Lorica Production in Choanoflagellates–A Model System for Investigating the Mechanics, Controls and Dynamics of Secretion

BIOMINERALIZATION is the process by which living organisms assemble structures from naturally occurring inorganic compounds. Mineral deposition is common and widespread amongst Protozoa and in most instances the mineralized structures provide skeletal support and protection for softer organic parts [10]. The 2 most common minerals to be deposited by Protozoa are silica and calcium carbonate. Groups of Protozoa that deposit silica, which we are concerned with here, include the diatoms, chrysophytes, choanoflagellates, Radiolar-ia, Heliozoa and testate amoebae [10]. In the majority of silica-depositing protista, silica is taken up from the medium in the form of monomelic orthosilicic acid Si(OH)₄ (soluble reactive silicate) and deposited as amorphous, polymerised biogenic silica or opal within membrane-bounded vesicles known as silica deposition vesicles (SDV). Often biogenic silica is characteristically patterned and ornamented and for most protozoan groups the morphology of silicified parts is of prime taxonomic importance. By far the most extensively studied group of silica-depositing organisms are the diatoms [1, 12, 13]. To date most of our knowledge of silica metabolism in protists has been based on investigations into this group. Diatoms require silica for the production of their frustules. Uptake and deposition of silica occurs within a closely denned portion of the cell cycle, between nuclear division and cell separation. It occupies about ± of the cell cycle and without an adequate supply of silica diatoms are unable to produce new frustule valves with the result that cell division cannot be completed. Diatoms, therefore, have an obligate requirement for silica and without this nutrient they cease to grow [11]. In contrast to diatoms a number of other silica-depositing protistan groups, such as loricate choanoflagellates and certain chrysophytes, have a facultative requirement for silica. In the past decade the ultras true ture, physiology and ecology of loricate choanoflagellates have been extensively studied by a number of different workers [7] and the significance of these studies to our understanding of the mechanisms, controls and dynamics of silica secretion is summarised and discussed here.     

Bioinspired silicification of silica-binding peptide-silk protein chimeras: comparison of chemically and genetically produced proteins

Novel protein chimeras constituted of "silk" and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](n)) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 28 equiv of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial-compatible conditions, the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared with the genetically derived variants. In all cases, the structure of the protein/chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk-based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source), and approach to synthesis (genetic or chemical) can be used to "tune" the properties of the composite materials formed and is a general approach that can be used to prepare a range of materials for biomedical and sensor-based applications.     

Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S-transferase (GST)

Silicatein from Suberites domuncula was known to catalyze silica deposition in vitro under near neutral pH and ambient temperature conditions. In this study, we employed GST–glutathione (GSH) interaction system to increase the production of silicatein and develop an efficient protein immobilization method. Recombinant silicatein fused with GST (GST-SIL) was produced in E. coli and the GST-SIL protein was employed on GSH-coated glass plate. GST-SIL bound surface or matrix can catalyze the formation of silica layer in the presence of tetraethyl orthosilicate as a substrate at an ambient temperature and neutral pH. During silicatein-mediated silicification, green fluorescent protein (GFP) or horseradish peroxidase (HRP) can be efficiently immobilized on the silica surface. Immobilized GFP or HRP retained their activity and were released gradually. This biocompatible silica coating technique can be employed to prepare biomolecule-immobilized surfaces or matrixes, which are useful for the development of biocatalytic, diagnostic and biosensing system, or tissue culture scaffolds.     

SCANNING ELECTRON MICROSCOPY AND ELECTRON MICROPROBE ANALYSIS OF SILICIFICATION PATTERNS IN INFLORESCENCE BRACTS OF AVENA SATIVA

Using scanning electron microscopy, we determined the kinds and distribution of epidermal cell types in Avena inflorescence bracts (glume, lemma, and palea). Electron microprobe analysis of silica deposition in these epidermal cells showed that silica cells constitute one of the important deposition sites. Probe ratio data indicate that the silica deposited is 74 % pure. Significant amounts of silica also become deposited in the trichomes and lesser amounts in the walls of long epidermal cells. None could be detected in the stomata. The possible functional significance of silica deposition in epidermal cells of these bracts is discussed.     

