Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice.

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods' axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.

Amelogenin phosphorylation regulates tooth enamel formation by stabilizing a transient amorphous mineral precursor.

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization in vitro, as observed in enamel rods. Phosphorylation of the sole AMELX phosphorylation site (Ser-16) in vitro greatly enhances its capacity to stabilize amorphous calcium phosphate (ACP), the first mineral phase formed in developing enamel, and prevents apatitic crystal formation. To test our hypothesis that AMELX phosphorylation is critical for amelogenesis, we generated and characterized a hemizygous knockin (KI) mouse model with a phosphorylation-defective Ser-16 to Ala-16 substitution in AMELX. Using EM analysis, we demonstrate that in the absence of phosphorylated AMELX, KI enamel lacks enamel rods, the hallmark component of mammalian enamel, and, unlike WT enamel, appears to be composed of less organized arrays of shorter crystals oriented normal to the dentinoenamel junction. KI enamel also exhibited hypoplasia and numerous surface defects, whereas heterozygous enamel displayed highly variable mosaic structures with both KI and WT features. Importantly, ACP-to-apatitic crystal transformation occurred significantly faster in KI enamel. Secretory KI ameloblasts also lacked Tomes' processes, consistent with the absence of enamel rods, and underwent progressive cell pathology throughout enamel development. In conclusion, AMELX phosphorylation plays critical mechanistic roles in regulating ACP-phase transformation and enamel crystal growth, and in maintaining ameloblast integrity and function during amelogenesis.

Trafficking and secretion of keratin 75 by ameloblasts in vivo.

A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER-Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER-Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.

Effects of seawater salinity and pH on cellular metabolism and enzyme activities in biomineralizing tissues of marine bivalves.

Molluscan shell formation is a complex energy demanding process sensitive to the shifts in seawater CaCO3 saturation due to changes in salinity and pH. We studied the effects of salinity and pH on energy demand and enzyme activities of biomineralizing cells of the Pacific oyster (Crassostrea gigas) and the hard-shell clam (Mercenaria mercenaria). Adult animals were exposed for 14 days to high (30), intermediate (18), or low (10) salinity at either high (8.0-8.2) or low (7.8) pH. Basal metabolic cost as well as the energy cost of the biomineralization-related cellular processes were determined in isolated mantle edge cells and hemocytes. The total metabolic rates were similar in the hemocytes of the two studied species, but considerably higher in the mantle cells of C. gigas compared with those of M. mercenaria. Cellular respiration was unaffected by salinity in the clams' cells, while in oysters' cells the highest respiration rate was observed at intermediate salinity (18). In both studied species, low pH suppressed cellular respiration. Low pH led to an upregulation of Na+/K+ ATPase activity in biomineralizing cells of oysters and clams. Activities of Ca2+ ATPase and H+ ATPase, as well as the cellular energy costs of Ca2+ and H+ transport in the biomineralizing cells were insensitive to the variation in salinity and pH in the two studied species. Variability in cellular response to low salinity and pH indicates that the disturbance of shell formation under these conditions has different underlying mechanisms in the two studied species.

X-ray Linear Dichroism in Apatite.

The recent observation in parrotfish teeth of X-ray linear dichroism motivated an in-depth investigation into this spectroscopic effect in various apatite crystals, including geologic hydroxyapatite (Ca5(PO4)3OH), fluorapatite (Ca5(PO4)3F), and their biogenic counterparts in human bone, mouse enamel, and in parrotfish bone, dentin, and enameloid, the equivalent of dental enamel in certain fish. These data are important because they now enable visualization of the nano- to microscale structure of apatite crystals in teeth and bone. Polarization-dependent imaging contrast (PIC) maps of lamellar bone, obtained with a new method that minimizes space-charge and charging effects, show the expected rotating apatite crystal orientations. PIC maps of mouse enamel reveal a complex arrangement of hydroxyapatite crystals perpendicular to the dentin-enamel junction, with rods arranged in a decussation pattern in inner enamel and nearly parallel to one another in outer enamel. In both inner and outer enamel crystal c-axes are not always aligned with the rod elongation direction.

Potential trade-offs between biomineralization and immunity revealed by shell properties and gene expression profiles of two closely related Crassostrea species.

