The shell matrix and microstructure of the Ram's Horn squid: Molecular and structural characterization.

Molluscs are one of the most diversified phyla among metazoans. Most of them produce an external calcified shell, resulting from the secretory activity of a specialized epithelium of the calcifying mantle. This biomineralization process is controlled by a set of extracellular macromolecules, the organic matrix. In spite of several studies, these components are mainly known for bivalves and gastropods. In the present study, we investigated the physical and biochemical properties of the internal planispiral shell of the Ram's Horn squid Spirula spirula. Scanning Electron Microscope investigations of the shell reveal a complex microstructural organization. The saccharides constitute a quantitatively important moiety of the matrix, as shown by Fourier-transform infrared and solid-state nuclear magnetic resonance spectroscopies. NMR identified ß-chitin and additional polysaccharides for a total amount of 80% of the insoluble fraction. Proteomics was applied to both soluble and insoluble matrices and in silico searches were performed, first on heterologous metazoans models, and secondly on an unpublished transcriptome of Spirula spirula. In the first case, several peptides were identified, some of them matching with tyrosinase, chitinase 2, protease inhibitor, or immunoglobulin. In the second case, 39 hits were obtained, including transferrin, a serine protease inhibitor, matrilin, or different histones. The very few similarities with known molluscan shell matrix proteins suggest that Spirula spirula uses a unique set of shell matrix proteins for constructing its internal shell. The absence of similarity with closely related cephalopods demonstrates that there is no obvious phylogenetic signal in the cephalopod skeletal matrix.

The shell matrix of the european thorny oyster, Spondylus gaederopus: microstructural and molecular characterization.

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids - fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

Environmentally Friendly Surface Modification Treatment of Flax Fibers by Supercritical Carbon Dioxide.

The present work investigates the effects of an environmentally friendly treatment based on supercritical carbon dioxide (scCO2) on the interfacial adhesion of flax fibers with thermoset matrices. In particular, the influence of this green treatment on the mechanical (by single yarn tensile test), thermal (by TGA), and chemical (by FT-IR) properties of commercially available flax yarns was preliminary addressed. Results showed that scCO2 can significantly modify the biochemical composition of flax fibers, by selectively removing lignin and hemicellulose, without altering their thermal stability and, most importantly, their mechanical properties. Single yarn fragmentation test results highlighted an increased interfacial adhesion after scCO2 treatment, especially for the vinylester matrix, in terms of reduced debonding and critical fragment length values compared to the untreated yarns by 18.9% and 15.1%, respectively. The treatment was less effective for epoxy matrix, for which debonding and critical fragment length values were reduced to a lesser extent, by 3.4% and 3.7%, respectively.

Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis - The saccharide moieties and their localization.

To construct calcium carbonate skeletons of sophisticated architecture, scleractinian corals secrete an extracellular skeletal organic matrix (SOM) from aboral ectodermal cells. The SOM, which is composed of proteins, saccharides, and lipids, performs functions critical for skeleton formation. Even though polysaccharides constitute the major component of the SOM, its contribution to coral skeleton formation is poorly understood. To this end, we analyzed the SOM of the massive colonial coral, Porites australiensis, the skeleton of which has drawn great research interest because it records environmental conditions throughout the life of the colony. The coral skeleton was extensively cleaned, decalcified with acetic acid, and organic fractions were separated based on solubility. These fractions were analyzed using various techniques, including SDS-PAGE, FT-IR, in vitro crystallization, CHNS analysis, chromatography analysis of monosaccharide and enzyme-linked lectin assay (ELLA). We confirmed the acidic nature of SOM and the presence of sulphate, which is thought to initiate CaCO3 crystallization. In order to analyze glycan structures, we performed ELLA on the soluble SOM for the first time and found that it exhibits strong specificity to Datura stramonium lectin (DSL). Furthermore, using biotinylated DSL with anti-biotin antibody conjugated to nanogold, in situ localization of DSL-binding polysaccharides in the P. australiensis skeleton was performed. Signals were distributed on the surfaces of fiber-like crystals of the skeleton, suggesting that polysaccharides may modulate crystal shape. Our study emphasizes the importance of sugar moieties in biomineralization of scleractinian corals.

