Insoluble Matrices Research Articles

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.

Enhancing quantum efficiency of Mn4+-doped fluoride phosphors through reverse doping strategy toward potential biological detection

Mn4+-doped fluoride red-emitting materials are renowned for their distinct characteristics, including fixed emission peaks and narrow spectral emission, rendering them highly favored in photoluminescent light-emitting diodes (LEDs). However, achieving ultra-high-concentration Mn4+ doping in fluoride phosphors is imperative to enhance quantum efficiency. Nevertheless, attaining such doping levels in metastable compound systems poses considerable challenges. In this study, we introduce an innovative reverse doping strategy, leading to the synthesis of Mn4+-doped cubic K2SiF6 phosphor (11.38 mol %) from hexagonal K2MnF6. Remarkably, this approach significantly improves the external quantum efficiency (EQE) to 66.7 % upon 450 nm excitation. Through systematic comparisons with conventional co-precipitation and ion exchange methods, our findings unequivocally demonstrate the superior efficacy of this approach in achieving high-concentration Mn4+ ion doping within relatively insoluble matrices. The successful application of our optimized phosphor enabled the fabrication of a red light-emitting diode (LED) that exhibits an exceptional photoelectric efficiency of 31.67 % at 350 mA, surpassing the performance of a commercial red LED chip. Furthermore, the stable red-light emission from the phosphor-converted LED (pc-LED) proves instrumental in rapid-response vascular imaging, showcasing the potential of our findings in advancing light sources for biological detection applications. This research provides valuable insights into enhancing red emission efficiency in slightly-soluble fluoride phosphors via novel synthetic strategy, unlocking possibilities for the development of highly efficient light sources in biological detection and imaging applications.

Effect of phosphoproteins on intracellular calcification of bacteria.

This study aimed to evaluate the effects of phosphoproteins on bacterial mineralization. Dental calculus formation is attributed to bacterial mineralization in the oral cavity; however, the influence of phosphoproteins (which are abundant in saliva) is not clear. The model bacterium Escherichia coli was suspended in a calcification solution containing casein as a model phosphoprotein. To evaluate mineralization independent of bacterial metabolism, bacteria killed by heat treatment at 70°C were compared with viable bacteria. After incubation at 37°C for 24h, the mode of calcification was observed using electron microscopy and energy dispersivex-ray spectroscopy. Solutions without casein produced precipitation in solution, which was identical to that in experiments without bacteria. In contrast, calcification solutions with 200ppm casein only produced calcium phosphate deposition intracellularly. Without heat treatment, intracellular calcification rarely occurred, even when casein was added. Thus, phosphoproteins promoted intracellular calcification of dead bacteria; this is similar to the calcification of insoluble matrices, such as collagen fibrils, promoted by acidic polymers. We concluded that intracellular calcification is caused by the collagen fibril-like behavior of dead bacteria. The promotion of intracellular calcification of dead bacteria by phosphoproteins suggested a basic principle of dental calculus formation.

Release kinetics of metronidazole from 3D printed silicone scaffolds for sustained application to the female reproductive tract

Sustained vaginal administration of antibiotics or probiotics has been proposed to improve treatment efficacy for bacterial vaginosis. 3D printing has shown promise for development of systems for local agent delivery. In contrast to oral ingestion, agent release kinetics can be fine-tuned by the 3D printing of specialized scaffold designs tailored for particular treatments while enhancing dosage effectiveness via localized sustained release. It has been challenging to establish scaffold properties as a function of fabrication parameters to obtain sustained release. In particular, the relationships between scaffold curing conditions, compressive strength, and drug release kinetics remain poorly understood. This study evaluates 3D printed scaffold formulation and feasibility to sustain the release of metronidazole, a commonly used antibiotic for BV. Cylindrical silicone scaffolds were printed and cured using three different conditions relevant to potential future incorporation of temperature-sensitive labile biologics. Compressive strength and drug release were monitored for 14d in simulated vaginal fluid to assess long-term effects of fabrication conditions on mechanical integrity and release kinetics. Scaffolds were mechanically evaluated to determine compressive and tensile strength, and elastic modulus. Release profiles were fitted to previous kinetic models to differentiate potential release mechanisms. The Higuchi, Korsmeyer-Peppas, and Peppas-Sahlin models best described the release, indicating similarity to release from insoluble or polymeric matrices. This study shows the feasibility of 3D printed silicone scaffolds to provide sustained metronidazole release over 14d, with compressive strength and drug release kinetics tuned by the fabrication parameters.

