Spatial localizing of skeletal proteins in biogenic minerals remains a challenge in biomineralization research. To address this goal, we developed a novel in situ mapping technique based on molecular ...recognition measurements via atomic force microscopy (AFM), which requires three steps: (1) the development and purification of a polyclonal antibody elicited against the target protein, (2) its covalent coupling to a silicon nitride AFM tip ('functionalization'), and (3) scanning of an appropriately prepared biomineral surface. We applied this approach to a soluble shell protein - accripin11 - recently identified as a major component of the calcitic prisms of the fan mussel Pinna nobilis1. Multiple tests reveal that accripin11 is evenly distributed at the surface of the prisms and also present in the organic sheaths surrounding the calcitic prisms, indicating that this protein is both intra- and inter-crystalline. We observed that the adhesion force in transverse sections is about twice higher than in longitudinal sections, suggesting that accripin11 may exhibit preferred orientation in the biomineral. To our knowledge, this is the first time that a protein is localized by molecular recognition atomic force microscopy with antibody-functionalized tips in a biogenic mineral. The 'pros' and 'cons' of this methodology are discussed in comparison with more 'classical' approaches like immunogold. This technique, which leaves the surface to analyze clean, might prove useful for clinical tests on non-pathological (bone, teeth) or pathological (kidney stone) biomineralizations. Studies using implants with protein-doped calcium phosphate coating can also benefit from this technology.
Our paper deals with an unconventional technical approach for localizing proteins that are occluded in biominerals. This technique relies on the use of molecular recognition atomic force microscopy with antibody-functionalized tips. Although such approach has been employed in other system, this is the very first time that it is developed for biominerals. In comparison to more classical approaches (such as immunogold), AFM microscopy with antibody-functionalized tips allows higher magnification and keeps the scanned surface clean for other biophysical characterizations. Our method has a general scope as it can be applied in human health, for non-pathological (bone, teeth) and pathological (kidney stone) biomineralizations as well as for bone implants coated with protein-doped calcium phosphate.
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Calcium carbonate skeletal tissues in metazoans comprise a small quantity of occluded organic macromolecules, mostly proteins and polysaccharides that constitute the skeletal matrix. Because its ...functions in modulating the biomineralization process are well known, the skeletal matrix has been extensively studied, successively via classical biochemical approaches, via molecular biology and, in recent years, via transcriptomics and proteomics. The optimistic view that the deposition of calcium carbonate minerals requires a limited number of macromolecules has been challenged, in the last decade, by high-throughput approaches. Such approaches have made possible the rapid identification of large sets of mineral-associated proteins, i.e., ‘skeletal repertoires’ or ‘skeletomes’, in several calcifying animal models, ranging from sponges to echinoderms. One of the consequences of this expanding set of data is that a simple definition of the skeletal matrix is no longer possible. This increase in available data, however, makes it easier to compare skeletal repertoires, shedding light on the fundamental evolutionary mechanisms affecting matrix components.
Many organisms incorporate inorganic solids into their tissues to improve functional and mechanical properties. The resulting mineralized tissues are called biominerals. Several studies have shown ...that nacreous biominerals induce osteoblastic extracellular mineralization. Among them,
Pinctada margaritifera
is well known for the ability of its organic matrix to stimulate bone cells. In this context, we aimed to study the effects of shell extracts from three other
Pinctada
species (
Pinctada radiata
,
Pinctada maxima
, and
Pinctada fucata
) on osteoblastic extracellular matrix mineralization, by using an in vitro model of mouse osteoblastic precursor cells (MC3T3-E1). For a better understanding of the
Pinctada
-bone mineralization relationship, we evaluated the effects of 4 other nacreous mollusks that are phylogenetically distant and distinct from the
Pinctada
genus. In addition, we tested 12 non-nacreous mollusks and one extra-group. Biomineral shell powders were prepared, and their organic matrix was partially extracted using ethanol. Firstly, the effect of these powders and extracts was assessed on the viability of MC3T3-E1. Our results indicated that neither the powder nor the ethanol-soluble matrix (ESM) affected cell viability at low concentrations. Then, we evaluated osteoblastic mineralization using Alizarin Red staining and we found a prominent MC3T3-E1 mineralization mainly induced by nacreous biominerals, especially those belonging to the
Pinctada
genus. However, few non-nacreous biominerals were also able to stimulate the extracellular mineralization. Overall, our findings validate the remarkable ability of CaCO
3
biomineral extracts to promote bone mineralization. Nevertheless, further in vitro and in vivo studies are needed to uncover the mechanisms of action of biominerals in bone.
Abstract
Massive corals of the genus Porites, common, keystone reef builders in the Indo-Pacific Ocean, are distinguished by their relative stress tolerance and longevity. In order to identify ...genetic bases of these attributes, we sequenced the complete genome of a massive coral, Porites australiensis. We developed a genome assembly and gene models of comparable quality to those of other coral genomes. Proteome analysis identified 60 Porites skeletal matrix protein genes, all of which show significant similarities to genes from other corals and even to those from a sea anemone, which has no skeleton. Nonetheless, 30% of its skeletal matrix proteins were unique to Porites and were not present in the skeletons of other corals. Comparative genomic analyses showed that genes widely conserved among other organisms are selectively expanded in Porites. Specifically, comparisons of transcriptomic responses of P. australiensis and Acropora digitifera, a stress-sensitive coral, reveal significant differences in regard to genes that respond to increased water temperature, and some of the genes expanded exclusively in Porites may account for the different thermal tolerances of these corals. Taken together, widely shared genes may have given rise to unique biological characteristics of Porites, massive skeletons and stress tolerance.