Tri-benzyl-ammonium chloride.

Single crystals of the title salt, C21H21NH(+)·Cl(-), were isolated as a side product from the reaction involving [(C6H5CH2)3NH]2[HPO4] and Sn(CH3)3Cl in ethanol. Both the cation and the anion are situated on a threefold rotation axis. The central N atom in the cation has a slightly distorted tetra-hedral environment, with angles ranging from 107.7 to 111.16 (10)°. In the crystal, the tri-benzyl-ammonium cations and chloride anions are linked through N-H⋯Cl and C-H⋯Cl hydrogen bonds, leading to the formation of infinite chains along [001]. The crystal studied was a merohedral twin.

Diverting the Use of Hand-Operated Tablet Press Machines to Bioassays: A Novel Protocol to Test 'Waste' Insoluble Shell Matrices.

To mineralize their shells, molluscs secrete a complex cocktail of proteins-collectively defined as the calcifying shell matrix-that remains occluded in the exoskeleton. Nowadays, protein extracts from shells are recognized as a potential source of bioactive substances, among which signalling molecules, bactericides or protease inhibitors offer the most tangible perspectives in applied sciences, health, and aquaculture. However, one technical obstacle in testing the activity of shell extracts lies in their high insolubility. In this paper, we present a protocol that circumvents this impediment. After an adapted shell protein extraction and the production of two organic fractions-one soluble, one insoluble-we employ a hand-operated tablet press machine to generate well-calibrated tablets composed of 100% insoluble shell matrix. FT-IR monitoring of the quality of the tablets shows that the pressure used in the press machine does not impair the molecular properties of the insoluble extracts. The produced tablets can be directly tested in different biological assays, such as the bactericidal inhibition zone assay in Petri dish, as illustrated here. Diverting the use of the hand-operated tablet press opens new perspectives in the analysis of insoluble shell matrices, for discovering novel bioactive components.

Genesis of amorphous calcium carbonate containing alveolar plates in the ciliate Coleps hirtus (Ciliophora, Prostomatea).

In the protist world, the ciliate Coleps hirtus (phylum Ciliophora, class Prostomatea) synthesizes a peculiar biomineralized test made of alveolar plates, structures located within alveolar vesicles at the cell cortex. Alveolar plates are arranged by overlapping like an armor and they are thought to protect and/or stiffen the cell. Although their morphology is species-specific and of complex architecture, so far almost nothing is known about their genesis, their structure and their elemental and mineral composition. We investigated the genesis of new alveolar plates after cell division and examined cells and isolated alveolar plates by electron microscopy, energy-dispersive X-ray spectroscopy, FTIR and X-ray diffraction. Our investigations revealed an organic mesh-like structure that guides the formation of new alveolar plates like a template and the role of vesicles transporting inorganic material. We further demonstrated that the inorganic part of the alveolar plates is composed out of amorphous calcium carbonate. For stabilization of the amorphous phase, the alveolar vesicles, the organic fraction and the element phosphorus may play a role.

Bis(cyclo-hexyl-ammonium) tetra-chlorido-(oxalato)stannate(IV).

The title salt, (C6H14N)2[Sn(C2O4)Cl4], was obtained as a by-product from the reaction between 2C6H14N(+)·C2O4 (2-)·1.5H2O and SnCl2·2H2O. The cyclo-hexyl-ammonium cation has a chair conformation. The complex anion consists of an oxalate anion chelating the SnCl4 moiety, resulting in a distorted octa-hedral coordination sphere of the Sn(IV) atom with the O atoms in equatorial cis positions. In the crystal, cations and anions are linked through N-H⋯O and N-H⋯Cl inter-actions into a layered arrangement parallel to (100).