Species of the Ostreidae family are key ecosystem engineers and many of them - including Crassostrea gigas and Crassostreavirginica - are commercially important aquaculture species. Despite similarities in their morphology and ecology, these two species differ in their ability to defend against pathogens, potentially reflecting species-specific differential specialization of hemocytes on immune defense versus biomineralization. To test this hypothesis, we investigated the expression levels of immune- and biomineralization-related genes as well as mineralogical and mechanical properties of the shells and the calcium sequestration ability of the hemocytes of C. gigas and C. virginica The expression of biomineralization-related genes was higher in C. virginica than in C. gigas in multiple tissues including the mantle edge and hemocytes, while the expression of immune genes was higher in the hemocytes of C. gigas Hemocytes of C. virginica contained more calcium (stored intracellularly as calcium carbonate mineral) compared with those of C. gigas Analysis of the adult shells showed that the crystallinity of calcite was higher and the laths of the foliated layer of the shell were thicker in C. virginica than in C. gigas Mechanically, the shells of C. virginica were stiffer, harder and stronger than those of C. gigas Taken together, our results show that the species-specific differences in physiology (such as disease resistance and exoskeleton properties) are reflected at the cellular and molecular levels in the differential specialization of hemocytes on potentially competing functions (immunity and biomineralization) as well as different expression profiles of other tissues involved in biomineralization (such as the mantle edge).

Biomineralization-related specialization of hemocytes and mantle tissues of the Pacific oyster Crassostrea gigas.

The molluscan exoskeleton (shell) plays multiple important roles including structural support, protection from predators and stressors, and physiological homeostasis. Shell formation is a tightly regulated biological process that allows molluscs to build their shells even in environments unfavorable for mineral precipitation. Outer mantle edge epithelial cells (OME) and hemocytes were implicated in this process; however, the exact functions of these cell types in biomineralization are not clear. Pacific oysters (Crassostrea gigas) were used to study differences in the expression profiles of selected biomineralization-related genes in hemocytes and mantle cells, and the functional characteristics of hemocytes such as adhesion, motility and phagocytosis. The specialized role of OME in shell formation was supported by high expression levels of the extracellular matrix (ECM) related and cell-cell interaction genes. Density gradient separation of hemocytes revealed distinct phenotypes based on the cell morphology, gene expression patterns, motility and adhesion characteristics. These hemocyte fractions can be categorized into two functional groups, i.e. biomineralization and immune response cells. Gene expression profiles of the putative biomineralizing hemocytes indicate that in addition to their proposed role in mineral transport, hemocytes also contribute to the formation of the ECM, thus challenging the current paradigm of the mantle as the sole source of the ECM for shell formation. Our findings corroborate the specialized roles of hemocytes and the OME in biomineralization and emphasize complexity of the biological controls over shell formation in bivalves.

Regulation of calcium phosphate formation by native amelogenins in vitro.

Our previous in vitro studies have shown that recombinant full-length porcine amelogenin rP172 can transiently stabilize amorphous calcium phosphate (ACP) and uniquely guide the formation of well-aligned bundles of hydroxyapatite (HA) crystals, as seen in the secretory stage of amelogenesis. This functional capacity is dependent on the hydrophilic C-terminal domain of full-length amelogenin. However, we have also found that native phosphorylated (single S-16 site) forms of full-length (P173) and C-terminal cleaved (P148) amelogenins can stabilize ACP for > 2 d and prevent HA formation. The present study was carried out to test the hypothesis that, at reduced concentrations, native full-length P173 also has the capacity to guide ordered HA formation. The effect of P148 and P173 concentrations (0.2-2.0 mg/ml) on the rate of spontaneous calcium phosphate precipitation was monitored via changes in solution pH, while mineral phases formed were assessed using TEM. At higher P173 concentrations (1.0-2.0 mg/ml), limited mineral formation occurred and only ACP nanoparticles were observed during a 48 h period. However, at 0.4 mg/ml P173, a predominance of organized bundles of linear, needle-like HA crystals were observed. At 0.2 mg/ml of P173, limited quantities of less organized HA crystals were found. Although P148 similarly stabilized ACP, it did not guide ordered HA formation, like P173. Hence, the establishment of the hierarchical enamel structure during secretory stage amelogenesis may be regulated by the partial removal of full-length amelogenin via MMP20 proteolysis, while predominant amelogenin degradation products, like P148, serve to prevent uncontrolled mineral formation.

Ultrastructural organization of dentin in mice lacking dentin sialo-phosphoprotein.

Dentin Sialophosphoprotein (DSPP) is the major non-collagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap, we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BS-SEM) of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of non-mineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpulpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.

