Colocation and role of polyphosphates and alkaline phosphatase in apatite biomineralization of elasmobranch tesserae

AbstractElasmobranchs (e.g. sharks and rays), like all fishes, grow continuously throughout life. Unlike other vertebrates, their skeletons are primarily cartilaginous, comprising a hyaline cartilage-like core, stiffened by a thin outer array of mineralized, abutting and interconnected tiles called tesserae. Tesserae bear active mineralization fronts at all margins and the tesseral layer is thin enough to section without decalcifying, making this a tractable but largely unexamined system for investigating controlled apatite mineralization, while also offering a potential analog for endochondral ossification. The chemical mechanism for tesserae mineralization has not been described, but has been previously attributed to spherical precursors, and alkaline phosphatase (ALP) activity. Here, we use a variety of techniques to elucidate the involvement of phosphorus-containing precursors in the formation of tesserae at their mineralization fronts. Using Raman spectroscopy, fluorescence microscopy and histological methods, we demonstrate that ALP activity is located with inorganic phosphate polymers (polyP) at the tessera–uncalcified cartilage interface, suggesting a potential mechanism for regulated mineralization: inorganic phosphate (Pi) can be cleaved from polyP by ALP, thus making Pi locally available for apatite biomineralization. The application of exogenous ALP to tissue cross-sections resulted in the disappearance of polyP and the appearance of Pi in uncalcified cartilage adjacent to mineralization fronts. We propose that elasmobranch skeletal cells control apatite biomineralization by biochemically controlling polyP and ALP production, placement and activity. Previous identification of polyP and ALP shown previously in mammalian calcifying cartilage supports the hypothesis that this mechanism may be a general regulating feature in the mineralization of vertebrate skeletons

Arctic Gypsum Endoliths: A Biogeochemical Characterization of a Viable and Active Microbial Community

Extreme environmental conditions such as those found in the polar regions on Earth are thought to test the limits of life. Microorganisms living in these environments often seek protection from environmental stresses such as high UV exposure, desiccation and rapid temperature fluctuations, with one protective habitat found within rocks. Such endolithic microbial communities, which often consist of bacteria, fungi, algae and lichens, are small-scale ecosystems comprised of both producers and consumers. However, the harsh environmental conditions experienced by polar endolithic communities are thought to limit microbial diversity and therefore the rate at which they cycle carbon. In this study, we characterized the microbial community diversity, turnover rate and microbe-mineral interactions of a gypsum-based endolithic community in the polar desert of the Canadian high Arctic. 16S/18S/23S rRNA pyrotag sequencing demonstrated the presence of a diverse community of phototrophic and heterotrophic bacteria, archaea, algae and fungi. Stable carbon isotope analysis of the viable microbial membranes, as phospholipid fatty acids and glycolipid fatty acids, confirmed the diversity observed by molecular techniques and indicated that present-day atmospheric carbon is assimilated into the microbial community biomass. Uptake of radiocarbon from atmospheric nuclear weapons testing during the 1960s into microbial lipids was used as a pulse label to determine that the microbial community turns over carbon on the order of 10 yr, equivalent to 4.4 gCm(-2) yr(-1) gross primary productivity. Scanning electron microscopy (SEM) micrographs indicated that mechanical weathering of gypsum by freeze-thaw cycles leads to increased porosity, which ultimately increases the habitability of the rock. In addition, while bacteria were adhered to these mineral surfaces, chemical analysis by micro-X-ray fluorescence (mu-XRF) spectroscopy suggests little evidence for microbial alteration of minerals, which contrasts with other endolithic habitats. While it is possible that these communities turn over carbon quickly and leave little evidence of microbe-mineral interaction, an alternative hypothesis is that the soluble and friable nature of gypsum and harsh conditions lead to elevated erosion rates, limiting microbial residence times in this habitat. Regardless, this endolithic community represents a microbial system that does not rely on a nutrient pool from the host gypsum cap rock, instead receiving these elements from allochthonous debris to maintain a more diverse and active community than might have been predicted in the polar desert of the Canadian high Arctic.Cancer Prevention and Research Institute of Texas (CPRIT) RP100934Kleberg Center for Molecular MarkersApoCell, IncEntertainment Industry Foundation SU2C-AACR-DT0209Geological Science

