Fungal biomineralization of toxic metals accelerates organic pollutant removal.

Fungal biomineralization plays an important role in the biogeochemical cycling of metals in the environment and has been extensively explored for bioremediation and element biorecovery. However, the cellular and metabolic responses of fungi in the presence of toxic metals during biomineralization and their impact on organic matter transformations are unclear. This is an important question because co-contamination by toxic metals and organic pollutants is a common phenomenon in the natural environment. In this research, the biomineralization process and oxidative stress response of the geoactive soil fungus Aspergillus niger were investigated in the presence of toxic metals (Co, Cu, Mn, and Fe) and the azo dye orange II (AO II). We have found that the co-existence of toxic metals and AO II not only enhanced the fungal biomineralization of toxic metals but also accelerated the removal of AO II. We hypothesize that the fungus and in situ mycogenic biominerals (toxic metal oxalates) constituted a quasi-bioreactor, where the biominerals removed organic pollutants by catalyzing reactive oxygen species (ROS) generation resulting from oxidative stress. We have therefore demonstrated that a fungal/biomineral system can successfully achieve the goal of toxic metal immobilization and organic pollutant decomposition. Such findings inform the potential development of fungal-biomineral hybrid systems for mixed pollutant bioremediation as well as provide further understanding of fungal organic-inorganic pollutant transformations in the environment and their importance in biogeochemical cycles.

Inhibiting Pathological Calcium Phosphate Mineralization: Implications for Disease Progression.

Pathological calcifications, especially calcium phosphate microcalcifications (MCs), appear in most early breast cancer lesions, and their formation correlates with more aggressive tumors and a poorer prognosis. Hydroxyapatite (HA) is a key MC component that crystallizes in the tumor microenvironment. It is often associated with malignant breast cancer lesions and can trigger tumorigenesis in vitro. Here, we investigate the impact of additives on HA crystallization and inhibition, and how precancerous breast cells respond to minerals that are deposited in the presence of these additives. We show that nonstoichiometric HA spontaneously crystallizes in a solution simulating the tumor microenvironmental fluids and exhibits lump-like morphology similar to breast cancer MCs. In this system, the effectiveness of poly(aspartic acid) and poly(acrylic acid) (PAA) to inhibit HA is examined as a potential route to improve cancer prognosis. In the presence of additives, the formation of HA lumps is associated with the promotion or only minimal inhibition of mineralization, whereas the formation of amorphous calcium phosphate (ACP) lumps is followed by inhibition of mineralization. PAA emerges as a robust HA inhibitor by forming spherical ACP particles. When precancerous breast cells are exposed to various HA and ACP minerals, the most influential factors on cell proliferation are the mineral phase and whether the mineral is in the form of discrete particles or particle aggregates. The tumorigenic effects on cells, ranging from cytotoxicity and suppression of proliferation to triggering of proliferation, can be summarized as HA particles < HA aggregates < ACP particles < ACP aggregates. The cellular response to minerals can be attributed to a combination of factors, including mineral phase, crystallinity, morphology, surface texture, aggregation state, and surface potential. These findings have implications for understanding mineral-cell interactions within the tumor microenvironment and suggest that, in some cases, the byproducts of HA inhibition can contribute to disease progression more than HA itself.

ROCK and the actomyosin network control biomineral growth and morphology during sea urchin skeletogenesis.

Biomineralization had apparently evolved independently in different phyla, using distinct minerals, organic scaffolds, and gene regulatory networks (GRNs). However, diverse eukaryotes from unicellular organisms, through echinoderms to vertebrates, use the actomyosin network during biomineralization. Specifically, the actomyosin remodeling protein, Rho-associated coiled-coil kinase (ROCK) regulates cell differentiation and gene expression in vertebrates' biomineralizing cells, yet, little is known on ROCK's role in invertebrates' biomineralization. Here, we reveal that ROCK controls the formation, growth, and morphology of the calcite spicules in the sea urchin larva. ROCK expression is elevated in the sea urchin skeletogenic cells downstream of the Vascular Endothelial Growth Factor (VEGF) signaling. ROCK inhibition leads to skeletal loss and disrupts skeletogenic gene expression. ROCK inhibition after spicule formation reduces the spicule elongation rate and induces ectopic spicule branching. Similar skeletogenic phenotypes are observed when ROCK is inhibited in a skeletogenic cell culture, indicating that these phenotypes are due to ROCK activity specifically in the skeletogenic cells. Reduced skeletal growth and enhanced branching are also observed under direct perturbations of the actomyosin network. We propose that ROCK and the actomyosin machinery were employed independently, downstream of distinct GRNs, to regulate biomineral growth and morphology in Eukaryotes.