Tris(cyclo-hexyl-ammonium) cis-di-chlorido-bis-(oxalato-κ(2) O (1),O (2))stann-ate(IV) chloride monohydrate.

The crystal structure of the title compound, (C6H14N)3[Sn(C2O4)2Cl2]Cl·H2O, contains three cyclo-hexyl-ammonium cations, one stannate(IV) dianion, one isolated chloride anion and one lattice water mol-ecule. The cyclo-hexyl-ammonium cations adopt chair conformations. In the complex anion, two bidentate oxalate ligands and two chloride anions in cis positions coordinate octa-hedrally to the central Sn(IV) atom. The cohesion of the mol-ecular entities is ensured by the formation of N-H⋯O, O-H⋯O, O-H⋯Cl and N-H⋯Cl inter-actions involving cations, anions and the lattice water mol-ecule, giving rise to a layer-like arrangement parallel to (010).

1-n-Butyl-3-methylimidazolium-2-carboxylate: a versatile precatalyst for the ring-opening polymerization of ε-caprolactone and rac-lactide under solvent-free conditions.

The ring-opening polymerization of ε-caprolactone (ε-CL) and rac-lactide (rac-LA) under solvent-free conditions and using 1-n-butyl-3-methylimidazolium-2-carboxylate (BMIM-2-CO2) as precatalyst is described. Linear and star-branched polyesters were synthesized by successive use of benzyl alcohol, ethylene glycol, glycerol and pentaerythritol as initiator alcohols, and the products were fully characterized by (1)H and (13)C{(1)H} NMR spectroscopy, gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). BMIM-2-CO2 acts as an N-heterocyclic carbene precursor, resulting from in situ decarboxylation, either by heating under vacuo (method A) or by addition of NaBPh4 (method B). Possible catalytic and deactivation mechanisms are proposed.

Crystal structure of di-benzyl-ammonium hydrogen (4-amino-phen-yl)arsonate monohydrate.

The title salt, C14H16N+·C6H7AsNO3 -·H2O or [(C6H5CH2)2NH2][H2NC6H4As(OH)O2]·H2O, (I), was synthesized by mixing an aqueous solution of (4-amino-phenyl)-arsonic acid with an ethano-lic solution of di-benzyl-amine at room temperature. Compound I crystallizes in the monoclinic P21/c space group. The three components forming I are linked via N-H⋯O and O-H⋯O inter-molecular hydrogen bonds, resulting in the propagation of an infinite zigzag chain. Additional weak inter-actions between neighbouring chains, such as π-π and N-H⋯O contacts, involving phenyl rings, -NH2 and -As(OH)O3 functions, and H2O, respectively, lead to a three-dimensional network.

Novel molluskan biomineralization proteins retrieved from proteomics: a case study with Upsalin.

The formation of the molluskan shell is regulated by an array of extracellular proteins secreted by the calcifying epithelial cells of the mantle. These proteins remain occluded within the recently formed biominerals. To date, many shell proteins have been retrieved, but only a few of them, such as nacreins, have clearly identified functions. In this particular case, by combining molecular biology and biochemical approaches, we performed the molecular characterization of a novel protein that we named Upsalin, associated with the nacreous shell of the freshwater mussel Unio pictorum. The full sequence of the upsalin transcript was obtained by RT-PCR and 5'/3' RACE, and the expression pattern of the transcript was studied by PCR and qPCR. Upsalin is a 12 kDa protein with a basic theoretical pI. The presence of Upsalin in the shell was demonstrated by extraction of the acetic-acid-soluble nacre matrix, purification of a shell protein fraction by mono-dimensional preparative SDS-PAGE, and by submitting this fraction, after trypsic digestion, to nano-LC-MS/MS. In vitro experiments with the purified protein showed that it interferes poorly with the precipitation of calcium carbonate. Homology searches also could not affiliate Upsalin to any other protein of known function, leaving open the question of its exact role in shell formation. An antibody raised against an immunogenic peptide of Upsalin was found to be specific to this protein and was subsequently assayed for immunogold localization of the target protein in the shell, revealing the ubiquitous presence of Upsalin in the nacreous and prismatic layers. Recently, with the application of high-throughput proteomic studies to shells, the number of candidate proteins without clear functions has been increasing exponentially. The Upsalin example highlights the crucial need, for the scientific community dealing with biomineralization in general, to dedicate the coming years to designing experimental approaches, such as gene silencing, that focus on the functions of mineral-associated proteins.

