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1.  Mechanical properties of nacre and highly mineralized bone. 
We compared the mechanical properties of 'ordinary' bovine bone, the highly mineralized bone of the rostrum of the whale Mesoplodon densirostris, and mother of pearl (nacre) of the pearl oyster Pinctada margaritifera. The rostrum and the nacre are similar in having very little organic material. However, the rostral bone is much weaker and more brittle than nacre, which in these properties is close to ordinary bone. The ability of nacre to outperform rostral bone is the result of its extremely well-ordered microstructure, with organic material forming a nearly continuous jacket round all the tiny aragonite plates, a design well adapted to produce toughness. In contrast, in the rostrum the organic material, mainly collagen, is poorly organized and discontinuous, allowing the mineral to join up to form, in effect, a brittle stony material.
PMCID: PMC1087608  PMID: 12123292
2.  Splice Variants of Perlucin from Haliotis laevigata Modulate the Crystallisation of CaCO3 
PLoS ONE  2014;9(5):e97126.
Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.
PMCID: PMC4019660  PMID: 24824517
3.  Origin of flaw-tolerance in nacre 
Scientific Reports  2013;3:1693.
Over the past decades, our understanding of nacre's toughening origin has long stayed at the level of crack deflection along the biopolymer interface between aragonite platelets. It has been widely thought that the ceramic aragonite platelets in nacre invariably remain shielded from the propagating crack. Here we report an unexpected experimental observation that the propagating crack, surprisingly, invades the aragonite platelet following a zigzag crack propagation trajectory. The toughening origin of previously-thought brittle aragonite platelet is ascribed to its unique nanoparticle-architecture, which tunes crack propagation inside the aragonite platelet in an intergranular manner. For comparison, we also investigated the crack behavior in geologic aragonite mineral (pure monocrystal) and found that the crack propagates in a cleavage fashion, in sharp contrast with the intergranular cracking in the aragonite platelet of nacre. These two fundamentally different cracking mechanisms uncover a new toughening strategy in nacre's hierarchical flaw-tolerance design.
PMCID: PMC3631768  PMID: 23603788
4.  On flaw tolerance of nacre: a theoretical study 
As a natural composite, nacre has an elegant staggered ‘brick-and-mortar’ microstructure consisting of mineral platelets glued by organic macromolecules, which endows the material with superior mechanical properties to achieve its biological functions. In this paper, a microstructure-based crack-bridging model is employed to investigate how the strength of nacre is affected by pre-existing structural defects. Our analysis demonstrates that owing to its special microstructure and the toughening effect of platelets, nacre has a superior flaw-tolerance feature. The maximal crack size that does not evidently reduce the tensile strength of nacre is up to tens of micrometres, about three orders higher than that of pure aragonite. Through dimensional analysis, a non-dimensional parameter is proposed to quantify the flaw-tolerance ability of nacreous materials in a wide range of structural parameters. This study provides us some inspirations for optimal design of advanced biomimetic composites.
PMCID: PMC3899869  PMID: 24402917
biomimetics; nacre; microstructure; flaw tolerance; strength
5.  Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells 
BMC Biophysics  2012;5:19.
Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed.
Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size.
In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519–17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types.
PMCID: PMC3507795  PMID: 22967319
6.  Organization pattern of nacre in Pteriidae (Bivalvia: Mollusca) explained by crystal competition 
Bivalve nacre is a brick-wall-patterned biocomposite of aragonite platelets surrounded by organic matter. SEM–electron back scatter diffraction analysis of nacre of the bivalve family Pteriidae reveals that early aragonite crystals grow with their c-axes oriented perpendicular to the growth surface but have their a- and b-axes disoriented. With the accumulation of successive lamellae, crystals progressively orient themselves with their b-axes mutually parallel and towards the growth direction. We propose that progressive orientation is a result of competition between nacre crystals at the growth front of lamellae, which favours selection of crystals whose fastest growth axis (b-axis) is oriented parallel to the direction of propagation of the lamella. A theoretical model has been developed, which simulates competition of rhombic plates at the lamellar growth front as well as epitaxial growth of crystals onto those of the preceding lamella. The model predicts that disordered nacre progressively produces bivalve-like oriented nacre. As growth fronts become diffuse (as is the common case in bivalves) it takes longer for nacre to become organized. Formation of microdomains of nacre platelets with different orientations is also reproduced. In conclusion, not only the organic matrix component, but also the mineral phase plays an active role in organizing the final microstructure.