Enhancing the degradation rate and biomineralization nature of antiferromagnetic Fe-20Mn alloy by groove pressing.

The Fe-Mn alloys are potential candidates for biodegradable implant applications. However, the very low degradation rates of Fe-Mn alloys in the physiological environment are a major disadvantage. In this study, the degradation rate of a Fe-20Mn alloy was improved using the groove pressing (GP) technique. Hot rolled sheets of 2 mm thickness were subjected to GP operation at 1000°C. Uniform fine-grained (UFG) Fe-Mn alloys were obtained using the GP technique. The influence of GP on the microstructure, mechanical properties, degradation behavior in simulated body fluid (SBF), surface wettability, biomineralization, and cytocompatibility was investigated and compared to the annealed (A Fe-Mn) and rolled (R Fe-Mn) sample. The groove-pressed Fe-Mn (G Fe-Mn) alloy had a grain size of approximately 40 ± 16 µm whereas the A Fe-Mn and R Fe-Mn samples had grain sizes of 303 ± 81 and 117 ± 14.5 µm, respectively. Enhanced strength and elongation were also observed with the G Fe-Mn sample. The potentiodynamic polarization test showed the highest Icorr, lowest polarization resistance, and lowest Ecorr for the G Fe-Mn sample among all other samples indicating its higher degradation rate. The weight loss data from immersion tests also shows that the percentage of weight loss increases with time indicating the accelerated degradation behavior of the sample. The static immersion test showed an enhancement in weight loss of 0.46 ± 0.02% and 1.02 ± 0.05% for R Fe-Mn and G Fe-Mn samples, respectively, than A Fe-Mn sample (0.31 ± 0.03%) after 56 days in immersion in SBF. The greater biomineralization tendency in UFG materials is confirmed by the G Fe-Mn sample's stronger hydroxyapatite deposition. When compared to the A Fe-Mn and R Fe-Mn samples, the G Fe-Mn sample has a better wettability, which promotes higher cell adhesion and vitality, showing higher biocompatibility. This study demonstrates that Fe-20Mn processed by GP has potential applications for the manufacture of biodegradable metallic implants.

Overcoming the inhibitory effects of urea to improve the kinetics of microbial-induced calcium carbonate precipitation (MICCP) by Lysinibacillus sphaericus strain MB284.

Among different microbial-induced calcium carbonate precipitation (MICCP) mechanisms utilized for biomineralization, ureolysis leads to the greatest yields of calcium carbonate. Unfortunately, it is reported that urea-induced growth inhibition can delay urea hydrolysis but it is not clear how this affects MICCP kinetics. This study investigated the impact of urea addition on the MICCP performance of Lysinibacillus sphaericus MB284 not previously grown on urea (thereafter named bio-agents), compared with those previously cultured in urea-rich media (20 g/L) (hereafter named bio-agents+ or bio-agents-plus). While it was discovered that initial urea concentrations exceeding 3 g/L temporarily hindered cell growth and MICCP reactions for bio-agents, employing bio-agents+ accelerated the initiation of bacterial growth by 33% and led to a 1.46-fold increase in the initial yield of calcium carbonate in media containing 20 g/L of urea. The improved tolerance of bio-agents+ to urea is attributed to the presence of pre-produced endogenous urease, which serves to reduce the initial urea concentration, alleviate growth inhibition, and expedite biomineralization. Notably, elevating the initial concentration of bio-agents+ from OD600 of 0.01 to 1, housing a higher content of endogenous urease, accelerated the initiation of MICCP reactions and boosted the ultimate yield of biomineralization by 2.6 times while the media was supplemented with 20 g/L of urea. These results elucidate the advantages of employing bio-agents+ with higher initial cell concentrations to successfully mitigate the temporary inhibitory effects of urea on biomineralization kinetics, offering a promising strategy for accelerating the production of calcium carbonate for applications like bio self-healing of concrete.