Crystal structure of the monoglycidyl ether of isoeugenol.

The title compound, C13H16O3 [GE-isoEu; systematic name: 2-({2-meth-oxy-4-[(E)-1-propen-1-yl]phen-oxy}meth-yl)oxirane], which crystallizes in the triclinic P space group, was synthesized in one step from iso-eugenol, a bio-based phenyl-propanoid, with an excess of epi-chloro-hydrin. Colourless prismatic crystals suitable for X-ray diffraction were obtained from a mixture of ethyl acetate and cyclo-hexane, during purification by column chromatography on silica gel. GE-isoEu, which corresponds to the trans isomer of the monoglycidyl ether of iso-eugenol, is based on a 1,2,4-tris-ubstituted benzene ring by diglycidyl ether, meth-oxy and 1-(E)-propenyl groups, respectively. In the crystal, mol-ecules are organized through offset π-stacking inter-actions. Chemically, GE-isoEu constitutes an inter-mediate in the synthesis protocol of 2-[3-meth-oxy-4-(2-oxiranylmeth-oxy)phen-yl]-3-methyl-oxirane (GEEp-isoEu), a di-epoxy-dized monomer used in the manufacturing of thermosetting resins and intended for the elaboration of bio-composites.

An alternative and effective method for extracting skeletal organic matrix adapted to the red coral Corallium rubrum.

Skeleton formation in corals is a biologically controlled process in which an extracellular organic matrix (OM) is entrapped inside the calcified structure. The analysis of OM requires a time-consuming and tedious extraction that includes grinding, demineralization, multiple rinsing and concentration steps. Here we present an alternative and straightforward method for the red coral Corallium rubrum that requires little equipment and saves steps. The entire skeleton is directly demineralized to produce a tractable material called ghost, which is further rinsed and melted at 80°C in water. The comparative analysis of the standard and alternative methods by electrophoresis, western blot, and FTIR of C. rubrum OM, shows that the 'alternative OM' is of higher quality. Advantages and limitations of both methods are discussed.

Variability of shell repair in the Manila clam Ruditapes philippinarum affected by the Brown Ring Disease: a microstructural and biochemical study.

For more than two decades, the Manila clam Ruditapes philippinarum has been regularly affected by Brown Ring Disease (BRD), an epizootic event caused by the bacterium Vibrio tapetis and characterized by the development of a brown deposit on the inner face of valves. Although BRD infection is often lethal, some clams recover by mineralizing a new repair shell layer, which covers the brown deposit and fully isolates it from living tissues. In order to understand this specific shell repair process, the microstructures of repaired zones were compared to those of shells unaffected by BRD. In addition, the organic matrix associated with unaffected shells and to repair patches were extracted and compared by biochemical and immunological techniques. Our results show that the repaired zones exhibit microstructures that resemble the so-called homogeneous microstructure of the internal layer, with some marked differences, like the development of crossed-acicular crystals, which form chevron-like patterns. In the three tested batches of repaired layers, the matrices exhibit certain heterogeneity, i.e., they are partially to widely different from the ones of shells unaffected by BRD, as illustrated by SDS-PAGE and by serological comparisons. Our results strongly suggest a modification of the secretory regime of calcifying mantle cells during the shell repair process. Polyclonal antibodies, which were developed against specific protein fractions of the shell, represent relevant tools for localizing by immunohistology the cells responsible for the repair.

The 'Shellome' of the Crocus Clam Tridacna crocea Emphasizes Essential Components of Mollusk Shell Biomineralization.