Preparation of β-cyclodextrin/polysaccharide foams using saponin.

Cyclodextrins, cyclic oligosaccharides with a hydrophobic cavity that form inclusion complexes with nonpolar molecules, can be used to functionalize other polysaccharides. Xanthan gum, locust bean gum or chitosan can be crosslinked using citric acid in the presence of β-cyclodextrin to produce insoluble matrices. In this work, polymeric foams based on those polysaccharides and saponin have been prepared using a green synthesis method to increase the porosity of the matrices. The saponin of soapbark (Quillaja saponaria) has been used to obtain foams using different procedures. The influence of the synthesis path on the porosity of the materials and their corresponding sorption capacities in the aqueous phase were evaluated.

Assessment of Optimal Conditions for Marine Invertebrate Cell-Mediated Mineralization of Organic Matrices.

Cellular strategies and regulation of their crystallization mechanisms are essential to the formation of biominerals, and harnessing these strategies will be important for the future creation of novel non-native biominerals that recapitulate the impressive properties biominerals possess. Harnessing these biosynthetic strategies requires an understanding of the interplay between insoluble organic matrices, mineral precursors, and soluble organic and inorganic additives. Our long-range goal is to use a sea anemone model system (Nematostella vectensis) to examine the role of intrinsically disordered proteins (IDPs) found in native biomineral systems. Here, we study how ambient temperatures (25–37 °C) and seawater solution compositions (varying NaCl and Mg ratios) will affect the infiltration of organic matrices with calcium carbonate mineral precursors generated through a polymer-induced liquid-precursor (PILP) process. Fibrillar collagen matrices were used to assess whether solution conditions were suitable for intrafibrillar mineralization, and SEM with EDS was used to analyze mineral infiltration. Conditions of temperatures 30 °C and above and with low Mg:Ca ratios were determined to be suitable conditions for calcium carbonate infiltration. The information obtained from these observations may be useful for the manipulation and study of cellular secreted IDPs in our quest to create novel biosynthetic materials.

Potassium incorporation and isotope fractionation in cultured scleractinian corals

Potassium (K) participates in coral biological activities and accumulates in their skeletons, driving the fractionation of stable K isotopes (41K/39K). Constraining the influences of biotic and abiotic controls on K isotope fractionation is important for interpreting coral records. However, the processes and mechanisms regulating K incorporation into coral skeletons and K isotope fractionation between seawater and coral skeletons remain unknown. Here, we combined isotopic and synchrotron-based spectroscopic analyses to evaluate the phase distribution and corresponding isotope variation of K in the skeleton of scleractinian (Porites australiensis) corals at a seawater temperature range from 20 to 29°C in aquaria culture experiments. Potassium in coral skeletons exists mainly as organic-K (K hosted in soluble and insoluble organic matrices) and carbonate-K (K incorporated into K2CO3 and aragonite) phases of various proportions. Coral δ41K values vary substantially in both direction and magnitude from the modern seawater δ41K composition (∼0.12‰), showing marked deviations (Δ41KCoral-Sea) from −2.00 to 0.67‰. As seawater temperature increases, the organic-K fraction increases, whereas δ41KCoral decreases. The variation in δ41KCoral reflects the relative proportions of organic-bound K to carbonate-associated K. In most cases, coral intracrystalline organic matrices preferentially sequester isotopically lighter K whereas carbonate phases prefer heavier K. Distinguishable inter-colony difference in skeletal δ41K of corals growing under the same culturing conditions reveals the influence of physiological controls on K partitioning and isotope fractionation. Although calcification rate correlates with temperature to different degrees in the studied corals, likely reflecting control of the difference in zooxanthellae density, we infer that calcification rate is not a major controlling factor on skeletal δ41K. Rather, skeletal δ41K correlates closely with K phase partitioning, which is ascribed to temperature-sensitive physiological modulation.