Hierarchical self-assembly of amelogenin and the regulation of biomineralization at the nanoscale.

Enamel is a highly organized hierarchical nanocomposite, which consists of parallel arrays of elongated apatitic crystallites forming an intricate three-dimensional microstructure. Amelogenin, the major extracellular matrix protein of dental enamel, regulates the formation of these crystalline arrays via cooperative interactions with forming mineral phase. Using cryoelectron microscopy, we demonstrate that amelogenin undergoes stepwise hierarchical self-assembly. Furthermore, our results indicate that interactions between amelogenin hydrophilic C-terminal telopeptides are essential for oligomer formation and for subsequent steps of hierarchical self-assembly. We further show that amelogenin assemblies stabilize mineral prenucleation clusters and guide their arrangement into linear chains that organize as parallel arrays. The prenucleation clusters subsequently fuse together to form needle-shaped mineral particles, leading to the formation of bundles of crystallites, the hallmark structural organization of the forming enamel at the nanoscale. These findings provide unique insight into the regulation of biological mineralization by specialized macromolecules and an inspiration for bottom-up strategies for the materials design.

Self-Assembly of Tooth Root Organoid from Postnatal Human Dental Stem Cells.

Challenges remain in simultaneously regenerating the multiple diverse tissues of the tooth root in a spatially organized manner. Previously, our research group has established that scaffold-free tissue engineering approaches enable dental pulp stem/progenitor cells (DPSCs) and periodontal ligament (PDL) stem/progenitor cells (PDLSCs) to self-assemble into dentin-pulp and PDL-cementum organoids, respectively. In this study, we leveraged the innate self-organizing capacity of DPSCs and PDLSCs to now engineer organoids that resemble the full tooth root. Scaffold-free engineered tissues were generated using a heterogeneous mixture of human DPSCs and PDLSCs. Within 2 days of construct formation, PDLSCs and DPSCs became spatially restricted to the periphery and center of the constructs, respectively, emulating their anatomical positions in the tooth root. Histological and microcomputed tomography analyses showed that organoids exhibited a striated mineral pattern with a central unmineralized core, surrounded by a mineralized tissue structure, enclosed within a second peripheral unmineralized tissue, similar to the natural tooth root. Interestingly, DPSCs gave rise to the central unmineralized tissue and the inner portion of the mineralized tissue, and PDLSCs generated the outer portion of the mineralized tissue and the peripheral soft tissue. Quantitative image analysis of immunofluorescent staining revealed increased dentin sialophosphoprotein expression in the region of mineralized tissue associated with DPSCs and increased cementum protein-1 expression in the portion formed by PDLSCs, demonstrating that tooth root organoids comprise two biochemically distinct mineralized tissues characteristic of dentin-like and cementum-like structures, respectively. In addition, PDL-associated protein-1 expression was localized to the peripheral soft tissue, suggesting the formation of a rudimentary PDL-like structure. This study demonstrates that DPSCs and PDLSCs have an inherent ability to orchestrate the formation of a full tooth root-like structure. These organoids present a biomimetic model system to study cellular dynamics driving dental tissue repair or could be utilized therapeutically as biological dental implants.

Role of amelogenin phosphorylation in regulating dental enamel formation.

Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELXS16A KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts). In the present investigation, our focus was on understanding the mechanisms of action of phosphorylated AMELX in amelogenesis. We have hypothesized that the absence of AMELX phosphorylation would result in a loss of controlled mineralization during the secretory stage of amelogenesis, leading to an enhanced rate of enamel mineralization that causes enamel acidification due to excessive proton release. To test these hypotheses, we employed microcomputed tomography (µCT), colorimetric pH assessment, and Fourier Transform Infrared (FTIR) microspectroscopy of apical portions of mandibular incisors from 8-week old wildtype (WT) and KI mice. As hypothesized, µCT analyses demonstrated significantly higher rates of enamel mineral densification in KI mice during the secretory stage compared to the WT. Despite a greater rate of enamel densification, maximal KI enamel thickness increased at a significantly lower rate than that of the WT during the secretory stage of amelogenesis, reaching a thickness in mid-maturation that is approximately half that of the WT. pH assessments revealed a lower pH in secretory enamel in KI compared to WT mice, as hypothesized. FTIR findings further demonstrated that KI enamel is comprised of significantly greater amounts of acid phosphate compared to the WT, consistent with our pH assessments. Furthermore, FTIR microspectroscopy indicated a significantly higher mineral-to-organic ratio in KI enamel, as supported by µCT findings. Collectively, our current findings demonstrate that phosphorylated AMELX plays crucial mechanistic roles in regulating the rate of enamel mineral formation, and in maintaining physico-chemical homeostasis and the enamel growth pattern during early stages of amelogenesis.