Structural diversity of crustacean exoskeletons and its implications for biomimetics.

The crustacean cuticle is a biological composite material consisting of chitin-protein fibres in a mineralized matrix. Recent research has revealed a surprising range of fibre architectures and mineral compositions of crustacean skeletal structures adapted to various mechanical demands. It is becoming increasingly clear that the organic fibres in the cuticle may be organized in patterns differing from the standard twisted plywood model. Observed fibre architectures in protruding skeletal structures include longitudinal and circular parallel fibre arrays. Skeletal minerals often include calcium phosphates in addition to calcium carbonates. Furthermore, skeletal properties are affected by protein cross-linking, which replaces mineralization as a stiffening mechanism in some structures. Several common structural motifs, such as the stiffening of the outer skeletal layers, the incorporation of non-mineralized cuticle in exposed structures, and interchanging layers of parallel fibres and the twisted plywood structure, can be identified in skeletal elements with similar functions. These evolutionary solutions have the potential for biomimetic applications, particularly as manufacturing technologies advance. To make use of this potential, we need to understand the processes behind the formation of the crustacean exoskeleton and determine which features are truly adaptive and worth mimicking.

Penicillium oxalicum induced phosphate precipitation enhanced cadmium (Cd) immobilization by simultaneously accelerating Cd biosorption and biomineralization.

Soil cadmium (Cd) is immobilized by the progressing biomineralization process as microbial induced phosphate precipitation (MIPP), which is regulated by phosphate (P) solubilizing microorganisms and P sources. However, little attention has been paid to the implications of Cd biosorption during MIPP. In this study, the newly isolated Penicillium oxalicum could immobilize 5.4-12.6 % of Cd2+, while the presence of hydroxyapatite (HAP) considerably enhanced Cd2+ immobilization in P. oxalicum and reached over 99 % Cd2+ immobilization efficiency within 7 days. Compared to P. oxalicum mono inoculation, MIPP dramatically boosted Cd biosorption and biomineralization efficiency by 71 % and 16 % after 96 h cultivation, respectively. P. oxalicum preferred to absorbing Cd2+ and reaching maximum Cd2+ biosorption efficiency of 87.8 % in the presence of HAP. More surface groups in P. oxalicum and HAP mineral involved adsorption which resulted in the formation of Cd-apatite [Ca8Cd2(PO4)6(OH)2] via ion exchange. Intracellular S2-, secreted organic acids and soluble P via HAP solubilization complexed with Cd2+, progressively mineralized into Cd5(PO4)3OH, Cd(H2PO4)2, C4H6CdO4 and CdS. These results suggested that Cd2+ immobilization was enhanced simultaneously by the accelerated biosorption and biomineralization during P. oxalicum induced P precipitation. Our findings revealed new mechanisms of Cd immobilization in MIPP process and offered clues for remediation practices at metal contaminated sites.

Genomic characterization of a novel ureolytic bacteria, Lysinibacillus capsici TSBLM, and its application to the remediation of acidic heavy metal-contaminated soil.

Soil heavy metal contamination is an essential challenge in ecological and environmental management, especially for acidic soils. Microbially induced carbonate precipitation (MICP) is an effective and environmentally friendly remediation technology for heavy metal contaminated sites, and one of the key factors for its realization lies in the microorganisms. In this study, Lysinibacillus capsici TSBLM was isolated from heavy metal contaminated soil around a gold mine, and inferred to be a novel ureolytic bacteria after phylogenomic inference and genome characterization. The urease of L. capsici TSBLM was analyzed by genetic analysis and molecular docking, and further applied this bacteria to the remediation of Cu and Pb in solution and acidic soils to investigate its biomineralization mechanism and practical application. The results revealed L. capsici TSBLM possessed a comprehensive urease gene cluster ureABCEFGD, and the encoded urease docked with urea at the lowest binding energy site (ΔG = -3.43 kcal/mol) connected to three amino acids threonine, aspartic, and alanine. The urease of L. capsici TSBLM is synthesized intracellularly but mainly functions extracellularly. L. capsici TSBLM removes Cu/Pb from the solution by generating heavy metal carbonates or co-precipitating with CaCO3 vaterite. For acidic heavy metal-contaminated soil, the carbonate-bound states of Cu and Pb increased significantly from 7 % to 16 % and from 23 % to 35 % after 30 days by L. capsici TSBLM. Soil pH improved additionally. L. capsici TSBLM maintained the dominant status in the remediated soil after 30 days, demonstrating good environmental adaptability and curing persistence. The results provided new strain resources and practical application references for the remediation of acidic heavy metal contaminated soil based on MICP.

