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Abstracts BIOMIN XV: 15Th International Symposium on Biomineralization 9–13 September 2019 • Munich, Germany

Abstracts BIOMIN XV: 15Th International Symposium on Biomineralization 9–13 September 2019 • Munich, Germany

Abstracts BIOMIN XV: 15th International Symposium on 9–13 September 2019 • Munich, Germany

1. Keynote lectures (K1 – K8) ...... 2

2. Talks (T1 – T89) ...... 4

3. Posters (P1 – P107) ...... 31

Design/Layout Layout: www.conventus.de Editorial Deadline: 31 August 2019

1 K 1 K 3 On ion transport and concentration toward formation Getting to the roots of apatite-based biomineralization of dental in larvae hard tissues: from and Cichlids to related Keren Kahi1, Neta Varsano1, Andrea Sorrentino2, Eva Pereiro2, Peter Rez3, bioinspired materials Steve Weiner1 and Lia Addadi*1 Elena V. Sturm*1 (née Rosseeva) 1Department of Structural Biology, Weizmann Institute of Science, Rehovot, 1Physical Chemistry, Zukunftskolleg, University of Konstanz, Konstanz, Israel Germany 2ALBA Synchrotron Light Source, MISTRAL Beamline−Experiments Division, Barcelona, Spain and especially represent the most highly advanced and 3Department of Physics, Arizona State University, Tempe, AZ, USA complex group of . The formation of their hierarchical apatite- organic based hard tissues is evolutionary optimized and exhibits high During mineralized tissue formation, organisms are faced with a major structural complexity on various length scales and amazing mechanical problem in ion transport and concentration. Suffice it to consider that marine performance. The major objective of our research is to explore the must concentrate calcium by >4 orders of magnitude relative to its biomineralization processes involved in the formation of dental (and dental- concentration in sea water to deposit or . Sea urchin like) apatite-based hard tissues of earliest and modern vertebrates. larvae are good model organisms for studying ion processing during Specifically, in my presentation I will focus on the detailed characterization formation, because of the extensive knowledge accumulated on the processes of morphology-structure-composition-property relationships of hard tissues related to mineral deposition. Sea urchin larvae have of feeding apparatus of Conodonts and Cichlid . We elaborate and composed of two calcitic spicules, deposited by primary mesenchymal combine exciting evolutionary model systems with cutting-edge spicule-forming cells (PMCs). PMCs take up seawater through spectroscopy, microscopy and diffraction techniques to analyze the endocytosis1 into a complex network of vacuoles. Within the PMCs, calcium structural, chemical and morphogenetic basis of the dental ions are translocated from the seawater vacuoles to various organelles and biomineralization process. Our new understanding of dentical and tooth vesicles where they accumulate, and subsequently precipitate as an structure in and Cichlids could also advance strategies for amorphous (ACC). The amorphous precipitates are finally synthesizing bioinspired and biomimetic materials and deepen our translocated to the spicule, where they crystallize. knowledge of their morphogenesis process. We address the question of the form in which calcium ions are stored in different locations in the cell, whether dissolved or solid and in which K 4 structural phase. In order to locate and characterize calcium content in Coral Biomineralization: linking pieces of the puzzle individual vesicles we performed cryo-soft X-ray microscopy (cryo-SXM) Sylvie Tambutté*1 on dispersed PMCs. The presence of concentrated calcium ions was detected 1Department of , Centre Scientifique de Monaco, Monaco by imaging the cells in the energy range before and after the calcium L- absorption edges. We characterized the chemical environment of the calcium Coral biomineralization is the process that leads to the formation of a calcium ions using X-ray absorption spectroscopy. We observe hundreds of particles carbonate . As for other biominerals, two essential questions are: containing Ca in each PMC. The particles are composed of different forms What do we know about the control of coral biomineralization? How can we of highly disordered phases of calcium salts, presumably carbonate. We also link biological control with physico-chemical processes? I will show how developed methods for quantitative evaluation of calcium ion concentrations. studies performed at the Centre Scientifique de Monaco since the 90’s have We observed concentrations diluted relative to ACC (19M), but concentrated provided insight into the coral biomineralization process from the whole relative to sea water (10mM). The spectroscopic and analytical data thus to the gene by combining molecular and physiological approaches. together indicate a transition through a series of amorphous calcium I will present how data obtained from experiments conducted in different carbonate phases in the low molar concentration range. These data shed light coral compartments: the tissues, the extracellular calcifying medium and the on the intracellular transport and concentration pathways of calcium ions in skeleton can be linked together and can help in deciphering where and how PMCs. This may well be relevant to other organisms, and thus lead to a biological control occurs. More specifically, I will present 1) the deeper understanding of biogenic mineral formation. measurements of pH, carbonate and calcium in the extracellular medium that 1. N. Vidavsky, S. Addadi, A. Schertel, D. Ben-Ezra, M. Shpigel, L. Addadi, we have obtained by developing in vivo approaches, and 2) the mechanisms S. Weiner, Calcium transport into the cells of the sea urchin larva in relation involved in transepithelial ion transport that we have characterized by to spicule formation. Proc. Natl. Acad. Sci. U.S.A, 113(45), 12637-12642, molecular and physiological approaches. Finally, I will show how changes 2016. 2. E. Beniash, J. Aizenberg, L. Addadi, S. Weiner, Amorphous in environmental parameters such as seawater pH influence biological calcium carbonate transforms into calcite during sea urchin larval spicule control and can provide information on coral biomineralization. growth. P. Roy. Soc. B-Biol. Sci., 264 (1380), 461-465, (1997).

K 5 K 2 Towards -on-a-chip: cell differentiation and extracellular Biomineralization in echinoderms: developmental mechanisms matrix organization and evolution. Sana Ansari1,2,3, Esther Cramer1,2, Johanna Melke2,3, Keita Ito2,3, Sandra Charles Ettensohn*1 Hofmann2,3, Nico Somemrdijk1,3, Anat Akiva*1,3 1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, 1Laboratory of Materials and Interface Chemistry and Center for Multiscale PA USA Electron Microscopy, Department of Chemical Engineering and Chemistry.

Eindhoven University of Technology, Eindhoven, The Netherlands. All adult echinoderms have a calcite-based . Embryonic and 2Institute for Complex Molecular Systems. Eindhoven University of larval patterns of skeletogenesis, however, vary greatly across the phylum, Technology, Eindhoven, The Netherlands. revealing a rich history of evolutionary modifications to the developmental 3Department of Biomedical Engineering. Eindhoven University of programs that underlie biomineral formation in this group. The formation of Technology, Eindhoven, The Netherlands. the skeleton has been particularly well studied in embryos of euechinoid sea urchins, which have served as a major experimental model for developmental Introduction biologists for more than a century. The cellular behaviors that underlie The formation of bone involves a cascade of biological and chemical events skeleton formation in sea urchin embryos have been described in detail and that control the process of mineralization in bone. In this process, many gene products that play essential roles in biomineral formation have bone cells named form a 3D organized collagen matrix and been identified. In addition, a complex transcriptional network that underlies secrete several non-collagenous proteins (NCPs) that control the skeletogenesis has recently been elucidated. This gene regulatory network mineralization process. However, the precise mechanism by which these links the early specification of embryonic skeletogenic cells (primary NCPs control the mineralization of collagen fibrils is not yet clear. In an mesenchyme cells, or PMCs) to their cellular behaviors and biomineral- attempt to get a direct observation on the time and place of the expression of forming properties. The PMC gene network is proving to be a powerful tool these proteins, and to study their role in collagen mineralization, we use an for understanding the genetic and molecular control of skeletogenesis in advanced in vitro model system. echinoderms and the evolution of biomineralization. Materials and methods. Here, human mesenchymal stromal cells (hMSCs) are seeded on 3D silk scaffold and are exposed to osteogenic medium and to continuous mechanical stimulation. Depending on the composition of the osteogenic medium and the specific mechanical load, we can control the differentiation of the hMSCs to osteoblasts and sequentially into osteocytes – the last step in differentiation. Results Using a unique bioreactor, we monitor the cellular and extracellular development of the system. The stem cells transformed from long and

2 elongated cells to round shaped cells, which are typical to osteoblasts. In this K 7 stage, the cells also expressed specific osteoblastic proteins. These proteins, The unique molecular physiologies of biomineralizing such as , , and where located intra and phytoplankton: , vegetable and mineral extracellular, embedded in the collagen matrix. In a later time point, when C. Brownlee*1 the cells are embedded in the collagen matrix, another morphological change 1Marine Biological Association, UK occured, the transformation into osteocytes as judged by the formation of University of Southampton, UK long processes. The osteocyte formation was further supported by the expression of unique osteocyte marker proteins such as DMP-1 and sclerostin. The marine eukaryotic phytoplankton account for around one quarter of Conclusion global productivity, roughly equivalent to the terrestrial rainforests. They In this study, we show for the first time that in vitro differentiation of hMSCs also underpin much of global biomineralization in the form of biogenic into osteoblast and osteocytes is achievable. The system shows the main silica () and calcite (coccolithophores). Despite the major characteristic features of bone, resembling the process of bone formation. importance of these groups in the Earth’s largest ecosystems, their This system will allow to obtain a better understanding of the role of specific physiologies remain relatively poorly studied. Combined functional proteins on collagen mineralization, in a system that is close as possible to in genomics and single cell biophysics approaches are beginning to reveal vivo bone. surprising novel features, including ion channels not previously found in eukaryotes. Their roles in excitability, signalling and cellular homeostasis K 6 will be discussed. The discovery of novel silicon transporters is also Bioinspired magnetite nanoparticles as smart drug shedding new light on mechanisms of coccolithophore biomineralization. nanocarriers and hyperthermia agents Together, these studies are providing new insights into the evolution of major classes of eukaryotic ion channels and membrane transporters. The C. Jimenez-Lopez*1, A. Peigneux1, Y. Jabalera1, F. Oltolina2, G. Iglesias3, rationale and approaches for studying single cell physiology in situ in the M. Prat2 oceans will also be discussed. 1Universidad de Granada (España), Department of Microbiology, Granada, Spain 2Università del Piemonte Orientale A. Avogadro, Dipartimento di Scienze K 8 della Salute, Novara, Patterns in the evolutionary acquisitions of mineralized 3Universidad de Granada (España), Department of Applied Physics, Granada, Spain S. Porter*1, J. Moore1, L. A. Riedman1, R. Wood1, R. Rickaby1 1University of California at Santa Barbara, Earth Science, Santa Barbara, United States Among the biologically controlled biomineralization process, few has raised so much expectations as the formation by magnetotactic . are composed of a magnetite (or greigite) Mineralized skeletons evolved many times within the eukaryotes, including enveloped by a lipidic bilayer and they are crucial for those bacteria survival. dozens of times within the Metazoa. These numerous independent Therefore, control magnetosome formation at the acquisitions provide an opportunity to hypotheses about the origin and genetic level, so the magnetite have the necessary characteristics to evolution of skeletons, including the factors controlling the initial choice of maximize their response to the magnetic field. Magnetosome research has mineralogy and the reasons why mineralized skeletons evolved in the first raised interest in several areas, from Astrobiology to Nanotechnology, since place. Earlier work in this area focused on the evolution of carbonate magnetosomes are the ideal magnetic nanoparticle that could be used in skeletons by metazoans, showing that acquisitions of aragonitic skeletons many applications, mainly clinics, both in diagnosis and in therapeutics. were clustered during times of aragonite seas (when the Mg/Ca ratio of However, the problem for their use is the extremelly low yields in which seawater favored aragonite precipitation) and those of calcitic skeletons magnetosomes are produced. during calcite seas. We have since expanded this dataset to include other In particular, the need for a directed targeted chemotherapy has become a skeletal mineralogies and other eukaryotic clades, including both extinct and matter of growing interest in order to increase efficiency, to reduce the cost extant taxa, and have identified more than 80 independent acquisitions of of systemic treatments and, even more important, to reduce the undesirable mineralized skeletons in eukaryotes, along with their time of first appearance, secondary effects related to the systemic distribution of the chemotherapeutic their mineralogy, and, for the earliest animals, their microstructures and molecule and thus, its interaction with healthy cells. This is not only true and habitats at the time they first evolved. crucial in tumor treatment, either by means of chemotherapy or Several interesting patterns have emerged from this preliminary dataset. immunotherapy, but also for local targetable diseases like local infections. In First, skeletal acquisitions among animals are clustered in time, with half this context, novel MamC-mediated biomimetic (magnetosome-like) appearing in the early , and another 25% in the through magnetic nanoparticles (BMNPs) are proposed as valuable carriers for periods. No acquisitions are recorded for the later Paleozoic. A targeted chemotherapy because of the size (36 ± 12 nm) and of surface smaller cluster of acquisitions occursed in the mid- to ; nearly properties conferred by MamC coating. They are superparamagnetic at room all of these later skeletons evolved within the cnidarians and annelids. In and body temperatures, have a large magnetic moment per particle, mediate contrast, skeletal acquisitions in non-metazoan eukaryotes are distributed hyperthermia, are cytocompatible, and, having a negative surface charge at throughout the Phanerozoic, with no obvious clustering in time. This physiological pH, can be efficiently coupled with a variety of molecules and supports the view that ecological factors affecting only animals (e.g., the antibodies directed against receptors overexpressed in target cells displaying appearance of carnivores), rather than physical factors affecting all marine coupling stability, while releasing DOXO at acidic pH. This release can be organisms (e.g., increased Ca2+), were a primary driver of skeletal enhanced by hyperthermia. As an example, the nanoassembly DOXO-mAb- biomineralization in animals. Second, acquisitions of phosphatic skeletons BMNPs has been characterized and demonstrated that it selectively are concentrated in the Neoproterozoic and Cambrian, perhaps reflecting recognizes the Met receptor (overexpressed in many cancers), binds higher levels of in the oceans at this time, though our count of efficiently to Met+ tumor cells, and discharges DOXO within their nuclei independent acquisitions may be inflated by post-mortem phosphatization of more efficiently than DOXO-BMNPs, exerting cytotoxicity. These data organic skeletons., In contrast with calcareous and siliceous skeletons, which represent proof of concept for in vivo experiments in which the controlled are distributed about equally among autotrophs and heterotrophs, phosphatic dual targeting (mAb-mediated and magnetic) approach and combined skeletons are only known to occur in heterotrophs, perhaps reflecting the (chemotherapy and hyperthermia) therapy are been studied. higher cost for autotrophs to use a limiting nutrient to form a skeleton. Peigneux, A., Oltolina, F., Colangelo, D., Iglesias, G. R., Delgado, A. V., Finally, the skeletal microstructures and habitats of the earliest carbonate Prat, M., Jimenez-Lopez, C. (2019) Functionalized Biomimetic Magnetic biomineralizers suggest phases of increasing biological control over Nanoparticles as Effective Nanocarriers for Targeted Chemotherapy. Particle biomineralization, with the earliest carbonate skeletons forming disorganized and Particle Systems Characterization. DOI:10.1002/ppsc.201900057. microstructures and inhabiting only carbonate environments. These were Nudelman, H,, Valverde-Tercedor, C., Kolusheva, S., Widdrat, M., succeeded in the latest Ediacaran and early Cambrian by calcifying animals Grimberg, N., Levi, H., Nelkenbaum, O., Davidov, G., Faivre, D., Jimenez- that lived in both carbonate and siliciclastic environments and formed more Lopez, C., Zarivach, R. (2016) Structure function studies of the magnetite- organized, though still relatively simple, fibrous microstructures. biomineralizing magnetosome-associated protein MamC. Journal of Structural Biology 194, 244-252.

3 T 1 process. We therefore characterized the carbonate chemistry environment at Nanostructure and growth of calcite crystals of the shell plates of the the shell-tissue interface using minute microsensors for pH, calcium and barnacle Austromegabalanus psittacus (Crustacea, Cirripedia, carbonate and find (3) that veliger larvae can elevate calcium carbonate Balanidae) saturation state of the space, which could be instrumental in A. Checa*1,2, E. Macías-Sánchez3, A. Sánchez-Navas4, A. Rodríguez- increasing rates of carbonate precipitation. However, larvae (4) are not able Navarro4, N. A. Lagos5 to maintain this regulatory effort when seawater pH decreases. This finding 1Universidad de Granada, Estratigrafía y Paleontología, Granada, Spain could explain reduced calcification and dissolution rates when larvae are 2CSIC-Universidad de Granada, Instituto Andaluz de Ciencias de la Tierra, exposed to simulated ocean acidification. In order to identify ion transport Armilla, Spain proteins that support calcification substrate transport and pH regulation, we 3Max Planck Institute of Colloids and Interfaces, Potsdam, Germany use substrate limitation assays in combination with transcriptomics and can 4Universidad de Granada, Mineralogy and Petrology, Granada, Spain (5) show that SLC4 and 26 family member candidate genes are probably vital 5Universidad Santo Tomás, Centro de Investigación e Innovación para el for bicarbonate transport and pH regulation. Cambio Climático, Santiago, Chile References: Ramesh et al. 2017 Nature Communications 8:1709 Ramesh et al. 2018 Journal of the Royal Society Interface 15: 20170723 The calcareous plates enclosing the body of the barnacle Austromegabalanus Ramesh et al. 2019 Ecology & Evolution (in press) psittacus are mainly made by calcite crystals. The crystals are arranged into bundles, within which all crystals are strictly co-oriented. The c-axes of T 3 crystals of different bundles are roughly perpendicular to the growth surfaces. Observations of crystals appearing on the free growth surfaces Terebratulide shell biomineralization by mantle reveal changes in size (from 1 to 5 µm) and morphology (from irregular to epithelial cells 1 2 1 1 2 euhedral rhombohedral). A remarkable feature of these crystals is their M. Simonet Roda* , A. Ziegler , E. Griesshaber , X. Yin , U. Rupp , D. 3 4,5 6 7 3 coarse nanogranular ultrastructure. The {104} rhombohedral faces are Henkel , V. Häussermann , J. Laudien , U. Brand , A. Einsenhauer , W. 1 distinctly much smoother than other types of surfaces. In turn, the W. Schmahl 1 nanogranules making up the crystals are arranged into rod-like structures Ludwig Maximilians Universität München, Department of Earth and (which we call lineations), which diverge at large angles from neat lines. Environmental Sciences, Munich, Germany 2 Examination of incomplete rhombohedra indicate that the lines from which University of Ulm, Central Facility for Electron Microscopy, Ulm, the lineations diverge are at large angles to the {104} faces and clearly in Germany 3 continuity with the rhombohedron edges. That is, they align in parallel to ˂- GEOMAR Helmhöoltz Centre for Ocean Research, Marine 441> directions of calcite. The complete sequence of growth from irregular Biogeochemistry/Marine Systems, Kiel, Germany 4 granules to {104} rhombohedra can be reconstructed. The {104} faces begin Pontificia Universidad Católica de Valparaíso, Facultad de Recursos to appear at or close to their centers and spread towards the edges by the Naturales, Escuela de Ciencias del Mar, Valparaíso, Chile 5 progressive lateral addition of nanogranule lineations. The last rhombohedral Huinay Scientific Field Station, Puerto Montt, Chile 6 elements completed are the edges. Rhombohedra are usually incomplete, Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und because the grains are quickly overgrown by other neighbors during plate Meeresforschung, Bremerhaven, Germany 7 growth. TEM observations reveal that the nanostructure consists of Brock University, Department of Earth Sciences, Ontario, Canada crystalline domains, which are surrounded by amorphous areas. This is in agreement with AFM observations, which show the existence of two phases The shell of modern rhynchonellide and terebratulide is a hybrid with different contrast. All the above observations give important insight into composite where an extracellular biopolymer matrix is reinforced by calcite. the growth mechanisms of these calcite biocrystals. We propose that during Both material components are secreted by outer mantle epithelium (OME) the transformation of ACC into calcite, due to the force of crystallization, cells. organic molecules are expelled from the crystal lattice. Growth of the In order to understand mineral transport pathways for shell secretion and to crystalline domains takes place preferentially along the directions, which are assess differences in cellular activity during mineralization, we imaged with the strongest periodic bond chains (PBCs) in calcite. Accordingly, organic TEM and FE-SEM ultrastructural characteristics of outer mantle epithelium molecules are barely incorporated along these directions and become (OME) cells of juvenile and live Magellania venosa shells. Imaging was expelled sidewards. This implies that the formation of nanogranules and their carried out on embedded/etched, chemically fixed/decalcified and high- arrangement into lineations in barnacle calcite crystals is a pressure frozen/freeze-substituted shell pieces taken from the commissure, crystallographically-controlled phenomenon and cannot be explained by a central shell portions and from puncta. Imaging results are complemented particle-aggregation process, as previously shown for molluscan biogenic with morphometric evaluations of volume fractions of membrane-bound aragonite. organelles (Simonet Roda et al. 2019a, Simonet Roda et al. 2019b). At the commissure, the OME is multi-cell layered, while in central shell T 2 regions it is single-cell layered. OME cells form at the commissure oblique Towards understanding larval bivalve calcification extensions that, in cross-section, are round below the primary layer and flat mechanisms in a changing ocean underneath the fibres. At central shell regions, OME cells are considerably F. Melzner*1, K. Ramesh2, M. Clark3, G. Nehrke4 thinner in comparison to cells at the commissure. 1GEOMAR, Marine Ecology, Kiel, Germany When actively secreting shell carbonate extrapallial space is lacking as OME 2University of Gothenburg, Gothenburg, Sweden cells are in direct contact with the calcite of the forming fibres. Upon 3British Antarctic Survey, Cambridge, United Kingdom termination of secretion, OME cells attach via apical hemidesmosomes to 4AWI Bremerhaven, Bremerhaven, Germany extracellular matrix membranes that line the proximal surface of fibres and tonofilaments connect apical to basal hemidesmosomes. This stabilizes the contact of epithelium and the fibres and keeps the mantle in place. Individual Marine larval bivalves (mussels, ) rapidly secrete first shells within fibres are secreted by several cells at the same time. This requires less than one day, thereby precipitating a calcium carbonate mass that communication and tight cooperation of neighbouring OME cells for the corresponds to their own body mass. This remarkable synthesis effort coordinated secretion of organic membrane and mineral, explaining the high requires massive transfer of inorganic carbon and calcium, as well as proton abundance of gap junctions between cells. removal from the site of calcification. These transport mechanisms are There is not any observation in the cell ultrastructure in our study that can be unknown so far. Similarly, it is not clear, whether amorphous precursor taken as evidence for a vesicular transport based mineralization process. On phases are formed intracellularly, or whether carbonate formation is located the contrary, the absent or very narrow (in the range of nanometers) space in dedicated extracellular spaces at the organism-mineral interface. A better between the epithelium and the growing fibres, together with the absence of mechanistic characterization of shell formation processes is important, as it significant differences in the volume fraction of vesicles between secreting allows us to understand the high vulnerability of larvae to ongoing ocean and non-secreting regions of the OME, as well as the extreme reduction in acidification. cell thickness at sites of mineral secretion suggests, that in modern We use mussel larvae (Mytilus) as model and utilize a variety of Magellania venosa (and likely in all Rhynchonellida and Terebratulida approaches, ranging from in vivo confocal microscopy, in vivo forming the fibrous microstructure) mineral transport to the sites of confocal Raman microscopy, microelectrode recordings to transcriptomic mineralization occurs via ion transport mechanisms through the cell approaches to learn more about early shell mineralization and how it is membrane and not by transport of mineral by organelles such as vesicles. impacted by ocean acidification. Simonet Roda, M., Ziegler, A., Griesshaber, E., Yin, X., Rupp, U., Greiner, Using in vivo calcein pulse-chase and confocal microscopy, we can show that M., Henkel, D., Häussermann, V., Eisenhauer, A., Laudien, J., Schmahl, (1) it is unlikely that biomineral precursors are formed in large vesicles in W.W., Terebratulide brachiopod shell biomineralization by mantle epithelial cells. This finding is supported by in vivo confocal Raman microscopical cells, Journal of Structural Biology, analysis of first secreted shell components at the trochophore stage, which https://doi.org/10.1016/j.jsb.2019.05.002 (2) exclusively indicates presence of aragonite in the shell. While it is Simonet Roda, M., Griesshaber, E., Ziegler, A., Rupp, U., Yin, X., Henkel, possible that very small intracellular carbonate particles are produced D., Häussermann, V., Laudien, J., Brand, U., Eisenhauer, A., Checa, A.G., intracellularly, it is equally possible that mineral formation is an extracellular

4 Schmahl, W.W., 2019b. Calcite fibre formation in modern brachiopod shells. exploring the presence and the role of extracellular vesicles to stabilize ACC Sci. Rep. 9, 598. and to address it to the mineralization site.

T 4 Results How hepatopancreas cells of a terrestrial crustacean take up In a first approach, we used real time qRT-PCR to assess the expression of and release calcium after ingestion of mineral from the old vesicular target genes in several tissues. The results confirmed a high shed cuticle expression of vesicular target genes (edil3, anxa1, anxa2, pdcd6ip) in oviduct portions where mineralization takes place. In this study, we have also U. Rupp*1, A. Ziegler1 explored the role of EDIL3 and MFGE8 proteins in chicken shell at key 1Ulm University, Central Facility For Electron Microscopy, Ulm, Germany stages of shell mineralization, and confirmed they could bind Ca2+ and vesicles, thanks to an EGF-like calcium-binding domain and a F5/8C The hepatopancreas of isopods serves for food digestion, storage of lipids phospholipid-binding domain. It was therefore suggested that both proteins and carbohydrates, storage of essential metals as well as accumulation and could be involved in the vesicular transport of calcium. In a second approach, detoxification of xenobiotic metals. Metal is accumulated in lysosomal metal electronic microscopy coupled with elementary analysis was used to observe containing granules in the two major cell types (S and B) of the the uterine fluid collected during eggshell biomineralization. Data obtained hepatopancreas (Dallinger and Prosi, 1989). A recent µCT study on mineral highlighted the presence of extracellular vesicles (~ 300 nm) containing shifts in moulting Porcellio scaber has shown that mineral is present within calcium carbonate. Finally, Western Blot analysis confirmed the presence of the hepatopancreas lumen, however, only when the animal has ingested the EDIL3, a key vesicular protein in the purified vesicle fraction. old shed cuticle (exuviae) after moult (Ziegler et al., 2017). This suggests Conclusions uptake of mineral from the ingested exuviae by the hepatopancreas and The results of this study showed for the first time the involvement of recycling for mineralisation of the new cuticle. extracellular vesicles in the transport of calcium carbonate for the The aim of this study was i) to reveal if the cells of the hepatopancreas biomineralization of hen"s eggshell. We proposed a model of calcification contain mineral from the exuviae, ii) if the metal-containing granules using vesicles to stabilize ACC and explaining the fast deposition of the contribute to the uptake of calcium and its release for mineral recycling, and crystalline calcite oriented layer in the shell. The proteins described in this iii) to find pathways for mineral/calcium transport from the exuviae to the study will have to be explored as biological markers for a selection of chicken hemolymph. We have therefore investigated the mineral distributions within layers with improved mechanical properties. the cytosol and organelles of hepatopancreas S- and B-cells. We used P. scaber at the postmoult stage that have ingested their exuviae after T 6 the moult, those that have not ingested their exuviae as a negative control, STIM1 a calcium sensor promotes the assembly of an ECM and animals at the intermoult stage, 9-10 days after exuviae ingestion that that contains extracellular vesicles and factors that modulate have a fully mineralized cuticle. mineralization We used STEM, TEM multi-image acquisition, EDX and EFTEM for the A. George*1, Y. Chen1 analysis of the ultrastructure and mineral distribution in high pressure frozen 1University of Illinois at Chicago, Oral Biology, Chicago, United States and freeze substituted hepatopancreas tissue. Ultramicrotomy of resin embedded samples was performed using a piezo-driven oscillating diamond knife and propane-1,3-diol as floatation medium for the sections to minimize Introduction loss of mineral. Biomineralization is a dynamic process in which living organisms deposit The cryofixed samples reveal numerous extracellular vesicles (exosomes) mineral in the extracellular matrix. Bone and development requires and many multivesicular bodies containing proexosomes in both cell temporal and spatial deposition of calcium phosphate mineral. Several types. After high-pressure freezing and freeze-substitution, granules in proteins work in coordination and contribute to this tightly regulated process. sections of S as well as B-cells contain well-preserved mineral deposits. In STIM1 (Stromal interaction molecule 1) is one such protein that has been animals that have ingested the exuviae, we found a significantly higher recently identified to function in bone and enamel mineralization. The calcium concentration in the metal granules of S cells in comparison to STIM1 protein is a calcium sensor localized to the membrane of the control animals. In B cells the calcium concentration in lysosomal granules endoplasmic cells and is well recognized for its physiological role in the was much lower and independent of moulting stage or exuviae ingestion. We endoplasmic reticulum. We have demonstrated earlier that DMP1 observe intracellular seams of mineral along the microvilli and the lateral stimulation of preosteoblasts lead to calcium release from internal Ca2+ plasma membranes, consisting of phosphorus co-localizing with calcium and stores and this store depletion is sensed by the ER Ca 2+ sensor STIM1. occasionally with calcium and zinc in S-cells and with iron or calcium in B- Store-operated calcium entry is one of the major Ca 2+ influx mechanisms cells. Small granules composed of calcium and phosphorous occur also following store depletion in the ER. between cells and within the basal lamina. In intermoult animals, such Objectives mineral seams and granules are less abundant and absent in To demonstrate a role for STIM1 in dentin matrix mineralization. control postmoult animals that have not ingested their cuticle. Materials and Methods The results indicate that calcium uptake from the ingested exuviae takes In order to understand the function of STIM1 during dentin mineralization, place across the apical plasma membrane of the cells and that we overexpressed STIM1 in dental pulp stem cells (DPSCs) to generate p- lysosomal metal granules in S-cells accumulate calcium for mineralization EF1α-STIM1 in which EF1a promoter drives the expression of STIM1. Cell of the new cuticle. Transport into the haemolymph occurs in the form of lines were characterized for the overexpression and knock down of STIM1. calcium phosphate via the basolateral membrane and the basal lamina. Co- The transgenic cells along with the control were cultured for 7, 14 and 21 localization of zinc with calcium suggests similar pathways for the two days in growth and differentiation media and the morphology of the cells and elements. topology of the matrix were examined by Field Emission Scanning Electron Prosi F. and Dallinger R. 1988. Cell Biol. and Toxicol. 4: 81–96. Microscopy. Further, we examined matrix mineralization by Alizarin red and Ziegler A. Neues F. Janáček J. Beckmann F. Epple. M. 2017. Arthropod von Kossa staining. Gene expression analysis by RT-PCR was performed to Struct. Dev. 46: 63–76. identify cell differentiation in the presence and absence of STIM1. Supported by the DFG ZI 368/11-1. Immunofluoresence was performed to confirm the localization of osteogenic markers in the presence and absence of STIM1. Results: Transgenic cell T 5 lines overexpressing and knockdown of STIM1 was successfully generated Identification of extracellular vesicles involved in the as assessed by fold changes in STIM1 mRNA expression. FESEM results biomineralization of the hen eggshell demonstrate that STIM1 overexpressing cells released large amount of L. Stapane1, N. Le Roy1, J. Gautron*1 extracellular microvesicles and matrix mineralization. Interestingly, 1French National Institute of Agricultural Research (INRA), Bird Biology knockdown of STIM1 resulted in fewer microvesicles and less mineralized and Poultry, Nouzilly, France matrix. Alizarin and von Kossa staining demonstrate the differentiation of stem cells into odontogenic phenotype with STIM1 overexpression. Conclusion Question STIM1 is a crucial molecule in biomineralization as it influences release of The eggshell is a critical barrier against mechanical stresses and microbial large amounts of extracellular vesicles and formation of mineralized matrix. penetration. Its integrity is essential to maintain the hygienic quality of this This was impaired in the absence of STIM1. Downregulation of basic human food and to limit the number of downgraded eggs. In such a differentiation markers suggest that STIM1 promotes differentiation context, we are looking for eggshell strength specific markers in order to probably by mobilizing intracellular calcium ions. As Ca2+ functions as optimize egg quality. second messengers, therefore their role in cellular differentiation suggest that The eggshell is made of 95% mineral phase (calcium carbonate on calcite STIM1 can promote intracellular Ca 2+ oscillations and thus provide a signal form) and an organic matrix (3.5%) mostly containing proteins. Eggshell for activation of downstream and upstream effectors that promote formation arises from an extra-cellular biomineralization process, which differentiation and matrix mineralization. takes place in a fluid that contains eggshell precursors and involves a transient phase of amorphous calcium carbonate (ACC). This work aims at

5 Acknowledgements fundamental thermodynamic and kinetic principles that drive the self- NIH-NIDCR DE 11657 and the Brodie Endowment Fund. assembly of .

T 7 T 9 Intraperiostracal calcification in the - tuning in to the Morphogenesis in the mollusc Atrina vexillum - an epitome for aerials of Catillopecten ideal coarsening E. Harper*1, G. Kamenev2, F. Varela- Feria3, J. Taylor4, E. Glover4, A. D. Zöllner*1, I. Zlotnikov1 Checa5 1B CUBE - Center for Molecular Bioengineering, TU Dresden, Dresden, 1University of Cambridge, Earth Sciences, Cambridge, United Kingdom Germany 2Russian Academy of Science, 2. National Scientific Center of Marine Biology, Vladivostok, Russian Federation The microstructure of polycrystalline materials is closely linked to many 3University of Seville, Seville, Spain materials properties. This fact makes the phenomenon of the migration of 4The Natural History Museum, Life Sciences, London, United Kingdom individual grain boundaries between neighboring crystals and therewith the 5Uniiversity of Granada, 5. Department of Stratigraphy and Paleontology, coarsening of the whole grain structure a major focal point in classical Granada, Spain materials science. Out of the different growth modes observed in natural and

synthetic materials—such as rocks, metals and alloys—the case of ideal Most studies of molluscan biomineralization concentrate on deposition of coarsening resp. grain growth is given special attention. Even though the shell layers onto and below the periostracum. However, in the last decade basic assumption for ideal coarsening—which is homogeneity of the physical there has been an increased appreciation of intraperiostracal calcification – properties of the boundaries—seems highly unrealistic, many analytical as where biominerals are secreted within the periostracum - producing often well as numerical investigations focus on this case due to the reduction in intricate and elaborate structures. Thus far most of this intraperiostracal problem complexity. As a result, the large variety of analytical models were calcification has been reported in the Bivalvia, where it is widespread in the rarely found to describe the microstructural evolution of experimentally Anomalodesmata, Palaeoheterodonta, Pteriomorphia and Imparidentia. This measured polycrystalline grain networks. In the present work, it is shown that distribution hints that this is either a plesiomorphic character or that there is biomineralization of the prismatic architecture in the shell of the mollusc such a facility within the Bivalvia for such intraperiostracal calcification that Atrina vexillum can be described qualitatively and quantitatively by it has evolved polyphyletically in different situations. conventional thermodynamic, kinetic and topological considerations from In this study we present a detailed study of recently discovered classical materials science. Hence, a biogenic polycrystalline material is intraperiostracal calcification in a clade in which it has not be observed presented as an epitome for ideal coarsening. before. Members of the Propeamussiidae, the "glass scallops", are a denizens of the deeper part of the oceans, where they produce fragile thin shells and pursue an actively carnivorous lifestyle. Amongst them, Catillopecten T 10 natalyae from the abyssal plain (4,860-5,680 m depths) adjacent to the Kuril- The role of residual stresses in biomineral morphogenesis Kamchatka and Japan trenches (Pacific Ocean) produce an extraordinary revealed by 3D dark-field x-ray microscopy ornament of intricate processes with multiple projections, looking like tiny V. Schoeppler*1, P. Cook2, I. Zlotnikov1 aerials and antennae. They are interesting because of their intricacy and the 1TU Dresden, B CUBE - Center for Molecular Bioengineering, Dresden, fact that they are calcitic rather than the more typical aragonitic nature of Germany intraperiostracal . Using a combination of SEM and EBSD we 2University of Natural Resources and Life Sciences, Institute of Physics and show that that these intricate structures are produced entirely within the Materials Science, Vienna, Austria periostracum and must form in a way that allows growth in a progressively outwards direction (i.e. entirely different from normal secretion whereby Residual internal stresses occur in numerous synthetic, geological and growth occurs inwards from the surface of the shell). Each aerial is biogenic crystals having desirable or undesirable effects on materials crystallographically continuous with the underlying prism. The stem of each performance. Specifically, a number of recent studies have demonstrated the aerial is shown to be parallel to the c-axis of calcite and the typical three-fold significance of residual stresses in the mechanical functionality of a number bifurcations of the antennae correspond with the three short diagonals of the of biomineralized tissues. However, the role of these forces in biomineral calcite rhombohedra. There may be multiple aerials from a single prism in morphogenesis was never previously examined, mainly due to the lack of an the outer layer of the shell, and those of a single prisms show a remarkable appropriate characterization approach, which requires spatial and co-orientation, whilst those on adjacent prisms show a slightly different crystallographic characterization of the mineral on several length scales. orientation. We will propose a model for the formation of these intricate Most of the state-of-the-art methods are either surface techniques yielding aerials and also to compare these with other forms of intraperiostracal 2D information or allow only limited 3D analysis of very small sample calcification in the Bivalvia. The function of these fascinating structures volumes. remains unknown! In this work, we employed the recently developed technique—dark-field x- We acknowledge Russian Foundation for Basic Research (grant no. 19-04- ray microscopy—to study the relationship between residual stresses and 00281-a) and CGL2017-85118-P (Spanish Ministry of Economy, Industry crystallographic properties of biogenic calcite in the prismatic ultrastructure and Competitivity) in the bivalves Pinna nobilis and Pinctada nigra in 3D. This method, developed at the European Synchrotron Radiation Facility (ESRF), utilizes T 8 magnifying refractive lenses to map the intensity profile of hard x-rays The dynamics of structural mesoscale dislocations in nacre diffracted from crystalline materials with an angular resolution of 0.01° and M. Beliaev*1, I. Zlotnikov1 allows to analyze millimeter sized samples with a spatial resolution of 50 nm. 1Technische Universitaet Dresden, B CUBE - Center for Molecular Whereas the prisms in P. nobilis have an almost perfect single crystalline Bioengineering, Dresden, Germany character, the growing prisms in P. nigra gradually change their crystallographic orientation and split into sub-prismatic domains. Due to the Molluscan shells ultrastructures are a paradigm of complex hierarchical high angular resolution of the method, we were able to obtain unprecedented biocomposite structures formed in the course of extracellular detail on the mosaicity of prisms in the two organisms and to demonstrate a biomineralization. Morphogenesis of these ultrastructures is known to follow correlation between internal lattice strains and local crystallographic thermodynamically driven self-assembly processes in accordance with the properties of biogenic calcite in 3D. By comparing the experimental data principles of classical materials science. Sheet nacre ultrastructure is a highly from the two species, we not only shed a new light on the relationship regular and periodic architecture composed of thin organic membranes and between structure and texture during the formation of biomineralized tissues, aragonitic layers, which are made of flat mesocrystalline platelets. Similar to but also demonstrate the role of internal stresses in biomineral classical crystal growth, nacre deposition is accompanied by incorporation of morphogenesis. two-dimensional defects, such as dislocations and twinning, that are integral to its formation process. However, in contrast to a generic atomic-scale T 11 lattice, in sheet nacre, these defects occur on the mesoscale level. Stabilizing the shell as an inducible defense - morphological, Specifically, twinning in aragonite is known to be responsible for the shape microstructural and chemical shell-modifications in the of nacre platelets and structural dislocations in the layered assembly are key freshwater snail Physella acuta in response to different crayfish to its morphogenesis. In the present work, we employ synchrotron-based species nanotomographic imaging combined with machine learning post-processing H. Eck*1, A. Schenk1, V. Grün1, C. Laforsch1 techniques to visualize and understand the nature and the dynamics of 1University of Bayreuth, Animal Ecology I, Bayreuth, Germany mesoscale dislocations in the nacre of the bivalve Unio Pictorum in 3D. By drawing an analogy to processes in classical materials science, we shed light on the role of structural dislocations and their interaction in sheet nacre Predator induced morphological defenses are a widespread phenomenon in formation. This work is a step towards a deeper understanding of the phylum of mollusks. It has been previously demonstrated that mollusks respond to shell-crushing crustacean predators with changes in their shell,

6 including shell size, weight and thickness. These changes in shell T 13 morphology are often associated with an increased shell stability, i.e. shell In-Situ TEM of calcium carbonate mineralization in the presence crush resistance. However, there is still very limited understanding of the of L-aspartic acid mechanism, by which such an adaptive increase in mechanical stability is M. Longuinho1, N. Peña2, D. Ihiawakrim2, M. Farina*3, A. Rossi4, O. achieved, e.g. microstructural or chemical modifications of the shells. Ersen2 Moreover, it remains elusive whether there is a specificity of predator 1Federal University of Rio de Janeiro, Rio de Janeiro, Brazil kairomones (semiochemicals released by the predator that allows prey 2Institut de Physique et Chimie des Matériaux de Strasbourg, Strasbourg, organisms to detect the predator), i.e. if mollusks can differentiate between France closely related crustacean predators and adjust the expression of inducible 3Federal University of Rio de Janeiro, Rio de Janeiro, Brazil morphological defenses accordingly. Therefore, we studied if freshwater 4Brazilian Center for Physics Research, Rio de Janeiro, Brazil., Rio de snails express different inducible morphological defenses when exposed to Janeiro, Brazil different crayfish species. Furthermore, the chemical and microstructural modifications, leading to an increasing crush resistance in shells of predator- exposed snails were analyzed. In this study, we particularly focus on the Introduction inducible morphological defenses in the acute bladder snail Physella acuta Biomimetic syntheses are widely performed in the development of new in response to the marbled crayfish Procambarus virginalis and the signal materials for pharmaceutical purposes and tissue regeneration, besides crayfish Pacifastacus leniusculus. The snails were exposed to kairomones seeking to reproduce in vitro biomineralization processes. A better from the two crayfish species until they reached maturity. Subsequently, the understanding on how mineralization occurs in biological environment morphology, weight and stability was compared across treatments. The shells allows the synthesis of materials with special properties, which ensure the were then analyzed for predator induced changes in microstructure and reliability of the production. Among the biominerals, calcium carbonates chemical composition using a wide range of analytical methods including (CaCO3) are the most abundant, being present in primitive to complex scanning electron microscopy (SEM), Raman- and Fourier-transform organism like the spicules in calcareos and in the inner ear infrared (FTIR) spectroscopy, powder x-ray diffraction (PXRD), small-angle of vertebrates. Therefore, CaCO3 minerals are of great interest for x-ray scattering (SAXS) and thermogravimetric analysis (TGA). Our results fundamental studies as a model for nucleation, growth and crystallization of demonstrate that P. acuta responds to both predators with the expression of biogenic minerals. A great challenge in the biomineralization field is the inducible defenses. Compared to non-exposed individuals, crayfish exposed understanding of the initial stages of the process, since the formation of a snails characteristically show an increased shell size and weight, a narrower critical nucleus, what occurs in a short period of time and at the molecular aperture of the shell and an increased crush resistance of the shell. level. This makes difficult the direct observation of this phenomenon with Interestingly, individuals from both predator treatments do not differ routine analytical imaging techniques. To overcome this restriction, we used significantly from each other. Regarding chemical modifications, shells of in situ techniques that allowed the observation of crystallization in the crayfish exposed snails exhibit higher volumes of carotenoids, while there aqueous medium in real time, like morphological changes, crystallinity and are no quantitative differences in the other components of the organic or structural dynamic. Specifically, in situ Transmission Electron Microscopy inorganic fraction. The microstructure of the shell did not show pronounced (in situ-TEM) enables acquisition of images and videos at Angstrom differences between non-induced and crayfish induced snails. Our study resolution, as chemical and structural information during the development of demonstrates that P. acuta expresses inducible morphological defenses in mineral phases. response to two different crayfish species. The observation that the Objectives expression of the defensive traits did not differ between the two crayfish The present work aims to understand how organic molecules, specifically species provides first indications that there may exist a universal crayfish cue. aminoacids, influence CaCO3 biomineralization. As described in the literature, there are specific proteins directly related to the biomineralization T 12 which are capable of influencing the structure and kinetic of CaCO3 Mural mechanics and morphology of shells - crystallization due to polarity, electrophilicity and presence of sulfate or adaptation or artifact? phosphate groups. Material & methods R. Lemanis*1, D. Stier1, I. Zlotnikov1 The experiment was carried out by mixing CaCl2 and Na2CO3 solutions, 1Technische Universität Dresden, B CUBE – Center for Molecular with and without L-aspartic acid, and then the mixture was dropped in a chip Bioengineering, Dresden, Germany of the in situ sample holder (Protochips) and analyzed in a JEOL-2100F TEM. have exploited their biomineralized skeleton over the course of Results & conclusion hundreds of millions of years for the purposes of buoyancy, hydrodynamics, Preliminary results show a region more electron dense, probably with and protection against predation. Over this time, they have explored a huge accumulation of ions where crystalline nuclei are formed. However, most of disparity of ultrastructures and shell geometries that reflect constructional them undergoes dissolution, before reaching a critical size (70 to 120 nm) in and ecological boundaries. Testing the biomechanics of these structures the samples without addition of the amino acid. The dissolution may have allows us to begin to untangle the evolutionary history and ecology of these happened because of the higher energy of the system leading to instability or animals. Here, we aim to test the potential function of mural modifications because of the energy of the electron beam during the analysis. In the in coleoid cephalopods using comparative computational mechanics. The experiment containing L-aspartic acid, it was also possible to observe spiral cephalopod phragmocone is a hollow tube divided into chambers by crystals with calcite-like morphology and organic vesicular-like structures mineralized walls; the attachment zone between these walls and the shell is containing crystalline material. The existence of nanocrystals inside the mural zone. The only extant coleoid with a spiral phragmocone, Spirula vesicular-like structures suggests that the amino acid can accumulate ions spirula, is imaged using high resolution computed tomography and used as a inside these structures, inducing the precipitation of crystals. Such basis to construct a representative coleoid shell model. Mechanical data from phenomena are described in micron and millimeter scale using polymers and nanoindentation from the Spirula shell are used to inform finite element polypeptides as polymer induced liquid phase (PILP), however, never models to test how the presence of mural modifications affect stress and described for small molecules like aminoacids forming nanoscale crystals. strain distributions due to hydrostatic pressure. Indentation data reveals a Further studies will be carried out to better understand of this mechanism. significant difference between the moduli of the shell and septa, however not one large enough to arrest cracks through the region. Furthermore, the septa T 14 show a stiffness gradient whose origins are not currently understood. Shells Affimer-directed control over calcium carbonate polymorphs with and without the mural flap show no differences in their response to the I. Sandei*1, T. Gaule 2, F. Meldrum 1 applied pressure, while the rear, adapical flaps seem to redistribute peak 1University of Leeds , Chemistry , Leeds, United Kingdom stress values away from the attachment site and onto the stiffer shell wall. 2University of Leeds, Biological Sciences , Leeds, United Kingdom We are able to show that mural modifications have only a minor effect on the mechanics of the shell. The mural flap, a defining character of Decabrachia, seems to have no apparent mechanical function. This result challenges prior A key factor in biomineralization is the use of organic molecules to direct the hypotheses that proposed the mural flap as an adaptation to deeper water formation of inorganic materials including and magnetite. Being depths that facilitated the ecological separation between Decabrachia and able to emulate this strategy synthetically and use organic molecules to belemnites. control the formation of inorganic materials is therefore a long-standing goal in materials synthesis and crystallisation studies. Significant efforts have therefore been made to identify molecules that can produce inorganic crystals with well-defined size, structure and morphology. One approach that has received particular attention is the screening of libraries of polypeptides to identify individual molecules that are active in directing mineralisation. There, polypeptides are typically displayed on phage, and active individuals are selected based on multiple bio-panning rounds to identify strongly- binding individuals. These are anticipated to have a strong effect on crystallisation.

7 Here, we use a phage-display approach to identify proteins that can direct T 16 calcium carbonate formation. In contrast to short polypeptides, these have Synthesis of pyrite nanoparticles using the matrix protein from well-defined conformations that can be expected to be important in 10 the scaly-foot, Chrysomallon squamiferum controlling crystallisation. A 1.3∙10 library of Affimers was employed, T. Yamashita1, H. Matsuda1, Y. Suzuki2, N. Ahsan3, Y. Okada3, M. where these comprise a small protein scaffold of 81 amino acids. They are Suzuki*1 extremely stable and displays significant beta sheet structure together with 1The University of Tokyo, Departiment of Applied Biological Chemistry, one or two nine amino acid variable regions that display two variable loops. Bunkyo-ku, Tokyo, Japan These were displayed on a modified form of the M13 phage major coat 2The University of Tokyo, Deapartment of Earth and Planetary Science, protein (pVIII), and multiple bio-panning rounds were conducted to identify Bunkyo-ku, Tokyo, Japan individuals that bound strongly to calcite and aragonite at different pH 3The University of Tokyo, Research Center for Advanced Science and conditions. Technology, Meguro-ku, Tokyo, Japan From the initial phage library, 14 different proteins were selected and purified. The ones that strongly bound to calcite are particularly rich in basic and nonpolar amino acids in both loops. At pH 8.5 there is a prevalence of Introduction negative charges (as the alpha carboxylic groups are mainly deprotonated) Chrysomallon squamiferum (scaly-foot) is a deep-sea snail which was with a great presence of histidine (H) and arginine (R) residues in Loop 1. discovered near the hydrothermal vent of the Central Indian Ocean Ridge For aragonite, we found a significant number of nonpolar amino acids, with "Kairei Field". This snail has scaly structures mineralized with iron sulfide fewer basic residues than calcite and the appearance of aspartate (D) and nanoparticles (pyrite, FeS2) on its foot, and the shell is covered with an iron glutamate (E) residues in one of the two loops. Moreover, tryptophan (W) is sulfide layer on the calcium carbonate layer. Pyrite nanoparticles have one of the main residues present, together with polar amino acids carrying - superior photovoltaic properties to be applied to photovoltaic power OH groups in the side chain. generation in the solar panel. However, the methods of its inexpensive and Experiments are currently underway to explore the function of these green chemical synthesis have not been established. Therefore, new proteins in directing calcium carbonate formation in bulk solution at industrial applications using the formation mechanism of pyrite nanoparticles different pH values, and in the presence of magnesium ions. The proteins in the scaly-foot are required. In this research, in order to identify the will also be tethered to surfaces in the form of a monolayer, and their substances that have key factors to synthesize the sulfide nanoparticles in influence on calcium carbonate precipitation determined. This will enable vitro, we tried to extract the organic molecules that bind to iron in the scaly- us to investigate whether the proteins behave differently in solution and foot, because the iron-binding organic molecules may have a function to when located on a solid substrate. prevent the aggregation of particles. Results and Discussion T 15 In this study, we analyzed the organic molecules extracted from the scaly Synthetic, prismatic-type CaCO3 films via seeded structure and the shell in the scaly foot and searched for substances related to mineralization the production of iron sulfide nanoparticles by interaction with iron. Pyrite in the scaly structure was dissolved by reducing with zinc and trivalent Y. Jiang*1 chromium under anaerobic conditions. We used HPLC post column chelator 1Xiamen University, Xiamen University, China method to identify the iron binding organic molecules. This result revealed

that large amount of low molecular weight organic matters having iron Introduction binding ability existed in the scaly structure. This low molecular weight Biominerals, by taking advantage of their hierarchical architecture, reconcile organic molecule was purified using cation exchange resin and reverse multiple functions which are otherwise contradictory in their synthetic phase-HPLC. We tried to determine its chemical structure using mass counterparts. From the synthetic point of view, the delivery of hierarchical spectrometry and NMR. architecture can be ascribed to the active roles of (multiple) soft matter, On the other hand, 1 M acetic acid was used to dissolve the calcium carbonate which turns the mineralization proceeding in organism into self-organization layer of the outer shell. Then, the insoluble iron sulfide layer in acetic acid processes. The control of a self-organization process for the delivery of life- was extracted using SDS-DTT. A specific protein band was detected from feathered synthetic minerals nonetheless remains technically challenging in the iron sulfide layer. LC-MS/MS analyses revealed that this band showed biomimetic mineralization. an amino sequence of protein. Heme is consisting of tetrapyrrole ring Objectives that can make the complex with iron in vivo. The primary focus of our work is to design specific microenvironment based The heme protein may have some roles for the formation of nano pyrite in on the organization of multiple types of soft matter for the delivery of vitro. We try to use the heme protein to synthesize an iron sulfide prismatic-type minerals with distinct architecture and remarkable mechanical nanoparticles using various conditions. performance. "Patients & methods" or "Materials & methods" We introduce seeded mineralization for the delivery of prismatic-type T 17 CaCO3 films. Oriented crystallization of single crystalline strontium sulfate Results in marine Acantharea The method starts with the deposition of granular CaCO3–polyacrylic acid V. Merk*1,2, P. Smeets2, J. Walker2, D. Joester2 hybrid films on polymer substrates like chitosan, silk fibroin, or polyvinyl 1Florida Atlantic University, Department of Chemistry & Biochemistry; alcohol. Next, an overgrowth procedure leads to prismatic-type CaCO3 Department of Ocean & Mechanical Engineering, Boca Raton, United overlayers. Our study shows that the presence of different additives can have States a tremendous impact on their morphological outcomes, while the selection 2Northwestern University, Materials Science and Engineering, Evanston, of polymer substrates determines the polymorphic form of the granular seed United States layer. In our very recent work, we study the impact of hydrogels on the structural information of the prismatic overlayers, though hydrogels Introduction themselves are not involved in the delivery of biogenic prismatic minerals. Simple organisms, such as sea urchins, calcerous sponges, coccolithophores, This prismatic CaCO3 films can be used for drug delivery applications. or magnetotactic bacteria, pursue highly sophisticated strategies for growing Conclusion single-crystalline skeletal units. Acantharia radiolarians present yet another To summarize, the synergistic effect of multiple types of soft matter and the prime example of single crystal engineering in nature. The marine protists proper use of biomimetic mineralization methods can create favored build intricate star-shaped endoskeletons from smoothly curved strontium microenvironments for the delivery of prismatic-type CaCO3 films in self- sulfate (SrSO4) spicules, whose long axes coincide with the crystallographic organization processes. These CaCO3 films exhibit comparable mechanical a-axis. Although Acantharia occur in the ocean's zooplankton from tropical performance to their biogenic counterparts. The rational design of to polar zones, the underlying crystallization mechanism remains virtually biomimetic mineralization systems helps for the mechanistic understanding unexplored. of biomineralization, and meanwhile, provides a practical tool to design Objectives proper materials for biomedical applications. In this paper, we anticipate addressing the question how crystal texture is controlled in a biological environment. The poor stability of Acantharia in culture renders time-resolved observations of the spiculogenesis impossible. To infer the crystallization mechanism of biogenic celestite, we analyzed the atomic- and nanostructure as well as the distribution of intercalated biomolecules on various length scales. Materials and Methods We investigated the atomic and nanostructure of Acantharian skeletons from the Solomon Islands using a combination of X-ray diffraction, Raman spectroscopy, electron-optical imaging and synchrotron-based techniques. The sample preparation for transmission electron microscopy (TEM)

8 included targeted focused-ion beam (FIB) lift-outs from Acantharian Life cell imaging: The pH sensitive dye BCECF_AM and BCECF-FA 10KD spicules, mostly along primary crystallographic axes. Synchrotron X-ray dextran were used to monitor intracellular as well as vesicular pH, fluorescence mapping and Sulfur K-edge XANES spectra were obtained at respectively. For cellular pH recordings the ectoderm was removed and sector 13-ID-E at the Advanced Photon Source (APS), Argonne National ratiometric pH recordings were performed. Laboratory. Scanning wide-angle X-ray scattering data were acquired at the Immunocytochemistry and westernblot analyses: Localization and P03 endstation, PETRA III at DESY. quantification of PMC specific acid-base transporters was performed using Results species-specific custom made antibodies. Acantharia grow highly ordered SrSO4 single crystals, whose crystal RT-qPCR and whole mount in situ hybridization: Expression levels of PMC structure is almost indistinguishable from geological and synthetic specific acid-base transporters were measured by qPCR and normalized to counterparts. As markers for intracrystalline organic matter, sulfur-bearing the housekeeping gene SpZ12. Whole mount in-situ hybridization was amino acids and di-sulfides were detected across the protist"s skeleton by performed to localize transcripts in the sea urchin larva. microbeam X-ray absorption spectroscopy (XANES) at the sulfur K-edge. Microinjection of GFP constructs and Morpholino knock-down: Micro- Highly anisotropic small-angle X-ray scattering signals correspond to injections were performed to insert morpholinos and BAC-GFP constructs nanoscale electron density variations parallel to the crystallographic a-axis, into the fertilized egg (one-cell stage). which are consistent with buried interphases in the SrSO4 biomineral. Results Similarly, scanning TEM Z-contrast imaging showed nanodomains with a Here we demonstrate the SLC4 HCO3- transporter family member lower atomic number around the spicule center, which do not seem to SpSlc4a10 to be critically involved in the formation of an elaborate calcitic correspond to light metals (e.g. Na, K, Ca, etc.). In the diffraction contrast endoskeleton. SpSlc4a10 is specifically expressed by calcifying primary image from the spicule cross-section, we observed concentric layers mesenchyme cells with peak expression during de novo formation of the reflective of fluctuating growth. skeleton. Knock-down of SpSlc4a10 led to pH regulatory defects Conclusions accompanied by decreased calcification rates and skeleton deformations. Re- Taken together, our findings suggest that biomolecules interact with specific calcification experiments demonstrated an increase in PMC pHi and planes and subsequently become introduced into the growing crystal. An substantial elevations in intracellular [HCO3-] during skeleton rebuild driven occlusion of specific biomolecules may guide the crystallographic by SpSLc4a10. Despite overall reductions in proton export capacities of orientation of primary seed crystals, indicating genetic regulation of the PMCs in this phase, large filopodial cells containing acidic vesicles connect crystal growth process. Furthermore, Acantharia present an excellent source to the PMC syncytium and locally elevate pH regulatory capacities. These of bio-inspiration for alkali earth sulfates with tailored mechanical, cells do not express ALX1 a specific marker for skeletogenic mesenchyme electronic, and optical properties. cells. The process of skeleton rebuild and vesicular acidification in PMC associated cells is sensitive to the V-Type-ATPase inhibitor bafilomycin. T 18 Conclusion Influence of stress on biogenic calcite growth Intracellular accumulation of bicarbonate is a fundamental mechanism of J. Colombani*1, B. Zareeipolgardani2, A. Piednoir1 calcification in PMCs of the sea urchin embryo to develop and rebuild the 1Université Claude Bernard Lyon 1, Institut Lumière Matière, calcitic endoskeleton. We propose that during this phase PMCs shift from a Villeurbanne, France NHE-based proton export mechanism to a vesicular storage of protons 2Université Grenoble Alpes, ISTerre, Grenoble, France generated through extensive precipitation of CaCO3. Non-PMC cells associated to the skeletogenic syncytium potentially play a critical role in storage and removal of protons during extreme calcification efforts. These Calcite, the most widespread crystalline form of calcium carbonate, is results highlight the importance to better understand pH regulatory processes ubiquitous both in nature and in the industry. In particular, its precipitation in calcifying systems including cell-cell interactions and acid-base transport by living organisms under the form of shells is the major CO2 sink inside the on the cellular and sub-cellular level. ocean, and it is used in multiple industrial sectors (ordinary cement manufacturing, paper bleaching, toothpastes, etc.). Therefore the knowledge T 20 of its mechanisms of growth, both organic and inorganic, are of foremost The formation of fenestrations in Eucidaris tribuloides importance, in the modelling of the carbon cycle, in the simulation of oil reservoirs, or as a promising new route for biomimetic materials engineering. embryonic spicules J. Walker*1, B. Moreno1, D. Joester1 One parameter, although almost always present during calcite growth, has 1 not been considered in most studies. When calcite grows from nuclei, they Northwestern University, Materials Science and Engineering, Evanston, eventually enter into contact, and stresses develop at their interfaces, United States particularly if the material grows in a confined environment, for example inside a living cell. So far, we have no idea on how this stress modifies the Skeletal patterning in sea urchin embryos is an important model system for growth kinetics, or the morphology of the grown surface. To address this investigating biomineralization of calcium carbonate. Harnessing a tightly issue, we have used an atomic force microscope (AFM), both to imply a local controlled growth mechanism, the organism is able to direct the formation of stress on a growing calcite surface, and to image it. We have found that the single crystals that have complex curved and branched morphologies with stress has a double influence: it slows down significantly the growth, and precise crystallographic directions. These single crystals of calcite, known as induces a change of the type of growing phase. Calcite biomineralization spicules, begin growing from a seed rhombohedral calcite crystal. The proceeds in presence of organic materials, among which amino acids are deposition of these spicules and patterning of the skeletal structure is widespread. Therefore we have investigated the influence of pentaglycine on controlled at the local level by the action of primary mesenchyme cells the growth kinetics. This amino acid shows the striking ability to cancel the (PMCs). They fuse to create a syncytium, or privileged space, in which the phase transition induced by the applied force. mineral is deposited and grows. The pattern of the skeleton is species specific, and PMCs have been shown to be able to autonomously direct T 19 skeletal design including the formation of features such as fenestrations. pH regulation in calcifying primary mesenchyme cells of the These are regularly spaced holes within the single-crystalline central rod that sea urchin larva is normal to the spicule direction, a property that is thought to increase the M. Hu*1 stiffness-to-weight ratios and fracture resistance of the structure. Replication 1University of Kiel, Institute of Physiology, Kiel, Germany of this ability to produce consistent periodic features at multiple length scales is vitally important for the creation of synthetic materials with specific and tunable properties. Question In this work we investigated the formation of fenestrations in the embryonic The sea urchin embryo develops an elaborate calcitic endoskeleton that has spicules of Eucidaris tribuloides, a tropical sea urchin, and the effect their been used by biologists to study the mechanisms of biomineralization. presence has on crystallographic and morphological features of the spicule. Amorphous calcium carbonate is formed in intracellular compartments and Spicule formation in both embryos of E. tribuloides and S. purpuratus were exocytosed into the syncycial cable formed by primary mesenchyme cells investigated. Embryos from each species were separately grown in culture. (PMCs). For intracellular precipitation of CaCO3, PMCs require HCO3- Spicules were extracted from the embryonic cultures and examined by SEM /CO32- concentrating as well as proton export mechanisms to promote at Northwestern University Atomic and Nanoscale Experimental Centre calcification. These processes are of fundamental importance in biological (NUANCE). The identified features were then further investigated by calcification, but remain relatively unexplored even for mammalian systems. synchrotron white beam X-ray diffraction mapping using a 250 nm beam Material & Methods size, which was obtained at beamline 34-ID-E at the Advanced Photon Larval cultures: For our experiments we used the purple sea urchin Source, Argonne National Laboratory. (Stongylocentrotus purpuratus) and larval cultures were maintained at 15°C SEM imaging of the extracted spicules at different time points allowed a in natural seawater for further experiments. detailed visualization of the morphology of the fenestrations as the spicule Recalcification assay: Skeletons of early pluteus larvae (3dpf) were develops. Investigation of an individual spicule using white beam X-ray dissolved in MES buffered natural seawater adjusted to pH 6.0 for 12 h. After diffraction mapping enabled us to correlate the fenestrations to changes of transfer to natural seawater (pH 8.1) larvae re-calcified their skeletons within direction of the lattice planes within the crystal. Using a beam size of 250 four days.

