Modulating chitin synthesis in marine algae with iminosugars obtained by SmI2 and FeCl3-mediated diastereoselective carbonyl ene reaction.

Strategies for synthesizing polyhydroxylated piperidines such as iminosugars have received broad attention. These substances are known to interact with carbohydrate related enzymes, glycosidases and glycosyltransferases, to which also the large enzyme families of chitin synthases and cellulose synthases belong. Many chemical and biological aspects of chitin synthases remain unexplored due to the fact that modulating substances are hardly available or expensive. Starting from enantiopure D- and L-amino acids, a series of iminosugars was prepared by a Lewis acid-catalyzed cyclization of amino acid-derived unsaturated aldehydes as key step. Therefore, different Lewis acids were tested. For samarium diiodide we observed a superior stereoselectivity in comparison to iron(III) chloride and methylaluminium dichloride. To increase water solubility for testing and measurement of enzyme activity, the cyclization products were further functionalized. We established a novel biological chitin synthesis test system which allows quantitative investigation of chitin synthesis in the chitin fiber producing diatom algae Thalassiosira in vivo under the light microscope. None of the compounds displayed cytotoxicity, but two of the four iminosugars increased the length of the chitin fibers produced. This is a strong indicator that these compounds mimic carbohydrates responsible for restarting chitin polymerization.

Mineralized scale patterns on the cell periphery of the chrysophyte Mallomonas determined by comparative 3D Cryo-FIB SEM data processing.

Unicellular protists can biomineralize spatially complex and functional shells. A typical cell of the photosynthetic synurophyte Mallomonas is covered by about 60-100 silica scales. Their geometric arrangement, the so-called scale case, mainly depends on the species and on the cell cycle. In this study, the scale case of the synurophyte Mallomonas was preserved in aqueous suspension using high-pressure freezing (HPF). From this specimen, a three-dimensional (3D) data set spanning a volume of about 25.6 µm × 19.2 µm × 4.2 µm with a voxel size of 12.5 nm × 12.5 nm × 25.0 nm was collected by Cryo-FIB SEM in 3 h and 24 min. SEM imaging using In-lens SE detection allowed to clearly differentiate between mineralized, curved scales of less than 0.2 µm thickness and organic cellular ultrastructure or vitrified ice. The three-dimensional spatial orientations and shapes of a minimum set of scales (N = 13) were identified by visual inspection, and manually segmented. Manual and automated segmentation approaches were comparatively applied to one arbitrarily selected reference scale using the differences in grey level between scales and other constituents. Computational automated routines and principal component analysis of the experimentally extracted data created a realistic mathematical model based on the Fibonacci pattern theory. A complete in silico scale case of Mallomonas was reconstructed showing an optimized scale coverage on the cell surface, similarly as it was observed experimentally. The minimum time requirements from harvesting the living cells to the final scale case determination by Cryo-FIB SEM and computational image processing are discussed.

Plant virus-based materials for biomedical applications: Trends and prospects.

Nanomaterials composed of plant viral components are finding their way into medical technology and health care, as they offer singular properties. Precisely shaped, tailored virus nanoparticles (VNPs) with multivalent protein surfaces are efficiently loaded with functional compounds such as contrast agents and drugs, and serve as carrier templates and targeting vehicles displaying e.g. peptides and synthetic molecules. Multiple modifications enable uses including vaccination, biosensing, tissue engineering, intravital delivery and theranostics. Novel concepts exploit self-organization capacities of viral building blocks into hierarchical 2D and 3D structures, and their conversion into biocompatible, biodegradable units. High yields of VNPs and proteins can be harvested from plants after a few days so that various products have reached or are close to commercialization. The article delineates potentials and limitations of biomedical plant VNP uses, integrating perspectives of chemistry, biomaterials sciences, molecular plant virology and process engineering.

Thermal decomposition of the amino acids glycine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine and histidine.