PMCID: PMC1560289  PMID: 16777720
biomineralization; microstructure; crystallography; shell; nacre; bivalves
7.  Multifunctional layered magnetic composites 
A fabrication method of a multifunctional hybrid material is achieved by using the insoluble organic nacre matrix of the Haliotis laevigata shell infiltrated with gelatin as a confined reaction environment. Inside this organic scaffold magnetite nanoparticles (MNPs) are synthesized. The amount of MNPs can be controlled through the synthesis protocol therefore mineral loadings starting from 15 wt % up to 65 wt % can be realized. The demineralized organic nacre matrix is characterized by small-angle and very-small-angle neutron scattering (SANS and VSANS) showing an unchanged organic matrix structure after demineralization compared to the original mineralized nacre reference. Light microscopy and confocal laser scanning microscopy studies of stained samples show the presence of insoluble proteins at the chitin surface but not between the chitin layers. Successful and homogeneous gelatin infiltration in between the chitin layers can be shown. The hybrid material is characterized by TEM and shows a layered structure filled with MNPs with a size of around 10 nm. Magnetic analysis of the material demonstrates superparamagnetic behavior as characteristic for the particle size. Simulation studies show the potential of collagen and chitin to act as nucleators, where there is a slight preference of chitin over collagen as a nucleator for magnetite. Colloidal-probe AFM measurements demonstrate that introduction of a ferrogel into the chitin matrix leads to a certain increase in the stiffness of the composite material.
PMCID: PMC4311584  PMID: 25671158
bio-inspired mineralization; biomineralization; chitin; ferrogel; hybrid materials; magnetite; nacre
8.  Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata 
PLoS ONE  2013;8(6):e66564.
The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo.
PMCID: PMC3680448  PMID: 23776687
9.  Organic–inorganic interfaces and spiral growth in nacre 
Nacre, the crown jewel of natural materials, has been extensively studied owing to its remarkable physical properties for over 160 years. Yet, the precise structural features governing its extraordinary strength and its growth mechanism remain elusive. In this paper, we present a series of observations pertaining to the red abalone (Haliotis rufescens) shell's organic–inorganic interface, organic interlayer morphology and properties, large-area crystal domain orientations and nacre growth. In particular, we describe unique lateral nano-growths and paired screw dislocations in the aragonite layers, and demonstrate that the organic material sandwiched between aragonite platelets consists of multiple organic layers of varying nano-mechanical resilience. Based on these novel observations and analysis, we propose a spiral growth model that accounts for both [001] vertical propagation via helices that surround numerous screw dislocation cores and simultaneous 〈010〉 lateral growth of aragonite sheet structure. These new findings may aid in creating novel organic–inorganic micro/nano composites through synthetic or biomineralization pathways.
PMCID: PMC2572677  PMID: 18753125
nacre; biomineralization; crystal growth
10.  Identification of genes associated with shell color in the black-lipped pearl oyster, Pinctada margaritifera 
BMC Genomics  2015;16(1):568.
Color polymorphism in the nacre of pteriomorphian bivalves is of great interest for the pearl culture industry. The nacreous layer of the Polynesian black-lipped pearl oyster Pinctada margaritifera exhibits a large array of color variation among individuals including reflections of blue, green, yellow and pink in all possible gradients. Although the heritability of nacre color variation patterns has been demonstrated by experimental crossing, little is known about the genes involved in these patterns. In this study, we identify a set of genes differentially expressed among extreme color phenotypes of P. margaritifera using a suppressive and subtractive hybridization (SSH) method comparing black phenotypes with full and half albino individuals.
Out of the 358 and 346 expressed sequence tags (ESTs) obtained by conducting two SSH libraries respectively, the expression patterns of 37 genes were tested with a real-time quantitative PCR (RT-qPCR) approach by pooling five individuals of each phenotype. The expression of 11 genes was subsequently estimated for each individual in order to detect inter-individual variation. Our results suggest that the color of the nacre is partially under the influence of genes involved in the biomineralization of the calcitic layer. A few genes involved in the formation of the aragonite tablets of the nacre layer and in the biosynthesis chain of melanin also showed differential expression patterns. Finally, high variability in gene expression levels were observed within the black phenotypes.