9 nm it was possible to investigate these microscale features and observe Key components of the genetic biomineralization toolkit were identified, changes to the (001) plane relating to the location in the spicule. Although including carbonic anhydrases, bicarbonate transporters and secreted spicule there was a high homogeneity in plane direction as would be expected for a matrix proteins. We observed changes in the spatial and temporal expression single crystal, small changes (>0.1 degrees) in rotation were identified in of these genes during the spicule formation process. Some matrix proteins regions surrounding a fenestration. are only produced by sclerocytes that form spicules of a specific shape. White beam X-ray diffraction mapping can be used to correlatively with Our results highlight the essential role of genetic control over the electron microscopy to demonstrate local changes in lattice planes in and biomineralization process even in as simple structures as the calcite spicules around micron-sized features. It can efficiently provide detailed of calcareous sponges. Comparative analyses with multiple species will help crystallographic information over a larger scale than other techniques such to reveal the genetic key-innovations obtained by the last common ancestor as TEM, which can narrow down the area of interest for investigation using of these class. higher resolution but more labour intensive techniques. We have used it to investigate lattice plane changes related to the formation of fenestration in T 23 the growing spicule of a sea urchin embryo. Crystallographic misorientation between spicules in the skeleton of the calcareous sponge sycettusa hastifera T 21 L. Souza Coelho1, M. Klautau2, M. Farina3, A. Rossi*1 Effects of seawater Mg2+/Ca2+ ratio and diet on the 1Brazilian Center for Research in Physics, Rio de Janeiro, Brazil biomineralization and growth of sea urchins - applicability of 2Federal University of Rio de Janeiro, Biology, Rio de Janeiro, Brazil echinoderms in paleoenvironmental reconstructions 3Federal University of Rio de Janeiro, Biomedical Science, Rio de Janeiro, D. Kołbuk*1, P. Dubois2, S. Di Giglio2, S. M’Zoudi2, J. Stolarski1, P. Brazil Gorzelak1 1Polish Academy of Sciences, Institute of Paleobiology, Warsaw, Poland 2 Introduction Université Libre de Bruxelles, Faculté des Sciences, Brussels, Belgium The skeleton of calcareous sponges (phylum Porifera, class Calcarea) is formed by Mg-calcite spicules ranging from approximately 10 to 2000 μm in It has been suggested that the skeletal Mg/Ca ratio of well-preserved length and usually composed of two, three or four (rarely five) conics rays calcifying marine invertebrates, including echinoderms, was predominantly joined at the base [1,2]. One individual spicule behaves as a monocrystal affected by the changes of Mg2+/Ca2+ ratio in the ancient seawater, which has (trigonal system) with only few degrees of misorientation (<1°) [1,3]. The varied between ~1.0 to 5.2 throughout the Phanerozoic. However, it has been species Sycettusa hastifera belong to the subclass Calcaronea. In this also demonstrated that the chemical composition of Recent and fossil subclass the unpaired actine of tri and tetractine spicules is elongated echinoderms can be modified by a number of other factors, such as approximately in the [211] direction. The crystallographic direction of the temperature, salinity, metabolic processes, diet, and diagenesis. In this work, actines are conserved through the evolution of the classes. In the class we addressed the problem of diet vs. seawater chemistry impact on skeletal Calcinea, the actines are elongated in the {210} directions. The spicules Mg/Ca ratio, in experimental conditions. Two phylogenetically distant normally have a specific position in the body of the sponge (cormus) and are echinoid species, Psammechinus miliaris and Prionocidaris baculosa, were used as a taxonomic character. S. hastifera has triactine and tetractine cultured in low Mg2+/Ca2+ (~2.5 and 1.5) artificial seawater, prepared by spicules in the cortical, subcortical, subatrial and atrial regions of the lowering the Mg2+ and increasing the Ca2+ concentrations. Before the sea choanosome of the sponge. urchins were exposed to low Mg2+/Ca2+ conditions, they were tagged with Objectives manganese in order to identify newly-formed skeleton areas under In this work, the crystallographic misorientations between spicules in the cathodoluminescence (CL) and calibrate their growth rates. After tagging, cortex, subcortical, subatrial and atrial regions were identified in different the echinoids were incubated for 21 days in seawater with different transversal sections of the tubular structure of the sponge (base, middle and Mg2+/Ca2+ ratios (~5.2, ~2.5, ~1.5 [mol/mol]) and fed with diets containing top of the sponge tube) and in different area of each section of the tube. The different amounts of magnesium (~0.3 wt% or ~20 wt%). Mg/Ca ratios in aim of this study is to relate the misorientation between spicules with the the newly formed skeleton in different types of echinoid ossicles (test plates, process of growth of the sponge skeleton and the resistance proprieties of the spines, teeth, and demipyramids) were determined using electron aquiferous system tubes. microprobe. Mg/Ca ratios in all ossicle types of both species decreased Materials and Methods proportionally with decreasing Mg2+/Ca2+ ratio of seawater, in which they Samples from S. hastifera were collected in Arraial do Cabo city, Rio de were growing. However, echinoids fed with magnesium-enriched diet Janeiro, Brazil. The sponges were dehydrated with increasing concentrations typically displayed higher skeletal Mg/Ca ratios. Growth rates in some of ethanol and infiltrated with epoxy resin. The sponge embedded in resin ossicles (test plates and demipyramids) significantly decreased with the was cut transversally to the tube direction using a diamond saw. Transversal reduction of ambient Mg2+/Ca2+ ratio. These results underscore the sections of the sponge were polished with alumina 1μm and then coated with importance of diet in modulating Mg/Ca ratio of echinoderm skeleton and a thin carbon layer. Electron Backscatter Diffraction (EBSD) were performed suggest that caution should be exercised when using the skeletal Mg/Ca ratio in a Scanning Electron Microscope Jeol 7100FT equipped with an Oxford of fossil echinoderms as a direct paleoseawater Mg2+/Ca2+ proxy. [This EBSD detector. Kikuchi line patterns were recorded using Aztec and research was funded by the National Science Centre (NCN) grant no. Channel 5 softwares. Each transversal section was divided into four areas (1, 2016/23/B/ST10/00990]. 2, 3 and 4) from where cortical, subcortical, subatrial and atrial spicules were studied. Stereographic projections were generated to study misorientation T 22 between spicules. Spicule types were identified using an inverted optical Genetic control over biomineralization in calcareous sponges microscope. O. Voigt*1, B. Fradusco1 Results 1LMU Munich, Department of Earth- and Environmental Sciences, The subcortical spicules were the less oriented spicule type from S. hastifera. München, Germany The absence of a preferential orientation may be related with the existence of pseudotriactine spicules which present similar morphology but different crystallographic orientation compared to a conventional triactine. All spicule The ability to produce biominerals has been a key innovation in animal types in the middle section of the sponge tube were more well oriented than evolution and allows animals to generate as essential structures as shells, in the base of the sponge. This result is related with the process of growth of teeth and skeletons. The genetic mechanisms underlying the the sponge tube. The transversal section of the sponge tube had an oval biomineralization process and their evolution are only poorly understood. It symmetry. The areas of the section superposed to the larger diameter seems that biomineralization evolved serval times in different animal linages, presented spicules more well oriented compared to the areas in the smaller but still utilize similar general principles and genetic pre-adaptations. diameter of the tube. The same spicule type may have different orientation in We study biomineralization in the class of calcareous sponges, which belong different area. to the oldest animal phylum. These sponges gained the ability to produce Conclusion calcite spicules of different shapes in contrast to other sponge classes, which The spicule orientation in the cormus of S. hastifera present more or less have siliceous spicules. They provide a simple system of misorientation depending on the transversal section of the sponge tube (base, biomineralization: Calcareous sponges produce numerous spicules in a short middle or top), the spicule type and the area of the transversal section. time (hours to days), and each is formed by only a few (e.g. two to seven) specialized cells. Therefore, we aimed to identify the genetic mechanisms that control the biomineralization system in calcareous sponges to understand general principles of biomineralization. We applied a combination of genomics, transcriptomics, proteomics and RNA in situ hybridization to identify key biomineralization genes of calcareous sponges and to obtain information about the spatial and temporal expression during the spicule formation process.

10 T 24 formation is very limited. In this study, we investigated the microstructure of Paradoxical mechanical properties of sponge spicules biosilicas from rice plants (Oryza sativa) by using scanning electron J. Werckmann*1, E. Bouzi2, E. Brodu2, M. Longuinho1, A. Porto Careiro3, microscopy (SEM) and Cryo-FIB SEM. In addition, we extracted organic D. Ihiawakrim4, A. Rossi1, M. Klautau5, M. Farina5 matrices from biosilicas and characterized by LC-MS/MS analysis. SEM 1Brazilian Center of Physic Researsh, COMAN, Rio de Janeiro, Brazil analyses revealed that the silicas of rice plants are composed of nanoparticles 2University, Metz, France that are several dozen nm in diameter, similar to silica and to siliceous 3University, Marseille, France spicules of sponge. We extracted a 11 kDa protein from silica from the leaf 4CNRS, Strasbourg, France blades and a 140 kDa protein was identified as the only organic component 5University Fédérale, Rio de Janeiro, Brazil contained in rice husk"s silica. Protein Database searches revealed that the 11 kDa proteins belong to plant defense proteins known as LTPs, and the 140 kDa protein has an amino acid sequence similar to glucanase. Both proteins Our study focuses on calcareous sponges. Their three-dimensional structure are cationic proteins present in abundance in higher plants and are known to is ensured by the entanglement of spicules within their soft cylindrical body, play important roles in resistance to abiotic and biotic environment stress. composed mainly of collagen. These spicules have the distinction of being Given their cationic nature, we hypothesize that they are involved in biogenic single crystals consisting of magnesium calcite. One of their regulating silica formation in the rice leaves and husks. Therefore, we are paradoxical properties is the conchoïdal fracture caused by mechanical stress, currently investigating their function in controlling silica biomineralization. whereas mineral calcite has fractures determined by {104} cleavage planes. The purpose of our work is to highlight the factors that are behind this T 26 property. Thus we have implemented several characterization techniques The biology of silica deposition in leaf silica cell - a first full associated to transmission electron microscopy. Specimens of the species Paraleucilla magna were immersed in a 2.5% sodium hypochlorite solution description of plant bio-mineralization process R. Elbaum*1, S. Kumar2 to completely remove the adhered organic tissue. The spicules were rinsed 1 with distilled water followed by 100% ethanol then air dried. Thin spicule Hebrew University of Jerusalem, Institute of Plant Sciences, Rehovot, Israel cross section were obtained by Focused Ion Beam (FIB). Briefly, the 2 spicules were dispersed on a silicon substrate and metalized with a 200 nm Weizmann Institute of Science, Plant & environmental sciences, Rehovot, gold layer for surface protection and electrical conductivity. To improve the Israel protection, a 1 µ Pt layer was in situ deposited. To avoid ion damages, progressive thinning process was implemented. To complete the process, a Biomineralization in animals is a very common strategy creating skeletal current of 30 pA at an energy of 5 keV was used to clean and reduce the elements. In plants, however, body design is flexible, and each organ is self- amorphous damaged layer produced during the former steps. Titan supported by cell walls. Plant minerals are typically utilized in defense transmission electron microscope operating at 300keV was used. The mechanisms. Silica is a very abundant mineral in plants that may constitute electron diffraction obtained on sections extracted on the same spicule, 10% of the dry weight of certain tissues. Most astonishingly, very little is whatever their orientation, are interpreted as originating a single crystal. In known about the control over silica formation in plants. We discovered the addition, the high resolution images of the external spicule-membrane mechanism for silica deposition in epidermal silica cells of sorghum interface show the presence of nanocluster attached or close to the surface of (Sorghum bicolor) leaves. Silica cell are almost completely filled with silica the spicule. Their FFT can be interpreted as coming from calcite when mature. We showed that silicification is confined to elongating leaves, nanocrystals. The dark field image obtained when the microscope operates in a well-defined active silicification zone (ASZ). The mineralization in Scanning Transmission Electron Microscopy (STEM) mode reveals the initiates in live cells. Silica starts to form at the cell"s periphery, adjacent to existence of significant porosity. Transmission Kikuchi diffraction (TKD) the primary cell wall. In contrast to other biomineralization processes, the was implemented in a scanning microscope working at 30keV for mapping mineral is not formed inside vesicles. Instead, the silica cell protoplasts the mosaicity. Before observation a thin layer of platinum was deposited to synthesize and secretes a unique protein that accelerates silica precipitation. enhance the electrical conductivity of the sample and to protect them to the The protein is packed in vesicles and is secreted to the extracellular space, beam damages. Spatial resolution was de of 10nm (pixel size) and the angular which is saturated with silicic acid. The silicic acid precipitates into silica, resolution reached about 0.01°. Thanks to this angular precision, the maps of creating a secondary wall inward to the primary cell wall, and restricting the disorientation obtained show that the domains of the mosaic are smaller than protoplast"s space. The thickening silica wall fills up the cell volume within or equal to 10 nm. Based on our experimental results and bibliography (1, 2) a few hours. Alongside this fast process, the silica cell goes through we conclude that the growth of spicules is a discontinuous process that leads programmed cell death, the cell contents are possibly evacuated to to the formation of a porous structure and that it is controlled by proteins that neighboring cells. This is a first description of silica deposition in plants. catalyze the formation of nanocrystals that are deposited almost epitaxially on the spicule in formation, resulting in the formation of a mosaic of very T 27 low disorientation (0.01°), undetectable by electron diffraction. During Imaging and analysis of internal silicon pools in diatoms growth a small amount of protein is included by chance in the spicules. These A. Gal*1 three factors: porosity, mosaicity and inclusion of proteins increase the 1Weizmann Institute of Science, Plant and Environmental Sciences, resistance of calcareous spicules to the mechanical stress entailing the Rehovot, Israel conchoidal fracture. This ensures a better resistance of the sponges to the mechanical stresses due to the ebb and flow of the sea and on the other hand a better resistance to the action of predators. Diatoms are abundant unicellular algae that cover themselves with a cell wall  1) Rossi et al. Acta Biomaterialia (2014) made of silica. The silicification process is under strict biological control, to the extent that the delicate nanoscale architecture and fine ornaments are  2) L. Freeman et al. Angewandte Chemie (2010) species-specific. The polymerization of silica from its soluble building blocks is thought to take place inside a specialized organelle that is T 25 responsible for precipitating the mineral phase and controlling its Rice plant biomineralization- Ultrastructure of biosilica, and morphology. Our understanding of the inorganic precipitation mechanism of defense protein diatom silica is still rudimentary, as conventional imaging and analytical N. Ozaki*1,2, R. Abiko1, K. Tsuji1, T. Ishida1, M. Suzuki3, F. Nudelman2 tools are inadequate to resolve the native-state structural information and 1Akita Prefectural University, Department of Biotechnology, Faculty of chemistry related to the silicification process. For example, it is unclear what Bio-resource Sciences, Akita, Japan are the mechanisms of silicon uptake and concentration from the 2The University of Edinburgh, School of Chemistry, Edinburgh, United environment, and what are the conditions inside the silicifying organelle that Kingdom give rise to the precise morphology. 3The University of Tokyo, Graduate School of Agricultural and Life We use a suite of cryo electron microscopy techniques in order to extract Sciences , Tokyo, Japan intracellular structural and chemical information with nanoscale resolution. 3D serial imaging of whole Thalassiosira pseudonana cells, using cryo focused-ion-beam scanning electron microscopy (cryoFIB-SEM), and Biologically formed amorphous silica (biosilica) are widely found in energy dispersive spectroscopy (EDS), shows that the cells maintain high bacteria, diatoms, marine sponges, terrestrial higher plants, some of which intracellular concentration of Si throughout the cell cycle. In addition, have been well characterized. Macromolecules in biominerals are known to scanning transmission electron tomography (STEM tomography), enables us control mineralization. Biosilicas in diatom and marine sponge, are formed to visualize in 3D the ultrastructure of the silica deposition vesicle along the under mild condition and neutral pH using unique macromolecules, such as silicification process. In the species Chaetoceros tenuissimus we followed silaffin, glassin, and long chain polyamines. The typical example of silicon the formation of long silica needles with cryo electron tomography (cryoET) accumulating higher plants, gramineous plants (rice plants), produces a large and discovered a new mechanism for the deposition of silica. Overall, our amount of biosilica in their leaf blades and rice husks. Although biosilicas direct approach to study the formation of diatom silica in situ by means of deposited in rice plants play various important roles, such as enhancing advanced microscopy tools is yielding detailed understanding of the cellular mechanical strength, improving disease resistance and photosynthetic controls that shape the silicification process in diatoms. activity, information on the molecular mechanisms involved in the biosilica

11 T 28 Conclusion Control of biosilica morphology and mechanical performance The stylus can bridge the gap of the vastly different mechanical properties of by the conserved diatom gene Silicanin-1 the hard head and the radula membrane by varying the amount of deposition S. Görlich1, D. Pawolski1, I. Zlotnikov1, N. Kröger*1 of the biomineral santabarabaraite. The discovered mineralized component, 1TU Dresden, B CUBE, Dresden, Germany the stylus of the , is a biological nanocomposite material that exhibits interweaved and co-aligned nanosized fibers with nanosized amorphous iron phosphate particles in a string-of-pearls manner. The Diatoms represent a large group of unicellular, eukaryotic microalgae that combination of iron phosphate and chitin has impressive mechanical are most well known for their ability to produce cell walls made of properties over a wide tunable range which surpass many man-made nanopatterned porous biosilica. The species-specifically patterned cell walls materials that require energy intensive manufacturing such as light-metal are paradigms for biological mineral morphogenesis and the evolution of alloys. Deliberate selection of synthesis conditions inspired by the results of lightweight materials with exceptional mechanical performance. The the biomineral provide us with an artificial that closely formation of biomineral building blocks often takes place within specialized resembles the stylus. The fabricated bulk AFP/chitosan is biodegradable, intracellular vesicles, which in diatoms are called Silica Deposition Vesicles non-toxic, and processed at room temperature. (SDV). Recently two families of SDV membrane proteins have been [1] L. R. Brooker, J. A. Shaw in Advanced topics in biomineralization, identified that are well conserved throughout all diatoms and may therefore IntechOpen, 2012, pp. 65–84. play a fundamental role in diatom cell wall formation. One promising [2] a) J. C. Weaver, Q. Wang, A. Miserez, A. Tantuccio, R. Stromberg, K. candidate is Silicanin-1 (Sin1), which was shown to have moderate silica- N. Bozhilov, P. Maxwell, R. Nay, S. T. Heier, E. DiMasi et al., Materials formation activity in vitro that is enhanced by the addition of long-chain Today 2010, 13, 42; b) H. A. Lowenstam, S. Weiner, On biomineralization, polyamines. It was therefore speculated that Sin1 may influence the assembly Oxford University Press, New York, 1989. of biosilica-forming biomolecules within the SDV lumen. Here we describe the CRISPR-Cas9 mediated gene knockout of Sin1 in Thalassiosira T 30 pseudonana. Although the mutants grew normally, the knockout cell lines Reinforcement and adaptation of the mantis shrimp spike- exhibit a reduced biosilica content and showed pleiotropic effects on silica How crustacean cuticle became a perfect harpoon? morphogenesis, which drastically compromised the strength and stiffness of Y. Delaunois*1, D. Ruffoni2, P. Compère1,3 their cell walls. These results identify Sin1 as a key player in the development 1ULiege, Biologie, écologie évolution, Liege, Belgium of specific structural features of the T. pseudonana cell wall and as essential 2Uliege, aerospace and mechanic, Liege, Belgium for the biogenesis of mechanically robust diatom cell walls, thus providing 3Uliege, Center for Applied Research and Education in Microscopy an explanation for the conservation of this gene throughout the diatom realm. (CAREM), Liège, Belgium

T 29 Investigation of the hybrid nanocomposites of the chiton tooth Introduction stylus to find inspiration for new material development In the field of bioinspired materials, the crustacean cuticle is an example of L. Stegbauer*1, E. E. Alp2, I. Moudrakovski3, P. Smeets1, R. Free1, S. natural organo-mineral biomaterial able to endure a variety of strong stresses Wallace1, M. Hersam1, D. Joester1 thanks to the combination of a complex fibre organization, adaptations and 1Northwestern University, Materials Science and Engineering, Evanston, controlled mineral deposition (Romano, Fabritius, & Raabe, 2007, Acta United States Biomateriala 3, 301-309). Stomatopoda is a crustacean order including two 2Argonne National Laboratory, Advanced Photon Source, Sector 3, Lemont, groups: smashing and spearing mantis shrimps. Hence, the first group is United States already well known for the mechanical abilities of its smashing limbs or 3Max Planck Institute for Solid State Research , Stuttgart, Germany "hammer" (Patek & Caldwell, 2005, Journal of Experimental biology 208, 3655-3664), this study rather focuses on spearing mantis shrimps. Their spearing appendages bear long spikes that impale in a fraction of second Introduction and are therefore designed to penetrate at high speed, to avoid escape of the Organisms possess unparalleled control over the structure and properties of prey but also to resist to bending forces during the capture. such as teeth and , creating curved single crystals Objective as well as tough and lightweight self-repairing skeletal structures. Organic The aim of this study is to determine the structural and compositional matrices play an integral role in the selective formation of metastable mineral adaptations in the spike cuticle of spearing mantis shrimps to endure the precursors and their transformation into the final biomineral. The chiton is a intense stress occurring during attacks. model system for the extracellular, matrix-mediated iron oxide.[1] Its radula Materials and methods is a ribbon-like rasping tongue with many rows of extremely hard, wear- Spikes from Lysiosquillina maculata specimens were cut in segments and resistant, and self-sharpening teeth designed to withstand the stresses of either 2.5%-glutaraldehyde- or ethanol-fixed for TEM observation (after grazing algae on rocks.[2] So far, there have been numerous studies on the OsO4–staining and resin embedding) and for µCTscan and SEM-observation cuspid heads of the tooth, which consists of one of the hardest and most wear- respectively. Ethanol-fixed samples were also resin-embedded, then polished resistant biogenic materials, a magnetite/chitin composite. The underlying for block-face imaging by BSE-SEM and EDAX under low vacuum base, also called stylus, has not been investigated. conditions (0.4 Torr) in a ESEM-FEG XL-30 fitted with Bruker 129eV SDD. Objectives Results In this study we aim to establish a better understanding of the biominerals in First, the µCTscan highlighted spike external features as curvature and the radula tooth stylus of the chiton Cryptochiton stelleri. Modern material serrations along both edges linked by grooves on the sides. When compared fabrication requires large amount of energy to synthesize materials for to the decapod exoskeleton (classic model) and to the regular stomatopod semiconductors, medicine, or construction, often employing extensive top- cuticle (cephalotoracic shield), the spike cuticle exhibits important down fabrication steps at high temperatures and pressures. Biominerals can modifications in its internal fibre architecture and mineralization. These serve as inspiration for finding innovative methods and new materials and main changes are the lack of inner epicuticle and its replacement by a hyper- may also allow to decrease the carbon footprint of these processes. Such mineralized exocuticle containing fluorapatite, and the alternating helicoidal knowledge could inform the synthesis of new nanocomposite materials with (twisted plywood) and nematic (longitudinal orientation) arrangements of outstanding properties. chitin-protein fibres in the deeper procuticle layers. Also present in the Materials and Methods smashing limbs, theses procuticle layers were previously named the outer A comprehensive characterization of the stylus was performed with a suite helicoidal layer, the striated layer and the inner helicoidal layer because no of techniques that provide insights into structure and compositions at clear subdivision between classical decapod layers (exo- and endocuticle) multiple length scales, including synchrotron Mößbauer spectroscopy was identified. The limit between exo- and endocuticle was identified in the (SMS), solid-state nuclear magnetic resonance (ss-NMR), X-ray thin outer helicoidal layer. In terms of composition, the endocuticle including spectroscopy and electron imaging. Mechanical properties such as hardness the 3 fibrous arrangements was seen to be mainly CaCO3-calcified with high and reduced modulus were tested by nanoindentation. phosphate (and some fluor) proportion. The phosphate content gradually Results: We find evidence that the cusp-adjacent stylus is mineralized by decreases toward the epidermis while inversely the substitution rates by Mg santabarbaraite, an amorphous iron hydroxy phosphate that was not known increases. Nanoindentation tests also revealed significant variation in the to occur as a biomineral. The mineral occurs in very small nanoparticles reduced modulus between the layers. adjacent to α-chitin nanofibers. The stylus possesses outstanding mechanical Conclusion properties that are comparable to steels. We propose that the organism During evolution, the spearing limbs of mantis shrimps acquired a establishes complex spatial gradients in terms of the degree of mineralization specialized exoskeleton strongly modified in shape, in internal fibre (10-25 wt%) and mechanical properties across the stylus. Based on our architecture and mineral composition. These changes suit the intense insights, we formulated bio-inspired inks for additive manufacturing, mechanical constraints, especially anisotropic stresses, resulting from the demonstrating that the mechanical properties can be tuned over a wide range spike shape and movement for rapid penetration in preys. They are also also in in vitro. thought to cope with cracks propagations.

12 Acknowledgments: The first authors is a fellow of the FNRS-FRIA Amelotin (AMTN) is an enamel protein, which is expressed specifically (Belgium) during the enamel maturation stage and was shown to have a direct promoting effect on mineral growth and formation both in vivo and in vitro. T 31 Since AMTN is secreted on a pre-existing mineral layer during Acid-induced demineralisation of enamel as a function of time amelogenesis, we have used nano- (HA) to mimic the native and pH environment in which AMTN exerts its mineral promoting properties. R. Harper*1, R. Shelton1, J. James1, E. Salvati2, C. Besnard2, A. Objectives Korsunsky2, G. Landini1 To investigate the ability of the combination of AMTN and Nano-HA to 1University of Birmingham, School of Dentistry, Birmingham, United improve mineralization in simulated body fluid (SBF) and remineralization Kingdom of demineralized dentin collagen. Hypothesis: combination of AMTN and 2University of Oxford, Department of Engineering Science, Oxford, United nano-HA improves mineral formation both in SBF and dentin collagen. Kingdom Method SBF was incubated with AMTN, nano-HA, or both at 37°C for up to 21 days. Light scattering was used to record the formation and growth of mineral Acid-induced enamel demineralisation affects many people either by particles. exposure to acidic diets, acidic gases/ particulates from pollution (dental Coronal dentinal sections of human extracted molars were fully erosion) or to dental plaque acids (dental caries) with the latter currently demineralized with 0.5M Ethylenediaminetetraacetic acid (EDTA). being the most prevalent disease in humans. This study aimed to develop in Demineralized specimens were treated with AMTN, nano-HA or both and situ micro-CT and light microscopy methods to determine progression of incubated in SBF at 37°C. Specimens were fixed and imaged with scanning enamel demineralisation and the dynamic relationship between acid pH and electron microscopy (SEM) to visualize mineral formation Results: The mineral density to aid in the development of improved prevention, diagnosis combination of AMTN and nano–HA promoted mineral formation in SBF and restorative treatments. significantly compared to controls (P<0.05) when tested by ANOVA. Intact human third molars extracted for therapeutic reasons with full ethical Minerals were visually identified on the surface of dentin disks as well as approval (National Research Ethics Committee; NHS-REC reference within the dentinal tubules on the samples treated with both AMTN and 09.H0405.33/ Consortium Reference BCHCDent332.1531.TB) were nano-HA in as little as 2 hours. Conclusion: combination of AMTN and longitudinally sectioned into 500µm thick slices. A flat tipped 300µm nano-HA can accelerate the mineralization of demineralized dentin diameter needle was clamped perpendicularly on the external enamel smooth Significance surface before coating the entire surface with nail varnish to generate an The accelerated mineral formation mediated by AMTN and nano-HA can be ~300µm diameter circular non-varnished area on the slice. Varnished slices further refined to engineer a complex that may enhance the durability of for micro-CT in situ imaging were placed in a radiolucent Kapton® holder dental restoration by creating a homogenous mineral interface between (5 x 4.5cm pieces glued at the edges) containing lactic acid (10%, 0.5mL, pH restorative material and native tooth tissue. 2.2) before sealing. Four samples were radiographically time-lapse imaged hourly for 85h using a SkyScan 1172 micro CT scanner. Greyscale (related T 33 to mineral density) loss was quantified for both the advance of the The hidden structure of mouse and human enamel demineralisation front and extent of mineral density reduction. Varnished P. Gilbert*1 slices for light microscopy in situ imaging were glued to the base of a petri 1Univeristy of Wisconsin - Madison, Physics, Madison, WI, United States dish, to which 5ml of lactic acid (10%, pH 2.2) was added before sealing. Three samples were then time-lapsed imaged (every 100 seconds) using a Zeiss Primotech D/A POL light microscope using a 5x objective. In addition, Introduction 18 varnished slices were incubated at 37°C in lactic acid (0.5%, 0.5mL) for Enamel is the hardest and most resilient tissue in any animal"s body. The three weeks at either pH 3.6, 4.0, 4.4, 4.8 and 5.2 whilst deionised water was morphology of human and mouse enamel is well established: it consists of used as a control and were tomographically imaged using micro CT with space-filling[1], aligned, parallel, ~50 nm wide, microns-long nanocrystals, lesion size and mineral loss quantified using an hydroxyapatite phantom. bundled into 5-micron-wide rods, previously known as prisms. The Hourly micro-CT time-lapse sequences showed the depth of enamel orientation and arrangement of enamel crystals, however, are poorly demineralisation progressed with time from the surface towards the dentine understood. following a power-law function, which was 21% faster than the lateral Question demineralisation progression after the 85h exposure to lactic acid (10%, pH 1. Are crystal orientations in human and mouse enamel as 2.2). The minimum greyscale remaining (related to the remaining mineral previously assumed based on morphology alone, from SEM density) within the induced enamel lesion followed an exponential decay, images? while the total greyscale loss with time was linear, which showed a constant 2. Do the observed crystal mis-orientations play a functional role in anisotropic mineral release within the enamel architecture. Polarised light toughening enamel? microscopy time-lapse sequences showed that once the demineralisation Methods front reached the Hunter-Schreger bands in enamel, there was preferential We use polarization-dependent imaging contrast (PIC) mapping[2,3], a demineralisation along those bands. Mineral density loss was linear with synchrotron method that took a decade to refine and optimize[4,5], so it can increasing pH acidity between pH 5.2 and pH 4.0 when incubated over a 3- finally be used to measure and display the crystal c-axis orientation of week period exposed to lactic acid (0.5%). At pH 4.0, there was complete carbonate or apatite biominerals. In a PIC map color quantitatively displays mineral loss at the centre of the demineralised area after the 3-week period the c-axis unit-vector orientation in polar coordinates, including the in-plane and the linear function intercepted the x-axis at pH 5.6, near the critical HA and off-plane angles, displayed as hue and brightness, respectively, both pH. referred to the polarization plane, not the image plane. The sample is The time dependent linear total greyscale loss, exponential minimum mounted vertically, and the beam illuminates it from the right with 30° greyscale remaining, power-law demineralisation front progression, grazing incidence. The polarization plane is the plane in which the preferential lateral demineralisation and linear relationship between pH and polarization vector rotates, which is perpendicular to the beam direction, thus mineral density loss showed the intrinsic enamel structure affected the the polarization plane intersects the sample surface plane at 60°, and only a progression of demineralisation. vertical c-axis (displayed as cyan with full brightness), lies in both the polarization and the image plane[5]. Radiation damage to enamel is T 32 minimal[6]. Before PIC mapping, enamel samples are embedded, polished, Remineralization potential of the enamel protein amelotin and coated[7]. nano-hydroxyapatite Results PIC maps of mouse enamel show that, within a rod, crystals are co-oriented M. Neshatian*1, J. Holcroft1, A. Kishen1, B. Ganss1 with one another but not with the long axis of the rod[8], in human enamel 1University of toronto, Dentistry, TORONTO, Canada they are not co-oriented with either: the c-axes of adjacent crystals are most frequently mis-oriented by 1°-30°, and their orientation gradually changes up Background to 30°-90° within each rod[9,5]. Molecular dynamics simulations Dental caries, which according to world health organization (WHO) is the demonstrate that mis-orientation of adjacent crystals induces crack most prevalent chronic disease worldwide, is caused by acid-mediated deflection[5], and thus toughens enamel. demineralization of dental tissues. Resin-based composites are the most Conclusions commonly used restoration system for dental caries lesions. The integrity of The newly observed mis-orientations contribute to make human enamel last the interface between the tooth and the adhesive used to bond composite resin a lifetime. to the tooth tissue is crucial to the longevity of the restoration. Currently used References resin bonded composites have an average life span of only 5.7 years. 1. L Yang, CE Killian, M Kunz, N Tamura, PUPA Gilbert, RSC-Nanoscale Therefore, remineralization of demineralized dentin, especially the (2011), DOI: 10.1039/C0NR00697A tooth/restoration interface, is of considerable interest in restorative dentistry. 2. PUPA Gilbert, A Young, SN Coppersmith, Proc Natl Acad Sci USA It may improve bond stability and delay or prevent restoration failures. (2011), DOI: 10.1073/pnas.1107917108

13 3. CE Killian, RA Metzler, YUT Gong, TH Churchill, IC Olson, V T 35 Trubetskoy, MB Christensen, JH Fournelle, F De Carlo, S Cohen, J High resolution mechanical, elemental and ultrastructural Mahamid, FH Wilt, A Scholl, A Young, A Doran, SN Coppersmith, characterizations during zebrafish caudal fin regeneration PUPA Gilbert, Adv Funct Mater (2011), DOI: 10.1002/adfm.201001546 P. Y. Chen*1, Y. R. Shih1, F. Bohns1, Y. J. Chuang2 4. RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer, 1National Tsing Hua University, Materials Science and Engineering, SN Coppersmith, PUPA Gilbert, Phys. Rev. Lett. (2007), DOI: Hsinchu, Taiwan 10.1103/PhysRevLett.98.268102 2National Tsing Hua University, Life Science, Hsinchu, Taiwan 5. E Beniash, CA Stifler, C-Y Sun, GS Jung, Z Qin, MJ Buehler, PUPA Gilbert, submitted (2018), 6. T Parasassi, O Sapora, AM Giusti, G De Stasio, G Ravagnan, In J Rad Introduction Biol (1991), DOI: 10.1080/09553009114550061 Mineralization process during zebrafish fin regeneration has been studied, 7. G De Stasio, BH Frazer, B Gilbert, KL Richter, JW Valley, mainly focused on the gene control mechanism, signal pathways and the roles Ultramicroscopy (2003), 10.1016/S0304-3991(03)00088-3 of collagen. Crystallization of mineral from amorphous to well-aligned 8. CA Stifler, N Kølln Wittig, M Sassi, C-Y Sun, MA Marcus, H Birkedal, crystal was evaluated by advanced X-ray analyses and cryo-electron E Beniash, KM Rosso, PUPA Gilbert, J Am Chem Soc (2018), DOI: microscopy techniques. However, the micro/nano-scale mechanical 10.1021/jacs.8b05547 properties of zebrafish fin during regeneration have not been 9. RT DeVol, C-Y Sun, MA Marcus, SN Coppersmith, SCB Myneni, comprehensively investigated. In this study, micro-/nano-mechanical PUPA Gilbert, J Am Chem Soc (2015), DOI: 10.1021/jacs.5b07931 properties at high spatial resolution of zebrafish caudal fin were investigated Funding sources by nanoindentation and PeakForce Quantitative Nanomechanical Mapping NSF: DMR-1603192; DOE: DE-FG02-07ER15899; DOE: DE-AC02- (PF-QNM). Mineralization and ultrastructure were evaluated by SEM, 05CH11231. Raman microscopy and electron probe X-ray microanalysis (EPMA) at different locations and stages during the regeneration process. By combining T 34 these techniques, the relationship between the degree of mineralization and Unique three-dimensional structure of a fish mandible bone mechanical properties during zebrafish caudal fin regeneration was subjected to unusually high mechanical loads elucidated. Materials & Methods E. Raguin*1, K. Rechav2, V. Brumfeld2, R. Shahar3, S. Weiner1 Wildtype AB strain zebrafish were used for the experiment. Fish caudal fins 1Weizmann Institute of Science, Structural Biology, Rehovot, Israel were amputated and examined after 7 days post amputation (dpa), 14dpa, 2Weizmann Institute of Science, Chemical Research Support, Rehovot, 21dpa and 28dpa. Nanoindentation (TI 980 TriboIndenter) was carried out to Israel probe localized mechanical properties (hardness and reduced elastic 3Faculty of Agriculture, Food and Environment, The Hebrew University of modulus). AFM(PF-QNM) (Dimension Icon, Bruker) was used to analyze Jerusalem, Koret School of Veterinary Medicine, Rehovot, Israel the nano-scale mechanical properties of the fin and characterize nanostructure simultaneously. SEM (SU8010, Hitachi) and EPMA (JXA- Introduction 8500F, JEOL) were performed to characterize micro-structural features and Much of our understanding of bone structure is based on two-dimensional high precision elemental analysis (Ca and P). In addition, by combing Raman techniques. However, these methods provide limited insight into the spectrometer (HORIBA, iHR550) and laser confocal microscope (Olympus, structural complexity found in these tissues, especially at the nanometer IX71), Raman signal mappings were generated to correlate degree of length scale. Here, we explore the structure-mechanics relations in 2D and mineralization and corresponding locations. 3D of a bone that is neither cortical nor trabecular at different hierarchical Results levels of organization. This bone is found in the jaws of a fish that uses its Nanoindentation results showed that the elastic modulus and hardness tooth-jaw complex to crush hard-shelled bivalve mollusks. The bone is thus gradually decreased from proximal to distal regions of regenerated ray. subjected to unusually high compressive loads during mastication. The tooth- Nanoindentation mapping of individual proximal ray revealed that elastic jaw complex is composed mainly of flat molar-like teeth characterized by the modulus at the central region reached ~15GPa and dropped to ~3GPa toward absence of roots. the edges and similar trends were observed in middle and distal rays. EPMA Objectives analyses showed the decrease of Ca and P concentration from proximal to To understand the structure-function relationships of the bone at the interface distal regions of the single ray and the mineral concentration declined from of the tooth and the mandible. the center to the edge within an individual ray, in agreement with Materials & methods nanoindentation results. Additionally, PF-QNM showed low DMT modulus Jaws of adult black drum fish (Pogonias cromis) from Texas were studied. at distal region and higher modulus at the central region of individual ray. Reflected light microscopy and back-scattered electron (BSE) microscopy of SEM and AFM both showed non-mineralized collagen fibrils at the edge of polished sections, as well as micro-computed tomography (micro-CT) were the ray while granular mineralized collagen fibrils were discovered at the used to assess at lower resolution the structural organization and density of center. Raman microscopy mapping showed a decrease of mineral signal the bone near the contact area in 2D and 3D. We then used focused ion beam from proximal to distal region of regenerated fins after 7, 14, 21, 28 dpa. with scanning electron microscopy (FIB-SEM) and the serial surface view Conclusion (SSV) method to examine both demineralized (treated) bone and also dried We have applied multi-scale approaches, including elemental analysis, mineralized bone (untreated) to characterize the high resolution structure. ultrastructural characterization and mechanical testing to establish a Both methods provide structural insights and the comparison shows that the comprehensive analyses platform for the regeneration of zebrafish caudal fin. untreated bone provides key structural information using FIB SEM, with less High precision EPMA elemental analysis and nanoindentation results both risk of artifacts. confirmed that the regeneration and mineralization processes of zebrafish fin Results start from the center and gradually spread to the edge of individual ray. This Micro-CT and 2D polished sections of the mandibular bone revealed a highly mechanical/materials science approach can not only explore bio- porous structure, with porosities ranging from tens to hundreds of microns in mineralization process, but also have the potential to be used for in vivo diameter. MicroCT also showed that the teeth are supported by a circular studies in the future. narrow boney rim. The bone at the bone-tooth interphase is defined by a small contact surface that is relatively more mineralized than most of the T 36 jawbone. The interphase structure is characterized by the presence of fewer Misorientation and enhanced hardness in pores of approximately 5 to 50 μm in diameter compared to the other C. Stifler*1, C. Y. Sun1, E. Beniash2, P. Gilbert1 mandibular bone. The nanoscale level presents the most unexpected 1University of Wisconsin-Madison, Physics, Madison, United States organization in both treated and untreated bones: rods of ordered collagen 2University of Pittsburg, Oral Biology, Pittsburg, United States fibrils with their long axes parallel to the load direction are embedded inside a disordered matrix. Untreated bones reveal the presence of nano-tubules with diameters of 40 to 60 nm, which are aligned with the mineralized Introduction collagen fibrils. Teeth are subjected to extreme, repetitive forces and wear on a daily basis. Conclusions Human enamel endures forces up to 770 Newtons, hundreds of times per day, We show an unexpected structure of bone at the tooth-bone interphase that and must remain functional for decades [1]. By comparison, great white is subjected to high mechanical loads: the collagen fibril nano-tubules fabric shark enameloid exerts 7400 N of force when biting, but they shed their teeth is a novel structural motif not identified to date in bone. While the orientation regularly [2]. The mechanical stress that the teeth undergo suggests that there of the mineralized collagen fibrils in the rods provides the greatest resistance are structural features in enamel that prevent catastrophic failure. While the to compression, the function of the tubules remains enigmatic. morphology of enamel is known to be space-filling [3], aligned groups of microns long, nanometers wide apatite crystals (called rods in humans and

bundles in sharks), the orientation of the crystals remains poorly understood. Question Is there a relationship between the degree of crystal misorientation in tooth enamel of diverse animals and the hardness and elastic modulus?

14 Methods introduces changes in crystal structure, hardness and whiteness of the enamel We used a method called polarization-dependent imaging contrast(PIC) [3]. Deciduous dental enamel has some specific microstructural features mapping [4,5] to reveal the crystal orientations within rods in human or which are different from permanent tooth enamel, e.g. the aprismatic outer mouse enamel and in bundles in sharkand parrotfish enameloid. PIC- layer, having a decisive role in mechanical response and also in corrosion mapping is a synchrotron method developed about a decade ago and has since resistance and caries evolution. been refined, so that it can quantitatively measure c-axis orientations in In this contribution we investigate structural and mechanical properties of carbonate and apatite biominerals [6,7]. To create PIC maps, a series of sound deciduous molar enamel as function of Mg2+ incorporation via ion- photoelectron emission microscopy (PEEM) images ranging in x-ray exchange reactions. Furthermore, to elucidate the Mg2+ interaction with Ca- polarization from parallel to perpendicular is acquired the calcium L-edge phosphate nanoparticles, the same Mg-exchange experiments were [8]. The intensity versus polarization curve that exists at every pixel in the performed on synthetic hydroxylapatite (HAP) and amorphous calcium stack is fit to a cosine squared curve according to Malus' law and orientation phosphate (ACP) nanopowders. information is extracted from the fit parameters.The colors in PIC maps Structural investigation is carried out using micro-X-ray diffraction (µXRD) correspond to the c-axis orientation relative to the polarization of the x-rays: scanning and transmission electron microscopy methods (SEM, TEM). hue is the in-plane angle and brightness is the out-of-plane angle.The enamel Mechanical testing was done using depth sensing nanoindentation. The ion samples are embedded, polished and coated before analysis, and the x-rays exchange experiments were monitored by X-ray photoelectron spectroscopy leave minimal damage to the sample [9,10]. (XPS) and energy dispersive spectroscopy (EDS) in the TEM. Results Untreated sound enamel exhibits strong correlation between Mg2+ Analysis of PIC maps from mouse enamel [8],human enamel [7], great white concentration, microstructure and nanohardness. The average (Ca+Mg)/P shark enameloid, and parrotfish enameloid [11] indicates that c-axis value is 1.6, close to stoichiometric apatites. According to XPS, ion- orientations of adjacent crystals are slightly misoriented by a few degrees, exchange experiments on sound enmel resulted an increased Mg2+ the median misorientation ranging from 1.5-4°. In the case of human enamel, concentration on the enamel surface up to 6 at% which stabilized at ca. 3 at% crystals within a single rod can be misoriented up to 30-90°, but this change with (Ca+Mg)/P =2.55 after 30 min sputtering time, corresponding to ca. 50 is gradual, similar to what was observed in nacre [7,12]. The observed nm depth. Synthetic HAP sample exhibited less increase of Mg2+ content, misorientation is positively correlated with the hardness and elastic modulus, from 0.17 at% up to 1.95 at% and (Ca+Mg)/P from 1.64 up to 1.94. In case with R=0.86 and R=0.75, respectively. of the ACP nanopowder, the formation of a new, probably amorphous phase Conclusions was observed with characteristic morphology and Ca:Mg:P =1 : 1 : 2 ratio. Increased crystal misorientation in enamel is related to, and possibly causes In case of HAP nanopowder, the Mg2+ incorporation occurs through surface enhanced hardness. hydration of the individual nanoparticles, as indicated by the increase of References (Ca+Mg)/P ratio. ACP nanopowder proved to be highly reactive in Mg-rich 1.S Varga, S Spalj, ML Varga, SA Milosevic, S Mestrovic, M Slaj, European environment. Enamel is a compact ceramic material, with crystallite size and Journal of Orthodontics, (2011), DOI: 10.1093/ejo/cjq097 morphology similar to HAP nanopowder but much less free crystalline 2.S Wroe, DR Huber, M Lowry, C McHenry, K Moreno, P Clausen, TL surfaces for hydration. The very high amount of incorporated Mg2+ even at Ferrara, E Cunningham, MN Dean, AP Summers, J. Zoology (2008), DOI: 50 nm from the surface and the (Ca+Mg)/P ratio is supposed to be related to 10.1111/j.1469-7998.2008.00494.x the low crystallinity material, probably with organic residue between the 3.L Yang, CE Killian, M Kunz, N Tamura, PUPA Gilbert, RSC-Nanoscale enamel forming apatite nanowires. This intercrystalline material can serve as (2011), DOI: 10.1039/C0NR00697A either chemical or physical channel for Mg2+ ions, by faster dissolution rate 4.PUPA Gilbert, A Young, SN Coppersmith, Proc Natl Acad Sci USA or the presence of nanofractures, respectively. The effect of Mg2+ content on (2011), DOI: 10.1073/pnas.1107917108 the nanohardess is also discussed. 5.CE Killian, RA Metzler, YUT Gong, TH Churchill, IC Olson, V [1] Ding, Pan et al. (2014) Cryst. Growth Des. 14, 763−769 Trubetskoy, MB Christensen, JH Fournelle, F De Carlo, S Cohen, J [2] La Fontaine, Zavgorodniy et al. (2016) Sci. Adv. 2, e1601145 Mahamid, FH Wilt, A Scholl, A Young, A Doran, SN Coppersmith, PUPA [3] Abdallah, Eimar, Basset et al. (2016) Acta Biomaterialia 37, 174–183. Gilbert, Adv Funct Mater (2011), DOI: 10.1002/adfm.201001546 6.RA Metzler, M Abrecht, RM Olabisi, D Ariosa, CJ Johnson, BH Frazer, T 40 SN Coppersmith, PUPA Gilbert, Phys. Rev. Lett. (2007), DOI: Osteoblast behavior and mineralization onto substrates with 10.1103/PhysRevLett.98.268102 controlled topographies 7.E Beniash, CA Stifler, C-Y Sun, GS Jung, Z Qin, MJ Buehler, PUPA R. Silva dos Santos1, P. Rougerie2, K. Anselme3, M. Farina*2 Gilbert, submitted (2018), 1Federal University of Rio de Janeiro, Institute of Biophysics Carlos 8.CA Stifler, N Kølln Wittig, M Sassi, C-Y Sun, MA Marcus, H Birkedal, E Chagas Filho, Rio de Janeiro, Brazil Beniash, KM Rosso, PUPA Gilbert, J Am Chem Soc (2018), 2Federal University of Rio de Janeiro, Institute of Biomedical Sciences, Rio DOI:10.1021/jacs.8b05547 de Janeiro, Brazil 9.T Parasassi, O Sapora, AM Giusti, G De Stasio, G Ravagnan, In J Rad Biol 3Université de Haute-Alsace, Mulhouse, France (1991), DOI:10.1080/09553009114550061 10.G De Stasio, BH Frazer, B Gilbert, KL Richter, JW Valley, Ultramicroscopy (2003), DOI:10.1016/S0304-3991(03)00088-3 Introduction 11.M Marcus, S Amini, CA Stifler, CY Sun, N Tamura, HA Bechtel, DY Topographical patterns can affect cell adhesion, proliferation, migration, Parkinson, HS Barnard, XXX Zhang, JQ Isaiah Chua, A Miserez, PUPA. differentiation and gene expression. Osteoblasts in vivo encounter large, Gilbert, ACS Nano (2017), DOI:10.1021/acsnano.7b05044 curved topographical patterns such as Howship"s lacunae walls. Studying 12.RT DeVol, C-Y Sun, MA Marcus, SN Coppersmith, SCB Myneni, PUPA whether and how curved topography affect osteoblast biology and bone Gilbert, J Am Chem Soc (2015), DOI:10.1021/jacs.5b07931 matrix production thus can help us to understand bone formation in vivo and Funding Sources enhance osteo-integrative processes in vivo. Interestingly, some studies NSF: DMR-1603192 report that artificial topographical elements like pillars or roughness can DOE: DE-FG02-07ER15899 improve matrix mineralization in vitro. However, these topographies are DOE: DE-AC02-05CH11231 usually irregular and/or contain edges. Therefore, they are of limited use to understand the reaction of osteoblast to curved surfaces more akin to what T 37 they encounter in vivo. The aim of this work is thus to evaluate the influence Investigation of Mg2+ incorporation into deciduous enamel and of curved substrate topography on osteoblast arrangement and its effect on the mechanical properties mineralization, and the possibility to harness it to reproduce in vitro matrix organization patterns observed in vivo, as in parallel fibered bone or . V. Kovacs Kis*1, A. Sulyok1, M. Hegedűs2, I. Kovács2,3, Z. Kovács2 Methods 1Centre for Energy Research Hungarian Academy of Sciences, Thin Film Polydimethylsiloxane (PDMS) scaffolds were produced presenting three Physics Laboratory, Budapest, Hungary types of cell-scale topographies: either edge-containing or smoothly curved 2Eötvös University, Department of Materials Physics, Budapest, Hungary anisotropic substrates (grooves and ridges) or smoothly curved isotropic 3Research Centre for Astronomy and Earth Sciences, HAS, Budapest, substrate (egg-box). The smoothly curved surfaces (isotropic or anisotropic) Hungary are expected to better mimic in vivo environments compared to more classic edge-containing surfaces. The scaffolds were subsequently coated with The substitution of bivalent cations exhibiting biological activity, such as fibronectin, and further used for seeding primary rat calvaria preosteoblasts Mg2+, in apatite, the main mineral reservoir of calcium and phosphorus in (F-OST cells) that were then induced to mineralization. Cells attachment and vertebrates, is an intriguing research topic in biomaterial synthesis and proliferation, quantity, composition and organization of the bone-like fundamental biochemistry as well. Mg2+ is known to inhibit the growth of extracellular matrix were evaluated. Mineralized deposits were assessed with apatite crystalline nuclei in biological environment [1]. Recently in has been Alizarin and Von Kossa stainings and cell distribution and orientation were revealed that a Mg-rich amorphous calcium phosphate phase exists between observed by staining for the actin cytoskeleton and laser scanning confocal the apatite nanocrystals in human dental enamel [2]. At the same time, it has microscopy. been demonstrated that Mg2+ incorporation into permanent tooth enamel

15 2- + Results temporal dynamics of pH, [CO3 ] and [Ca2 ] in the ECM and observe Osteoblast cells aligned with the scaffold axes in all anisotropic substrates interrelations in the different ion concentrations. although it was more evident in the topography with straight edges. Our study provides the most comprehensive in vivo characterization of Mineralization occurred after two weeks without induction factors and after directly measured ECM carbonate chemistry parameters in a single live coral a few days upon induction, apparently also following the orientation of the species to date, and highlights the role of active transport mechanisms for the ridges and grooves, and it was more intense on the scaffold with straight biological control of ECM carbonate chemistry. Moreover, the novel insights edges. The mineral deposits initiated in concave regions in all topographies, into the temporal dynamics of the different ion concentrations improve showing an alignment of the matrix with the substrates, which was not understanding of carbonate chemistry dynamics during CaCO3 formation in observed in the control (flat PDMS). After approximately 10 days the the ECM. deposits tended to occupy the whole substrate. From confocal imaging we were able to generate topographical maps and 3D T 43 reconstruction of our surfaces and determined the osteoblast positioning Modelling coral calcification - isotope and element fluxes in a relative to the local curvature at different stages of their proliferation and tightly constrained system maturation. F. Böhm*1, I. Taubner1, A. Eisenhauer1, M. Bleich2 Conclusions 1Geomar, Kiel, Germany Osteoblasts cultivated over anisotropic surfaces orientate along the 2University of Kiel, Institute of Physiology, Kiel, Germany topography axis, indicating a topography-induced cell guidance and suggesting a possible anisotropy of the produced mineralized matrix. We found also that biomineralization initiated at concave regions. Putting Isotopes are useful tools for studying biomineralization, where mineral together, these findings can help in the design of topography inducing formation mechanisms, material sources and transport pathways are reflected scaffolds for different purposes in biomineralization and bioengineering, as in the isotopic composition of biominerals. On the other hand, applications the in vitro production of bone like tissue. of isotopes as environmental proxies recorded in biominerals can profit siginificantly from a better understanding of the processing of elements and T 41 isotopes during biomineralization. Influence of mineral properties and organic matrix We develop a numerical model of elemental and isotopic fluxes in composition and structure on bone mineral dissolution scleractinian corals. Based on two recently published coral boxmodels (1-3) our model quantifies ionic and molecular fluxes between seawater and coral A. Rodriguez-Navarro*1, N. Dominguez-Gasca1, C. Benavides-Reyes1, compartments: oral/aboral epithelia, coelenteron, calcifying medium, E. Sanchez-Rodriguez1, M. Greiner2, W. W. Schmahl2 skeleton. It includes carbonic and boric acid chemistry and isotopes, Ca2+ and 1Universidad de Granada, Granada, Spain its isotopes, and O . Trans- and para-cellular fluxes are implemented. 2Ludwig-Maximilians-Universität, Department of Earth and Enviromental 2 The ion permeability of the oral epithelium was tuned to 45Ca tracer data and Sciences, Munich, Germany Ussing chamber results (4, 5). Diffusive ion permeabilities of the aboral epithelium and the skeleton are constrained by electro-physiological data Bone mineral dissolution is a highly complex process due to large chemical from Ussing chamber experiments on Stylophora pistillata colonies (6). The and structural heterogeneity of bone tissue. Bone mineral dissolution is para- and trans-cellular model fluxes were adjusted to yield calcification rates facilitated by the small crystal size of apatite crystals and large amount of equivalent to an extension on the order of 1 cm/year. ionic substitutions. It is also dependent on the associated collagen matrix in We find that the aboral epithelium is extremely tight. Para-cellular fluxes (2) which apatite crystals are integrated and on other proteins absorbed on crystal and diffusion through the skeleton are of minor importance. Trans-cellular surfaces. To better understand this process, we have studied in detail how transport is the only significant source of ions for calcification. bone mineral chemistry and structure changes during demineralization using While small molecules like CO2 and B(OH)3 diffuse through cells, trans- - 2+ 2+ - different analytical techniques such as electron microscopy, 2D X-ray membrane transport is necessary for ions like HCO3 , Ca , Sr or B(OH)4 . diffraction and infrared spectroscopy. We show that bone mineral and Constraints from isotopes are used to design possible transport pathways in organic matrix characteristics of different types of bone tissues (i.e., bovine the model: While B/Ca ratios observed in coral skeletons can be achieved enamel, bovine and avian cortical bone, avian medullary bone) strongly with trans-cellular boric acid diffusion, the resulting boron isotope influence bone mineral solubility and dissolution behavior. For instance, composition disagrees with known coral values. However, correct isotope - medullary bone mineral which is poorly crystalline and its organic matrix is ratios are achieved assuming co-transport of borate ions with HCO3 through non-collagenous, dissolves much more rapidly than any other type of bone. the cell membranes. Consequently, boron isotopes in corals are influenced It is also shown than during demineralization, there is a selective dissolution by pH and by bicarbonate transport rates. of poorly crystalline disorganized bone mineral rich in labile carbonate.The (1) Hohn S., Merico A. (2012) Biogeosci. 9, 4441-4454. (2) Hohn S., Merico information gathered in this work provides a more complete picture of how A. (2015) Front. Earth Sci. 2, 37. (3) Nakamura T. et al. (2013) Coral Reefs bone mineral dissolution occurs which is needed to understand basic but 32, 779-794. (4) Furla P.et al. (2000) J. exp. Biol. 203, 3445-3457. (5) highly important processes such bone formation and resorption during the Tambutté" E. et al. (2012) Proc. Royal Soc. B, 279, 19-27. (6) Taubner et al. bone remodeling. (2017) Limnol. Oceanogr. Meth., 15, 753-765.