CryoTEM study of effects of phosphorylation on the hierarchical assembly of porcine amelogenin and its regulation of mineralization in vitro.

Amelogenin, the major extracellular enamel matrix protein, plays a critical role in regulating the growth and organization of enamel. Assembly and mineralization of full-length native (P173) and recombinant (rP172) porcine amelogenins were studied by cryogenic Transmission Electron Microscopy (cryoTEM). The cryoTEM revealed that both native and recombinant porcine amelogenins undergo step-wise self-assembly. Although the overall structural organization of P173 and rP172 oligomers was similar and resembled oligomers of murine recombinant amelogenin rM179, there were subtle differences suggesting that a single phosphorylated serine present in P173 might affect amelogenin self-assembly. Our mineralization studies demonstrated that both P173 and rP172 oligomers stabilize initial mineral clusters. Importantly, however, rP172 regulated the organization of initial mineral clusters into linear chains and guided the formation of parallel arrays of elongated mineral particles, which are the hallmark of enamel structural organization. These results are similar to those obtained previously using full-length recombinant murine amelogenin (Fang et al., 2011a). In contrast to that seen with rP172, phosphorylated P173 strongly inhibits mineralization for extended periods of time. We propose that these differences might be due to the differences in the structural organization and charge distribution between P173 and rP172. Overall our studies indicate that self-assembly of amelogenin and the mechanisms of its control over mineralization might be universal across different mammalian species. Our data also provide new insight into the effect of phosphorylation on amelogenin self-assembly and its regulation of mineralization.

Nanoscale confinement controls the crystallization of calcium phosphate: relevance to bone formation.

A key feature of biomineralization processes is that they take place within confined volumes, in which the local environment can have significant effects on mineral formation. Herein, we investigate the influence of confinement on the formation mechanism and structure of calcium phosphate (CaP). This is of particular relevance to the formation of dentine and bone, structures of which are based on highly mineralized collagen fibrils. CaP was precipitated within 25-300 nm diameter, cylindrical pores of track etched and anodised alumina membranes under physiological conditions, in which this system enables systematic study of the effects of the pore size in the absence of a structural match between the matrix and the growing crystals. Our results show that the main products were polycrystalline hydroxapatite (HAP) rods, together with some single crystal octacalcium phosphate (OCP) rods. Notably, we demonstrate that these were generated though an intermediate amorphous calcium phosphate (ACP) phase, and that ACP is significantly stabilised in confinement. This effect may have significance to the mineralization of bone, which can occur through a transient ACP phase. We also show that orientation of the HAP comparable, or even superior to that seen in bone can be achieved through confinement effects alone. Although this simple experimental system cannot be considered, a direct mimic of the in vivo formation of ultrathin HAP platelets within collagen fibrils, our results show that the effects of physical confinement should not be neglected when considering the mechanisms of formation of structures, such as bones and teeth.

Environmental salinity modulates the effects of elevated CO2 levels on juvenile hard-shell clams, Mercenaria mercenaria.