Structure-radical scavenging activity relationships of flavonoids

The objective of this work is to establish the structural requirements of flavonoids for appreciable radical-scavenging activity (RSA) and elucidate a comprehensive mechanism that can explain their activity. To this end, the RSA of 52 flavonoids against 2,2-diphenyl-1-picrylhydrazyl was determined. The relative change in energy (DeltaH(f)) associated with the formation of various flavonoidal and other phenolic radicals and also the spin distribution in these radicals were determined using computational programmes. By correlating experimental data with DeltaH(f), structural features that affect activity have been identified and considered in perspective. It was shown with compelling evidences that the RSA of flavonoids could be mapped to one of their ring systems, making it possible to study their RSA by dissecting their structures and designing representative simpler models. Consequently, hydroxytoluene units were demonstrated to successfully account for the RSA of flavonoids due to ring B and also to satisfactorily do so for activities due to ring A. Further, a comprehensive model for the radical scavenging reactions of flavonoids (and in general, phenolic compounds), which could account for hydrogen atom donation and the termination of aroxyl radicals, was proposed. Finally, prediction of structural features that could endow flavonoids with appreciable radical scavenging capability was made by considering the stability data and the ease of termination. In conclusion, the underlying molecular phenomena of the RSA of flavonoids could be explained by the ease of hydrogen atom abstraction and the ease of the termination of the flavonoidal aroxyl radicals.     

Silica deposition in abaxial epidermis before the opening of leaf blades of Pleioblastus chino (Poaceae, Bambusoideae)

BACKGROUND AND AIMS Silica deposition is one of the important characteristics of the family Poaceae. The distribution, deposition process and physiology of silica in this family have been extensively investigated. Bamboos among members of Poaceae have leaves with a fairly long life span, and the leaves continuously accumulate silica in their tissues throughout their life, not only during the course of leaf opening, but also after opening. It has been revealed that the silica deposition process in relation to ageing of the bamboo leaf after opening differed depending on the cell types comprising the tissues. However, silica deposition has never been examined during the development and maturation periods of bamboo leaves. Hence, to clarify the silica deposition process in a developmental stage of the bamboo leaf, distribution of silica was observed in the abaxial epidermis before the opening of the leaf blades of Pleioblastus chino. METHODS: Abaxial epidermal tissues of leaves were examined using a scanning electron microscope equipped with an energy dispersive X-ray microanalyser. KEY RESULTS: Among seven cell types comprising the abaxial epidermis, three types of cells, guard cells, prickle hairs and silica cells, deposited silica conspicuously, and another four types, cork cells, long cells, micro hairs and subsidiary cells, deposited only a little silica. Among the former group of cell types, silica cells and guard cells deposited silica over their entire surfaces, while prickle hairs deposited silica only in the point-tips. Silica deposition was detected firstly in prickle hairs, and then in silica cells and guard cells. Only silica cells were assumed to deposit silica conspicuously before leaf opening but not conspicuously after opening. CONCLUSIONS: Cell types in leaf epidermis of bamboo are classified into three groups according to the silica deposition pattern. Silica deposition in silica cells may be positive as a part of the physiological activities of leaves.     

Delayed induced silica defences in grasses and their potential for destabilising herbivore population dynamics

Some grass species mount a defensive response to grazing by increasing their rate of uptake of silica from the soil and depositing it as abrasive granules in their leaves. Increased plant silica levels reduce food quality for herbivores that feed on these grasses. Here we provide empirical evidence that a principal food species of an herbivorous rodent exhibits a delayed defensive response to grazing by increasing silica concentrations, and present theoretical modelling that predicts that such a response alone could lead to the population cycles observed in some herbivore populations. Experiments performed under greenhouse conditions revealed that the rate of deposition of silica defences in the grass Deschampsia caespitosa is a time-lagged, nonlinear function of grazing intensity and that, upon cessation of grazing, these defences take around one?year to decay to within 5?% of control levels. Simple coupled grass-herbivore population models incorporating this functional response, and parameterised with empirical data, consistently predict population cycles for a wide range of realistic parameter values for a (Microtus) vole-grass system. Our results support the hypothesis that induced silica defences have the potential to strongly affect the population dynamics of their herbivores. Specifically, the feedback response we observed could be a driving mechanism behind the observed population cycles in graminivorous herbivores in cases where grazing levels in the field become sufficiently large and sustained to trigger an induced silica defence response.     