T 42 T 44 Coral skeleton formation ? Concentrations and temporal Ion transporter gene expression is linked to the thermal 2- + dynamics of pH, [CO3 ] and [Ca2 ] in the extracellular sensitivity of coral calcification calcifying medium of a live tropical coral C. Bernardet1, E. Tambutté1, N. Techer1, S. Tambutté1, A. Venn*1 D. S. Sevilgen*1, A. Venn1, V. Planas-Bielsa1, E. Tarnbutté1, D. Zoccola1, 1Centre Scientifique de Monaco, Marine Biology Department, Monaco, S. Tarnbutté1 Monaco 1Centre Scientifique de Monaco, Monaco, Monaco Reef coral biomineralization is sensitive to temperature, which is one reason

To build their skeleton, stony corals precipitate calcium carbonate (CaCO3 in why coral reef ecosystems are threatened by climate change, but the the form of aragonite) within an extracellular calcifying medium (ECM) mechanisms underlying the thermal sensitivity of corals are poorly which is located between the skeleton and the coral tissue. Increases in understood. Furthermore, light is also a key factor in modulating important calcification parameters such as pH, calcium concentration [Ca2+] biomineralization rates but a mechanistic understanding of how light 2- and dissolved inorganic carbon species (DIC, e.g. [CO3 ]) in the ECM can enhances coral calcification is lacking. We carried out a controlled laboratory facilitate the formation of CaCO3. Indeed, elevation of these parameters study that characterized the thermal performance curve (TPC) of increases the aragonite saturation state (Ωarag), a determinant that describes calcification in light and darkness in the widely-used model coral species the favorability of CaCO3 to form from solution. Stylophora pistillata. Using the TPC to target low and high temperatures at The objectives of our study were to access the ECM of live coral directly which calcification rates were depressed, we used gene expression analysis with microsensors and conduct real time in vivo measurements in the ECM to investigate the role of ion transport mechanisms in the coral"s thermal of the growing edge of Stylophora pistillata microcolonies. The aim was to response. We focused the study on genes with functions in the transport and 2- + + evaluate if and to which extend pH, [CO3 ] and [Ca2 ] are elevated. regulation of dissolved inorganic carbon, calcium and H . Our findings Furthermore, we aimed at resolving temporal dynamics of the different ion reveal a high degree of coherence between physiological responses (e.g. concentrations. Microsensor measurements were facilitated by inverted calcification and respiration) with distinct gene expression patterns to the brightfield- and confocal microscopy and images of the studied coral region different temperatures, and also to day and night conditions. At high and low were taken frequently throughout the experiments. temperatures, a core gene expression pattern emerged, pointing to thermal Our data showed that all parameters within the ECM, as well as derived DIC effects on dissolved inorganic carbon transport mechanisms linked to concentrations and Ωarag, were significantly elevated above seawater values. reductions in calcification rate. At high temperature but not low temperature, Furthermore, by combining different approaches, we were able to follow light stimulated calcification and the response of a more functionally diverse group of genes. Overall, our results highlight biological control via ion

16 transport mechanisms linked to the thermal sensitivity of calcification, which Objectives is a subject of growing interest to emerging fields of study that seek to The aim is to fully characterize the structure and crystal orientations in improve the resilience of corals to climate change. diverse coral skeletons and to reveal the mechanism of spherulitic growth in general. T 45 Materials and Methods DNP-enhanced NMR of metamorphosing young corals shows Twelve different coral species analyzed here include different clades, that skeleton construction entails modulation of organic different growth morphologies, and different geographic origins. material Polarization-dependent Imaging Contrast (PIC) mapping was used to measure quantitative crystal orientations. PIC mapping was done using the S. Nasser1, M. Neder2, B. Uluca3, U. Akbey3, H. Heise3, T. Mass2, G. Photoelectron Emission spectroMicroscopy (PEEM) at ALS, LBNL. PIC Goobes*1 mapping utilizes X-ray linear dichroism, which, for coral, is maximum at the 1Bar Ilan University, Chemistry, Ramat Gan, Israel O K-edge π* peak (534 eV), and is done by rotating the linear polarization 2University of Haifa, Marine Biology, Haifa, Israel of the illuminating X-rays from horizontal to vertical in 5º steps, acquiring a 3Juelich Research Institute, Institute of Complex Systems, Juelich, Germany PEEM image at each polarization, and then fitting the intensity variation versus polarization angle to a cosine-square curve, from which the c-axis for The ability of corals to maintain homeostasis and mineralize has been each pixel can be deduced [3-5]. Electron backscattered diffraction and X- compromised by ocean acidification and temperature rise causing reefs to ray microdiffraction were done for further grain boundary analyses. recede and even vanish. Therefore, corals have been serving lately as Phase-field simulation models with an orientation field [6] were used to important proxies of environmental impact on marine life. Early in their life, support the proposed formation mechanism. corals undergo transformation from a swimming organism (planula) to an Results benthic immobile one (polyp), that lives in colonies and forms exoskeleton Quantitative PIC maps confirmed that all coral skeletons form aragonite for protection. During this metamorphosis process, they are vulnerable spherulites. In addition, we discovered that 4 out of the 12 species show a leaving them exposed to detrimental external changes. It is therefore very different growth form of aragonite with tiny (0.2-2 µm), randomly oriented, important to carefully characterize the two developmental states – planula equant crystals, we termed "sprinkles". With quantitative PIC mapping, we and polyp- and to be able to assist corals withstand ongoing hazardous confirm that sprinkles are not spherulites due to their much larger c-axis variations in their surroundings. misorientation angles across grain boundaries. We propose that the randomly Recently, we have shown using 13C MAS NMR on whole 13C-labeled oriented sprinkles are the initially nucleated crystals, which later coarsen into young Stylophora pistillata corals that mineralization starts before the coral spherulites: crystals with radially oriented c-axes have more space to grow settles. We also found using 2D 13C DARR that Glu-rich proteins bind soft and thus become larger, at the expense of smaller, randomly oriented amorphous mineral in the planula and Asp-rich proteins bind aragonite sprinkles. Phase-field simulations support our proposed mechanism: in all crystals in the polyp. Regulation of the two mineral states by the disparate spherulites the first nucleated crystals are randomly oriented sprinkles, which proteins suited well the needs of the coral changing from motile to sessile. then coarsen and become only radially oriented crystals. Here, we expand investigations of the metamorphosis using DNP-enhanced Conclusions MAS NMR measurements, obtaining favorable signal enhancements of With quantitative crystal orientation analysis, we discovered randomly about 16- to 48-fold. Using 2D 13C DQ-SQ and PDSD measurements on the oriented sprinkles in coral skeletons. We propose that sprinkles are the early- intact corals, labeled either via 13C carbonate/ or via 13C6 stage nucleated crystals in all spherulites, whether biogenic, synthetic, or glucose/glycine, we follow the changes in organic level production related to geologic, and then radially oriented crystals coarsen at the expense of non- the transformation and the onset of aragonite precipitation. We monitor radially oriented ones. Phase-field theory confirms that this is indeed the contribution of symbiotic dynoflagellates via carbonate by using case. the latter as a food source. [1] Yang 2011, DOI: 10.1039/C0NR00697A The carbon fingerprint changes observed relate to increased carbohydrate [2] Sun 2017, DOI: 10.1021/acsnano.7b00127 production and certain proteins which were not detected without [3] Metzler 2007, DOI: 10.1103/PhysRevLett.98.268102 enhancement. These changes are indicative of the transitions entailing onset [4] Gilbert 2011, DOI: 10.1073/pnas.1107917108 of colonization and expedited mineralization effort initiated after settling on [5] Killian 2011, DOI: 10.1002/adfm.201001546 the bottom of the ocean. [6] Gránásy 2005, DOI: 10.1103/PhysRevE.72.011605

T 46 T 47 How inorganic spherulites grow- learning from corals Anisotropic lattice distortions caused by photosymbiosis in C. Y. Sun*1,2, L. Gránásy3, C. Stifler1, T. Zaquin4, R. Chopdekar5, N. scleractinian corals Tamura5, J. Weaver6, J. Zhang1, S. Goffredo7, G. Falini8, M. Marcus5, T. I. Coronado*1, J. Stolarski1 Pusztai3, T. Mass4, P. Gilbert1,2,9 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 1University of Wisconsin - Madison, Physics, Madison, United States 2University of Wisconsin - Madison, Materials Science, Madison, United States The symbiosis between scleractinian corals (host) and unicellular 3Wigner Research Centre for Physics, Institute for Solid State Physics and zooxanthellae (dinoflagellate algae, Symbiodinium spp), is the key to the Optics, Budapest, Hungary success of modern reefs. Symbionts deliver to coral host nutrients, resulting 4University of Haifa, Marine Biology, Haifa, Israel from photosynthesis, and promote the light-enhanced skeletal calcification. 5Lawrence Berkeley National Laboratory, Advanced Light Source, Coral-algae symbiosis has had an important role in the reef evolution and Berkeley, United States allowed them to thrive in different environments through the geological 6Harvard University, Wyss Institute for Biologically Inspired Engineering, record. Unfortunately, the identification of symbiosis in the fossil record is a Cambridge, United States paramount challenge. Several criteria have been applied with the purpose to 7University of Bologna, Biological, Geological and Environmental differentiate Recent and fossil symbiotic and asymbiotic corals on the basis Sciences, Bologna, Italy of their skeletons due to the lack of fossilization of zooxanthellae. 8University of Bologna, Chemistry, Bologna, Italy Morphological, bio-geochemical, and lately microstructural differences have 9University of Wisconsin - Madison, Chemistry, Geoscience, Madison, been applied in order to distinguish the presence or absence of United States photosymbionts. High-resolution X-ray diffraction (HR-XRD) studies have shown that crystal lattice of bio-aragonite, in the molluscs and coral skeletons, is anisotropically Introduction distorted as compared with that of geological counterparts. It has been Modern coral skeletons are commonly assumed to consist of aragonite suggested that these distortions result from the intercalation of organic crystals that grow spherulitically, that is, acicular crystals radiating from molecules into the crystallographic lattice during the biomineralization common centers and fill space [1]. Although the name "spherulite" comes process. It was assumed by our hypothesis that distinct biogeochemical directly from its shape, morphology alone can be misleading. In an earlier differences found between symbiotic and asymbiotic corals should also be study, we determined quantitatively and found that spherulitic aragonite reflected in changes of lattice parameters of bio-aragonite of these two crystals, both synthetic and biogenic from coral skeletons, exhibit a 0-35º groups. In order to test this, we sampled a large suite of coral skeletons (30 misorientation of c-axes across grain boundaries [2]. Previously coral symbiotic and 24 asymbiotic taxa) representing different phylogenetic lines. skeletons were only been identified as spherulitic from morphological High-resolution X-ray powder diffraction measurements were carried out at observations, now we set out to characterize the crystal orientation MCX beamline of the Elettra Synchrotron Radiation Facility. The structural distributions in coral skeletons from various genera and species to reveal parameters were refined by Rietveld analysis and the lattice distortions (as their structural differences. Δd/drf), crystallite size and microstrain analyses were performed. To correlate the lattice distortions data with information about inorganic and organic molecules trapped into the crystals we collected also X-ray powder diffraction data of isochronously annealed and bleached in block at different

17 time treatments of skeletons of selected taxa of both ecological groups. We References also performed thermogravimetric analyses under an N2 atmosphere and 1 Akiva A, Neder M, Kahil K, Gavriel R, Pinkas I, Goobes G, and Mass T, mass-spectroscopy measurements (using ICP-MS). Minerals in a Pre-Settled Coral Stylophora pistillata Crystallize via Protein A linear variation between the lattice parameters of symbiotic and asymbiotic and Ion Changes, Nature Communications, 9, 1880; DOI: 10.1038/s41467- corals was observed. Orthorhombic constant ratios clustered both ecological 018-04285-7 (2018) groups, confirming our original hypothesis. In addition, significant 2 Neder M, Laissue P P, Akiva A, Akkaynak D, Albéric M, Spaeker O, Politi differences in the biogeochemical composition were found, showing a Y, Pinkas I*, and Mass T*, Mineral formation in the primary polyps of complex correlation with anisotropic distortions. The observed pocilloporoid corals, Acta Biomaterialia, DOI:10.1016/j.actbio.2019.07.016 crystallographic vital effect seems to be related to physiological changes (2019) resulting from the interaction between symbiont and coral host but also by the subsequent ecological variations of the studied samples. T 49 Acknowledgements: This work was supported by the National Science Coral biomineralization toolkit differs in octocorallian vs Centre (Poland) grant 2017/25/B/ST10/02221. The authors acknowledge the hexacorallian Elettra Synchrotron Radiation Facility (Trieste, Italy) for provision of MCX P. Ganot*1, N. Le Roy1,2, M. Fritz3, T. Rausch3, D. Aurelle4, A. beamline and synchrotron facilities (proposal: 20170388). Haguenauer4, M. Arenda5, V. Benes3, D. Allemand1, S. Tambutté1 1Centre Scientifique de Monaco, Monaco, Monaco T 48 2INRA, Nouzilly, France In-vivo and in-situ micro-Raman spectroscopy and imaging 3EMBL, Heidelberg, Germany study of mineral formation in the primary polyps of 4CNRS, Marseille, France pocilloporoidea corals 5KAUST, Thuwal, Saudi Arabia I. Pinkas*1, M. Neder2, P. P. Laissue3, A. Akiva4, D. Akkaynak5, M. Albéric6, O. Späker6, Y. Politi6, T. Mass2 1 Introduction Weizmann Institute of Science, Chemical Research Support, Rehovot, Corals are calcifying organisms represented in diverse taxa of Cnidaria, Israel including Hexacorallia (e.g. reef-building corals), and Octocorallia (e.g. 2University of Haifa, Department of Marine Biology, Haifa, Israel 3 precious red corals). Corallium rubrum, the Mediterranean precious red University of Essex, School of Biological Sciences, Colchester, United coral, produces two types of biomineralized structures, the sclerites and the Kingdom 4 axial skeleton. Both are made of high Mg calcite precipitates and an organic Eindhoven University of Technology, Chemical Engineering and matrix (OM) which is secreted by the scleroblast cells and the calcifying Chemistry, Eindhoven, Netherlands 5 epithelium, respectively. It is noteworthy that the red coral axial skeleton is Princeton University, Department of Ecology & Evolutionary Biology , the material for one of the oldest forms of jewelry known to man. Princeton, United States 6 Objectives Max Planck Institute of Colloids and Interfaces, Biomaterials, Potsdam, Hexacorallia and Octocorallia diverged around 550-700 Mya, before the first Germany mineralized cnidarian fossil dating. How evolutionary conserved are the processes controlling biomineralization between the reef-building corals and Introduction the precious red corals? Although calcification in hexacorallia has been the In reef-building corals, the earliest stages of mineral formation are critical to subject of several investigations, little is known on octocorallian the construction of reefs and their survival potential. The short time window calcification. of coral settlement, when massive, rapid calcification occurs, provides a Mat&met unique opportunity to study a range of bio-carbonate morphologies and to We combined microdissection technics with omics approaches to shed light understand the transformation of mineral phases into the mature coral on the synthesis and composition of the red coral biominerals. Proteomic exoskeleton. analysis of the decalcified biominerals allowed us to identify over 100 Objectives different proteins composing the sclerites and axial skeleton OMs. To this end, we study two members of the pocilloporoid stony Transcriptomic analysis of the calcifying tissues enabled us to ascertain corals, Stylophora pistillata and Pocillopora acuta, during the first few days whether the OM proteins were specific to the calcification process or after settlement. Following our discovery that mineralization starts even partaken with other regular extracellular matrix processes. before the polyp settlement and that the deposition of minerals is controlled Results by specific proteins1, we show that the initial mineral phase after settlement Our results show that 1/ many (but not all) OM proteins are shared between is nascent magnesium calcite (Mg-Calcite), with rod-like structures in P. the sclerites and the axial skeleton; 2/ in comparison to the known reef- acuta, and with dumbbell-like structures in S. pistillata. building coral OM proteins, octocorals seem to have evolved different Materials and methods strategies: a marked utilization of collagen fibers but also, many novel The project utilizes several techniques to uncover the mechanism of skeleton proteins; 3/ only half of the red coral OM proteins are specifically expressed building in the coral larvae. Among them, SEM, EDS, Confocal fluorescence by the calcifying cells; 4/ many of calcifying specific proteins appear to be microscopy, XRD, XRF, molecular biology, bioinformatics, and in- co-opted from already existing extracellular matrix gene families. vivo and in-situ micro-Raman spectroscopy and imaging. The organisms Conclusion were collected from the Red Sea over several nights in February-May 2016 Coral is a vernacular name: calcification in different cnidarian taxa, although and 2017 by covering the corals with nets following peak releases of larvae. using an evolutionary conserved "tool kit" (e.g. Carbonic Anhydrases), Results appears to result from independent recruitments of extracellular matrix genes Our measurements reveal that there are basic growth structures with similar involved in other processes. characteristics for the two coral species that consist of Mg-Calcite (rods and dumbbells). We verify this by both micro-Raman spectroscopy and T 50 XRD/XRF. Confocal microscopy reveals vesicles filled with materials Biomineralization in cephalopods- first proteomic data on consisting of divalent ions (Ca2+, Mg2+, Sr2+) within the coral cell layers. Spirula spirula and Argonauta hians These rod and dumbbell-shaped structures, which, most likely, form the M. OUDOT*1, E. FARA1, P. NEIGE1, F. MARIN1 centers of calcification (CoC), grow and merge to build the basic skeletal 1CNRS / Université de Bourgogne - Franche-Comté, DIJON, France units of the polyp, the basal plate and the septa, in a spherulitic growth pattern of aragonite needle shaped crystals. As these initial shapes (dumbbells) have also been observed in calcium carbonate precipitation by bacteria, we have Cephalopods constitute a major class of molluscs, from which only a part of verified that these do not exist in the coral samples by fluorescence imaging the living representatives possesses a calcified shell. The macro-evolutionary with fluorophores sensitive to bacteria, and by RNA sequencing of the history of this clade shows a tendency to shell reduction, internalization and samples, showing that no relevant bacterial community is associated with the finally, complete loss, from "basal" forms (nautilus) to the most derived ones dumbbell structures. (octopus). Although phylogenetic relationships between living shell-bearing Conclusions cephalopods are rather well established, molecular mechanisms of shell Based on our data, we suggest that mineralization in these corals occurs in formation are still poorly understood. In particular, skeleton-associated three phases: first, ion rich vesicles of amorphous calcium carbonate (ACC) proteins, which are supposed to constitute the 'molecular toolbox' for shell are formed intracellularly. Then, the vesicles are transferred to the formation, have been partly characterized, and only in two cephalopod calcification site, forming rod/dumbbell-shaped structures composed of genera, the nautilus and the cuttlefish. nascent Mg-Calcite. During the third phase, the Mg-Calcite outer surface is The objective of the present study is to sketch the outlines of the skeletal used for the growth of needle-shaped aragonite crystallites in a spherulitic matrix in cephalopods, to check whether representatives of this class use growth pattern forming the septa and basal plate of the coral. These processes similar or totally different 'molecular toolboxes'. In addition, our study aims take place at each center of calcification, starting at different times so that at describing precisely the microstructural characteristics observed in the one can observe all of them in the same polyp. We suggest a reason behind models. Our ongoing study is primarily delimited to two species, the Ram's this complex process2. horn squid Spirula spirula and the paper nautilus Argonauta hians. Note that

18 in this latter case, the shell is a calcitic eggcase secreted only by females and very hard, PEEK has mechanical properties very similar to bone which is an constitutes a remarkable example of a derived character, unique to argonautid advantage. However, while titanium is at least partly accepted by the bone clade. building osteoblast cells, PEEK is completely bioinert. This leads to a bad or To this end, we have extracted - after two cleaning procedures - the shell missing connection of the implant to the surrounding bone tissue and revision calcifying matrices of the two studied species and performed proteomic surgeries are often necessary with a chance of 15 – 40 % depending on the characterization followed by in silico investigations. The saccharidic implant. This is painful for the patient and causes enormous costs. signature of soluble fractions has also been investigated by ELLA tests using We have therefore developed a strategy to covalently coat the implant with a a large set of lectins. In the case of S. spirula, all our investigations rest upon bone mimetic layer. We use a coupling chemistry to covalently bind gelatin two geographically different batches: one from Canary Islands, one from or collagen to a PEEK surface or alternatively to titanium. Afterwards, this Brazil. In parallel, we have studied the shell microstructural features of S. layer is mineralized with calcium phosphate resulting in an only 50 – 100 nm spirula and of A. hians by conventional SEM observations on polished thick bone mimetic surface layer. Comparative cell tests show that sections. Osteoblasts like the bone mimetic layer and secrete collagen as a first step to The shell of S. spirula has a complex microstructural organization. In building new bone already within the first 12 h. Animal tests with a sheep particular, SEM observations evidence a peculiar feature, also described model show the superior performance of the osseointegrative bone implant among Spirula"s ancestors and defined as "mural flap": at the junction of the surface in comparison to the common implant materials. septa with the shell wall, the lamello-fibrillar structure of the septa is inserted We have extended this work to covalently bound polysaccharide-based between two sub-layers of the internal microstructure of the wall, in the form coatings using the same approach to bind and mineralize Hyaluronic acid. of a bevel. From a biochemical viewpoint, the organic matrix of S. spirula is Also, fully synthetic bone implant surface modifications are presented. essentially proteinaceous and saccharidic with macromolecules of discrete and non-discrete molecular weight. The soluble fraction reacts with WGA, T 53 LEL, jacalin and concanavalin A lectins. Our proteomic investigations Multiscale characterization of bone matrix changes in a pre- identified several protein hits. However, most of them occur with one peptide metastatic mouse model of breast cancer only and do not match with already known mollusc skeletal proteins. This C. Liu*1, A. E. Chiou2, I. Moreno1, T. Tang1, W. Wagermaier1, M. Dean1, strongly suggests that the shell matrix of the ram"s horn squid is quite P. Fratzl1, C. Fischbach2 different from that of other shell-bearing molluscs studied so far. We come 1Max Planck Institute of Colloids and Interfaces, Department of to a similar preliminary conclusion by analyzing the proteomic data obtained Biomaterials, Potsdam, Germany from the shell matrix of Argonauta hians, also characterized by a majority of 2Cornell University, Meinig School of Biomedical Engineering, Ithaca, 'one-peptide' hits. Our study emphasizes the diversity of shell matrix United States components in cephalopods, which may put into question a single molecular model for describing shell formation in this mollusc class. Introduction T 51 Metastatic breast cancer often spreads to bone, resulting in incurable Tools for understanding proteins of unknown function in osteolytic lesions and poor clinical outcome. While bone degradation is a biomineralization hallmark of bone metastasis, recent studies indicate that early-stage bone metastasis may depend on tumor cell adhesion to osteogenic regions. A. Skeffington*1, A. Ohara2, A. Milentyev3, C. Heintze2, S. Görlich2, N. Increasing evidence suggests that primary tumors can prepare metastatic Poulsen2, A. Fischer1, M. Brzezinka1, M. Gorka1, A. Graf1, N. Kröger2, A. niches via circulating tumor-derived factors, but it remains unclear whether Scheffel1 this also occurs in breast cancer bone metastasis, and if changes in the bone 1MPIMP, Organelle Biology and Biotechnology, Potsdam-Golm, Germany matrix play a role in this process. 2B CUBE Center for Molecular Bioengineering, Dresden, Germany Objectives 3MPI of Molecular Cell Biology and , Dresden, Germany To test the hypothesis that primary breast cancer can alter bone matrix across various length scales prior to metastasis, we have applied multiscale analysis Proteins have been found to be intimately associated with biominerals from techniques to characterize the bones of a mouse model of pre-metastatic a wide range of organisms. Many roles for these proteins have been breast cancer. postulated, including the stabilisation of amorphous phases, generation of Methods hydrogels and supramolecular templates, control of crystal nucleation and To model the effects of circulating tumor-derived factors, 3-week female the control of crystal morphology. When examining the results of a nude mice (n=5) received daily intraperitoneal injections of either tumor cell proteomics experiment designed to identify proteins associated with a given conditioned media (TCM), collected from the human breast cancer cell line biomineral, studies often focus on those proteins with similarity to proteins MDA-MB-231, or blank media (control), for a period of 3 weeks. Because or domains of known function, which have normally been characterised in the proximal tibia and distal femur are common sites of bone metastasis in organisms that do not biomineralize. However a number of the proteins mice, the hindlimbs were harvested for analyses. For each mouse, one tibia identified in biomineralization proteomic experiments are normally was fixed in 70% ethanol and subjected to micro-computed tomography annotated as proteins of unknown function and indeed we would expect (micro-CT), backscattered electron (BSE) imaging, confocal laser scanning proteins important for a particular mode of biomineralization to be largely microscopy (CLSM), Raman microspectroscopy and laboratory-based small- restricted to those taxa that engage in this process. Thus, it is clear that to angle X-ray scattering (SAXS). The contralateral tibia was fixed, decalcified, understand taxon specific biomineralization processes we need to understand and processed for histological analysis. Distal femurs were used to harvest the proteins of as yet unknown function associated with biominerals. RNA from trabecular bone for sequencing (RNAseq). Molecular genetic tools for many biomineralizing organisms are in the early Results stages of development, meaning that experimental investigations into the Micro-CT analysis of trabecular bone in the proximal tibia of TCM-injected roles of proteins of unknown function are highly laborious. Thus, mice revealed that bone volume fraction, trabecular thickness, and trabecular computational tools are required to investigate the properties of the proteins separation increased significantly relative to control mice, while trabecular and thereby guide and focus experimental work. In this presentation I will number decreased. BSE agreed with these changes, and further indicated that describe a range of user-friendly computational tools for the analysis of the increase in trabecular bone area for TCM-injected mice was most proteins of unknown function. This includes tools to examine the statistical prominent within 200 µm to the metaphyseal growth plate. SAXS analysis properties of sequences, including local and global biases in amino acid within this region demonstrated that the thickness of mineral crystals does composition, the degree of intrinsic disorder and sequence complexity. In not change, but their degree of orientation was slightly decreased in TCM- addition, the tools can be applied to find overrepresented motifs and can be injected mice compared to control mice. Bone dynamic histomorphometry used for the visualisation and exploration of these data. I will also describe a showed the cortical bone in TCM-injected mice has higher mineral new method for the identification of novel domains independent of multiple apposition rate compared to the control. Raman data showed increased sequence alignment methods. To illustrate the utility of these tools, I will use mineral-matrix and carbonate-mineral ratios in the endocortical bone of the our own proteomic data, focussing on the silica associated proteins of TCM group. Ongoing experiments examine differences in bone remodeling diatoms and the calcite associated proteins of coccolithophores. I will also cells via immunostaining, collagen organization via second harmonic demonstrate that new information can be found in previously published data generation imaging and gene expression profiles via RNAseq. sets using these methods, focussing on data from molluscs and echinoderms. Conclusion Together, these results suggest that circulating tumor-derived factors T 52 modulate both trabecular and cortical bone structures from the micro- to Osseointegrative one implant coatings nano-scales in sites prone to breast cancer bone metastasis. Changes in the H. Cölfen*1, J. Knaus1, M. Gießl1, D. Schaffarzcyck2 bone microstructure, degree of mineral crystal orientation, mineral 1Universität Konstanz, Physikalische Chemie, Konstanz, Germany apposition rate and carbonate-mineral ratio suggest that bone remodeling and 2stimOS GmbH, Konstanz, Germany osteogenic activity may increase due to tumor-derived factors, which may provide a favorable metastatic niche in bone, even before their arrival at bone tissues. Implants are important materials to replace bone. The today commonly used materials are titanium and polyetheretherketone (PEEK). While titanium is

19 T 54 The defects were induced in the rat tibia diaphysis for 07, 14 and 21 days. Effect of mother-of-pearl feeding on bone loss due to Microspheres (400-600 μm in diameter) composed of nanostructured ovariectomy in the rat carbonated hydroxyapatite (CHA) and sodium alginate were implanted in K. D. Nguyen1, N. Laroche1, A. Vanden Bossche 1, Y. Bertache1, M. T. group 2 for the same experimental periods. Linossier 1, M. Thomas 1, S. Peyroche 1, M. Normand 1, L. Vico 1, M. SR-μCT analyses of the tibia defects were conducted in the IMX beamline Rousseau*1,2 of the Brazilian Synchrotron Light Laboratory (LNLS) in Campinas/Brazil. 1U1059 Inserm - Sainbiose (Santé INgéniérie Biologie St-Etienne) Campus The reconstructed 3D images were segmented and skeletonized with Avizo Santé Innovation, Université Jean Monnet, Saint-Priest-en-Jarez, France (FEI, Oregon, USA), Fiji/ImageJ (http://fiji.sc/Skeletonize3D), PNExtract ( 2UMR5510 Mateis, CNRS/Lyon University/INSA-Lyon, Lyon, France Imperial College and ITA App (Weizmann Institute of Science) software.

Introduction The 3D reconstruction of the trabecular network and its skeletonization Previous in vivo and in vitro studies showed that mother-of-pearl or nacre, a were used to determine and quantify morphological parameters such as the natural material of marine origin, influence bone health. Nevertheless, number of edges and nodes, length and thickness distribution, planarity, limited studies investigated the effects of oral nacre supplementation in bone intra-trabecular angles and the amount biomaterial surface covered with loss model. In addition, the model of bilaterally ovariectomized (OVX) rats, new bone. After seven days surgery, a trabecular network consisting of which mimics the acceleration of bone loss observed in postmenopausal small trabeculae (≈ 5 μm) was already formed in group 1 and progressed to women due to estrogen deficiency, is well established in investigations of the center of the gap, which was covered with new trabecular bone in 21 osteoporotic therapies. days. The trabecular architecture was based on geometrical motives Objective consisting of 3, 4 and 5 trabecular edges interconnected to a node (3N, 4N This study was aimed to investigate the pharmacological effect of oral and 5N, Nathaly et. al, 2016). The distribution of inter-planar angles (ITA) administration with powdered nacre on ovariectomy-induced had a maximum at 118 °, 106 ° and 96°, for 3N, 4N and 5N motifs, postmenopausal osteoporosis in female rats. respectively. After 21 of the CHA implantation, new trabecular bone was Materials & Methods: Eight-week-old 58 femal Wistar rats were purchased adhered to 70% of the spheres fragments surface and occupies the majority in the study. At 16 weeks of age, the Baseline group (n = 10) was sacrificed; of the gap and the tibia medullary cavity. The presence of the biomaterial 48 rats were either sham- operated (Sham group, n=12) or ovariectomized inhibited the trabecular growth (length and thickness) but did not modify (n=36). These 36 ovariectomized rats were randomly divided into three the inter-trabecular angle distribution. The trabecular architecture is lost subgroups (n = 12 each): OVX group (the standard diet), OVX CaCO₃ group when new bone is very close (50-150 μm) and in contact with the (250 mg CaCO₃ / kg body weight / day in the standard diet) and OVXB Nacre biomaterial surface. SEM-FIB and TEM analyses showed that group (250 mg Nacre / kg body weight / day in the standard diet). Their bone/biomaterial interface was composed of disordered mineral tissue corresponding treatments were administrated orally for 4 weeks. This study grown close to the surface, and into the surface pores smaller than 1 μm. was reviewed and approved by the Institutional Animal Care and Use The results demonstrated that bone repair induced in rat tibia defects Committee (IACUC) of St-Etienne Univ (Approval No.: #13401- initiated from interconnected edges and nodes structures, with triangular 2018020615412128 v6). Changes in body weight as well as those in the planar, tetrahedral and pentahedral geometries. Since the tibia diaphysis uterus were measured. PIXImus measured BMD and % fat mass in vivo. The was not submitted to extensive mechanical load during the 07 days of the bone microstructure of the proximal tibia was assessed by μCT imagery in animal life, we may conclude that trabecular topology was an intrinsic vivo and ex vivo. The level of mRNA expression of genes involved in bone characteristic of the trabecular bone formation in rats. This inherent feature formation and resorption was analyzed by qRT-PCR. The Kruskal-Wallis of the trabecular bone was not altered with the implantation of the and Mann-Whitney U-tests with the FDR adjustment were used to analyze biomaterial, and the presence of numerous nanostructured fragments that the different data. occupied the defect volume and interacted with the newly formed bone. Results Mineral tissue may be grown in sub-micron porosity of the biomaterial Changes in body and uterine weights confirmed the OVX model inducing surface. estrogen deficiency (p <0.001). The increase in body weight of the OVX Nacre group was significantly limited after 2 weeks and 4 weeks compared T 56 with OVX group (p <0.01). The presence of extensive trabecular In-situ hydrostatic compression and diffraction reveal deterioration (μCT in vivo) of the three OVX groups confirmed the onset of increased stiffness of ashed dentine apatite mineral bone loss. The ex vivo study showed that mother-of-pearl treatment improves J. B. Forien*1, C. Fleck2, C. Krywka3, A. C. Deymier4, P. Zaslansky5 the trabecular bone volume. Representative genes for bone formation and 1Lawrence Livermore National Laboratory, Livermore, United States resorption increased significantly in OVX groups compared to the Sham 2Technical Universit Berlin, Berlin, Germany group (p <0.01). Some genes (e.g. OPN, RANK) are overexpressed in OVX 3Helmholtz-Zentrum Geesthacht, Geesthacht, Germany Nacre group compared with OVX and OVX CaCO₃ group (p <0.05). 4University of Connecticut, Farmington, Germany Conclusion 5Department for Operative and Preventive Dentistry, Charité - The results obtained provide evidence of the protective effect of mother-of- Universitätsmedizin Berlin, Berlin, Germany pearl on weight gain related to estrogen deficiency. We also found an improvement in the trabecular parameters which determined bone Tooth dentine is a bone-like material containing carbonated hydroxyapatite microstructure. In conclusion, our study is the first step to advance towards nanoparticles (cHAp) within and surrounding a network of collagen fibrils the development of a new osteoporosis therapy based on mother-of-pearl. with submicrometer diameters. The role of the mineral particles is to stiffen the nanocomposite and to enhance both stiffness and toughness of dentine so T 55 that it can sustain decades of intense daily, cyclic mechanical stress. The Trabecular bone growth and architecture in tibia defects nanometer size of the mineral particles makes the measurement of their implanted with carbonated hydroxyapatite microspheres elastic properties technically challenging. Consequently, it is widely assumed A. Malta Rossi*1, V. Martinez-Zelaya2, N. Lopes Archilha2, M. that the elastic properties of biogenic are identical to those Calasans-Maia3, M. Farina4, A. Linhares Rossi1 of geological apatite. However, in a previous study we found that pristine 1Brazilian Center for Research in Physics, Condensed Matter, Applied dentine apatite particles are about 20% softer than geological and synthetic Physics and Nanoscience, Rio de Janeiro, Brazil apatites and that the mineral has an average bulk modulus K=82.7 GPa. 2Brazilian Synchrotron Light Laboratory, Campinas, Brazil, Campinas, In a new series of measurements, we investigate the effect of dentine heat Brazil treatment on the response of the apatite phase of the composite to 3Federal Fluminense University,Niteroi, Brazil compression. X-ray diffraction combined with in situ hydrostatic water- 4Federal University of Rio de Janeiro, Rio de Janeiro, Brazil mediated pressurization were used to track changes in the c and a crystal lattice parameters. Dentine samples were measured wet under pristine The trabecular bone architecture and its dependence on the mechanical load condition, after annealing for 1 hour at 250 deg, and after ashing for 10 hours have been intensively reported in the literature. In recent work, Nathaly at 550 deg. Reznikov et al. (2016) introduced the concept of inter-trabecular angle to Results show a decrease of ~3% in Young"s modulus from pristine to describe the architecture of the trabecular network in adult human femora. partially annealed dentine (250 deg, 1 hr), and an increase of ~7% from The authors concluded that the trabecular topology present geometrical pristine to fully annealed dentine (550 deg, 10 hrs). Similarly, bulk modulus motifs with tensegrity. decrease by ~3% and increase by about 7% from pristine to partially and fully In the present work, we used Synchrotron Radiation-based X-ray annealed dentine, respectively. The change of properties in cHAp upon heat microtomography (SR-μCT), SEM-FIB (Slice and View) and transmission treatment is attributed to an initial partial degradation of the organic phase electron microscopy (TEM) to investigate the trabecular architecture induced followed by ashing leading to loss of carbonate impurities. in rat tibia diaphysis defects in the early times of bone growth, in the absence (group 1) and presence of a nanostructured biomaterial (group 2).

20 T 57 agglomerates of thin mineral particles surrounding unmineralized spaces 30 Heat-induced changes in bone mineral structure and to 50 nm in diameter3-5. Both the filamentous (longitudinal) and lacy pattern chemistry: transformation from carbonate-apatite to (transverse) are thought to be different projections of the same ultrastructure, hydroxyapatite but the underlying 3D mineral arrangement is yet unclear. Objectives M. Greiner*1, A. B. Rodríguez-Navarro2, M. F. Heinig1, K. Mayer3, B. In order to understand the formation of these mineral deposition patterns, we Kocsis1, A. Göhring3, A. Toncala3, G. Grupe3, W. W. Schmahl1 studied early stages of mineralization using a simplified but equivalent model 1Ludwig-Maximilians-Universität München, Department of Earth- and system to bone: the avian leg . Its collagen fibrils, arranged in a Environmental Science, Munich, Germany parallel fashion, mineralize as the animal ages and permit the mineralization 2Universidad de Granada, Departamento Mineralogía y Petrología, process to be monitored precisely. Study aims were to increase understanding Granada, Spain of collagen fibril mineralization and to compare resulting data with recently 3Ludwig-Maximilians-Universität München, Fakultät für Biologie, described bone mineral patterns2-5. Anthropologie und Humangenomik, Planegg-Martinsried, Germany Materials and methods The present work combines Transmission Electron Microscopy (TEM), Bone is a complex hierarchically structured composite material constituted Scanning Transmission Electron Microscopy (STEM), and Selected Area of carbonated apatite (bioapatite) nanocrystals mineralizing collagen (type I) Electron Diffraction (SAED) to elucidate crystal distribution and orientation microfibrils. Biological apatite chemical composition is crucially different through the mineralization process. Elemental analysis by means of Energy from stoichiometric hydroxyapatite being particularly susceptible to marked Dispersive X-ray Spectroscopy (EDS) was used to characterize the organo- changes in ionic substitutions on the cationic, phosphate and channel anionic mineral interface of structures occurring during mineral formation and to sites during heating. asses Ca/P molar ratio. Experimental incineration experiments were conducted in order to better Results understand changes in the complex biomineral during heating. The Early mineral aggregates were found in extrafibrillar collagen spaces, experiments were motivated by archeological and forensic research following the contour of the fibrils. Crystallites then propagated within fibrils questions, that can also shed important light on our understanding of the bone (at sites where mineral was also outside the same sites) following curved mineral structure and properties. trajectories. As these foci of mineralization grew, they appeared as radially- Bovine cortical bone pieces were heated at 100, 200, 300…1000 °C for 150 oriented clusters of relatively large crystal aggregates. Mineral foci expanded minutes. In addition, short-time incinerations (10, 20, 30… 60 minutes) at until they coalesced, encompassing several fibrils and forming a continuous 650 °C and 700 °C were performed to understand the time-dependence of the mineral network. The resulting pattern strikingly resembles the lacy pattern reaction in a critical temperature range. Original and heated bone samples described for bone ultrastructure2-5. were investigated using complementary analytical methods such as X-ray Conclusion powder diffraction (XRPD) with advanced profile deconvolution by Rietveld These analytical observations support the concept that early stages of refinement, Fourier-transform infrared spectroscopy (FTIR), and infrared- mineralization in avian leg tendon lead to formation of deposition patterns coupled thermogravimetric analysis (TGA-FTIR). that are similar to those documented in bone. This result would suggest a No hydroxyl ions are discernible in FTIR spectra of original bone mineral possibly common mineralization mechanism for type I collagen-based 2- while CO3 and H2O bands are prominent. Original bone mineral has a XRD materials. crystallite size with an average of 65 Å in the ab plane and 174 Å along the 1. Landis, Song, Leith, McEwen, McEwen. 1993. J. Struct. Biol. 110, 39- c-axis. A pronounced increase of crystallite size at 700 °C after 30 minutes 54 of heat treatment can be observed, from 205 Å along [001] and an average of 2. Rubin, Jasiuk, Taylor, Rubin, Ganey, Apkarian. 2003. Bone 33, 270-282 134 Å in the (001) plane (700 °C/30 min) to 392 Å along [001] and an 3. McNally, Schwarcz, Botton, Arsenault. 2012 PLosOne 7(1): e29258 average of 266 Å in the (001) plane (700 °C/40 min). 4. Grandfield, Vuong, Schwarcz. 2018. Calcif. Tiss. Int. 103, 606-616 Rather than a simple recrystallization, this process can be better described as 5. Reznikov, Bilton, Lari, Stevens, Kröger. 2018. Science 360, eaao2189 2- a reaction with a decrease of CO3 and H2O and an increase of hydroxyl groups in the apatite lattice, as detected by FTIR. Thus, the bovine bone T 59 mineral should definitely be referred to as "carbonate-hydro-apatite" rather Infrared imaging analysis of collagen and hydroxyapatite in than hydroxyapatite. sclerotic aortic valve tissue The carbonate content in bone decreases further with higher treatment A. Mieting*1, C. Dittfeld1, A. Jannasch1, K. Plötze1, S. M. Tugtekin1, K. temperatures and only weak carbonate signals are observed in bone heated at Matschke1, G. Steiner2 1000 °C. Above 800 °C, buchwaldite (CaNaPO4) is formed from the Na 1Dresden University of Technology, Faculty of Medicine Carl Gustav component of the bone mineral. Signals of combusted organic compounds in Carus, Department of Cardiac Surgery, Dresden, Germany the TGA-FTIR spectra clearly diminish at temperatures higher than 650 °C. 2Dresden University of Technology, Faculty of Medicine Carl Gustav The time-dependence of the recrystallization-reaction at 650°-700°C is much Carus, Clinical Sensoring and Monitoring, Dresden, Germany more complex than a simple Boltzmann/Arrhenius exponential relation. From our data we can conclude that the recrystallization reaction from bioapatite to hydroxyapatite accelerates dramatically after the complete Introduction combustion of all organic compounds of the bone, i.e., when the apatite Aortic valve (AV) fibrosis is accompanied by collagen remodelling which grains come into direct contact to each other (Greiner et al. 2019). promotes accumulation of hydroxyapatite, resulting in a pathological Greiner, M., Rodríguez-Navarro, A., Heinig, M.F., Mayer, K., Kocsis, B., biocomposite. Biomineralization is the main characteristic of calcified aortic Göhring, A., Toncala, A., Grupe, G., Schmahl, W.W. (2019) Bone valve disease (CAVD) and in glutaraldehyde cross-linked AV bioprostheses incineration: An experimental study on mineral structure, colour and but pathophysiology is still not fully understood. Infrared (IR) spectroscopic crystalline state. Journal of Archaeological Science: Reports, 25, 507- 518. imaging yields spatial information e.g. of the collagen and mineralic composition due to unsupervised chemical information of the spectroscopic T 58 data set by multivariate chemometrics. An IR-spectrum of biomineralization shows vibrational band assignments for phosphate of the apatite structure and Mineral deposition and structure in the avian leg tendon -1 1 2 1 1 3 for the carbohydrate moieties of collagen at 950-1200 cm and for the E. Macias-Sanchez* , Z. Zou , T. Tang , L. Bertinetti , N. Tarakina , W. J. -1 Landis4, P. Fratzl1 secondary structure of collagen by amid bands at 1500-1700 cm . 1Max Planck Institute of Colloids and Interfaces, Biomaterials, Potsdam, Objective Germany Aim of the study is the investigation and association of different chemical 2Wuhan University of Technology, State Key Laboratory of Advanced properties of hydroxyapatite and amid I and II alterations in biomineralized Technology for Materials Synthesis and Processing, Wuhan, China collagen of calcification nodules in CAVD tissues by IR-spectroscopic 3Max Planck Institute of Colloids and Interfaces, Colloid Chemistry, imaging followed by multivariate chemometrics. Potsdam, Germany Materials & methods: Tissue samples of calcified human AV and 4The University of California, San Francisco, United States bioprostheses were cryosectioned and transferred on CaF2 slides. IR- microscopy: FT-IR spectrometer Vertex 70; infrared microscope Hyperion 3000; MCT focal plane array detector. Before multivariate analysis, the Introduction spectra were evaluated for outliers, corrected for baseline and area Mineral deposition in bone and mineralized tendon has been well described normalized (Matlab). Representative parts of the sections were defined from in electron microscopy of longitudinal tissue sections, based on the banding visible light image. K-means clustering analysis was used to group the pattern of collagen and mineral platelets observed mainly parallel to the preprocessed infrared spectra. Principle component analysis (PCA) was collagen fiber long axis1. Deposition pattern in transverse sections2-5 is calculated on correlation matrix of the imaging data set in the spectral ranges more uncertain and remains controversial. In particular, the question to which of 950-1200 cm-1 and 1480-1800 cm-1. extent mineral particles are predominantly intra- or extrafibrillar is still Results debated3-5. The cross-section deposition pattern has been described in terms Spectral information of initial sample set were grouped by cluster analysis in of a "lacy pattern" that consists of groups of concentrically arranged, curved spatially distinct mineralic and organic regions with characteristic spectral

21 band features for each tissue group. PC1-PC4 of amid I and II bands of excretion. Here, the results suggest that this inhibition of COM does not only mineralized tissue areas reveal an increased content of β-sheet collagen occur at the microscale but upon the very nanoscale nucleation of CaOx. -1 conformation demonstrated by signals at 1623, 1673 and 1690 cm . In addition a signal at 1515 cm-1 is detected referring to an increased content of T 61 tyrosine residues in mineralized tissue areas of the human AV. The non- Precipitation mechanisms in renal calcium phosphate plaques mineralized organic AV tissue is characterized by α-helical collagen -1 and their relevance for the growth of calcium oxalate kidney conformation assigned to 1558 and 1662 cm . In comparison, glutaraldehyde stones cross-linked pericardial tissue of bioprosthesis exhibits additionally signals I. Sethmann*1, G. Wendt-Nordahl2, T. Knoll2, H. J. Kleebe1 at 1532 cm-1 for anti-parallel β-sheets and signals at 1684 cm-1 for β-sheets 1Technische Universität Darmstadt, Institut für Angewandte of collagen. In contrast, changes in the range of 950-1200 cm-1 are similar in Geowissenschaften, Darmstadt, Germany AV and bioprosthesis: non-mineralized tissue is characterized by 2Klinikum Sindelfingen-Böblingen, Urologische Klinik Sindelfingen, carbohydrate groups of collagen at 973, 1030 and 1075 cm-1 and mineralized 3- -1 Sindelfingen, Germany tissue is characterized by PO4 of apatitic structures at 996 cm and an assignment for poorly crystalline apatites at 1110 cm-1. Conclusion Introduction Collagen conformation is modified in calcified AV tissue and in Calcium oxalate monohydrate (COM) kidney stones often occur attached to bioprosthesis due to chemical pretreatment or due to the origin of the calcium phosphate (CaP) plaques that form in the interstitium of the renal pericardial tissue. An increased β-sheet collagen content and a higher inner medulla and can come in contact with urine at lesions of the epithelium tyrosine content in collagen of mineralized human AV tissue is detected by of the renal pelvis. These CaP precipitates, called Randall"s plaque (RP), IR-spectroscopy in an initial dataset. Also in pericardial bioprosthetic tissue seem to be a precondition for the formation of these COM stones. RP, as a an increased β-sheet content was monitored that may promote degeneration form of pathological biomineralization, forms in the course of ion re- by calcification. absorption in the nephrons at high levels of ion concentration in the interstitial fluid and possibly significantly increased levels of CaP T 60 supersaturation.[1] Comprehensive knowledge of the conditions and In situ nanoscale biomineralization of calcium oxalate kidney mechanisms of the formation of RP may be the key to inhibiting the stone in presence of molecular modifiers precipitation of CaP and preventing the growth of COM renal stones. T. Shokuhfar*1, R. Shahbazian 1 Objective 1University of Illinois at Chicago, Bioengineering, Chicago, United States The aim of this study was a microstructural investigation of RP attached to COM stones in order to conclude on conditions and processes that lead to RP precipitation and initial COM stone growth. Since direct observation of Calcium oxalate (CaOx) is the primary constituent of kidney stones, calcium plaque formation and analyses of the local conditions at the formation sites storage medium in plants, and may play a role in plant-mediated capture of is hardly possible, model experiments mimicking the precipitation process atmospheric carbon. Here, CaOx nucleation was studied in real-time with have to be designed to enable monitoring the mechanisms involved. The nanoscale resolution via graphene liquid cell transmission electron synthesis of structural analysis and experimental simulation is expected to microscopy. We observed the co-existence of both classical nucleation and lead to a better understanding of RP formation. non-classical nucleation pathways by which the structure and morphology of Materials and Methods CaOx crystals show distinct characteristics. Interestingly, we observed that RP attached to surgically removed COM kidney stones was investigated the addition of citrate to the CaOx solution significantly reduced the using scanning electron microscopy (SEM) and integrated energy-dispersive thermodynamic stability of CaOx nuclei, interfering with CaOx nucleation, X-ray (EDX) spectroscopy. Model experiments were performed using and promoted the formation of CaOx dihydrate (COD). This study shows that hydrogels with simulated body fluid in a double diffusion set-up for CaP manipulation of nanoscale formation pathways may influence macroscale precipitation mimicking the formation of RP. The precipitation process and properties dependent on the mineral morphology, crystal structure, and its products are currently being characterized using vibrational spectroscopy, hydration state, such as mechanical properties, solubility, and biological cell X-ray diffraction and SEM techniques. attachment of CaOx. Results In Situ GLC-TEM Imaging In RP, the mesh size of the interstitial tissue appeared to be positively In situ TEM imaging was achieved via encapsulation of 0.1M CaCl2 and correlated to the local size of the CaP particles. Larger particles showed 0.1M NaOx in control samples and 0.1M CaCl2, 0.1M NaOx, and 0.1M spherical morphologies and laminated internal structures suggesting an initial citrate in the citrate treated samples. The samples were added to precipitation as metastable amorphous calcium phosphate (ACP) which may microcentrifuge tubes and mixed. 0.5µl of the solution was added to a have crystallized at a later stage.[2] In COM stones, crystals in contact with graphene coated grid. A secondary graphene coated grid was placed RP showed morphologies different from the bulk of the stones and in many graphene side down on the liquid sample. The grid was then placed into a places they were encrusted with a thin layer of CaP.[2] Model experiments TEM sample holder. The TEM holder was placed into a vacuum pump to show that the density of the gel network is negatively positively correlated remove any encapsulated liquid from the TEM holder and the sample. The with the waiting time for precipitation and negatively correlated with the sample was then imaged in TEM at 80kV. The increase in the contrast of the spreading rate of the cloud of CaP particles. Further characterization of the particle throughout the video was measured as a function of the grey scale of precipitates is in progress. the image, where C is the measured greyscale: Conclusions ∆C=C_particle/C_bacground -1. We conclude that locally differing spatial confinements in the tissue and the The electron dose rate was calculated by: related restrictions of ion diffusion have an impact on particle sizes and Ψ=(10^5 SI)/(πa^2 ) arrangements in RP. Furthermore, we propose that CaP-(super)saturated Where S is the stopping power ((MeVcm^2)/g), I the current, a the radius of interstitial fluid may diffuse through RP and into the urine, where it could the electron beam (m), and 105 converts units SI units to Grays/s. trigger COM and CaP precipitation at the RP–urine interface and, hence, The STEM- data was acquired using a Hitachi HD2300 STEM initiate stone growth. microscope operated at 200 kV. The energy range of 270-577.2 eV was References examined with 0.30 eV energy dispersion and EELS aperture of 3mm. [1] A.P. Evan, F.L. Coe, J. Lingeman, S. Bledsoe, E.M. Worcester, Am. J. Energy windows of 50 eV were used to compare the ratio of O:Ca using the Physiol. Renal Physiol. 315 (2018) F1236–F1242. O K edge and Ca L edge. Electron exposure was set to 15 seconds. The EELS [2] I. Sethmann, G.Wendt-Nordahl, T. Knoll, F. Enzmann, L. Simon, H.-J. data collection electron dose rate was between of 0.08 to 38.20e-/Å2s while Kleebe, Urolithiasis 45 (2017) 235–248. the total dose was between 1.16 to 573 e-/Å2. CONCLUSIONS T 62 Here, real-time nanoscale studies show the nanoscale nucleation of CaOx via Molecular structure of osteopontin fragment at the interface two pathways. Classical nucleation causes formation of rhombohedral COM, with calcium ion interaction and within calcium oxalate the most thermodynamically favorable crystal structure of CaOx, whereas monohydrate composite rectangular COM forms via non-classical nucleation. Further, formation of 1 2 3 2 3 1 both ex situ and in situ nanoscale COM via classical nucleation indicates the H. Lu* , S. Alamdari , S. Roeters , J. Pfaendtner , T. Weidner , M. Bonn 1Max Planck Institute for Polymer Research, Molecular Spectroscopy, lower ∆G and 〖∆G〗^‡ of COM as compared to COD. Citrate increases the Mainz, Germany 〖∆G〗_COM^‡ over the relatively high 〖∆G〗_COD^‡ to induce the 2University of Washington, Department of Chemical Engineering, Seattle, Washington, United States formation of COD via cyclic aggregation of CaOx rather than via classical 3 nucleation. The inhibition of CaOx formation by citrate occurs at the Aarhus University, Department of Chemistry, Aarhus, Denmark nanoscale, which prevents formation of COM, but with high mixing this energy barrier is overcome to form COD in bulk solutions. Introduction . Since COD does not bind to cell walls as well as COM, citrate may prevent Calcium oxalate monohydrate (COM) is the major inorganic mineral kidney stone formation by preferentially forming COD to promote CaOx component of kidney stone – the chronic human disease threatening peoples's

22 lives for years. Understanding molecular mechanisms of biological control Results over COM crystallization will promote development of effective stone One of the most efficient urease producing and Pb tolerant bacterial strain, disease therapies and stimulate novel strategies for synthesizing biologically identified as Lysinibacillus sp. was selected in this research. During seven inspired materials. The most potent kidney stone inhibitor is osteopontin days of experiment, urease production was significantly more in media with (OPN). OPN protein and its peptide fragments are considered to be poly-Lys with maximum activity on the fifth day and pH was also on the rise. intrinsically disordered and they have significant flexibility to bind calcium The concentration of Pb decreased significantly on the fifth day with removal ions. Despite previous extensive literatures all attest the great importance of efficiency of 81% and 74% without poly-Lys, when the initial Pb direct interaction between OPN (or OPN peptides) and COM mineral surface concentration was 50 and 100 mg/L, respectively. The addition of poly-Lys – due to the general difficulty in probing protein (peptide) surface structure led to further decrease in Pb concentration, with removal efficiency of 87% – the exact interaction mechanism and molecular picture of OPN at its and 83%, respectively. The FTIR spectra revealed number of peaks mineralization interface are all the time missing. indicative of CO3 bonding and formation of carbonate compounds in MICP Objectives samples; however, such peaks were more intense in the presence of poly-Lys. Here we focus on the functional OPN peptide domain (i.e. OPN62-85 XRD identified peaks of calcite, in addition to confirmation on H2NSNESHDHMDDMDDEDDDDHVDSQDCOOH). We combine immobilization of Pb in the form of PbCO3. surface specific sum frequency generation (SFG) spectroscopy and Conclusion molecular dynamics simulations to probe peptide structure at the interface The presented study improved biomineralization efficiency of carbonate with calcium ion interaction and within its inherent COM composite. precipitation in metal immobilization with poly-Lys addition. Materials & methods OPN peptides firstly absorbed at air-water interface, by injecting calcium T 64 cations and subsequent oxalate anions, we were capable to use SFG to in situ Biosynthesis of copper nanoparticles from mine waste probe the peptide at the interface with calcium ion interaction and within J. Ordóñez*1, L. S. Wong-Pinto1 mineral composite. SFG spectroscopy is a second order non linear optical 1Universidad de Antofagasta, Department of chemical and mineral process spectroscopy by overlapping visible and infrared laser pulse. Its selection engineering, Antofagasta, Chile rules dictate that SFG spectroscopy only probes the "interface" without interference from bulk solutions. Molecular dynamics simulations were performed to complement SFG results. Calculated SFG spectra from Introduction the simulated structure were compared with experimental spectra, thereby Chile is one of the most important worldwide producers of copper, which further verifying the simulated peptide structure. results in the generation of a large amount of waste, such as ore tailings. Results Latest reports indicate that eight billion tons of tailings have been generated OPN peptides restructure strongly with Ca ions at their interface prior to from the copper mining industry. In tailings remain a broad spectrum of calcium oxalate nucleation. In contrary to one disordered motif, we disclose species among them copper, that. On the other hand, nanosized materials that – induced by the bidentate binding Ca ions – OPN peptides adopt highly have unique properties compared to the macromaterial counterparts, due to ordered beta turn motif. The ordered peptide structure is retained after COM their quantum confinement. For this, the application of nanoparticles (NPs) mineralization. in strategical, medical and renewable energy devices are in full development. Conclusion Cheaper and more eco-friendly techniques for nanoparticles synthesis is the Our results, on the one hand, provide direct molecular picture of OPN key current challenge to increase the application of these materials. fragment at their inherent relevant interfaces, and on the other hand, Objective demonstrate the strength of combining SFG spectroscopy and molecular This work aims to synthesise copper nanoparticles (CuNPs) from copper ore simulation in probing weakly folded peptides at their functional interfaces. tailings, using bacterial biomass of Pseudomonas stutzeri. Material and methods T 63 Samples of ore tailings were collected from porphyry copper deposits located Improved biomineralization of lead in aqueous media by in active mining operations in northern Chile. Mineralogical and chemical characterisation was carried out by X-ray diffraction (XRD), microbial carbonate precipitation assisted with cationic microfluorescence short-wave infrared reflectance spectrometry (SWIR), polypeptide and by atomic absorption spectroscopy (AAS). 1 1 1 J. He , W. Li , V. Achal* Biosynthesis of CuNPs was performed using 10 g/L biomass of P. stutzeri in 1 Guangdong Technion Israel Institute of Technology, Environmental two different solutions: i) CuSO4 6 mM, and ii) leached tailings (copper Engineering, Shantou, China concentration of 1.4 mM), stirred at 150 rpm for 24 h. After biosynthesis, biomass was discarded by centrifugation, and the supernatant containing Introduction CuNPs was then analysed by microfluorescence, Fourier transformed Ever increasing urban expansion, industrial development and anthropogenic infrared spectroscopy (FTIR), atomic force microscopy (AFM), X-ray activities are major sources of polluting environment with heavy metals. photoelectron spectroscopy (XPS) and field-emission scanning electron Posing with serious environmental problems, heavy metals are the microscopy coupled with energy dispersity X-ray spectroscopy environmental priority pollutants and require ultimate remediation to protect (FESEM/EDX). both health and environment. In recent years, biomineralization involving Results ureolytic bacteria producing enzyme urease that leads to carbonate Tailings have high levels of copper remaining about 1000 ppm, mainly as precipitation, chiefly calcite, was found effective in combating heavy metal chalcopyrite (CuFeS2). The absence of copper oxides and the predominance toxicity. This biocalcification process, widely known as "microbially of kaolinite in the clays was verified, which was useful to define the leaching induced carbonate precipitation (MICP)" has the ability to immobilize heavy conditions. CuNPs were obtained both synthetic CuSO4 solution and leached metals efficiently by precipitating them during urea hydrolysis. tailing. Preliminary results suggest that the use of fresh biomass show best Objectives biogenic activity than lyophilised one. Analysis of structural variations in the Lead (Pb) is widely recognized by its highly toxic and mobile nature, also cell wall indicates biosorption and eventually, bioreduction, activity in both brings severe negative effect on human health and the environment. Thus, treatments, related to hydroxyl, methylene and carboxyl groups. Various the process of MICP was adopted in presented research to immobilize Pb. characterisation techniques confirmed bioreduction; microfluorescence analysis revealed that after biosynthesis, on the surface of biomass, Cu, Fe Further, considering the role of cationic polypeptide in CaCO3 crystallization, study aimed to improve the MICP efficiency to immobilize and S were detected. AFM images showed the formation of agglomerated Pb significantly. CuNPs. Finer visualisation by FESEM/EDX shows that CuNPs are spherical Materials & methods with sizes between 20-250 nm. XPS spectra confirmed that CuNPs are not 0 1+ 2+ To get Pb tolerant bacterial strains, bacteria were isolated from metals only Cu but also as Cu and Cu , being possible the coexistence of copper contaminated industrial sludge samples. Urease selection pressure media oxides and sulphides with the metallic form. were further used to screen urease producing bacteria. The efficient urease Conclusions producing Pb tolerant bacterial strain was identified based on 16S rRNA gene Biomass of P. stutzeri can biosynthesise CuNPs from tailings deposits, which sequencing. The bacterial strain was grown in optimized MICP nutrient is a novel biotreatment of diluted industrial effluents that allow their valorisation in an eco-friendly way and extracting value from mining waste. media containing urea, CaCl2, and poly-Lys, supplemented with 50 mg/L and 100 mg/L of Pb, for a week. The experiments were also conducted in the One of the challenges that must be faced is the separation of the nanoparticles absence of poly-Lys. Urease activity, pH, Pb concentration were measured and the reduction of the agglomerated mass. at regular interval. The concentration of Pb was analyzed by ICP-MS. The FTIR spectra were recorded in order to know the functional groups involved in Pb immobilization. The morphological and elemental analyses were carried out with SEM-EDX. The biominerals formed in the process of remediation of Pb was identified by XRD.

23 T 65 Materials and Methods Bacterial effect on the crystallization of mineral phases in the Unleached samples of bornite, chalcocite and covellite were collected from human urinary system (based on biomimetic syntheses) the mines of HCM in Cyprus, and placed in different test tubes containing 30 A. Izatulina*1, A. Nikolaev1,2, M. Kuz’mina1, O. Frank-Kamenetskaya1, V. ml of the corresponding growth medium and a total of 10% inoculation of Malyshev3 cell suspensions from stock cultures collected from the mine at pH 1.8. FTIR 1St. Petersburg State University, Crystallography, St. Petersburg, Russian and Raman microspectroscopy. Federation Results 2Grebenshchikov Institute of Chemistry of the Russian Academy of We report the μm-FTIR and Raman microspectroscopic detection of bornite [Cu5FeS4] -, chalcocite [Cu2S] -, and covelitte [CuS] -bacterial interactions Sciences, St. Petersburg, Russian Federation + 3S.M. Kirov Military Medical Academy, Microbiology, St. Petersburg, by a consortium of microorganisms. The absorption signals of amide I, K - Russian Federation jarosite and of the produced extracellular polymeric substances (EPS) from the mixed culture as a function of position on the surfaces of the bioleached bornite, chalcocite and covellite demonstrated their heterogeneity within the Urolithiasis is an example of pathogenic mineral formation in the human surface of the minerals. To our knowledge this is the first combined body. It is well known that the presence of a variety of bacteria in the urine application of μm-FTIR and Raman microspectroscopy for the bioleaching is very likely and bacterial inflammation often accompanies stone formation. behaviour of bornite, chalcocite and covellite and the comparison with other Under the conditions of model experiments, the effect of bacteria that are bioleached systems such as chalcopyrite [CuFeS2] provides valuable present in human urine (, Pseudomonas aeruginosa, information on the whole bio-hydrometallurgy Cu/Fe/S system. Klebsiella pneumoniae and Staphylococcus aureus) on the formation of the Conclusions renal stone mineral phases, such as brushite, struvite, vitlocite, octacalcium We report for the first time the ν(Cu-S) of bornite and chalcocite which are phosphate, apatite, whewellite, and weddellite, was studied in systems intermediates in the bioleaching of chalcopyrite and the bioleaching behavior simulating the composition of human urine and using two types of nutrient of bornite, chalcocite and covellite by a consortium of microorganisms media (Muller–Hinton Broth and Meat–Peptone Broth). Multidirectional present in HCM by Raman and FTIR microspectroscopies and compare it changes in the pH values of the solutions were analyzed, which are the result with that previously reported for chalcopyrite. Based on our results we of all system components" interactions with the crystallization process. It was propose a mechanism for the sequential steps for chalcopyrite bioleaching in shown that the presence of bacteria has a different effect on the phosphate which chalcopyrite is converted initially to Cu2S, and subsequently to CuS. and oxalate phases" formation. The presence of and nutrient media significantly affect the precipitant phase composition and the position of the T 67 resulting phosphate phase"s crystallization boundaries, which can shift both Influence of bacterial EPS on mineral organization in EPS- to more acidic (struvite, apatite) and more alkaline (brushite) areas. Under hydrogel-calcite composite aggregates - the chelating effect of conditions of oxalate mineralization, bacteria accelerate the nucleation of calcium oxalates by almost two times and also increase the amount of oxalate Bacillus subtilis, Mycobacterium phley, Mycobacterium precipitates along with and stabilize the calcium oxalate smegmatis, and Pseudomonas putida EPS 1 1 1 2 dihydrate to weddellite. As it can be seen from the reported results and the X. Yin* , F. Weitzel , E. Griesshaber , C. Jiménez-López , L. Fernández- 3,4 5 6 1 available literature data, the bacterial effect on oxalate and phosphate phase Díaz , A. Ziegler , A. Rodríguez-Navarro , W. W. Schmahl 1 formation is different. Thus, in the case of oxalate mineralization, primarily Ludwig-Maximilians-Universität München, Department für Geo- und (most likely), the inflammatory process will contribute to the decrease of Umweltwissenschaften, München, Germany 2 oxalate supersaturation in urine due to calcium oxalate crystallization. In the Universidad de Granada, Departamento de Microbiología, Granada, case of phosphate mineralization, the change in urine pH and the products of Spain 3 bacterial metabolism will be of major importance. Studies aimed at Universidad Complutense de Madrid, Departamento de Mineralogía y identifying the specific action of certain microorganisms on the Petrología, Madrid, Spain 4 crystallization of certain mineral phases should serve to develop individual Universidad Complutense de Madrid, Instituto de Geociencias (ICMM, methods of treatment and prevention of urolithiasis. The obtained results CSIC), Madrid, Spain 5 could be regarded as the scientific basis for understanding the mechanisms Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, of bacterial participation stone formation in the human urinary system and Germany 6 the creation of biotechnological methods for the prevention of this disease. Universidad de Granada, Departamento de Mineralogía y Petrología, This work was supported by the Russian Science Foundation (no. 18-77- Granada, Spain 00026). The XRD studies have been performed at the X-ray Diffraction Centre of St.Petersburg State University. Mineralized structures generated under biological control are hierarchical composites that consist of two distinct materials: biopolymer matrix that is T 66 reinforced by mineral(s). The biopolymer matrix is developed within the Bio-dynamics of copper sulfide minerals and K+-jarosite biological hard tissue as membranes or network of fibrils. The biopolymers probed by Raman and FTIR microspectroscopy affect mineral organization as well as material properties of the biological C. Varotsis*1 composite material. Hydrogel systems can be regarded to some extent as 1Cyprus University of Technology, Environmental Science and Technology, model systems for understanding the influence of biopolymer matrices in Lemesos, Cyprus biologic structural materials on nucleation, crystal growth, mineral orientation, and hard tissue organization. The fibrous fabric of agarose and gelatin gels forms compartments with specific diffusion rates, local Introduction concentrations, and supersaturation of solutes. Hence, an environment is Environmental friendly approaches for the proper management of copper formed in an artificial system that shows some common features to those that production including processing of low grade ore and tails have been are present at sites of mineralization in biological hard tissues. However, developed. Heap bioleaching is the appropriate technology to handle low despite many similarities, major differences still remain. grade copper sulfide ores bearing chalcopyrite, idaite, bornite, chalcocite and Microbial cells surround themselves by a self-produced matrix of hydrated covellite. A variety of chemical and biological processes have been applied extracellular polymeric substances (EPS), which protects the cells and towards establishing the most efficient environmental friendly bio- enhances their physiological activities. Bacterial EPS is not a denaturalized hydrometallurgy treatment technique of low-grade copper mixed ores. High product if compared to agarose or gelatin hydrogels. To understand the copper extractions are achieved in environmental acceptable conditions and directing influence of biopolymers on mineral organization and composite in low cost, under optimum redox potentials with isolated and mixed cultures material formation, we synthesized with Bacillus subtilis, Mycobacterium of A. ferroxidans and A. thiooxidans, The understanding of synergistic effects phley, Mycobacterium smegmatis, and Pseudomonas putida EPS hydrogel- which have resulted from the presence of mixed ores in the bioleaching calcite composite aggregates and investigated the influence of EPS matrices procedure is important for understanding the bioleaching behavior between on aggregate formation, growth and calcite organization. EPS composition single and mixed ores and the origin of the existing differences. was investigated with FTIR, aggregate morphologies and hierarchical Objectives mineral organization were characterized with scanning electron microscopy Raman and FTIR microspectroscopies are structure sensitive techniques and (FE-SEM) and electron backscatter diffraction (EBSD). Bacterial EPS and have been applied towards our understanding of the characterization of the agarose hydrogel distribution were visualized with selective etching structure as well the structure-function relationship in minerals. The procedures, micro-Raman and kernel misorientation analysis derived from combined application of the techniques for the bioleaching behavior of EBSD data. bornite, chalcocite and covellite and the comparison with other bioleached Relative to reference aggregates devoid of bacterial EPS, aggregates that Cu/Fe/S systems such as chalcopyrite provides valuable information on the contain bacterial EPS are reduced in size and, for the EPS of a specific whole bio-hydrometallurgy system and overall visualizations of the bacterium, have distinctive crystal morphologies. Polymer (bacterial dynamics of the bioleached mineral ore. EPS/agarose hydrogel) distribution is highly inhomogeneous in aggregates that contain EPS. In the case of P. putida and M. phley, the occluded polymer (EPS/agarose hydrogel) is mainly present as membranes, while for M.