Our results revealed that three main genetic processes were involved in color polymorphisms: the biomineralization of the nacreous and calcitic layers and the synthesis of pigments such as melanin, suggesting that color polymorphism takes place at different levels in the shell structure. The high variability of gene expression found within black phenotypes suggests that the present work should serve as a basis for future studies exploring more thoroughly the expression patterns of candidate genes within black phenotypes with different dominant iridescent colors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1776-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4521380  PMID: 26231360
Differential expression; Biomineralization; Nacre; Pearl; Pigmentation; Albino
11.  Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells 
Molecules and Cells  2015;38(3):267-272.
Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I–IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s.
PMCID: PMC4363727  PMID: 25666352
bone morphogenetic protein; human bone marrow stromal cells; mesenchymal stem cells; nacre; nacre soluble matrix proteins; osteoinduction
12.  Spatial analysis of biomineralization associated gene expression from the mantle organ of the pearl oyster Pinctada maxima 
BMC Genomics  2011;12:455.
Biomineralization is a process encompassing all mineral containing tissues produced within an organism. One of the most dynamic examples of this process is the formation of the mollusk shell, comprising a variety of crystal phases and microstructures. The organic component incorporated within the shell is said to dictate this architecture. However general understanding of how this process is achieved remains ambiguous. The mantle is a conserved organ involved in shell formation throughout molluscs. Specifically the mantle is thought to be responsible for secreting the protein component of the shell. This study employs molecular approaches to determine the spatial expression of genes within the mantle tissue to further the elucidation of the shell biomineralization.
A microarray platform was custom generated (PmaxArray 1.0) from the pearl oyster Pinctada maxima. PmaxArray 1.0 consists of 4992 expressed sequence tags (ESTs) originating from mantle tissue. This microarray was used to analyze the spatial expression of ESTs throughout the mantle organ. The mantle was dissected into five discrete regions and analyzed for differential gene expression with PmaxArray 1.0. Over 2000 ESTs were determined to be differentially expressed among the tissue sections, identifying five major expression regions. In situ hybridization validated and further localized the expression for a subset of these ESTs. Comparative sequence similarity analysis of these ESTs revealed a number of the transcripts were novel while others showed significant sequence similarities to previously characterized shell related genes.
This investigation has mapped the spatial distribution for over 2000 ESTs present on PmaxArray 1.0 with reference to specific locations of the mantle. Expression profile clusters have indicated at least five unique functioning zones in the mantle. Three of these zones are likely involved in shell related activities including formation of nacre, periostracum and calcitic prismatic microstructure. A number of novel and known transcripts have been identified from these clusters. The development of PmaxArray 1.0, and the spatial map of its ESTs expression in the mantle has begun characterizing the molecular mechanisms linking the organics and inorganics of the molluscan shell.
PMCID: PMC3191542  PMID: 21936921
13.  Material properties of brachiopod shell ultrastructure by nanoindentation 
Mineral-producing organisms exert exquisite control on all aspects of biomineral production. Among shell-bearing organisms, a wide range of mineral fabrics are developed reflecting diverse modes of life that require different material properties. Our knowledge of how biomineral structures relate to material properties is still limited because it requires the determination of these properties on a detailed scale. Nanoindentation, mostly applied in engineering and materials science, is used here to assess, at the microstructural level, material properties of two calcite brachiopods living in the same environment but with different modes of life and shell ultrastructure. Values of hardness (H) and the Young modulus of elasticity (E) are determined by nanoindentation. In brachiopod shells, calcite semi-nacre provides a harder and stiffer structure (H∼3–6 GPa; E=60–110/120 GPa) than calcite fibres (H=0–3 GPa; E=20–60/80 GPa). Thus, brachiopods with calcite semi-nacre can cement to a substrate and remain immobile during their adult life cycle. This correlation between mode of life and material properties, as a consequence of ultrastructure, begins to explain why organisms produce a wide range of structures using the same chemical components, such as calcium carbonate.
PMCID: PMC2358955  PMID: 17015292
nanoindentation; brachiopod; biomineral; shell ultrastructure; ecology
14.  Multifunctional, supramolecular, continuous artificial nacre fibres 
Scientific Reports  2012;2:767.
