A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic-inorganic hybrid materials in sponge primmorphs.

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).

Utilization of metabolic energy in treatment of ocular surface disorders: polyphosphate as an energy source for corneal epithelial cell proliferation.

Impaired regeneration of the corneal epithelium, as found in many ocular surface diseases, is a major clinical problem in ophthalmology. We hypothesized that corneal epithelial regeneration can be promoted by the physiological, energy-delivering as well as "morphogenetically active" polymer, inorganic polyphosphate (polyP). Corneal limbal explants (diameter, 4 mm) were cultivated on collagen-coated well plates in the absence or presence of polyP (chain length, ∼40 Pi units; 50 µg ml-1) or human platelet lysate (hp-lysate; 5% v/v). Cell outgrowth and differentiation were analyzed after staining with DRAQ5 (nuclei) and rhodamine phalloidin (cytoskeleton), as well as by environmental scanning electron microscopy (ESEM). Cell growth/viability of hCECs was assessed by XTT assay. The expression of SDF-1 was quantitated by qRT-PCR. Exposure to hp-lysate (also containing polyP) increased cell migration already at day 1. Even stronger was the effect of polyP. This effect was blocked by a mast cell serine protease. The formation of cell multilayers was enhanced by hp-lysate or even more by polyP. ESEM revealed continuous cell junctions and prominent microvilli on the surface of adjacent cells exposed to polyP; those structures were only rarely seen in the controls. The hp-lysate and, more potently, polyP increased the proliferation of hCECs, as well as SDF-1 expression. The findings indicate the potential usefulness of the natural polymer, polyP, for topical treatment of corneal epithelial defects. Future studies are directed to develop suitable formulations of polyP, such as biomimetic polyP nano/microparticles showing an adjustable release kinetics.

Uptake of polyphosphate microparticles in vitro (SaOS-2 and HUVEC cells) followed by an increase of the intracellular ATP pool size.

Recently two approaches were reported that addressed a vitally important problem in regenerative medicine, i. e. the successful treatment of wounds even under diabetic conditions. Accordingly, these studies with diabetic rabbits [Sarojini et al. PLoS One 2017, 12(4):e0174899] and diabetic mice [Müller et al. Polymers 2017, 9, 300] identified a novel (potential) target for the acceleration of wound healing in diabetes. Both studies propose a raise of the intracellular metabolic energy status via exogenous administration either of ATP, encapsulated into lipid vesicles, or of polyphosphate (polyP) micro-/nanoparticles. Recently this physiological polymer, polyP, was found to release metabolic energy in form of ATP into both the extra- and also intra-cellular space. In the present work the uptake mechanism of the amorphous polyP microparticles "Ca-polyP-MP" has been described and found to be a clathrin-dependent endocytosis import, based on inhibition studies with the inhibitor trifluoperazine, which blocks the clathrin-dependent endocytosis import. The experiments had been performed with SaOS-2 cells, by studying the uptake and distribution of the electron-dense particles into the cells, and with HUVEC cells, for analysis of the intracellular accumulation of polyP, visualized by fluorescent staining of polyP. Concurrently with the uptake of particular polyP the intracellular ATP level increased as well. In contrast to "Ca-polyP-MP" the soluble polyP, administered as "Na-polyP[Ca2+]", did not cause an increase in the intracellular Ca2+ level, suggesting a different mode of action of these two forms of polyP. Based on existing data on the effect of polyP and ATP on the induction of vascularization during wound repair, both groups (Sarojini et al. and Müller et al.) propose that the acceleration of wound repair is based on an increased metabolic energy supply directly to the regenerating wound area.

An evolutionary perspective on the role of mesencephalic astrocyte-derived neurotrophic factor (MANF): At the crossroads of poriferan innate immune and apoptotic pathways.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a recently discovered family of neurotrophic factors. MANF can be secreted but is generally resident within the endoplasmic reticulum (ER) in neuronal and non-neuronal cells, where it is involved in the ER stress response with pro-survival effects. Here we report the discovery of the MANF homolog SDMANF in the sponge Suberites domuncula. The basal positioning of sponges (phylum Porifera) in the animal tree of life offers a unique vantage point on the early evolution of the metazoan-specific genetic toolkit and molecular pathways. Since sponges lack a conventional nervous system, SDMANF presents an enticing opportunity to investigate the evolutionary ancient role of these neurotrophic factors. SDMANF shares considerable sequence similarity with its metazoan homologs. It also comprises a putative protein binding domain with sequence similarities to the Bcl-2 family of apoptotic regulators. In Suberites, SDMANF is expressed in the vicinity of bacteriocytes, where it co-localizes with the toll-like receptor SDTLR. In transfected human cells, SDMANF was detected in both the organelle protein fraction and the cell culture medium. The intracellular SDMANF protein level was up-regulated in response to both a Golgi/ER transport inhibitor and bacterial lipopolysaccharides (LPS). Upon LPS challenge, transfected cells revealed a decreased caspase-3 activity and increased cell viability with no inducible Bax expression compared to the wild type. These results suggest a deep evolutionary original cytoprotective role of MANF, at the crossroads of innate immune and apoptotic pathways, of which a neurotrophic function might have arisen later in metazoan evolution.