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules - in particular proteins - that are essential for molluscan shell formation.

New Eco-Friendly Synthesized Thermosets from Isoeugenol-Based Epoxy Resins.

Epoxy resin plays a key role in composite matrices and DGEBA is the major precursor used. With the aim of favouring the use of bio resources, epoxy resins can be prepared from lignin. In particular, diglycidyl ether of isoeugenol derivatives are good candidates for the replacement of DGEBA. This article presents an effective and eco-friendly way to prepare epoxy resin derived from isoeugenol (BioIgenox), making its upscale possible. BioIgenox has been totally characterized by NMR, FTIR, MS and elemental analyses. Curing of BioIgenox and camphoric anhydride with varying epoxide function/anhydride molar ratios has allowed determining an optimum ratio near 1/0.9 based on DMA and DSC analyses and swelling behaviours. This thermoset exhibits a Tg measured by DMA of 165 °C, a tensile storage modulus at 40 °C of 2.2 GPa and mean 3-point bending stiffness, strength and strain at failure of 3.2 GPa, 120 MPa and 6.6%, respectively. Transposed to BioIgenox/hexahydrophtalic anhydride, this optimized formulation gives a thermoset with a Tg determined by DMA of 140 °C and a storage modulus at 40 °C of 2.6 GPa. The thermal and mechanical properties of these two thermosets are consistent with their use as matrices for structural or semi-structural composites.

'Palaeoshellomics' reveals the use of freshwater mother-of-pearl in prehistory.

The extensive use of mollusc shell as a versatile raw material is testament to its importance in prehistoric times. The consistent choice of certain species for different purposes, including the making of ornaments, is a direct representation of how humans viewed and exploited their environment. The necessary taxonomic information, however, is often impossible to obtain from objects that are small, heavily worked or degraded. Here we propose a novel biogeochemical approach to track the biological origin of prehistoric mollusc shell. We conducted an in-depth study of archaeological ornaments using microstructural, geochemical and biomolecular analyses, including 'palaeoshellomics', the first application of palaeoproteomics to mollusc shells (and indeed to any invertebrate calcified tissue). We reveal the consistent use of locally-sourced freshwater mother-of-pearl for the standardized manufacture of 'double-buttons'. This craft is found throughout Europe between 4200-3800 BCE, highlighting the ornament-makers' profound knowledge of the biogeosphere and the existence of cross-cultural traditions.

Crystal structure of the diglycidyl ether of eugenol.

The diep-oxy monomer, C13H16O4 {DGE-Eu; systematic name: 2-[3-meth-oxy-4-(oxiran-2-ylmeth-oxy)benz-yl]oxirane}, was synthesized from eugenol by a three-step reaction. It consists of a 1,2,4-tris-ubstituted benzene ring substituted by diglycidyl ether, a meth-oxy group and a methyl-oxirane group. The three-membered oxirane rings are inclined to the benzene ring by 61.0 (3) and 27.9 (3)°. The methyl-ene C atom of one of the two terminal epoxide rings is positionally disordered [refined occupancy ratio = 0.69 (1):0.31 (1)]. In the crystal, mol-ecules are linked by C-H⋯O hydrogen bonds, forming layers parallel to the ab plane. The layers are linked by C-H⋯π inter-actions, forming a three-dimensional network.

Crystal structure of 2-methyl-1H-imidazol-3-ium hydrogen oxalate dihydrate.

Single crystals of the title mol-ecular salt, C4H7N2 (+)·HC2O4 (-)·2H2O, were isolated from the reaction of 2-methyl-1H-imidazole and oxalic acid in a 1:1 molar ratio in water. In the crystal, the cations and anions are positioned alternately along an infinite [010] ribbon and linked together through bifurcated N-H⋯(O,O) hydrogen bonds. The water mol-ecules of crystallization link the chains into (10-1) bilayers, with the methyl groups of the cations organized in an isotactic manner.