Removal of arsenic from water by an iron-loaded resin prepared from Pinus pinaster bark tannins

Arsenic is a toxic and carcinogenic metalloid naturally present in water bodies. However, its concentration can be exacerbated by anthropogenic activities (e.g. mining, industry, agriculture). Thus, removal of arsenic from contaminated waters is a crucially important goal. Tannins are ubiquitous and inexpensive natural biopolymers that are easy to extract and convert into insoluble matrices. The aim of this work was to assess the potential of Pinus pinaster bark tannin resins to take up arsenic from aqueous solutions. Methods of tannin extraction and polymerization, oxidation and iron-loading were optimized. An extraction efficiency of 24.2 ± 0.1% was achieved and 166 ± 2 mg of formaldehyde-condensable phenols were extracted per gram of bark used. Polymerization of tannins, under the best conditions, presented an efficiency around 80%. Oxidation and iron loading yielded a tannin resin with an iron content of approx. 20 mg g–1. Adsorption of As(V) was optimal at pH 3 with a removal of approx. 90% from an initial solution containing 5 mg L–1. Adsorption assays with As(III) presented negligible results. As(V) adsorption was somewhat fast, with equilibrium being achieved in 1–4 h. Equilibrium data obtained for As(V) adsorption showed a maximum adsorption capacity of 0.72 ± 0.03 mg g–1 (pH 3, smaller than 150 µm). Iron leaching was high at low pH but could be drastically reduced by limiting the contact time. This work attempts to show that a forest residue, usually undervalued, can be converted into an adsorbent and can help in dealing with arsenic-contaminated waters, which is an environmental problem affecting many countries in the world.

Whole grain cereal fibers and their support of the gut commensal Clostridia for health

During processing of refined cereal grain foods, cell wall fibers present in whole grains are diminished and are often replaced with dissimilar soluble fibers. Contrary to a view that soluble fibers are generally preferable for health and that the removed insoluble bran cell wall fibers of whole grains provide bulk for laxation, a body of evidence now shows that these insoluble fibers support key mucosal-associated commensal Clostridial bacteria important for gut and whole-body health. Commensal Clostridia possess cellulosomes, a specialized extracellular apparatus with enzymes, that can degrade the physical form of the insoluble fiber matrices. They show a preference to matrix fibers, and when the same insoluble bran fiber polymers are made soluble, Clostridia are outcompeted by other bacteria. Clostridial group members are major producers of butyrate, an important fermentation metabolite impacting human health. Butyrate suppresses localized and systemic inflammation, maintains and promotes good epithelial barrier function, and provides energy to the colonocytes, among other associated benefits. This article proposes that an important aspect of whole grain fiber consumption is to support these beneficial gut bacteria, and not just simply providing a higher amount of dietary fiber. This view differs from the regulatory perspective where all fibers, regardless of structure, are labeled as “dietary fiber”, and where the addition of a soluble fiber to a refined cereal product is the same as the removed insoluble matrix fiber.

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 β-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.

Release kinetics of the model protein FITC-BSA from different polymer-coated bovine bone substitutes

BackgroundControlled release of proteins bound to conventional bone substitutes is still insufficient. Therefore, this study evaluates in-vitro release kinetics of the model protein FITC-BSA (fluorescein conjugated bovine serum albumine) from insoluble bovine collagenous bone matrices (ICBM) with different polymer coatings. Analyzes aim at comparing FITC-BSA release from uncoated versus coated ICBM over time to find bone substitute coatings with consistent release profiles.MethodsRelease kinetics of FITC-BSA from uncoated as well as coated ICBM with five different polymers (RESOMER R 203 H, RG 503 H, RG 504 H, RG 505, L 206 S) were measured over a period of 11 days (d). Measurements were conducted after 6 h (h), 12 h, 24 h, 3 d, 5 d, 7 d, 9 d and 11 d with six samples for each coated ICBM. Two groups were formed (1) with and (2) without medium change at times of measurement. For each group ANOVA with post-hoc Bonferroni testing was used. Scanning electron microscopy assessed morphologic differences between ICBM coating.ResultsIn group 1 approx. 70% of FITC-BSA release from uncoated ICBM occurred after 6 h compared to approx. 50% in group 2. Only polymers with medium inherent viscosity, i.e. RESOMER RG 503 H, constantly showed significantly more FITC-BSA release throughout 11 d than uncoated ICBM (p = 0.007). The same was found for group 2 (p = 0.005). No significant differences between PLA and PLGA polymers were found. Scanning electron microscopy results indicate a weak adhesion of polymer coatings to ICBM explaining its rather weak retentive effect on overall FITC-BSA release.ConclusionsMedium molecular size polymers reduce the overall released FITC-BSA from ICBM over time. In clinical practice these polymers may prove ideal for bone substitute materials.