Deterioration phenomenon of Pb-contaminated aqueous solution remediation and enhancement mechanism of nano-hydroxyapatite-assisted biomineralization.

Modern metallurgical and smelting activities discharge the lead-containing wastewater, causing serious threats to human health. Bacteria and urease applied to microbial-induced carbonate precipitation (MICP) and enzyme-induced carbonate precipitation (EICP) are denatured under high Pb2+ concentration. The nano-hydroxyapatite (nHAP)-assisted biomineralization technology was applied in this study for Pb immobilization. Results showed that the extracellular polymers and cell membranes failed to secure the urease activity when subjected to 60 mM Pb2+. The immobilization efficiency dropped to below 50% under MICP, whereas it due to a lack of extracellular polymers and cell membranes dropped to below 30% under EICP. nHAP prevented the attachment of Pb2+ either through competing with bacteria and urease or promoting Ca2+/Pb2+ ion exchange. Furthermore, CO32- from ureolysis replaced the hydroxyl (-OH) in hydroxylpyromorphite to encourage the formation of carbonate-bearing hydroxylpyromorphite of higher stability (Pb10(PO4)6CO3). Moreover, nHAP application overcame an inability to provide nucleation sites by urease. As a result, the immobilization efficiency, when subjected to 60 mM Pb2+, elevated to above 80% under MICP-nHAP and to some 70% under EICP-nHAP. The findings highlight the potential of applying the nHAP-assisted biomineralization technology to Pb-containing water bodies remediation.

Evolution and diversity of biomineralized columnar architecture in early Cambrian phosphatic-shelled brachiopods.

Biologically-controlled mineralization producing organic-inorganic composites (hard skeletons) by metazoan biomineralizers has been an evolutionary innovation since the earliest Cambrian. Among them, linguliform brachiopods are one of the key invertebrates that secrete calcium phosphate minerals to build their shells. One of the most distinct shell structures is the organo-phosphatic cylindrical column exclusive to phosphatic-shelled brachiopods, including both crown and stem groups. However, the complexity, diversity, and biomineralization processes of these microscopic columns are far from clear in brachiopod ancestors. Here, exquisitely well-preserved columnar shell ultrastructures are reported for the first time in the earliest eoobolids Latusobolus xiaoyangbaensis gen. et sp. nov. and Eoobolus acutulus sp. nov. from the Cambrian Series 2 Shuijingtuo Formation of South China. The hierarchical shell architectures, epithelial cell moulds, and the shape and size of cylindrical columns are scrutinised in these new species. Their calcium phosphate-based biomineralized shells are mainly composed of stacked sandwich columnar units. The secretion and construction of the stacked sandwich model of columnar architecture, which played a significant role in the evolution of linguliforms, is highly biologically controlled and organic-matrix mediated. Furthermore, a continuous transformation of anatomic features resulting from the growth of diverse columnar shells is revealed between Eoobolidae, Lingulellotretidae, and Acrotretida, shedding new light on the evolutionary growth and adaptive innovation of biomineralized columnar architecture among early phosphatic-shelled brachiopods during the Cambrian explosion.

Calcium isotope fractionation by intracellular amorphous calcium carbonate (ACC) forming cyanobacteria.