24 smegmatis and B. subtilis the EPS/hydrogel mixture is occluded within the Anthemiphyllia (the basalmost position in Complexa clade), and aggregate as membranes and as network of fibrils. Relative to reference Letepsammia and Gardineria, both representing basal scleractinians. Large aggregates devoid of EPS, where subunit formation is either absent or phylogenetic distances on molecular tree topology and unique negligible, subunit formation in aggregates containing bacterial EPS is microstructural skeletal organization of each taxon suggest that the extensive. Subunits vary significantly in shape, size, and mode of composition of skeletal matrices may differ accordingly to the phylogenetic organization within the aggregate. For M. smegmatis and B. subtilis subunit position. organization is radial to spherulitic, while for P. putida subunit organization Following thorough cleaning of the skeleton, the organic content was within the aggregate is almost random, while for M. phley subunit extracted, divided into soluble and insoluble fractions and submitted to organization is highly co-oriented. proteomic analysis. Since no genomic/transcriptomic data are available on In conclusion, bacterial EPS changes the microstructure and texture of the the 4 species, the in silico analysis was performed against a large data set mineral in a specific manner. This is a specific characteristic for a given comprising all "non proteins" available and the transcriptome of bacterium and is a feature that can be used as a tool for the recognition and the coral Porites australiensis. Aliquots of the soluble fractions were identification of bacterially mediated calcification in present environments analyzed on SDS-PAGE and screened against a set of 21 lectins in order to as well as in the geological record. obtain their saccharidic signature. For each sample, several peptides were identified, matching with a large T 68 number of protein hits (32 to 109). Many of them occured with P. The red algae mineralized tissue- a low weight high strength australiensis proteins; hits with "other metazoans" proteins (including material Acropora millepora) were significantly reduced. Only 8 to 29% of the hits N. Bianco Stein*1, B. Pokroy1, P. Zaslansky2 corresponded to 2 or more peptide matches. In spite of potential bias, our 1Technion, Materials Science and Engineering, Haifa, Israel data suggest that the protein matrices of Desmophyllum and Letepsammia are 2Charité - Universitätsmedizin Berlin, Department for Operative and more similar between them than from the matrices of Gardineria and Preventive Dentistry, Berlin, Germany Anthemiphyllia. The saccharide analysis indicates that only 5 (Desmophyllum, Anthemiphyllia), 7 (Gardineria) and 8 (Letepsammia) lectins reacted. The lectin profiles of all samples were rather similar and Biomineralization, the formation of minerals by living organisms, has long almost superimposable for Gardineria and Anthemiphyllia. In all cases, the attracted researchers' attention due to the enhanced properties of biominerals highest reactivity was obtained with LEL (an N-acetylglucosamine-binding compared to their synthetic and geological counterparts. For example, the lectin); STL lectin gave high reactivity with Desmophyllum and toughness of the lenses on the arm plates of the brittle star O. wendtii was Letepsammia and moderate with Gardineria and Anthemiphyllia. found to be twice as high compared to geological calcite. The composition of organic matrices may provide some background Calcite, the most thermodynamically stable polymorph of calcium carbonate, information about the evolution of biomineralization in scleractinian corals. is an abundant structural component in the skeleton of many marine The protein similarity between Desmophyllum and Letepsammia that seem organisms. In many cases Mg is incorporated into the calcite lattice with only distantly phylogenetically related or similar lectin profiles of levels reaching as high as 45 mol%. Gardineria and Anthemiphyllia provide a hint to seek deep In this study we investigate the structure of the coralline red algae at various homology/convergence of their biomineralization paths. Lack of close length-scales. Their mineralized tissue is composed of high-Mg calcite. In similarity between two members of basal clade (Gardineria and addition, an organic phase comprised of polysaccharides serves as a template Letepsammia) may point to their early evolutionary divergence. for crystallization. The structure of these algae has not been widely studied Acknowledgments: This work was supported by the National Science Center before and information on their nanostructure is highly missing. (Poland) grants 2011/03/N/ST10/06471 (KJ) and 2017/25/B/ST10/02221 Structural characterization was performed utilizing various high-end (JS). techniques such as synchrotron radiation macro- tomography and nano- tomography. In addition, we used high resolution SEM and high resolution T 70 TEM for the study of the morphology and the nanostructure. Molecular and skeletal fingerprints of scleractinian coral Coralline red algae exhibit a remarkable macrostructure. We have discovered biomineralization along the depth gradient that the structure of these algae is in fact a highly porous structure, with T. Mass*1, A. Malik1, S. Einbinder1, P. Zaslansky2, B. Pokroy3, I. porosity reaching as high as 70vol%. In addition, we have shown that their Polishchuk3, J. Stolarski4 structure is hierarchical with several orders from the nano to the macro scale, 1University of Haifa, Marine Biology, Haifa, Israel formed by crystals with diameter in the nanometric size. Moreover, we have 2Charité - Universitätsmedizin, Department for Operative and Preventive discovered unique structural elements that have not been previously reported. Dentistry, Berlin, Germany We have shown that these unique structural elements provide the algae with 3Technion - Israel Institute of Technology, Department of Materials Science greatly enhanced mechanical properties that allow them to endure stresses & Engineering, Haifa, Israel applied by the sea waves present in shallow waters. 4Polish Academy of Sciences, Institute of Paleobiology,, Warszawa, Poland The combination of high porosity along with a unique macrostructure allows these algae to obtain a low weight structure with enhanced mechanical properties. This study can lead toward a better understanding of the structure Reef building corals, the major producers of biogenic calcium carbonate, function relation of biomineralized structures and toward the synthesis of form skeletons in plethora of morphological forms. The skeleton shapes and novel materials with improved mechanical properties. sizes are distinct for individual species but show also phenotypic plasticity along environmental gradients. A fundamental question remains how these T 69 phenotypic skeletal modifications are reflected in molecular Deep-water scleractinian corals and their skeletal organic biomineralization program of the coral organisms? This study provides the matrices: A source of phylogenetic data? first comprehensive macro- and microstructural skeletal analysis of J. Stolarski1, K. Janiszewska1, I. Coronado1, T. Takeuchi2, L. Ravet3, J. scleractinian coral Stylophora pistillata (clade 4) collected across the depth Thomas3, F. Marin*3 gradient from the sea surface to mesophotic depths of 60m and reciprocally 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland transplanted from 5->60 and 60->5 m in the Gulf of Eilat (Red Sea, Israel). 2Okinawa Institute of Science and Technology Graduate University, Marine Morphological study was combined with expression pattern of distinct gene Genomics Unit, Onna, Japan ontology (GO) and of biomineralization "tool kit" genes. Macro- and 3University of Burgundy-Franche-Comté, Dijon, France microstructure of S. pistillata colonies significantly change over a depth gradient from spherical (surface depths) to flat and branching morphotypes (mesophotic depths) and from intense (surface depths) and weak (mesophotic The reef-building scleractinian corals contribute massively to the global depths) development of fine-scale regions of enhanced skeleton growth formation of biogenic calcium carbonate. Although the physiology of ("calcification centers"). In parallel, the trascriptome composition shifts from calcification in corals has been well studied, the molecular mechanisms overrepresentation of genes associated with oxygen stress response and DNA underlying this process are still poorly understood. Similarly to other repair (shallow-water), to coral-biomeniralization, cilia, extracellular matrix, mineralizing metazoans, scleractinian corals build their exoskeleton by and immune response (mesophotic depths). Interestingly, at both the secreting inorganic precursors of calcification together with an organic phenotypic and the gene level, transplanted corals partly adapt the typical matrix, which consists of proteins and polysaccharides. This matrix - the depth-specific properties. Accordingly, we show that organic matrix fraction main regulator of the deposition of calcium carbonate - remains occluded in is enriched in the skeleton at deep-water, in parallel with overrepresentation the growing biocrystals. In recent years, high-throughput screening of biomineralization "tool-kit" structural extracellular genes in deep water techniques have allowed the identification of a large number of skeletal colonies. Our results provide insights into molecular mechanisms of coral proteins. To date, however, these data were retrieved only from shallow- calcification in changing environment which are also encoded in the water, photosymbiotic taxa such as Acropora, Stylophora or Porites. skeleton. As such they provide exciting perspective of new paleogenomic The main objective of this study was to supplement the skeletal database with interpretations of fossil corals that preserve fine-scale skeletal features. data from deep-water, asymbiotic scleractinians. The selected samples represent 3 major clades i.e., Desmophyllum (the Robusta clade)

25 T 71 these bodies, 52 (about 15%) are located between the two inner organic Modern corals vs. ancient oceans layers. We suggest that these MgCaP-rich bodies are secreted into the outer N. Conci*1, S. Vargas1, E. Griesshaber1, W. W. Schmahl1, G. Wörheide1,2,3 matrix and are part of a Ca-concentrating or transport mechanism. 1Ludwig Maximilian University, Munchen, Germany Calcite formation occurs inside the outer matrix via multiple independent 2SNSB — Bayerische Staatssammlung für Paläontologie und Geologie, nucleation events. Based on 2D and 3D cryo-electron microscopic datasets München, Germany we show that individual calcite crystals grow with preferred orientation into 3GeoBio-Center, Ludwig-Maximilians-Universität München, München, a dense reticular network that forms the calcitic cyst. In the final calcification Germany stage the main structural motif are calcite plates with evenly arranged pores that form the highly regular, porous calcite shell. Conclusion Biomineralization is a taxonomically ubiquitous process by which organisms In summary, our results show the presence of a multifunctional vacuole. In form minerals that they use for support, protection, or nutrient storage, such calcifying cells of L. granifera and C. operosum aff. vacuolar MgCaP-rich as shells, skeletons, and bones. Among corals (Class Anthozoa, Phylum bodies seem to be part of a Ca-concentrating or transport mechanism, Cnidaria) biomineralization is widespread and both aragonite and calcite indicating there is a common biomineralization pathway in the family structures can be observed. Coral biomineralization has been extensively Thoracosphaeraceae: (1) Uptake of Ca and other cations through the studied, primarily due to the ecological role these organisms play in the membranes. (2) Deposition of Mg and Ca ions inside a disordered MgCaP- formation and accretion of coral reefs. Nevertheless, how corals acquired the body. (3) Secretion of these bodies to the extracellular space between the ability to form skeletons characterized by different calcium carbonate outer membranes. (4) Formation and growth of calcite bodies into a dense (CaCO3) polymorphs, i.e. calcite and aragonite, remains elusive. One reticulate network that forms the mature calcitic shell. standing question revolves around whether corals can biologically control the deposition of either polymorph. This has important evolutionary implications T 73 as changes in seawater chemistry through the Earth"s history, especially the Marine temperature recorded in bivalve shell ultrastructures - Mg/Ca ratio, appear to favor the deposition of either one polymorph or the other. proxy potential and current limitations N. Höche*1, E. O. Walliser1, M. Peharda2, B. R. Schöne1 To address this question, we have set-up calcite-inducing, -like 1 marine aquaria experiments to grow coral species from both clades of Johannes Gutenberg University Mainz, Dept. of Applied and Analytical Paleontology, Mainz, Germany biomineralizing anthozoans, namely the subclass Octocorallia (Heliopora 2 coerulea) and the order Scleractinia (Montipora digitata). We employed a Institute of Oceanography and Fisheries, Split, Croatia diverse array of mineralogical analyses, including electron backscatter diffraction (EBSD) and energy dispersive spectroscopy (EDS), to determine Introduction and examine the presence of environmentally-induced modifications within Bivalve shells are increasingly exploited as high-resolution paleoclimate the coral skeleton. We then used RNA sequencing to investigate the archives. These mollusks are globally distributed in aquatic ecosystems, transcriptional response of the corals to different polymorph favoring occur in large numbers and individuals of some species are very long-lived. environments. In conjunction, these data provide insights on how the Periodic growth patterns can be used to precisely date each shell portion. diversity of skeletal structures in corals evolved and may inform our Furthermore, the shells form in equilibrium with the oxygen isotope 18 predictions about the response of these ecologically important organisms to signature of the ambient water, so that δ Oshell provides information on future changes in ocean chemistry . temperature during growth. However, oxygen isotope-based temperature estimates can be challenging if the isotope value of the water is unknown or T 72 if the isotope signal is diagenetically altered. Evidently, there is a strong need Biomineralization pathways in calcifying dinoflagellates- for independent temperature proxies. One potential candidate is the shell uptake, storage in MgCaP-rich bodies and formation of the ultrastructure, specifically the size and shape of individual biomineral units shell (BMUs). So far, BMU-based temperature reconstructions have only been A. Jantschke*1, I. Pinkas2, A. Hirsch2, A. Schertel3, L. Addadi2, S. Weiner2 tested in a few species from a limited number of localities. In addition, the 1Technical University Dresden, Bioanalytical Chemistry, Dresden, recognition and morphometric analysis of BMUs (in SEM images) needs to Germany automated through suitable image-processing algorithms. 2Weizmann Institute of Science, Rehovot, Israel Objective 3Carl Zeiss Microscopy GmbH, Oberkochen, Germany In this study, we assessed the effect of temperature on BMUs of ultrastructures, which have hitherto not been studied (i.e. crossed-lamellar, homogenous and crossed-acicular). To advance objective and quantitative Introduction temperature reconstructions, we developed new image-processing Dinoflagellates are one of the most important contributors to both marine and algorithms for automated BMU recognition. freshwater productivity. They have two main life stages, the thecate/motile Material and methods stage and the cyst/resting stage. One family of dinoflagellates, the We analyzed the marine bivalves Glycymeris bimaculata and G. nummaria Thoracosphaeraceae, produce calcitic shells in their cyst stage. from the Adriatic Sea, and Arctica islandica from Iceland and the Baltic Sea. Objectives The two glycymerids were used to study potential species-specific Although dinoflagellates are the second most important calcifying differences, and A. islandica was used to evaluate potential locality-specific phytoplankton group after coccolithophores, their biomineralization differences in recording environmental variables. mechanism is still not fully understood and considered as one of the most Results serious gaps in knowledge. For this reason, the main objectives are to gain All studied species showed the same relationship between temperature and insight in the mineral architecture and stabilization mechanism, subcellular ultrastructure as reported in previously studied bivalves, i.e., larger and more structures that may play a role in biomineralization, as well as into cyst elongated BMUs were formed in warmer waters. We calculated transfer formation mechanism. functions for crossed-lamellar ultrastructures of Glycymeris spp. enabling Materials & methods quantitative temperature estimates. The homogenous outer shell layer of A. We investigate calcitic cyst formation in two representative members islandica is challenging to interpret. Specimens from the Baltic Sea showed (L. granifera and C. operosum aff.) from different clades of calcareous significantly larger variation in BMU size than specimens from Iceland. dinoflagellates using cryo-electron microscopy (cryo SEM and cryo FIB Conclusions SEM) in combination with various spectroscopic techniques (FT-IR, Raman, Studied bivalve species recorded temperature changes in their shell Fluorescence, EDS). These imaging techniques allow investigation of cells ultrastructure: BMUs were larger and more elongated when formed in as close as possible to the natural state. warmer waters. Transfer functions are species-specific. SEM-based Results quantification of BMU morphometry is challenging in case of the Only calcein AM and not calcein enters these cells, indicating active uptake homogenous ultrastructure of A. islandica. of calcium and other divalent cations. In both species, we observed vacuoles containing crystalline inclusions using cryo-SEM. So far, crystalline deposits in the family Thoracosphaeraceae were assigned to calcite and it was assumed that they are involved in the calcitic shell formation. Surprisingly, using in situ Raman spectroscopic imaging we could identify these crystalline inclusions as anhydrous guanine in the biogenic β-form using their low-wavenumber Raman signature. Live-cell imaging of cells stained with Calcein-AM, the use of cryo- sectioning and cryo-EDS shows the presence of small MgCaP-rich mineral bodies within the same vacuolar enclosures. 3D cryo-FIB-SEM serial block face imaging of a calcifying cell of C. operosum aff. shows a remarkably large number (353) of these bodies distributed in the cell volume. Out of

26 T 74 the epigenetic response of an ecologically and commercially important Ocean acidification impacts European abalone (Haliotis aquaculture species - Crassostrea hongkongensis to OA. tuberculata) shell microstructure and mechanical properties Objectives S. Auzoux-Bordenave*1,2, S. Avignon1, P. Dubois3, N. Richard1, M. Mantle tissue specialises in biomineralisation and is conserved throughout Coheleach4, A. Badou1, S. Di Giglio3, L. Malet5, S. Martin2,6, S. Roussel4, molluscs. Thus, in our study we focus over the epigenetic response of the S. Huchette7 mantle tissue under OA. The objectives are: 1Museum national d'histoire naturelle, Station de Biologie Marine, 1) Understanding the epigenetic response of mantle to OA (gene expression Concarneau, France and DNA methylation) with a focus over ion transporters and shell matrix 2Sorbonne Université, Paris, France proteins. 3Université Libre de Bruxelles, Laboratoire de Biologie Marine, Brussels, 2) Characterising the changes in the shell quality under OA via shell Belgium microstructure, density, Mg/Ca ratio and hardness. 4Université de Brest, CNRS, IRD, Ifremer, LEMAR, Plouzané, France Materials & methods 5Université Libre de Bruxelles, Service 4Mat, Brussels, Belgium Oyster spats (one day post settlement) were grown under ambient pH (8.1) 6Station Biologique de Roscoff, CNRS/CU, Roscoff, France and OA conditions (pH 7.4 and 7.7) for five months, a significantly long 7Ecloserie France-Haliotis, Plouguerneau, France duration for an OA study. mRNA-seq and Methyl-RAD techniques were used for gene expression and DNA methylation analysis. Shell properties were analysed using Scanning Electron Microscope (SEM), SEM-EDS, Ocean acidification (OA) is of major concern for marine organisms, Micro-CT and Vicker's Hardness test. especially for calcifying species such as mollusks, with their shell made of Results calcium carbonate. Indeed, OA has been shown to reduce survival and The shell growth under OA was surprisingly maintained, like the ambient growth, alter morphology and/or impair shell formation and mineralization conditions, throughout the experimental duration. The Mg/Ca ratio and in marine molluscs. The European abalone Haliotis tuberculata is a hardness also remains unchanged. However, there was complete dissolution commercially and ecologically important gastropod species with a calcified of the prismatic layer under pH 7.4. In-spite of severe dissolution, the overall shell mostly composed of CaCO3 under aragonite form. Since aragonite is shell growth was maintained. more susceptible to dissolution compared to calcite, the abalone shell Preliminary analysis of the RNA-seq data shows that the expression of well- provides a relevant model to study the impact of OA on known biomineralisation related genes such as alkaline phosphatase, biomineralization. This study investigated the effects of pCO2 induced OA carbonic anhydrase and tyrosinase were maintained. Among the on shell microstructure, calcification and resistance in adult abalone. differentially expressed genes, above 80% of the genes where According to the most pessimistic scenario (-0.3 pH unit by 2100), adult 3.5 downregulated. The top downregulated genes under OA are related to year-old abalone were exposed to two pH conditions (pHT = 7.71 ± 0.06, mitosis, ribosomes and various energy metabolism such as amino acid, pCO2 = 988 ± 143 µatm, vs pHT = 8.01 ± 0.05, pCO2 = 449 ± 63 µatm) carbohydrates and glycan. Among the few upregulated genes, the top during four months by increasing pCO2 into five experimental tanks per upregulated genes are related to plasma membrane and calcium binding. The condition. Shell surfaces as well as cross sections were examined by notable genes among the upregulated were voltage dependent calcium Scanning Electron Microscopy (SEM) to assess whether lowering the pH had channels. Preliminary analysis of the DNA methylation data shows that top an influence on shell microstructure. The thickness of the shell layers (i.e. down-regulated methylation sites are also related to voltage gated periostracum, spherulitic and nacre) was determined on cross-section images calcium/sodium channels, indicating a strong epigenetic base for adaptation using Image J software. Biomechanical tests (such as compression tests and in oysters. nanoindentation measurements) were performed to compare the shell Conclusion properties (strength, hardness and elasticity) between the two pH treatments. Crassostrea hongkongensis maintains biomineralisation even under extreme After four month of exposure to low pH condition, the periostracum of ocean acidification scenarios. The adaptation strategy involves: 1) abalone shell exposed to low pH appeared lighter and exhibited a corroded upregulating the ion transporter system, for supplying required substrate and surface compared to those exposed to control condition. This corrosion was proton homeostasis. 2) The energy spent on upregulating ion transporters is confirmed by the decreased thickness of the periostracum observed at pH 7.7, compensated by undergoing metabolic depression. while no difference in spherulitic and nacre layers thickness were observed. SEM observation of nacre microstructure revealed irregularities with a T 76 heterogeneous aragonite platelets and a corroded texture surface in abalone Effect of polycarboxylated eggshell membrane on in-vitro shells exposed to low pH. The shell resistance of abalone exposed to pH 7.7 mineralization was significantly reduced by 29 % compared to those exposed to pH 8.0. In J. L. ARIAS*1, K. SILVA1, A. NEIRA-CARRILLO1, L. ORTIZ1, N. addition, nanoindentation assays revealed significant differences in shell BUTTO1, M. S. FERNANDEZ1 hardness and elasticity for abalone exposed to pH 7.7. Shell corrosion and 1University of Chile, SANTIAGO, Chile microstructure changes would likely result from indirect effects of OA either on the carbonate chemistry of the calcification site or on physiological processes driving CaCO3 deposition. Biomineralization concerns not only inorganic ions formation, but also the Our results highlighted that OA negatively impacted the shell microstructure regulatory role of an organic matrix mainly related to the action of anionic and resistance in adult abalone, leading to more fragile shell. In their natural groups such as sulfate in proteoglycans or aspartate and glutamate in environment, abalone may be at greater risk under future pH condition as proteins. The eggshell membrane (ESM) is a network of a fibrillar their shells may not offer sufficient protection from predators and other biopolymer organized as two layers of protein fibers. Main protein of the environmental stressors. As a consequence for the abalone industry, OA ESM is type X collagen. Mineralization of chicken eggshell started on might represent two challenges (i) by reducing abalone protection and specialized negative charged sites of the ESM referred to as mammillae, threatening wild population stocks, and (ii) by enhancing the cost of its associated to the occurrence of mammillan, a keratan sulfate-rich aquaculture due to an increased time to reach a marketable size. proteoglycan. Growth of calcite crystals occurs from the mammillae upwards in association with the sequential secretion of ovoglycan, a dermatan sulfate- T 75 rich proteoglycan. Although 22% of the ESM aminoacids are glutamic plus Oyster biomineralisation is maintained under ocean aspartic, there is no mineralization on the surface of ESM fibers, but only on acidification the afore mentioned sulfate-rich mammillae. Therefore, as a possible K. Chandra Rajan*1, T. Vengatesan1 explanation we hypothesize that the occurrence of a competitive effect 1The University of Hong Kong, Swire Institute of Marine Science, School of between sulfated and carboxylic groups on the surface of the ESM could be Biological Sciences, Hong Kong Island, Hong Kong involved in driving calcium carbonate crystal nucleation to privileged sites. In order to answer this question, an experimental enrichment of ESM with polycarboxylic groups was tested on its ability to crystallize calcium Introduction carbonate in vitro. Ocean acidification (OA), the on-going reduction of oceanic pH and the Polycarboxylated ESM was obtained by coupling polyaspartic or carbonate saturation state (Ω), is known to affect biomineralisation in several polyglutamic acids to ESM fibrils crosslinked with water-soluble marine invertebrates. Ion transport is an important aspect of maintaining carbodiimide. Selective protection and cleavage of carboxylic groups during biomineralisation under OA. There are two concepts, related to ion synthesis were followed by FTIR spectroscopy. Crystallization assays were homeostasis, that explain the negative effect of OA on marine calcifiers: based on a gas-diffusion chamber method consisting of a chamber built with 1) Substrate limitation theory, where the reduction in the carbonate saturation a plastic Petri dish having a central hole in its bottom and glued to a plastic state is considered as the main negative stressor. cylindrical vessel. Polystyrene microbridges were settled on the bottom of 2) Proton flux limitation model, where more energy will be required for the petri dish. Microbridges were filled with 200 mM dihydrate calcium active transport of H+ ions from the site of biomineralisation when the H+ in chloride solution in 200 mM Tris buffer, pH 9.0. The cylindrical vessel the ocean increases. contained 25 mM ammonium carbonate solution. Intact or polycarboxylated However, little is known about the epigenetic ability of the marine calcifiers ESM strips were deposited on the top of each microbridge with the to adapt to these two challenges. Hence, in this study we have investigated mammillary side facing down. Five replicates of each experiment were

27 carried out inside the chamber at 20 °C for 24 h. After the experiments, Raman spectroscopy techniques. Such "biogenic" organization of the post- eggshell strips were taken out of the microbridges, air-dried at room mortem formed calcite is explained by the inheritance of the original temperature, mounted on aluminum stubs with scotch double-sided tape, and macromolecular framework, which acts as an organic template in precisely coated with gold. Crystal morphology was observed and number and size orchestrated dissolution-co-precipitation process. The finding has several estimated in a Jeol JSM-IT300 EDS-Oxford Instrument scanning electron implications for traditional, structural criteria in interpreting the pristine microscope. structure of biominerals in paleontology and astrobiology. When functionalized ESM were located upside down on the calcification Acknowledgments: Acknowledgments: This work was supported by the solution on the top of the microbridges for 24 hours, higher amount of small National Science Center (Poland) grant 2017/25/B/ST10/02221. 2-3 µm rhombohedral calcite crystals adhered to the fibers were observed when compared with the intact ESM where 7-10 µm calcite crystals were T 79 located only on the mammillae. Spinodal decomposition in the formation of biogenic single It is safe to conclude that the occurrence of polycarboxylic groups crystals of magnesium calcite immobilized on ESM is a strong attractor for calcium carbonate E. Seknazi1, S. Kozachkevich1, I. Polishchuk1, N. Bianco Stein1, P. crystallization on functionalized ESM used as a template scaffold. Funded Zaslansky2, A. Katsman1, B. Pokroy*1 by FONDECYT 1180734 1Technion Israel Institute if Technology, Materials Science and Engineering, Hiafa, Israel T 77 2Charite, Berlin, Germany Fungi-induced mineralization in vitro- controlling the crystal morphology and polymorph As organisms can form crystals only under ambient conditions, they I. Polishchuk*1, A. Livne1, B. Pokroy1 demonstrate fascinating strategies to overcome this limitation. Recently, we 1Technion - Israel Institute of Technology, Materials Science and reported a previously unknown biostrategy for toughening brittle calcite Engineering, Haifa, Israel crystals by means of pre-compression of the material, using coherently incorporated Mg-rich nanoprecipitates arranged in a layered manner in the Fungi live within diverse environments and survive well under extreme lenses of a brittlestar, Ophiocoma wendtii. Here we propose the mechanisms conditions, which are usually beyond the tolerance of many other organisms. of formation of this functional hierarchical structure under conditions of In various environments fungi are known to induce precipitation of a wide ambient temperature and limited solid diffusion. For the first time, we range of minerals. Specifically, it has been shown that various species of propose that formation proceeds via a spinodal decomposition of a liquid or fungi facilitate calcium carbonate mineralization. Inspired by examples of gel-like magnesium amorphous calcium carbonate (Mg-ACC) precursor into needle-fiber calcite formed via fungal biomineralization typically observed Mg-rich nanoparticles and a Mg-depleted amorphous matrix. In a second in soils and sediments, herein we utilized active and inactivated fungus to step, crystallization of the decomposed amorphous precursor leads to the induce mineralization synthetically. For the first time we report the ability of formation of high-Mg particles-rich layers. The model was supported by our fungi mycelium to serve as a template for growth of chlorhydroxy-apatite experimental results in synthetic Mg-calcite, which reinforce the concept of tubes and hopeite cylinders. We also demonstrate the feasibility of growing a spinodal decomposition in the amorphous precursor. We also show that aragonite needles from a fungal source in vitro. The obtained needles are organics suppress the spinodal decomposition and that most probably the curved, display hexagonal facets and demonstrate high aspect ratios close to paucity of organics present in the brittlestar"s mineralized tissue allows it to 60. The size and the shape of the synthetic needles match that of the natural occur in this system. These new insights have significant implications for fungus" mycelium. The morphology, micro- and nanostructure of the grown fundamental understanding of the role of Mg-ACC material transformation crystals were studied utilizing high-resolution characterization techniques. during crystallization and its subsequent stability. The presented findings demonstrate that fungus present in the crystallization environment can induce the formation of high aspect ratio fibers and stabilize T 80 metastable polymorphs. Bionic synthesis of a magnetic calcite skeletal through living T 78 G. Magnabosco1, H. Hauzer2, C. Albonetti3, V. Morandi4, J. Erez2, G. Post-mortem recrystallization of biogenic amorphous calcium Falini*1 carbonate and crystal tailoring by the inherited 1Alma Mater Studiorum - Università di Bologna, Chemistry "Giacomo macromolecular framework Ciamician", BOLOGNA, Italy J. Stolarski*1, I. Coronado1, G. Luquet2, M. Potocka3, M. Mazur4, A. 2The Hebrew University of Jerusalem, Institute of Earth Sciences, Baronnet5, O. Grauby5, A. Meibom6,7 Jerusalem, Israel 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland 3National Research Council, Institute for Nanostructured Materials, 2Muséum National d'Histoire Naturelle, UMR BOREA, Paris, France Bologna, Germany 3Institute of Biochemistry and Biophysics , Department of Antarctic 4National Research Council, Institute for Microelectronics and Biology, Warsaw, Poland Microsystems, Bologna, Italy 4University of Warsaw, Department of Chemistry, Warsaw, Poland 5 CNRS - Aix Marseille Université, CINaM - UMR 7325, Marseille, France The peculiar functional properties of calcium carbonate biominerals, such as 6Ecole Polytechnique Fédérale de Lausanne , Lausanne, Switzerland 7 shells, echinoderm spines and brittle stars, have stimulated both fundamental Université de Lausanne, Center for Advanced Surface Analysis, Lausanne, research in biomineralization and in vitro bio-inspired synthetic processes in Switzerland material science. As a consequence of the latter, bio-materials having different/additional properties with respect to the natural ones have been Assembly of mineral deposits from transient, disordered and nanometric-size produced by cell-free laboratory activities. Despite this effort, the production precursor particles is a widespread skeleton-formation strategy of many of materials having functional properties even similar to the natural ones groups of organisms (e.g., corals, mollusks, echinoderms). It enables remains elusive. Here we demonstrate that new nano-composite materials efficient cellular transport of mineral components to the skeleton formation can be prepared in vivo exploiting the special mineralization pathway of the site and their aggregation within biologically confined space. The disordered foraminifer Amphistrigina lessoni, a calcifying organism producing a calcitic particles enclosed by the organic matrix are then transformed through local skeleton by vacuolization of seawater. Accordingly, a bionic skeleton dissolution–co-precipitation processes into stable and crystalline phases. possessing a chamber entrapping magnetic nanoparticles has been obtained Consequently, biologically-controlled minerals, in contrast to abiotic growing the organism in seawater containing such nano-particles. Such a (synthetic/geological) crystals, are organo-mineral nanocomposites with bionic synthetic approach differs and goes beyond the biologically inspired uniform particle sizes, high levels of spatial organization, complex synthetic processes and the biosynthesis of nano-particles by bacteria. It morphologies, controlled aggregation and texture, preferential represents the first research in which a bionic calcified tissue has been crystallographic orientation and form higher-order hierarchical structures. prepared in vivo. This represents a new powerful tool for the preparation of Such criteria are often used to distinguish biogenic and abiogenic minerals in nano-materials exploiting the capability of organisms to control the the fossil record. calcification pathway with an accuracy that is unparalleled in laboratory cell In our experiment, we induced the formation of crystalline mineral phase free synthetic processes. from lobster gastroliths, originally formed mainly by biogenic amorphous calcium carbonate (ACC). In natural conditions (inside the lobster body) the amorphous gastrolith mineral does not crystallize and is assimilated by the organism immediately after molting. Inducing crystallization of the gastrolith amorphous phase, which acted as a crystallization "precursor", we found that the resulting mineral is calcite, which shows all aforementioned properties of biominerals, including highly elaborated hierarchical organization. These newly formed structures were characterized by FESEM, AFM, EBSD,

28 T 81 enamel prisms of hydroxyapatite are regarded as a bundle of nanometer-scale Biomimetic precipitation of carbonates in tubular materials fibers that are elongated in the c direction. Curving parts were observed in formed in flow conditions the radial arrangements of the nanofibers around the root of the prisms. A C. I. Sainz-Diaz*1, E. Esacmilla-Roa2, J. Cartwright1 gradual stepwise change of the c direction is found to be essential for the 1Instituto Andaluz de Ciencias de la Tierra, CSIC-UGR, Armilla-Granada, formation of the curving parts. Spain We produced highly ordered arrays of c-axis-elongated aragonite nanorods 2Lulea University of Technology, Lulea, Sweden ~100 nm diameter similar to the microstructure in lamellae of gastropods in a supersaturated solution containing specific organic molecules. We also fabricated enamel-like fluorapatite nanorod arrays on an organic substrate in Biomineral tubes are often secreted around an organism, or part of it, that an aqueous solution system that was based on the simulated body fluid. A forms a template about which the tube assembles. Another method of gradual change in the growth direction along the c axis was induced through forming biomineral tubes can be templating mineral tube formation around a a stepwise growth behavior. The direction of an ion flow is deduced to affect fluid jet. Such self-assembled fluid-flow-templated tubes are found in the a gradual change in the growth orientation of nanometer-scale crystalline physical sciences, in the so-called chemical gardens[1], and also in such rods. geophysical cases as volcanoes, soda straws in caves, and brinicles under sea Conclusion ice. Chemical gardens of carbonates have been prepared at laboratory We characterized aragonite helix of a pteropod shell and hydroxyapatite forming different nano-morphology and crystallographic transformations by around the enamel-dentin junction of a bovine incisor. We produced nanoscale precipitation of carbonates and other metal oxides grown in the biomimetic curving morphologies with calcium carbonate and calcium surfaces of tubular materials generated in flow conditions out of equilibrium. phosphate in artificial systems. The crystallographic orientation in the The formed materials were characterized observing nanocrystals with curving crystals in biominerals and biomimetic minerals was changed in a different morphology and chemistry depending on the growth conditions. stepwise fashion along nanometer-scale rods and fibers. The interaction of several organic polymers has been explored during the precipitation of carbonates. T 83 Molecular modeling calculations have been performed related with the Minerobiolization - biomineralization at the origin of life interaction of organic molecules on mineral solid surfaces. The adsorption of J. Cartwright*1 organic compounds showed to be an exothermic process. This indicates that 1CSIC, Granada, Spain these nanomaterials can be good absorbants for organic compounds. These results open interesting applications of these materials to clean polluted soils and as nanocarriers of bioactive compounds for therapeutic and If, as seems likely, life developed in hydrothermal vents it first used mineral environmental applications. membranes within the vents. The first photo-cells must have learnt to [1] Barge, L. M.; Cardoso, S. S. S.; Cartwright, J. H. E.; Cooper, G. J. T.; manipulate the mineral membranes that formed their compartments in order Cronin, L.; De Wit, A.; Dolobo↵, I. J.; Escribano, B.; Goldstein, R. E.; to control their metabolism. There must have occurred a biological takeover Haudin, F. et al. From chemical gardens to chemobrionics. Chem. Rev. 2015, of the self-assembled mineral structures in the first proto-cells, with the 115, 8652–8703. incorporation of proto-biological molecules within the mineral membranes to alter their properties for life"s purposes, so that, for example, passive T 82 osmosis in a mineral membrane gradually became active chemiosmosis in a Curving crystals in biominerals and biomimetic minerals proto-cell membrane. This biological takeover of a mineral system is what H. Imai*1, Y. Yukimasa1, M. Takasaki1, M. Suzuki1, Y. Oaki1, T. Sasaki2 we term minerobiolization, in contradistinction to the usual 1Keio University, Applied Chemistry, Yokohama, Japan biomineralization in which the biology controls and assembles the mineral. 2The University of Tokyo, The University Museum, Tokyo, Japan T 84 Insights into the evolution of biomineral (calcium and silicon) Introduction transporters in viridiplantae A wide variety of biominerals are comprised of micrometric and nanometric M. A. Nawaz*1, I. Zemchenko1, X. Lin2, A. Zakharenko1, R. Muhammad building blocks. We frequently observed unusual curving crystalline units as Atif3, T. F. Chan2, K. S. Golokhvast1 a building block in the biogenic hierarchical architectures. However, details 1Far Eastern Federal University, Education and Scientific Center of of the curving morphologies including their crystallographic structures and Nanotechnology, Vladivostok, Russian Federation their formation mechanism have not been clarified sufficiently. The 2The Chinese University of Hong Kong, Shatin, Hong Kong clarification of the detailed structures would be important for understanding 3University of Agriculture, US-Pakistan Centre for Advanced Studies in of biogenic mineralization and development of biomimetic material Agriculture and Food Security, Faisalabad, Pakistan processing. Objective We characterized several kinds of curving crystals in biominerals to clarify Introduction their crystallographic structure. Here we focused on aragonite helix of a Biomineralization is a ubiquitous adaptive strategy in living organisms for pteropod shell and hydroxyapatite around the enamel-dentin junction of a sustenance and structural integrity. Members of viridiplantae and especially bovine incisor as typical examples of curving morphologies. Moreover, we the vascular plants have mastered the art of transporting and accumulating tried to produce biomimetic curving morphologies with calcium carbonate silicon and calcium biominerals. To this respect silicon transporters (SITs), and calcium phosphate in artificial systems to discuss the formation NOD26-like major intrinsic proteins (NIPs) are responsible for silicon mechanism of the specifically designed crystals. transport and Ca2+ ATPases (pumps) and Ca2+ exchangers (CAXs) are Materials and method primarily responsible for calcium transport. A petropod shell of C. globulosa was used for structural analysis of aragonitic Question helical structures. Bovine incisors of cattle were used for characterization of To resolve the sequestration mechanisms behind the deposition of these hydroxyapatite in tooth enamel. We fractured the teeth with a hammer and minerals i.e. the formation of phytoliths and cystoliths, it is essential to prepared small pieces of the specimen that exposed their cross section for understand the transport mechanism and especially how the transporters electron microscopies. Several pieces were treated with solutions of sodium evolved in plants and diverged functionally. hypochlorite or disodium ethylenediaminetetraacetate to reveal their Methods microstructures. The morphology of the shell surfaces and grown crystals We performed comparative genomic studies supported by phylogenetic was observed using scanning microscopes. The crystallographic structures reconstruction, gene structure analysis, duplication analysis, divergence were revealed by transmission electron microscope with selected area analysis, codon bias analysis, molecular evolution analysis, co-expression electron diffraction using cross-sectional samples prepared using focused ion networking and phylostratigraphic analysis to provide evolutionary insights. beam milling. Results and Conclusions Rod-like crystals of calcium carbonate and calcium phosphate were prepared Here, we identified SITs, NIPs, Ca2+ pumps and CAXs in viridiplantae. Our on several kinds of organic and inorganic substrates in supersaturated evidence suggests that segmental duplication was a prevalent evolutionary aqueous solutions containing specific organic molecules. Curving force for the expansion of viridiplantae biomineral transporters. Non- morphologies consisting of rod-like crystals were successfully produced with terrestrial plants lost many members of biomineral transporters suggesting change in the direction of an ion flow. lower biomineralization need/capacity. We identified that angiosperms Results and discussion experienced speciation followed by species/lineage-specific gene We observed the parallel assembly of curving aragonite fibers ~200−300 nm duplications and similar gene modules existed across monocots and dicots. in width on the mildly etched shells of C. globulosa. The rod-like building The duplicated gene pairs have resulted in substantial neo-functionalization. blocks 1−2 mm in length were found in the curving fibers as parts of the Silicon transport-related genes expanded after embryophyte split. Dicots had helical architecture. The curving fibers are deduced to be composed of higher biomineral transporter genes owing to lineage-specific and species several rod-like building blocks whose a and b axes of the orthorhombic specific whole genome duplications. crystal are intermittently deviated. We also characterized the crystallographic structures in the enamel-dentin junction region of bovine incisors. The

29 T 85 T 87 Evolution of planktonic gastropod calcification over short and The shell microstructure of the species of Gigantoproductus long timescales (), the giants of the phylum brachiopoda P. Ramos-Silva*1, D. Wall-Palmer1, L. Mekkes1,2, F. Marin3, F. Marlètaz4, L. Angiolini*1, G. Crippa1, K. Azmy2, G. Capitani3, G. Confalonieri4, G. K. Peijnenburg1,2 Della Porta1, E. Griesshaber5, D. Harper6, M. Leng7, L. Nolan8, M. Orlandi3, 1Naturalis Biodiversity Center, Marine Biodiversity, Leiden, Netherlands R. Posenato9, W. W. Schmahl5, V. Banks7, M. Stephenson7 2University of Amsterdam, Institute for Biodiversity and Ecosystem 1Università degli Studi di Milano, Scienze della Terra A. Desio, Milano, Dynamics (IBED), Amsterdam, Netherlands Italy 3University of Burgundy-Franche-Comté, Biogéosciences UMR CNRS 2Memorial University of Newfoundland, St. John's, Canada 6282, Dijon, France 3Università degli Studi Milano Bicocca, Milano, Italy 4Okinawa Institute for Science and Technology, Molecular Genetics Unit, 4Università degli Studi di Torino, Torino, Italy Okinawa, Japan 5Ludwig-Maximilians Universität München, Munchen, Germany 6Durham University, Durham, United Kingdom 7British Geological Survey, Keyworth, United Kingdom Calcification by marine organisms can be drastically affected by ocean 8University of Leicester, Leicester, United Kingdom acidification (OA) due to a reduced availability of calcium carbonate in the 9Università di Ferrara, Ferrara, Italy seawater. Pteropods and heteropods are planktonic gastropods believed to be among the most vulnerable organisms to the effects of OA because they live at the ocean surface and build thin shells of aragonite. Shelled pteropods have The species of Gigantoproductusreach over 30 cm in width and are received considerable attention and are reported to decrease calcification considered giants within the Palaeozoic sedentary marine benthos. Their rates and experience shell dissolution under high CO2 conditions. Shelled shell thickness is remarkable, reaching over 1 cm and consisting of a thin heteropods have received much less attention, but are expected to be equally pseudopunctate laminar secondary layer and a very thick columnar tertiary vulnerable. Both groups are proposed as bioindicators to monitor the impacts layer, made of a few hundreds of micrometres long, substructured columnar of global change on open ocean ecosystems. However, their vulnerability is units. The microstructure of several specimens has been investigated by based on short-term exposures to extreme OA conditions. Similar to other multiple analyses [petrography, cathodoluminescence (CL), Scanning mollusks, planktonic gastropods build shells through a biomineralization Electron Microscopy (SEM), Electron Backscatter Diffraction (EBSD), process, which is biologically controlled at the molecular level. Still, little is Transmission Electron Microscopy (TEM)]. The analysed shells are known about their evolutionary potential in the long-term. generally well preserved, but locally altered by authigenic silica replacement Using shell proteomics and transcriptomics we are currently identifying the of brownish, fibrous chalcedony and anhedral to subhedral microquartz "biomineralization toolkit" in pteropod and heteropod species. Extensins, forming concentrically laminated spherulites, and euhedral megaquartz and whey acidic domain proteins are part of the repertoire of shell crystals with hexagonal basal section, which embed calcite crystals and matrix proteins (SMPs) that is being identified in planktonic gastropods for growth lines of the brachiopod shells. Petrographic and isotope analyses the first time. Next, SMPs are used to understand how biomineralization show that silica replacement of the gigantoproductid outer shell occurred evolved in the two independent plankton groups over short and long during early diagenetic phase. Despite this, EBSD analyses show that the timescales. Over long timescales includes the evolutionary analysis of SMPs shells retain the presence of large pristine columnar units with crystallites using a fossil-calibrated phylogenomics tree. Over short timescales involves being highly co-oriented. MUD values are 41 to 71, except for occasionally studies at the population-level by measuring gene expression under past, overprinted shell margins with low MUD values. TEM analyses also show present and future concentrations of CO2. Combining macro- with micro- that the columnar layer is formed by nanoscopic, biocomposite mesocrystal evolutionary approaches will shed light on the processes that drive diversity calcite, that is built up by grains approximately co-oriented and that it and evolution of calcification in planktonic gastropods and will allow more contains nanometres inclusions, with a dark contrast, forming trails between realistic predictions of the consequences of global change on marine calcite grain borders similar to those observed in Recent brachiopod shells. calcifiers. These inclusions were analysed by Nuclear Magnetic Resonance (NMR), and Gas Chromatography Mass Spectrometry (GC-MS) analyses, which showed T 86 that an occluded organic fraction is preserved in these ancient fossil shells, Biomineral growth kinetics and thermodynamics as an and its preserved amino acid composition is comparable with that observed architectural constraint on the evolution of molluscan shells in Recent brachiopod taxa. I. Zlotnikov*1, V. Schoeppler1, R. Lemanis1 Finally, the analyses of the carbon- and nitrogen-isotopic compositions of the 1B CUBE - Center for Molecular Bioengineering, Dresden, Germany occluded organic matrix within their calcite shells allowed to explain the gigantic size and thick carbonate skeleton of these Palaeozoic benthic brachiopods, as the result of a mixotroph lifestyle, by which they could rely Molluscan shells are a classical model system to study formation-structure- on the energy and nutrients derived both from photosymbiotic microbes and function relationships in biological materials and the process of from suspension feeding. biomineralized tissue morphogenesis. Typically, each shell consists of a number of highly mineralized ultrastructures, each characterized by a T 88 specific three-dimensional mineral-organic architecture. Surprisingly, in How did the carrier shell Xenophora crispa build its shell? some cases, despite the lack of a mutual biochemical toolkit for Evidence from the recent and fossil record biomineralization or evidence of homology, shells from different G. Crippa*1, G. Pasinetti1, M. Dapiaggi1 independently evolved species contain similar shell ultrastructures. In the 1University of Milan, Milano, Italy current study, using a recently developed physical framework, which is based on an analogy to the well known process of directional solidification, we compare the process of ultrastructural morphogenesis of shells from three The animal kingdom offers several examples of organisms forming their major molluscan classes: a bivalve Unio pictorum, a cephalopod Nautilus selecting and agglutinating objects from the surrounding pompilius and a gastropod Haliotis asinina. First, we demonstrate that the environment. The most famous and spectacular among these agglutinating fabrication of these highly biomineralized tissues is guided by the organisms organisms is probably represented by the carrier shell Xenophora Fischer von by regulating the chemical and physical boundary conditions that control the Waldheim, 1807. The Xenophora comprises species of marine growth kinetics of the mineral phase. Second, we expand our understanding gastropods, known from the Cretaceous to the Recent, which are able to of the theoretical macroscopic morphospace of possible molluscan shell agglutinate fragments of different origins to form their shells; they show shapes (Raup's concept of morphospace) to the level of possible different agglutination potentials, from species lacking attachments to ultrastructures that comprise them. Finally, we shed a new light on the species completely covered by agglutinated materials, as the Mediterranean evolutionary aspect of molluscan shell ultrastructural fabrication within the Xenophora crispa. Here, we analyse Recent and fossil specimens of framework of Seilacher's Constructional Morphology and Morphodynamics. Xenophora crispa from the Mediterranean area at the Scanning Electron We suggest that the repeated "discovery" of some mineral morphologies Microscope and X-Ray Powder Diffraction, to better understand its reflects a series of architectural constraints provided specifically by biomineralization pattern and the mechanisms leading to the agglutination of biomineral growth kinetics. shells and bio/lithoclasts. Also, we provide new data on the poorly described microstructures which knowledge is generally limited to a relatively small number of specific taxonomic groups. We conclude that: a) most of the Xenophora crispa shell is composed by an aragonitic crossed lamellar fabric, but a fibrous to spherulitic prismatic fabric seemingly of calcite has been found in the columella and in the peripheral edge, i.e. the thickest parts of the shell; b) the attachment of objects is mediated by a prismatic microstructure, indicating that this is the most functional fabric in attachment areas in molluscs; c) the functional meaning of the agglutination in Xenophora crispa is related to a snowshoe strategy to

30 successfully colonize muddy substrates. Indeed, this species secretes in the References columella and in the peripheral edge a less dense, less hard and more organic 1 T. Vos, A. A. Abajobir et. al., Lancet, 2017, 390, 1211–1259. rich calcitic fabric, to lighten the thickest parts of the shells in order not to 2 Y. Lei, T. Wang, J. W. Mitchell, L. Zaidel, J. Qiu and L. Kilpatrick- sink in soft sediments. Liverman, RSC Adv., 2014, 4, 49053–49060.

T 89 P 02 Molecular evolution of the matrix proteins of shells and darts The effects of protein incorporation and crosslinking on the in terrestrial snail Euhadra quaesita mechanical properties of mineralized chitin matrices K. Shimizu*1,2, K. Kimura3,4, Y. Isowa5, K. Oshima2, M. Ishikawa2,6, H. J. Elias*1, L. Gower1 Kagi2, K. Kito5, M. Hattori2,7, S. Chiba3, K. Endo2 1University of Florida, Materials Science and Engineering, Gainesville, 1University of Exeter, College of Life and Environmental Sciences, Exeter, United States United Kingdom 2The University of Tokyo, Tokyo, Japan Many natural protective systems, such as the exoskeletons of various 3Tohoku University, Sendai, Japan arthropods, use specialized organic templates and additives to guide the 4Kyungpook National University, Bukgu, South Korea mineralization of calcium phosphate, calcium carbonate or other mineral 5Meiji University, Kawasaki, Japan species. These natural mechanisms create mineral-organic composite 6Yamazaki University of Animal Health Technology, Hachioji, Japan structures with attractive mechanical properties tailored to specific 7Waseda University, Tokyo, Japan environments, making them a subject of interest for many materials researchers. Even though there have been many attempts to mimic the Mollusks are among the most diverse calcifying animals and are well studied structure and properties of the crustacean cuticle in synthetic materials, there for various aspects of their biomineralization. Although many shell matrix are still interactions that are not fully understood because of the limitations proteins (SMPs) have already been reported in mollusks, most reports have of studying natural organisms. In vitro model systems, however, can provide focused on marine mollusks, and the SMPs of terrestrial snails remain for the modification and isolation of system variables, making them valuable unclear. In addition, some terrestrial stylommatophoran snails have evolved for the understanding of various natural systems. The goals of this proposal a novel calcified apparatus, known as "love dart", used for mating behavior. are to: 1) Synthesize ordered chitin templates, then incorporate silk fibroin We identified 54 SMPs in the terrestrial snail Euhadra quaesita. The SMP of or methacrylated silk fibroin into the templates to create chitin-fibroin the highest abundance index value in E. quaesitais similar to the most structures with varying degrees of crosslinking, 2) mineralize templates with abundant SMP in the other hitherto studied pulmonate Cepaea nemoralis. calcium carbonate using a polymer-induced liquid precursor (PILP) process These proteins contain Pro- and Gly-rich regions, but have no known specific and evaluate the effects of fibroin incorporation and crosslinking on the domains and have no homologous proteins in the GenBank non-redundant mineralized structures, and 3) determine the effect of fibroin incorporation protein database except for the proteins reported from pulmonates. These on the hardness, modulus, and fracture toughness of mineralized templates novel proteins may have evolved in the last common ancestor of pulmonates using nanoindentation. The liquid crystalline ordering and protein with a key role in their shell mineralization under terrestrial or freshwater stabilization is expected to produce structures that mimic the helicoidal environments. We then identified four dart matrix proteins (DMPs) and ordering of the chitin-protein phase in the , and the found that two of them are the same proteins as those identified as SMPs in biomimetic mineralization of these templates can create structures that mimic this species. Our results suggest that some DMPs possibly have evolved by the crustacean cuticle composite structure. This research can provide insight independent gene co-option from SMPs during dart evolution events. These into the possible mechanisms used by biological organisms to create unique results provide a new perspective on the evolution of SMPs in the terrestrial hierarchical structures as well as investigating unique mechanisms that may environment and the novel calcified feature of "love darts" in land snails. be used as inspiration for the synthesis of new composite structures.

P 01 P 03 Self-assembling block copolymers in the nucleation of Influence of bioaragonite microstructure in the kinetics of its hydroxyapaptite pseudomorphic replacement by apatite Y. Jhons*1, N. Judge1, C. Fowler2, F. Nudelman1 L. Fernández-Díaz*1,2, M. Greiner3, E. Griesshaber3, D. Reinares4, M. 1University of Edinburgh, Chemistry, Edinburgh, United Kingdom Zenkert3, X. Yin3, A. Ziegler5, S. Veintemillas-Verdaguer4, W. W. 2GlaxoSmithKline, GSK Consumer Healthcare, Weybridge, United Schmahl3 Kingdom 1Universidad Complutense de Madrid, Mineralogy and Petrology, Madrid, Spain 2Institute of Geosciences (IGEO) , Geomaterials, Madrid, Spain Dental erosion is a worldwide pandemic with over 3.6 billion people 3Ludwig-Maximilians-Universität, Geo-und Umweltwissenschaften, suffering from tooth decay, with 486 million children with caries on their Munich, Germany permanent teeth.1 Enamel is the outer layer of the teeth, is highly mineralised 4Institute of Material Sience of Madrid (ICMM, CSIC), Madrid, Spain with around 95 % (by volume) of the structure being formed of carbonated 5University of Ulm, Central Facility for Electron Microscopy, Ulm, hydroxyapatite (HAP), a form of mineralised calcium phosphate (CAP). The Germany exposure of enamel to acids from bacterial fermentation or from our diets results in dissolution of the mineral and the formation of irreversible lesions that can result in tooth loss if left untreated. To address these issues, polymers Numerous features of carbonate biological hard tissues are inspirational for that are able to bind to enamel and prevent demineralization have been the development of new functional materials. Their characteristic internal investigated, however the effect of the polymer structure on the growth of hierarchically arranged porosity and their microstructure are among these HAP have not yet been studied.2 features. Both can partially or totally be preserved during the transformation The aim of this project is to design and synthesise self-assembling of carbonate biominerals into apatite after interaction with boiling phosphate- phosphorous-containing polymers that either prevent demineralisation or bearing aqueous solutions. This carbonate-apatite transformation involves promote controlled nucleation and growth of HAP in enamel. We the development of interface coupled dissolution-crystallization reactions hypothesize that controlling the self-assembling morphology can result in (ICDR) and commonly takes place with the preservation of the biomineral controlled HAP growth, which could be key to remineralizing enamel. Here external shape as well. In this work we study the kinetics of the reversible addition fragmentation chain transfer (RAFT) polymerisation has pseudomorphic replacement by apatite of the bioaragonite of the been used to synthesize phosphorous-based triblock copolymers. Using of the cephalopod Sepia officinalis and the different portions of the shells of dynamic light-scattering (DLS) and scanning electron microscopy (SEM), the bivalves Arctica islandica and Hyriopsis cumingii. All these biominerals we observed that the triblock copolymer forms hollow spheres 300 – 700 nm have distinct microstructures and distribution of occluded organic in size at concentrations above 6.2 x 10-3 mg cm-3. When incubated in tris biopolymers. The kinetics of the transformation is fastest for Sepia 4 º buffer pH 7.4 containing 2 mM of CaCl2 and 1 mM of K2HPO at 37 C, DLS officinalis cuttlebone, which after only 10 hours of interaction with a and SEM demonstrate that the polymer at concentrations above 7.5 x 10-3 mg (NH4)2HPO4 boiling aqueous solution is converted in a 90 wt % apatite cm-3 promotes the precipitation of calcium phosphate. Control experiments scaffold [1, 2]. This conversion is accompanied by a significant increase in without additives or using similar polymers that are either devoid of the the biomineral surface area, which is attributed to the generation of new phosphate group or that do not self-assemble did not result in calcium porosity that facilitates the progress of the ICDR. All other biominerals phosphate precipitation. In conclusion, we synthesized a triblock copolymer transform into apatite at a much slower rate [2]. Both, the inner and outer that promotes the precipitation of calcium phosphate. Both the self-assembly layers of the shell of Arctica islandica transform into apatite at similar rates, into micelles and the presence of phosphate groups in the polymer are needed but after 14 days of interaction with the phosphate-bearing solution only to promote calcium phosphate precipitation. Next steps will be to use reach transformation percentages as small as 10 wt% and 15 wt%, transmission electron microscopy to study their 3D morphology and respectively. The prismatic portion of the shell of Hyriopsis cumingii is more characterize the form of calcium phosphate precipitated. reactive than the shell of Arctica islandica, with transformation percentages above 25 after 14 days interaction with the phosphate bearing solution. In

31 contrast, the aragonite of the nacreous portion of this shell is extremely The Kinetics of the Replacement Reaction of Biological Aragonite to unreactive and remains virtually pristine during the whole duration of the Apatite. Minerals 2018, 8, 315. interaction experiment (14 days). We interpret that the distinctly different kinetics of the into apatite transformation reflect the different accessibility of P 05 the phosphate-bearing aqueous solution to the mineral component of the hard Synergetic organic-inorganic interactions regulate tissues studied. Differences in fluid accessibility arise from the mineralization characteristics of each hard tissue regarding their porosity, their content of Y. C. Huang*1, B. Wu2, M. Drechsler3, S. J. Huang4, A. Rao5, J. C. C. biopolymers and their ultra- and microstructure as well as the specific Chan4, D. Gebauer1,6 evolution of each of these features as the ICDR progresses. 1University of Konstanz, Chemistry, Konstanz, Germany References 2Forschungszentrum Jülich, Jülich Centre for Neutron Science, Garching, [1] Reinares-Fisac, D.; Veintemillas-Verdaguer, S.; Fernández-Díaz, L. Germany Conversion of biogenic aragonite into hydroxyapatite scaffolds in boiling 3University of Bayreuth, Bayreuth Institute for Macromolecular Chemistry, solutions. CrystEngComm, 2017, 19, 110-116. Bayreuth, Germany [2] Greiner, M.; Fernández-Díaz, L.; Griesshaber, E.; Zenkert, M.; Yin, X.; 4National Taiwan University, Chemistry, Taipei, Taiwan Ziegler, A.; Veintemillas-Verdaguer, S.; Schmahl, W. W. Biomineral 5University of Twente, Faculty of Science and Technology, Enschede, reactivity: The kinetics of the replacement reaction of biological aragonite to Netherlands apatite. Minerals, 2018, 8(8), 315. 6Leibniz University of Hannover, Institute of Inorganic Chemistry, Hannover, Germany P 04 Mineral replacement of bioaragonite by apatite – differences between symbiotic and asymbiotic corals Biomineralization is regulated by biomolecular and ionic species in complex physiological environments. However, the underlying mechanism of how P. Forjanes*1, M. Greiner2, E. Griesshaber2, L. Fernández-Díaz1,3, I. organisms precisely exert control over mineralization still remains elusive. Coronado4, J. Stolarski4, M. Zenkert2, D. Joester5, L. Stegbauer5, U. In the present study, we address the fundamental aspects of nucleation and Rameshbabu5, S. Veintemillas-Verdaguer6, W. W. Schmahl2 crystallization of calcium carbonate (CaCO ), a vital biological and 1Universidad Complutense de Madrid , Mineralogía y Petrología, Madrid, 3 geological mineral. The bidirectional relations between specific protein Spain moieties (CTL domains) of the sea urchin spicule proteome and distinct 2LMU Munich, Department of Environment and Earth Sciences, Munich, inorganic entities in the course of mineralization are elucidated. Given the Germany pH-dependent speciation of (bi)carbonate ions, a potentiometric titration 3Institute of Geosciences (CSIC, UCM), Madrid, Spain methodology was implemented for quantitatively investigating CaCO 4Institute of Palaeobiology,, Warsaw, Poland 3 mineralization at near-neutral pH levels (pH 7.5–9.0). Notably, our 5Northwestern University, Materials Science and Engineering, Evanston, investigation brings forth the mechanistic contributions of HCO - ions in United States 3 mineral nucleation as active soluble species interacting with ionic 6Institute of Materials Science (CSIC), Madrid, Spain components and structural constituents of the emergent solid amorphous phases. Moreover, exploring the influences of recombinant proteins Scaffolds for bone tissue engineering can be obtained through the associated with sea urchin skeletons (CTL proteins), biophysical properties pseudomorphic mineral replacement of carbonate skeletons of invertebrates involving ion-complexation and self-association are found to impact by apatite upon interaction with boiling phosphate-bearing aqueous solutions mineralization. In particular, in the presence of Mg2+ ions at lower pH levels, [1, 2]. These skeletons exhibit porosity at different length-scales, which is the investigated CTL proteins exert enhanced control over mineralization. preserved during the mineral transformation process. Their characteristic This implies that in physiological scenarios, with the synergy of "spectator" 2+ - pore sizes and pore distribution are closely matching those ideals for bone ion species (e.g. Mg and HCO3 ions), minute quantity of biomolecules can grafting. In this work, we assessed the kinetics of the aragonite-apatite profoundly govern mineral nucleation. Collectively, our findings suggest that transformation of skeletons of four coral taxa: Acropora, Porites, Lophelia, biomineralization, emerging as a biologically programmed multistep and Madrepora. Coral skeletons are organic-inorganic composites that show crystallization reaction, is a bidirectional process which encompasses (i) distinct function-related characteristics, regarding their microstructure, ionic and biomolecular additives that regulate the nucleation and skeletal density, original porosity compositional and distributional crystallization of inorganics and (ii) distinct mineral entities that tune the differences in organic biopolymers and geochemistry. These distinct self-association of biomolecules. features, which are significantly different in some symbiotic (Acropora and Porites) and asymbiotic (Lophelia and Madrepora) corals, have a complex P 06 influence on the skeleton reactivity. Skeletons of Acropora and Porites Green synthesis of silver nanoparticles using Crocus sativus contain larger volumes of macro- and mesoscale porosities and have rougher corms aqueous extract and evaluation of their antibacterial surfaces. In contrast, the skeletons of Lophelia and Madrepora are compact activity and cytotoxic effect on human ovarian cancer cell line and show smooth surfaces. Moreover, the bioaragonite of symbiotic corals is (A2780cp) Mg-richer and Sr-poorer than that of asymbiotic corals. Further differences 1 2 3 regard the characteristics of their organic matrix. The highest content of A. Taghva* , M. Entezari , S. Ghafoori 1Islamic Azad University Farahan Branch, Microbiology, Tehran, Iran organics is found in the skeleton of Madrepora oculata, with 6.05%, 2 followed by Lophelia pertusa, with 4.93%. The two symbiotic corals, Islamic Azad University Tehran Medical Sciences branch, Biology, Tehran, Iran Acropora sp. and Porites sp., show lower contents of organics, with 2.64% 3 and 2.10%, respectively. Payame Noor University, Biotechnology, Tehran, Iran The skeletons of Madrepora and Lophelia transform at a slower rate in contrast to Porites and Acropora into apatite during the first 4 days of Introduction interaction with the phosphate-bearing solution. This is followed by a Recently, the application of nanoparticles is grown in various fields such as subsequent latent period, without apatite replacement, that lasts 7 to 9 days biotechnology, nanotechnology, physics, chemistry, materials science, as and afterwards, the transformation rate rapidly increases. The fastest well as other new commercial applications. Noble metal nanoparticles such transformation kinetics corresponds to the skeletons of the two symbiotic as gold, silver, and platinum present unique physicochemical properties, corals Porites and Acropora, whose apatite content after 14 days of which are not observed in larger metal nanoparticles, therefore, the metal interaction reaches 39 wt% and 60 wt%, respectively. After this period the nanoparticles are used in different fields such as optical devices, catalysis, skeleton of Lophelia contains only 15.3 wt% of apatite. Interestingly, the biological labelling, drug delivery system, and cancer therapy. skeleton of Madrepora is the least reactive, reaching only a 2.2 wt% of Objectives apatite at the end of the experiment. We conclude that the transformation of In the present investigation, biosynthesizing silver nanoparticles (AgNPs) via coral bioaragonite into apatite is facilitated by the higher porosity and Crocus sativus corm aqueous was explored. Then, antibacterial and rougher surfaces of the skeletons of symbiotic corals. The higher content of mutagenicity potential of the silver nanoparticles was investigated by Agar organics of asymbiotic coral skeletons seems to prevent aragonite Well Diffusion and Ames methods, respectively. Finally, the cytotoxic effect transformation. Further differences in aragonite into apatite transformation of AgNPs against human ovarian cancer cells was examined by the MTT kinetics might be modulated by both, biopolymer decomposition rate and method. composition-related differences in aragonite solubility. Materials and methods References Biosynthesis of silver nanoparticles has done by subjecting Crocus sativus [1] D. Reinares-Fisac, S. Veintemillas-Verdaguer, L. Fernández-Díaz. corm aqueous extract and 0.001 Mm silver nitrate solution. After color Conversion of biogenic aragonite into hydroxyapatite scaffolds in boiling changing, nanoparticle production has investigated via spectrophotometry, solutions. CrystEngComm 2017, 19, 110-116. XRD, and TEM. Then, antibacterial properties of produced silver [2] M. Greiner, L. Fernandez-Diaz, E. Griesshaber, M.N. Zenkert, X. Yin, A. nanoparticles have investigated by Agar Well Diffusion and MIC Ziegler, S. Veintemillas-Verdaguer, W.W. Schmahl, Biomineral Reactivity: determining Methods. Also, mutagenicity potential of produced silver nanoparticles has determined by Ames test. Finally, cytotoxic effects of the

32 created the nanoparticles on A2780cp cell line has checked by cell culture P 09 and MTT method. Biosynthesis and evaluation of the characteristics of silver Results nanoparticles using Cassia fistula fruit aqueous extract and its The results indicated that nanoparticles were produced in 45 minutes by antibacterial activity reducing silver nitrate ions and color changing occurred during this time. S. Ghafoori*1, A. Taghva2, Z. Tayebi3, M. Hashemi4 Spectrophotometry assay showed an absorbance peak at 425 nm wavelength. 1Payame Noor University, Biotechnology, Tehran, Iran Also, XRD test was confirmed nanoparticles production. TEM microscopy 2Islamic Azad University, Farahan Branch, Microbiology,, Farmahin, Iran indicated that the size of produced AgNPs was ranging from 5 to 25 nm with 3Islamic Azad University, Tehran Medical Sciences, Faculty of Medicine, a spherical shape. Antibacterial properties of silver nanoparticles were Microbiology, Tehran, Iran approved by both Agar Well Diffusion and MIC test methods. Moreover, No 4Islamic Azad University Tehran Medical Sciences branch, Genetics, mutagenic activity was seen in produced AgNPs. MTT assay after treatment Tehran, Iran of human ovarian cancer cell line (A2780cp) with AgNPs in 24 h showed that there is a dose-dependent cytotoxic activity in produced AgNPs against cancer cell line. Introduction Conclusion There are several ways of nanoparticles production, but the biological It is clear that silver nanoparticles have a high potential to be used in industry method of nanoparticles production is under the attention of researchers due and medicine. However, they should be examined from various aspects such to its eco-friendly and energy saving properties. as safety, toxicity, and so on. Objectives In the present study biosynthesis of silver nanoparticle by Cassia fistula fruit P 07 extracts were examined and mutagenesis potential of nanoparticles was Enhancement in the photobiological hydrogen production of investigated. Furthermore, the antibacterial effect of the produced chlorella-material hybrids by dimethyl sulfoxide nanoparticles was investigated on In vitro and In vivo. Y. Zhao*1, L. Shu1, C. Shao1, W. Xiong2, R. Tang1 Materials and methods 1Zhejiang University, Hangzhou, China In order to nanoparticles production, the fruit extract was subjected to the 2Nanjing University, Nanjing, China silver nitrate aqueous solution at the final concentration of 1 mMolar. After nanoparticles production, the color changed reaction mixture was used for characterization with spectrophotometry, X-ray diffraction analysis (XRD), Photobiological production of hydrogen is low energy consumption and Transition electron microscope (TEM) microscopy and DLS. Then, the environmental friendliness, ensuring the generation of clean and renewable antibacterial effect of the produced nanoparticles was investigated by agar energies. It has been reported that Chlorella aggregates, which is induced by well diffusion method against three bacterial pathogenic strains. Mutagenesis silica-based materials, can continuously produce hydrogen photobiologically effect of silver NP was investigated by the Ames test. At the final, wound under normal aerobic conditions. Even so, the yield is relatively low, which healing repair of silver Nano NP in vivo was examined. equals only 0.42 % of the light‐to‐H2 energy‐conversion efficiency. Results However, we find that a simple addition of dimethyl sulfoxide (DMSO) into After nanoparticles production, the color of the plant extract was converted an aqueous environment (to 0.5 vol %) can significantly promote the H2 yield to dark green attributed to the surface plasmon resonance band (SPR) of the of Chlorella aggregates, reaching 0.69 % of the light‐to‐H2 energy‐ silver nanoparticles. Visible spectra of the color changed extract had conversion efficiency. The improvement is explained by a DMSO-induced maximum absorption peaks around 418 nm. Furthermore, the presence of the increase in the cellular respiration rate. This will lead to a decrease of the silver nanoparticles was confirmed by the XRD. TEM analysis revealed that oxygen content within the aggregates, and subsequently resulting in the the obtained silver nanoparticles were triangle, hexahedron and spherical in activation of more hydrogenases. In general, this strategy exhibits a their shapes. DLS test has shown that average sizes of nanoparticles were functional enhancement by a combination of small molecules and organism- around 3/6-4/5nm. Antibacterial assays revealed that the produced material hybrids. nanoparticles had suitable effects against all of the three bacterial strains. Mutagenic effects of nanoparticles were not observed. The silver P 08 nanoparticles have a suitable effect on preventing wound infection in mice Non-classical crystallization of calcium carbonate towards were tested. single crystal formation Conclusion Z. Liu*1, H. Pan1, Z. Zhang2, Z. Wang3, B. Jin1, R. Tang1, J. J. De Yoreo3 It seems that the biological production of nanoparticles with the usage of 1Zhejiang University, Chemistry, Hangzhou, China these plant extracts is able to enhance their medicinal effects. In this study, it 2Xiamen University, Xiamen, China is indicated that the antibacterial property of the extracts containing 3Pacific Northwest National Laboratory, Richland, WA, United States nanoparticles was promoted considerably. Promoting other effects of these nanoparticles can be prospective for future studies.