Ocean acidification due to increasing atmospheric CO2 concentrations results in a decrease in seawater pH and shifts in the carbonate chemistry that can negatively affect marine organisms. Marine bivalves such as the hard-shell clam, Mercenaria mercenaria, serve as ecosystem engineers in estuaries and coastal zones of the western Atlantic and, as for many marine calcifiers, are sensitive to the impacts of ocean acidification. In estuaries, the effects of ocean acidification can be exacerbated by low buffering capacity of brackish waters, acidic inputs from freshwaters and land, and/or the negative effects of salinity on the physiology of organisms. We determined the interactive effects of 21 weeks of exposure to different levels of CO2 (~395, 800 and 1500 µatm corresponding to pH of 8.2, 8.1 and 7.7, respectively) and salinity (32 versus 16) on biomineralization, shell properties and energy metabolism of juvenile hard-shell clams. Low salinity had profound effects on survival, energy metabolism and biomineralization of hard-shell clams and modulated their responses to elevated PCO2. Negative effects of low salinity in juvenile clams were mostly due to the strongly elevated basal energy demand, indicating energy deficiency, that led to reduced growth, elevated mortality and impaired shell maintenance (evidenced by the extensive damage to the periostracum). The effects of elevated PCO2 on physiology and biomineralization of hard-shell clams were more complex. Elevated PCO2 (~800-1500 µatm) had no significant effects on standard metabolic rates (indicative of the basal energy demand), but affected growth and shell mechanical properties in juvenile clams. Moderate hypercapnia (~800 µatm PCO2) increased shell and tissue growth and reduced mortality of juvenile clams in high salinity exposures; however, these effects were abolished under the low salinity conditions or at high PCO2 (~1500 µatm). Mechanical properties of the shell (measured as microhardness and fracture toughness of the shells) were negatively affected by elevated CO2 alone or in combination with low salinity, which may have important implications for protection against predators or environmental stressors. Our data indicate that environmental salinity can strongly modulate responses to ocean acidification in hard-shell clams and thus should be taken into account when predicting the effects of ocean acidification on estuarine bivalves.

Interactive effects of elevated temperature and CO(2) levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria).

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO2 levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO2 levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO2 levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27°C) and CO2 levels (the present day conditions of ~400ppm CO2 and 800ppm CO2 predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22-27°C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+5°C) and hypercapnia (~800ppm CO2). This indicates that long-term exposure to moderately elevated CO2 and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO2 and temperature must be explained by other cellular mechanisms.

Interactive effects of elevated temperature and CO2 levels on energy metabolism and biomineralization of marine bivalves Crassostrea virginica and Mercenaria mercenaria.

The continuing increase of carbon dioxide (CO2) levels in the atmosphere leads to increases in global temperatures and partial pressure of CO2 (PCO2) in surface waters, causing ocean acidification. These changes are especially pronounced in shallow coastal and estuarine waters and are expected to significantly affect marine calcifiers including bivalves that are ecosystem engineers in estuarine and coastal communities. To elucidate potential effects of higher temperatures and PCO2 on physiology and biomineralization of marine bivalves, we exposed two bivalve species, the eastern oysters Crassostrea virginica and the hard clams Mercenaria mercenaria to different combinations of PCO2 (~400 and 800µatm) and temperatures (22 and 27°C) for 15weeks. Survival, bioenergetic traits (tissue levels of lipids, glycogen, glucose and high energy phosphates) and biomineralization parameters (mechanical properties of the shells and activity of carbonic anhydrase, CA) were determined in clams and oysters under different temperature and PCO2 regimes. Our analysis showed major inter-species differences in shell mechanical traits and bioenergetics parameters. Elevated temperature led to the depletion of tissue energy reserves indicating energy deficiency in both species and resulted in higher mortality in oysters. Interestingly, while elevated PCO2 had a small effect on the physiology and metabolism of both species, it improved survival in oysters. At the same time, a combination of high temperature and elevated PCO2 lead to a significant decrease in shell hardness in both species, suggesting major changes in their biomineralization processes. Overall, these studies show that global climate change and ocean acidification might have complex interactive effects on physiology, metabolism and biomineralization in coastal and estuarine marine bivalves.

Identification of stages of amelogenesis in the continuously growing mandiblular incisor of C57BL/6J male mice throughout life using molar teeth as landmarks.

Continuously growing mouse incisors are widely used to study amelogenesis, since all stages of this process (i.e., secretory, transition and maturation) are present in a spatially determined sequence at any given time. To study biological changes associated with enamel formation, it is important to develop reliable methods for collecting ameloblasts, the cells that regulate enamel formation, from different stages of amelogenesis. Micro-dissection, the key method for collecting distinct ameloblast populations from mouse incisors, relies on positions of molar teeth as landmarks for identifying critical stages of amelogenesis. However, the positions of mandibular incisors and their spatial relationships with molars change with age. Our goal was to identify with high precision these relationships throughout skeletal growth and in older, skeletally mature animals. Mandibles from 2, 4, 8, 12, 16, and 24-week-old, and 18-month-old C57BL/6J male mice, were collected and studied using micro-CT and histology to obtain incisal enamel mineralization profiles and to identify corresponding changes in ameloblast morphology during amelogenesis with respect to positions of molars. As reported here, we have found that throughout active skeletal growth (weeks 2-16) the apices of incisors and the onset of enamel mineralization move distally relative to molar teeth. The position of the transition stage also moves distally. To test the accuracy of the landmarks, we micro-dissected enamel epithelium from mandibular incisors of 12-week-old animals into five segments, including 1) secretory, 2) late secretory - transition - early maturation, 3) early maturation, 4) mid-maturation and 5) late maturation. Isolated segments were pooled and subjected to expression analyses of genes encoding key enamel matrix proteins (EMPs), Amelx, Enam, and Odam, using RT-qPCR. Amelx and Enam were strongly expressed during the secretory stage (segment 1), while their expression diminished during transition (segment 2) and ceased in maturation (segments 3, 4, and 5). In contrast, Odam's expression was very low during secretion and increased dramatically throughout transition and maturation stages. These expression profiles are consistent with the consensus understanding of enamel matrix proteins expression. Overall, our results demonstrate the high accuracy of our landmarking method and emphasize the importance of selecting age-appropriate landmarks for studies of amelogenesis in mouse incisors.