The formation of intracellular amorphous calcium carbonate (ACC) by various cyanobacteria is a widespread biomineralization process, yet its mechanism and importance in past and modern environments remain to be fully comprehended. This study explores whether calcium (Ca) isotope fractionation, linked to ACC-forming cyanobacteria, can serve as a reliable tracer for detecting these microorganisms in modern and ancient settings. Accordingly, we measured stable Ca isotope fractionation during Ca uptake by the intracellular ACC-forming cyanobacterium Cyanothece sp. PCC 7425. Our results show that Cyanothece sp. PCC 7425 cells are enriched in lighter Ca isotopes relative to the solution. This finding is consistent with the kinetic isotope effects observed in the Ca isotope fractionation during biogenic carbonate formation by marine calcifying organisms. The Ca isotope composition of Cyanothece sp. PCC 7425 was accurately modeled using a Rayleigh fractionation model, resulting in a Ca isotope fractionation factor (Δ44Ca) equal to -0.72 ± 0.05‰. Numerical modeling suggests that Ca uptake by these cyanobacteria is primarily unidirectional, with minimal back reaction observed over the duration of the experiment. Finally, we compared our Δ44Ca values with those of other biotic and abiotic carbonates, revealing similarities with organisms that form biogenic calcite. These similarities raise questions about the effectiveness of using the Ca isotope fractionation factor as a univocal tracer of ACC-forming cyanobacteria in the environment. We propose that the use of Δ44Ca in combination with other proposed tracers of ACC-forming cyanobacteria such as Ba and Sr isotope fractionation factors and/or elevated Ba/Ca and Sr/Ca ratios may provide a more reliable approach.

In-depth investigation of FAM20A insufficiency effects on deciduous dental pulp cells: Altered behaviours, osteogenic differentiation, and inflammatory gene expression.

AIM: Loss-of-function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel-renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. METHODOLOGY: DDP were obtained from a FAM20A-AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A-related genes, osteogenic genes, and inflammatory genes was analysed using real-time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein-protein interaction networks. RESULTS: The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL-1ß and TGF-ß1, whereas IL-6 and NFκB1 expression was significantly reduced. CONCLUSION: The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology.

Tropomyosin induces the synthesis of magnesian calcite in sea urchin spines.

Calcium carbonate is present in many biominerals, including in the exoskeletons of crustaceans and shells of mollusks. High Mg-containing calcium carbonate was synthesized by high temperatures, high pressures or high molecular organic matter. For example, biogenic high Mg-containing calcite is synthesized under strictly controlled Mg concentration at ambient temperature and pressure. The spines of sea urchins consist of calcite, which contain a high percentage of magnesium. In this study, we investigated the factors that increase the magnesium content in calcite from the spines of the sea urchin, Heliocidaris crassispina. X-ray diffraction and inductively coupled plasma mass spectrometry analyses showed that sea urchin spines contain about 4.8% Mg. The organic matrix extracted from the H. crassispina spines induced the crystallization of amorphous phase and synthesis of magnesium-containing calcite, while amorphous was synthesized without SUE (sea urchin extract). In addition, aragonite was synthesized by SUE treated with protease-K. HC tropomyosin was specifically incorporated into Mg precipitates. Recombinant HC-tropomyosin induced calcite contained 0.1-2.5% Mg synthesis. Western blotting of sea urchin spine extracts confirmed that HC tropomyosin was present in the purple sea urchin spines at a protein weight ratio of 1.5%. These results show that HC tropomyosin is one factor that increases the magnesium concentration in the calcite of H. crassispina spines.

In situ/In vivo Optical Microspectroscopy to Probe the Emergence of Morphology.

Morphology governs function. Yet, understanding and controlling the emergence of morphology at the molecular level remains challenging. The difficulty in studying the early stage of morphology formation is due to its stochastic nature both spatially and temporally occurring at the nanoscale. This nature has been particularly detrimental for the application of optical spectroscopy. To overcome this problem, we have been developing new in situ/in vivo optical spectroscopy tools, which are label-free and non-invasive. This account highlights several examples of how optical spectroscopy can become an important tool in studying the birth of morphology.

Tumor-targeted delivery of copper-manganese biomineralized oncolytic adenovirus for colorectal cancer immunotherapy.