Crystallization by particle attachment or two step nucleation has been widely P 10 observed in both natural and synthetic environments. However, this non- SEM/EDS analysis of microbial induced calcium carbonate classical crystallization towards single crystal formation cannot be explained by classical nucleation theory, and many mysteries are still remained to crystal formation in agarose hydrogels B. Christgen*1, H. Mitrani1, M. Zhang2 understand this process. We herein use calcium carbonate (CaCO3), which is 1 a typical geological and biological mineral, as a model to investigate its Newcastle University, School of Engineering, Newcastle upon Tyne, crystallization. It is revealed that a random particle attachment followed by a United Kingdom 2Northumbria University, Health and Life Sciences, Newcastle upon Tyne, self-orientation could occur on CaCO3 particles, leading to single crystal formation. And this is attributed by its surface stress induced grain-boundary United Kingdom migration. Besides, we find the crystallization pathway of amorphous CaCO3 can be controlled by magnesium ion (Mg), which is a widely observed Introduction impurity in biominerals. In most cases, the crystallization of amorphous Microorganism mediated processes are present in every environment on the CaCO3 is dominated by dissolution/re-precipitation process, and the resulting Earth. Understanding how bacteria interact with and use their environment crystal exhibit expected morphologies. Only at high Mg concentration, the to survive is essential to translate to and use this knowledge in sustainable crystallization occurs in the absence of a morphological change to give biotechnologies. Microbial induced calcite precipitation (MICP) as a spheroidal single crystalline Mg-calcite. It is explained by the high water potential low cost and environmental sustainable process has gathered content in Mg doped amorphous CaCO3, in which the reorganization of extensive interest in the geotechnical and construction community for soil molecules in solid-phase becomes available. Our findings promote the improvement and as a sustainable construction material in recent years. understanding of crystallization in nature, implying some potential Underground carbon storage, sustainable biobricks, and healing and mechanisms in biomineralization. Most importantly, these results can shed corrosion protection of cracks in cement based building materials are light on the construction of functional crystals with controllable crystalline examples of the multitude of applications investigated. Hydrogels have long orientation and morphology, by design. been used to investigate chemical crystal growth and morphology for calcium carbonate. Additionally hydrogels can be used as soil analogues to study bacterial growth and MICP in situ in a more confined and easily accessible environment. Objectives In this study we investigated the influence of variations in the cementation media on micro-scale calcite growth and distribution in agarose hydrogels over time using S. pasteurii as a model organism.

33 Materials and Methods to oxytetracycline (OTC) which was less than 15%. The selective capturing S. pasteurii was grown over night in liquid culture before suspension in efficiency towards OTC in both buffer and tap water was about 85% and agarose hydrogels which contained a cementation media of nutrient broth, 73%, respectively. Moreover, these bio-hybrid mineral microparticles were urea, CaCl2 (at concentrations between 30 mM and 480 mM), NH4Cl and found to be stable, even after 5 repeated usages, maintaining the initial NaHCO3. The hydrogels were grown between two to nine days at 30 °C and capturing efficiency of 72%. were then analyzed by SEM/EDS and the calcium carbonate content was Conclusion measured. SEM specimen were prepared by flash freezing in liquid nitrogen Using the biomineralized and highly nanostructured CaCO3 microparticles to avoid structural deformations during the freeze drying process and then for biosensing and bioseparation, we could successfully detect and capture freeze dried for 24 hours at -80 °C under vacuum. Before SEM analysis, the the target molecules with improved performances. With the advantages of specimen were sputter coated with a 5 nm layer of platinum using a high biominerals, these new studies could open up new possibilities for resolution sputter coater. The specimen were visualized at low voltage (1.8 biotechnological applications of biominerals. kV) to avoid damaging the hydrogel structure while the EDS X-ray spectrum for elemental analysis was obtained using 20 kV afterwards. P 12 Results Calcium carbonate crystals formation under the influence of SEM analysis shows spherical crystal growth after three days days incubation otolithic and otoconial matrix collagen-like protein otolin-1 of the hydrogels at 30 °C. Crystal size varied between 10µm and 300µ in K. Bielak*1, A. Zoglowek1, J. Stolarski2, A. Ożyhar1, P. Dobryszycki1 different specimen depending on incubation time and CaCl2 concentration. 1Politechnika Wrocławska, Department of Biochemistry, Wrocław, Poland Higher calcium chloride concentrations in the cementation media led to more 2Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland and larger crystal formation in the hydrogel. EDS X-ray spectra of the crystals confirmed the presence of carbon, oxygen and calcium, the elements that make up CaCO3 which verifies that the formed crystals are calcite. The creation of mineralized structures in invertebrates and vertebrates Control hydrogel samples incubated without bacteria and/or without calcium requires cellular regulations, where the action of distinctive proteins control chloride which were treated the same as the main specimen showed no crystal the deposition of biominerals as they have an influence on the nucleation, formation. growth, localization and morphology of the growing structure. Many of them Conclusion belong to intrinsically disordered proteins (IDPs) considered as the major SEM and EDS anlysis have been used to investigate calcite formation in regulators of the process. However, significant role of matrix proteins in the hydrogels from MICP induced by S. pasteurii and cementation media over process of biomineralization cannot be neglected. Matrix proteins provide an time. EDS confirmed that the spherical crystals observed in the hydrogels organic scaffold and a surface for mineral deposition as in an epitaxial were calcite and growth depended on incubation length as well as the nucleation. The possible interactions between matrix proteins and inorganic concentration of calcium chloride in the cementation media used. elements suggest that also crystals supporting proteins may have an influence on the polymorphic form as well as biomineral morphology. P 11 In the formation of fish otoliths and higher vertebrates otoconia, a protein Use of biomineralized microsilica and highly nanostructured named otolin-1 is involved as the scaffolding and possible tethering element of these calcium carbonate structures of the inner ear. Otolin-1 is one of a CaCO3 particles for biosensors and bioseparation few non-IDPs proteins involved directly in the process of ear stones and ear S. Kim1, M. B. Gu*1 dust biomineralization. 1Korea University, Department of Biotechnology, College of Life Sciences In this study, we would like to test the influence of recombinant homologues and Biotechnology, Seoul, South Korea of Danio rerio and Homo sapiens otolin-1 on the formation of calcium carbonate crystals in slow diffusion method. Introduction Recombinant zebrafish and human otolin-1 was expressed in the bacterial The calcium carbonate (CaCO3), which is most abundant biomineral found system composed of Escherichia coli Arctic Express cells transformed with in nature, is commonly used as exoskeletons of many life forms such as algae, pQE80L plasmid containing cDNA of zOtol1 and hOtol1 separately. Protein clam, and sea urchins. Recently, there has been a growing interest in using purification and sample preparation involved affinity chromatography to CaCO3 for diverse applications due to their versatility and unique properties. cobalt or nickel cations followed by size exclusion chromatography. Calcium Especially, CaCO3 can be promising materials in biotechnological areas. carbonate crystals were grown in the range of protein concentrations in 96- Furthermore, the various hierarchical structures of biominerals make CaCO3 wells plate-based biomineralization chambers with the use of a slow based materials more attractive. diffusion method. Objectives The proposed method of production and purification of recombinant zOtol1 We report the application of CaCO3 based biomineral in the biosensing and and hOtol1 was sufficient to obtain highly pure samples in the yields ranging bioseparation using aptamer as a bioreceptor. For biosensing application, from 0.86 mg up to 1 mg of the protein from one liter of bacterial culture. CaCO3 structure derived from unicellular algae was utilized to enhance the zOtol1 and hOtol1 affected the size and morphology of calcium carbonate analytical performance of the biosensor. For bioseparation application, crystals obtained by slow diffusion method. Micro-Raman microscopy aptamer conjugated magnetically separable silica coated CaCO3 determined the polymorphic form of these biominerals. microparticles were developed for antibiotics removal. Concluding, the presence of and otoconia matrix recombinant Materials and Methods collagen-like otolin-1 has an impact on the formation of calcium carbonate For biosensing application, CaCO3 microparticle derived from algae was crystals in the slow diffusion system for Danio rerio and Homo sapiens applied for surface modification of electrode. Briefly, 1 μl of algae-derived homologues. CaCO3 structure (45 mg/ml) was applied to the substrate, and the gold Acknowledgments: This work was supported by the National Science Center working electrode was formed by sputtering. Sandwich-type binding (Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity aptamers were applied for sensing type-2-diabetes. For bioseparation subsidy from the Polish Ministry of Science and High Education for the application, aptamer conjugated magnetically separable silica coated calcium Faculty of Chemistry of Wroclaw University of Science and Technology. carbonate microparticles were synthesized by simple procedures. Briefly, 20 mM of CaCl2 in Tris-HCl (1 M, pH 8.3) solution with Poly-acrylic acid (0.1 P 13 mg/ml) and 100 μL of magnetic nanoparticles was stirred in a CO2 chamber Cryo-TEM study of the growth and crystallization processes of and coated with silica by the modified sol-gel. Finally, oxytetracycline calcium phosphate binding aptamers were immobilized on to the particle for specific removal G. Dalmonico1, M. Farina2, A. Rossi*1 oxytetracycline. 1Brazilian Center for Research in Physics, Rio de Janeiro, Brazil Results 2Federal University of Rio de Janeiro, Biomedical Science, Rio de Janeiro, The morphological, electrochemical characteristics and the performances of Brazil the newly developed biosensor were investigated by SEM, EDAX, cyclic voltammetry, and chronoamperometry. The electroactive surface area was increased about 3.8-fold compared to a commercial screen-printed electrode Introduction (SPGE) which is likely due to the roughened surface provided by the Calcium orthophosphates are a class of materials that attracts strong interest nanostructured CaCO3 microparticles. The analytical performance of the in many research areas in the fields of chemistry, physics, biology and newly developed biosensor was enhanced, when compared to the SPGE, by medicine. They are one of the main inorganic constituents of the calcified 1.16-fold and 3-fold in buffer and serum condition, respectively. The tissues of vertebrates (bones and teeth) and can be used in bone implants due improved analytical performance could be explained by the surface to their remarkable biocompatibility and bioactivity. The synthesis of HA in properties of algae-derived CaCO3 microparticles which increased a controlled manner is an important requirement for different applications electroactive surface area affecting the diffusion of electrolytes and analytes. and can also be used for elucidating the mechanism of mineralization in Aptamer-conjugated magnetically separable silica coated calcium carbonate biological systems (biomineralization). microparticles for antibiotics removal were successfully synthesized and Objective characterized by SEM, FT-IR, EDAX, BET, and CLSM. About 6 μm sized In this work, the morphology and structural characteristics of calcium aptamer modified CaCO3 microparticles showed low non-specific adsorption phosphate (CaP) nanoparticles were investigated at different steps of the

34 synthesis process by analytical cryo-transmission electron microscopy Acknowledgment (Cryo-TEM) in combination with other analytical techniques that need large The authors are grateful for the funding provided by Fondecyt, Project N° amounts of material. 1171520. Materials and Methods The synthesis of HA was performed by dropwise addition of phosphoric acid P 15 to calcium hydroxide solution in order to obtain a final solution with a Ca/P Biomimetic supertough and strong hybrid macrofibers ratio of 1.67. Calcium and phosphate ions react to form CaP and water. The Y. Yu*1, R. Tang1 whole synthesis (dropwise addition and aging) occurred over a period of 24 1Zhejiang University, Department of Chemistry, hangzhou, China h. The reaction was performed at room temperature (~25°C), and the pH was not kept constant. Samples were collected after different periods of time (5 min, 10 min, 30 min, 1 h, 2 h, 7 h, 11 h, 15 h, 19 h and 24 h) for the study of Inspired by the special organic-inorganic hierarchical nanostructure of bone, the process of HA growth and crystallization. The samples were ultrafast- the block copolymer with crystalline and amorphous protein regions as well frozen to be analysed by transmission electron microscopy or freeze-dried to as special structural orientation of natural spider silk and their excellent be analysed by XPS, XRD and FTIR. mechanical strength and toughness, here we show a simple down-up self- Results and Discussion assembly method to prepare organic-inorganic hybrid macrofibers with We showed that the process of drying the sample, instead of analyzing the ordered mineralized polymer chain alignment. Owing to the coexistence of sample in vitreous ice, did not significantly alter the morphology of the crystalline and amorphous domains and the special rivet structure in the nanoparticles but did induce significant changes in their crystallinity. As hybrid nanostructured macrofibers, the resultant hybrid macrofibers exhibits shown by Cryo-TEM, crystallization of the nanoparticles in the frozen superhigh tensile strength to ~950 MPa which is close to natural spider silk hydrated state was detected only after a long period of synthesis. In the first (1150 ± 200 MPa), special toughness to 221 J g-1 which is proved to be the 5 min of reaction, which corresponds to a low amount of phosphate ions in world"s highest special toughness among the PVA based materials and solution, we observed nanoparticles characterized by a high Ca/P ratio that surpasses natural spider silk (195 J g-1) and large stretchability (80.6%) as transformed after 30-60 min into needle-like amorphous nanoparticles with well as excellent knitting properties and dyeability. This supertough and a comparatively lower Ca/P ratio. The mechanism of HA formation using strong biomimetic hybrid macrofibers hence has promising applications in these chemical routes will also be discussed in the presentation regarding textile fields such as flexible ballistic fabric. This down-top biomimetic new paradigms of (bio)mineralization on the basis of a nonclassic mechanism preparation approach of nanostructured macrofibers with excellent of nucleation induced by nanocluster aggregation, amorphous calcium mechanical properties is simple, scalable, and cost-effective, representing a phosphate and multiple stages of crystallization. promising direction for the development of fiber industry.

P 14 P 16 Stabilization of amorphous calcium oxalate (ACO) particles The synergic effect of Sr2+ and Mg2+ on the stabilization of with poly(acrylic) acid in pre-nucleation essays amorphous calcium phosphate 1 1 F. Diaz-Soler*1, M. Weber1, A. Neira-Carrillo1 W. Jin* , R. Tang 1 1Universidad de Chile, Santiago, Chile Zhejiang Unniversity, Hangzhou, China

Introduction Amorphous calcium phosphate (ACP) is a meta-stable precursor phase for Calcium Oxalate (CaOx) is one of the most widely distributed biominerals in bone formation and it can transform into the thermodynamic stable mineral nature and recently a non-classical crystallization route (CNC) via ionic phase, hydroxyapatite (HAP, the main inorganic phase in bone), under aggregates (clusters) as amorphous precursors particles has been reported. It physiological conditions. In biological system, it has been documented that 2+ is well known that acidic biomolecules present a key role in classical Mg can effectively stabilize ACP to ensure a regulation of crystallization (Biomineralization) and pathological mineralization of CaOx, biomineralization kinetics. In this study, we investigate the effect of another 2+ present in plants and animals. Thus, poly(acrylic) acid (PAA) is an acidic alkaline earth metal ion, Sr , on ACP stabilization. In the human body, 2+ 2+ synthetic polymer, described as an inhibitor, widely used to understand how 99.9% Sr is located in bones, and administered Sr is almost exclusively 2+ biomolecules modulate nucleation and the stages of aggregation and precipitated in bone. Sr is vital to young bone tissues formation. The role 2+ crystalline growth of biogenic materials. However, the effect of PAA on the of Sr on ACP formation and its transformation needs to be clarified. The kinetics of the CaOx CNC is currently unknown. precipitation of ACP and the crystallization of HAP are accompanied by an 2+ General Objective abrupt drop in pH. We find that Sr itself have less stabilization effect on 2+ 2+ To investigate the kinetics and PAA concentration effect on the CNC of ACP in comparison with Mg pH curves. However, the presence of Sr can 2+ CaOx through in vitro pre-nucleation assays. significantly enhance the stabilization of Mg on ACP due to a synergic 2+ Materials and methods effect. The chemical analysis reveals more Mg should be excluded from 2+ The CaOx pre-nucleation assays were performed using a computationally ACP to initiate the crystallization of HAP when Sr ions are co-existed in controlled automatic titration system (Titrando 907, Metrohm) and the amorphous phase. This change results in additional energy barriers for commercial computational software (Tiamo ™, current version: 2.3). CaOx the solid phase transformation to provide a better stabilization effect on ACP, pre-nucleation assays were performed by using an automated titration of which benefit bone formation. The revealing of multiple ions effects on the 5mM sodium oxalate solutions with 20mM calcium chloride solutions at a phase transformation of amorphous minerals might shed lights on the rate of 0.06 ml per minute in the absence and presence of PAA at understanding of biomineralization process as well as the fabrication of concentrations of 10, 50 and 100 mg/L. During the whole pre-nucleation test, stable amorphous phase, which has wide applications in drug delivery, the monitoring of free calcium ion concentration was carried out with a vaccine reservations, environment treatments, and lithium ion battery.This selective ion electrode kept constant at pH 6.7. The determination and finding highlights the process of dopant ion exclusion in ACP and its control, characterization of amorphous precursors (cluster) was studied by using which enriches our understanding on the bioinspired regulation of dynamic light scattering system (Nanosizer), transmission electron crystallization by using the cooperation of multiple ions. microscopy (TEM) with selected area electron diffraction and Raman and FTIR spectroscopic techniques. P 17 Results Understanding the phase transformation from amorphous to Preliminary trials showed that the concentration of free calcium detected in crystalline calcium phosphate by in situ transmission electron the absence or presence of PAA differs from the amount of calcium added to microscopy the solution. When PAA was used as an additive at concentration of 100 B. Jin*1, C. Shao1, Z. Liu1, Z. Mu1, R. Tang1 mg/L, there is a decrease in the slope of the kinetic curve of the CNC prior 1Zhejiang University, Hangzhou, China to nucleation point respect to the kinetic curves obtained in the absence of additives. PAA acted as an inhibitor of CaOx crystallization, stabilizing the pre-nucleation cluster, delaying the nucleation stage at longer times, which In nature, the growth front of delicate mineral structures is covered with an was proportional to the additive concentration. In addition, PAA allowed to amorphous continuous layer which is regarded as a precursor phase of tolerate a higher concentration of free calcium, increasing its super- crystallized phase. For example, amorphous calcium phosphate (ACP) saturation. Finally, PAA induces the formation of more soluble solid phases widely exists in the new bone where ACP can transform into hydroxyapatite after nucleation. Ultra-structural characterization by HR-TEM is under (HAP). Although the local rearrangement mediated direct phase progress. transformation mechanism from ACP to HAP has been reported, the detailed Conclusion phase transformation process still remains to be a great challenge due to the The current experimental results regarding to the kinetics of CNC of CaOx lack of direct experimental evidences. Here, we use in situ transmission suggest that PAA has the ability to stabilize in vitro amorphous precursors electron microscopy (TEM) to investigate the phase transformation formed during the pre-nucleation assays. We believe that the electrostatic mechanism by skillfully designing a system in where spherical ACP particle interaction of the acid groups of PAA with the ionic CaOx clusters would be closely contacts with HAP. It is found that the phase transformation is stabilized by steric repulsion, preventing their aggregation. achieved via epitaxial growth. Furthermore, in situ high resolution TEM results show that the epitaxial growth induced crystallization advances via

35 the formation of kinks and steps. It is proposed that these steps and kinks are Precipitates of 2nd series do not contain anatase by XRD data. However, after formed by the local rearrangement of posner"s clusters within ACP. This annealing samples at 700 °C for 6 hours peaks of anatase appear at diffraction finding highlights the key role of physicochemical effects in phase patterns. It evidences of formation of amorphous TiO2 during precipitation, transformation, contradicting the prior assumed epitaxial match between the also confirmed by EDX data. After annealing amorphous TiO2 transits into structural organic matrix and the new produced mineral, which provides an anatase. Samples obtained from solutions with Ti/Ca=0.01 – 0.03 do not important implication in biomimetic mineralization and materials science. contain any titanium dioxide phases by all used methods. The increase of Ti concentration is assotiated with cell parameters variations. P 18 For 1st series, in the interval of Ti/Ca=0 – 0.13 titanium enters at Ca-sites Influence of Equisetum arvense extract on calcium oxalate and in the interval of Ti/Ca =0.13 – 0.15 titanium enters at P-sites. For 2nd crystallization series, titanium enters at both Ca and P-sites, but substitution of P is S. POLAT*1, P. SAYAN1 prevalent. Maximum concentration of Ti in 2nd series (9.5 wt%) is 1Marmara University, Chemical Engineering, Istanbul, Turkey significantly higher than in 1st series (5.4 wt%). Diffuse reflection spectra of precipitates become more similar to anatase spectra (KRONOClean 7050) with increase of Ti concentration. Band gap Introduction energy estimated by Tauc method using Kubelka-Munk transformation of Calcium oxalate crystallization is of great interest in medicine because it is experimental diffuse reflectance spectra R(λ) ranges from 4,34 to 3.25 eV the main constituent in the majority kidney stones. Calcium oxalate exists in and is less than band gap energy of pure hydroapatite (Eg = 6 eV). nature as calcium oxalate monohydrate (COM, whewellite), calcium oxalate The obtained data confirm that Ti-containing hydroxyapatites can show dihydrate (COD, weddellite) and calcium oxalate trihydrate (COT, caoxite). photocatalytic activity which depends on the entrance of titanium into Calcium oxalate monohydrate and dihydrate forms are the major constituents apatite. The next step of our work is estimating their photocatalytic properties in most kidney stones. In this study, the effects on Equisetum arvense extract through decomposition of organics. on calcium oxalate crystallization were investigated detailed. The research was supported by RFBR grant №19-55-45019 IND_a and Objectives following resource centers of SPBU: Geo-Environmental Research and The main aim of this study was to investigate the potential inhibition effect Modelling (GEOMODEL), X-ray Diffraction Studies, Nanophotonics, on Equisetum arvense extract on calcium oxalate crystallization to prevent Optical and Laser Matetials Research, Microscopy and Microanalysis kidney stone formation. References Materials & methods Wakamura, M., Hashimoto, K., Watanabe, T. Photocatalysis by Calcium The calcium oxalate crystallization as a result of the reaction between Hydroxyapatite Modified with Ti(IV): Albumin Decomposition and calcium chloride and sodium oxalate was carried out in a cylindrical glass Bactericidal Effect. Langmuir, 2003, 19, 3428-3431. crystallizer. The experiments were performed in batch crystallization mode Tsukada, M., Wakamura M., Yoshida, N., and Watanabe, T. Band gap and and the conditions were set at 37°C and pH 7.4 at a constant stirring rate. The photocatalytic properties of Ti-substituted hydroxyapatite: Comparison with crystallizer was kept at a constant temperature using a thermostat. During the anatase-TiO2, J. Mol. Catal. A: Chem., 2011, 338, 18–23. experiment, a pH meter was used to monitor the solution pH. To investigate the effects Equisetum arvense, the extract was fed into P 20 crystallizer via a syringe pump. In this study, the concentration of the Immobilization of Amino Acid Dehydrogenases by GO-PEI-IL aqueous Equisetum arvense extract used was 10 wt%. The end-products were Hybrid Material induced Biomineralization characterized by X-ray diffraction (XRD), Fourier-transform infrared K. Liu1, S. Wang*1 spectroscopy (FTIR), thermogravimetric analysis (TGA), and scanning 1Xiamen University, Department of Chemical and Biochemical electron microscopy (SEM) were used. Engineering, Hamburg, Germany Results XRD results showed that the crystals prepared in pure media include only the calcium oxalate monohydrate form. According to the SEM analysis, the Introduction crystals prepared in pure medium had a hexagonal prismatic morphology. L-Homophenylalanine (L-HPA) is a chiral unnatural amino acid, which is a The calcium oxalate monohydrate crystals had very fine structures and their key intermediate of several pharmaceuticals. Biosynthesis of L-HPA surfaces were slightly rough. The surface morphology of the crystals catalyzed by phenylalanine dehydrogenase (PheDH) is an ideal route. There prepared in the presence of the Equisetum arvense was changed. In addition, are problems with limited substrate solubility in aqueous system and enzyme the functional groups and thermal properties of the crystals were investigated denaturation in organic solvents. in Equisetum arvense extract medium. Objectives Conclusion Immobilization of halophilic phenylalanine dehydrogenase with hybrid Equisetum arvense has been showed to affect the crystal structure and materials based on biomineralization in order to create biocompatible morphology of calcium oxalate using in vitro experiments. The structural and microenvironment and improve its stability and catalytic efficiency. morphological transformations were confirmed using XRD, SEM, and FTIR Materials & methods spectroscopy. Our results are useful and will contribute to the research efforts Halophilic phenylalanine dehydrogenase (PheDH) from Bacillus on kidney stone formation, a significant clinical disease and important area nanhaiensis is expressed in E.coil and purified. PheDH was absorbed to GO- of research in the field of biomineralization. PEI-IL and formed GO-PEI-IL-PheDH-TiO2 with the addition of titanium bis(ammonium lactato)-dihydroxide (Ti-BALDH). Enzyme activity were P 19 studied with 2-oxo-4-phenylbutyrate as substrate and NADH as cofactor. Synthesis, characterization and photocatalytic properties of Ti- Results bearing hydroxyapatites The effect of ionic liquid on [EMIM]BF4, [BMIM]BF4, [BMIM]Cl enhanced A. Korneev*1, O. Frank-Kamanetskaya1, M. Kuz'mina1, V. Ryabchuk2, E. enzyme activity by 2.1, 1.5 and 4.6 folds, respectively, while enzyme Sturm3 entrapment efficiency was 92.3%, 82.9% and 99.0%. ILs not only increased 1Saint Petersburg State University, Department of Crystallography, Saint the substrate solubility and also activated the enzyme activity, which was Petesburg, Russian Federation accord to the salt active property of PheDH from Bacillus 2Saint Petersburg State University, Department of Photonics, Saint nanhaiensis. Compared with free PheDH, GO-PEI-PheDH, GO-PEI-IL- Petersburg, Russian Federation PheDH, thermostability were greatly enhanced by induced biomineralization 3University of Kontanz, Department of Chemistry, Konstanz, Germany of Ti-BALDH. Titania provided a rigid cage pocket for the protection from structure unfolding. A mechanistic illustration of the formation of hybrid nanoparticles were proposed based on the multi-level interactions of enzyme, According to [Wakamura et al., 2003; Tsukada et al., 2011] Ti-containing PEI and ILs. hydroxyapatites have photocatalytic activity comparable to activity of Conclusion anatase. However, there are doubts if this activity related to incorporation of Polyethylenimine (PEI) and ionic liquids (ILs) functioned graphene oxide Ti-ions into hydroxyapatite or to small concentrations of TiO2 in precipitate, (GO) were applied for PheDH immobilization by induced biomineralization which can be not detected by X-ray diffraction. of TiO2 with improved stability and activity for biosynthesis of L- Two series of hydroxyapatites were synthesized from solutions containing Ti homophenylalanine. Hybrid materials GO-PEI-IL-TiO2 with synergistic ions. The 1st series was precipitated from CaNO3, (NH4)2HPO4 and TiCl3 effect is are highly potential for enzyme immobilization, especially for solutions, the 2nd series was precipitated from Ca(OH)2, H3PO4 and bioelectrocatalysis, due to its biocompatibility and conductivity. C12H28O4Ti. Synthesized precipitates were studied with a wide set of methods (X-ray powder diffraction, Raman spectroscopy, SEM, EDX, TEM, diffuse reflection spectroscopy). According to the study results, hydroxyapatite has been formed in all synthesis. Also precipitates of 1st series contain anatase impurity. Formation of anatase starts at atomic ratio Ti/Ca=0.56 in solution by XRD data, and at Ti/Ca=0.01 by Raman spectroscopy.

36 P 21 24%), high (about 90%) and a wet state, where destilled water was sprayed Biomimetic synthesis of bovine serum albumin and cysteine onto the ACC tablet. Transformation experiments lasted for one, two, six and modified ubiquitin-templated fluorescent inorganic nine weeks. We characterize our reference and newly-formed carbonate nanocomposites material with XRD, FE-SEM, and electron backscatter diffraction (EBSD). We find that in the low humidity environment, even with low Mg contents, O. Akyüz*1, H. Cölfen1 ACC is highly stabilized and, for time periods up to six weeks, does not 1Konstanz University, Chemical Biology, Konstanz, Germany transform to calcite or aragonite. The most significant transformation takes place when ACC contains 5 wt% Mg but is exposed for a long time to a high Introduction humidity environment. Spraying the surface of the ACC tablet with water Biomineralization which is one of the interesting strategy found in nature for induces transformation and crystalline carbonate formation, however, to a the fabrication of advanced inorganic materials, is a source of inspiration for lesser extent as it is the case when the ACC tablet is exposed high humidity many scientists searching methods for the synthesis of functional and bio- for a long time period. friendly nanomaterials. Proteins which possess well defined three- dimensional structures and self-assembly properties based on their specific P 23 binding sequences have been used as templates for biomineralization of Size matters- nucleation kinetics of amorphous calcium various noble metals to functional nanostructures. By combination of the carbonate in confinement functionalities of nanostructures with the biomineralization strategies; D. Joester*1, N. Metoki1, J. Cavanaugh1, M. Whittaker1, J. S. Evans1, K. fabrication of convenient functional and bio-friendly protein-templated Alvares1 fluorescent inorganic nanocomposites (FINCs) has been possible to use them 1Northwestern University, Materials Science and Engineering, Evanston, in various biomedical applications[1]. Illinois, United States To sum up, proteins are promising biomolecules serve as scaffolds for the fabrication of FINCs with marvelous functions via the biomineralization process. Therefore, developing new strategies for the synthesis of FINCs are Introduction crucial to extend their properties and potential usages in bio nanotechnology. Biomineralizing organisms routinely assemble materials with sophisticated Objectives design and advanced functional properties, often using amorphous precursors Synthesis of FINCs has been getting attention by many scientists due to their to access compositional and structural states far from equilibrium. Organic attractive functions and features. However, the traditional chemical synthesis macromolecules and inorganic additives are thought to play an integral role routes of such FINCs require harsh reaction conditions which are the in controlling phase transformations in these systems. However, it has proven drawbacks for their activities in biological applications. Therefore, extremely challenging to accurately describe pathways and determine developing biomimetic synthesis strategies for the fabrication of FINCs mechanisms, even for extensively studied system such as amorphous calcium which are templated with natural and recombinant proteins under mild carbonate (ACC).[1] A particular problem is the determination of accurate reaction conditions, is pivotal to improve their functionalities. nucleation rates for the transformation of metastable precursors such as ACC The purpose of this study is biomimetic synthesis of FINCs (gold into the final, crystalline minerals.[2] nanoclusters (AuNCs), cadmium selenide and lead sulfide quantum dots Objectives (CdSeQD and PbSQD)) by using genetically engineered and natural proteins Inspired by work in protein crystallization,[3] and guided by theoretical as biotemplates and investigating their properties via spectroscopic and considerations,[4] we set out to expand our earlier work on nucleation and microscopic methods. growth in liposomes[5] by developing a platform for the determination of Materials & Methods nucleation rates in confinement based on droplet microfluidics.[6] Bovine serum albumin (BSA), Ubiquitin (Ub) and Cysteine Modified Materials and Methods Ubiquitin (CMUb) were used as natural and recombinant proteins for the We designed and fabricated microfluidic devices that enable in situ synthesis of AuNCs, CdSeQD, and PbSQD in aqueous solutions at observation of the crystallization of amorphous precursors confined in physiological temperature under alkaline conditions. The proposed emulsion droplets. Using time lapse polarized light microscopy, XRD, and stabilizing/reducing mechanism behind the protein-templated biomimetic Raman spectro-microscopy, we estimated the timepoint of individual mineralization of FINCs is as follows: (1) proteins containing abundant metal nucleation events in hundreds of droplets. Finally, statistical analysis of binding sites, such as cysteine (Cys), histidine residues can covalently nucleation events allowed us to determine steady state nucleation rates for interact with the metal ions, (2) when the pH of protein environment is the conversion of ACC to vaterite and calcite, and the impact of adjusted to alkaline conditions by adding NaOH, i) the conformation of the biomacromolecules and inorganic ions on these rates. protein changes and forms such a cage-like structure which is suitable for the Results accumulation of metal ions (nucleation), ii) the phenolic groups on tyrosine In the absence of additives, statistical analysis revealed kinetics consistent residue can be activated to a phenoxide ion at alkaline pH and then reduces with classical nucleation theory. A steady-state nucleation rate of 1.2 cm-3s- metal ions to metal nanoclusters (reduction). 1 for the crystallization from ACC was determined. This low rate has Results important implications for phase transformation in biological systems. We The biomineralization experiments with the natural Ub and recombinant find that protein extracts from the sea urchin tooth (S. purpuratus) greatly CMUb have clarified the pivotal role of the presence of Cys in protein accelerate the nucleation rate. Recombinant spicule matrix protein sequence by resulting in unstable and stable FINCs, respectively. As a result, (rSM30B/C) shows complex, concentration-dependent impact on BSA and CMUb stabilized, functional, and stable FINCs possess crystallization rates. To our surprise, this includes a polymorph switch at photoluminescence and crystal size between 570-700 nm and 3-4 nm, elevated concentrations. Finally, we find that the presence of barium cations respectively, have been synthesized and characterized. in droplets has a dramatic impact on nucleation mechanisms and rates. Conclusion Conclusion Green biomimetic synthesis strategies have been used to direct the formation Droplet microfluidic devices are powerful means to dissect nucleation and of FINCs having sub nanometer size and stable photoluminescence. growth of amorphous precursors, and the impact of additives relevant in [1] J.Huang, L. Lin, D. Sun, H. Chen, D. Yang, Q. Li, Chem Soc Rev biology and bio-inspired materials chemistry. I will discuss implications of 2015,44,6330-6374 the absolute rates we determined for biological systems as well as mechanistic insights into the acceleration by rSM30B/C and barium cations. P 22 References The coupled influence of Mg content and humidity on the [1] J. J. De Yoreo, P. U. P. A. Gilbert, N. A. J. M. Sommerdijk, R. L. Penn, transition from amorphous calcium carbonate to calcite S. Whitelam, D. Joester, H. Zhang, Jeffrey D. Rimer, A. Navrotsky, J. F. Banfield, A. F. Wallace, F. M. Michel, F. C. Meldrum, H. Cölfen, P. M. R. Thuemmler*1, B. Purgstaller2, E. Griesshaber1, M. Zenkert1, M. Dietzl2, Dove, Science 2015, 349. [2] L. M. Hamm, A. J. Giuffre, N. Han, J. Tao, D. W. W. Schmahl1 Wang, J. J. De Yoreo, P. M. Dove, PNAS 2014, 111, 1304-1309. [3] S. V. 1Ludwig-Maximillian Universitaet, Muenchen, Germany Akella, A. Mowitz, M. Heymann, S. Fraden, Crystal Growth and Design 2University of Technology, Institute of Applied Geosciences , Graz, Austria 2014, 14, 4487-4509. [4] R. P. Sear, CrystEngComm 2014, 16, 6506- 6522. [5] C. C. Tester, M. L. Whittaker, D. Joester, Chem Commun 2014, 50 Amorphous calcium carbonate (ACC) shows a remarkable stability, which is 5619 - 5622. [6] J. Cavanaugh, M. L. Whittaker, D. Joester, RSC Chemical initiated by incorporation of impurities, e.g. ions or/and organic ligands. Science 2019, available online. Some impurities, e.g. Mg, stabilizes amorphous calcium carbonate to a high degree and prevents effectively its transformation to crystallized calcite or aragonite. In this study we discuss the coupled influence of Mg and humidity on the transformation of ACC to crystalline carbonate. We describe for ACC stabilized with 5 and 50 wt% Mg contents the newly-formed carbonate phase and the mode and speed from the amorphous to crystalline transformation. Experiments were carried out in three humidity environments: low (about

37 P 24 P 26 Polymorph control of anhydrous guanine crystals Novel nanoscopic pathways in crystallisation and selfassembly Y. Ma*1, F. Chen1 of barium carbonate nanoparticles in microemulsions 1Beijing Institute of Technology, School of Chemistry and Chemical W. Sager*1 Engineering, Beijing, China 1Forschungszentrum Jülich, Peter Grünberg Institute - Microstructure reserch (PGI-5), Jülich, Germany Amorphous guanine was proposed to be the mineralization precursor of biological anhydrous guanine β phase in vesica of the Koi fish scales1. Different nanoscopic paths to witherite nanoparticle formation via the birth Guanine is one of the most widespread organic crystal existing in organisms and deterioration and/or transformation of meta-stable amorphous and, so far to produce structural colors. Guanine monohydrate and two anhydrous unreported, crystalline barium carbonate nanoparticles using nonionic water- guanine phases (AG), aand b , are three well known crystalline phases of in-oil microemulsions as precipitation media have been investigated in detail guanine. The excellent optical performances of guanine crystals are mainly by transmission electron microscopy and selected area diffraction. Compared attributed to the crystalline AG β micro-platelets exposed (100) plane and to precipitation from homogeneous media, the employment of high refractive index (1.83) 2. Herein, a novel crystal phase of anhydrous microemulsions allows for a more precise control of the ongoing nucleation guanine consisting of N9-G purine ring was obtained by dehydration of and growth processes, not only via adjusting the concentration of the reacting guanine monohydrate (GM) at 111 °C, termed as dehydrated-GM, which is species involved and temperature, but also by tuning the size and the the first reported tautomeric polymorph of AG (N7-G purine ring). The exchange kinetics of the isolated and compartmentalised nanometre-sized Dehydrated-GM has very good water harvest ability (8 wt%) with a fast rate water domains. Here we present a comprehensive study on how tuning the ( in 30 min) even at relatively low room humidity (below 20 %) and low properties of the parental microemulsion can be implemented in tailoring the temperature (40 °C). We realized the synthesis of three polymorphs of crystal structure, morphology and self-assembling properties of nucleating anhydrous guanine, AG a, AG b and dehydrated-GM in this work. A pure and growing nanoparticles. At low water content a variety of different meta- phase of the anhydrous guanine (AG) β form was obtained via transformation stable nanoparticles forms that ranges from in the electron beam amorphous of a hydrated amorphous guanine phase (HAmG), demonstrating short-range filaments to monoclinic (m) cubes and hexagonally shaped, probably trigonal order in solvents such as formamide, DMSO and DMF. Solid-state NMR (t), thin platelets, which assemble into micrometre-long stacks. Slowing (ssNMR) characterization indicates that the HAmG precursor has similar down of the exchange kinetics permits us to study the early stages of particle short-range order as AG β, which might be the reason for the formation of formation and to gain full insight into unrevealed crystal transformation and AG β instead of the thermodynamically more stable AG α. The AG β nano- re-crystallisation processes. The genesis of the, at ambient conditions platelets obtained in DMSO expose (100) plane when polyvinylpyrrolidone thermodynamically stable, orthorhombic (o) polymorph (witherite) evolves (PVP) was applied as additive. The sizes of AG β nano-platelets were about either via direct transformation (e.g. a→o transition from filaments into rods) 100 nm in length, 40 nm in width, and 10 - 20 nm in thickness. The delicate or via dissolution/re-crystallisation processes. In the latter case the crystalline control on the polymorph and morphology of guanine crystals via an pre-structure particles dissolve, thereby freeing material for the re-nucleation amorphous phase strategy may inspire the formation of highly ordered of the witherite phase (m/t→o transition), emphasising the double role of the hierarchical structures of guanine crystals with unique optical properties. microemulsion domains as nucleation site and host and thus transport References medium for water-soluble species. Depending on the different crystallisation [1] D. Gur, Y. Politi, B. Sivan, P. Fratzl, S. Weiner, L. Addadi, Angew. Chem. paths taken and the growth conditions encountered, a rich variety in final Int. Ed. 2013, 52, 388. nanoparticle morphologies is obtained. The ubiquitous microemulsion [2] A. Levy-Lior, B. Pokroy, B. Levavi-Sivan, L. Leiserowitz, S. Weiner, L. droplets induce attractive depletion forces between the forming nanoparticles Addadi, Cryst. Growth Des., 2008, 8, 507. leading to different types of particle selfassembly, such as columnar stack [3] F. H. Chen, Y. R. Ma*, Y. X. Wang, and L. M. Qi*, Cryst. Growth Des., formation of nanoplatelets that is additionally characterised by small angle 2018, 18, 6497. x-ray scattering and electron tomography. [4] F. H. Chen, B.B. Wu, N. Elad, A. Gal, Y.N. Liu, Y. R. Ma * and L. M. Qi, CrystEngComm, 2019, DOI: 10.1039/C9CE00245F. P 28 Organization of prismatic layers in mollusk shells - Pinctada vs. P 25 Pinna The incorporation of organic and inorganic impurities into the Y. Dauphin*1, E. Zolotoyabko2, A. Berner2, E. Lakin2, C. Rollion-Bard3, lattice of metastable vaterite J. P. Cuif4, P. Fratzl5 E. Seknazi*1, B. Pokroy1 1Museum national d'histoire naturelle, ISYEB UMR 7205, Paris, France 1Technion - Israel Institute of Technology, Material Science and 2Technion-Israel Institute of Technology, Department of Materials Science Engineering, Haifa, Israel and Engineering, HAIFA, Israel 3Université Sorbonne Paris Cité, IPGP UMR 7154, Paris, France 4Museum national d'histoire naturelle, CR2P UMR 7207, Paris, France Most of calcitic marine biominerals were shown to incorporate 5Max Planck Institute of Colloids and Interfaces, Department of intracrystalline organic and inorganic inclusions. Inspired by these previous Biomaterials, Potsdam, Germany findings, biomimetic calcite, containing organic or inorganic intracrystalline inclusions was successfully synthesized in various routes. In the present Introduction study, we consider this feature in another calcium carbonate polymorph, Cross-sections of calcitic prismatic layers in mollusk shells, cut namely vaterite. Vaterite is the least thermodynamically stable and the least perpendicular to growth direction, reveal well-defined polygonal shapes abundant anhydrous crystalline phase of calcium carbonate. It is also the least clearly resolved by light and electron microscopy. These observations are well understood, as its crystal structure is still a matter of debate. typical for the prisms of Pinna, Pinctada, Atrina, and . Taking into In this study, we show that vaterite is able to sustain high lattice strains and account the rhombohedral symmetry of calcite, often presented in hexagonal incorporate Mg, Ba, and aspartic acid in its lattice. We report, for the first axes, this led to the long-standing opinion that calcitic prisms grow along the time, the formation of substituted Mg- and Ba-vaterite. We characterized and c-axis of calcite. In other words, the c-axis of calcite is thought to be quantified the structural distortions caused by incorporation of aspartic acid, perpendicular to the surface of the shell. This paradigm is mostly based on Mg or Ba into the vaterite hexagonal pseudo-cell by means of high-resolution the long-term studies (since 1840) of Pinna nobilis shells, revealing the synchrotron X-ray diffraction and micro-Raman spectroscopy. We show that largest prism size; however other shells are much less investigated. Mg substituted for up to 12% of Ca, and Ba for up to 2.5% of Ca, to form In this research, we address the question whether or not the crystallography crystals of Mg-vaterite and Ba-vaterite with lattice distortions of up to 0.9% of calcitic prisms in Pinctada shells is similar to that well established in and 0.14%, respectively. Pinna. As working example, we focused on the structure of Pinctada Interestingly, this study proves that the presence of Mg and organics in margaritifera shells. biogenic vaterite can be parameters which affect its reported structural Materials & methods variability. Therefore, this study, if accompanied by analyses of the Mg and The shells of Pinctada margaritifera were collected in French Polynesia, organics present in biogenic vaterite, could help clarify the true structure of while those of Pinna nobilis came from the Mediterranean Sea. The shell vaterite. morphologies and finer structural features were imaged by optical microscopies, SEM and AFM; crystallographic dissimilarities were analyzed by X-ray diffraction and EBSD; in situ composition was studied using Raman and FTIR spectroscopies, EPMA, XANES and X-ray fluorescence. Results: There are two kinds of prisms in Pinctada margaritifera: small simple prisms and large prisms comprising several sectors. Only simple prisms are known to exist in Pinna and Atrina shells. All prisms are built of nano-granular calcite (still being nearly single-crystalline) and organic matrix. In contrast to Pinna nobilis, we find that in both large and small prisms of Pinctada margaritifera the c-axis is mostly perpendicular to their

38 long morphological axis, i.e., parallel to the surface of the shell. Furthermore, Seamount. This result strongly implies that most previously recognized the degree of this preferred orientation is higher in large prisms. "species" in fact belong to one species that is highly morphologically plastic We relate these growth dissimilarities to the striking differences in the in shell form. The shell microstructure is the micro-scale morphological trait organic contents. In fact, the soluble organic matrix of Pinna nobilis is rich of molluscan shells (Carter, 1990) and can represent a strong tool to estimate in acidic sulphated polysaccharides, whereas that of Pinctada margaritifera phyletic relationships and physiology. Therefore, the purpose of this study is is rich in proteins. Lipid contents of Pinna and Pinctada also differ, as well to assemble how genetic constraints and environmental factors affect the as Mg and S concentrations. In addition, small prisms have higher organic expression of shell microstructure of pectinodontid . Shell and organics-related sulfur contents than the large ones. In particular, atomic microstructures of pectinodontids were observed by SEM and their mineral percentage of sulfur in Pinna nobilis is three and twenty times higher than in composition was detected by Raman spectrophotometry. As a result, we were small and large prisms, respectively, of Pinctada margaritifera. able to estimate specimens to genus level classification based on their shell Conclusion microstructural compositions. Furthermore, the proportion of shell We conclude that, despite their morphological similarities and common microstructures with two different mineral compositions (aragonite or mineralogy, the prisms of Pinctada and Pinna, both Pteriomorpha, are far calcite) clearly corresponds to differences in chemosynthetic habitat. Our from being identical. The comparison of the organic contents reveals study reports that mollusks regulate the mineral polymorphism in response significant differences in their nature and quantities. Based on these findings, to their chemosynthetic habitat. This phenotypic plasticity in shell we suggest that in mollusk shells, the morphology of calcitic prisms is not mineralogy is significant not only in the context of the adaptive radiation of primarily determined by the crystallographic structure of calcite, but largely pectinodontid linages to chemosynthetic environments, but also to shed light by organic components, the presence or absence of which drastically change on mineralogical aspects regarding the biomineralization mechanism of the the growth mode. molluscan shells.

P 29 P 31 Biomimetic cross-lamellar structures fabricated by ordered Immunological detection and LC-MS/MS analysis of the shell self-assembly of CaCO3 nanorods matrix protein ICP-1 in brachiopods M. Takasaki*1, M. Tago1, T. S. Suzuki2, Y. Oaki1, H. Imai1 Y. Isowa*1, K. Kito2, H. Sawada1, K. Endo3 1Keio University, Yokohama, Japan 1Nagoya university, Sugashima Marine Biological Laboratory, Toba, Japan 2National Institute for Materials Science, Tsukuba, Japan 2Meiji University, Department of Life Sciences, Kawasaki, Japan 3University of Tokyo, Department of Earth and Planetary Science, Tokyo, Japan Introduction

Biominerals have excellent mechanical and optical properties as compared Introduction with geologic balky crystals. Especially, cross-lamellar structures of Brachiopods are sessile marine invertebrates belonging to Lophotrochozoa, consisting of CaCO -based nanorods have received attention as an 3 and have two valves composed of calcium carbonate or calcium phosphate. excellent model for lightweight and tough structural materials because of Recently, proteomic analyses of shell matrix proteins have been performed their superior mechanical properties derived from oriented architectures. on several brachiopod species to understand the molecular mechanisms and Ordered self-assembly of nanocrystals is a non-classical way to fabricate evolution of brachiopod shell formation. In a previous shell proteomic analogues of biogenic oriented architectures. However, artificial cross- study of the rhynchonelliform brachiopod Laqueus rubellus, we identified a lamellar structures have not been produced by the self-assembly technique. complete sequence of ICP-1, which was originally characterised as a Objectives pigment-carrying intracrystalline protein from the three rhynchonelliform The aim of our study is fabrication of biomimetic cross-lamellar structure by brachiopods Neothyris lenticularis, Calloria inconspicua, and Terebratella an evaporation-driven assembly method. We utilized calcite nanorods as a sanguinea using Edman degradation. We showed that it is the most building block and an intense magnetic field to enhance their oriented abundant protein in the shell of L. rubellus by LC-MS/MS analysis. attachment. However, further details of ICP-1 remained to be clarified. Materials & methods Calcite nanorods ~50 nm in diameter that were elongated in the c direction were obtained by oriented attachment of calcite nanograins synthesized by Objectives carbonation of Ca(OH)2 aqueous dispersion. We demonstrated a millimeter- Antibodies are useful for localization of proteins within tissues. As a first scale ordered assembly of calcite nanorods dispersed in ethanol by step to localize shell matrix proteins in the shells, we prepared an antibody combining the arrangement with an evaporation-driven capillary force and against ICP-1 in this study, and verified the binding specificity of the alignment under an intense magnetic force. Layer-by-layer accumulation of antibody. In addition, we carried out LC-MS/MS analysis to characterize the ordered assembly of the calcite nanorods was performed with a rotation post-translational modifications of ICP-1. of the substrate at a 90-degree angle. Materials & methods Results The shell extracts from L. rubellus were subjected to SDS-PAGE, and Calcite films ~10 μm in thickness were formed by oriented assembly with Western blotting was performed using the antibody against a synthetic lateral stacking of the nanorods on a substrate. The orientation of the peptide designed from the ICP-1 sequence of L. rubellus. LC-MS/MS nanorods in the films was improved by application of an intense magnetic analysis was also performed against three major bands in the SDS-PAGE. field at 12 T. The single-crystalline feature of the films similar to biogenic Results cross-lamellae was characterized by X-ray and electron diffraction. Artificial Western blotting showed a single band of approximately 60 kDa in size. This cross-lamellar structures with crystal direction switching at 90-degree were is substantially higher than the molecular mass expected from the theoretical obtained by layer-by-layer accumulation of the ordered calcite films. amino acid sequence of ICP-1 (17.5 kDa). We inferred that LrICP-1 Conclusions undergoes post-translational modifications. Indeed, searches using Biomimetic cross-lamellar structures were fabricated through oriented NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc) indicated attachment of calcite nanorods by evaporation-driven self-assembly. Our that LrICP-1 has one potential glycosylation site. Meanwhile, LC-MS/MS results suggest that the self-assembly techniques are applicable for analysis showed that a peptide of ICP-1 was identified from a band under 6.5 fabrication of excellent analogues of biogenic oriented architectures. kDa, which is smaller than the molecular mass expected from the theoretical amino acid sequence of ICP-1. P 30 Conclusion The phenotypic plasticity in shell microstructures of vent and The synthetic peptide used for antibody generation is located in the N- seep pectinodontid limpets terminal region, while the peptide identified by LC-MS/MS is located in the K. Sato*1, C. Chen2, R. G. Jenkins3, H. K. Watanabe2 C-terminal region. Therefore, one possibility is that ICP-1 undergoes post- 1Waseda University, Department of Earth Sciences, School of Education, translational cleavage, and the N-terminal fragment undergoes other post- Tokyo, Japan translational modifications. To confirm this hypothesis, we plan to perform 2Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan additional LC-MS/MS analysis to identify peptides in the N-terminal region 3Kanazawa university, Kanazawa, Japan of ICP-1.

Gastropods often show intraspecific variation in phenotypes among different habitats. Recently, the authors carried out molecular phylogenetic analyses of pectinodontid limpets in two genera, Bathyacmaea and Serradonta, commonly associated with vent and seep environments in the west Pacific (Nakano & Sasaki, 2011). Instead of revealing distinct lineages corresponding to morphological identification, the analyses showed that all individuals from both genera were mixed in a single nested monophyletic clade, except for one undescribed species from the South Chamorro

39 P 32 information was obtained by using optical observation (mostly epi- Exploring biomineral formation at the nanoscale by electron polarisation), SEM on both secondary and back-scattered modes, energy loss spectroscopy (EELS) synchrotron based XANES method (ESRF Grenoble France) and M. de Frutos*1, O. Stéphan1, W. Ajili2, T. Azaïs2, N. Nassif2, S. Auzoux- fluorescence (SOLEIL Saint-Aubin France). Bordenave3, D. Bazin4, M. Daudon5, E. Letavernier5 Results 1CNRS Université de Paris Sud, Laboratoire de Physique des Solides, In Pinna nobilis etching shows that prisms are built by closely associated 6 Orsay, France to 8 µm thick sub-units (about 8 to 15 for every prism depending on 2CNRS Sorbonne Université, LCMCP, Paris, France specimens) whose overall orientations is slightly oblique to longitudinal 3CNRS Sorbonne Université, BOREA, Paris, France growth axis. Close correspondence of these mineral sub-structures between 4CNRS Université de Paris Sud, Laboratoire de Chimie Physique, Orsay, adjacent prisms shows that their formation at the growing edge of the mantle France is related to the pulsed stepping extension of the shell. 5Hopital Tenon AP-HP, Service des Explorations Fonctionnelles, Paris, Prisms of the Pinctada margaritifera exhibit important morphological and France microstructural changes during growth. After an initial monocrystalline status increase in prism diameter is correlated to passage to a polycrystalline status. Additionally, orientation of the crystallographic c axes becomes Understanding of the hierarchical organization of biominerals and the perpendicular to growth direction, a surprising contrast to common views. mechanisms involved in their formation implies to unveil their chemical Shift from single crystal to polycrystalline structure of the prisms, then composition and structure at different scales. Of particular relevance is the regression of the diameter and biochemical change in the final stages suggest characterization of the organo-mineral interface to understand how the a continuous evolution of the mineralizing phase related to shell overall organic fraction regulates the initiation of the calcification (nucleation) and elongation and aging of the mineralizing cell layer. direct the crystal growth. Conventional TEM (transmission electron Conclusion microscopy) is limited, since it cannot easily distinguish the nature of the These two prisms exhibit very distinct and contrasting internal sub- different components. Compared to other spectroscopic approaches, EELS structures, revealing the biological control exerted over crystallization by the in a STEM (scanning transmission electron microscope) offers the advantage mineralizing secretions of the epithelial cells during shell growth. From a of an outstanding spatial resolution (at the nanometer scale) in both chemical methodological view point, note can be made that such biological control of analysis and imaging. the structural and crystallographic patterns rises question regarding models To illustrate the interest of EELS for biomineral studies, we will present based on simple physical process (e.g. crystalline competition as well as selected results related to two different systems: kidney calcifications and predictive capability of thermodynamic grain growth theory and self-similar abalone nacre. EELS was used to analyze the specimens with a resolution crystallization). defined by the diameter of the electron probe (typically better than a nanometer). P 34 Kidney samples were prepared by ultramicrotomy from small pieces of Correlative 3D Raman imaging- a powerful method for human kidney chemically fixed and embedded in epoxy resin. Nacre thin comprehensive studies of biomineralized samples section samples were obtained by Focused Ion Beam (FIB) on a selected area U. Schmidt1, J. Englert1, A. Richter1, M. Böhmler1, T. Olschewski1, K. of the nacre layer of one-year-old Haliotis tuberculata shell. EELS data were Hollricher1, H. Fischer*1 recorded on a VG HB01 STEM operated at 100 keV. The measurements were 1WITec GmbH, 89081, Germany obtained using a nitrogen-cooled sample stage and with the minimum electron dose to limit the radiation damage induced by the electron beam. During an acquisition, the electron beam was scanned over the specimen and Introduction an EELS spectrum was acquired for each position, giving access to an Raman microscopy is a useful tool for analying biominerals, as it can reveal elemental mapping. the chemical composition of composite materials. Even more information EELS data were compared with different reference samples, including can be obtained by correlating the acquired images with data from other mineral and organic compounds, in order to identify the chemical species methods, such as Atomic Force Microscopy (AFM) or Scanning Electron present in calcified tissues. The relative distributions of mineral and organic Microscopy (SEM). Such approaches can yield information that would not fractions were obtained from the signal of the calcium and phosphorus edges be available with only one technique. Thus, Raman Imaging in combination and by the gaussian fitting of the peaks associated to carbonate and organic with AFM or SEM can provide new insights into the fundamental processes compounds on the carbon K-edge. Two main minerals were found: calcium by which organisms produce biocomposites. carbonate in nacre and calcium phosphate, sometimes associated to Objectives carbonate, in kidney calcifications. The biological fraction were clearly In this contribution we will introduce the principles of state-of-the-art distinguished from the embedding resin. confocal Raman Imaging as a tool for analyzing the chemical and molecular Our study shows that STEM-EELS allows a nanoscale analysis of the characteristics of a sample. We will show how this technique can be used in chemical composition of biominerals in different calcified tissues giving combination with AFM and SEM to correlate chemical information with access to the mineral/organic interface. These data provides new insights to structural properties. The aim of this contribution is to describe and highlight elaborate possible mechanisms involved in biominerals formation. the unique features of such combined scientific analysis instruments, based Acknowledgements: This work was partially funded via the CNRS-CEA on examples from various nano-biomaterials. "METSA" French network (FR CNRS 3507) and the EU grant ESTEEM. Materials & Methods For this study we use a Raman Imaging system which combines a confocal P 33 microscope with a highly sensitive Raman spectrometer. It delivers an Sub-structures in bivalve simple prisms - a record of species- excellent depth resolution that enables the acquisition of 3D Raman images. specific control over crystallization during shell growth Raman microscopy allows the analysis of the distribution of the chemical J. P. Cuif*1, Y. Dauphin2 components in a sample without requiring specialized sample preparation or 1Museum Histoire Naturelle, CR2P-Paleontology, Paris, France risking damage to the sample. It can easily be combined with other 2Museum National Histoire Naturelle, umr ISYEB, Paris, France techniques, such as AFM or SEM for correlative measurements. Results SEM provides information on morphology, elemental composition and Introduction crystalline structure of a sample. Confocal Raman Imaging of the same Since their very first observation in the shell of the Pteriomorphid Bivalve sample area reveals the chemical composition as well as polymorphism, Pinna nobilis, (Bowerbank 1844) "simple prism" as a microstructural stress states and anisotropies. The combination of SEM and confocal Raman category has been used for shell description in many biological taxa, with microscopy in a single instrument allows the nondestructive characterization taxonomic and evolutionary purposes. Most of the comparative studies are of biominerals with the highest resolution. Measurements of pearls and nacre based on static description focusing on morphological patterns and three illustrate the benefits of combining these methods for investigating dimensional arrangements of the crystal-like units observed in adult shells. biominerals. For example, a biomineralization defect in pearls was studied, Here attention is drawn to diversity of mineral phase organizations and which leads to loss of its pearlescence ("milky pearl"). growth mode variations in two simple prisms: P. nobilis and Pinctada Conclusion margaritifera. This submission highlights the power of correlative Raman techniques, such Materials and Methods as Raman-AFM and Raman Imaging and Scanning Electron (RISE) Series of Pinna shells were kindly provided by Pr. N. Vicente, head of the Microscopy, for the analysis of composite materials. Les Embiez laboratory at Toulon-Six-Fours (France). Shells of Pinctada margaritifera were collected in Polynesia during a five years research project involving the research department of the Direction des Ressources Marines at Tahiti. Structural patterns were made visible through polished surfaces prepared on variously located areas of the prismatic layers followed when necessary by appropriate etchings. From these specifically prepared surfaces,

40 P 35 technics and examined with electrophoresis. Proteins compositions were Asymmetric distribution of the crystal particle implies the analyzed with LC-MS/MS. Their structural properties were investigated by potential mechanism of aragonite tablets formation in the CD. Influence of isolated proteins on calcium carbonate crystallization was nacreous layer of Pinctada fucata studied by a slow diffusion method. Crystals morphology and polymorph were investigated by SEM and micro-Raman microscopy. Moreover, post- T. Jiang*1, J. Huang1, L. Li1, L. Xie1, R. Zhang1,2 translational modifications, such as phosphorylation and glycosylation, were 1Tsinghua University, MOE Key Laboratory of Protein Sciences, School of also studied. Life Sciences, Beijing, China Developed methods allowed the initial separation of otolith's proteins. 2Yangtze Delta Region Institute of Tsinghua University, Department of Besides previously studied otolith proteins, proteomics identified several Biotechnology and Biomedicine, Jiaxing, China new ones that could be connected to biomineralization. Isolated macromolecules strongly affect the morphology of calcium carbonate The structure of the pearl oyster shell is intriguing because it implies that crystals. Appropriate staining of separated fractions indicates a possible high there are two biomineralization strategies co-existed in one system. The percentage of proteoglycans and glycosaminoglycans in the ear stones. In thermodynamic aspect of the prismatic layer formation has already been addition, one protein that has a particularly strong affinity to the discussed but that of the nacreous layer seems lost in the whole picture. The hydroxyapatite column was isolated. In vitro studies of this protein indicate, mechanism governing the formation of the nacreous layer is convoluted. that it occurs in phosphorylated form in otoliths. The protein changes calcium However, the discussion about the pivotal component in the process is still carbonate crystals polymorph from calcite to vaterite. Our results pointed out enlightenment. Here, with the help of the scanning electron microscope that the protein is present in high-molecular-weight aggregates and peptides (SEM) and the transmission electron microscope (TEM), the crystal particle identified with MS/MS demonstrated that it might be Starmaker-like protein. distribution on the shell surface and the arrangement of aragonite tablets in In conclusion, obtained results indicate that otolith biomineralization process Pinctada fucata have been carefully examined. Strikingly, contrary to might be far more complex than previously claimed. Our research allowed intuition, particles do not uniformly accumulate outward from the center isolation, initial separation and characterization of individual components of which might imply the dominant role played by the Marangoni effect. The the organic fraction. Most probably it was the first time when the Starmaker- thickness of the aragonite tablets is not identical. The organic frame has like protein was successfully isolated from otolith matrix. Our results evenly shifted among different layers. Taken together, these results imply the revealed that proteins from common carp could be especially prone to existence of the Marangoni effect in the process of the nacreous layer phosphorylation and glycosylation. formation and the asymmetric distribution of the particle may lead to the even Acknowledgments: This work was supported by the National Science Center shift of the organic frame. (Poland) [UMO-2015/19/B/ST10/02148] and in a part by statutory activity subsidy from the Polish Ministry of Science and High Education for the P 36 Faculty of Chemistry of Wroclaw University of Science and Technology. Microstrured biomineral particles from Emiliania huxleyi as a raw material P 38 M. Chairopoulou*1, M. Herrmann2, U. Teipel1 Proteomic investigation of the blue mussel larval shell organic 1TH Nürnberg Georg Simon Ohm, Mechanical Process Engineering, matrix Nürnberg, Germany A. Carini*1, T. Koudelka2, A. Tholey2, E. Appel3, S. Gorb3, F. Melzner4, 2Fraunhofer-Institut für Chemische Technologie, Pfinztal, Germany K. Ramesh5 1The University of Hong Kong, School of Biological Sciences, Hong Kong, Hong Kong Microstructured particles have a number of interesting assets that can be 2University of Kiel, Institute for Experimental Medicine - Systematic beneficial for certain applications. To that counts for example the specific Proteomics and Bioanalytics, Kiel, Germany surface area, which tends to increase with decreasing particle size, the pore 3University of Kiel, Department of Functional Morphology and structure, promising for material loading, and also the wetting behavior of Biomechanics, Institute of Zoology, Kiel, Germany fine powder materials. When such structures are pursuit in natural systems 4GEOMAR, Marine Ecology, Kiel, Germany the algae family of Coccolithophorida pose a good representative. The 5University of Gothenburg, Sven Lovén Centre for Marine Infrastructure, sophisticated structures, formed according to strict biomineralization Fiskebäckskil, Sweden mechanisms, are called coccoliths and are composed mainly of calcium carbonate (CaCO3). Since CaCO3 is broadly used in numerous applications the potential of coccoliths is anticipated to be promising. However, in order Introduction to use those uniquely formed particles in any future applications basic Shell matrix proteins (SMPs) are occluded within molluskan shells and are questions and challenges still need to be improved. In an effort to address fundamental to the biological control over mineralization. While many challenges associated with their separation process this study focuses on studies have been performed on adult SMPs, those of larval stages remain recovering coccoliths from freshly cultivated media. The biogenic broth for largely undescribed. the experimental part was taken from laboratory cultivations of an Emiliania Objectives huxleyi strain in a 5 L fed-batch cultivation system. To that system, strategies Therefore, this study aimed to characterize the larval shell proteome of the to clean and isolate the particles were followed according to literature blue mussel for the first time and to compare it to adult mussel shell references and were tested for the first time for the same system. Through proteomes. characterization of the treated particles, the applied methods could be Methods compared and two separation concepts could be distinguished. For the two Following development of a method for cleaning minute larval shells of concepts further optimization steps were followed concluding in still open tissue contaminants, the whole blue mussel larval shell proteome was challenges that need to be phased in the coccolith separation chapter. extracted and subsequently sequenced using shotgun proteomics. Results P 37 Forty-nine SMPs were identified in total. Twenty-one proteins were common Proteins involved in Cyprinus carpio otoliths biomineralization to all samples including: the blue mussel shell protein, a peroxidase domain- M. Kalka*1, A. Ożyhar1, J. Stalarski2, P. Dobryszycki1 containing sequence, a laminin G domain-containing sequence, a ZIP 1Wroclaw University of Science and Technology, Department of domain-containing sequence and a ferric chelate reductase 1-like sequence. Biochemistry, Wrocław, Poland Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, 2Institute of Paleobiology, Polish Academy of Science, Department of chitin-binding type 3, thyroglobulin and EF-hand. This study demonstrates Environmental Paleobiology, Warsaw, Poland that the mussel larval shell proteome is unique: while key predictable mollusk shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized. Further, comparison to adult mussels reveals Otoliths, the ear stones of teleost fish, are biomineralized structures whose that only four domains are conserved among species and developmental major role is sound transduction and sensation of linear acceleration. They stages and nine domains are specific to mussel larvae. are mainly composed of calcium carbonate, which is deposited onto an Conclusion organic matrix. The organic matrix, which consists of proteins, The bivalve larval shell proteome is not a subset of the adult one: blue mussel polysaccharides and lipids, accounts for only a few mass percent of an larvae assemble a unique and novel shell proteome that serves specific otolith. Nevertheless, the organic fraction, especially proteins, have been physiological needs for their development. Further, the observed differences shown to be essential for proper biomineralization of otoliths. Our recent in shell proteomes between species and life stages support the idea that strong proteomics studies have revealed that otoliths contain more proteins than species-specific and ontogenetic variation exists in molluskan shell previously thought. proteomes. The aim of our work was the isolation and characterization of macromolecules found in common carp otoliths. We paid particular attention to structural properties and biomineralization activity of isolated proteins. In order to isolate proteins, asteriscus otoliths were decalcified with EDTA. Then, obtained macromolecules were separated by various chromatographic