Bacterial Adhesion under Static and Dynamic Conditions

The deposition of various pseudomonads and coryneform bacteria with different hydrophobicities (water contact angles) and negative cell surface charges on negatively charged Teflon and glass surfaces was investigated. The levels of deposition varied between 5.0 × 10⁴ and 1.6 × 10⁷ cells cm^-2 and between 5.0 × 10⁴ and 3.6 × 10⁷ cells cm^-2 for dynamic column and static batch systems, respectively, indicating that there was a wide variation in physicochemical interactions. Batch and column results were compared in order to better distinguish between hydrodynamic and other system-dependent influences and method-independent physicochemical interactions. Despite the shorter suspension-solid contact time in columns (1 h) than in batch systems (4 h), the level of deposition (expressed as the number of cells that adhered) divided by the applied ambient cell concentration was 4.12 ± 1.63 times higher in columns than in batch sytems for 15 of 22 strain-surface combinations studied. This demonstrates that transport of microbial particles from bulk liquid to surfaces is more efficient in dynamic columns (transport dominated by convection and diffusion) than in static batch systems (transport by diffusion only). The relative constancy of this ratio for the 15 combinations shows that physicochemical interactions affect adhesion similarly in the two systems. The deviating deposition behavior of the other seven strain-surface combinations could be attributed to method-dependent effects resulting from specific cell characteristics (e.g., to the presence of capsular polymers, to an ability to aggregate, to large cell sizes, or to a tendency to desorb after passage through an air-liquid interface).     

DIATOM SILICA BIOMINERALIZATION: AT NANOSCALE LEVEL A CHEMICALLY UNIFORM PROCESS

Using a high-brilliance synchrotron X-ray source, combined small- and wide-angle X-ray scattering (SAXS and WAXS) was applied to study nanoscale characteristics, in particular pore size in the range of 3 to 65 nm, of a variety of unialgal cultures of centric and pennate diatoms, and of mixed diatom populations sampled in the field. Results of scattering analysis were compared with details of pore size, structure and orientation visible at the electron microscopic level. WAXS patterns did not reveal any crystalline phase or features of microcrystallinity (resolution 0.07 to 0.51 nm), which implies a totally amorphous character of the SiO₂ matrix of the frustule material. SAXS data (resolution 3 to 65 nm) provided information on geometry, size, and distribution of pores in the silica. Overall, two pore regions were recognized that were common to the silica of all samples: the smallest (d less than 10 nm) regularly spaced and shaped spherically, the larger (up to 65 nm) being cylinders or slits. Apparently, at a nanoscale level diatomaceous silica is quite homologous among species, in agreement with the chemical principles of silica polymerization under the conditions of pH and precursor concentrations inside the silicon deposition vesicle. The final frustule "macro"-morphology is of course species-specific, being determined genetically. Synthetically-derived MCM-type silicas have a similarly organized pore distribution in an amorphous silica matrix as we found in all diatom species studied. We therefore suggest that organic molecules of a kind used as structure-directing agents to produce these artificial silicas play a role in the nucleation of the silica polymerization reaction and the shaping of pore morphology inside the silicon deposition vesicle of diatoms. Structure-directing molecules now await isolation from the SDV, followed by identification and characterisation by molecular techniques.     