A cautionary (spectral) tail : red-shifted fluorescence by DAPI–DAPI interactions

The fluorescent dye DAPI is useful for its association with and consequent amplification of an ∼460 nm emission maximum upon binding to dsDNA. Labelling with higher DAPI concentrations is a technique used to reveal Pi polymers [polyphosphate (polyP)], with a red-shift to ∼520–550 nm fluorescence emission. DAPI–polyP emissions of ∼580 nm are also generated upon 415 nm excitation. Red-shifted DAPI emission has been associated with polyP and RNA and has more recently been reported with polyadenylic acid (polyA), specific inositol phosphates (IPs) and heparin. We find that amorphous calcium phosphate (ACP) also demonstrates red-shifted DAPI emission at high DAPI concentrations. This DAPI spectral shift has been attributed to DAPI–DAPI electrostatic interactions enabled by molecules with high negative charge density that increase the local DAPI concentration and favour DAPI molecular proximity, as observed by increasing the dye/phosphate ratio. Excitation of dry DAPI (∼360 nm) confirmed a red-shifted DAPI emission. Whereas enzymatic approaches to modify substrates can help define the nature of DAPI fluorescence signals, multiple approaches beyond red-shifted DAPI excitation/emission are advised before conclusions are drawn about DAPI substrate identification.ACP, amorphous calcium phosphate; CD, circular dichroism; DAPI, 4′,6-diamidino-2-phenylindole; IP, inositol phosphate; HAp, hydroxyapatite; polyA, polyadenylic acid; polyP, polyphosphat

High solids density gypsum production through an improved neutralization process for zinc plant effluent

A common wastewater treatment process practiced by zinc production facilities is the single-stage mixing of acidic wastewaters with slaked lime, inducing the reactive precipitation of fine (∼1 mum) gypsum (CaSO4.2H 2O) and other solids with a solids density less than 10%. These solids report to a tailings pond for containment.Tailings pond life would be increased if the solids density of the precipitated solids was improved. Previous work at McGill University suggested that a staged neutralization process with solids recycle and seeded with gypsum would produce large-sized gypsum crystals with a high solids density. A continuous lab-scale process run with synthetic zinc plant effluent produced large (∼100 mum) gypsum crystals with a solids density of 50 +/- 3%.Meissner's method of calculating mean activity coefficients allowed for the calculation of gypsum solubility in mixed, strong sulphate electrolyte solutions

Polar endoliths - an anti-correlation of climatic extremes and microbial diversity

We examined the environmental stresses experienced by cyanobacteria living in endolithic gneissic habitats in the Haughton impact structure, Devon Island, Canadian High Arctic (75° N) and compared them with the endolithic habitat at the opposite latitude in the Dry Valleys of Antarctica (76° S). In the Arctic during the summer, there is a period for growth of approximately 2.5 months when temperatures rise above freezing. During this period, freeze–thaw can occur during the diurnal cycle, but freeze–thaw excursions are rare within higher-frequency temperature changes on the scale of minutes, in contrast with the Antarctic Dry Valleys. In the Arctic location rainfall of approximately 3 mm can occur in a single day and provides moisture for endolithic organisms for several days afterwards. This rainfall is an order of magnitude higher than that received in the Dry Valleys over 1 year. In the Dry Valleys, endolithic communities may potentially receive higher levels of ultraviolet radiation than the Arctic location because ozone depletion is more extreme. The less extreme environmental stresses experienced in the Arctic are confirmed by the presence of substantial epilithic growth, in contrast to the Dry Valleys. Despite the more extreme conditions experienced in the Antarctic location, the diversity of organisms within the endolithic habitat, which includes lichen and eukaryotic algal components, is higher than observed at the Arctic location, where genera of cyanobacteria dominate. The lower biodiversity in the Arctic may reflect the higher water flow through the rocks caused by precipitation and the more heterogeneous physical structure of the substrate. The data illustrate an instance in which extreme climate is anti-correlated with microbial biological diversity