41 P 39 recrystallization (secondary calcite or silicic nodules) that contributes to Deciphering the intricate 3D Structure of growing calcite destroying or partly strengthening the fossil shells. This aspect can be due to crystals during coccolith formation bacterial activity using the organic matrices as food supply, presence of E. M. Avrahami*1, L. Aram1, A. Gal1 microboring organisms that induce dissolution and recrystallization, and 1Weizmann Institute of Science, Plant and Environmental Sciences, weathering. Rehovot, Israel P 41 High resolution study of nacre formation and organo-mineral Coccolithophores are a widespread and ecologically important group of interface in the shell of the European abalone Haliotis unicellular calcifying phytoplankton. They are distinguished by a complex tuberculata exoskeleton composed of multiple interlocking calcite plates, known as 1,2 1 1 1 3 coccoliths. Coccoliths are produced intracellularly within a specialized S. Auzoux-Bordenave* , W. Ajili , N. Menguy , I. Estève , M. De Frutos , K. Benzerara1, N. Nassif1, T. Azaïs1 membrane-bound organelle, the coccolith vesicle, and are subsequently 1 exocytosed to the cell surface. Although extensive research in the past Sorbonne University, Paris, France 2Muséum national d’Histoire naturelle, Concarneau, France century has contributed greatly to our understanding of coccolithophore 3 physiology and ecological significance, not much is known about the University Paris-Sud, Physics, Orsay, France mechanisms by which they precipitate and control coccolith assembly. Nearly thirty years ago, the V-R model for crystal nucleation, orientation, The European abalone Haliotis tuberculata is a commercially and and growth was suggested based on light- and electron microscopy, and to ecologically important gastropod species and represents a key-model to study this day it is the ruling paradigm. However, the path by which crystals grow the basic mechanisms of shell formation. The abalone shell is composed of into their final morphology remains unclear. In this work, we chose two calcified layers underlying the periostracum, i.e. an outer spherulitic Cacidiscus leptoporus as a model organism for exploring the growth of layer and an inner nacreous layer. The nacreous layer is exclusively intracellular coccoliths, given that this species forms relatively large composed of aragonite platelets, 6–8 μm in diameter, horizontally aligned in coccoliths in comparison to the commonly studied species Emiliania huxleyi layers and vertically in columns, arranged with a small amount of organic and Pleurochrysis carterae, simplifying their analysis. A simple, yet material. This typical architecture provides to nacre exceptional mechanical effective, protocol was established for extracting developing coccoliths at properties with potential uses in jewellery and . The organic initial formation stages while preserving the inorganic phases and their phase, which represents less than 5 wt.% of nacre, is proposed to guide ultrastructure. These coccoliths were then imaged ex vivo using several nucleation and control the growth and orientation of the mineral phase. electron microscopy techniques, including high-resolution STEM. By Although the nacre structure is studied since many decades, the formation recording a tilt-series of the crystals, a tomogram was created, and a 3D mechanisms and the interaction between organic and mineral components model of the interlocking crystals was constructed. This model has allowed still need to be clarified. us to visualize the native interlocked R and V units with clear In this study, we used juvenile shell of one-year-old abalone H. tuberculata crystallographic planes. Applying this methodology for multiple samples to investigate nacre formation and organo-mineral interface through high- will enable us to reconstruct the entire sequence of crystal growth, thus resolution (HR) microscopies, at the nanometer scale. A growing nacre area linking shape control to the crystallographic driving forces, and yielding a was selected in juvenile shell for further investigation of nacre surface using mechanistic understanding about the control organisms have on biomineral scanning electronic microscopy (SEM) and Field Emission Gun (SEM- formation. FEG). HR imaging of the juvenile abalone shell resulted in an in-depth characterization of the growing nacre, from the spherulitic edge to the mature P 40 zone of the nacre surface, providing a detailed sequence of nacre formation. Diagenetic alterations reshape the hierarchical organization of At a higher magnification, the tablet surface exhibits typical nanograins and Cretaceous brachiopod shells - from nanoscale to higher scales early stacks of aragonite platelets appeared separated by a porous layer of D. Gaspard*1, J. Nouet2, C. Rountree3 organic matrix. Additional focused ion beam (FIB) coupled with SEM 1Muséum National d'Histoire Naturelle - Sorbonne Université, allowed the observation of the 3D organization of the aragonite platelets Département Origines & Evolution, Centre de Recherche en Paléontologie stacks at early stages of their formation. de Paris, UMR 7207, CR2P, Paris, France Additional techniques such as high-resolution transmission electronic 2Université Paris-Saclay, Earth Sciences, Orsay, France microscopy (HR-TEM), scanning transmission X-ray microscopy (STXM) 3SPEC, CEA, Gif-sur-Yvette, France and electron energy loss spectroscopy (EELS) were performed onto thin FIB section of aragonite platelets columns to gain local information on the nature and atomic composition of the organo-mineral interface at the nanometer Introduction/objectives scale. In growing nacre, large disordered organic inclusions were evidenced After the attempt to reveal and understand the hierarchical organization of in the center of each aragonite platelet. These organic inclusions are oriented the inner layers of the calcite shell of modern rhynchonelliform brachiopods along the c-axis direction, extending from one tablet to another through (Gaspard, 2006, Gaspard & Nouet, 2016), the purpose here is to highlight the organic bridges. It is suggested that these organic inclusions act as a vertical modifications introduced in Cretaceous shells since the death till the state of "backbone" controlling the longitudinal growth of the stacked tablets column fossil. Conservation, alterations in different palaeo-environmental situations along the c-axis. In the case of lateral growth, a 20 nm thick disordered layer for these witnesses of old times are to be uncovered from the nanoscale to was evidenced by HR-TEM at the surface of growing aragonite platelet. This continuum scale. disordered layer appeared mainly composed of an organic fraction where Materials and Methods carbonates ions appear to be absent. However, the presence of both calcium Two and three-layered shells [cf. Sellithyris cenomanensisGaspard, and phosphate suggests the presence of calcium-binding acidic and Moutonithyris dutempleana (d"Orbigny) among others], from different phosphorylated proteins involved in the control of lateral growth of nacre. Cretaceous Western Europe locations, were observed with comparable Together, these results bring new insights to the fundamental mechanisms of modern ones (Liothyrella neozelanica, Aerothyris kerguelenensis, Tichosina nacre formation and on the interaction between organic and mineral cubensis, Notosaria nigricans…) using complementary throughput components involved in the control of aragonite platelets growth. techniques (scanning electron microscopy (SEM), mapping elements, including in the foremost rank the Bruker atomic force microscopy (AFM). P 42 Results Growth dynamics and fine scale characterization of calcareous The observations reveal that from the death of the organisms, the exoskeleton granules of the annelid Lumbricus terrestris (biomaterial) composed of low-Mg calcite and organic matrices is subject of 1 1 2 1 chemical and physical aggressions leading to light or heavy alterations. More S. Mandera* , I. Coronado , M. Mazur , J. Stolarski 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland often the organic matrices are partly or entirely destroyed. First, after the 2 disappearance of the organic sheathes, several fibrous elements merge. As University of Warsaw, Department of Chemistry, Warsaw, Poland well, after partial disappearance of the intracrystalline organic matrix in intimate relation with the individual homogeneous nanoparticles (in the Calciferous glands of some species of earthworms (Annelida, Oligochaeta) ordinary course of shell construction) in extant shells, these later become secrete amorphous calcium carbonate (ACC) particles that are transported more angular and amalgamate to shape blocky calcite and heterogeneous toward a pair of oesophageal pouches where they coalesce and crystallize. structures in fossil shells. AFM Peak Force tapping modes provide The final product is a solid calcareous granule that is excreted by the topographical and nanomechanical information. Nanomechanical organism to the soil. Spherical or sub-elliptical CaCO3 granules from 0.5 to information includes adhesion, deformation…measurements highlighting 2 mm in diameter are composed mostly of calcite, although vaterite, the most viscoelastic components (organic compounds) with respect to the aragonite and ACC have also been found. The purpose of formation of such nanoparticles. mineralized structures still remains uncertain and possible hypotheses Conclusion include: pH buffering of the internal body, adjustment of respiratory CO2, As a matter of fact, the hierarchical organization observed at sub-micrometric and elimination of excess of Ca derived from diet and even spurious levels in the modern shells can be more or less heavily rubbed out by a mineralization. Little is known on the processes that control the final mineral

42 product secreted by earthworms, even though metals uptake by earthworms snails. Such calcified egg shells may supply the snail embryo with calcium granules as a potential use for bioremediation have been studied in-depth and ions to enable the formation of the earliest shell before hatching, provide the ACC formation by the calciferous glands have been characterized. The mechanical support, and potentially (not confirmed) reduce the water main purpose of the current study is to elucidate the mechanisms of evaporation from the egg. It was shown, that in contrast to almost invariably and the growth dynamics during granules formation. aragonite mineralogy of adult gastropod shells, the egg membranes are Earthworm"s granules were obtained from laboratory culturing of fully mineralized with calcite crystals, often showing tetrahedral morphology. clitellate specimens of Lumbricus terrestris. Specimens were kept in artificial This suggests a lack of strong biological control on this type of soil at a constant temperature, neutral pH, and controlled moisture. In order mineralization. Nonetheless, some authors noted a surprising variation of to characterize the growth dynamics of the granules, an Mn-labelling ultrastructural features among more heavily calcified eggs. experiment in an amended artificial soil was carried out using two Mn- Here, we have examined with SEM, micro-Computed Tomography, Raman sources in separate lines of trials: 1) Mn-bearing CaCO3 (at 50 and 100 ppm, and FT-IR spectroscopy), various structural and compositional features of respectively), and 2) a solution of MnSO4 (100 ppm of Mn). 15 specimens egg membrane mineralization in representatives of a highly diversified were cultured during 28 days, 3 control specimens in an Mn-free soil and 12 family of door snails (Clausiliidae). The eggs were taken from oviparous specimens in amended soil (4 for each composition). Specimens were (Caspiophaedusa, Oospira), viviparous (Idyla), and taxa with egg-retention exposed to amended soils and Mn-free soils every 2 to 7 days. An ulterior reproductive strategy (Pontophaedusa, Zaptyx, and Formosana). The structural, biogeochemical and crystallographic characterization of the mineralized egg membranes show different patterns of crystal distribution, granules was carried out, using petrographic microscopy, which invariably are composed of calcite. The calcite crystals are typically cathodoluminescence (CL), SEM, AFM, EMPA, EBSD, and FTIR embedded in the organic multilayered membrane (mixture of proteins and spectroscopy. polysaccharides), which can sandwich the crystal stacks. No evidence of Growth dynamics experiment reveals that control specimens produce amorphous precursors was found (common in systems of biologically between 6-9.8 mg of CaCO3 in 2-5 granules per week. Granules show a radial controlled mineralization), however distinct patterns of distribution of texture formed mostly by calcite crystals with variable habits and some crystals point to biological coordination of this process. A unique, spiral evidence of original concentric growth. The crystal habits are prismatic, lath, arrangement of crystal clusters is typical to eggs of Caspiophaedusa, more and foliated–lamellar crystals, which occasionally are arranged forming homogenous distribution of tetrahedral crystals is characteristic of eggs of cross-lamellar-like microstructures. Moreover, some opaque, in transmitted Idyla and Formosana, whereas in eggs of Zaptyx and Oospira, the calcite light, and microgranular patches have been found. Organic matrix is crystals form pyramidal aggregates. In Pontophaedusa homogenously arranged, mostly, parallel to the growth axis of crystals and some calcite distributed calcite crystals form a dense cover. crystals surfaces exhibit nanogranular textures. CL data show a hidden Biological patterning that governs the calcite crystal distribution in door concentric sub-daily banding, with a thrombolite-like texture and kinked snails egg membranes points to the potential use of this character in a zonation inside the calcite crystals. These features reveal a complex phylogenetic context. In fact, well-differentiated molecular clades (such as mechanism of formation that depends on physicochemical conditions Caspiophaedusa, Pontophaedusa) show distinct crystal distribution patterns, generated at microscale during the granule formation. irrespective of reproductive strategy. Further proteomic and organic content Acknowledgments: This work was supported by the National Science Centre analyses are required to elucidate the precise biological mechanism of crystal (Poland) grant 2017/25/B/ST10/02221. growth coordination. Acknowledgments: This work was supported by the National Science Center P 43 (Poland) grant 2017/25/B/ST10/02221 (to JS) and 2016/21/B/NZ8/03086 (to Towards identification of the protein machinery of coccolith ASD). formation in the coccolithophore Pleurochrysis carterae E. Zschieschang*1, A. Skeffington1, M. Gorka1, A. Scheffel1 P 45 1Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Crystallographic texture and microstructure of modern Germany Glycymeris shells G. Crippa*1, E. Griesshaber2, A. Checa3, E. Harper4, W. W. Schmahl2 1University of Milan, Milano, Italy Coccolithophores are marine unicellular algae that produce mineralized 2Ludwig-Maximilians Universität München, Munich, Germany scales called coccoliths, which are 3D arrays of complex-shaped calcite 3Universidad de Granada, Granada, Spain crystals. How the arrangement of the crystals is guided and how crystal 4University of Cambridge, Cambridge, United Kingdom morphogenesis is controlled is of interest for bioinspired materials synthesis. Coccolith formation involves an insoluble organic structure called the base plate that templates calcite nucleation, as well as soluble acidic Mollusk shells are biocomposites made of calcium carbonate crystals polysaccharides. Recent work on the coccolithophore Pleurochrysis carterae (calcite, aragonite or both) and intra- and intercrystalline organic matrix, (recently reassigned to Chrysotila carterae) suggests that the base plate resulting in a lightweight product of highly elaborate architectures, endowed provides an attractive model system for the identification of macromolecules with unique structural properties (stiffness, fracture toughness, tensile controlling the crystal nucleation pattern. These base plates are composed of strength). Calcite and/or aragonite crystals show different crystallographic polysaccharides and proteins. The exact molecular composition and arrangements, which are hierarchically organized at the nano-, micro- and identities of these components remain unknown. Here we tested different meso-scale, forming different microstructures. extraction approaches to isolate individual components of the base plates of The most common fabric among mollusk shells is represented by the P. carterae coccoliths. One extract was subjected to proteomic analysis. As aragonitic crossed lamellar one, which forms the shells of both marine and the proteomic analysis of the coccolith base plates is at its infancy, we tested continental taxa, thus showing a great adaptive potential and evolutionary different proteases to generate peptides. For protein identification we used success; this is one of the hardest microstructures which has the ability to the publicly available protein database of P. carterae, which has been dissipate and stop cracks through the shell. generated from short Illumina reads and is likely to contain incorrect protein Species belonging to the genus Glycymeris Da Costa, 1778 are excellent taxa models due to misassembly of the reads. To improve our confidence in the to investigate for this type of microstructure, as they have an aragonitic shell identifications we also used a database which we generated from long-read with an outer and an inner crossed lamellar layers. Also, they are widespread cDNA PacBio data. A comparison of the different data sets will be presented. and common in fossil faunas and their applicability in the fossil record spans The workflow we have established will greatly aid attempts to elucidate the from paleoecology, paleoclimatology, sclerochronology to protein machinery involved in coccolith formation. archaeomalacology. Although Glycymeris shells and the crossed lamellar fabric have been widely P 44 study and documented, up to now a detailed characterization of the Biological patterning in calcareous biomineralization of crystallography of both this taxon and this microstructure with Electron terrestrial gastropod eggs Backscatter Diffraction (EBSD) is lacking. Here, we analyzed modern J. Stolarski*1, I. Coronado1, M. Mazur2, A. Sulikowska-Drozd3 Glycymeris shells combining the use of the traditional and easily available 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland Scanning Electron Microscopy with the more advanced EBSD technique, to 2University of Warsaw, Department of Chemistry, Warsaw, Poland improve the knowledge of the crossed lamellar microstructure and of its 3University of Lodz, Department of Invertebrate Zoology and relationship with other shell layers (i.e., myostraca). With the present Hydrobiology, Lodz, Poland contribution we will be able to provide: i) a modern unaltered reference sample for detecting diagenetic alteration in fossil shells, which, due to the widely use of Glycymeris fossil shells in paleoclimatic and Gastropod mollusks are known from the formation of elaborated calcium paleoenvironmental reconstructions, is of crucial importance, and ii) new carbonate shell patterns. Calcified protoconchs (larval shells) and complex data to better understand mollusk biomineralization processes, as well as the hierarchical structures of adult shells (teloconch) have been described among formation of the crossed lamellar microstructure and its correlation with various gastropod clades that include marine, fresh-water and terrestrial taxa. myostracal layers, here characterized in detail for the first time. Much less is known about the earliest developmental phase of calcification associated with the formation of eggs, especially among the pulmonate land

43 P 46 secretion may interacts mutually not only in the direction of mantle edge but Formation of biomimetic agar gel carbonate composites – also in the perpendicular direction. Second, we proposed a new model assuming 2D-distributed secretory organs Influence of Mg and Sr and simulated the model numerically. Then, the results were compared with S. Rudin1, M. Greiner*1, E. Griesshaber1, L. Fernández-Díaz2,3, W. W. actual shell patterns. The new model was based on a reaction-diffusion Schmahl1 system. A simulation of the model generated triangles and stripes on surface, 1Ludwig-Maximilians-Universität München, Department of Earth- and which appear different patterns on cross sections respectively. The generated Environmental Science, Munich, Germany patterns corresponded with observed patterns of C. textile shells. 2Universidad Complutense de Madrid, Departamento de Cristalografía y The correspondence between the observation and the simulation suggests Mineralogía , Madrid, Germany that Conidae species have mutually interacting secretory organs of pigment 3Ciudad Universitaria, Instituto de Geociencias (UCM, CSIS), Madrid, which are 2D-distributed, and form 3D pigmentation patterns. The new Spain model for 3D patterns provides a new interpretation of morphogenesis of pigmentation patterns. Hydrogels are excellent models for understanding extracellular matrix microenvironments and are therefore highly suitable for mimicking P 48 biomineralization processes. Recent studies (Greiner et al. 2018) showed the When predators become prey- shell repair patterns in Nucella combined influence of reagent solution concentration and agar solid content lapillus (Linnaeus, 1758) on the formation of calcite aggregates grown in double-diffusion systems. D. Mayk*1,2, L. Peck2, E. Griesshaber3, W. W. Schmahl3, E. Harper1 Biologic calcite hard tissues e.g. sea urchin teeth can incorporate up to 45 1University of Cambridge, Earth Sciences, Cambridge, United Kingdom mol% Mg (Ma & Qi 2010). Coral skeletons, both aragonitic or calcitic, 2British Antarctic Survey, Cambridge, United Kingdom incorporate small amounts of Sr (Smith et al. 1979). 3Ludwig-Maximillian Universitaet, Muenchen, Germany We present results on the effect of additives (Mg, Sr) in agar gel on the formation of carbonate agar composites in double-diffusion systems. We vary the additive concentration, the agar solid content and the concentration Introduction of reagent solutions and compare our results to previous findings where The common intertidal gastropod Nucella lapillus (also referred to as dog additives were not used. whelk) is a predatory species feeding on e.g., blue mussels and barnacles. It The aggregates are characterized by X-ray powder diffraction (XRPD), builds a massive calcium carbonate shell that shelters it from predators and scanning electron microscopy (SEM), electron backscatter diffraction other environmental hazards. Young dog whelks which have not been able (EBSD) and energy dispersive X-ray spectroscopy (EDX). to grow thick shells yet may be subject to attacks from crabs, birds and even The presence of magnesium results in the formation of dumbbell shaped and conspecifics. If an attack turns out to be non-lethal it is of great importance spherical calcite aggregates with a vast variation of surface morphologies for the individual to repair the damaged shell as fast as possible to be fit for containing a spherulitic inner structure, whose subunits are crystals with a potential future attacks. It is very likely that the need for rapid repair goes slight mosaic structure. The incorporated magnesium is (almost) evenly jointly with an increase in the energy requirement per unit calcium carbonate distributed within the composites; occasionally it is slightly enriched at the formed due to [1] an elevation of the saturation state of the calcifying fluid rim of the aggregates. Crystallization under high Mg to Ca ratios in the or [2] an increase in the amount of organics used. Previous studies have aqueous solution (0.1M Mg: 0.5M Ca or higher) appears to promote the shown that increased calcification rates at damaged sites often result in a stabilization of amorphous calcium carbonate (ACC) and the formation of disturbed microstructure and crystal alignment which possibly affects the aragonite. shell integrity. The presence of strontium in the growth medium leads to the formation of Objectives calcite aggregates with predominantly spherical morphologies. These We investigate the ability of N. lapillus specimens from a broad shell size aggregates are polycrystals. Individual subunits of the aggregates show a low range (i.e., juvenile to adult specimens), to repair artificially damaged shells mosaic-spread (high co-orientation). Incorporated Sr shows a zoned pattern and compare the microstructure of the repaired regions with those of within the calcite aggregates, regardless of the Sr concentration in the agar naturally occurred damage in conspecifics. We investigate the rate and mode hydrogel. For Sr to Ca ratios of 0.01M Sr:0.1M Ca and 0.1M Sr:0.5M Ca we of shell repair in an attempt to link the capability of shell repair and mortality observe calcite composites overgrown by strontianite. A high agar solid rate of the specimens to energy availability among shell size/age groups. content (2 wt%) promotes this overgrowth compared to a low agar solid Materials & methods content (0.5 wt%). 120 specimens of N. lapillus were collected from the rocky shore in Whitby, Mg or Sr in the growth medium promotes misorientation in calcite-agar gel UK in spring 2019 and reared in a seawater aquarium setup at the British composites. This misorientation most likely arises from split growth Antarctic Survey [BAS] in Cambridge. Specimens were starved throughout phenomena as dislocations generate to release Mg or Sr incorporation-related the experiment. After an acclimation phase of two weeks, long notches were lattice strain. cut perpendicular to the aperture rim into the shells of ~60 specimens using Greiner, M., Yin, X., Fernández-Díaz, L., Griesshaber, E., Weitzel, F., a milling bit. This was done with great care to prevent damages of the Ziegler, A., Veintemillas-Verdaguer, S., Schmahl, W.W. (2018) Crystal specimen's tissue. Notch repair increments were monitored weekly. Growth and Design, 18, 1401-1414. Subsequent analysis of the repaired notch regions of sections under the Ma, Y. & Qi, L. (2010) Biomineralization of sea urchin teeth. Frontiers of scanning-electron microscope [SEM] and microstructure orientation Chemistry in China, 5, 299-308. mapping by means of electron-backscatter diffraction [EBSD] was carried Smith, S.V., Buddemeier, R.W., Redalje, R.C., Houck, J.E. (1979) out. Strontium-calcium thermometry in coral skeletons. Science, 204, 404-407. Preliminary results Our results suggest that smaller individuals (shell length < 1.5 cm) are readily

P 47 capable of repairing shell damage and start the repair earlier than larger Observation and simulation of 3D pigmentation patterns of individuals. Absolute shell repair measured as length, however, is greater in larger/older individuals. The mortality rate during the experiment was > 10 molluscan shells times higher in < 1.5 cm long specimens than in > 1.5 cm long specimens H. Sato*1, T. Sasaki2 suggesting that energy reserves in the younger dog whelks are smaller and 1The University of Tokyo, Earth and Planetary Science, Tokyo, Japan faster depleted by the repair process than in larger animals. The shells of N. 2The University of Tokyo, The University Museum, Tokyo, Japan lapillus consist of three layers, namely cross-lamellar aragonite, homogeneous calcite, and the periostracum. The microstructure analysis Molluscan shells show highly diverse, variable and complex pigmentation showed that N. lapillus produces a more regulated shell structure in the patterns. Morphogenesis of the patterns can be understood by a mathematical repaired regions than is normally secreted in the undisturbed shell. We model which represents dynamics of pattern formation. Several models have distinguish four steps observed in the repair process: [1] the damaged shell been proposed so far. The previous models assume that secretory organs of surface is lined with a thin layer of cross-lamellar aragonite, [2] a mound- pigment are distributed in line along mantle edge, and the pattern on shell are shaped calcite structure is built on to the aragonite lining exhibiting a specific regarded as a track of a temporally changing pattern of pigment secretion upward grading of crystal sizes from small to large and with a highly co- along mantle edge. However, an observation of mantle edge suggests that the orientated crystal orientation, [3] homogeneous and disorientated calcite is secretory organs are two-dimensionally (2D) distributed; hence, the patterns built on top in a rather elongated way with a strongly frayed surface to the in shells should be three-dimensional (3D). Here, we confirmed if 2D- outside. This step happens rather quickly and covers up most of the damaged distributed secretory organs generated 3D pigmentation patterns in shells, by area. [4] A lining of cross lamellar aragonite is laid over the inside of the observation and simulation. entire notch covering the inner surface. First, we observed pigmentation patterns on cross sections of Conidae shells Outlook to describe 3D patterns. We found that some species had 3D pigmentation Thermogravimetry measurements of the notch material will be carried out to patterns and different surface patterns had different patterns on cross investigate the organic content of the repaired shell regions. Results will be sections, which cannot be explained by simple dynamics such as simple compared to the organic content of the shell layers in undisturbed shells. diffusion in the direction of inner shell surface. This suggests that pigment

44 Further SEM and high-resolution EBSD measurements will be carried out to the composite hard tissue with complex hierarchical structures and further characterize N. lapillus' microstructure of the repair margins. architectures. In this study we investgate the interlinkage between mineral organization P 49 (microstructure, texture), biopolymer matrix distribution and biomaterial Shell-muscle attachment in the bivalves Ostrea stentina properties for some marine gastropods (Haliotis glabra, Patella vulgata, (Payraudeau, 1826) and Anomia ephippium (Linnaeus, 1758) Tegula sp.) and some marine bivalves (Mytilus edulis, Chama arcana). The J. D. Castro-Claros1, C. Salas*1, C. Lucena2, A. Checa3 hard tissues of all these organisms consist of calcite and aragonite, however 1Universidad de Malaga, Biologia Animal, Malaga, Spain in characteristic organizational patterns. Haliotis laevigata and Mytilus 2Universidad de Malaga, SCAI, Malaga, Spain edulis contain two shell layers: an outer calcitic and an inner aragonitic shell 3Universidad de Granada, Estratigrafia y Paleontologia, Granada, Spain region. The aragonite in both organisms is nacreous aragonite, in Haliotis laevigata the calcite is prismatic, while in Mytilus edulis it is fibrous. In Patella vulgata and Chama arcana the aragonitic shell portion is sandwiched Introduction between two calcitic shell layers. These two calcitic shell portions have The muscles of the mantle in bivalves attach to the shell valves and retract distinct microstructures; are neither prismatic (as it is the case in Haliotis the mantle edges. Among them, the adductors are particularly important for laevigata) nor fibrous (as it is the case in Mytilus edulis). Tegula sp. also has the survival of the animal. Most studies have addressed the type and an outer calcitic and an inner aragonitic shell portion. However, the aragonite morphology of the muscular fibers and filaments (Paniagua et al 1996), and in Tegula sp. is not developed in a nacreous microstructure; it is an assembly a few have focused on the attachment of these muscles to the shell (Nakahara of aragonitic prisms that increase in size towards the soft tissue of the animal. & Bevelander 1970). However, the mechanism of transport through the Microstructure and texture characterization was done with SEM imaging and muscle to the myostracum was never addressed. The goal of this research is electron backscatter diffraction (EBSD), biopolymer content was analyzed to describe ultrastructure and the transport of material across the adductor with Thermo-Gravimetric- Analysis (TGA), biopolymer distribution within muscle-myostracum attachment in Ostrea stentina and Anomia ephippium. the biological hard tissue was visualized with various etching protocols and Material and methods SEM imaging, biomaterial properties were measured for two hierarchical We examined 10 specimens of O. stentina and 10 specimens of A. ephippium, levels with instrumented nanoindentation and microindentation. collected in the littoral of Málaga (south Spain). Specimens were fixed in We find that biopolymer contents is fairly similar for the hard tissues of the 2.5% glutaraldehyde (4 °C), decalcified in 2% EDTA, post-fixed in OsO4 investigated species, however, mineral organization differs significantly (2%) and embedded in epoxy resin Epon 812 (EMS). Samples for calcium between the species, for both, the calcite and the aragonite. Accordingly, for detection were post-fixed in a mixture of OsO4 (2%) and potassium the selected hard tissues, mineral organization exerts the strongest effect on hexahydroxoantimonate (2%) in PBS. Ultra-thin sections (50 nm) were the observed variation of nano- and micro-hardness as well as elastic stained with uranyl acetate (2%). They were observed in a TEM JEOL- modulus. With the wide range of selected microstructures we aim to highlight JEM1400 and a TEM-STEM FEI Talos 200X, with EDX analyzer. in this contribution the relation between specific microstructural construction Collagenase was used to test the presence of collagen. One specimen of each principles, biomaterial properties and biomaterial functions. species was embedded in methacrylate (Technovit 7200 VLC). These sections (50 μm) and the semi-thin sections (~0.5 μm) were stained with P 51 toluidine blue (1%) Pattern formation of interlocking calcite fibre hybrid Results composite in brachiopod shells The adductor muscles in both species are composed of "smooth" and W. W. Schmahl*1, E. Griesshaber1, A. Ziegler2, M. Simonet Roda1, D. "striated" muscles. The outer epithelium of the mantle continues across the Henkel3 adductor muscle area and their cells contain many vesicles. Bundles of 1LMU Munich, Earth and Environmental Sciences, Munich, Germany filaments connect the muscular cells with the extrapallial space through 2University of Ulm, Central Facility of Electron Microscopy, Ulm, hemidesmosomes. The extrapallial space (ca.100-150 nm thick) is filled with Germany organic secretions from the vesicles of the mantle cells. Additional bundles 3GEOMAR Helmholtz Centre for Ocean Research , Marine of microfilaments of collagen fibers cross the extrapallial sheet, from the Biogeochemistry/Marine Systems, Kiel, Germany hemidesmosomes to inside the myostracum, where they form a network. EDX analysis shows the presence of calcium inside vesicles from the mantle cells, the extrapallial sheet and the myostracum network. Fibrous biological hybrid composites are an important class of materials. Conclusion Aragonite or calcite fibers are embedded in a pliant biopolymer matrix, the The presence of a layer of cells between the muscular cells and the shell was latter being always cross-linked within the hard tissue. Most biological indicated by Nakahara & Bevelander (1970), who called them "adhesive carbonate hard tissues are subject to compressive, bending and shearing cells". Subsequently, Bubel (1984) called them "tendon cells". Our forces. As fibers within a matrix cannot be reorganized once they endure observations confirm the existence of this cell layer, which is the these forces, they must be properly packed and oriented within the hard tissue continuation of the outer mantle epithelium across the adductor muscle. The from the onset of their formation. This is accomplished by the formation of extrapallial space is replenished with secretions of the mantle cells and stacks of parallel fibers with interlocking concave/convex cross-sectional collagen fibers, seemingly originated in the hemidesmosomes. The presence shapes. On the next hierarchical level, the stacks are twisted in a plywood- of numerous vesicles extruding from the mantle cells were probably mistaken like arrangement. This ensures that all components of the composite are as a microvillous border by Nakahara & Bevelander (1970). However, no interleaved in three dimension and on all length scales. microvilli have been observed in the basal mantle cell membranes. The The fibrous shell layer of modern brachiopod shells is such a hybrid presence of calcium inside some vesicles suggests that part of the calcium is composite material. The interlocked concave-convex packing morphology of transported thereby. It would be interesting to study whether calcium is the calcite fibres is more sophisticated than the simple "brick and mortar" transported as amorphous calcium carbonate or as a crystalline phase. pattern of mollusc nacre or the random cellular polygonal convex cross- Bubel A. 1984. Epidermal cells. In: Bereiter-Hahn et al. (eds.) Biology of the sectional of columnar calcite of many invertebrates. In the fibrous calcite Integument 1, Springer: 400-447. composite, the convex surface of each fibre is lined by an organic membrane, Nakahara H., Bevelander G. 1970. Texas Rep. Biol. Med. 28(3): 279-286. which is intricately connected to the fibre calcite. The four concave surfaces Paniagua R., Royuela M., García-Anchuelo R.M., Fraile B. 1996. Histol. are adjacent to the linings of the convex sides of neighboring fibers. Histopathol. 11(1): 181-201. When brachiopod fibre and nacreous tablet formation are compared, significant differences emerge in biopolymer/mineral deposition and, hence, P 50 microstructure generation. In the case of modern brachiopod fibers, during Structure - material property relationships in marine bivalve secretion, mantle epithelium cells are always in direct contact with the and gastropod calcite and aragonite mineral, whereas in molluscs the nacreous tablets are never in direct contact Z. Tang*1, E. Griesshaber1, E. Harper2, S. Zaefferer3, M. Zenkert1, N. with epithelial cells. There is always an interlamellar (in bivalves) or surface Lagos4, W. W. Schmahl1 (in gastropods) membrane between secreting mantle cells and the growing 1Ludwig-Maximilians-Universität München, Department of Geoscience and aragonite platelets. When brachiopod fibers form, secretion of the Environmental Sciences, Munich, Germany biopolymer membrane covering the convex surface of the fibre is the last and 2University of Cambridge , Department of Earth Sciences, Cambridge, terminal step in fibre growth. In contrast, when nacre forms, aragonite tablet United Kingdom formation starts with the consecutive self-assembly by liquid crystallization 3Max-Planck-Institut für Eisenforschung, Department of Microstructure of interlamellar membranes. This leads to the formation of compartments that Physics and Alloy Design, Düsseldorf, Germany become successively infiltrated by aragonite. Accordingly, we find for 4Universidad Santo Tomás, Centro de Investigación e Innovación para el modern brachiopod shell and molluscan nacre development two divergent Cambio Climático, Santiago, Chile microstructure generation processes. One is biologically controlled through direct cellular contact and activity with the mineral as it is the case for brachiopods, the other is physically controlled through the self-organization Carbonate biological hard tissues are composite materials consisting of of extracellular matrix membranes as it is the case for molluscan nacre. biopolymer and biomineral components. Both material classes are present in

45 P 52 were placed back into the tanks for repairing their shell. Most specimens Formation and mosaicity of coccolith segment calcite of the survived and, thus, coped well with injured shells. They repaired damaged marine algae shell parts quickly, within three to five months. X. Yin*1, A. Ziegler2, K. Kelm3, R. Hoffmann1, P. Watermeyer3, P. Alexa1, Calloria inconspicua secrets two-layered shells, a primary, outer, and a C. Read2,4, L. Schlüter5, T. B. H. Reusch5, E. Griesshaber1, P. Walther2, W. fibrous, inner, shell portion with, in the latter, well-aligned calcite fibres (Ye W. Schmahl1 et al. 2018, Journal of Structural Biology). As brachiopod fibres occlude very 1Ludwig-Maximilians-Universität München, Department für Geo- und minor amounts of biopolymers, calcite crystallites within the fibres are Umweltwissenschaften, München, Germany highly co-aligned (e.g. Casella et al. 2018, Biogeosciences). At repair of the 2Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, hole drilled into the shell, the mantle tissue secrets first a thin layer of calcite Germany that is added onto inner shell surfaces at still undisturbed shell portions. 3Deutsches Zentrum für Luft- und Raumfahrt, Institut für Werkstoff- Relative to shell calcite secreted under normal living conditions, the calcite Forschung, Köln, Germany that forms first at repair has an almost random microstructure and texture. It 4Universitätsklinikums Ulm, Institut für Virologie, Ulm, Germany is a thin layer of calcite that appears to serve as stabilization for the 5GEOMAR Helmholtz-Zentrum für Ozeanforschung Kiel, Marine subsequently secreted calcite, the calcite that covers the hole drilled into the Evolutionary Ecology, Kiel, Germany shell. The microstructure and texture of the first-formed shell calcite is significantly different to that secreted under normal conditions. In the former (the first-formed calcite) typical morphologies of mineral units forming the Coccolithophores belong to the most abundant CaCO3 mineralizing primary and the fibrous shell layers are entirely absent as well as the high co- organisms.Coccolithophore calcification is a complex and highly regulated orientation strength of calcite crystallites within a particular mineral unit. process, resulting in a biogenic product that differs in both, morphology and MUD values (indication for the strength of crystallite co-orientation, Casella chemical composition from the abiogenically produced equivalent. Unlike et al. 2018, Biogeosciences) of undisturbed shell portions of modern extracellularly formed biological carbonate hard tissues, coccolithophore Callorica inconspicua range between 60 to 70, while for the first-formed calcite is neither a hybrid composite, nor is it distinguished by a hierarchical calcite and the calcite that covers the hole drilled into the shell MUD values microstructure. This is remarkable, as the key to optimize crystalline are between 18 and 20. biomaterials for mechanical strength and toughness lies in the composite Despite low MUD values microstructure recovery starts soon when the shell nature of the biological hard tissue and the specific microstructure. is repaired. For the shell portion that covers the hole drilled into the shell, we To get insight into the pathway of biomineralization of E. huxleyi coccoliths can clearly distinguish between primary and fibrous layer mineral units. With we examine intracrystalline nanostructural features of the coccolith calcite in ongoing repair these gain more and more their normal morphologies. Recent combination with cell ultrastructural observations related to the formation of studies of cell ultrastructure secreting the calcite of the modern terebratulid the calcite in the coccolith vesicle within the cell. With TEM diffraction and brachiopod Magellania venosa (Simonet Roda 2019a, Nature Scientific annular dark field (ADF) imaging, we prove the presence of planar Reports; Simonet Roda 2019b: Journal of Structural Biology) showed that at imperfections in the calcite crystals such as planar mosaic-block-boundaries. active secretion outer mantle epithelium cells are in direct contact to the As at these only minor misorientations occur, we attribute them to dislocation forming fibre ant the primary layer calcite. Taking this into account it can be networks creating small-angle boundaries. Intracrystalline occluded assumed that the mantle portion beneath the drill-hole only secretes the biopolymers are not observed. Hence, in E. huxleyi calcite mosaicity is not calcite that is needed for repair of that shell portion. caused by occluded biopolymers, as it is the case in extracellularly formed hard tissues of marine invertebrates, but by planar defects and dislocations P 54 which are typical for crystals formed by classical atom-by-atom growth The role of innate immunity of aortic valve cells in human mechanisms. Using cryo-preparation techniques for SEM and TEM we found aortic valve calcification that the membrane of the coccolith vesicle and the outer membrane of the X. Meng*1, L. Ao1, D. Fullerton1 nuclear envelope are in tight proximity, with a well-controlled constant gap 1University of Colorado Denver, Surgery, Aurora, United States of about 4 nm between them. We describe this conspicuous connection as a not yet described inter-organelle junction, the "nuclear envelope junction". Chronic inflammation and progressive calcification of the aortic valve The narrow gap of this junction likely facilitates transport of Ca2+ ions from leaflets lead to calcific valvular heart disease (CVHD) that is common in the the nuclear envelope to the coccolith vesicle. On the basis of our elderly. Currently, pharmacological prevention of CVHD progression is observations, we propose that formation of the coccolith utilizes the nuclear unavailable although there is a wide window for intervention. In addition, the envelope – endoplasmic reticulum Ca2+-store of the cell for the transport of interaction between the pro-inflammatory and pro-osteogenic mechanisms in Ca2+ ions from the external medium to the coccolith vesicle and that E. aortic valve calcification is not well understood. Aortic valve interstitial cells huxleyi calcite forms by ion-by-ion growth rather than by a nanoparticle (AVICs) are actively involved in valvular calcification. Initial studies by our accretion mechanism. group demonstrate that human AVICs express pro-osteogenic proteins (including BMP-2 and TGF-β1) in response to stimulation of Toll-like P 53 receptor (TLR) 2 or 4. Further, the TLR-mediated innate immune response Skeletal repair of modern brachiopod shells in human AVICs leads to pro-osteogenic reprogramming characterized by 1 2 3 4 E. Griesshaber* , J. H. Robinson , E. M. Harper , M. Lamare , M. the expression of Runx2 and alkaline phosphatase, and formation of calcium 4 5 5 Williams , M. Zenkert , W. W. Schmahl deposits. These studies uncovered a novel mechanistic role of the AVIC 1 Ludwig-Maximilians University Munich, Department of Earth and innate immunity in aortic valve calcification. Our recent work identified Environmental Sciences , Munich, Germany several endogenous factors that can elicit the osteogenic response in human 2 University of Otago, Department of Geology, Dunedin, New Zealand AVICs through TLR2/4, including oxidized low-density lipoprotein, 3 University of Cambridge, Department of Earth Sciences, Cambridge, biglycan and matrilin 2. These endogenous factors (damage-associated United Kingdom molecular patterns, DAMPs) induce the osteogenic response in human 4 University of Otago, The Department of Marine Sciences, Dunedin, New AVICs mainly through the NF-κB and ERK1/2 pathways. Together, our Zealand findings demonstrate that DAMPs are capable of inducing osteogenic 5 LMU Munich, Department of Earth and Environmental Sciences, Munich, response in human AVICs and that the innate immune receptors have novel Germany functions in modulating the osteogenic response in human aortic valve cells. These findings suggest that AVIC TLRs may play an important role in the Invertebrates, brachiopods, molluscs, echinoderms, are known to repair pathogenesis of CVHD and that modulation of the signaling pathways damage to their shells. This is either inflicted by predators or by physical utilized by TLRs may have therapeutic potential for suppression of CVHD environmental impact, e.g. ice blocks or, in high surf regions, e.g. rocks. progression. Shells often show various and distinct repair traces. This indicates that a specific organism is often attacked by a range of animals as well as different environmental impacts. In this contribution we describe new shell formation at damaged shell portions for the modern terebratulid brachiopod. We investigate how Calloria inconspicua copes with an injured shell, how quickly it repairs its shell and how much control the animal exerts on the organization of the newly-formed mineral, the mineral that is secreted for repair. Optical and electron microscopical techniques were used for imaging the shells, electron backscatter diffraction analysis (EBSD) was applied for the determination of calcite organization and measurement of calcite crystal co-orientation strength. Live Calloria inconspicua specimens were collected at Otago harbor, New Zealand and were placed into aquaria. Animals were damaged mechanically either by drilling 1 to 2 mm sized holes into their shells or by cutting slices off the shell. Subsequent to damage formation, brachiopods

46 P 55 and high-resolution EBSD (electron backscattered diffraction) and Structural characterization of acellular components in transmission Kikuchi diffraction (TKD); differences in structural Ariolimax californicus (; Stylommatophora) organization of the mineral were imaged with FE-SEM and STEM. We D. Montroni*1,2, X. Zhang1, J. Leonard3, M. Kaya4, C. Amemiya5, G. characterize carbonate assembly in the buoyancy device of the cephalopod Falini2, M. Rolandi1 Spirula spirula, the shell of the molluscs Mytilus edulis and Haliotis glabra, 1University of California Santa Cruz, Department of Electrical and the calcite occlusions within the cuticle of the isopods Tylus europaeus Engineering, Santa Cruz, California, United States and Porcellio scaber. 2Alma Mater Studiorum Università di Bologna, Department of Chemistry The cephalopod Spirula spirula possesses a light-weight internal shell that is “Giacomo Ciamician”, Bologna, Italy divided into discrete chambers. These are separated from each other by 3University of California Santa Cruz, Joseph M. Long Marine Laboratory, partitions, the septa. A thin organic siphuncle runs through all chambers and Institute of Marine Science, Santa Cruz, California, United States facilitates that liquid and gas diffuse into and out of the chambers, hereby 4Aksaray University, Department of Biotechnology and Molecular Biology, allowing for buoyancy adjustments. Spirula sp. covers daily almost 1 km Faculty of Science and Letters, Aksaray, Turkey difference in altitude as the animal spends the night in deep waters, while for 5University of California, School of Natural Science, Merced, CA, United the day it traverses the water column up to about 200 to 300 km depth below States the water surface. The shell is coiled in a single plane, resembling a logarithmic spiral, the coils do not touch one another. Both the shell wall and the septum are mineralized with the walls being almost devoid and the septa Biological materials such as the chiton"s tooth, the squid"s beak, and the consisting of a large proportion of organic matter. The septa allow the shell byssal threads of bivalves have inspired the development of new materials. to function as a buoyancy device. They are curved, with the concave side In this regard, we have characterized the acellular components of the pointing towards the first chamber and consist of organic laminae enforced terrestrial slug Ariolimax californicus (banana slug), which are three matrices by intercalated nanometric granular aragonite. In contrast, aragonite crystals in the buccal mass and the internal shell. that form the shell walls are micrometer sized and are irregular in size and The buccal mass is an apparatus, very similar in function to the human mouth, morphology. Their arrangement is structured, such that large crystals line the whose role is processing food. The components observed in the buccal mass outer sides, while an assembly of small aragonite crystallites form the inner were the radula, the jaw, and the odontophore. In many marine mollusks the regions of the shell wall. Crystal axes co-orientation strengths is high for both radula is a well studied organ, but no study has ever been done on terrestrial the aragonite of the walls as well as for the granular aragonite within the septa soft food eating mollusks. Moreover, the jaw and the odontophore have never (MUD values scatter between 150 and 200). Aragonite c*-axes are been described before under a structural point of view. perpendicular to the shell vault and rotate with its curvature. The other matrix studied was the internal shell. Along with the evolution, The shell of the molluscs Mytilus edulis and Haliotis glabra consist of three slugs have lost their external shell to adapt to calcium poor environments, but distinct layers separated from each other by either organic membranes or, for some of them maintained a vestigial internal shell. Differently from mollusks a given genera, by characteristic transition zones. The mineralized shell shells, which role, composition and mechanisms of formation have been portions consist of dense assemblages of carbonate mineral units. For both widely investigated, the internal shell of slugs has been poorly studied and organisms calcite forms the seaward pointing layer, while nacreous aragonite many aspects are still unknown. It has only been observed that mineralizes constitutes the shell part that is next to the soft tissue of the animal. In Mytilus and demineralizes depending on the life cycle of the animal. edulis the calcitic shell portion comprises calcite fibres, each of them The research involved the sectioning of the matrices and the study of their sheathed in organic substance. In Haliotis glabra the calcitic shell section is morphological structures using electron scanning and optical microscopy. formed by irregularly sized and shaped calcite units that decrease Spectroscopy, X-ray diffraction, and chemical analysis were also used to consistently in size towards the periostracum, the outermost layer of the shell. investigate the composition and the structure of the material in its different For a given age, shell thicknesses are comparable for the two animals. regions. Mytilus edulis lives attached to the substrate, in very shallow waters and in In the radula, calcium-rich denticles were tightly interlocked one to the other high surf regions, while the benthic Haliotis glabra lives in water depths on top of a nanofibrous chitin membrane. The jaw was observed having a between 50 to 80 m. Mytilus edulis developed a thin, flexible and tough shell, nanostructured morphology made of chitin to achieve compression resistance while Haliotis glabra protects its soft tissue with a hard and stiff shell. The and is directly linked to the foregut cuticle, which has a protective calcitic shell portion is rigid in Haliotis glabra, while it is ductile in Mytilus nanofibrous structure. Finally, in the odontophore, we observed a structurally edulis. For adult specimens and within the central portion of a cross-section elastic microstructure that interfaces soft tissues with a highly stressed radula through the shell in Haliotis glabra we find an about 60 to 70 mm thick layer membrane. Based on those observations, we discussed the interaction of prismatic calcite and of an about 280 to 300 mm thick layer of nacreous between these components and highlighted how the materials in these aragonite. In a comparable section of the shell the calcitic shell region of components have evolved together to perform their tasks. Mytilus edulis comprises about 150 to 160 mm of fibrous calcite and an about The study of the internal shell showed two different calcite crystal 80 mm thick layer of nacreous aragonite. Thus, in Haliotis glabra nacreous morphologies on the two faces of the shell. Those crystal phases grew at the aragonite prevails, while in Mytilus edulis fibrous calcite forms the major interface of three chitin-protein based layers: a thick internal layer and two part of the hard tissue. The outer shell portion of Haliotis glabra with its external membranes on each side of it. In the ventral face spherulite crystals irregularly shaped and sized mineral units (arranged with a low degree of co- were observed, while lamellar crystals were observed in the dorsal face. This orientation; MUD below 20) provides the necessary stiffness, while in polymorphic asymmetry was observed also in the organic matrix hosting the Mytilus edulis the (highly co-oriented; MUD above 300) calcite fibre crystals. Those observations led to the hypothesis that this matrix act as arrangement, with each fibre being sheathed by biopolymers, provides the calcium storage with a face working as short-term calcium reserve, while the tensile strength and ductility. other as a long-term one. The common rough woodlouse Porcellio scaber and the sand burrowing In conclusion, considering the structural characterization of the A. species Tylos europaeus reinforce the exo- and endocuticle layers with californicus" matrices in relation to their functions, this study could give a mineral, amorphous calcium carbonate, calcite and phosphate. The amount new perspective on fabricating bioinspired materials. of incorporated calcite as well as the pattern of calcite organization differs significantly between the species. While the calcite layer within the P 56 exocuticle of Tylos europaeus is thick and consists of a multitude of highly Structure-function relationships in carbonate endo- and misoriented calcite domains, the calcite in the exocuticle of Porcellio scaber exoskeletons is significantly thinner and is composed of a few large domains containing E. Griesshaber*1, A. Checa2, A. Ziegler3, M. Zenkert1, W. W. Schmahl1 highly co-oriented calcite crystallites. Differences in mineral incorporation 1Ludwig-Maximilians University Munich, Department of Earth and and patterns of crystal assembly are relatable to functional and habitat Environmental Sciences , Munich, Germany adaptations: The tergite of Porcellio scaber is thin and flexible. Upon 2Departamento de Estratigrafía y Paleontología Facultad de Ciencias predation the animal either runs away or clings still and firmly to the Universidad de Granada Avenida Fuentenueva s/n Granada, Granada, substrate, hence it needs a lightweight and highly flexible cuticle. In contrast, Spain the beach dwelling isopod Tylos europaeus rolls into a sphere upon threat 3University of Ulm, Central Facility for Electron Microscopy, Ulm, and the animal relies on its thick cuticle for protection of its soft body tissue. Germany

In this contribution we highlight and discuss differences in mineral organisation of biocarbonate hard tissues used by organisms for diverse purposes: as a buoyancy device, for protection of the soft tissue and as enforcement of the crustacean cuticle. We observe a huge diversity of microstructure and texture patterns that range from almost unaligned over graded to co-aligned crystal assemblies. Hence, a high order as well as a high disorder in mineral organization is advantageous and is fabricated by the organism. Carbonate organization patterns were measured with conventional

47 P 57 the polymorphic transformation process and the thermal decomposition Nanoscale structuring of modern brachiopod calcite fibres behavior of calcium carbonate. E. Griesshaber*1, R. Schmidt2, T. Schmidt2, R. Steffen2, H. Gnägi3, M. Materials & methods Simonet Roda1, A. Ziegler1, W. W. Schmahl1 Calcium chloride dihydrate and sodium carbonate were used as the reactants 1Ludwig-Maximilians University Munich, Department of Earth and in the calcium carbonate precipitation process. The experiment was Environmental Sciences , Munich, Germany conducted in a 1-L double-jacketed crystallizer. The temperature was 2Hitachi High-Technologies Europe GmbH, Krefeld, Germany controlled using a thermostat and the experiments performed at 30 °C. 3Diatome Ltd, Biel-Bienne, Switzerland During the experiments, the pH of the solution was monitored consistently and fixed at pH 8.5. The solution was mixed at 500 rpm using a three-blade propeller and mechanical stirrer. The calcium carbonate suspensions Organization of nanosized entities across many length scales poses a major obtained as a result of the experiments were removed from the crystallizer, challenge in the development and production of man-made materials with filtered and then dried. The as-prepared products were characterized by X- advanced functions. In contrast, in biologically formed hard tissues, this ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), design feature and its formation principle is intrinsic. It began already with thermogravimetric analysis (TGA), and scanning electron microscopy the emergence of first skeletal hard parts in late Precambrian times and, since (SEM) were used. then, it was diversified by evolutionary adaptation. Results Modern terebratulid and rhynchonellid brachiopod shells consist of up to To investigate the structure of the calcium carbonate formed in the absence three mineralized shell layers: the outermost primary, the inner fibrous, and, and the presence of additive, calcium carbonate crystal forms were where developed, an innermost columnar layer. In two-layered shells the investigated using XRD and FTIR spectroscopy. All the diffraction peaks for fibrous layer forms an extensive part of the shell. The fibers are hundreds of the crystals obtained in pure media were attributed to the existence of calcite micrometers long and are almost single-crystalline mineral units. The shape crystals with a rhombohedral structure. The calcite form was fully converted of brachiopod fibers is unique and was already developed in Lower Cambrian into the vaterite form in eggshell media, which was in agreement with the times. In recent brachiopods, the morphology and dimension of fibers is FTIR results. characteristic for a given brachiopod species and is evolutionary adapted to SEM and particle size analysis were performed to investigate the effect of the animal"s habitat. the eggshell on the morphology and the size of the calcium carbonate. The The aim of our study was to detect and visualize a possible nanostructuring crystals formed in pure media mainly consisted of uniform crystals with a of modern brachiopod fibres. We investigated the fibrous layer of the modern cubic-like shape and smooth surface. When using eggshell in the brachiopod Magellania venosa from micrometer to nanometer scale levels crystallization media, nearly all of the crystals occurred in vaterite form and with FE-SEM and EBSD, STEM imaging and TKD. Conventional EBSD their appearance was spherical and the specific surface area of calcium measurements prove a strong co-orientation strength of calcite crystallites carbonate increased significantly. within the fibres and document on micrometer scales their clossness to being The filtration characteristics of the calcium carbonate crystals were single crystals. However, with AFM and STEM imaging and transmission determined and the mean specific cake resistance and the mean cake porosity Kikuchi diffraction (TKD) measurements we are able to demonstrate the of the crystals were calculated based on Darcy"s Law. In addition, the internal nanoscale structuring of the fibres. Co-oriented nanosized calcite thermal decomposition of the calcium carbonate samples was analyzed using crystals are stringently assembled to individual rows, a multitude of the latter thermogravimetric analysis. comprise individual fibres. Conclusion Hence, the almost single crystalline calcite brachiopod fibres are The present study investigated the effect of eggshell on the precipitation of substructured internally. They comprise highly co-oriented calcite calcium carbonate. In the eggshell medium, the diffraction peaks of the crystallites that are placed in a thin network of biopolymer fibrils, a finding calcite crystals disappeared and only vaterite crystals were observed. SEM that has not yet been shown for modern brachiopod calcite fibres. Individual images illustrated that eggshell significantly improved the rounded and fibres in modern brachiopod shells are not single crystals. spherical crystals. This study demonstrated the modification of the structure and morphology of calcium carbonate as well as the formation of its vaterite P 58 form were possible using eggshell. Diversity and function of biominerals in Ciliates M. L. Lemloh*1 P 60 1University of Stuttgart, Materials Testing Institute, Stuttgart, Germany Crustacean cuticular matrix in transition- spatial and temporal diversity Eukaryotic single-celled organisms like Ciliates (Protista) represent an N. Žnidaršič*1, P. Mrak1, J. Štrus1, K. Žagar Soderžnik2, M. Čeh2 excellent model system to discover biomineralization principles with respect 1University of Ljubljana, Department of Biology, Biotechnical faculty, to intracellular mechanisms involved in ion enrichment, vesicular mineral Ljubljana, Slovenia transport and biomineral formation. There are over 8,000 species of the 2Jožef Stefan Institute, Department for Nanostructured Materials, Phylum Ciliophora and since Ciliates naturally occur in freshwater, brackish, Ljubljana, Slovenia and marine habitats as well as in extreme environments, they can be used to study the impact of different environmental conditions, e.g. available mineral Biological mineralized matrices display inhomogenity in structure and sources. Although it is known that Ciliates form diverse intracellular mineral composition. Coexistance of different mineral forms in well defined structures, little is understood about their biomineralization processes. functional layers was characterized in detail in several calcified and other Therefore, we combine dynamic in vivo studies of mineral forming Ciliates biomineralized structures. Complex assemblages of hierarchically organized together with high-resolution methods like analytical electron microscopy, to organic macromolecules in biomineralized matrices also show structural identify common principles and to distinguish diverse mechanisms with alterations in distinct regions. Additional complexity of biomineralized specific adaptations involved in mineral formation. The presentation will matrices refers to the time scale alterations in structure and composition due outline selected examples of biomineralizing Ciliates with respect to the to tissue differentiation during animal development and during replacement. characteristics and various functions of the formed minerals. To address the questions in biomineralized matrix elaboration it is necessary to determine the ultrastructure and composition of the sample at the specific P 59 site(s) and at the specific time. In addition, for microscopical characterization Effect of eggshell on polymorphic transformation of calcium it is beneficial to preserve both, mineral and organic components. carbonate We have applied a correlative microscopic approach that combines structure S. Polat*1, P. Sayan1 determination with elemental and molecular localization data to characterize 1Marmara University, Chemical Engineering, Istanbul, Turkey the crustacean cuticle differentiation during development and to characterize the interface between calcified and non-calcified layers in adult animals. Localizations with specific ligands, transmission electron microscopy and Introduction block face imaging in BSE mode by scanning electron microscopy were Calcium carbonate precipitation has drawn a lot of research attention over supplemented by EDXS analyses. Different specimen preparation the last few decades due to its widespread industrial applications and procedures were implemented, including methanol fixed and resin embedded scientific value in the field of biomineralization. Calcium carbonate crystals specimens. This enables to obtain information on elemental composition in can exist as three different forms, namely, calcite, vaterite, and aragonite. selected regions of the sample that was not exposed to water solutions and to Different polymorphs tend to have different chemical and physical correlate this chemical information with ultrastructure as revealed by SEM properties, which can strongly influence the properties of the products; thus, imaging of the block surface and by TEM imaging of the corresponding it is important to have control of the crystal form. This study investigated ultrathin section. We have focused to characterize the interface between the precipitation of calcium carbonate in the presence of eggshell. two horizontal regions of the cuticle in the adult specimens, i.e. between the Objectives calcified endocuticle and non-mineralized membranous layer that faces The purposes of this study were to determine the influence of eggshell on the epidermal cells. This is a complex transition zone from the architectural and morphology of calcium carbonate and to describe the effects of eggshell on compositional point of view. Next, the cuticle of different developmental

48 stages was analysed to determine the modifications of exoskeletal cuticle distance determination between hetero nuclei. (REDOR: Rotational Echo during differentiation. The earliest stage with an epidermal matrix displaying DOuble Resonance) typical structure of adult exoskeletal cuticle is the prehatching embryo. In Materilas & methods early postembryonic stages a new cuticle is formed and calcium sequestration Within the present contribution, the biosilica of Cyclotella cryptica, a centric in the matrix is evident. Major alterations of epidermal matrix correspond to maritime diatom, is studied. The silica phase of the purified cell walls can be the transit from embryonic to postembryonic period that is accompanied by characterized using 29Si solid-state NMR spectroscopy whereas the organic embryo hatching from the vitelline membrane. compounds are investigated by 13C, 15N, and 31P solid-state NMR spectroscopy. The sensitivity of the experiments can be greatly enhanced P 61 through isotope-labeling of the diatoms during cultivation in isotope- Crystallographic and biomineral organization of the cuticle of enriched culture medium. Moreover, DNP-supported solid-state NMR Devonian - tailored armours of the past experiments (DNP: Dynamic Nuclear Polarization) provide further signal I. Coronado*1, J. Esteve2, J. A. Cruz3, J. Stolarski1 enhancement, which is especially interesting for 2-dimensional NMR 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland experiments like PDSD (Proton-Driven Spin Diffusion) or DQSQ (Double 2Los Andes University, Geosciences Department, Bogotá, Colombia Quantum Single Quantum). 3Complutense University, GEODESPAL, Madrid, Poland Results One important feature of C. cryptica is a massive insoluble organic matrix. Solid-state NMR spectroscopy is used to investigate the insoluble organic Systematic characterization of well-preserved can help to trace back matrix in C. cryptica as well as the silica/organic interface. The different evolutionary changes in biomineralization processes that occurred along the organic compounds like sugars as well as proteins and LCPAs are studied by Earth history, but also to provide inspiration to create biomaterials of unique the observation of hetero nuclei like 13C, 15N and 31P. In addition, the distance properties. These tasks can be reached insofar as the original crystallo- between different organic compounds and the silica phase is determined chemical properties of biominerals have not been obliterated by diagenesis. using a 1H-13C-29Si REDOR experiment. It can be shown, that the shortest Biominerals formed in biologically controlled process by marine and distances between silica and organic compounds appear for different signals terrestrial organisms are hierarchical organo-mineral composite in the 13C-chemical shift range of 40 – 60 ppm, which is typical for LCPAs. nanomaterials, with distinctive microstructures and well constrained This indicates a very close contact between the LCPAs and the biosilica. crystallographic arrangements. Moreover, DNP-supported NMR measurements confirm the presence of Trilobites are marine fossil arthropods that inhabited seas from Cambrian to different sugars. Especially, the presence of chitin is verified using a PDSD , which have a characteristic articulated carapace. cuticle is experiment. The calculated enhancement factors for different signals and heavily mineralized assembled by layered structures that form a dense thus, for different organic compounds can help to get an idea for the framework, providing them tailored armours for protection. Cuticles moult supramolecular architecture of the biosilica. Furthermore, a comparison to during ontogeny (creating exuviae) and they represent majority of trilobite the biosilica of Thalassiosira pseudonana reveals both, similarities as well finds, whereas only exceptionally the full skeletons are preserved. Although as differences between the organic matrices of these diatom species. microstructure of trilobite cuticles have been vastly described in the Conclusion literature, still, little is known about the mechanisms that ruled their Our studies show that solid-state NMR is one powerful technique to learn formation and their characteristic biomineral properties. The purpose of this more about biomolecules and their arrangement in diatom biosilica. NMR work was assessed for the first time, the structure of Devonian trilobite experiments help to identify different organic compounds. Moreover, we get cuticles at micro- and nano-scale levels, as well as the crystallographic a deeper insight into the supramolecular arrangement of the different arrangement of selected parts of carapace. biomolecules in the biosilica. Two complete trilobite carapaces belonging to the genera Phacops sp. and Scabriscutellum sp. from Middle Devonian of Hamar Laghdad Formation P 63 (Erfoud, Morocco) were studied. The material was observed under Biosilicification- phytolith formation in plants cathodoluminescence (CL) with a special emphasis to the preservation state. M. A. Nawaz1, I. Zemchenko*1, A. Zakharenko1, K. S. Golokhvast1 Micro- and nanostructure was characterized by SEM and AFM and 1Far Eastern Federal University, School of Engineering, Vladivostok, subsequently crystallographic arrangement of four skeletal parts was studied Russian Federation (EBSD): cranidia, pygidia, articulated thoracic segments and hypostome. The calcitic cuticle is a hierarchical self-assembled structure composed by a taxa-specific microstructure, dense packaging, forming curved and Silica is deposited extra and intracellularly in plants in solid form as interwoven textures. Although, some evidence of diagenesis have been phytoliths. Phytoliths have emerged as accepted proxies for reconstructing observed in Scabriscutellum sp. cuticle (CL and EBSD data), most of ancient flora, agricultural economies, environment, climate, and taxonomic microcrystals are well preserved in both taxa, and they are composed of tools. The discovery of silicon transporter genes has aided in understanding nanogranules coated by dark envelopes, at nanoscale. Cuticles exhibit the c- the mechanism of silicon transport within the plant body and reconstruct axis of crystals oriented perpendicular to skeletal surface and although the plant phylogeny based on the ability of plants to accumulate silica. However, analysis shows a common crystallographic arrangement between both taxa, a clear understanding of silica deposition and the formation of phytoliths is small differences have been observed in the supra-specialized structures. still an enigma and the information on the proteins involved in plant External structures of carapace exhibit a dense and well-constrained biosilicification is still scarce. With the observation of various shapes and crystallographic organization in comparison with internal ones. Such morphologies of phytoliths, it is essential to understand which factors control structural organization of trilobite cuticle may explain its mechanical this mechanism. During the last two decades, much research has been done resistance and contribute to a better understanding of the success of this in this regard and silicon research has expanded as an earth-life science group during almost 350 million years. superdiscipline. We integrated the recent knowledge and new concepts on Acknowledgments: This study was supported by the Spanish "Ministerio de the evolution of biosilicification in plants; uptake, transport and deposition, Economía y Competitividad" (research projects CGL2016‐78738‐P). shape, size, and chemistry. We also consider the questions such as how phytoliths of fixed shape are biogenically produced and discuss their P 62 implications in , palaeoenvironment, and palaeoflora. Finally, the Solid-state NMR studies of 13C, 15N,29Si-enriched biosilica of applications of phytoliths in the fields of agricultural and biomedical the diatom Cyclotella cryptica nanotechnology are discussed. F. Kolbe*1, H. Ehren2, M. Baldus2, E. Brunner1 1TU Dresden, Faculty of Chemistry and Food Chemistry, Dresden, P 64 Germany Regioselective immobilization of an enzyme cascade on diatom 2Utrecht University, Bijvoet Center for Biomolecular Research, Utrecht, biosilica Netherlands E. kumari*1, N. Kröger1 1Technische Universitaet Dresden (BCUBE), Dresden, Germany Introduction Diatoms are unicellular algae producing micro- and nano-structured cell Introduction walls consisting of amorphous silica. The shape and pattern of these cell Diatom biosilica is a favorable support material for enzyme immobilization, walls is species-specific. Although some compounds which are involved in as its hierarchical mesoporous structure provides a large surface area for the the process of biomineralization like polycationic peptides called silaffins or attachment and allows the efficient reactant diffusion. The immobilization of long-chain polyamines (LCPAs) are known, the process of biomineralization multi-enzyme systems is an emerging technology that enables the rapid is not fully understood until today. multi-step conversion of a substrate into a desired product. Previously it was Objectives shown that the activity of immobilized glucose oxidase is strongly influenced Solid-state NMR spectroscopy is used to gain a deeper insight into the by the species-specific diatom biosilica structures. However, it has remained interactions at the organic/inorganic interface of the diatom cell wall. Special unknown, whether the activity of an enzyme cascade is influenced by its solid-state NMR methods, i.e. the REDOR experiments, can be used for positioning within structurally different regions of the biosilica from the same diatom species. Thalassiosira pseudonana biosilica, which served as a