Organic Matrix Derived from Host-Microbe Interplay Contributes to Pathological Renal Biomineralization.

Matrix stones are a rare form of kidney stones. They feature a high percentage of hydrogel-like organic matter, and their formation is closely associated with urinary tract infections. Herein, comprehensive materials and biochemical approaches were taken to map the organic-inorganic interface and gather insights into the host-microbe interplay in pathological renal biomineralization. Surgically extracted soft and slimy matrix stones were examined using micro-X-ray computed tomography and various microspectroscopy techniques. Higher-mineral-density laminae were positive for calcium-bound Alizarin red. Lower-mineral-density laminae revealed periodic acid-Schiff-positive organic filamentous networks of varied thickness. These organic filamentous networks, which featured a high polysaccharide content, were enriched with zinc, carbon, and sulfur elements. Neutrophil extracellular traps (NETs) along with immune response-related proteins, including calprotectin, myeloperoxidase, CD63, and CD86, also were identified in the filamentous networks. Expressions of NETs and upregulation of polysaccharide-rich mucin secretion are proposed as a part of the host immune defense to "trap" pathogens. These host-microbe derived organic matrices can facilitate heterogeneous nucleation and precipitation of inorganic particulates, resulting in macroscale aggregates known as "matrix stones". These insights into the plausible aggregation of constituents through host-microbe interplay underscore the unique "double-edged sword" effect of the host immune response to pathogens and the resulting renal biominerals.

Interactive effects of salinity and elevated CO2 levels on juvenile eastern oysters, Crassostrea virginica.

Rising levels of atmospheric CO(2) lead to acidification of the ocean and alter seawater carbonate chemistry, which can negatively impact calcifying organisms, including mollusks. In estuaries, exposure to elevated CO(2) levels often co-occurs with other stressors, such as reduced salinity, which enhances the acidification trend, affects ion and acid-base regulation of estuarine calcifiers and modifies their response to ocean acidification. We studied the interactive effects of salinity and partial pressure of CO(2) (P(CO2)) on biomineralization and energy homeostasis in juveniles of the eastern oyster, Crassostrea virginica, a common estuarine bivalve. Juveniles were exposed for 11 weeks to one of two environmentally relevant salinities (30 or 15 PSU) either at current atmospheric P(CO2) (∼400 µatm, normocapnia) or P(CO2) projected by moderate IPCC scenarios for the year 2100 (∼700-800 µatm, hypercapnia). Exposure of the juvenile oysters to elevated P(CO2) and/or low salinity led to a significant increase in mortality, reduction of tissue energy stores (glycogen and lipid) and negative soft tissue growth, indicating energy deficiency. Interestingly, tissue ATP levels were not affected by exposure to changing salinity and P(CO2), suggesting that juvenile oysters maintain their cellular energy status at the expense of lipid and glycogen stores. At the same time, no compensatory upregulation of carbonic anhydrase activity was found under the conditions of low salinity and high P(CO2). Metabolic profiling using magnetic resonance spectroscopy revealed altered metabolite status following low salinity exposure; specifically, acetate levels were lower in hypercapnic than in normocapnic individuals at low salinity. Combined exposure to hypercapnia and low salinity negatively affected mechanical properties of shells of the juveniles, resulting in reduced hardness and fracture resistance. Thus, our data suggest that the combined effects of elevated P(CO2) and fluctuating salinity may jeopardize the survival of eastern oysters because of weakening of their shells and increased energy consumption.