New Insights on the Diurnal Mechanism of Calcification in the Stony Coral, Stylophora pistillata

Scleractinian corals are evolutionary-successful calcifying marine organisms, which utilize an endo-symbiotic relationship with photosynthetic dinoflagellate algae that supply energy products to their coral hosts. This energy further supports a higher calcification rate during the day in a process known as light enhanced calcification. Although this process has been studied for decades, the mechanisms behind it are still unknown. However, photosynthesis and respiration also cause daily fluctuations in oxygen and pH levels, resulting in the coral facing highly variable conditions. Here we correlated gene expression patterns with the physiological differences along the diel cycle to provide new insights on the daily dynamic processes, including circadian rhythm, calcification, symbiosis, cellular arrangement, metabolism, and energy budget. During daytime, when solar radiation levels are highest, we observed increased calcification rate combined with an extensive up-regulation of genes associated with reactive oxygen species, redox, metabolism, ion transporters, skeletal organic matrix, and mineral formation. During the night, we observed a vast shift toward up-regulation of genes associated with cilia movement, tissue development, cellular movement, antioxidants, protein synthesis, and skeletal organic matrix formation. Our results suggest that light enhanced calcification is related to several processes that occur across the diel cycle; during nighttime, tissue might elevate away from the skeleton, extending the calcifying space area to enable the formation of a new organic framework template. During daytime, the combination of synthesis of acid-rich proteins and a greater flux of ions to the sites of calcification facilitate the conditions for extensive mineral growth.

Applications of Cryogenic Electron Microscopy in Biomineralization Research

Biological mineralization is a natural process manifested by living organisms in which inorganic minerals crystallize under the scrupulous control of biomolecules, producing hierarchical organic-inorganic composite structures with physical properties and design that galvanize even the most ardent structural engineer and architect. Understanding the mechanisms that control the formation of biominerals is challenging in the biomimetic engineering of hard tissues. In this regard, the contribution of cryogenic electron microscopy (cryo-EM) has been nothing short of phenomenal. By preserving materials in their native hydrated status and reducing damage caused by ion beam radiation, cryo-EM outperforms conventional transmission electron microscopy in its ability to directly observe the morphologic evolution of mineral precursor phases at different stages of biomineralization with nanoscale spatial resolution and subsecond temporal resolution in 2 or 3 dimensions. In the present review, the development and applications of cryo-EM are discussed to support the use of this powerful technique in dental research. Because of the rapid development of cryogenic sample preparation techniques, direct electron detection, and image-processing algorithms, the last decade has witnessed an exponential increase in the use of cryo-EM in structural biology and materials research. By amalgamating with other analytic techniques, cryo-EM may be used for qualitative and quantitative analyses of the kinetics and thermodynamic mechanisms in which organic macromolecules participate in the transformation of mineral precursors from their original liquid state to amorphous and ultimately crystalline phases. The present review concentrates on the biomineralization of calcium phosphate mineral phases, while that of calcium carbonate, silica, and magnetite is only briefly mentioned. Bioinspired organic matrix–mediated inorganic crystallization strategies are discussed from the perspective of tissue regeneration engineering.

The Mechanical Consequences of the Interplay of Mineral Distribution and Organic Matrix Orientation in the Claws of the Sea Slater Ligia pallasii

Exposed regions of the arthropod exoskeleton have specialized structure and mineral composition. Their study can provide insights into the evolutionary optimization of the cuticle as a material. We determined the structural and compositional features of claws in the crustacean Ligia pallasii using X-ray micro-computed tomography, scanning electron microscopy (SEM), and analytical scanning transmission electron microscopy (STEM). In addition, we used nanoindentation to determine how these features fine-tune the mechanical properties of the claw cuticle. We found that the inner layer of the claw cuticle—the endocuticle—contains amorphous calcium phosphate, while the outer layer—the exocuticle—is not mineralized and contains elevated amounts of bromine. While the chitin–protein fibers in crustacean exoskeletons generally shift their orientation, they are aligned axially in the claws of L. pallasii. As a consequence, the claw cuticle has larger elastic modulus and hardness in the axial direction. We show that amorphous calcium phosphate mineralization and the brominated cuticle are widespread in isopod crustaceans inhabiting terrestrial habitats. We discuss how the features of the claw cuticle may aid in minimizing the likelihood of fracture. Ultimately, our study points out the features that increase the durability of thin skeletal elements.