Plant‐Inspired Pyrogallol‐Containing Functional Materials

AbstractPyrogallol‐containing molecules are ubiquitous in the plant kingdom. The chemical synthesis of these molecules remains challenging. Thus, they are obtained via purification from heterogeneous mixtures of plant extracts. Previous studies have focused on their biological roles, such as antioxidants. Additionally, the molecules are used as ink colorants and in tanning processes for leather. Recently, many disciplines have paid attention to adhesiveness of pyrogallol‐containing molecules, including the control of interface properties in energy storage/generation and medical devices, as well as the changes in wettability related to membrane technologies. In particular, pyrogallol‐containing molecules act as “molecular glues,” binding to virtually all biomacromolecules, for example, DNA/RNA, soluble proteins, insoluble extracellular matrices, and peptides. Furthermore, the cohesion of pyrogallol by forming pyrogallol‐to‐pyrogallol covalent bonds is useful for the preparation of bulk hydrogels and thin films. The content of this review focuses on interactions with biomacromolecules used as molecular glues, used as modifiers in material‐independent surface chemistry, and applied as chemical moieties to form covalent linkages to fabricate hydrogels and related biomaterials. Future perspectives include the development of new pyrogallol‐containing materials, the understanding of chirality in adhesion, and the improvement of the mechanical stability for applications in various biomedical, energy, and industrial devices.

Abstract 2532: High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation

Abstract Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by H&E-staining. Many human samples provide outstanding quality nuclei suspensions, including cryopreserved cell lines, PBMCs, bone marrow, liver, brain, breast, colon, lung, prostate and melanomas. Fixed and preserved specimens also yield nuclei readily interrogated by targeted DNA sequencing panels. Nuclei suspensions are readily processed with the Tapestri Platform for single-cell DNA analysis. Leveraging droplet microfluidics and barcoding, the workflow enables high uniformity of ~90%, low ADO of ~10%, and typical nuclei recovery rates exceed 10%. Up to 20,000 nuclei can be interrogated in each run with catalog or custom panels for any tumor type. Here, a 59-gene tumor hotspot panel was used to study five melanoma metastases. The single-cell data enabled the unique reconstruction of tumor sample clonal phylogeny unresolved by bulk analysis. Also, low prevalence metastatic subclones masked in bulk NGS data were detected in normal liver samples. In summary, we show that diverse types of archived tumor tissues are readily dissociated with this universal nuclei protocol. Researchers now have a highly sensitive, targeted, customizable solution for revealing genomic variation and clonal propagation in complex archived solid tumor samples. Citation Format: David Ruff, Pedro Mendez, Daniel Mendoza, Nianzhen Li, Adam Sciambi, Kaustubh Gokhale, Dalia Dhingra, Keith Jones, Dennis Eastburn. High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2532.

Porous ZrC-carbon microspheres as potential insoluble target matrices for production of 188W/188Re

New microsphere sorbents are reported, which could find application in demanding radiation environments and especially as targets for the production of nuclear medicines by neutron irradiation. An easily-synthesized Zr anionic complex was introduced into quaternary amine-functionalised polystyrene-divinylbenzene-based anion-exchange resins by batch adsorption. Upon carbothermal reduction, the precursors were converted to porous carbon matrices containing particles of ZrC and ZrO2 polymorphs. The most phase-pure material, ZrAX-1, possessed high surface area, multi-scale porosity and high mechanical strength. Adsorption of Re and W was investigated and its possible deployment as a reusable host for the production of 188W/188Re is discussed.

Structured Growth of Metal–Organic Framework MIL-53(Al) from Solid Aluminum Carbide Precursor

The conventional synthesis of metal-organic frameworks (MOFs) through soluble metal-salt precursors provides little control over the growth of MOF crystals. The use of alternative metal precursors would provide a more flexible and cost-effective strategy for direction- and shape-controlled MOF synthesis. Here, we demonstrate for the first time the use of insoluble metal-carbon matrices to foster directed growth of MOFs. Aluminum carbide was implemented as both the metal precursor and growth-directing agent for the generation of MIL-53(Al). A unique needle-like morphology of the MOF was grown parallel to the bulk surface in a layer-by-layer manner. Importantly, the synthesis scheme was found to be transferrable to the production of different linker analogues of the MOF and other topologies. Given the variety of metal carbides available, these findings can be used as a blueprint for controlled, efficient, and economical MOF syntheses and set a new milestone toward the industrial use of MOFs at large-scale.