Oncolytic viral therapy (OVT) is a novel anti-tumor immunotherapy approach, specifically replicating within tumor cells. Currently, oncolytic viruses are mainly administered by intratumoral injection. However, achieving good results for distant metastatic tumors is challenging. In this study, a multifunctional oncolytic adenovirus, OA@CuMnCs, was developed using bimetallic ions copper and manganese. These metal cations form a biomineralized coating on the virus's surface, reducing immune clearance. It is known that viruses upregulate the expression of PD-L1. Copper ions in OA@CuMnCs can decrease the PD-L1 expression of tumor cells, thereby promoting immune cell-related factor release. This process involves antigen presentation and the combination of immature dendritic cells, transforming them into mature dendritic cells. It changes "cold" tumors into "hot" tumors, further inducing immunogenic cell death. While oncolytic virus replication requires oxygen, manganese ions in OA@CuMnCs can react with endogenous hydrogen peroxide. This reaction produces oxygen, enhancing the virus's replication ability and the tumor lysis effect. Thus, this multifunctionally coated OA@CuMnCs demonstrates potent amplification in immunotherapy efficacy, and shows great potential for further clinical OVT. STATEMENT OF SIGNIFICANCE: Oncolytic virus therapy (OVs) is a new anti-tumor immunotherapy method that can specifically replicate in tumor cells. Although the oncolytic virus can achieve a therapeutic effect on some non-metastatic tumors through direct intratumoral injection, there are still three major defects in the treatment of metastatic tumors: immune response, hypoxia effect, and administration route. Various studies have shown that the immune response in vivo can be overcome by modifying or wrapping the surface protein of the oncolytic virus. In this paper, a multifunctional coating of copper and manganese was prepared by combining the advantages of copper and manganese ions. The coating has a simple preparation method and mild conditions, and can effectively enhance tumor immunotherapy.

Formation of Sb2O3 microcrystals by Rhodotorula mucilaginosa.

Antimony (Sb) pollution seriously endangers ecological environment and human health. Microbial induced mineralization can effectively convert metal ions into more stable and less soluble crystalline minerals by extracellular polymeric substance (EPS). In this study, an efficient Sb-resistant Rhodotorula mucilaginosa (R. mucilaginosa) was screened, which can resist 41 mM Sb(III) and directly transform Sb(III) into Sb2O3 microcrystals by EPS. The removal efficiency of R. mucilaginosa for 22 mM Sb(III) reached 70% by converting Sb(III) to Sb2O3. The components of supernatants as well as the effects of supernatants and pH on Sb(III) mineralization verified that inducible and non-inducible extracellular protein/polysaccharide biomacromolecules play important roles in the morphologies and sizes control of Sb2O3 formed by R. mucilaginosa respectively. Sb2O3 microcrystals with different morphologies and sizes can be prepared by the regulation of inducible and non-inducible extracellular biomacromolecules secreted by R. mucilaginosa. This is the first time to identify that R. mucilaginosa can remove Sb(III) by transforming Sb(III) into Sb2O3 microcrystals under the control of EPS. This study contributes to our understanding for Sb(III) biomineralization mechanisms and provides strategies for the remediation of Sb-contaminated environment.

A transporter that allows phosphate ions to control the polymorph of exoskeletal calcium carbonate biomineralization.

The SLC20A2 transporter supplies phosphate ions (Pi) for diverse biological functions in vertebrates, yet has not been studied in crustaceans. Unlike vertebrates, whose skeletons are mineralized mainly by calcium phosphate, only minute amounts of Pi are found in the CaCO3-mineralized exoskeletons of invertebrates. In this study, a crustacean SLC20A2 transporter was discovered and Pi transport to exoskeletal elements was studied with respect to the role of Pi in invertebrate exoskeleton biomineralization, revealing an evolutionarily conserved mechanism for Pi transport in both vertebrates and invertebrates. Freshwater crayfish, including the study animal Cherax quadricarinatus, require repeated molt cycles for their growth. During the molt cycle, crayfish form transient exoskeletal mineral storage organs named gastroliths, which mostly contain amorphous calcium carbonate (ACC), an unstable polymorph long-thought to be stabilized by Pi. RNA interference experiments via CqSLC20A2 dsRNA injections reduced Pi content in C. quadricarinatus gastroliths, resulting in increased calcium carbonate (CaCO3) crystallinity and grain size. The discovery of a SLC20A2 transporter in crustaceans and the demonstration that knocking down its mRNA reduced Pi content in exoskeletal elements offers the first direct proof of a long-hypothesized mechanism by which Pi affects CaCO3 biomineralization in the crustacean exoskeleton. This research thus demonstrated the distinct role of Pi as an amorphous mineral polymorph stabilizer in vivo, suggesting further avenues for amorphous biomaterial studies. STATEMENT OF SIGNIFICANCE: • Crustaceans exoskeletons are hardened mainly by CaCO3, with Pi in minute amounts • Pi was hypothesized to stabilize exoskeletal amorphous mineral forms in vivo • For the first time, transport protein for Pi was discovered in crayfish • Transport knock-down resulted in exoskeletal CaCO3 crystallization and reduced Pi.