49 model diatom for this project, is composed of two morphologically distinct turris exists almost exclusively as Fe2O3 clusters/nanoparticles which are 29 building blocks: girdle bands and valves. Girdle bands biosilica rings which tightly associated to the cell wall. T1 measurements in Si MAS NMR a smooth surface and perforated by uniformly sized mesopores (20 nm experiments revealed that only a minor amount (below 5 %) is dispersed over diameter). In contrast, valves have very rough surfaces with a hierarchical the biosilica. That means that iron does not replace silicon atoms at pore patterns. tetrahedral sites as it is known for aluminum incorporation. Objective Conclusion The aim of the present work is to test the hypothesis that silica structure is a Iron is not mainly coprecipitated in dispersed form within the biosilica during key parameter for the activity of an immobilized enzyme cascade. cell wall synthesis of Stephanopyxis turris. It rather exists as Fe2O3 clusters. Furthermore, we investigated whether the activity of the cascade is The obtained material is thus not suitable for technical applications due to influenced by the relative positioning of the two enzymes on the biosilica the low amount of iron incorporation. A suitable material may therefore be surface. an iron-silica-nanocomposite. Materials and methods We have used an in vivo method (LiDSI) to immobilize on biosilica the P 66 enzymes glucose oxidase and horseradish peroxidase. Together they New data on the structure of the giant basal spicule of constitute a minimal enzyme cascade. LiDSi is based on introducing into the monorhaphis sponge diatom T. pseudonana synthetic genes that encode the desired enzymes fused A. Pisera*1, M. Łukowiak1, K. Tabachnick2 to a fluorescent proteins and a regioselective silica targeting peptide. The 1Institute of Paleobiology, PAS., Warszawa, Poland enzymes were localized on the biosilica surface of transformant diatoms 2P.P. Shirshov Institute of Oceanology, Russian Academy of Sciences, using confocal fluorescence microscopy. After biosilica isolation the Moscow, Russian Federation catalytic activity of the immobilized cascade was determined through a colorimetric assay. Result The skeletal elements of sponges are called spicules and are made of silica We generated four T. pseudonana strains each carrying the same enzyme or calcium carbonate. They are usually small (micrometers to millimetres) cascade, but with different locations of the two enzymes inside the biosilica. and situated inside the sponge body. But the basal (siliceous) spicule of deep The four enzyme cascade configurations exhibited striking differences in water hexactinellid sponge Monorhaphis can reach almost 1 cm in diameter specific catalytic activities (i.e. catalytic activity per immobilized enzyme and over 2 meters of length being probably the largest single biosilica-based molecule). Transformants with both enzymes immobilized in the valve structure on Earth. The spicule protrude from the sponge body and anchors exhibited the lowest specific activity, whereas the specific activity of it in the sediment. transformants containing both enzymes in the girdle band region was 3- to 6- This giant spicule became a model of hexactinellid spicules structure and fold higher. Further investigations into the cause for the observed differences formation. According to previous studies (Wang et al. 2011 with references) in the cascade activity ruled out influences of quantity, surface exposure and the majority of the spicule is composed of regular smooth siliceous laminae, stability of the enzymes. but two most external layers differ in morphology and structure. The most Conclusion external layer called "banded ribbon layer" was first described as made of a Our results confirm the remarkable, yet little understood, influence of solid fibrous collagen but later its siliceous composition has been proven. biosilica architecture on enzyme activity. The sculpture of the second layer, occurring directly below, was described as depressions with elevated rims that housed sclerocytes. P 65 The aim of the study was to better understand the structure of this spicule, In vivo incorporation of iron oxide into diatom biosilica and to interpret the observed features. S. Machill*1, J. Kaden1, S. I. Brückner1, L. Köhler1, R. Reichenbächer1, M. We have examined several spicules of Monorhaphis along the whole length Schumacher1, E. Brunner1 under a light microscope. Then the fragments which were different in 1TU Dresden, Chair of Bioanalytical Chemistry, Faculty of Chemistry and appearance were studied under SEM. Mineral composition of the studied Food Chemistry, Dresden, Germany samples was determined with EDAX. Our studies have shown that the two most external layers are not occurring along the whole spicule length. All three structurally different units Introduction superimposed occur only in the lower part of the spicules, the uppermost part Diatom biosilica is a species-specific, micro- and nanostructured material display only central core of smooth solid silica. We have confirmed that the which consists of and tightly attached organic compounds. banded ribbon layer is siliceous, not organic, but, composed of granular silica Diatoms can accumulate "foreign" elements like germanium, aluminum or and porous, in opposition to a solid glassy structure of all more internal gold and incorporate or attach them to the silica. These in vivo synthesized lamellae. The structure of the penultimate layer is exactly the same as the nanocomposite materials can be used as catalysts, adsorbents or core of the spicule. i.e. glassy and without visible porosity. We also biosensors. The role of iron in the biosilification of diatoms is not fully demonstrated that this layer is covered with regularly developed and understood. Iron is an essential nutrient in the metabolism of diatoms and distributed elevations, not with depressions as previously thought. For this plays a central role for photosynthesis. Genomic analyses indicate a close reason they cannot house sclerocytes; additionally in late ontogenetic stages connection between the metabolism of silicon and iron in diatoms. Therefore, in hexactinellids occurs only sclerosyncytium. The elevations in penultimate the role of iron in biosilification should be further elucidated. layer are developed by progressively stronger folding of the surface of Objectives superimposed thinner layers. They fit tightly into depressions of the banded The aim of this work is to examine weather or not iron can be accumulated ribbon layer, developed its lower surface. by diatoms and to determine the chemical state it is associated with the It is not clear why and how the banded ribbon and tuberculated layers siliceous diatom cell walls. In order to quantify the maximum amount of of Monorhaphis basal spicule have developed, but we speculate that the incorporated iron, we offered excessive iron concentrations up to the banded ribbon surface may be an adaptation to help the sponge to stay fixed tolerance limit in the culture medium during diatom growth. The morphology on the spicule, not to glide down on very smooth silica surface. The and the structure of the obtained materials were characterized by microscopic elevations of the penultimate layer that fit into depressions of the lower and spectroscopic techniques to answer the question if iron is dispersed in surface of banded ribbon layer help to stabilize (fix together) the two most the biosilica or preferentially clustered. As a defined reference material, silica external layers. precipitates with different amounts of iron were also studied. References Materials & methods 1. X. H. Wang, M. Wiens, H. C. Schröder, K. P. Jochum, U. Schloßmacher, The diatom species Stephanopyxis turris and Thalassiosira pseudonana were H. Götz, H. Duschner, W. E. G. Müller, J. Experimental Biol. 214 (2011) cultured in an artificial seawater medium with increased iron concentrations. 2047-2056. To remove the physically bound organic matter and ions, the cell walls were Financial support by the Polish National Science Center Grant extracted by a lysis buffer. The obtained biosilica as well as samples of the 2016/21/B/ST10/02332 to A. Pisera. reference material were analyzed by ICP-OES in order to determine total Fe:Si ratios. For morphological studies, scanning electron microscopy (SEM) was used. The chemical state of iron was characterized by Fourier transform infrared (FTIR), Raman, 29Si magic angle spinning (MAS) nuclear magnetic resonance (NMR), as well as electron paramagnetic resonance (EPR) spectroscopy. Results The amount of iron incorporated in the biosilica of Stephanopyxis turris does not linearly increase with the amount of iron offered in the growth medium. It is concluded that iron deposition in biosilica is obviously regulated and limited. The spectroscopic investigations revealed that iron within the silica framework of the synthetic silicagels is preferentially dispersed leading to Si- O-Fe bond formation. In contrast, biosilica-associated iron in Stephanopyxis

50 P 67 varying amounts of aluminum to have Si and Al precursors simultaneously The accumulation of aluminum in diatom and in the culture. After 12 days of cultivation, cells were harvested and washed modifications of the resulting alumosilicate by centrifugation and kept at -20 for future characterizations. In order L. Köhler*1, S. Machill1, A. Werner2, C. Selzer2, S. Kaskel2, E. Brunner1 to monitor the frustules formation inside the silica deposition vesicle (SDV) 1Chair of Bioanalytical Chemistry, Faculty of Chemistry and Food with fluorescence microscopy, synchronized growth was Chemistry, Technische Universität Dresden, Dresden, Germany initiated by repleting the culture with a final concentration of 100 µM silicic 2Chair of Inorganic Chemistry I, Faculty of Chemistry and Food Chemistry, acid and 1 µM PDMPO as a staining agent. Technische Universität Dresden, Dresden, Germany Results Fluorescence microscopy images show valve formation starts from the central nodule and proceeding toward the outer valve edges. Following one Introduction hour the full two-dimensional size of the new valve has been reached. After Diatoms stand out from other unicellular algae through their uniquely 6 hours the fluorescence intensity of the SDV reaches the maximum, patterned, siliceous cell walls. These so-called frustules contain mainly implying at this point the two cells are relatively separated and cell division amorphous SiO2, but also foreign elements such as metalloid and metal is taking place. EDX measurements of P.sp and T.p unveil the presence of atoms. Aluminum is a particularly interesting constituent because it directly aluminum in the frustules. SEM images of P.sp show that the pore diameters replaces silicon in the silica framework, forming an alumosilicate.1,2 This seem to decrease by increasing the concentration of aluminum in the aluminum insertion leads to a negative charge, which is probably cultures. The Solid-state NMR spectra show that the sample contained both counterbalanced by cations like calcium.1 four and six coordinated Al (III) as indicated by peaks at 7.99 and 55.03 ppm, Objectives respectively. Six coordinated Al is the most predominant Al (III) species in In the present study, several ways of aluminum addition to the living diatom our sample which could be from our precursor bonded Al to organic cultures were evaluated. The structure model from literature1 was reviewed compounds inside and outside of the cells or adsorbed Al (III) on the surface with respect to the coordination of aluminum and the type of counter ions. of frustules. However, the peak at 55.03 ppm shows the presence of four-fold Varying growth parameters were assessed to tailor materials properties of the coordinated Al suggesting it is incorporated inside the silica networks of the frustules, i.e., the molar ratio of aluminum to silicon. This characteristic frustules. property affects possible future applications in sorption and catalysis. Conclusion Besides the changes in vivo, frustules were further modified in vitro to EDX measurements show the presence of aluminum in both species. SEM enhance porosity and acidic strength. images of P.sp show that the pore size decreased by increasing the AlCl3 in Materials & methods the growth medium. Cell division in P.sp as shown in fluorescence The fast growing and resistant diatom species Thalassiosira pseudonana was microscopy images takes six hours. Solid State 27Al NMR spectra show four cultivated in aluminum-enriched media. After harvest, frustules were cleaned and six coordinated aluminum in the structure of T.p. In the future, we aim with lysis buffer and calcined. This biosilica was characterized by various to perform 29Si and 27Al solid-state NMR of the to not only methods including infrared, nuclear magnetic resonance and optical emission evaluate the coordination environment of aluminum but also to pinpoint the spectroscopy as well as nitrogen and ammonia sorption. To exchange the effect of aluminum on cross-linking of silica. In addition, some mechanical counter ions created by aluminum incorporation, the biosilica material was properties of intact cells and frustule will be measured to investigate the stirred in an ammonia solution and subsequently heated. Furthermore, effect of aluminum on the mechanical characteristic of diatoms. frustules were etched with a mild base to increase the specific surface area. Catalytic activity was investigated by an acid-catalyzed alkylation of P 69 aromatic compounds, which was monitored by gas chromatography coupled Highly efficient encapsulation of biocatalyst in diatom-inspired with mass spectrometry. silica nanoparticleforenzymatic CO2 capture and utilization Results B. H. Jo*1 Diatom incorporated aluminum into their frustules, resulting in n(Al):n(Si) 1Gyeongsang National University, Division of Life Science, Jinju, South ratios up to the scale of 1:10. With further in vitro modification steps, acidity Korea was more than doubled to 320 µmol NH g-1 and the specific surface slightly 3 increased to 70 m2g-1. Ion-exchanged biosilica was shown to be catalytically This study reports on the development and characterization of a carbonic active in contrast to non-modified biosilica.3 anhydrase (CA)-based biocatalyst encapsulated in a biosilica matrix for use Conclusion in CO capture an utilization. Encapsulation occurred simultaneously with The current study shows that the siliceous cell walls of Thalassiosira 2 autonomous silica synthesis by diatom-derived silica-condensing R5 peptide, pseudonana can be aluminum enriched in vivo while preserving their which was genetically fused to recombinant CA. The encapsulation structure. Through in vitro modification, the acidity and specific surface area efficiency was greater than 95%, and the encapsulated CA was not leached of the biosilica can be enhanced. As successful catalytic tests show, diatoms from the silica matrix, demonstrating the highly efficient R5-mediated auto- are a possible source for "green" catalysts. encapsulation process. The catalytic efficiency for CO hydration was pH (1) Gehlen, M.; Beck, L.; Calas, G.; Flank, A.-M.; Bennekom, A. J. Van; 2 dependent, suggesting that proton transfer from silica to water is a rate Beusekom, J. E. E. Van. Geochim. Cosmochim. Acta 2002, 66 (9), 1601– limiting step. In addition to good reusability, the encapsulated CA exhibited 1609. outstanding thermostability, retaining 80% activity after 5 days at 50˚C. The (2) Machill, S.; Köhler, L.; Ueberlein, S.; Hedrich, R.; Kunaschk, M.; thermoactivity was also remarkable, showing ~10-fold higher activity at Paasch, S.; Schulze, R.; Brunner, E. BioMetals 2013, 26 (1). 60˚C compared to that at 25˚C. The physical structure was observed to be (3) Köhler, L.; Machill, S.; Werner, A.; Selzer, C.; Kaskel, S.; Brunner, E. highly compact with a low surface area, stressing the importance of the Molecules 2017, 22 (12). outermost surface for catalytic performance. We also demonstrated the applicability of the silica nanoparticle to the sequestration of CO in P 68 2 carbonate minerals. The rate of CaCO3 precipitation was remarkably The role of aluminum in diatom biosilicification accelerated by the encapsulated biocatalyst. Thus, this silica-CA 1 1 1 1 2 M. Soleimani* , S. Maddala , A. Akiva , L. Rotten , I. Zlotnikov , R. nanocomposite, efficiently synthesized via a biomimetic green route, can be 1 1 1 A.T.M. van Benthem , H. Friedrich , N. A. J. M. Sommerdijk successfully used as a robust biocatalyst for biomimetic sequestration of the 1 Eindhoven University of Technology , chemical engineering and chemistry greenhouse gas CO2. , Eindhoven, Netherlands 2 CUBE Center for Molecular Bioengineering, Dresden, Germany P 70 Design of biosilica-enveloped R5 ferritin cage for dual drug Introduction delivery system development Diatoms are unicellular photosynthetic algae which live in nearly every M. R. Ki1, M. A. A. Mohamed1, K. B. Yeo1, S. H. Kim1, K. H. Min1, S. P. aquatic territory. Their unique morphological characteristic is the silica cell Pack*1 wall (frustule), which is formed from two halves, like a petri dish, joined 1Korea University, Biotechnology and Bioinformatics, Sejong, South Korea together by girdle bands. Besides silicon, diatoms incorporate aluminum into their silica cell wall, impacting the solubility and dissolution rate of the frustule. Due to the complexity of the structural arrangement of diatoms, the Silica has unique properties such as good biocompatibility, excellent pH and chemical connectivity between aluminum and silicon inside the frustule has thermal stability, as well as high porosity, low toxicity, and superior not yet been discovered. Here, we use various analytical techniques to get mechanical stability. Consequently, silica-based nanomaterials have gained insights into how aluminum plays a role in frustule formation. considerable interest for their potential biotechnological applications such as Materials and methods in drug delivery, enzyme immobilization, and biocatalysis. However, the Pinnularia sp (P.sp) and Thalassiosira pseudonana (T.p) were grown in classic approaches to silica synthesis require harsh reaction conditions such filtered, autoclaved seawater amended with nutrients according to Guillard"s as extreme pH, high temperature, and toxic chemicals. One approach to f/2-recipe. To synchronize cells at cytokinesis arrest, silicon starvation was alleviating these limitations is to mimic the silica mineralization seen in applied with an incubation time of 48 h in Si free medium. Starvation was biological systems. In one of well-known examples, silaffin polypeptides terminated by adding Guillard"s (F/2) marine water enrichment solution and derived from diatom biosilica are a class of massively post-translationally

51 modified (PTM) proteins that are responsible for initiating silica formation P 72 at ambient temperature and pressure. Interstingly, an unmodified silaffin R5 Interactions between gold nanoparticles and the diatom peptide is capable of mediating silica precipitation in vitro under specific Stephanopyxis turris conditions. Using recombinant DNA technology, R5 allows for N. Pytlik*1, S. Machill1, B. Klemmed2, A. Eychmüller2, E. Brunner1 in situ formation of biosilica matrices containing the R5-fused recomabinant 1Technische Universität Dresden, Bioanalytical Chemistry, Dresden, proteins. Here, we designed R5 peptide-fused ferritin (R5FT) for the Germany developement of an advanced dual drug delivery system (dDDS). Since the 2Technische Universität Dresden, Physical Chemistry, Dresden, Germany fused R5 can mediate biosilica deposition on the ferritin surface (SiO2/R5FT), we could load two types of molecules into the core inside (ferritin cage) and the shell outside (biosilica matrix), respectively. One Introduction model (D#1) was loaded into the cage by the reassembly of R5FT to obtain Diatoms are unicellular algae, which contribute very significantly to the R5FT(D#1), and then the other model drug (D#2) was captured in situ by global carbon fixation by photosynthesis. They are especially famous for biosilica matrix formation to prepare SiO2(D#2)/R5FT(D#1). The their cell wall made from amorphous biosilica which presents a spectacular captured D#2 in the shell exhibited a short-term release, while D#1 in the example for the beauty of biomineralization. The cell walls are highly core showed a long-term sustained release. This dDDS system with the structured and exhibit regular pore patterns at micro- and nanoscale. Each additional release of D#2 decreased the IC50 value of D#1 by two-fold diatom species is characterized by a unique cell shape and pore pattern. compared to the use of only D#1 in dDDS. We further optimized the size and However, the understanding of the complex process of three-dimensional cell performance of biosilica-enveloped ferritin cage in terms of drug delivery wall formation is still limited.1 system. The developed dDDS using biosilica-enveloped ferritin cage can Objectives provide more efficient combinational drug therapies. Diatoms, furthermore, find increasing research interest in the field of catalysis, solar cells or as templates. For several potential applications, the P 71 cells are combined with gold nanoparticles either in vivo or in vitro. Biological silica formation in diatoms - mineralization outside However, only few information is available about the interactions and effects the box? of gold nanoparticles on the diatom cells. Materials & methods B. Mayzel*1, S. Wolf2, A. Gal1 We have recently exploited the potential of the gold nanoparticles to generate 1Weizmann Institute of Science, Department of Plant & Environmental a SERS (surface enhanced Raman spectroscopy) effect for an in vivo Sciences, Rehovot, Israel monitoring. Using a 3D setup, it was shown that gold nanoparticles, which 2Weizmann Institute of Science, Department of Chemical Research Support, were biosynthesized by the diatom Stephanopyxis turris, can even occur Rehovot, Israel inside the diatom cells.2 Consequently, these intracellular gold nanoparticles must either a) be taken up by the cell after extracellular gold ion reduction or Introduction b) be produced inside the cell after the uptake of gold ions.2 To answer the Diatoms are unicellular algae, abundant in all aquatic environments. The question whether or not gold nanoparticles can generally be taken up by hallmark of diatoms is their mineralized cell wall, made of amorphous silica. diatom cells, chemically synthesized gold nanoparticles were added to living The morphology of the cell wall is species-specific, forming distinct 3D diatom cells and their interactions were observed by 3D SERS. micro- and nano-metric architectural features. The prevailing paradigm to Results explain the biological control over the formation of the inorganic phase is Gold nanoparticles with diameters ≥ 50 nm could clearly be localized inside that each silica element is formed intracellularly inside a silica deposition the cells, whereas smaller gold nanoparticles were never detected in the cell vesicle (SDV), and once completed it is exocytosed. The complex cellular interior. This indicates a size-dependent uptake mechanism that comes along processes within the SDVs are thought to regulate the mineralization process. with different toxicities.3 Chaetoceros tenuissimus is a diatom species with a cell size of 5µm in Conclusion diameter, which is characterized by four long extensions, called the setae, The consequences of gold nanoparticle contact with diatom cells have not radiating from the main cell body. The setae are 15-30µm long and 250- yet been fully understood and long-term effects for the organism itself as well 300nm in diameter and are covered by a silica shell. Seta growth commences as for the environment are hardly predictable. Having in mind the raising after cell division and continues at the seta tip until its full length is reached. gold nanoparticle implementation in industry, our findings point out the Therefore, the growing tip of the C. tenuissimus seta is a unique system to responsibility to intensify research concerning nano-bio interactions – study the cellular process of silica formation. especially focusing on diatoms, as an essential biomineralizing organism for Objective our ecosystem. Identifying the cellular organelles and structural elements responsible for 1 N. Pytlik and E. Brunner. MRS Commun. 8, 2018, 322-331. silica mineralization of C. tenuissimus setae. 2 N. Pytlik et al. Algal Res. 28, 2017, 9-15. Methods 3 N. Pytlik et al. Algal Res. 39, 2019, 101447. C. tenuissimus cells were grown in culture and their cell cycle synchronized to increase the abundance of growth-stage seta in cells. CryoTEM P 73 tomography was used to study the 3D structure of C. tenuissimus setae at Macrobiomineralogy- principles of biomineralization in giant various growth stages. Samples were plunge-frozen in liquid ethane to whale bones and perspectives for bioinspired materials science preserve the native structure of the setae. Tomograms of seta tips were M. Wysokowski*1 acquired using the 200kV Tecnai F20 and the 300kV Titan Krios G3i 1Poznan University of Technology, Faculty of Chemical Technology, microscopes. IMOD software was used to reconstruct the tomograms from Poznan, Poland the data. Results 24 tomograms of setae were generated, 11 are assigned to growth-stage setae. Introduction These tomograms clearly show the complex helical architecture of the thin Nowadays, biomineralization is defined as ancient fundamental biological silica fibers enveloping the seta. A thin extra-cellular polysaccharide layer process with high dynamics by which living organisms produce minerals covering the mineralized silica cell wall was observed. The cytoplasmic with multifunctional properties including to harden existing subcellular extension within the growing setae contained a single microtubule filament organic matrices and tissues, to produce protective armors and shells against inside the cell membrane. In some cases, small vesicles were noted in the external damages of diverse origins (predators, UV irradiation, toxic metals, cytoplasm. Importantly, none of the tomograms showed an SDV or other etc.), as well as to carry out magnetic navigation. Diverse biominerals and associated cellular structures at the tip of the setae. biomineral-based skeletal structures are occurring in nature on nano-(virus Conclusions and bacteria), micro- (diatoms, coccolothophores) and macro- (up to 7- These results suggest a silicification mechanism in diatoms that is not SDV meter-long whale bones) levels. The mechanisms of biomineralization (silica deposition vesicle) dependent, but rather a continuous process of remain hotly debated. The best way to understand the basic principles of extracellular silicification. This mechanism may explain other silica elements biomineralization on both molecular and macro-scale levels is by a coherent in diatoms and solve fundamental unknowns about the biological process of synergetic collaboration using explicit reasoning and well-tested explanatory silicification. principles of multidisciplinary experience, knowledge and new technologies. Giant bones of whales (Cetacea) represent the largest biomineral-based constructs known. The existence of such macrobiominerals with still unknown mechanisms of their origin and development is an example of a ground-breaking phenomenon unexplored in nature. In this study, the concept of macrobiomineralization is proposed for the first time. Objectives The main objective is to discover the fundamental mechanisms of biomineralization in selected giant whale bones. This includes the analysis of the structure-function relationship between organic (collagen, lipids) and

52 inorganic mineral phases in selected lipid-rich whale bones (mandibles, ribs Conclusions and vertebrae). 1) Secondary structural characteristics of different protein extracts were Materials & methods studied using various biophysical techniques. The presence of IDPs in the Selected whale bones from registered museum collections have been gentle majority was confirmed within these protein extracts. demineralized using EDTA-based solution at pH 7.2 at 37°C to extract 2) We have also investigated the effects of these protein extracts on corresponding organic phases and to carry out their identification using mineralization of calcium phosphate through in vitro biomineralization assay modern bioanalytical tools including GC, HPLC, ESI-MS, FTIR, Raman, followed by size-based measurement analysis via Nanoparticle tracking CARS, 13C solid state NMR, XPS, XRD, HR-TEM and NEXAFS. Modern analysis (NTA). 3D stereo microscopy together with fluorescence microscopy and BET This is the first time the effect of the whole protein extract was taken into measurements are used for additional characterization of porosity, inner account which in our view more closely imitates the in vivo process of tooth structural organization and special surface area of the biomaterials studied. biomineralization. Results Biomineralizers like whales, which are able to produce hydroxyapatite P 75 (HAP)-based skeletal structures in lipid-rich (hydrophobic) environment, are Brachiopod shells can be folded when wet of crucial scientific interest. For the first time we show that millimeter-sized F. Nudelman*1, J. Ihli2, A. Schenk3, M. Holler2, K. Wakonig2, M. Duer4, M. fibers of collagen remain to play significant role in formation of highly Cusack5 carboxylated HAP, especially in vertebrae. Strong difference with respect to 1University of Edinburgh, School of Chemistry, Edinburgh, United organic phases has been shown between the whale vertebrae and ribs using Kingdom NEXAFS spectroscopy. Phospholipids in ribs fraction have been found 2Paul Scherrer institut, Villigen, Switzerland tightly bound to collagen fibres. It is suggested that in contrast to human 3University of Bayreuth, Faculty of Biology, Chemistry and Earth Sciences, bones,calcium oleate can be the precursor of HAP in the lipid-rich bones of Bayreuth, Germany whales. 4University of Cambridge, Chemistry, Cambridge, United Kingdom Conclusion 5University of Stirling, Biological and Environmental Sciences, Stirling, The fact that giant whale bones represent examples of large-scale United Kingdom biocomposites which has been synthesized in situ at 36.6 °C is very motivating to resolve the outstanding questions. Consequently, based on obtained results the objective with regard to "large-scale biomimetics" Question includes the goal to develop the key way for industrial production and design For hundreds of millions of years, nature has evolved a large assortment of at ambient conditions of "3D Ca-phosphate-lipid-collagen composites" using organic-inorganic hybrid materials that are optimally adapted for a wide naturally occurring sources of each component. range of functions, including navigation, protection, mechanical support and protection. These materials not only exhibit exceptional material properties P 74 but also display multifunctionality, including features such as adapting, The unstructured proteins in biological structures- the case of sensing and self-healing. Among the most remarkable biominerals found in nature are the shells of the phosphatic brachiopod Discinisca tenuis. We human teeth from a protein chemist's perspective empirically observed, for the first time, that the mechanical properties of V. Sharma*1, A. Srinivasan2, A. Roychoudhary3, F. Nikolajeff4, S. Kumar1 1 these shells vary according to their water content. While they are hard and All India Institute of Medical Sciences, Biophysics, New Delhi, India stiff when dry, they become flexible when hydrated, to the point that the shell 2Jamia Hamdard University, Biochemistry, New Delhi, India 3 can be folded in 2 without fracturing. Such capability of an organic-inorganic All India Institute of Medical Sciences, Oral and Maxillofacial Surgery, composite to switch reversibly between stiff and flexible and in real time, New Delhi, India 4 adapting to changes in the environment that demand a different set of Uppsala University, Engineering Sciences, Uppsala, Sweden mechanical properties, is truly unique among both biological and synthetic materials. The aim of this research was to characterise, from the molecular Introduction to the micron-scale, how the structure the brachiopod shells changes as a Proteins are the biomacromolecules that work downstream of genes in every function of hydration, leading to changes in mechanical properties. living system. It is these protein molecules that are responsible for a plethora Methods of functions in our body, ranging from building skeleton tissues to We used cryo-ptychographic X-ray computed tomography (PXCT) on dry, transmitting signal from one place to others. There was a period when partially hydrated (exposed to 70 % relative humidity) and fully hydrated biologists have the view that for a protein to function, the structure is of shells (exposed to 100 % relative humidity) to characterise how the structure supreme importance. Nevertheless, this structure-function paradigm is fairly of the shell at the sub-micron and micron levels changed as a function of obeyed by most of the proteins, if not all of them. Last two decades have hydration. Cryo-scanning electron microscopy (cryoSEM) measurements witnessed the increased reports of proteins that do not have a stable three- were performed on dry and wet shells to analyse changes at the shell nano- dimensional structure but are very much indispensable for the cellular and structure as a function of hydration. Solid-state NMR (SSNMR) was used to biological activities. These unstructured proteins are now known as determine the effect of hydration on the molecular conformation of the "Intrinsically disordered proteins" (IDPs). Interestingly, these IDPs function organic components. not only in isolation (e.g. transcription factors) but helps in the formation of Results hard tissues like teeth, bone, and mollusk shell, by governing a process called PXCT measurements demonstrated the organic-rich layers in the shell biomineralization. It is the process by which living organisms produce expanded in thickness, increasing from ~160 nm in the dry sample to ~180 minerals, often to harden or stiffen existing tissues. Such tissues are called nm in the partially hydrated and ~340 nm in the fully hydrated sample. mineralized tissues. The process of biomineralization is still considered as a CryoSEM suggests that the organic components of the shell swell upon scientific puzzle. hydration. Using SSNMR, we identified that the macromolecular chains of Aim proteins, and in particular the methyl groups in proximity to the mineral, To investigate the structural differences of protein extracts from four types become more mobile with hydration. of human teeth (i.e. Molar, Premolar, Canine, and Incisor) and their Conclusions implication in regulating the calcium phosphate mineralization in vitro. Our results show that the changes in the mechanical properties of the shell Methods as it absorbs water arise from modifications in the mobility of the organic 1) Isolation and purification of proteins from different types of human teeth. components and structural changes, at the sub-micron and micrometer 2) Characterization of tooth proteins by (a) Fourier transform Infrared levels, caused by the swelling of the organic layers. Spectroscopy(FTIR); (b) Circular Dichroism (CD) 3) In vitro calcium phosphate biomineralization assay followed by size P 76 measurement experiments by Nanoparticle tracking analysis and analysis by Ion substitutions in apatites of human urinary stones scanning/transmission electron microscopy. A. Korneev*1, A. Izatulina1, O. Frank-Kamanetskaya1 1Saint Petersburg State University, Department of Crystallography, Saint Results and Discussion Petesburg, Russian Federation Protein extract was successfully purified from different types of human teeth (i.e. Molar, Premolar, Incisor, and Canine) which were quantified and run on an SDS PAGE and subsequently stained by silver staining. FTIR was done Hydroxyapatite is the main mineral component of phosphate stones in human to understand the differences present among the different teeth extract and urinary system according to the powder XRD data: it also occurs as an there secondary structure characteristics. CD was done to validate the protein impurity in oxalate and urate stones. According to our collection of renal secondary structure details obtained by FTIR. We have also investigated the stones of St. Petersburg and the Leningrad region residents, apatite is present effects of these protein extracts on mineralization of calcium phosphate in 64,7% stones. Using SEM and EDX it was revealed that individual apatite through in vitro biomineralization assay followed by size-based grains are present almost in all oxalate urinary stones, which confirms the measurement analysis via Nanoparticle tracking analysis (NTA). hypothesis of initializing calcium oxalates crystallization by hydroxyapatite. Due to the low degree of crystallinity of bioapatites the conclusion of their composition can be made according to the detailed EDX analysis and values

53 of lattice parameters (Frank-Kamenetskaya et al. 2011). According to our data values of the a unit cell parameter of urinary stones apatites vary from P 78 9.395(2) to 9.457(4) Å, i.e. can be higher or lower than that of stoichiometric Correlation of bone morphology and quality with melatonin hydroxyapatite (a = 9.418, JCPDS № 9-432). Values of the c unit cell intake in pregnant and lactating rats parameter (6.849– 6.885(4) Å) can be significantly lower than in N. Ishikawa*1, H. Mishima2, A. Hattori3, Y. Maruyama3, N. Suzuki4, Y. stoichiometric hydroxyapatite (c = 6.884 Å, JCPDS № 9-432). The increase Matsumoto5 of a parameter of urinary stone apatites, are associated with substitutions of 1 - Kagawa University, graduate school of Agriculture, Division of applied OH ions by H2O molecules in channels of crystal structure, the decrease - by - biological and rare sugar sciences, MIki, Kita gun, Kagawa prefecture, F ions. EDX data show that fluorine is present in trace amounts in apatite of Japan phosphate kidney stones, and in apatite of oxalate stones its concentration 2Tsurumi University School of Dental Medicine, Dental Engineering, significantly higher (1.6 – 4.2 wt.%). Reduction of c parameter relative to Yokohama, Japan stoichiometric hydroxyapatite is due to the large portion of vacancies in Ca 3 - Tokyo Medical and Dental University, Biology, Ichikawa, Japan sites. Their appearance can be associated with substitutions of OH ions by 4 3- 2- 2- Kanazawa University, Nature and Environmental technology, Housu-gun, H2O molecules and PO4 ions by CO3 and HPO4 ions. Japan The range of urinary stones apatite lattice parameters variations is 5Kagawa University faculty of agriculture, Life Science Course, MIki, significantly higher than in other pathogenic apatites that are formed in Japan human organism, which indicates the essential variation of their forming conditions. Introduction This work was supported by the Russian Science Foundation (no. 18-77- Melatonin (MEL) is a hormone that plays a role in the circadian rhythm. 00026). The XRD studies have been performed at the X-ray Diffraction MEL increases bone density and inhibits the differentiation of osteoclasts Centre of St. Petersburg State University. Scanning electron microscopy and (Koyama, 2002; Hattori, 2017). In 6-day-old rats, MEL participates in the EDX analysis have been performed at «Center for Geo-Environmental formation of incremental lines and calcification of dentin (Mishima et al., Research and Modeling (GEOMODEL)» of St. Petersburg State University. 2018). However, the effects of MEL intake during osteogenesis on bone References formation in rats have not been clarified. Therefore, we analyzed the bone Frank-Kamenetskaya O., Kol'tsov A., Kuz'mina M., Zorina M., Poritskaya morphology and quality based on MEL intake in lactating rats. L. Ion substitutions and non-stoichiometry of carbonated apatite-(CaOH) Objectives synthesised by precipitation and hydrothermal methods // Journal of The present study evaluated the effects of MEL intake in lactating rats on the Molecular Structure. 2011. Vol. 992. P. 9-18. bone morphology and quality during lactation. Materials and methods P 77 Six SD pregnant rats were divided into 3 groups: (1) control group (Con; TEM study of compositional and structural changes of bone 0.5% alcohol-containing water), (2) low-concentration group (Low; 0.5% crystals by heating alcohol + 20 µg/mL MEL-containing water), and (3) high-concentration M. Yosikawa*1, M. Kakei2 group (High; 0.5% alcohol + 100 µg/mL MEL-containing water). MEL was 1Meikai University School of Health Sciences, Department of Oral Health administered for 9 days, the last 3 days of pregnancy and the 6 days after Sciences, Urayasu, Japan birth. Slaughter was carried out at midday and midnight on 6-day-old. The 2Medical English Institute[NPO], Saitama, Japan concentrations of plasma melatonin metabolites (serotonin: 5HT, N-acetyl serotonin: NAS, melatonin: MEL, and 6-hydroxymelatonin: HaMT) were measured by LC-MS/MS. The trabecular area ratio was observed and Introduction analyzed using a fluorescence microscopy. The calcification, crystallinity Octacalcium phosphate (OCP) is considered to be a candidate for the central and collagen maturity of bone were analyzed by FTIR. The bone composition dark line (CDL) which is observed in tooth enamel, dentin and bone crystals. was analyzed by SEM-EDS. On the other hands, it has been reported that the formation of huntite minerals Results occurred prior to the nucleation of apatite crystal (Casciani et. al., 1979). 5HT was significantly higher in the MEL-treated group at midday and Regarding the physical property of OCP, it has been reported that OCP was midnight (p < 0.05). MEL was significantly higher in the High group than decomposed completely up to 150 ℃ (Bigi et. al., 1990), while the thermal that of the Con group at midnight (p < 0.05). HaMT value increased in the decomposition of huntite minerals has been reported to begin from around proportion with the MEL dose. The trabecular area ratio was significantly 550 ℃ (Földvárl, M. 2011). From the ultrastructural viewpoint, transmission higher in the Low group than that of the Con group at midday (p < 0.05). electron microscopic (TEM) study has shown that after heating at about 600 Calcification was significantly higher in the High group than that of the Con ℃, a small number of crystals still preserved CDLs in their structures as group at midday (p < 0.05). The crystallinity and collagen maturity were reported previously (Kakei et. al., 2005). This finding suggested that CDL significantly lower in the MEL-treated group at midnight (p < 0.05). The known as the nucleation site of biological apatite crystals might consist from mass ratios of Ca and P were significantly higher in the MEL-treated group huntite minerals and a subsequent induced initial lattice line of apatite. at midday (p < 0.05). At midnight, the mass ratio of P was significantly lower Furthermore, the lattice line of CDL did not create two lattice lines of apatite, in the High group than that of the Con group (p < 0.05). meaning that CDL is not identical to OCP. Based on the previous findings, Conclusion the present study was aimed to further clarify the involvement of huntite In analysis, 5HT is secreted in the breast milk during lactation, thus we minerals to the CDL formation. Also, we conducted to examine the structural think that 5HT was transferred from breast milk to rat pups and NAS was change of crystals by heating at over 700 ℃. confirmed in rat pups plasma. Further, we think that MEL metabolism was Objective activated by MEL intake and 5HT in breast milk increased. The value of The present study is conducted to distinguish between CDL in apatite crystal MEL and HaMT in plasma suggested the transfer of MEL to rat pups through and OCP, and examined the structural changes of apatite crystals by heating. breast milk. In the MEL-treated group, the trabecular area increased, and Material and Methods calcification was promoted, however bone was immature. Thus, bone To clarify the composition of CDL, the results of both thermal analysis of maturation probably did not catch up with bone formation in the MEL-treated huntite minerals and TEM observation of bone crystals treated by heating at group. The SEM-EDS analysis suggests that MEL may have promoted bone different temperatures were compared. After removing the surrounding soft formation and affected bone composition. The present results suggest that tissues, rat calvaria were treated with a plasma reactor to remove organic MEL intake in the pregnant rats was transferred to rat pups through breast substances. Thermogravimetry/Differential thermal analysis (TG/DTA) milk to promote bone formation and calcification. From Mishima's (2018) analysis of huntite minerals was conducted in air atmosphere with heating at finding that MEL changes the structure of dentin apatite crystals and collagen 20 °C/min. For TEM study, samples were heated at 600, 700 and 1,000 ℃, fibers, the results of this study suggest that the mechanism of MEL effects respectively, for one hour in the muffle furnace. on the bone calcification may differ from the dentin calcification. Results Fig.1 showed the TG and DTG derivative curves of huntite minerals. The decomposition of huntite minerals began from around 500 ℃ (Fig. 1). TEM study of the control of calvaria showed numerous tiny crystals showing the presence of CDL (inset) (Fig. 2). After heating at 600 ℃, each crystal seemed to be obscure and some crystals amalgamated with each other, though some crystals still preserved CDL (Fig. 3). After heating at 700 and 1,000 ℃, respectively, TEM observations revealed that large crystals were formed (Figs 4a and b). CDLs disappeared completely. Conclusion The preservation of CDLs by heating at around 600 ℃ clearly reveals that CDL is not identical to octacalcium phosphate, meaning that two different mechanisms for apatite formation exist. Creating larger crystals by heating at above 700 ℃ may contribute to the increase of crystallinity.

54 P 79 mechanosensory response in 3D in vitro osteocyte cultures. Variations in Crystallographic and chemical vital effects in Dendrophyllia osteocyte shape between bone regions with different loading modes raise the skeleton - a by-product of the biocrystallization question whether osteocyte shape represents an adaptive response. I. Coronado*1, A. Pérez-Huerta2, J. A. Cruz3, J. O. Cáceres4, S. Moncayo5, V. Motto-Ros5, F. Trichard5, G. Panczer5, F. Pelascini6, J. Stolarski1 Materials & methods 1Institute of Paleobiology, Polish Academy of Sciences, Warsaw, Poland Osteocyte morphology will be modified by 3D bioprinting of polymers into 2The University of Alabama, Department of Geological Sciences, patterns that control lacunar shape. After we have differentiated the osteocyte Tuscaloosa, United States cultures in these networks, we apply mechanical compression load 3Complutense University, GEODESPAL, Madrid, Spain (nanoindentation) and fluid-flow shear stress to measure their ability to 4Complutense University, Departamento de Química Analítica, Madrid, produce a mechanosensory response. Lacunar shape will be digitized using Spain high-resolution X-ray tomography in a Synchrotron. The shape of the 5Institut Lumière Matière, UMR5306 – UCBL- CNRS, Lyon University, lacunae will be quantified in 3D using geometric morphometrics. The Villeurbanne, Germany canalicular network will be analysed in terms of connectivity, canalicular 6CRITT Matériaux Alsace, Schiltigheim, France thickness, canalicular spacing, etc. Multivariate statistics will be used to assess the correlation between osteocytic mechanosensation and morphology in extant vertebrates. Extreme changes in mechanical loading at the Coral skeletons are natural archives of the climate variability and a potential evolutionary transition from land to water will be used to test whether proxy for paleoenvironmental reconstructions. Paleoclimate reconstructions osteocyte shape is an adaptive response. are based on changes in isotopic ratios and trace element uptake by bio- Results aragonite skeletons, as a response to environmental changes such as We hypothesise that osteocyte morphology is predictive of mechanosensory temperature and ocean circulation . Unfortunately, corals are not longer ability of the bone. We expect to find that osteocyte markers (e.g., sclerostin, considered as passive recorders of environmental changes because the RANKL, osteoprotegerin, and extracellular vesicles) will vary with lacunar geochemical composition of their skeletons does not precipitate in morphology and canalicular network topology in extant vertebrates. We also thermodynamic equilibrium with seawater. This desequilibrium has been expect to find a correlation between the morphological parameters and the attributed to vital effects. marker variations, as well as differences between secondary aquatic Two classes of vital effects have been recognized: those derived from vertebrates and their land inhabiting counterparts. physiological processes and those related to biocrystallization, from crystal Conclusion nucleation to stabilization of the mineral phases. Those vital effects linked to If our hypotheses prove to be correct, we will have established that osteocyte biocrystallization have not been studied in-depth in corals.Their shape is an adaptive response to mechanical loading. We also expect to interpretation requires the incorporation of groundbreaking knowledge from establish that the osteocyte and the canalicular network change across recent discoveries in coral biomineralization i.e., identification of amorphous extreme changes in loading modes, such as during the land to water transition precursor phases that fulfills a new paradigm of crystallization by particle of secondary aquatic vertebrates.This information could be used to interpret attachment (CPA) and biocomposite nature of coral skeleton. Therefore, in the loading regimes of fossils. order to retrieve a "purely" environmental signalfrom coral skeletons, chemical variations linked to precursor phases and the organic templates P 81 involved in bio-aragonite crystallization have to be understood. First finding of mineralized primary layer in Lingula anatina The purpose of this study is to identify and characterize the vital effect (Lamarck, 1801) Brachiopoda, Lingulida derived from biocrystallization processes. We performed a detailed study of A. Madison*1, T. Kuzmina2 a cold-water scleractinian coral species Dendrophyllia ramea skeletons, 1Paleontological Institute, Moscow, Russian Federation analyzing the micro- and nanostructures (using SEM and AFM), the 2Lomonosov Moscow State University, Moscow, Russian Federation crystallographic arrangement and lattice parameters (using EBSD and XRD), and the biogeochemical composition (using FTIR of isolated organic matrices, Raman mapping, TG-analysis and a multi-elemental imaging using The shell secretion of recent brachiopods remains poorly known, especially LIBS). if compared with the studied in detail biomineralization processes in recent The Dendrophyllia skeleton has a hierarchical mesocrystalline organization mollusks. The lingulids are best known of all brachiopods in microstructural formed by aragonite fibers in the Thickened Deposits (TD) and sense as they possess the unique shell composed of alternating microgranular aggregates of amorphous nature in the Rapid Accretion biomineralized and organic layers that remained structurally stable Deposits (RAD). We recognize several structural and biogeochemical throughout the Phanerozoic. Three parts are distinguished in the lingulide heterogeneities in the different skeletal parts derived from biocrystallization shell: a periostracum and primary and secondary layers. The mineralized part processes: a variable amount and composition of organic matrix and water in of the linguloid shell consists of up to 10 nm in diameter nanogranules of septa and coenosteum, which may cause marked distortions in the apatite, specifically the fluorapatite francolite, aggregated into up to several orthorhombic constant ratios. Also, the edge layers of coenosteum (in microns in size spheroids or variously long rods. Though the superfamily contrast to those formed earlier) are poorly crystallized, and show Linguloidea appeared in the Early Cambrian, only two genera, Lingula enrichments of Mg, Sr and H. Bruguière, 1797 and Glottidia Dall, 1870, survived up to recently. Studies on Acknowledgments: This work was supported by the National Science Centre the linguloid primary layer provided quite controversial data on its presence (Poland) grant 2017/25/B/ST10/02221. and structure. Watabe and Pan (1984) described 40–50 µm thick primary layer composed of aggregates of spherulites for Glottidia pyramidata P 80 (Stimpson, 1860). However, Iwata (1981) did not find any mineralized The role of lacuna and canalicular network morphology in primary layer in Lingula unguis (L.) (this species was considered as a osteocyte mechanosensation synonym of Lingula anatina Lamarck, 1801 by Emig (1982). Williams et al. A. H. van Heteren*1, Y. Nakajima2, T. Näreoja3 (1994) reported a 40 µm thick organic primary layer in L. anatina composed 1Ludwig-Maximilians-Universität München, Department Biologie II, mainly of glycoaminoglycans (GAGs). München, Germany We studied ten specimens of L. anatina from Vietnam and the Philippines. 2Tokyo City University, Department of Natural Science, Tokyo, Japan The Vietnam specimens were preserved in a 4% formaldehyde solution in 3Karolinska Institutet, Department of Laboratory Science, Huddinge, filtered sea water and the Philippine specimens were dried without any Sweden chemical treatment. All specimens were studied with SEM first with periostracum and then after bleaching with collagenase/proteinase mixture. The shell substance was studied externally and along the fractures. In many Introduction places on the shell outer surface after bleaching preserved a well-ordered There are differences in bone structure between anatomical sites and species sheets of about 0.5 µm high and 0.2 mm thick cylindroids directed and these are reflected in canalicular networks and the lacunar shape of perpendicularly to the valve surface. Such dolioform crystals were previously osteocytes. For example, bone microanatomy is different in terrestrial and unknown for linguliform brachiopods. The sheets look as a thin light film in aquatic vertebrates. In bone, osteocytes are intertwined between type-1 SEM images in contrast with the underlying compact lamina and cover the collagen lamella and hydroxyapatite crystals that reform countless times as whole shell surface including the protegulum and brephic shell. On the the bone adapts to loading applied on it. Differently shaped or oriented brephic and adult shells, the cylindroids are neatly packed and their outer and osteocyte lacunae likely have a different volumetric deformation under a inner ends are obtuse while on the protegulum they are more random and specific load, which will change the load-induced fluid flow that osteocytes fusiform and somewhat resemble the acicular primary layer of feel (e.g., round osteocytes are more mechanosensitive than flat ones). The rhynchonelliform brachiopods with carbonate shell. The external surface of precise influence of the canalicular network has not yet been assessed either. the sheets of cylindroids bear reflections of the radial striation of Objectives periostracum and thus this is the first shell layer underlying the periostracum, Our primary goal is to establish how lacunar and osteocyte cell shape impact i.e. the primary layer. As it preserved after the bleaching, it is mineralized. mechanosensation, using long bones of extant vertebrates, as well as Thus three types of the primary layer are known for recent linguloids: 40–50 palaeontological specimens for a deep-time perspective. We aim to test the µm thick primary layer composed of aggregates of spherulites in G.

55 pyramidata (Watabe and Pan 1984), about 40 µm thick primary layer epithelial calcium phosphate transport in comparison to those in CaCO3 composed of GAGs in L. anatina from Japan (Williams et al. 1994) and about transport. 1 µm thick layer of cylindroids in L. anatina from Vietnam and the We used TEM and STEM to investigate the ultrastructure of epithelial cells, Philippines. One of the possible explanations for strongly differing primary and X-ray spectral mapping to detect mineral containing organelles within layers in one species is that in fact they are different taxa but additional data cells. For elemental mapping, we used high pressure frozen material, freeze- on the molecular phylogenetics of these brachiopods are needed in order to substituted in acetone containing uranyl acetate and glutaraldehyde. Thin resolves this problem. sections of resin embedded material were floated on propane-1,2 diol instead of water to minimize loss of diffusive elements within the sample. P 82 The results show that the hypodermis of the PI consists of two different cell Guided mineral growth on amelogenin scaffolds promoted by types. The P-cells and the C-cells that secrete and mineralize the calcium amelotin phosphate and calcium carbonate containing part of the PI cuticle, B. Ganss1, A. Danesi1, A. Mansouri1, A. Phen1, J. Holcroft*1, J. Bonde1, K. respectively. The P-cells have up to 400 µm long extensions that originate Carneiro1 from the cell somata that are situated in the base region and corpus and 1University of Toronto, Faculty of Dentistry, Toronto, Canada extending to the forming cuticle of the middle region. The extensions, of P- cells contain numerous clathrin-coated vesicles and non-coated vesicle along microtubules. Near the cuticle, the extensions contain calcium phosphate Introduction granules up to 1µm in diameter, likely of endosomal origin, that are filled Enamel, the outermost layer of the tooth, is composed of intertwined with calcium phosphate. The element composition of these granules hydroxyapatite crystals; it is the hardest, most highly mineralized tissue in resembles that of the ACP in the PI cuticle. The C-cells have no large the body and incapable of regeneration. Amelogenesis is the matrix-guided extensions and contain no mineral granules. They resemble the hypodermis process of enamel formation, but its detailed mechanism is not well of the sternal integument for which the cellular mechanism for epithelial elucidated. The most abundant protein present in the developing enamel calcium transport are well studied. matrix is amelogenin (AMEL). AMEL acts as a scaffolding protein that The results suggest that within the PI the cellular mechanism for templates hydroxyapatite (HA) crystal organization. In vitro, recombinant mineralization of the cuticle follows two different pathways. In P-cells AMEL self-assembles into nanoribbons in the presence of calcium and mineral formation takes place within organelles of the endosomal pathway phosphate ions. Amelotin (AMTN), a recently discovered enamel matrix that contribute to cuticle mineralization, while in C-cells mineral is protein essential for proper enamel mineralization and has been shown to transported across the epithelial cells by ion channels and carriers, and promote mineral formation in vitro and in vivo. The functional relationship intracellular compartmentation of calcium ions within the endoplasmic between AMTN and AMEL as the major enamel matrix protein has yet to be reticulum. studied. We hypothesize that AMTN can promote mineralization on AMEL Huber J, Fabritius HO, Griesshaber E, Ziegler A (2014) Journal of Structural structures. Biology 188, 1-15 Objectives Supported by DFG ZI 368/11-1. This study aims to 1) determine the self-assembly behavior of AMTN and 2) determine the effect of self-assembled AMTN and AMEL, alone and in P 84 combination, on calcium-phosphate mineral formation and growth. Physical and anatomical variation of mammalian bone Materials & Methods bioapatite structure and composition Human recombinant AMEL and AMTN proteins were expressed in B. Foley*1, M. Greiner1, G. McGlynn2, W. W. Schmahl1 Escherichia coli. Proteins were self-assembled in a calcium phosphate 1Ludwig-Maximilians-Universität München, Department of Earth- and solution for up to 28 days before being co-incubated for 20 minutes. Self- Environmental Science, München, Germany assembled AMEL and AMTN, as well as AMEL-AMTN were characterized 2Staatssammlung für Anthropologie und Paläoanatomie München, by Atomic Force Microscopy (AFM) and Transmission Electron Microscopy München, Germany (TEM). Results AMTN self-assembled into ribbon-like nanostructures over a period of up to Bone is a hierarchical composite material primarily consisting of an 3 weeks. . Mineral deposits formed on top of these ribbons 14 days after inorganic calcium phosphate phase—a widely substituted variant of apatite assembly. Co-incubation of self-assembled AMTN with AMEL resulted in commonly referred to as bioapatite—dispersed in an organic collagen matrix. 3- the formation of needle-like crystals along AMEL nanoribbons. No Structural variation of the bioapatite lattice occurs with substitution of PO4 mineralization was observed on AMEL alone. and OH- by carbonate ions, which results in changes of lattice parameters Conclusion (Handschin et al. 1995). Changes in structure and chemical composition of The co-incubation of self-assembled AMTN to self-assembled AMEL bioapatite have been linked to aging and nutrition (Boskey et al. 2010). nanoribbons promotes the formation of guided mineral growth along the Lattice parameters, crystallinity and ionic substitution of bioapatite also vary AMEL template. This combined activity provides new insights on with anatomical location, pathological conditions, and physical stress in vivo mechanistic details of enamel biomineralization. Understanding this and with the environment during fossilization and cremation post mortem combined effect will open opportunities for developing biological and/or (Greiner et al. 2018). As such, detailed investigation of bone bioapatite synthetic regeneration strategies for dental enamel. structure and chemical composition is essential to uncovering the stories of forensic and archaeological bone samples and understanding P 83 processes in contexts such as disease pathology and tissue engineering. Calcium phosphate granules in hypodermal cells participate in Human bone samples were analyzed by X-ray powder diffraction and the mineralization of crustacean mandible incisors Fourier-transform infrared spectroscopy (FTIR) to investigate bone A. Ziegler*1, B. Nutz1, U. Rupp1 bioapatite structure and composition. Specifically, Rietveld refinement of X- 1University of Ulm, Central Facility for Electron Microscopy, Ulm, ray diffraction data was used to determine lattice parameters and crystallite Germany size. Samples of the femur, clavicle, sternum, talus, calcaneus, and parietal bone were analyzed from the skeletal remains of three middle-aged human subjects: an average-build male, a robust-build male, and a petite-build The incisive regions of the mandibles, the pars incisivae (PI), of the terrestrial female. Lattice parameters and crystallite size were shown to vary with crustacean Porcellio scaber can be subdivided into three regions, according respect to anatomical position, gender, and build. Bone samples from the to differences in mineral content. The distal tip of the PI is not mineralized, robust-build male showed consistently larger a (=b) lattice parameters than the middle region is mineralized with amorphous calcium phosphate (ACP) did samples from the average-build male and petite-build female for all and the base, that connects the incisive region with the corpus of the anatomical positions. FTIR spectra of analyzed samples showed consistent mandible, with calcium carbonate (Huber et al., 2014). The presence of both, ratios of organic to inorganic content. ACP in the middle region and CaCO3 in the adjacent base and corpus is an Preliminary results show consistent proportion of organic content but ideal situation to study mineral phase specific differences in epithelial structural variation with respect to physical build. calcium transport pathways. Which intracellular compartments are involved in calcium transport is Boskey, A., & Coleman, R. (2010). Aging and Bone. Journal of Dental currently the most important question to understand the contribution of cells Research,89(12), 1333-1348. doi:10.1177/0022034510377791 in biological mineralization processes. Knowing the pathway of how Greiner, M., Kocsis, B., Heinig, M. F., Mayer, K., Toncala, A., Grupe, G., & intracellular calcium is directed through the cell is indispensable for Schmahl, W. W. (2018). Experimental Cremation of Bone: Crystallite Size understanding, which molecular transport mechanisms are possibly involved and Lattice Parameter Evolution. Biomineralization,21-29. doi:10.1007/978- in biomineralization. How mineralization with calcium phosphate instead of 981-13-1002-7_3 calcium carbonate affects the structural differentiation of the mandible Handschin, R., & Stern, W. (1995). X-ray diffraction studies on the lattice epithelial cells and transport of mineral through it, is completely unknown. perfection of human bone apatite (Crista Iliaca). Bone,16(4). Therefore, we investigated the hypodermis cells of the PI with the aim to doi:10.1016/s8756-3282(95)80385-8 describe calcium compartmentation and cell structure differentiations for

56 P 85 the supernatant became neutral. The biosilica pellet was suspended in 6 M Infrared nanoscopy of biomaterials NH4F solution to dissolve the silica. After incubating at room temperature A. Cernescu*1, S. Amarie1, A. Huber1, F. Keilmann2 for 30 min, the solution was repeatedly dialyzed against 200 mM ammonium 1neaspec GmbH, Haar, Munich, Germany acetate to remove NH4F. The solution was then freeze-dried to remove the 2Ludwig-Maximilians-University, Munich, Germany solvents and the remaining substances were dissolved in a small amount of distilled water. Finally, the extract was analyzed by SDS-PAGE and mass spectrometry. Introduction Results Scattering-type scanning near-field optical microscopy (s-SNOM) has The extract from the biosilica showed a low-molecular-weight band on SDS- become a key technology to study the chemical composition of inorganic and PAGE gels. Mass spectrometric analysis revealed that this band contained organic materials at the nanoscale. long-chain polyamines (LCPAs) with long repeats of the -CH2CH2CH2NH- Objectives unit. Notably, LCPAs were also identified in silica-accumulating eukaryotes The investigated samples are nanocomposite biomaterials, namely human such as diatoms and siliceous sponges, although their chemical structures are bone sections [1], specimens [2] and mollusk shell which slightly different from those of B. cereus LCPAs. These findings strongly contain mineral nanocrystals in organic matrices [3]. The mineral parts are suggested that LCPAs play a common and important role in silica formation highlighted by their resonantly enhanced contrast due to phonons. in these organisms. Moreover, the chemical structure of B. cereus LCPAs Materials & methods strongly suggested that they were synthesized via repetitive aminopropyl- Our method is surface-sensitive, probing to a depth of about 20 nm. transfer reactions. We also identified a plausible candidate for LCPA Spectroscopic near-field imaging is enabled by combining 20nm-resolving synthase via homology searches using bacterial aminoproplytransferases as tip-scattering near-field microscopy (s-SNOM) with an infrared continuum query sequences. Furthermore, gene disruption experiments showed that the source. Specific contrasting of biomineral components is enabled by simply candidate gene is indeed essential for LCPA synthesis. Unexpectedly, the choosing the appropriate "fingerprint" infrared region that as in traditional disruptant lacking LCPAs formed biosilica around the spore coat, similar to FTIR (Fourier-transform infrared spectroscopy) identifies virtually any the wild type, when cultivated in the presence of silicic acid, thereby chemical compound. Hence nano-FTIR stands for the successful realization suggesting that LCPAs are not essential for biosilicification in B. cereus. of combining s-SNOM and FTIR [3]. Conclusion Results In this study, we demonstrated the presence of LCPAs in the B. cereus Mineral distribution across dentine/enamel interface is resolved with 20nm biosilica and found a putative gene encoding LCPA synthase. Despite the spatial resolution showing a gradient in the hydroxyapatite concentration. common presence of LCPAs in different evolutionary lineages of silica- Relatively large Ca2PO4 crystals (size >100 nm) are observed in both dentin accumulating organisms, gene disruption analysis unexpectedly showed that and enamel regions, as well on the human bone samples (Fig. 1). LCPAs are not essential for biosilicification in this bacterium. Fig. 1: Minerals in human bone. High resolution IR nanoscopy image obtained simultaneously with the AFM topography shows the nanoscale P 87 distribution of Calcium phosphate and collagen within the cross section of a Crystallization of calcium oxalates inherent in mineralized human bone lamella extracted from a hip joint. Imaging at different wavelengths maps different minerals within the tissue, which reveals A. Rusakov*1, M. Kuz’mina1, O. Frank-Kamenetskaya1 important information about the bone structural integrity and could provide 1 details about the process of demineralization due to diseases, such as Saint Petersburg State University, Crystallography, Saint Petersburg, osteoporosis. Russian Federation Interestingly, phosphate nano-crystals are also found in mollusk shell specimens, close to the interface between calcite and aragonite biominerals. Biofilms containing colonies of microscopic fungi (micromicetes) can often Conclusion be found on the surface of carbonate rocks (marble, limestone etc.) in urban The benefits of using the s-SNOM technology are the nanoscale resolution environment. Micromycetes excrete substantial amounts of organic acids combined with the chemical spectroscopic identification of molecules. (oxalic, citric, malic, succinic, fumaric, lactic etc.) in the course of their Straightforwardly applicable in many fields of biomaterials, general metabolism. These acids solubilize carbonate rocks and induce calcium mineralogy and solid state research, this technology is becoming one of the oxalate (calcium oxalate monohydrate - whewellite, calcium oxalate most powerful tools in nanoscale analytics. dihydrate - weddellite) crystallization. These minerals as well as micromicete References metabolites and several other chemical additives which come from the [1] T. Geith et. al, Visualisation of methacrylate-embedded human bone environment form the so called oxalate patina. The aim of this work was to sections by infrared nanoscopy, J. of Biophotonics (2012); study the influence of different organic and inorganic components on [2] T. Sui et. al, Structure-Function Correlative Microscopy of Peritubular phase composition and the morphology of the formed calcium oxalates in and Intertubular Dentine, Materials (2018); vitro. [3] S. Amarie et al, Nano-FTIR chemical mapping of minerals in biological Calcium oxalate crystallization was performed by decantation of a mixture materials, Beilstein J. Nanotechnol. (2012). of sodium oxalate and calcium chloride aqueous solutions with pH variation (4.0-7.0) at room temperature (22-25С). Na+, K+, Mg2+, Fe3+, PO43- P 86 ,SO42-, CO32- ions were added to the solution as the impurity components Discovery of long-chain polyamines and their biosynthetic as well as the organic acids, excreted by micromicetes. The compound enzyme in the biosilicifying bacterium Bacillus cereus concentrations were close to the ones observed in the biofilms.