MICROMORPHOGENESIS DURING DIATOM WALL FORMATION PRODUCES SILICEOUS NANOSTRUCTURES WITH DIFFERENT PROPERTIES1

Micromorphogenesis within the silica deposition vesicle (SDV) of the diatom Pinnularia viridis (Nitzsh) Ehrenb. resulted in distinct silica nanostructures and layers within forming valves and girdle bands. These siliceous components were similarly disclosed following alkaline etching of mature valves/girdle bands, where their different susceptibilities to dissolution over time resulted from apparent differences in silica density and/or chemistry. The bulk of silica appeared to be deposited at the interface of the forming valve or girdle band with the silicalemma and occurred by the outward expansion of microfibrils of silica that aligned perpendicularly to the silicalemma. Microfibrils originated from both sides of the “silica lamella,” the first nanostructure formed within the SDV, and several silica species of distinct nanostructure and density resulted, including distinctive inner and outermost silica “coverings” of mature valves/girdle bands and the central and terminal nodules. Not all silica deposition and micromorphogenesis occurred in contact with the expanding silicalemma, but was somehow directed within the SDV cavity, and resulted in the distinct silica layers that lined the raphe fissures and poroids. Following alkaline etching, the inner surfaces of valves/girdle bands, as well as the silica layers lining the raphes, poroids, and slits, were determined to be significantly more resistant to alkaline etching than the exterior surfaces, while the outer silica coating and the nodules were quickly dissolved. The processes of micromorphogenesis must have exerted precise control over the chemical nature of the silica formed at different positions within the SDV and affected the overall structure and function of the diatom wall.     

Impact of substituents on the enantioseparation of racemic 2-amidotetralins on polysaccharide stationary phases. I. chiralcel OD

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and π-π interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R_S), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. Chirality 8:574–578, 1996. © 1997 Wiley-Liss, Inc.     

Abiotic–biotic controls on the origin and development of spicular sinter: in situ growth experiments, Champagne Pool, Waiotapu, New Zealand

Abiotic–biotic mechanisms of microstromatolitic spicular sinter (geyseritic) initiation and development were elucidated by in situ growth experiments at Champagne Pool (75 °C, pH 5.5). Siliceous sinter formed subaerially on glass slides placed along the margin of the hot spring. Environment–silica–microbe interactions were revealed by periodic collections of incremental sinter growth that formed under a range of environmental conditions including quiescence vs. wave turbulence, and wind–evaporation vs. steam–condensation. Sinter surfaces were intermittently colonized by voluminous networks of filamentous micro‐organisms, with submicron diameters, that provided an extensive surface area for silica deposition. The subaerial distribution of sinter and its textures reflected micron‐ to centimetre‐scale differences in environmental conditions, particularly relating to the balance between wave‐supplied dissolved silica and its precipitation, forced by cooling and evaporation. A continuum of sinter textures formed, representing rates of silica precipitation that either out‐paced biofilm growth or regulated the structural development of biofilms, and hence also the nature of microbially templated sinter. Massive laminae of porous, filamentous‐network sinter and/or fenestrae (up to 10's of microns in thickness and diameter) formed at relatively low rates of silica deposition (approximately 0.2 mg slide^?1 day^?1). At high rates (>1.9 mg slide^?1 day^?1), densely packed, granular or nonporous sinter formed, with filament networks disappearing into the siliceous matrix and becoming imperceptible under scanning electron microscopy (SEM). Furthermore, spicules were nucleated by filamentous microcolonies, where their discrete conical morphologies were preserved by accretion of thin sinter laminae. Microstromatolitic spicular growth ensued at fluctuating low to high rates of silica precipitation. Greater apical sinter build‐up, and hence upward polarity, resulted from focused microbial recolonization and progressively greater subaerial exposure at microspicule tips. The biogenic origin of spicular sinter at Champagne Pool clearly demonstrates that micron‐scale biofilms, displaying self‐organization patterns common to both biofilms and microbial mats, can be an essential factor in shaping characteristic centimetre‐scale sinter macrostructures. These findings suggest that a biogenic origin for geyserites elsewhere should also be considered. Moreover, results corroborate the supposition that microbially generated surface roughness may be significant for stromatolite morphogenesis in cryptic Precambrian carbonates.     