Nature has created amazing materials during the process of evolution, inspiring scientists to studiously mimic them. Nacre is of particular interest, and it has been studied for more than half-century for its strong, stiff, and tough attributes resulting from the recognized “brick-and-mortar” (B&M) layered structure comprised of inorganic aragonite platelets and biomacromolecules. The past two decades have witnessed great advances in nacre-mimetic composites, but they are solely limited in films with finite size (centimetre-scale). To realize the adream target of continuous nacre-mimics with perfect structures is still a great challenge unresolved. Here, we present a simple and scalable strategy to produce bio-mimic continuous fibres with B&M structures of alternating graphene sheets and hyperbranched polyglycerol (HPG) binders via wet-spinning assembly technology. The resulting macroscopic supramolecular fibres exhibit excellent mechanical properties comparable or even superior to nacre and bone, and possess fine electrical conductivity and outstanding corrosion-resistance.
PMCID: PMC3479450  PMID: 23097689
15.  Layer-by-Layer Proteomic Analysis of Mytilus galloprovincialis Shell 
PLoS ONE  2015;10(7):e0133913.
Bivalve shell is a biomineralized tissue with various layers/microstructures and excellent mechanical properties. Shell matrix proteins (SMPs) pervade and envelop the mineral crystals and play essential roles in biomineralization. Despite that Mytilus is an economically important bivalve, only few proteomic studies have been performed for the shell, and current knowledge of the SMP set responsible for different shell layers of Mytilus remains largely patchy. In this study, we observed that Mytilus galloprovincialis shell contained three layers, including nacre, fibrous prism, and myostracum that is involved in shell-muscle attachment. A parallel proteomic analysis was performed for these three layers. By combining LC-MS/MS analysis with Mytilus EST database interrogations, a whole set of 113 proteins was identified, and the distribution of these proteins in different shell layers followed a mosaic pattern. For each layer, about a half of identified proteins are unique and the others are shared by two or all of three layers. This is the first description of the protein set exclusive to nacre, myostracum, and fibrous prism in Mytilus shell. Moreover, most of identified proteins in the present study are novel SMPs, which greatly extended biomineralization-related protein data of Mytilus. These results are useful, on one hand, for understanding the roles of SMPs in the deposition of different shell layers. On the other hand, the identified protein set of myostracum provides candidates for further exploring the mechanism of adductor muscle-shell attachment.
PMCID: PMC4517812  PMID: 26218932
16.  The Intrinsically Disordered C-RING Biomineralization Protein, AP7, Creates Protein Phases That Introduce Nanopatterning and Nanoporosities into Mineral Crystals 
Biochemistry  2014;53(27):4317-4319.
We report an interesting process whereby the formation of nanoparticle assemblies on and nanoporosities within calcite crystals is directed by an intrinsically disordered C-RING mollusk shell nacre protein, AP7. Under mineralization conditions, AP7 forms protein phases that direct the nucleation of ordered calcite nanoparticles via a repetitive protein phase deposition process onto calcite crystals. These organized nanoparticles are separated by gaps or spaces that become incorporated into the forming bulk crystal as nanoporosities. This is an unusual example of organized nanoparticle biosynthesis and mineral modification directed by a C-RING protein phase.
PMCID: PMC4215888  PMID: 24977921
17.  Ubiquitylation Functions in the Calcium Carbonate Biomineralization in the Extracellular Matrix 
PLoS ONE  2012;7(4):e35715.
Mollusks shell formation is mediated by matrix proteins and many of these proteins have been identified and characterized. However, the mechanisms of protein control remain unknown. Here, we report the ubiquitylation of matrix proteins in the prismatic layer of the pearl oyster, Pinctada fucata. The presence of ubiquitylated proteins in the prismatic layer of the shell was detected with a combination of western blot and immunogold assays. The coupled ubiquitins were separated and identified by Edman degradation and liquid chromatography/mass spectrometry (LC/MS). Antibody injection in vivo resulted in large amounts of calcium carbonate randomly accumulating on the surface of the nacreous layer. These ubiquitylated proteins could bind to specific faces of calcite and aragonite, which are the two main mineral components of the shell. In the in vitro calcium carbonate crystallization assay, they could reduce the rate of calcium carbonate precipitation and induce the calcite formation. Furthermore, when the attached ubiquitins were removed, the functions of the EDTA-soluble matrix of the prismatic layer were changed. Their potency to inhibit precipitation of calcium carbonate was decreased and their influence on the morphology of calcium carbonate crystals was changed. Taken together, ubiquitylation is involved in shell formation. Although the ubiquitylation is supposed to be involved in every aspect of biophysical processes, our work connected the biomineralization-related proteins and the ubiquitylation mechanism in the extracellular matrix for the first time. This would promote our understanding of the shell biomineralization and the ubiquitylation processes.
