for aquatic organisms.

Deep sexual dimorphism in adult medaka fish liver highlighted by multi-omic approach.

Sexual dimorphism describes the features that discriminate between the two sexes at various biological levels. Especially, during the reproductive phase, the liver is one of the most sexually dimorphic organs, because of different metabolic demands between the two sexes. The liver is a key organ that plays fundamental roles in various physiological processes, including digestion, energetic metabolism, xenobiotic detoxification, biosynthesis of serum proteins, and also in endocrine or immune response. The sex-dimorphism of the liver is particularly obvious in oviparous animals, as the female liver is the main organ for the synthesis of oocyte constituents. In this work, we are interested in identifying molecular sexual dimorphism in the liver of adult medaka fish and their sex-variation in response to hepatotoxic exposures. By developing an integrative approach combining histology and different high-throughput omic investigations (metabolomics, proteomics and transcriptomics), we were able to globally depict the strong sexual dimorphism that concerns various cellular and molecular processes of hepatocytes comprising protein synthesis, amino acid, lipid and polysaccharide metabolism, along with steroidogenesis and detoxification. The results of this work imply noticeable repercussions on the biology of oviparous organisms environmentally exposed to chemical or toxin issues.

Gender-Specific Toxicological Effects of Chronic Exposure to Pure Microcystin-LR or Complex Microcystis aeruginosa Extracts on Adult Medaka Fish.

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to other organisms. However, their overall and specific implications for the health of aquatic organisms that are chronically and environmentally exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with other bioactive compounds have still not been clearly established and remain difficult to assess. The medaka fish was chosen as the experimental aquatic model for studying the cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing or nonproducing cyanobacterial biomasses, and of a Microcystis aeruginosa natural bloom. Our results showed a higher susceptibility of females to the different treatments compared to males at both the cellular and the molecular levels. Although hepatocyte lysis increased with MC-containing treatments, lysis always appeared more severe in the liver of females compare to males, and the glycogen cellular reserves also appeared to decrease more in the liver of females compared to those in the males. Proteomic investigations reveal divergent responses between males and females exposed to all treatments, especially for proteins involved in metabolic and homeostasis processes. Our observations also highlighted the dysregulation of proteins involved in oogenesis in female livers. These results suggest that fish populations exposed to cyanobacteria blooms may potentially face several ecotoxicological issues.

Purification and partial characterization of a lectin protein complex, the clathrilectin, from the calcareous sponge Clathrina clathrus.

Carbohydrate-binding proteins were purified from the marine calcareous sponge Clathrina clathrus via affinity chromatography on lactose and N-acetyl glucosamine-agarose resins. Proteomic analysis of acrylamide gel separated protein subunits obtained in reducing conditions pointed out several candidates for lectins. Based on amino-acid sequence similarity, two peptides displayed homology with the jack bean lectin Concanavalin A, including a conserved domain shared by proteins in the L-type lectin superfamily. An N-acetyl glucosamine - binding protein complex, named clathrilectin, was further purified via gel filtration chromatography, bioguided with a diagnostic rabbit erythrocyte haemagglutination assay, and its activity was found to be calcium dependent. Clathrilectin, a protein complex of 3200kDa estimated by gel filtration, is composed of monomers with apparent molecular masses of 208 and 180kDa estimated on 10% SDS-PAGE. Nine internal peptides were identified using proteomic analyses, and compared to protein libraries from the demosponge Amphimedon queenslandica and a calcareous sponge Sycon sp. from the Adriatic Sea. The clathrilectin is the first lectin isolated from a calcareous sponge and displays homologies with predicted sponge proteins potentially involved in cell aggregation and interaction with bacteria.

Shell matrix proteins of the clam, Mya truncata: Roles beyond shell formation through proteomic study.

Mya truncata, a soft shell clam, is presented as a new model to study biomineralization through a proteomics approach. In this study, the shell and mantle tissue were analysed in order to retrieve knowledge about the secretion of shell matrix proteins (SMPs). Out of 67 and 127 shell and mantle proteins respectively, 16 were found in both shell and mantle. Bioinformatic analysis of SMP sequences for domain prediction revealed the presence of several new domains such as fucolectin tachylectin-4 pentraxin-1 (FTP), scavenger receptor, alpha-2-macroglobulin (α2 M), lipocalin and myosin tail along with previously reported SMP domains such as chitinase, carbonic anhydrase, tyrosinase, sushi, and chitin binding. Interestingly, these newly predicted domains are attributed with molecular functions other than biomineralization. These findings suggest that shells may not only act as protective armour from predatory action, but could also actively be related to other functions such as immunity. In this context, the roles of SMPs in biomineralization need to be looked in a new perspective.

Characterisation of the mantle transcriptome and biomineralisation genes in the blunt-gaper clam, Mya truncata.

Members of the Myidae family are ecologically and economically important, but there is currently very little molecular data on these species. The present study sequenced and assembled the mantle transcriptome of Mya truncata from the North West coast of Scotland and identified candidate biomineralisation genes. RNA-Seq reads were assembled to create 20,106 contigs in a de novo transciptome, 18.81% of which were assigned putative functions using BLAST sequence similarity searching (cuttoff E-value 1E-10). The most highly expressed genes were compared to the Antarctic clam (Laternula elliptica) and showed that many of the dominant biological functions (muscle contraction, energy production, biomineralisation) in the mantle were conserved. There were however, differences in the constitutive expression of heat shock proteins, which were possibly due to the M. truncata sampling location being at a relatively low latitude, and hence relatively warm, in terms of the global distribution of the species. Phylogenetic analyses of the Tyrosinase proteins from M. truncata showed a gene expansion which was absent in L. elliptica. The tissue distribution expression patterns of putative biomineralisation genes were investigated using quantitative PCR, all genes showed a mantle specific expression pattern supporting their hypothesised role in shell secretion. The present study provides some preliminary insights into how clams from different environments - temperate versus polar - build their shells. In addition, the transcriptome data provides a valuable resource for future comparative studies investigating biomineralisation.