Characterization of Degraded Proteins in Paintings Using Bottom-Up Proteomic Approaches: New Strategies for Protein Digestion and Analysis of Data

Chemical hydrolysis assisted by microwave irradiation has been proposed as an alternative method for the analysis of proteins in highly insoluble matrices. In this work, chemical hydrolysis was applied for the first time to detect degraded proteins from paintings and polychromies. To evaluate the performance of this approach, the number of identified peptides, protein sequence coverage (%), and PSMs were compared with those obtained using two trypsin-based proteomics procedures used for the analysis of samples from cultural heritage objects. It was found that chemical hydrolysis allowed the successful identification of all proteinaceous materials in all paint samples analyzed except for egg proteins in one extremely degraded sample. Moreover, in one sample, casein was only identified by chemical digestion. In general, chemical hydrolysis identified more peptides, more PSM's, and greater sequence coverage in the samples containing caseins, and often also in animal glue, highlighting the great potential of this approach for the rapid digestion and identification of insoluble and degraded proteins from the field of the cultural heritage.

Azide‐substituted Poly(N‐ethyl‐3‐vinylcarbazole) as a Thermally Cross‐Linkable Hole‐Transporting Material for Polymeric Light‐Emitting Diodes

We have successfully synthesized a series of thermally cross‐linkable azide‐substituted poly(N‐ethyl‐3‐vinylcarbazoles) P1–P6. These polymers can be self‐cross‐linked by heat treatment under atmospheric conditions to form insoluble polymer matrices. Their electronic properties can be tuned by using various triphenylamine‐substituted alkyne‐based curing agents as additives. The polymeric materials can be simply coated on a substrate by spin coating or dip coating. Through the Huisgen 1,3‐dipolar cycloaddition reaction, the polymeric film can be cured by alkyne‐based curing agents effectively under copper‐free conditions. This approach can be utilized in the fabrication of multilayer polymeric light‐emitting devices (PLEDs). Under optimized conditions, we have demonstrated that the PLED of ITO/cured P5/PVK‐Ir(ppy)3‐t‐PBD/Mg–Ag shows a turn‐on voltage of 10.1 V (1 cd/m2), with the maximum brightness of 7851 cd/m2 and current efficiency of 12.8 cd/A.

Antioxidants Bound to an Insoluble Food Matrix: Their Analysis, Regeneration Behavior, and Physiological Importance.

Dietary antioxidants play an important role in human health by counteracting oxidative stress and preventing chronic diseases. Most common dietary antioxidants in foods are vitamins, carotenoids, phenolic compounds, sulfur-containing compounds, and neoformed antioxidants. Antioxidants may be present in free soluble or bound insoluble forms in foods. Antioxidants bound to insoluble food matrices have gained the spotlight because they exert their antioxidant effects much longer than free soluble ones. A direct procedure called QUENCHER has been shown to accurately measure the antioxidant capacity of antioxidants bound to insoluble matrices. This procedure overcomes the drawbacks of extraction-dependent classical assays leading to underestimation of the total antioxidant capacity (TAC) of foods. This review focuses on antioxidants that are found naturally in foods or are formed in foods during processing specifically the antioxidants bound to the insoluble food matrices. The literature gap on the importance of bound antioxidants, their physiological relevance, and methods for measurement of their antioxidant capacity will be filled by this comprehensive review. In particular, chemical properties and health effects of food antioxidants, measurement of the TAC of foods by the QUENCHER method, digestion behavior of bound insoluble antioxidants, and their interactions with free soluble antioxidants are discussed throughout this review.

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).

An Overview of the Medical Applications of Marine Skeletal Matrix Proteins.

In recent years, the medicinal potential of marine organisms has attracted increasing attention. This is due to their immense diversity and adaptation to unique ecological niches that has led to vast physiological and biochemical diversification. Among these organisms, marine calcifiers are an abundant source of novel proteins and chemical entities that can be used for drug discovery. Studies of the skeletal organic matrix proteins of marine calcifiers have focused on biomedical applications such as the identification of growth inducing proteins that can be used for bone regeneration, for example, 2/4 bone morphogenic proteins (BMP). Although a few reports on the functions of proteins derived from marine calcifiers can be found in the literature, marine calcifiers themselves remain an untapped source of proteins for the development of innovative pharmaceuticals. Following an overview of the current knowledge of skeletal organic matrix proteins from marine calcifiers, this review will focus on various aspects of marine skeletal protein research including sources, biosynthesis, structures, and possible strategies for chemical or physical modification. Special attention will be given to potential medical applications and recent discoveries of skeletal proteins and polysaccharides with biologically appealing characteristics. In addition, I will introduce an effective protocol for sample preparation and protein purification that includes isolation technology for biopolymers (of both soluble and insoluble organic matrices) from coralline algae. These algae are a widespread but poorly studied group of shallow marine calcifiers that have great potential for marine drug discovery.