Infusion of Bone Marrow Mononuclear Cells Reduces Lung Fibrosis but Not Inflammation in the Late Stages of Murine Silicosis

We hypothesized that infusion of bone marrow mononuclear cells (BMMCs) in the late stages of silica-induced damage would reduce the remodelling process in a murine model of silicosis. C57BL/6 mice were assigned to 2 groups. In the SIL group, mice were instilled with a silica particle suspension intratracheally. Control (C) mice received saline under the same protocol. On the 40th day, some of the animals from both groups were killed. The others were treated with either saline or BMMCs (1×10⁶cells) intravenously (C+BMMC and SIL+BMMC), and the mice were killed 70 days after the start of the protocol. In the mice in the SIL+BMMC group, collagen deposition, the presence of silica particles inside nodules, the presence of macrophages and cells reactive for inducible nitric oxide synthase were reduced. Lung parameters also improved. Beyond that, the total and differential cellularity of bronchoalveolar lavage fluid, immunoexpression of transforming growth factor-β, the number of T regulatory cells and apoptosis were increased. However, the presence of male donor cells in lung tissue was not observed using GFP+ cells (40d) or Y chromosome DNA (70d). Therefore, BMMC therapy in the late stages of experimental silicosis improved lung function by diminishing fibrosis but inflammatory cells persisted, which could be related to expansion of T regulatory cells, responsible for the beneficial effects of cell therapy.     

Estimated yield of double-strand breaks from internal exposure to tritium

Internal exposure to tritium may result in DNA lesions. Of those, DNA double-strand breaks (DSBs) are believed to be important. However, experimental and computational data of DSBs induction by tritium are very limited. In this study, microdosimetric characteristics of uniformly distributed tritium were determined in dimensions of critical significance in DNA DSBs. Those characteristics were used to identify other particles comparable to tritium in terms of microscopic energy deposition. The yield of DSBs could be strongly dependent on biological systems and cellular environments. After reviewing theoretically predicted and experimentally determined DSB yields available in the literature for low-energy electrons and high-energy protons of comparable microdosimetric characteristics to tritium in the dimensions relevant to DSBs, it is estimated that the average DSB yields of 2.7 × 10(-11), 0.93 × 10(-11), 2.4 × 10(-11) and 1.6 × 10(-11) DSBs Gy(-1) Da(-1) could be reasonable estimates for tritium in plasmid DNAs, yeast cells, Chinese hamster V79 cells and human fibroblasts, respectively. If a biological system is not specified, the DSB yield from tritium exposure can be estimated as (2.3 ± 0.7) × 10(-11) DSBs Gy(-1) Da(-1), which is a simple average over experimentally determined yields of DSBs for low-energy electrons in various biological systems without considerations of variations caused by different techniques used and obvious differences among different biological systems where the DSB yield was measured.     

VALVE MORPHOGENESIS IN THE PENNATE DIATOM NAVICULA CUSPIDATA1

The deposition of siliceous valves during asexual reproduction of the pennate diatom, Navicula cuspidata Kütz., is described with emphasis on the cytoplasmic components involved. The events accompanying valve secretion are similar to those already known from other pennate species. After mitosis, the microtubule centre (MC) moves to the center of the cleavage furrow where silica deposition is initiated inside a tubular silicalemma, and it remains associated with the prospective central nodule during valve growth. Microtubules (MTs), emanating from the MC, run parallel to the prospective raphe and together with the raphe fibres, appear to be involved with raphe development. Multiple raphe fibres occupy the maturing raphe fissure, in contrast to the single fibre of Pinnularia viridis, P. maior and Hantzschia amphioxys. The fibers exhibit a periodic substructure and are often opposed to the silicalemma where they may inhibit silica deposition and control the shaping of the raphe fissure. In contrast with the above species, in N. cuspidata MTs are clustered strictly opposite the raphe and lose their association with the MC which degenerates before the valves are mature. The primary role of MTs may be the stabilization of the cytoplasmic region where initial silicification occurs. Mitochondria and endoplasmic reticulum are not involved in molding valve growth in this species. Evidence for vesicle involvement in silica transport and deposition was limited. The possible contributions provided by comparative studies on the ultrastructure of valve morphogenesis towards elucidating the control of valve formation and the taxonomy of diatoms are discussed briefly.     