BACKGROUND: The pathways of thermal instability of amino acids have been unknown. New mass spectrometric data allow unequivocal quantitative identification of the decomposition products. RESULTS: Calorimetry, thermogravimetry and mass spectrometry were used to follow the thermal decomposition of the eight amino acids G, C, D, N, E, Q, R and H between 185 °C and 280 °C. Endothermic heats of decomposition between 72 and 151 kJ/mol are needed to form 12 to 70% volatile products. This process is neither melting nor sublimation. With exception of cysteine they emit mainly H2O, some NH3 and no CO2. Cysteine produces CO2 and little else. The reactions are described by polynomials, AA→a NH3+b H2O+c CO2+d H2S+e residue, with integer or half integer coefficients. The solid monomolecular residues are rich in peptide bonds. CONCLUSIONS: Eight of the 20 standard amino acids decompose at well-defined, characteristic temperatures, in contrast to commonly accepted knowledge. Products of decomposition are simple. The novel quantitative results emphasize the impact of water and cyclic condensates with peptide bonds and put constraints on hypotheses of the origin, state and stability of amino acids in the range between 200 °C and 300 °C.

Aqueous ball milling of nacre constituents facilitates directional self-assembly of aragonite nanoparticles of the gastropod Haliotis glabra.

A ball-milling approach was developed to investigate the constituents of isolated nacre tablets of the gastropod Haliotis glabra in aqueous suspension without additional chemical additives. The obtained particle mixtures were characterized using X-ray crystallography as well as scanning and transmission electron microscopy. Aragonite nanoparticles retained their crystal structure even after 14 h of ball milling. The long-term stability of the particle mixtures varied as a function of the ball-milling duration. An increased milling time led to rod-like stable assemblies of aragonite nanoparticles. Selected area electron diffraction investigations revealed that the longitudinal axes in about one-third of these nanoparticle rods were oriented along the crystallographic c-axis of aragonite, indicating oriented attachment of the aragonite nanoparticles. These in vitro observations support the idea that a two-stage process, separated into crystallization of nanoparticles and oriented assembly of nanocrystals, could also occur in vivo.

Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization.

Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO2 mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

Datasets from a vapor diffusion mineral precipitation protocol for Dictyostelium stalks.

Datasets from a slow carbonate vapor diffusion and mineral precipitation protocol for Dictyostelium ECM and cellulose stalks show examples for composite materials obtained by an in vitro approach, which differs substantially from the in vivo approach reported in The Journal of Structural Biology, doi: 10.1016/j.jsb.2016.03.015 [1]. Methods for obtaining the datasets include bright field transmitted light microscopy, fluorescence microscopy, LC-PolScope birefringence microscopy, variable pressure scanning electron microscopy (VP-SEM/ESEM), and Raman imaging spectroscopy.

In vivo modified organic matrix for testing biomineralization-related protein functions in differentiated Dictyostelium on calcite.

This work reports an in vivo approach for identifying the function of biomineralization-related proteins. Synthetic sequences of n16N, OC-17 and perlucin with signal peptides are produced in a novel Gateway expression system for Dictyostelium under the control of the [ecmB] promoter. A fast and easy scanning electron microscopic screening method was used to differentiate on the colony level between interplay effects of the proteins expressed in the extracellular matrix (ECM). Transformed Dictyostelium, which migrated as multicellular colonies on calcite crystals and left their ECM remnants on the surface were investigated also by energy-dispersive X-ray spectroscopy (EDX). Calcium minerals with and without phosphorous accumulated very frequently within the matrix of the Dictyostelium colonies when grown on calcite. Magnesium containing phosphorous granules were observed when colonies were exposed on silica. The absence of calcium EDX signals in these cases suggests that the external calcite crystals but not living cells represent the major source of calcium in the ECM. Several features of the system provide first evidence that each protein influences the properties of the matrix in a characteristic mode. Colonies transformed with perlucin produced a matrix with cracks on the length scale of a few microns throughout the matrix patch. For colonies with OC-17, almost no cracks were observed, regardless of the length scale. The non-transformed Dictyostelium (Ax3-Orf+) produced larger cracks. The strategy presented here develops the first step toward an efficient eukaryotic screening system for the combinatorial functionalization of materials by bioengineering in close analogy to natural biomineralization concepts.

Real-time monitoring of calcium carbonate and cationic peptide deposition on carboxylate-SAM using a microfluidic SAW biosensor.