Guinea fowl eggshell structural analysis at different scales reveals how organic matrix induces microstructural shifts that enhance its mechanical properties.

Guinea fowl eggshells have an unusual structural arrangement that is different from that of most birds, consisting of two distinct layers with different microstructures. This bilayered organization, and distinct microstructural characteristics, provides it with exceptional mechanical properties. The inner layer, constituting about one third of the eggshell thickness, contains columnar calcite crystal units arranged vertically as in most bird shells. However, the thicker outer layer has a more complex microstructural arrangement formed by a switch to smaller calcite domains with diffuse/interlocking boundaries, partly resembling the interfaces seen in mollusk shell nacre. The switching process that leads to this remarkable second-layer microstructure is unknown. Our results indicate that the microstructural switching is triggered by changes in the inter- and intracrystalline organic matrix. During production of the outer microcrystalline layer in the later stages of eggshell formation, the interactions of organic matter with mineral induce an accumulation of defects that increase crystal mosaicity, instill anisotropic lattice distortions in the calcite structure, interrupt epitaxial growth, reduce crystallite size, and induce nucleation events which increase crystal misorientation. These structural changes, together with the transition between the layers and each layer having different microstructures, enhance the overall mechanical strength of the Guinea fowl eggshell. Additionally, our findings provide new insights into how biogenic calcite growth may be regulated to impart unique functional properties. STATEMENT OF SIGNIFICANCE: Avian eggshells are mineralized to protect the embryo and to provide calcium for embryonic chick skeletal development. Their thickness, structure and mechanical properties have evolved to resist external forces throughout brooding, yet ultimately allow them to crack open during chick hatching. One particular eggshell, that of the Guinea fowl, has structural features very different from other galliform birds - it is bilayered, with an inner columnar mineral structure (like in most birds), but it also has an outer layer with a complex microstructure which contributes to its superior mechanical properties. This work provides novel and new fundamental information about the processes and mechanisms that control and change crystal growth during the switch to microcrystalline domains when the second outer layer forms.

Composite material in the sea urchin Cidaris rugosa: ordered and disordered micrometre-scale bicontinuous geometries.

The sponge-like biomineralized calcite materials found in echinoderm skeletons are of interest in terms of both structure formation and biological function. Despite their crystalline atomic structure, they exhibit curved interfaces that have been related to known triply periodic minimal surfaces. Here, we investigate the endoskeleton of the sea urchin Cidaris rugosa that has long been known to form a microstructure related to the Primitive surface. Using X-ray tomography, we find that the endoskeleton is organized as a composite material consisting of domains of bicontinuous microstructures with different structural properties. We describe, for the first time, the co-occurrence of ordered single Primitive and single Diamond structures and of a disordered structure within a single skeletal plate. We show that these structures can be distinguished by structural properties including solid volume fraction, trabeculae width and, to a lesser extent, interface area and mean curvature. In doing so, we present a robust method that extracts interface areas and curvature integrals from voxelized datasets using the Steiner polynomial for parallel body volumes. We discuss these very large-scale bicontinuous structures in the context of their function, formation and evolution.

A Comprehensive Study of Meningioma Biomineralization: Morphological, Crystallographic, and Immunohistochemical Aspects.

Biomineralization of brain tissues occurs both in normal and pathological conditions. Dura mater biomineralization is widespread and occurs in 1-72% of cases, depending on the patient's age and research method. The amount of biomineral deposits under the conditions of tumor growth in the meninges only increases, reaching 100% in the case of psammomatous meningiomas. Since calcifications are often found in the meninges, the problem of differential diagnosis with calcified meningiomas arises. A total of 30 samples of meningiomas with signs of biomineralization-dense structure, characteristic crunch, psammoma bodies (group I) and 30 samples of meningiomas without any signs of biomineralization were examined as controls (group II). To detect pathological biomineralization, the meningioma tissue was studied using the methods of macroscopic description, histology, histochemistry, and immunohistochemistry, scanning electron microscopy with microanalysis, and transmission electron microscopy. A significantly higher level of caspase3 and features of the expression of osteoblastic markers (a lower level of OPG expression and a higher level of the presence of RANKL in group I, the absence of fluctuations in the expression of SPARC) may indicate a dystrophic type of development of biomineral deposits in meningiomas.