T. Ikeda*1, K. Yamamoto1, R. Hirota1, A. Kuroda1 It was found that whewellite crystallizes in the presence of all inorganic and 1Hiroshima University, Hiroshima, Japan organic components. Weddellite formed in the presence of citric ions of varying concentrations with no dependance on the presence of inorganic Introduction components, as well as in the presence of three acids (malic, succinic and Although silica biomineralization (biosilicification) has been intensively fumaric) with an equal ratio of their concentrations in the absence of investigated in several eukaryotes, such as diatoms, sponges, and higher inorganic components. Weddellite also solely formed when Ca2+ ions to the plants, prokaryotic biosilicification was not studied until recently. In 2010, oxalate ions concentration was set close to stoichiometry (1.5:1) at pH≥6, we reported that biosilicification occurs in the gram-positive, spore-forming and at an increased ratio of more than 7.5:1 at pH≥5. The addition of iron bacteria, Bacillus cereus and its close relatives, and that silica is deposited in cations to the system significantly increases the amount of weddellite in the and around a spore coat layer as a protective coating against acids (Hirota et sediment, while the admixture of carbonate ions or phosphate ions, on the al. 2010 J. Bacteriol. 192, 111). In a recent study, we demonstrated that the contrary, decreases the amount of weddellite in the sediment. The presence spore coat protein, CotB1, which carries a characteristic C-terminal of phosphate and carbonate ions in the crystallization medium can lead, in zwitterionic sequence, plays an essential role in biosilicification; however, appropriate conditions, to the formation of very small amounts of additional the underlying mechanism was not elucidated (Motomura et al. 2016 J. mineral phases, such as brushite and calcite, for example. Bacteriol. 198, 276). Whewellite crystallized in the following forms: small elongated lamellar Objectives crystals with side faces not very pronounced (in syntheses without any This study aimed to further investigate bacterial biosilicification additional impurities); large aggregates consisting of oval petals (in systems mechanisms. Since most eukaryotic silica-precipitating peptides and proteins with additional inorganic impurities); small spherical spherulites consist of were discovered by dissolving biosilica in hydrofluoric acid (HF) or small curved plates (in systems citrate ions); small spherical spherulites (in systems with citrate ions and inorganic impurities); elongated cylindrical or ammonium fluoride (NH4F), we hypothesized that the organic compounds embedded in the B. cereus biosilica also play an important role in silica "kidney-shaped" aggregates which consist of thin plates (with the addition of formation. additional organic acids). Materials & methods Weddellite crystals had tetragonal dipyramid facetes (in systems with citrate A suspension of B. cereus spores was mixed with nitric and sulfuric acid and ions and inorganic impurities), and with the increase of Ca2+ concentration then boiled to degrade organic matter. Insoluble biosilica were harvested by (while C2O42- concentration was set to 3 mmol/l) a transition from ultracentrifugation and repeatedly washed with distilled water until the pH of dipyramidal to skeletal crystals was observed. In systems with Fe3+, Mg2+,

57 PO43- ions weddellite precipitated as real crystals with small tetrahonal P 89 dipyramid in the center of a large tetrahonal plate with an uneven edge. The Cyanobacteria-related carbonate sedimentation in modern formation of prism facets of weddellite crystals was observed only in the rivers, Leningrad region, Russia presence of citrate ions and MgSO4 in the solution. O. Rodina*1, O. Vereshchagin2, D. Vlasov1, M. Nikitin3, M. Zelenskaya1 The results contribute to the research of the oxalate patina formation and give 1Saint Petersburg State University, Biological, Saint Petersburg, Russian evidence on the specific patina components which influence the mineral Federation composition and the crystal morphology. 2Saint Petersburg State University, Institute of Earth Sciences, Saint The obtained results allow to recreate the original pattern of biofilm Petersburg, Russian Federation mineralization based on the morphology of calcium oxalate crystals found in 3The Bonch-Bruevich Saint-Petersburg State University of biofilms. Telecommunications, Institute of Military Education, Saint Petersburg, Acknowledgements: This work was supported by the Russian Science Russian Federation Foundation (no. 19-17-00141). The laboratory researches were carried out in the Research Resource Centers of Saint Petersburg State University: SEM investigations - in the "Resource Center Microscopy and Microanalysis Introduction (RСMM)" and "Interdisciplinary Resource Center for Nanotechnology" Today, cyanobacteria (CB) continue to play a central role in the carbon and "XRD measurements – in the «X-ray Diffraction Centre". nitrogen cycle. They are considered to be the pioneers of the colonization of mineral substrata. CB are not only biodegradation agents, but also they take P 88 important place in biomineralization of carbonate in fresh water. CB are Modifying the marble surfaceinfluenced bybacteria Bacillus common habitants of modern rivers riched in carbonates of the south of the spp. and bacterial-fungal associations Leningrad region, Russia. However, cyanobacteria-carbonates relation is still in focus of scientific debates. K. Sazanova*1, O. Frank-Kamenetskaya2, D. Vlasov1,2, A. Vlasov2, M. Objectives Zelenskaya2, A. Rusakov2 Minerals composition of carbonate sediments from modern rivers of the 1Komarov Botanical research institute RAS, laboratory of analytical south of the Leningrad region was studied. The CB diversity was described. phytochemistry, Saint-Patersburg, Russian Federation An experiment of the community growing in cumulative culture for studying 2Saint-Petersburg State University, Saint-Petersburg, Russian Federation cyanobacteria-carbonates relation was performed. Materials & methods Metabolism products secreted by the microbial lithobiont community are a Thirty one samples of layers (biofilms) from the stone surfaces were selected powerful factor in modern mineral formation. Most of the time rock surfaces in Leningrad region. Samples were dried for 3 month. X-ray diffraction in natural systems are inhabited by the multi-species communities (mainly patterns (XRD) were recorded on a Rigaku Miniflex II diffractometer. The fungi and bacteria). The biochemical activity of microorganisms in chemical composition and micromorphology was studied by means of an communities differs significantly from monocultures. The patterns of Hitachi S-3400 N scanning electron microscope equipped with an Oxford microbial crystallizationunder influence of several species of Instruments AzTec Energy X-Max 20. For the identification of CB, direct microorganisms are practically unexplored. microscopy was carried out after their settling in distilled water for a month. The aim of this study was to receive a mophogenetic patterns of calcium The cumulative culture of the microorganism community was grown in carbonates and oxalates crystallization on marble surface induced by distilled water for a year. Identification of species was carried out using light metabolism of bacteria and bacterial-fungal associationsin experimental microscopy (Leica DM 1000). conditions Results Two series of experiments were performed with microorganisms: 1- The mineral composition of carbonate deposits is represented by calcite and monoculture of bacteria Bacillus spp., 2 - co-culture of Bacillus subtilis and aragonite. The appearance of aragonite in the sediment is associated with an fungus Aspergillus niger. increased content of magnesium in calcite (MgO> 1 wt.%). CB recovered Cultivation of microorganisms was carried out in a liquid medium and in wet and started to grow in solution only from samples, which contain aragonite. chamber.Czapek-Dox medium with different glucose concentration (1, 5, 10, Twenty-nine taxa were identified in these samples. CB taxa are belonging to 30 g/l) was used as a nutrient medium. In the experiments in liquid medium 5 orders, 10 families, 17 genera. The most widely distributed species were marble blocks were put on the bottom of Petri dishes and 15 ml liquid from the genus Phormidium. Only Calothrix (C.) sp. from one sample has Czapek-Dox medium were added. The cultivation time: 14, 21, 30, 60 mucous cover with mineral crystals on it. This sample was used for further days. In experiments in wet chamber microorganism cell suspension was community growing in cumulative culture. The sample of the community applied on the marble block surface.The cultivation period for different with C. sp. started to be green after one week settling in distilled water and marble blocks was 21 and 90 days. after that, it grew at room temperature in a 100 ml glass beaker under a glass Scanning electron microscopy (SEM) and energy-dispersive X-ray cover (ventilated) for a year. After a year of cultivation, the C. sp. began to spectroscopy (EDXS) were used for the identification of phase composition dominate in the community, and it was almost completely covered with of crystallization products and examination of crystal morphology. The mineral particles. Result of XRD showed that Mg-calcite is main phase of determination of the metabolite composition was carried out in a liquid this cover. However, no aragonite was found. culture of microorganisms using gas chromatography-mass spectrometry Conclusion (GC-MS) by use of an Agilent mass spectrometer (MSD5975 mass selective The communities of CB associated with the deposition of carbonates in the detector), column HP-5MS, 30 m 9 0.25 mm. The analysis of the EPS content modern rivers of Leningrad region was described. The fact of the association in the liquid monoculture of Bacillus subtilis and in co-culture of Bacillus of mineral particles with mucous covers of C. sp. is confirmed by means of subtilis and Aspergillus niger was performed by precipitation in cold ethanol, optical and SEM microscopy, as well as by XRD. The natural samples of centrifugation and weighing. carbonate sediments with CB contain calcite and aragonite. However, only In a liquid medium with slightly alkaline pH values, the release of EPS by the mineral case of cyanobacteria grown in distilled water is composed of bacteria leads to crystallization of calcite, the intensity of which increases calcite. It is necessary to conduct further experiments for establishing the with increasing sugar content in the crystallization medium. In oligotrophic relationship between cyanobacteria and carbonates. The authors thank the conditions of a moist chamber the acidifying activity of Bacillus Resource Center for X-ray diffraction studies, Geomodel Resource Centre, subtilis prevales which leads to crystallization of calcium oxalate dehydrate Resource Center for Microscopy and Microanalysis (RСMM) of Saint (weddellite). The metabolic activity of B. subtilis and A. niger association is Petersburg State University for providing instrumental and computational vastly different from the activity of the separate monocultures. The metabolic resources. This work was supported by the Russian Science Foundation activity of micromycetes can suppress the formation of bacterial EPS and (project No. 19-17-00141). prevent the formation of calcite crust. In a liquid medium, as the sugar content increases, the acidifying activity of the fungus increases which leads P 90 to a shift of pH of the medium to the acidic region, carbonate crystallization Biodegradation of 2 - methoxyethanol by a new bacterium attenuation, and oxalate crystallization activation - the formation of calcium isolate Pseudomonas sp. strain VB under aerobic conditions oxalate monohydrate and dihydrate (whewellite and weddellite). F. O. ekhaise*1 The phase composition of microbial crystallization products is vastly 1University of Benin, Microbiology, Benin, Nigeria different in association of microorganisms from the activity of monocultures. In the case of Bacillus subtilis and co-culture of Bacillus subtilis-Aspergillus niger it determined by the ratio of the concentrations of EPS and oxalic acid Microbial biodegradation of 2-methoxyethanol also known as Methyl glycol in the crystallization medium. (MG) under anaerobic conditions has received much attention during the past This work was supported by the Russian Science Foundation (project No. 19- decade. However, not much is known about the aerobic degradation of 2- 17-00141) methoxyethanol. Samples from various environmental niches were enriched to isolate and determine bacterial isolates capable of utilizing 2- methoxyethanol as a sole source of carbon and energy under aerobic conditions. A 2-methoxyethanol degrading bacterium was isolated from anaerobic sludge of a municipal sewage from a treatment plant in Bayreuth,

58 Germany by selective enrichment techniques. The isolate was designated building materials industry are under extensive investigation and gradually strain VB after it was shown by the 16S rRNA phylogenetic sequence being disclosed. One of the known challenges for reaching an effective analysis as belonging to the genus Pseudomonas. Under aerobic conditions application of this kind of biotechnology in the concrete industry has been Pseudomonas sp. strain VB was capable of mineralizing 2-methoxyethanol the dramatical loss of cell growth and ureolytic activity under the extreme and its intermediary metabolites. Stoichiometrically, the strain utilized one alkaline environment compatible with concrete sustainability. Obtaining mole of oxygen per one mole of 2-methoxyethanol instead of four mole microorganisms naturally adapted to conditions of extreme alkalinity could oxygen per one mole of 2-methoxythanol for the total oxidative metabolism facilitate the adaptation process and improve the in situ mineralization performance on the concrete matrix. The aim of this study was to obtain and P 91 adapt novel microbial consortia, native from extreme ambient of Chile, with 2+ Formation of humboldtine-group oxalates Me (C2O4) 2H2O improved capacities to produce CaCO3 by biologically induced and (Me = Fe, Mn, Mg) under the influence of fungus Aspergillus controlled mineralization under extreme alkalinity conditions (pH > 12) and niger useful for in situ concrete repairing and improving. Materials and methods M. Zelenskaya*1, O. Frank-Kamenetskaya2, A. Izatulina2, A. Rusakov2, D. Ten microbial consortia with proven ability of ureolytic activity at laboratory Vlasov1 scale were obtained from different extreme alkaline lagoons (pH > 8.5), salt 1St. Petersburg State University, Department of Botany, Saint Petersburg, flats and soils from the Altiplano, Chile, and immediately characterized by Russian Federation massive sequencing of 16S-rRNA gene. Consortia were enriched in both 2St. Petersburg State University, Crystallography , Saint Petersburg, Tryptic Soy Broth (TSB) and specific ureolytic culture media. Enriched Russian Federation consortia were characterized again by DGGE. A gradual adaptation to extreme alkalinity conditions from pH 8.0 to 12.5 was performed for seven The interaction between the products of lichen metabolism and the rocks months. Urease activity, pH and cell growth were constantly monitored. One underlying the lichens leads to processes of modern mineral formation. The of the best representative species of CaCO3 biomineralizing microbes, present work describes the results of the laboratory experiments on the Sporosarcina pasteurii (DSM 33), was used as a positive control. Mixes of formation of humboldtine-group oxalates under the influence of the fungus the native consortia were performed in an attempt to increase the adaptation Aspergillus niger on the surface of todorokite (Ca,Sr)0.3- ability. Sand conglomeration and concrete repair ability for the best alkaline 0.5(Mn4+,Mn3+,Mg)6O12•3-4.5H2O, kutnohorite Ca (Mn,Mg,Fe)(CO3)2, adapted and most efficient mineralizer consortium were evaluated. Obtained siderite (Fe,Mg,Mn)CO3, as well as ankerite Ca(Fe,Mg)CO3. CaCO3 mineral was characterized by X-ray Diffraction and Scanning The experiments were held at room temperature in liquid Czapek-Dox Electron Microscopy. medium. The synthetic products were studied under scanning electron Results and Conclusions microscope, as well as by EDX analysis and X-Ray powder diffraction Natural consortia obtained from extreme alkalinity zones presented an methods. interesting microbial diversity and confirm the microbial adaptation to these Magnesium oxalate dihydrate (glushinskite) formed under the influence of extreme environments. Microbial consortia enriched with TSB medium was Aspergillus niger in the initial stages of the experiment with todorokite and more diverse than those enriched with ureolytic medium. Species that grow kutnohorite. The sequence of transformations of todorokite and kutnohorite in ureolytic specific medium are not necessarily present in TSB cultures or by fungus Aspergillus niger, leading to the formation of insoluble Mn2+ natural samples meaning that some ureolytic identified species are present in oxalates of different water content (mycogenic analogues of the minerals natural environments but with low abundance. New native strains falottaite and lindbergite), is different and depends on the valence of of Sporosarcina, Bacillus and Lysinibacillus species were obtained from manganese ions in the underlying mineral substrate. Due to the dissolution ureolytic cultures. All ureolytic consortia were active until pH 11.5 but only of todorokite by organic acids excreted by fungi, manganese ions (mostly two of them were able to actively growing and do hydrolyze urea at pH > 12. Mn3+ and Mn4+) get into the crystallization medium, where are then reduced Moreover, the mix of these two native microbial consortia achieved a higher to Mn2+ along with a gradual pH increase, which all leads to oxalate ureolytic and mineralizing performance under all the evaluated conditions crystallization. Kutnohorite on the other hand initially contains only Mn2+ showing an even better adaptive ability respect to each separate consortia and manganese ions. The formation of manganese oxalate here is preceded by the respect to control, S. pasteurii. According to the authors, this is the first work oxidation of Mn2+ ions to Mn3+,4+ during the initial stage of the experiment performing successfully biologically induced CaCO3 mineralization at the and the following crystallization of micogenic Mn-oxide (a todorokite extreme alkaline conditions analyzed, with a great perspective in the concrete analog). Under the action of fungi micogenic todorokite immediately begins repair application. to dissolve, and only after the reduction of manganese back to Mn2+ the oxalate crystallization begins. During todorokite transformation, manganese P 93 oxalate trihydrate (falottaite) appears in the medium first followed by Bacterial choreography- designing interactions through manganese oxalate dihydrate (lindbergite), which we can explain by biological induced mineralisation falottaite dehydration which feeds lindbergite crystallization. In the case of T. H. Arnardottir*1 the mycogenic transformation of kutnohorite, falottaite was not formed and 1Newcastle University, Architecture, Newcastle, United Kingdom the crystallization of lindbergite occurs without the participation of falottaite. We can assume that the formation of manganese oxalates of different water content is regulated by chemical compounds of their crystallization media, This research explores the potential design role of bacterial-induced compositions of which are substantially different for todorokite and biomineralisation. It sits within the speculations regarding our changing kutnohorite. relationship with nature through engineered biological systems and new Iron oxalate dihydrate (humboldtine) was first obtained in the experiments material processes. In the convergence of design and biological fabrication, with fungus Aspergillus niger on the surface of iron carbonates: siderite and the role of the designer is shifting from assuming the role of the sculptor to ankerite. It formed plate-like crystals, close in habit to glushinskite: on adopting the perspective of the cultivator in the study and production of new siderite surface it had an admixture of magnesium and manganese, on living material assemblages. In this intersection, we are moving away from ankerite surface - an admixture of magnesium. the mass production of fast, cheap and repetitive elements by enabling the Acknowledgements: This work was supported by the Russian Science production of biologically made materials. Much like properties of natural Foundation (no. 19-17-00141). The laboratory researches were carried out in materials such as bones, which are not designed but instead and are shaped the Research Resource Centers of Saint Petersburg State University: SEM by a set of natural constraints, biomineralisation has opened up the possibility investigations - in the "Resource Center Microscopy and Microanalysis of utilising nature"s intelligence for the built environment. Such process has (RСMM)" and XRD measurements – in the «X-ray Diffraction Centre». precise applications in the fabrication of materials as there has been extensive research on utilising bacteria to induce biological cementation through P 92 engineering solutions, such as crack repair in concrete, soils" improvement or forming energy-efficient bricks. By harnessing the biomineralisation Improved CaCO3 biomineralization under extreme alkalinity conditions of two native microbial consortia from extreme process, bacterial-induced fabrication can be used in the creation of new materials with functionally graded and variable properties. Chilean ecosystem 1 2 1 1 This research aims to explore material- and fabrication methods that S. Marín* , V. Zetola , S. Olivares , C. Demergasso* incorporate living cells as an inherent part of the process and to outline 1Universidad Católica del Norte, Biotechnology Center, Antofagasta, Chile 2 parameters that facilitate the synthesis of this biomaterial. It focuses on the Universidad Católica del Norte, Facultad de Ciencias de Ingeniería y assemblages of biologically fabricated matter through the interaction of Construcción, Antofagasta, Chile bacterial agents in a complex system, by challenging the designer"s thinking from the application of an ideal form on an inert matter, to the shaping of a Introduction bottom-up emerging form. The goal is to set out material and fabrication Biologically induced, controlled and influenced mineralization are three processes to enable designers to engage with these living systems as avowed microbe-mineral interactions with noted implication in ecology, resources. astrobiology, biogeochemistry and paleontology sciences. In the last decade, This research concentrates on structuring a biofabrication process whose the benefits of microbe-mineral interactions for the biocementation and purpose is to partly control the physical geometry of a microbial induced

59 mineralised structure. The experimental approach entailed an exploration of microbiologically induced calcium carbonate precipitation (MICP). Here the established procedures and the testing of novel physical apparatuses that carbonate formed in presence of the bacteria will react with calcium in the were part bioreactors and part moulding vessels. These enabled the surrounding, resulting in precipitation of CaCO3. In order to obtain a specific biomaterial synthesis through the alteration and control of chemical, spatial CaCO3–based structure using these strains, the bacteria have to be placed in and structural compositions of the environment. In this setting, the bacteria a shape of interest. In this study, Sporosarcina pasteurii bacteria, formerly Sporosarcina pasteurii was mixed in with sand in nutrient solution and known as Bacillus pasteurii, are embedded in bio-based polymer hydrogel grown overnight. This allowed the culture to situate itself within the grain matrices (e.g. sodium alginate) together with the appropriate nutrients. The before being flushed at intervals with the cementation media of nutrient hydrogel should possess certain properties, such as biocompatibility, broth, urea and CaCI2 over a few days. porosity and high water content, optimised for the survival of the bacteria The change in compositions led to a framework to reach biomineralisation at and production of mineral crystals. The bacteria have to be provided with given points and, by optimising the biofabrication process, obtaining fully molecules specific to the bacterial strain, as well as supplementary products, cemented pieces through the hybrid moulding/bioreactor technique. such as calcium sources, that are necessary for the mineralisation process. Based on these findings, this design framework can enable designers to The mineral formation is distinctively affected by different components visualise and generate interruptions and interactions with the mineralised added to the matrix and by the residing bacteria. Examining liquid media material. Designers can think of this process as an interactive, alive network cultures, this is observed as variations in S. pasteurii induced CaCO3 that can be used to choreograph parameters to produce form alongside polymorphisms upon addition of different calcium sources. Additionally the microbes. By utilising this induced material, the framework can demonstrate ratio and transformation from one polymorph to the other are investigated different ways the design paradigm can potentially shape living matter and over time using several analysis methods, e.g. XRD and SEM. Finally, the inherently evolve our relationship with biological design in new ways. bacteria are embedded in the hydrogel and are shown to also induce CaCO3 formation in this condition. P 94 The directing effect of bacterial EPS on calcite organization P 96 and EPS-carbonate composite aggregate formation Mineral deposition by lichens X. Yin*1, F. Weitzel1, E. Griesshaber1, C. Jiménez-López2, L. Fernández- F. Bachmair*1, G. Lehrberger2, A. Beck3, M. Zenkert1, E. Griesshaber1, Díaz3, A. Rodríguez-Navarro4, A. Ziegler5, W. W. Schmahl1 W. W. Schmahl1 1LMU Munich, Department of Earth and Environmental Sciences, Munich, 1Ludwig-Maximilians University Munich, Department of Earth and Germany Environmental Sciences , Munich, Germany 2Universidad de Granada, Departamento de Microbiología, Granada, 2Ingineurgeologie TU Munich, Munich, Germany Spain 3Botanical State Collection Munich, Munich, Germany 3Universidad Complutense de Madrid, Departamento de Mineralogía y Petrología, Madrid, Spain 4 Litchens deposit a variety of minerals, in particular, calcium oxalates in two Universidad de Granada, Departamento de Mineralogía y Petrología, hydration states: the monohydrate whewellite and the dehydrate weddelite. Granada, Spain 5 Litchens release oxalic acid that reacts with the substrate mineral. Hence, Universität Ulm, Zentrale Einrichtung Elektronenmikroskopie, Ulm, litchens etch and weather the surfaces of the rocks on which they grow. Germany Etching rock minerals results in the extracellular formation of new mineral deposits that most probably have no organic matrix associated with the Mineralized structures generated under biological control are hierarchical crystals. composites that consist of two distinct materials: a compliant biopolymer The purpose of our study is to identify and characterize those minerals that matrix that is reinforced by stiff and hard minerals. The biopolymer matrix are formed with the activity of litchens. In particular we aim to distinguish in the biological structural material is developed as a membrane or as a between grains that constitute the substrate and the crystals that are deposited network of fibrils and has structural as well as functional roles for the through litchen activity. We investigated Caloplaca falvescens litchens that architecture and material properties of the composite hard tissue. colonize limestone of the Jurassic Kehlheimer Kalkstein formation present Microbial cells surround themselves with a fibrous biopolymer matrix (EPS) in the surroundings of Regensburg, Germany. Mineral characterization was for protection, orientation of cells and enhancement of physiological performed with FE-SEM imaging, EBSD, EDX, XRD and FTIR. With the activities. For understanding the influence of biopolymer matrices on mineral applied combination of methods we detected both, chemical and organization and composite material formation we conducted growth morphological differences between minerals and grains forming the substrate experiments with the extracellular polymeric substance (EPS) of the gram and the minerals that were secreted by the litchens. While the limestone negative bacteria Pseudomonas putida. We synthesized EPS-agarose substrate is composed of large, irregularly oriented carbonate grains, hydrogel-calcite composite aggregates and characterized aggregate substrate portions where the litchens settled are infused with a multiude of morphologies, EPS/hydrogel distribution, mineral organization and co- minute holes containing organic substance. In the latter regions we find orientation strength. circular structures consisting of layered calcite, with the layers being We find that P. putida EPS exerts a tremendous influence on aggregate assembled of minute calcite crystallites. morphology, pattern of polymer distribution and mode of mineral In conclusion, the distinctness of grain morphologies and crystallite sizes organization. Contrasting to reference aggregates that are devoid of bacterial coupled with some chemical characteristics enabled the clear distinction EPS, in aggregates that occlude EPS the pattern of polymer distribution is between original sedimentary carbonate deposits and carbonate minerals highly inhomogenous and is developed mainly as membranes. Accordingly, formed as a mineralization product of litchens when colonizing and etching subunit formation in these is extensive. Subunits are irregular in shape, size the substrate mineral components. and distribution and are highly misoriented to each other. Aggregates that contain P. putida EPS are radial mosaic polycrystals, while the reference P 97 aggregates, devoid of EPS, are branched dendrites with the branches being Examination of the periodicity of incremental lines observed in highly stepped and the calcite well co-oriented. the otolith fossils of fishes from Nobori formation, Japan Incorporation of P. putida EPS into calcite changes the microstructure and H. Mishima*1, Y. Kondo2, F. Ohe3, Y. Miake4 texture of the mineral in a specific manner. This is a characteristic that can 1Tsurumi University School of Dental Medicine, Department of Dental be developed and used as a tool for the recognition and identification of Engineering, Yokohama, Japan bacterially mediated calcification. 2Kochi University, Science and Technology Unit, Natural Sciences Cluster, Kochi, Japan P 95 3Nara National Research Institute of Cultural Properties, Nara, Japan 4 Development of CaCO3 materials through bacteria embedded Tsurumi University School of Dental Medicine, Oral anatomy, Yokohama, hydrogels Japan R. Boons*1, G. Freitas1, G. Nyström1, A. Studart2, T. Zimmermann1 1EMPA, Dübendorf, Switzerland 2 Introduction ETH Zürich, Zürich, Switzerland The main component of the otolith in fishes is composed of a crystal of calcium carbonate (CaCO3). As compared with the teeth and bone composed Numerous types of mineral-based structures are used nowadays in a variety of apatite crystal, the otolith is a different hard tissue. However, the of applications, ranging from construction to medicine. One possible way to incremental lines in the tissue of the otolith are formed in the same manner create a mineral formed structure is through a biomineralization process, as the teeth and bones. It has reported that the periodicity of the incremental which is utilised in this research to create mineral-based products. lines of otolith has a daily (circadian rhythm), a tidal, a lunar, a seasonal, and Biomineralisation is a widespread phenomenon in several phylogenetic an annual periodicity (circannual) (Pannella, 1980). The ages of the fish were groups including bacteria that are able to control, influence and induce estimated through the annual incremental lines. Little information is mineralisation. Examples of the latter are certain Bacillus strains which are available regarding the ultrastructure, chemical composition and the known to induce calcium carbonate formation. This process is called periodicity of incremental lines of otolith fossils of fish.

60 Objectives P 99 The purpose of the present study is to examine the structure and composition The evolution of Theicideid brachiopods shell microstructure of incremental lines in the otolith fossils of fishes through the histological M. Simonet Roda*1, S. Milner2, E. Griesshaber1, H. Jurikova3, C. Rollion- and analytics studies. Bard2, L. Angiolini4, F. Ye4, A. Ziegler5, A. Bitner6, D. Henkel3, A. Materials and methods Einsenhauer3, W. W. Schmahl1 In this research, the otolith fossils of fishes (Nobori formation, Pliocene, 1Ludwig Maximilians Universität München, Department of Earth and Muroto, Kochi Prefecture, about 3 million years ago) were used. The ten Environmental Sciences, Munich, Germany otolith fossils samples were the different fish genera habitats and used the 2Institut de Physique de Globe de Paris, Department of Geochemistry and following samples (different fish genera). Cosmochemistry, Paris, France (1) 1) spp. indet., 2) Cetonurus noboriensis (Aoki and Baba), 3GEOMAR Helmholtz Centre for Ocean Research, Marine 3) Caelorinchus anatirostris Jordan et Gilbert, 4) Ventrifossa sp. A, 5) Biogeochemistry/Marine Systems, Kiel, Germany Coryhaenoides cinereus Gilbert. The habitat was continental shelf and slope 4University of Milan, Department of Earth Sciences "A. Desio", Milan, Italy at a depth of 200m or deeper. 5University of Ulm, Central Facility for Electron Microscopy, Ulm, (2) 1) Myctophidae spp. Indet., 2) Lobianchia gemellarii (Cocco), 3) Diaphus Germany gigas Gilbertt. The main habitats were the diurnal vertical movement. 6Polish Academy of Sciences, Institute of Palaeobiology, Warsaw, Poland (3) Sebates scythropus (Jordan and Starks). The habitat was continental shelf to upper slope (water depth 300m). (4) Congriscus megastomus (Günther). The habitat was continental shelf - The interpretation of geochemical proxies allows the reconstruction of past upper slope habitat. and present seawater chemical conditions and evolution with time. As proxy Polyester resin-embedded samples and non-embedded samples were used in data are obtained from geological archives, e.g. carbonate shells of marine this study. One-sided polish specimens and ground specimen were observed invertebrates, a substantial understanding of shell microstructures is of and analyzed using light microscopy, digital microscopy, polarizing immense importance. With that knowledge a better understanding and microscopy, scanning electron microscopy（SEM), SEM-EDS analysis, interpretation of geochemical proxy data is possible. electron-probe microanalyzer (EPMA), laser Raman microprobe Their abundance in the geological record together with the chemical and spectrometry, and X-ray diffraction method. structural stability of their low-magnesium calcite shells render brachiopods Results to be an important group within the invertebrates. Further, within the phylum The otolith crystals were aragonite by the X-ray diffraction method and the Brachiopoda, the investigation of thecideidine shell morphology and laser Raman microprobe spectrometry. The inside of the otolith was found to structure, histological characteristics of the mantle and metabolism in general consist of needle-like crystals crossed by incremental lines. The circadian has been the subject of research for several decades and revealed the incremental lines (2-5 μm), and several long periods of incremental lines distinctiveness of this group of brachiopods relative to species of other (tidal rhythm: about 14 days interval, monthly rhythm: about 28 days extinct and modern brachiopod genera (e.g. Pajaud, 1970, Grant, 1972, interval, circannual rhythm) were observed in the otolith. By SEM images, Williams, 1972). Previous work suggests a progressive loss of the fibrous the incremental lines were observed as the dark bands. The incremental lines shell layer and the development of a "granular" primary layer, a process that had content of C, O, and Ca, and additionally contained Si as a trace element. started in the Jurassic and carries on until now. Comparing FE-SEM, EBSD Both Lobianchia gemellarii and Diaphus gigas were unclear in the circadian and AFM results, on shell microstructure and texture, between modern incremental lines. Their habits were diurnal vertical movement. By rhynchonellide, terebratulide and thecideide brachiopod shells show that comparison, Ventrifossa sp., Cetonurus nobonesis, Sebates scythropus, and mineral unit organization in modern thecideide brachiopod shells is entirely Congriscus megastomus were evident in the circadian incremental lines. different than the one present in the other groups. The study was completed Their habitats were the slope from the continental shelf. with stable isotope and element analyses. Conclusion The objective of this study is to discuss in detail patterns of shell It is possible that the periodicity of the incremental lines of otolith changed microstructure of fossil and modern representatives of theicideid brachiopods with the ecosystems. and trace the evolution of shell organization and microstructure development from the Triassic to the present time. Instead of the clear distinction between

P 98 an outer primary and an inward fibrous shell layer and the presence of stacks of fibers, as it is the case in modern rhynchonellide and terebratulide Misorientation driven morphological evolution of the prismatic brachiopod shells, we do not find any obvious mineral unit organization in ultrastructure in mollusc shells the shell of modern thecideides. The presence of fibers is clearly visible for 1 1 1 D. Stier* , D. Zöllner , I. Zlotnikov the Triassic and Jurassic specimens. It disappears subsequently, and from the 1 TU Dresden, B CUBE - Center for Molecular Bioengineering, Dresden, Cretaceous, to modern times granular microstructures prevail. The latter Germany consist of highly irregularly shaped mineral units with calcite being assembled with a low degree of co-orientation. Larger calcite granules, Molluscs are a well-established model system to study biomineralization and acicular crystallites, and pseudo-polygonal mineral units are embedded in a the process of biomineral morphogenesis. In addition to our growing matrix of small crystallites. understanding of the different biochemical mechanisms that are responsible In modern species as Pajaudina atlantica and Kakanuiella chathamensis we for mineral formation, a number of recent studies suggest that the deposition observe within the shell an extracellular organic matrix; however, this of the various shell ultrastructures is a thermodynamically driven self- biopolymer matrix does not have a regular structure such as that of organic assembly process. Specifically, it was shown that the formation of the sheaths encasing the fibers. We consider the granular microstructure of prismatic ultrastructure, consisting of elongated mineral columns glued thecideide shells as a recently evolved feature, followed its development with together by an interprismatic organic membrane, can be quantified by time, and discuss its interlinkage with the fibrous fabric from a classical models of grain growth and coarsening. In this model, microstructural point of view. morphological evolution of the entire prismatic ultrastructure, which Grant R. E. 1972. The lophophore and feeding mechanism of the proceeds parallel to the long axis of the prisms, is driven by the reduction of Productidina (Brachiopoda). Journal of Paleontology, 46, 213- 249. the amount of interfaces that separate the individual prisms. However, so far, Pajaud D. 1970. Monographies des Thecidees (Brachiopodes). Memoire the energy of those interfaces was considered to be uniform. Therefore, the Societe Geologique Francaise, 49, 112, 1-349. influence of the crystallographic misorientation between adjacent prisms on Williams A. 1972. The secretion and structural evolution of the shell of the interface energy and thus, the morphogenesis of the corresponding Thecideidine brachiopods. Phil. Trans. Roy. Soc. B, 439-478. prismatic units, was never previously considered. In the current study, we examine the prismatic ultrastructure in three mollusc species from the genus P 100 Pinctada. In these bivalves, the prismatic assemblies not only demonstrate a Pineal gland calcification under hypoxic conditions coarsening behavior, which is predicted by the recently developed self- M. Kopani*1 Bronislava Vraníková1, Daniel Kosnáč1, Michal Zeman2, assembly models, but also exhibit gradual changes in the crystallographic Vladimír Šišovský3 orientation of the individual prisms. Using electron backscatter diffraction *1Comenius University, Institute of Pathological Anatomy, Faculty of analysis (EBSD) and synchrotron-based X-Ray tomography to follow the Medicine, Bratislava, Slovakia crystallographic properties and the shape of the prisms at different stages of 1 Institute of Medical Physics, Biophysics, Informatics and Telemedicine, growth, respectively, we show a correlation between the textural and Faculty of Medicine, Comenius University, Bratislava, Slovakia morphological evolution of the prismatic ultrastructures. In fact, we 2 Department of Animal Physiology and Ethology, Faculty of Natural demonstrate the key role of misorientation between neighboring mineral Science, Comenius University, Bratislava, Slovakia building blocks in the growth kinetics of biocomposite architectures. 3 Institute of Pathological Anatomy, Faculty of Medicine, Comenius University, Bratislava, Slovakia

Introduction The pineal gland (glandula pinealis) is neuroendocrine gland secreting melatonin. This hormone is involved in many physiological processes

61 including regulations/controls the circadian rhythm, body temperature, XRD, particle size distribution measurement, ion chromatography, calcium immunity and antioxidant activity. sensitive electrode, ultra-sonic wave measurement and heat flow calorimetry. Objectives The different techniques are needed to understand the process in detail. The aim of this study is investigated the effect of hypoxia on the occurrence The influence of betain on the crystallisation of portlandite was checked in a of pineal gland calcification. Distribution of calcerous material by light (LM) crystallisation experiment. Portlandite was produced from CaCl2 and NaOH and transmission electron microscopy (TEM) was investigated. Chemical during a diffusion experiment with and without betain. The formed crystals composition of foreign material by scanning electron microscopy with without betain are needle-like with a hexagonal shaped base. With betain energy-dispersive microanalysis (SEM-EDX) was done. Melatonin thinner layers of those crystals grew together in different steps turned to each concentrations in blood plasma by direct radioimmunoassay were measured. other. The crystals morphology was investigated by ESEM and the sorption Materials & methods of betain by RAMAN spectroscopy. The used techniques can also be applied The experiments were performed on 24 adult male Wistar rats. First group to understand the crystal growth of different crystals in the presence of was exposed to prenatal hypoxia for 12 h at 20th day of development (n = 9), organic additives. The influence of different organic substances which can second group to prenatal hypoxia for 2x8 h at 19th and 20th days of be found in bio-minerals should be investigated in future. development (n = 5) and third group was control group (n = 10). All procedures were conducted in accordance with the Declaration of Helsinki. P 102 Results Reading between the lines in biomineralizations reaching out Scattered vacuoles and large focal calcium-rich particles in the pineal gland for clues of environmental impact - case reports from studies and at the surface of pineal gland by LM were observed. The size of particles based on analytic spectroscopy, isotope geochemistry and is around 3 µm, sporadically up to 30 µm. SEM-EDX reveals that they proteomics methods consist of Ca, P, Na, S and Fe. TEM reveals vacuoles in the cytoplasm of 1 1 1 1 1 pinealocytes type I filled with both flocculent and fibrous material in 12h S. Berland* , J. Arivalagan , E. Feunteun , A. Bartolini , C. Thaler , A. Marie1 hypoxia group. No significant differences in melatonin concentration among 1 groups were found. MNHN, Adaptation du Vivant, Paris, France Conclusion Pineal gland consists of pinealocytes type 1 and type 2. Their function is Living organisms and mineral collectively speak in a rich and ancient story. secretion of melatonin. It was observed that the cytoplasm of pinealocytes Along evolution, complex biomineralized structures have appeared in a range contains vacuoles filled with flocculent and fibrous material. Welsh (1984) of species. These structures develop during ontogenesis under the control of showed that vacuoles play important role in calcification process. Our TEM metabolism. The morphology and chemical composition of these structures results confirmed the presence of flocculent and fibrous material in the are known to be shaped by environmental (external) factors under the limits vacuoles. Our previous study revealed that calcerous material was mainly of functional outcomes. Here we present cases that illustrate calcium amorphous with the presence of calcite, aragonite and vaterite (nano)crystals carbonate biomineralisation responses to environmental factors, with special in the samples (Tofail et al. 2019). It is suggested that mineralization process emphasis on technological investigations range, limits and stumbling blocks. of pineal gland is similar like bone mineralization and hypoxia favors Calcareous tests of foraminifera were investigated. Among biomineralizing calcification under hypoxic conditions (Tan et al. 2018). Physical properties organisms, foraminifera (unicellular eukaryotes) have been producing (pyro-, piezo- and ferroelectricity) of crystals may play the important role in carbonates since geological time. Their tests (or shells) of calcium carbonate the function of pineal gland. trace environmental signals, such as ocean temperature, oxygen level, References contamination, salinity, etc. Our approach was to investigate the molecular TAN DX, CHEN LD, POEGGELER BEA. Melatonin: a potent, endogenous and cellular machinery of biocalcification based on a benthic calcitic hydroxyl radical scavenger. Endocr J 1: 57– 60, 1993. formainifera, protozoan unicellular model. Coupling geochemical and TOFAIL SAM, MOURAS R, McNAMARA K, PATYK-KAZMIERCZAK biological perspectives on foraminifera will enhance interpretation of the E, GEANEY H, ZAWOROTKO M, RYAN KM, SOULIMANE T, SILIEN proxies used for environmental and climatic reconstructions. C, KOPÁNI M. Multimodal surface analyses of chemistry and structure of The second case report arise from the Sub-Antarctic islands, in which severe biominerals in rodent pineal gland concretions. Appl Surf Sci. 469: 378-386, environmental conditions drive discrete ecosystems at meso-scale level. In 2019 the native land snail Notodiscus hookeri specific populations have evolved WELSH MG. Cytochemical analysis of calcium distribution in the into two ecophenotypes suited with shell either mineral-rich or organic-rich superficial pineal gland of the Mongolian gerbil. J Pineal Res. 1: 305-316, flexible shells. The soil environment and especially the availability of 1984. exchangeable calcium was the driving factor for ecotypes. Several methodologies were applied: scanning electron microscopy of the shell, X- P 101 Ray diffraction analysis of the soil, Solid-state Nuclear Magnetic Resonance Influence of organic additives on the crystallisation of gypsum and shell proteomics. This model enables to follow up the time over which and portlandite such adaptive response may have occurred towards phenotypic radiation. C. Pritzel*1,2 In the context of global change, Baltic Sea was considered as a workshop 1Building and Materials Chemistry University of Siegen, Chemistry, Siegen, area to study how native bivalve molluscs (Mytilus sp) can form shells under Germany lower calcium carbonate saturation states. A proteomic approach was 2University of Siegen, Chemistry, Siegen, Germany performed on the shells to learn about the composition of the nested matrix proteins, the local conductor of biomineralization. Result have shown patterns of modulation in shell proteins which could be correlated to different Gypsum (CaSO4*2H2O) and portlandite (Ca(OH)2) are used in inorganic shell phenotypes in relationship with shell capacity to offer protection to the binding materials, in those materials organic additives are used to influence individuals. technical properties. Most of the used additives are influencing the Finally stable isotopes geochemistry (oxygen and carbon isotopes) in otolith morphology of the formed crystals. This influence of organic additives on of glass eels (post larvae of European eels) was used with nanoscale mass the morphology of gypsum or portlandite was tested in crystal growth spectrometry techniques (NanoSIMs) to track changes of the environment experiments. Gypsum was prepared from Na2SO4 and CaCl2 or from along the early life history of this diadromous teleost fishes. The otolith calcium sulphate hemihydrate with and without different additives like betain contains visible morphological checks that indicate life cycle key event, e.g (used as foaming agent), fruit acids (used as retarder), poly carboxylate ethers hatching, first feeding, and metamorphosis. Minute differences in oxygen (used as superplasticizers) for example. The different additives influence the isotopic ratios of oxygen (d18O) in otolith allow addressing the changes in crystals morphology, because they are occupying different crystals surfaces temperature encountered by the larvae along their early life history. preferred. In case of citric acid the crystals morphology is changed from long Therefore, these analyses provide environmental information to trace needle like crystals with a large aspect ratio and having more ramifications spawning places and dissemination routes of the larvae from the with the lack of acid to shorter, narrower crystals and less branches. The spawning places to the estuaries of Europe. smaller diameter of the crystals can be explained because the c-axis is firstly At the scale of the individual, community or species, biominerals stand as a covered by citric acid and as the consequence the fastest growing axis grows repository of environmental information alongside with evolution much slower. Owing to the collection the Ca2+ ions from the crystallization arrangements from lineage or acquisition. Enclosed time-frame range for the solution leads to the lower ratio Ca/SO4 and the fewer branches can be process to apply needs to be looked for now. observed. These phenomena caused the first forming SO4-backbone which References results in the fewer branches in the crystals afterward. The citric acid Sabbatini A., Bédouet L., Marie A., Bartolini A., Landemarre L. , Weber M. adsorbed on the c-axis was shown by Raman-microscopy and AFM. The X., Gusti Ngurah Mahardika I., Berland S., Zito F., Vénec-Peyré M.T. (2014) calcium sorption was proved by Ca-ion selective electrode. The changed Biomineralization Of Schlumbergerella Floresiana, A Significant Carbonate- morphology of the created gypsum crystals is decreasing the strength of the Producing Benthic Foraminifer. Geobiology: 12, 289-307 formed gypsum stone and increasing the forming accuracy. It was found that Charrier M, Marie A, Guillaume D, Bédouet L, Le Lannic J, Roiland C., the Polycarboxylate ethers are influencing the crystal morphology in the Berland S., Pierre J.S. , Le Floch M. , Frenot Y., Lebouvier M. (2013) Soil same way. The crystallisation of gypsum with and without additives from Calcium Availability Influences Shell Ecophenotype Formation in the Sub- hemihydrate was investigated with in-situ optical microscopy, ESEM, in-situ Antarctic Land Snail, Notodiscus hookeri. PLoS ONE 8(12): e84527.

62 Arivalagan, J., Yarra, T., Marie, B., Sleight, V. A., Duvernois-Berthet, E., and the origin of biominerals under extreme conditions (high pressure and Clark, M., Berland S. (2016). Insights from the shell proteome: salinity, low temperatures, high content of hydrogen sulfide and methane, biomineralization to adaptation. Molecular Biology and Evolution, vol.34, etc.) in the aquatic geosystems. pp. 66-77 Biominerals were studied during scientific surveys in the Arctic and the Antarctic, as well as the Caspian, the Black and the Aral seas. Biominerals P 103 in ferromanganese nodules of the Clarion-Clipperton Fracture Zone (Pacific Role of biogenic carbonates in the sediment balance of Ocean) were also studied [1]. The sampling of biominerals was carried out accumulative beaches of the Anapa Bay Bar (the Black Sea) from the ice cover, water column, bottom and coastal sediments; temperature A. Kosyan*1 and salinity of the water column were measured in situ. Laboratory research 1A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of included light and scanning electron microscopy, electron probe Sciences, Laboratory of morphology and ecology of marine invertebrates, microanalysis, X-ray diffraction, geochemical analyses, Moscow, Russian Federation micropaleontological and microbiological studies. It was found out that specific associations of authigenic biominerals are formed as a result of biogenic and abiogenic interactions in each studied Question geosystem. However, some biominerals, such as opal, framboidal pyrite (FP) Sandy beaches of the Anapa Bay Bar are unique nature landscapes, and some Fe-Mn oxyhydroxides behave like «cosmopolitans». Opal is possessing great esthetic and recreational value, as well as essential formed under different extreme conditions and in different mineral significance for the economy of the Krasnodar region (Russia). The stability associations, including cryogenic and chemogenic minerals. Thus, opal of the beaches is determined by balance and dynamics of sediments. Biogenic frustules of diatoms are formed in the ice cover and under-ice water of the sediments (carbonates) are provided by populations of coastal shelly Central Arctic Ocean [2]. In the hypersaline residual basins of the Aral Sea mollusks, mainly bivalves. In some sites, the share of biogenic carbonates (the Lake Tshchebas and the Chernyshev Bay) with a high content of reaches 90%. hydrogen sulfide and methane [3], opal is actively generated in the frustules Within the framework of the long-scale monitoring of the Anapa Bay Bar, of euryhaline diatoms in the water mass and involved in the structure of having been conducted by the researchers from IO RAS and IPEE RAS and coastal microcrystalline cortical deposits composed of chemogenic crystals aimed to complex studying of lytho- and hydrodynamic processes, we of konyaite, thenardite, blödite, eugsterite, halite, and gypsum. assessed the quantitative role of mollusks in the sediment balance of the Bar. FP was found in bottom sediments of the areas under the variety of extreme Methods factors, such as low temperatures, high concentrations of hydrogen sulfide We studied samples of benthic mollusks at five sections with stations at 2, 6 and methane [2, 4-6]. Influence of the hydrological and biogeochemical and 10 m depths (deeper mollusks cannot be transferred to the shore even by conditions on the depth of formation, size, morphology, and structure of FP, stormy waves), taken in 2016-2018. Selected mollusks were counted and as well as the type of mineral/organic substrate, was determined for each measured. The age was determined by the lines of shell external growth. studied area. Determination of carbonate and mineral components of the sand samples was «Endemical» biominerals, such as discovered vivianite and polycrystalline carried out using 30% hydrochloric acid solution. Mg-calcite are formed in very specific conditions during the diagenetic Results transformation of organic and mineral matter. Thus, in association with The productivity of the two most widespread and abundant in the area species aragonite and FP, Mg-calcite forms microcrystalline cement of the carbonate of bivalves Chamelea gallina and Donax trunculus is not the same in concretions at a cold methane seep site in the Laptev Sea [6]. different years at different sections and depths. The contribution of C. The results of the study can be used in the interpretation of the oceanographic gallina shells to the total carbonate sediments at depths of its mass data, paleogeographic reconstructions, environmental monitoring, mining of development 6-10 m was 327.2 g/m2 in 2016, 127.1 g/m2 in 2017, and 179.3 the oceanic ore deposits and development of new biocomposite materials. g/m2 in 2018; the contribution of D. trunculus shells at depths 2-10 m was Acknowledgments. The work has been realized in the framework of the state 123.7, 35.9 and 31.3 g/m2, respectively. Gastropods Rapana venosa had an assignment (theme № 0149-2019-0004). order less number and biomass than bivalves, the biomass of other mollusk References species was negligibly low. Carbonates content in the bottom sediments [1] Reykhard, L.Ye., Shulga, N.А. (2019). Ore Geology Reviews. Vol. 110. varied from 3 to 30 % (average 11%). An increase in carbonates in bottom 102933 sediments was not directly related to an increase in biomass of living [2] Reykhard, L.Ye. et al. (2018) Acta Cryst. A74, e250 mollusks on the same depths (r = -0.22). Thus, the distribution of carbonates [3] Izhitskaya, E.S. et al. (2019). Environmental Research Letters. ERL- is rather explained by the movement of sediments, than the distribution of 105929.R2 live mollusks at the bottom. The data on the size-age structure of the mollusk [4] Novichkova, Ye.A. et al. (2017). Doklady Akademii Nauk, Vol. 474, No. population and earlier observations (Kosyan, 2016) show that the vast 3, pp. 365–369 majority of C. gallina within the Anapa Bay-bar (60%) die as a result of [5] Kozina, N.V. et al. (2018). Russ. J. Earth. Sci., 18, ES6003 rapana predation at an age of 2-3 years; most D. trunculus die at an age of [6] Kravchishina, M.D. et al. (2017). Oceanology. V. 57. No. 1. P. 174–191 one year (94% of the population). The width of the zone of mass development of C. gallina at depths of 6–10 m is approximately 400 m, the width of such P 105 zone of D. trunculus (2–10 m) is 900 m. Taking all above mentioned into Biomineralization plasticity can maintain mechanical stability account, we may calculate the annual income of the carbonates of biogenic of scallop shells exposed to ocean acidification and warming origin on the studied 45 km long site of the Anapa Bay Bar: 7834.5, 2227.5, N. Lagos*1, A. Rodriguez-Navarro2, J. Vivanco3, C. Garcia4, C. Duarte5, M. and 4257 tons in 2016, 2017 and 2018 respectively, or on average 4773 tons Lardies6 per year. There are about 1.0 to 4.0 million tons of sediments in constant 1Universidad Santo Tomas, Centro de Investigación e Innovación para el motion over the underwater coastal slope, thus restoring of the beaches by Cambio Climático, Santiago, Chile means of shelly material amounts from 0.1 to 0.5%. 2Universidad de Granada, Petrology and Mineralogy, Granada, Spain Conclusions 3Universidad Adolfo Ibañez, Bioingenieria, Viña del Mar, Chile The annual contribution of shelly material to the total sediment balance of 4Universidad de Santiago de Chile, Ingenieria Mecanica, Santiago, Chile the Anapa Bay Bar is on average 4773 tons per year which comprises from 5Universidad Andres Bello, Dept. Ecología y Biodiversidad, Santiago, 0.1 to 0.5% of total amount of moving sediments. This indicates the Chile importance of taking measures to protect and restore the population of 6Universidad Adolfo Ibañez, Fac. Ingeniería y Ciencias, Santiago, Chile mollusks living on the underwater coastal slope. The work was supported by RFBR grant No. 19-45-230001. Ocean acidification (OA) is projected to impact the physiology and shell P 104 carbonate precipitation in mollusks. However, warming may confer Biominerals in aquatic geosystems under extreme conditions resistance to these impacts, and mollusks may trade-off growth and L. Reykhard*1, N. Shulga1, N. Kozina1, Y. Novichkova1, O. Dara1, A. calcification for maintain shell functionality under the influence of climate Boev1, P. Sapozhnikov1, A. Izhitskiy1, N. Belyaev1, V. Gordeev1, O. stressors. In this study, we test this hypothesis by assessing the organic Kalinina1, A. Reikhard2 composition, crystallography and mechanical properties in shells of 1Shirshov Institute of Oceanology, Russian Academy of Sciences, Moscow, Argopecten purpuratus juvenile scallops exposed to increased temperature Russian Federation and pCO2-driven ocean acidification. Shell organic matter (%, TGA) 2State Secondary General School № 2086, Moscow, Russian Federation increased at cold (14°C) and acidified (7.7) seawater condition. Discriminant function analysis indicates that organic composition of the shells shown systematic differences among treatments, with high levels of success (93%) Authigenic biominerals and their associations with minerals of other origin in reclassifiyng the individuals to the combination of are used as indicators of the geological, hydrological, and ecological temperature/acidification treatment at which were exposed based on their characteristics of ancient and modern aquatic geosystems. Extreme organic composition data (ATR-FTIR). Amide and carbonate groups showed environments of biomineralization lead to the appearance of specific significant increments under warming (18ºC) and acidification conditions, structural and chemical features of minerals that must be considered when while sulphates and polysaccharides decreased under the same conditions. using the biomineral indicators. This work aimed to identify the properties

63 Crystallographic orientation (XRD) of mineral phase was also variable, P 107 showing a significant reduction in the angular spread at the Fluorescence lifetime- sensor components warming/acidification treatment. In spite these changes in the biopolymer I. Tobehn-Steinhäuser*1, A. Winzer1, C. Möller1, C. Heinze1, H. G. and mineral phases of the shells of A. purpuratus, the structural resistance Ortlepp1, T. Ortlepp1 (elastic module) do no was affected, but a significant reduction in 1CiS Forschungsinstitut für Mikrosensorik, Design, Erfurt, Germany microhardness was recorded in individuals shell exposed to cold/acidification conditions. SEM and micro-CT observations indicate that exposure to these conditions promotes the erosion of the shell periostracum Introduction and dissolution of microstructures associated to shell ribs of the outer surface. Determination of fluorescence lifetime is a key tool in the investigation of Our results suggest that plasticity in both organic composition as mineral cell metabolism and cell growth. The time decay of the fluorescence of phases of the A. purpuratus shells could be a general compensatory functionalized dyes facilitates the understanding for instance of intracellular mechanism to confront climate stressors in order to maintain the shell macro- temperature fluctuations originating from chemical processes within the structure functionality, but micro-scale dissolution and lesser hardness may cells. compromise further ecological functions and the overall adaptive response. Objectives For the development of our sensor components we establish the following

P 106 key requirements: Impedance-spectroscopy in biology and health-science  The excitation of the fluorescent dyes must be done with light I. Tobehn-Steinhäuser*1, A. Winzer1, T. Frank1, S. Herbst1, S. Görland1, T. pulses as short as 1 nanosecond. And the excitation wavelength Ortlepp1 must be about 370 nm, i.e. suitable functionality confirmation 1CiS Forschungsinstitut für Mikrosensorik, Design, Erfurt, Germany with a dye.  Our design of the optics module makes it possible to measure an array of probe chambers in titer plates in parallel. Therefore the Introduction We are partners for industrial research and development of silicon-based width must not exceed 8 mm. sensors. The main areas of focus are MEMS and MOEMS with highest  As detectors silicon photomultipliers (SiPM) are used which also stability and reliability and solutions for special photonic detectors and enable for miniaturization and thus for parallel measurements. detector arrays, as well as radiation and particle detectors. You will receive  An application-specific integrated circuit (ASIC) allows for a fast customized solutions for your sensor systems as well as production in small and accurate collection of the fluorescence decay statistics. series and the qualification of reliability and service life. Materials & methods The contributions show the possibilities of impedance spectroscopy (IS) and The optical behavior of the lighting module was simulated by a ray tracing fluorescence in the field of microsystems technology with regard to software tool (ZEMAX). For this purpose, a model of the UV 370nm LED biological and medical applications. was established and adjusted to the measured LED radiance. For beam Objectives shaping, UV antireflection-coated quartz lenses were used. The mounting of With the help of IS three projects were worked on. The first involved the the optical elements (lenses, filters and aperture) was done with 3D printed online determination of the diclofenac concentration in wastewater without brass housing. As excitation filter a commercial bandpass filter is used. major laboratory work (BioSam). Another project was the online detection A UV enhanced photodiode was integrated on the pulse excitation of mastitis pathogens in cow's milk directly at the milking process (Mastitis). component for monitoring the radiated power of the LED. In addition, a project was conducted to enable the testing of new products for For the fast determination of the fluorescence lifetime, we designed an ASIC biocompatibility using IS and mouse fibroblasts (3DCellSens). for the SiPM detectors. Materials & methods The functionality of the excitation module as well as the detection module is To detect toxins, simple biological systems such as yeast cells are suitable demonstrated with the fluorescence dye ATTO 390. (BioSam). These can be genetically modified so that they emit fluorescent Results light when certain substances appear. To properly quantify this detectable The possibility of generating nanosecond pulses with a LED is known in the light, the number of yeast cells must be known. For this purpose one uses in literature. However, the existing concepts need 200 to 400 V as supply this case the IS. In order to be able to combine both sensor principles, voltage for the avalanche transistors used in the circuitry. Our solution only interdigital structures made of ITO were realized on glass substrate, so that needs 9 V of supply voltage, which is of benefit for assembling a compact the light can be detected simultaneously throw the optically transparent array of such sensors. The design allows a pitch of the PCB of 8 mm. structure. The generated light pulse of the LED was experimentally determined via a In the project Mastitis the pathogens should be detected directly at the time-correlated single photon counting (TCSPC) measuring. The FWHM of milking process. without too much laboratory infrastructure. The structure the LED pulse determined from the measurement is around 1 ns. consists of different variants of a switchable electrode array. It is possible to The functional test of the excitation component yielded a time constant of interconnect random patterns to adapt the electric field to the measuring task τ = 4.6 ± 0.3 ns for the used fluorescent dye ATTO 390. This value agrees (cell geometry). The switchable electrodes enable a fast changeover of the very well with τ = 5.0 ns given by the manufacturer. electrode geometry. This makes it possible on the one hand to hide the The ASIC performance was determined and improved by simulations. Which properties of the cells from the measurement signal (small electrode suggest a high suitability for fluorescent lifetime measurements. Key distances) and, on the other hand, to measure the polarization of the cells parameter is a time measurement accuracy of 80 ps and a measurement time directly (interconnection to electrode blocks). of up to 20 ns. From the behavior of certain biological cell cultures on different Conclusion environmental influences and substances, conclusions can be drawn and on An illumination unit for fluorescence lifetime determination operates at a the interactions in other applications. An important task here is the wavelength of 370 nm and has a pulse width of around 1 ns. The width determination of the biocompatibility of materials. The given new method (8 mm) of the entire module is suitable for parallel measurements on titer allows the evaluation of biocompatibility already during the experiment. The plates. As a confirmation example, the fluorescence lifetime of the dye measuring device consists of a sensor with evaluation electronics and records ATTO 390 was determined. Further, an ASIC for the TCSPC measurement the data by means of IS. The basis for this is formed by adhesion coupling with a SiPM was developed. The ASIC specifications as well as the first real between the cells and the sensor. Vital cells behave like an insulator in the test results are discussed. nutrient medium, which binds to the cell carrier through adhesion. Upon the onset of cell death, the cell membrane breaks and, as a result, the adhesive contacts disapear. The cell no longer acts as an insulator. As a result the impedance changes. 4. Results Fully functional samples or prototypes could be produced in all three projects. The results will be shown during the conference. Conclusion IS, also in combination with other measuring methods, is one powerful method to caracterise biological and medical systems. With our contribution, we may be able to show a way to solve an existing measurement task.

64 P 108 Extracellular vesicles involved in the guinea fowl eggshell quantitative proteomics yield new findings related to its unique structure N. Le Roy1, L. Combes-Soia2, V. Labas2, A. Rodriguez-Navarro3, M. Hincke3,4, Y. Nys1, J. Gautron*1 1French National Institute of Agricultural Research (INRA), Bird Biology and Poultry, Nouzilly, France 2French National Institute of Agricultural Research, Physiology of Reproduction and behaviour, Nouzilly, France 3Universitad of Granada, Departemento de mineralogia y petrologica, Granada, Spain 4University of Ottawa, Department of cellular and molecular medicine, Ottawa, Canada

Question The Guinea fowl (Numida meleagris) eggshell is a bioceramic material with the remarkable mechanical property of being twice as strong as the chicken eggshell. In both species, the eggshell is composed of 95% mineral in the form of calcite polymorph and 3.5% organic matrix including shell membranes. However, the eggshell ultrastructure and microstructure are significantly different between these species. In the chicken, the eggshell is made of columnar calcite crystal units arranged vertically. In the Guinea fowl, the same crystal architecture is observed in its inner half, followed by a dramatic change in crystal size and orientation. The unique ultrastructural characteristics of Guinea fowl eggshell confer a superior resistance to breakage compared to eggshells from other bird species. In order to understand the underlying mechanisms controlling the formation and structural organization of this highly resistant material at five key stages of the mineralization process (4 h, 10 h, 11 h, 12 h and 18 h post-ovulation). Methods FormingGuinea fowl Eggs were sampled at five stages: 4 h (n=5), 10 h (n=6), 11 h (n=6), 12 h (n=6) and 18 h (n=6) post-ovulation (p.o., i.e. time after the previous egg) when the nucleation sites appear and early mineralization starts (4 h p.o.), or just before (10 h p.o.), during (11-12 h p.o.) and after the shift in crystal orientation (18 h p.o.). The organic matrix was extracted from each eggshell and analyzed by a bottom-up quantitative proteomics approach. We the used statistical and bioinformatics tools to determine the protein related to the main structural shift and their functiuons. Results The present work is the first Guinea fowl eggshell proteomic study, which allowed the identification of 149 proteins. Comparison of the Guinea fowl eggshell proteome with that of other bird species leads to the identification of 9 proteins that are only present in Guinea fowl. Among them Protein S100- A6 and GDF6 are notable as they exhibit potential functions related to shell mineralization and especially at the point of the modification in crystal size and orientation that confers remarkable strength on the Guinea fowl eggshell. In addition to these two proteins, we also report that 61 proteins are more abundant during the secondary nucleation events associated with the change in crystal orientation and the formation of the new layer. Additionally, we identify the most abundant proteins involved in the different phases of Guinea fowl shell formation, from the first events of biomineralization until the deposition of the new layer. Our study showed that proteins associated with early mineralization are similar in chicken and Guinea fowl, but also revealed candidate proteins, which may be involved in a dramatic shift in eggshell microstructure that is unique to Guinea fowl. The proteins more abundant during the shift were ANXA2, S100A6, CALB1, TSKU, FAM20C, GPC-4, DCA-1-like and GDF6. Amongst them are calcium binding proteins, protein cores of proteoglycans, proteins involved in the regulation of proteins driving the mineralization. Conclusion This is the first proteome survey in Guinea fowl eggshell which exhibits exceptional mechanical proeprties. These data enriched the huge number of matrix proteins identified in various bird"s eggshell proteomics studies and will allow genomic improvements and will give insights for material sciences. Genes coding matrix proteins will be used as biological markers for genomic selection to reinforce eggshell breaking strength. The corresponding transcripts will be associated with published and private SNPs and mapped in QTLs related to shell quality. They will constitute candidate genes to gain precision for genomic selection to reinforce shell mechanical properties. Industrial ceramics are made in high temperature and pressure. Material science explores the biomineralization to investigate how living organisms build their shell in physiological conditions. Amongst various biominerals, the bird"s eggshell are the most widely documented. Information on shell matrix proteins and how they contribute to the mechanical properties, gives a chance to establish a list of natural organic compounds of benefit usable in the fabrication of calcium carbonate materials/ceramics.

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