A microfluidic biosensor with surface acoustic wave technology was used in this study to monitor the interaction of calcium carbonate with standard carboxylate self-assembled monolayer sensor chips. Different fluids, with and without biomolecular components, were investigated. The pH-dependent surface interactions of two bio-inspired cationic peptides, AS8 and ES9, which are similar to an extracellular domain of the chitin synthase involved in mollusc shell formation, were also investigated in a biological buffer system. A range of experimental conditions are described that are suitable to study non-covalent molecular interactions in the presence of ionic substances, such as, mineral precursors below the solubility equilibrium. The peptide ES9, equal to the mollusc chitin synthase epitope, is less sensitive to changes in pH than its counterpart AS8 with a penta-lysine core, which lacks the flanking acidic residues. This study demonstrates the extraordinary potential of microfluidic surface acoustic wave biosensors to significantly expand our experimental capabilities for studying the principles underlying biomineralization in vitro.

Peptide induced crystallization of calcium carbonate on wrinkle patterned substrate: implications for chitin formation in molluscs.

We here present the nucleation and growth of calcium carbonate under the influence of synthetic peptides on topographically patterned poly(dimethylsiloxane) (PDMS) substrates, which have a controlled density of defects between the wrinkles. Experiments with two lysine-rich peptides derived from the extracellular conserved domain E22 of the mollusc chitin synthase Ar-CS1, AKKKKKAS (AS8) and EEKKKKKES (ES9) on these substrates showed their influence on the calcium carbonate morphology. A transition from polycrystalline composites to single crystalline phases was achieved with the peptide AS8 by changing the pH of the buffer solution. We analyzed three different pH values as previous experiments showed that E22 interacts with aragonite biominerals more strongly at pH 7.75 than at pH 9.0. At any given pH, crystals appeared in characteristic morphologies only on wrinkled substrates, and did not occur on the flat, wrinkle-free PDMS substrate. These results suggest that these wrinkled substrates could be useful for controlling the morphologies of other mineral/peptide and mineral/protein composites. In nature, these templates are formed enzymatically by glycosyltransferases containing pH-sensitive epitopes, similar to the peptides investigated here. Our in vitro test systems may be useful to gain understanding of the formation of distinct 3D morphologies in mollusc shells in response to local pH shifts during the mineralization of organic templates.

On the function of chitin synthase extracellular domains in biomineralization.

Molluscs with various shell architectures evolved around 542-525 million years ago, as part of a larger phenomenon related to the diversification of metazoan phyla. Molluscs deposit minerals in a chitin matrix. The mollusc chitin is synthesized by transmembrane enzymes that contain several unique extracellular domains. Here we investigate the assembly mechanism of the chitin synthase Ar-CS1 via its extracellular domain ArCS1_E22. The corresponding transmembrane protein ArCS1_E22TM accumulates in membrane fractions of the expression host Dictyostelium discoideum. Soluble recombinant ArCS1_E22 proteins can be purified as monomers only at basic pH. According to confocal fluorescence microscopy experiments, immunolabeled ArCS1_E22 proteins adsorb preferably to aragonitic nacre platelets at pH 7.75. At pH 8.2 or pH 9.0 the fluorescence signal is less intense, indicating that protein-mineral interaction is reduced with increasing pH. Furthermore, ArCS1_E22 forms regular nanostructures on cationic substrates as revealed by atomic force microscopy (AFM) experiments on modified mica cleavage planes. These experiments suggest that the extracellular domain ArCS1_E22 is involved in regulating the multiple enzyme activities of Ar-CS1 such as chitin synthesis and myosin movements by interaction with mineral surfaces and eventually by protein assembly. The protein complexes could locally probe the status of mineralization according to pH unless ions and pCO2 are balanced with suitable buffer substances. Taking into account that the intact enzyme could act as a force sensor, the results presented here provide further evidence that shell formation is coordinated physiologically with precise adjustment of cellular activities to the structure, topography and stiffness at the mineralizing interface.

GFP facilitates native purification of recombinant perlucin derivatives and delays the precipitation of calcium carbonate.