Mollusc shells are organic-inorganic composites that are often preserved in the fossil record. However, the way the organic fraction, also called shell matrix, gets fossilized remains an unsolved ...question, in spite of several old and more recent studies. In the present paper, we have tried to mimic a diagenetic process by constantly heating for ten days at 100°C fresh nacre powder samples of the Polynesian pearl oyster Pinctada margaritifera. Each day, aliquots of nacre powder were sampled and the matrix was subsequently extracted. It was further analysed by direct weigh quantification, by immunological techniques and by proteomics. Our preliminary data suggest that nacre proteins, when heated at 100°C in dry condition, degrade rather slowly. We evidenced a differential degradation pattern of the soluble and insoluble fractions, and showed that some nacre proteins of the insoluble fraction are stable after ten days of heating. Factors that influence the diagenetic stability of some shell proteins are discussed.
Denaturing 1D electrophoresis on acrylamide gels, also referred as SDS-PAGE, is a classical technique for fractionating and visualizing the macromolecular constituents of matrices associated to ...calcified tissues. This technique has been widely used in association with the subsequent silver nitrate staining. But because matrices associated to calcified tissues are very often glycosylated and constituted of numerous polydisperse macromolecules, the obtained pattern is frequently ‘smeary’ and discrete bands, when present on the gel, are often blurred and thickened. In this paper, we present a simple protocol that can circumvent this drawback and ‘clean’ the gels. In short, after the classical migration step of the matrix macromolecules, the gel is electro-blotted on a PVDF membrane, similarly to a Western blot, but for a shorter time (partial transfer, i.e., one hour or less). It is subsequently stained with silver nitrate. The likely effect of the transfer is to partly remove polydisperse macromolecules and to ‘sharpen’ the discrete bands. We think that this extra-step may improve in several cases the gel pictures, particularly when they are blurred. We illustrate this phenomenon with two examples taken from brachiopod and mollusc shell matrices.
The teeth of sea urchins are highly complex composite structures, composed predominantly of high magnesium calcite, and of a minor heterogeneous assemblage of organic macromolecules that are occluded ...within the mineral. The organic matrix fulfils important functions in mineralization, in addition to giving the mineral phase peculiar mechanical properties, different from that of purely inorganic calcite. Nevertheless, the composition and function of individual components of the organic matrix still remains largely unknown. Up to now, the detailed protein repertoire of teeth from a single sea urchin species (Strongylocentrotus purpuratus, order Camarodonta) was investigated. In this study, we characterized for the first time the teeth skeletal matrix of another sea urchin, Arbacia lixula (order Arbacioida). The acetic acid soluble and acetic acid insoluble matrices, namely ASM and AIM respectively, were extracted and characterized with different biochemical methods including mono-dimensional SDS-PAGE, FT-IR spectroscopy, HPAE-PAD for monosaccharide analysis, and finally, proteomics. In spite of the paucity of peptide data, several of them displayed a high abundance of hydrophobic residues, i.e., alanine, glycine and valine, and of the apolar proline. We assert that the alanine- and proline-rich domains are important features of some of the matrix proteins associated to the teeth of sea urchins. None of the known skeletal matrix proteins from S. purpuratus teeth were identified in the organic matrix of A. lixula teeth. This might suggest major differences in teeth matrix protein repertoires of these two species belonging to orders that diverged in the Mesozoic times.
Generalized Minimum Variance Control (GMVC) is one of the predictive control method for the time delay system. Most of the GMVC are designed for a Single-Input Single-Output (SISO) system. However, ...there are a lot of Multi-Inputs Multi-Outputs (MIMO) systems, such as the chemical plants, which have the multiple time delays of different length in regard of inputs and outputs pair. As a result, there is a problem that the response time still mutual interference. This paper proposes two new techniques for the MIMO system with the multiple time delays. The first is GMVC with a Cross-Controller method. The second is GMVC with the skeletal matrix. Comparing the two methods, we demonstrate the advantages of the matrix skeleton. Also, the closed-loop system is structured of Continuous-Time GMVC (CGMVC).
Generalized Minimum Variance Control (GMVC) is one of the predictive control method for the time delay system. Most of the GMVC are designed for a Single-Input Single-Output (SISO) system. However, ...there are a lot of Multi-Inputs Multi-Outputs (MIMO) systems, such as the chemical plants, which have the multiple time delays of different length in regard of inputs and outputs pair. As a result, there is a problem that the response time still mutual interference. This paper proposes two new techniques for the MIMO system with the multiple time delays. The first is GMVC with a Cross-Controller method. The second is GMVC with the skeletal matrix. Comparing the two methods, we demonstrate the advantages of the matrix skeleton. Also, the closed-loop system is structured of Continuous-Time GMVC (CGMVC).
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