PMCID: PMC3338455  PMID: 22558208
18.  Identification of Proteins with Potential Osteogenic Activity Present in the Water-Soluble Matrix Proteins from Crassostrea gigas Nacre Using a Proteomic Approach 
The Scientific World Journal  2012;2012:765909.
Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM). We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2) were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.
PMCID: PMC3361287  PMID: 22666151
19.  The expanded amelogenin polyproline region preferentially binds to apatite versus carbonate and promotes apatite crystal elongation 
The transition from invertebrate calcium carbonate-based calcite and aragonite exo- and endoskeletons to the calcium phosphate-based vertebrate backbones and jaws composed of microscopic hydroxyapatite crystals is one of the great revolutions in the evolution of terrestrial organisms. To identify potential factors that might have played a role in such a transition, three key domains of the vertebrate tooth enamel protein amelogenin were probed for calcium mineral/protein interactions and their ability to promote calcium phosphate and calcium carbonate crystal growth. Under calcium phosphate crystal growth conditions, only the carboxy-terminus augmented polyproline repeat peptide, but not the N-terminal peptide nor the polyproline repeat peptide alone, promoted the formation of thin and parallel crystallites resembling those of bone and initial enamel. In contrast, under calcium carbonate crystal growth conditions, all three amelogenin-derived polypeptides caused calcium carbonate to form fused crystalline conglomerates. When examined for long-term crystal growth, polyproline repeat peptides of increasing length promoted the growth of shorter calcium carbonate crystals with broader basis, contrary to the positive correlation between polyproline repeat element length and apatite mineralization published earlier. To determine whether the positive correlation between polyproline repeat element length and apatite crystal growth versus the inverse correlation between polyproline repeat length and calcium carbonate crystal growth were related to the binding affinity of the polyproline domain to either apatite or carbonate, a parallel series of calcium carbonate and calcium phosphate/apatite protein binding studies was conducted. These studies demonstrated a remarkable binding affinity between the augmented amelogenin polyproline repeat region and calcium phosphates, and almost no binding to calcium carbonates. In contrast, the amelogenin N-terminus bound to both carbonate and apatite, but preferentially to calcium carbonate. Together, these studies highlight the specific binding affinity of the augmented amelogenin polyproline repeat region to calcium phosphates versus calcium carbonate, and its unique role in the growth of thin apatite crystals as they occur in vertebrate biominerals. Our data suggest that the rise of apatite-based biominerals in vertebrates might have been facilitated by a rapid evolution of specialized polyproline repeat proteins flanked by a charged domain, resulting in apatite crystals with reduced width, increased length, and tailored biomechanical properties.
PMCID: PMC4227485  PMID: 25426079
amelogenin; hydroxyapatite; calcium carbonate; polyproline repeat proteins; vertebrate evolution
20.  The Role of Acidic Phosphoproteins in Biomineralization 
Connective tissue research  2014;55(1):34-40.
Biomineralization is the process by which living organisms deposit mineral in the extracellular matrix. In nature, almost 50% of biominerals are calcium-bearing minerals. In addition to calcium, we also find biominerals formed from silica and magnetite. Calcium containing biominerals could be either calcium phosphate as in apatite found in vertebrates or calcium carbonate as in calcite and aragonite found in many invertebrates. Since all biomineralization is matrix mediated, an understanding of the nature of the proteins involved is essential in elucidating its mechanism. This review will discuss some of the proteins involved in the process of biomineralization involving calcium. Two proteins, dentin matrix protein 1 and dentin phosphoprotein (Phosphophoryn) will serve as models for the vertebrate system, and two others - P16 and phosphodontin will serve as models for the invertebrate system.