Silica Deposition in the Grass Leaf in Relation to Transpiration and the Effect of Dinitrophenol

Characteristic opal phytolith (‘silica body’) formationwas demonstrated in detached leaves of Sieglingia decumbens(Heath Grass), cultured in 100 ppm dissolved silicon (silicondioxide), previously, the leaves were free from intracellulardeposits as a result of silica-minimal tiller growth. The formertechnique allowed the study of the leaf deposition processesindependently of apical and root tissues, under growth-cabinetand glasshouse conditions. Deposition in excised leaves wascompletely suppressed by a surface, monomolecular coating, thusindicating that total net water loss was a limiting factor,however, evaporation from the recipient, epidermal tissues perse was not a requirement for this in situ deposition Generally,apart from an apparent, cell site shift in one treatment, phytolith-formationwas unaffected by the presence of the metabolic inhibitor 2,4-dinitrophenol. Also, some evidence was obtained of the influxof germanium dioxide into epidermal ldioblasts, which indicateda non-specificity of the host cell for silica. These results and those of earlier studies suggest that passive,non-metabolic mechanisms could account for the transport, influx,and cell lumen polymerization of silica in the grass leaf.     

BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions

We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems.     

APPLICATION OF THE PDMPO TECHNIQUE IN STUDYING SILICA DEPOSITION IN NATURAL POPULATIONS OF FRAGILARIA CROTONENSIS (BACILLARIOPHYCEAE) AT DIFFERENT DEPTHS IN A EUTROPHIC RESERVOIR1

At weekly intervals from July to October 2006, we measured silica deposition in the summer diatom assemblage at various depths in the eutrophic ?ímov Reservoir (Czech Republic) using PDMPO, the 2‐(4‐pyridyl)‐5{[4‐(2‐dimethylaminoethyl‐aminocarbamoyl)‐methoxy]phenyl}oxazole labeling technique. Fluorescence microscopy coupled with image analysis allows quantifying silicon (Si) deposition over time and a simple distinction between cells that are actively depositing Si and those that are not. Diatom assemblage was exclusively dominated by Fragilaria crotonensis Kitton, which formed pronounced subsurface maxima (2–6.5 m). Concentrations of the main nutrients (Si and phosphorus, P) were low over the whole season; however, at depth, the nutrient availability was higher than at the surface. Fragilaria silica deposition rates were eight times higher at the surface than at depth. Half the population was involved in silica deposition at the surface, while only 20% active cells were doing so at depth. At the surface, silica deposition was limited by P deficiency; the effect of dissolved Si (DSi) was not statistically significant. Silica deposition at depth was significantly constrained by low light availability despite the 1% average light attenuation at depth, which is supposed sufficient for photosynthesis. This study represents the first attempt to employ the PDMPO technique coupled with quantitative image analysis of PDMPO fluorescence in freshwater ecology. On the basis of our results, PDMPO probe appears to be an appropriate proxy for the study of resource limitation in natural diatom populations.     

An overview of the fundamentals of the chemistry of silica with relevance to biosilicification and technological advances

Biomineral formation is widespread in nature, and occurs in bacteria, single-celled protists, plants, invertebrates, and vertebrates. Minerals formed in the biological environment often show unusual physical properties (e.g. strength, degree of hydration) and often have structures that exhibit order on many length scales. Biosilica, found in single-celled organisms through to higher plants and primitive animals (sponges), is formed from an environment that is undersaturated with respect to silicon, and under conditions of approximately neutral pH and relatively low temperatures of 4-40 °C compared to those used industrially. Formation of the mineral may occur intracellularly or extracellularly, and specific biochemical locations for mineral deposition that include lipids, proteins and carbohydrates are known. In most cases, the formation of the mineral phase is linked to cellular processes, an understanding of which could lead to the design of new materials for biomedical, optical and other applications. In this contribution, we describe the aqueous chemistry of silica, from uncondensed monomers through to colloidal particles and 3D structures, that is relevant to the environment from which the biomineral forms. We then describe the chemistry of silica formation from alkoxides such as tetraethoxysilane, as this and other silanes have been used to study the chemistry of silica formation using silicatein, and such precursors are often used in the preparation of silicas for technological applications. The focus of this article is on the methods, experimental and computational, by which the process of silica formation can be studied, with an emphasis on speciation.     

Label-free detection of proteins using SERS-based immuno-nanosensors

This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.