Comparative Study of Sample-Preparation Techniques for Quantitative Analysis of the Mineral Composition of Humic Substances by Inductively Coupled Plasma Atomic Emission Spectroscopy

Five sample-preparation techniques were compared to quantify 31 elements in coal humic substances (HS) by ICP–AES from the viewpoints of complete isolation and speciation of elements. They include, for bulk composition, preparation of an aqueous colloidal HS solution followed by direct injection of the sample without decomposition and ashing followed by metaborate fusion; for element speciation, preparation of an aqueous colloidal HS solution followed by centrifugation and direct analysis without decomposition for water-soluble species; treatment with boiling nitric acid; and microwave-assisted treatment with nitric acid at 250 °C for acid-isolated species. The results of analysis significantly depend on the selected method of sample preparation due to specific features of HS, the simultaneous presence of many inorganic components in wide concentration ranges, and a significant fraction of the organic matrix; therefore, the total mineral composition of HS, both macro- and microcomponents, requires a combination of decomposition methods.

The architecture of Recent brachiopod shells: diversity of biocrystal and biopolymer assemblages in rhynchonellide, terebratulide, thecideide and craniide shells

Biological hard tissues are a rich source of design concepts for the generation of advanced materials. They represent the most important library of information on the evolution of life and its environmental conditions. Organisms produce soft and hard tissues in a bottom-up process, a construction principle that is intrinsic to biologically secreted materials. This process emerged early on in the geological record, with the onset of biological mineralization. The phylum Brachiopoda is a marine animal group that has an excellent and continuous fossil record from the early Cambrian to the Recent. Throughout this time interval, the Brachiopoda secreted phosphate and carbonate shells and populated many and highly diverse marine habitats. This required great flexibility in the adaptation of soft and hard tissues to the different marine environments and living conditions. This review presents, juxtaposes and discusses the main modes of mineral and biopolymer organization in Recent, carbonate shell-producing, brachiopods. We describe shell tissue characteristics for taxa of the orders Rhynchonellida, Terebratulida, Thecideida and Craniida. We highlight modes of calcite and organic matrix assembly at the macro-, micro-, and nano-scales based on results obtained by Electron Backscatter Diffraction, Atomic Force Microscopy, Field Emission Scanning Electron Microscopy and Scanning Transmission Electron Microscopy. We show variation in composite hard tissue organization for taxa with different lifestyles, visualize nanometer-scale calcite assemblies for rhynchonellide and terebratulide fibers, highlight thecideide shell microstructure, texture and chemistry characteristics, and discuss the feasibility to use thecideide shells as archives of proxies for paleoenvironment and paleoclimate reconstructions.

Experimental Study on Planting Soil Preparation by Improving River & Lake Silt with Plant Wastes

The physicochemical properties of river & lake silt are complex, and whether it can be directly used as planting soil is worth studying. The calliopsis pot experiment is carried out with planting soil prepared by amendment material, i.e. the organic matrix which is made by fermentation of high-nutrient sludge of a river in Nanjing, the dry excavating sludge in a lake and its flocculated and dewatered sludge together with plant wastes such as wood chips, to study the effects of different types of amendment materials and compounding ratio on plant growth. The results showed that the basic properties and fertility index of the planting soil could be adjusted directionally by adding wood chips or matrix. The overall growth of calliopsis in the planting soil formed by the high-nutrient silt in a river and its compound is the best, but some of the fertility indexes of the planting soil are too high and need to be further adjusted before use; the growth of calliopsis in the improved soil made of dry-excavation silt in a lake is better than that in the original silt, such situation is positively correlated with the amount of improved materials mixed; the difference between the growth of calliopsis in the flocculated silt in a lake and that in its improved planting soil is not significant, but some of the fertility indexes are higher than the standard indexes, and such silt can be slightly adjusted and improved into the planting soil. The field cultivation experiment study of calliopsis is carried out with the dry-excavation silt in a lake mixed with 4% wood chips and the original loess soil in the experimental field, and the growth of calliopsis planted in the dry-excavation silt in a lake is better compared with that of calliopsis planted in original loess soil. The research results can provide ideas and basis for the study on improving river & lake silt into planting soil with plant wastes.