Insolubility is one of the possible functions of proteins involved in biomineralization, which often limits their native purification. This becomes a major problem especially when recombinant expression systems are required to obtain larger amounts. For example, the mollusc shell provides a rich source of unconventional proteins, which can interfere in manifold ways with different mineral phases and interfaces. Therefore, the relevance of such proteins for biotechnological processes is still in its infancy. Here we report a simple and reproducible purification procedure for a GFP-tagged lectin involved in biomineralization, originally isolated from mother-of-pearl in abalone shells. An optimization of E. coli host cell culture conditions was the key to obtain reasonable yields and high degrees of purity by using simple one-step affinity chromatography. We identified a dual functional role for the GFP domain when it became part of a mineralizing system in vitro. First, the GFP domain improved the solubility of an otherwise insoluble protein, in this case recombinant perlucin derivatives. Second, GFP inhibited calcium carbonate precipitation in a concentration dependent manner. This was demonstrated here using a simple bulk assay over a time period of 400 seconds. At concentrations of 2 µg/ml and higher, the inhibitory effect was observed predominantly for HCO(3) (-) as the first ionic interaction partner, but not necessarily for Ca(2+). The interference of GFP-tagged perlucin derivatives with the precipitation of calcium carbonate generated different types of GFP-fluorescent composite calcite crystals. GFP-tagging offers therefore a genetically tunable tool to gently modify mechanical and optical properties of synthetic biocomposite minerals.

Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells.

BACKGROUND: Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 µm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. RESULTS: Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. CONCLUSIONS: In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130:17519-17527]. Polarized optical microscopy revealed unprecedented super-structures in the calcitic shell part. This bears, in principle, the potential for in vivo studies, which might be useful for investigating the growth modes of nacre and other shell types.

Functional expression of Ca²âº dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.

Transmembrane myosin chitin synthase involved in mollusc shell formation produced in Dictyostelium is active.

Several mollusc shells contain chitin, which is formed by a transmembrane myosin motor enzyme. This protein could be involved in sensing mechanical and structural changes of the forming, mineralizing extracellular matrix. Here we report the heterologous expression of the transmembrane myosin chitin synthase Ar-CS1 of the bivalve mollusc Atrina rigida (2286 amino acid residues, M.W. 264 kDa/monomer) in Dictyostelium discoideum, a model organism for myosin motor proteins. Confocal laser scanning immunofluorescence microscopy (CLSM), chitin binding GFP detection of chitin on cells and released to the cell culture medium, and a radiochemical activity assay of membrane extracts revealed expression and enzymatic activity of the mollusc chitin synthase in transgenic slime mold cells. First high-resolution atomic force microscopy (AFM) images of Ar-CS1 transformed cellulose synthase deficient D. discoideumdcsA(-) cell lines are shown.

Plasticity of two structural proteins: alpha-collagen and beta-keratin.

Although the microstructures of Type-I collagen in bone and F-keratin in avian feathers are very different, their plastic behaviour is similar. In both plasticity is thermally activated, with the activation enthalpy H=1.1 eV in bone and 1.75 eV in feather. The activation volumes are v=0.6 nm3 in bone and v=0.83 nm3 in feather. This indicates that the rate controlling process in both is the breaking of electrostatic bonds.

Calcium and silicon mineralization in land plants: transport, structure and function.

Plant biomineralization involves calcium and silicon transport and mineralization. Respective analytical methods and case studies are listed. Calcium carbonate is deposited in cystoliths, calcium oxalate in idioblasts. Silicon is deposited in phytoliths. Biomineralization is a coordinated process.

The peacock's train (Pavo cristatus and Pavo cristatus mut. alba) II. The molecular parameters of feather keratin plasticity.

Thermal activation analysis of plastic deformation of peacock tail feathers, by temperature changes and stress relaxation, gave for the keratin cortex an activation enthalpy of 1.78 ± 0.89 eV and an activation volume of 0.83 ± 0.13 nm³, for both the blue and the white subspecies. These values suggest that breaking of electrostatic bonds is responsible for plasticity in feather keratin. These might be bonds between keratin and nonkeratinous matrix or keratin-keratin cross-links. The mechanical properties of the rachis cortex are surprisingly uniform along the length of the feathers.

The peacock's train (Pavo cristatus and Pavo cristatus mut. alba) I. structure, mechanics, and chemistry of the tail feather coverts.

The feathers in the train of the peacock serve not for flying but for sexual display. They are long, slender beams loaded in bending by their own weight. An outer circular conical shell, the cortex, is filled by a closed foam of 7.6% relative density, the medulla, both of feather keratin. Outer diameter and thickness of the cortex decrease linearly from the body toward the tip. This self-similar geometry leads to a division of labor. The cortex (longitudinal Young's modulus 3.3 GPa, transverse modulus 1 GPa) provides 96% of the longitudinal strength and bending rigidity of the feather. The medulla (Young's modulus 10 MPa) provides 96% of the transverse compressive rigidity. Fracture stress of the cortex, both longitudinal and transverse, is 120 MPa.