PMCID: PMC4005356  PMID: 24437603
DMP1; DSPP; sea urchin P16; Phosphodontin
21.  Characteristics and Causes for Non-Accrued Clinical Research (NACR) at an Academic Medical Institution 
The impact of non-accrued clinical research (NACR) represents an important economic burden that is under consideration as the U.S. Department of Health and Human Services looks into reforming the regulations governing IRB review. NACR refers to clinical research projects that fail to enroll subjects. A delineation of the issues surrounding NACR is expected to enhance subject accrual and to minimize occurrence of NACR. The authors assessed demographics, characteristics, and reasons for NACR at an academic medical center, including time trends, funding source, research team (principal investigator, department), IRB resource utilization (IRB level of review, number of required IRB reviews, initial IRB turn-around time, and duration of NACR).
The authors analyzed data from 848 clinical research study closures during 2010 and 2011 to determine proportion, incidence, and characteristics of NACR. Studies with subject enrollment during the same time period were used as a comparative measure.
Data from 704 (83.0%) study closures reported enrollment of 1 or more subjects while 144 (17.0 %) reported NACR (zero enrollment). PI-reported reasons for NACR included: 32 (22.2%) contract or funding issues; 43 (30.0%) insufficient study-dedicated resources; 41 (28.4%) recruitment issues; 17 (11.8%) sponsor-initiated study closure and 11 (7.6%) were “other/reason unreported”.
NACR is not uncommon, affecting about one in six clinical research projects in the study population and reported to be more common in some other institutions. The complex and fluid nature of research conduct, non-realistic enrollment goals, and delays in both the approval and/or accrual processes contribute to NACR. Results suggest some simple strategies that investigators and institutions may use to reduce NACR, including careful feasibility assessment, reduction of institutional delays, and prompt initiation of subject accrual for multi-center studies using competitive enrollment. Institutional action to support investigators in the conduct clinical research is also encouraged to reduce likelihood of NACR.
PMCID: PMC3651069  PMID: 23671544
Clinical research; Recruitment issues; Accrual
22.  Saline systems of the Great Plains of western Canada: an overview of the limnogeology and paleolimnology 
Saline Systems  2005;1:10.
In much of the northern Great Plains, saline and hypersaline lacustrine brines are the only surface waters present. As a group, the lakes of this region are unique: there is no other area in the world that can match the concentration and diversity of saline lake environments exhibited in the prairie region of Canada and northern United States. The immense number of individual salt lakes and saline wetlands in this region of North America is staggering. Estimates vary from about one million to greater than 10 million, with densities in some areas being as high as 120 lakes/km2.
Despite over a century of scientific investigation of these salt lakes, we have only in the last twenty years advanced far enough to appreciate the wide spectrum of lake types, water chemistries, and limnological processes that are operating in the modern settings. Hydrochemical data are available for about 800 of the lake brines in the region. Composition, textural, and geochemical information on the modern bottom sediments has been collected for just over 150 of these lakes. Characterization of the biological and ecological features of these lakes is based on even fewer investigations, and the stratigraphic records of only twenty basins have been examined.
The lake waters show a considerable range in ionic composition and concentration. Early investigators, concentrating on the most saline brines, emphasized a strong predominance of Na+ and SO4-2 in the lakes. It is now realized, however, that not only is there a complete spectrum of salinities from less than 1 ppt TDS to nearly 400 ppt, but also virtually every water chemistry type is represented in lakes of the region. With such a vast array of compositions, it is difficult to generalize. Nonetheless, the paucity of Cl-rich lakes makes the northern Great Plains basins somewhat unusual compared with salt lakes in many other areas of the world (e.g., Australia, western United States). Compilations of the lake water chemistries show distinct spatial trends and regional variations controlled by groundwater input, climate, and geomorphology. Short-term temporal variations in the brine composition, which can have significant effects on the composition of the modern sediments, have also been well documented in several individual basins.
From a sedimentological and mineralogical perspective, the wide range of water chemistries exhibited by the lakes leads to an unusually large diversity of modern sediment composition. Over 40 species of endogenic precipitates and authigenic minerals have been identified in the lacustrine sediments. The most common non-detrital components of the modern sediments include: calcium and calcium-magnesium carbonates (magnesian calcite, aragonite, dolomite), and sodium, magnesium, and sodium-magnesium sulfates (mirabilite, thenardite, bloedite, epsomite). Many of the basins whose brines have very high Mg/Ca ratios also have hydromagnesite, magnesite, and nesquehonite. Unlike salt lakes in many other areas of the world, halite, gypsum, and calcite are relatively rare endogenic precipitates in the Great Plains lakes. The detrital fraction of the lacustrine sediments is normally dominated by clay minerals, carbonate minerals, quartz, and feldspars.