Raman and XANES Spectroscopic Study of the Influence of Coordination Atomic and Molecular Environments in Biomimetic Composite Materials Integrated with Dental Tissue

In this work, for the first time, the influence of the coordination environment as well as Ca and P atomic states on biomimetic composites integrated with dental tissue was investigated. Bioinspired dental composites were synthesised based on nanocrystalline calcium carbonate-substituted hydroxyapatite Ca4ICa6IIPO46−xCO3x+yOH2−y (nano-cHAp) obtained from a biogenic source and a set of polar amino acids that modelled the organic matrix. Biomimetic composites, as well as natural dental tissue samples, were investigated using Raman spectromicroscopy and synchrotron X-ray absorption near edge structure (XANES) spectroscopy. Molecular structure and energy structure studies revealed several important features related to the different calcium atomic environments. It was shown that biomimetic composites created in order to reproduce the physicochemical properties of dental tissue provide good imitation of molecular and electron energetic properties, including the carbonate anion CO32− and the atomic Ca/P ratio in nanocrystals. The features of the molecular structure of biomimetic composites are inherited from the nano-cHAp (to a greater extent) and the amino acid cocktail used for their creation, and are caused by the ratio between the mineral and organic components, which is similar to the composition of natural enamel and dentine. In this case, violation of the nano-cHAp stoichiometry, which is the mineral basis of the natural and bioinspired composites, as well as the inclusion of different molecular groups in the nano-cHAp lattice, do not affect the coordination environment of phosphorus atoms. The differences observed in the molecular and electron energetic structures of the natural enamel and dentine and the imitation of their properties by biomimetic materials are caused by rearrangement in the local environment of the calcium atoms in the HAp crystal lattice. The surface of the nano-cHAp crystals in the natural enamel and dentine involved in the formation of bonds with the organic matrix is characterised by the coordination environment of the calcium atom, corresponding to its location in the CaI position—that is, bound through common oxygen atoms with PO4 tetrahedrons. At the same time, on the surface of nano-cHAp crystals in bioinspired dental materials, the calcium atom is characteristically located in the CaII position, bound to the hydroxyl OH group. The features detected in the atomic and molecular coordination environment in nano-cHAp play a fundamental role in recreating a biomimetic dental composite of the natural organomineral interaction in mineralised tissue and will help to find an optimal way to integrate the dental biocomposite with natural tissue.

UNDERSTANDING THE ETIOPATHOGENESIS AND PATHOPHYSIOLOGY OF RENAL CALCULI

Calculus (Stone) is a polycrystalline aggregate made up of different quantities of the crystalloid and organic matrix. Urine calculus is a stone-like formation made up of urine salts held together by a colloid matrix or organic elements. It has a nucleus or nidus around which concentric layers of urinary salts are formed, giving it a stone-like appear- ance. Urolithiasis (from Greek oûron, "urine," and lithos, "stone") is a urinary system pathology in which urinary crystalloids clump together anywhere in the urinary tract, from the kidney to the bladder. The kidneys play a critical role in excreting waste products from the body, but various problems can disrupt the urinary system's crucial activ- ities and cause illnesses, one of which is urolithiasis. Urinary calculi are worldwide in distribution but are particu- larly common in some geographic locations such as in parts of the United States, South Africa, India and South- East Asia. Renal calculi are characterised clinically by colicky pain (renal colic) as they pass down along the ureter and manifest by haematuria. This article focuses on the etiopathogenesis of Renal Stone, predisposing factors, and its pathophysiology for a better understanding of the disease so that its formation can be prevented, and the formed calculi can be treated with better knowledge. Keywords: Urinary Stones, Oxalates, Predisposing factors: urinary crystalloids, Hyperoxaluria, Hypercalciuria, Super-Saturation Theory, Nucleation Theory, Randall’s plaque

Lessons from Biomineralisation: the Role of Post-translational Modification

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>