Sediment accumulation in these salt lakes is controlled and modified by a wide variety of physical, chemical, and biological processes. Although the details of these modern sedimentary processes can be exceedingly complex and difficult to discuss in isolation, in broad terms, the processes operating in the salt lakes of the Great Plains are ultimately controlled by three basic factors or conditions of the basin: (a) basin morphology; (b) basin hydrology; and (c) water salinity and composition. Combinations of these parameters interact to control nearly all aspects of modern sedimentation in these salt lakes and give rise to four 'end member' types of modern saline lacustrine settings in the Great Plains: (a) clastics-dominated playas; (b) salt-dominated playas; (c) deep water, non-stratified lakes; and (d) deep water, "permanently" stratified lakes.
PMCID: PMC1315329  PMID: 16297237
23.  CO2-Driven Ocean Acidification Alters and Weakens Integrity of the Calcareous Tubes Produced by the Serpulid Tubeworm, Hydroides elegans 
PLoS ONE  2012;7(8):e42718.
As a consequence of anthropogenic CO2-driven ocean acidification (OA), coastal waters are becoming increasingly challenging for calcifiers due to reductions in saturation states of calcium carbonate (CaCO3) minerals. The response of calcification rate is one of the most frequently investigated symptoms of OA. However, OA may also result in poor quality calcareous products through impaired calcification processes despite there being no observed change in calcification rate. The mineralogy and ultrastructure of the calcareous products under OA conditions may be altered, resulting in changes to the mechanical properties of calcified structures. Here, the warm water biofouling tubeworm, Hydroides elegans, was reared from larva to early juvenile stage at the aragonite saturation state (ΩA) for the current pCO2 level (ambient) and those predicted for the years 2050, 2100 and 2300. Composition, ultrastructure and mechanical strength of the calcareous tubes produced by those early juvenile tubeworms were examined using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and nanoindentation. Juvenile tubes were composed primarily of the highly soluble CaCO3 mineral form, aragonite. Tubes produced in seawater with aragonite saturation states near or below one had significantly higher proportions of the crystalline precursor, amorphous calcium carbonate (ACC) and the calcite/aragonite ratio dramatically increased. These alterations in tube mineralogy resulted in a holistic deterioration of the tube hardness and elasticity. Thus, in conditions where ΩA is near or below one, the aragonite-producing juvenile tubeworms may no longer be able to maintain the integrity of their calcification products, and may result in reduced survivorship due to the weakened tube protection.
PMCID: PMC3418283  PMID: 22912726
24.  The quest for stiff, strong and tough hybrid materials: an exhaustive exploration 
How to arrange soft materials with strong but brittle reinforcements to achieve attractive combinations of stiffness, strength and toughness is an ongoing and fascinating question in engineering and biological materials science. Recent advances in topology optimization and bioinspiration have brought interesting answers to this question, but they provide only small windows into the vast design space associated with this problem. Here, we take a more global approach in which we assess the mechanical performance of thousands of possible microstructures. This exhaustive exploration gives a global picture of structure–property relationships and guarantees that global optima can be found. Landscapes of optimum solutions for different combinations of desired properties can also be created, revealing the robustness of each of the solutions. Interestingly, while some of the major hybrid designs used in engineering are absent from the set of solutions, the microstructures emerging from this process are reminiscent of materials, such as bone, nacre or spider silk.
PMCID: PMC3808548  PMID: 24068176
biological materials; bioinspired composites; optimization; hybrid materials; bone; nacre
25.  Novel Matrix Proteins of Pteria penguin Pearl Oyster Shell Nacre Homologous to the Jacalin-Related β-Prism Fold Lectins 
PLoS ONE  2014;9(11):e112326.
Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of Pteria penguin oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of β subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and in vitro CaCO3 crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of P. penguin nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of P. penguin mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation.
PMCID: PMC4223035  PMID: 25375177

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