Two-Armed Activation of Bone Mineral Deposition by the Flavones Baicalin and Baicalein, Encapsulated in Polyphosphate Microparticles.

In this study, we investigated the effect of the two flavonoids, baicalin (baicalein 7-O-[Formula: see text]- d-glucuronic acid) and its aglycone, baicalein (5,6,7-trihydroxyflavone), after encapsulation into amorphous calcium polyphosphate (Ca-polyP) microparticles on mineralization of primary human osteoblasts (phOSB). Both flavonoids, which come from root extracts of Scutellaria baicalensis Georgi, are used in Traditional Chinese Medicine, and are nontoxic in cells up to a concentration of 3[Formula: see text][Formula: see text]g/ml. The morphogenetically active, energy-rich Ca-polyP particles with a stoichiometric P:Ca ratio of 1:2 are degraded by cellular alkaline phosphatase (ALP) to ortho-phosphate used for bone hydroxyapatite formation. Here we show that the flavone-loaded Ca-polyP microparticles are readily taken up by phOSB, resulting in the accumulation of polyP around the nuclei and the formation of intracellular vesicles containing the ALP. In addition, we demonstrate that baicalin/baicalein causes a rise of the intracellular calcium [Ca[Formula: see text]]i a level which markedly is augmented after encapsulation into Ca-polyP, through activation of the phospholipase C. Moreover, both flavones, either alone or associated with Ca-polyP microparticles, upregulate the expression of the osteoblast calcium efflux channel, the plasma membrane Ca[Formula: see text]-ATPase (PMCA), while the expression of ALP, which promotes bone mineralization, is induced by Ca-polyP and by the flavones only if present in the Ca-polyP-microparticle-associated form. As a result, the extent of bone mineralization is markedly enhanced. Based on the two-armed activating function, new applications of baicalin/baicalein as a component of nutriceuticals for osteoporosis prevention or bone implants can be envisaged.

Self-assembly and photocatalytic activity of branched silicatein/silintaphin filaments decorated with silicatein-synthesized TiO2 nanoparticles.

The fundamental mechanisms of biomineralization and their translation into innovative synthetic approaches have yielded promising perspectives for the fabrication of biomimetic and bioinspired organic-inorganic hybrid materials. In siliceous sponges, the enzyme silicatein catalyzes the polycondensation of molecular precursors to nano-structured SiO2 that is deposited on self-assembled filaments consisting of the two silicatein isoforms (silicatein-α and -ß) and the scaffold protein silintaphin-1. Due to its broad substrate specificity silicatein is also able to convert in vitro various other precursors to non-biogenic materials (e.g., hydrolysis of titanium bis(ammonium lactato)-dihydroxide [TiBALDH] and subsequent polycondensation to titania [TiO2]). In the present approach, silicatein was bioengineered to carry a protein tag (Arg-tag) that confers binding affinity to TiO2. Then, by combining Arg-tagged silicatein-α with silicatein-ß and silintaphin-1, self-assembled branched hybrid protein microfilaments were fabricated. Upon subsequent incubation with TiBALDH the filaments were decorated with TiO2 and assayed for photocatalytic activity through photodegradation of the dye methylene blue. This is the first approach that considers concomitant application of two silicatein isoforms for the synthesis of bioinspired organic-inorganic hybrid materials. It is also the first time that the biocatalytic activity of the enzymes has been combined with both the structure-providing properties of silintaphin-1 and a TiO2 affinity protein tag to fabricate self-assembled branched protein filaments as template for a silicatein-synthesized TiO2 photocatalyst. The TiO2-decorated filaments might be explored as a practical alternative to approaches where biotemplates have to be laboriously isolated from their original biological source prior to TiO2 immobilization.

Characterization and osteogenic activity of a silicatein/biosilica-coated chitosan-graft-polycaprolactone.

Several attempts have been made in the past to fabricate hybrid materials that display the complementary properties of the polyester polycaprolactone (PCL) and the polysaccharide chitosan (CHS) for application in the field of bone regeneration and tissue engineering. However, such composites generally have no osteogenic activity per se. Here we report the synthesis of a chitosan-graft-polycaprolactone (CHS-g-PCL) and its subsequent characterization, including crystallinity, chemical structure and thermal stability. Upon surface-functionalization of CHS-g-PCL with osteogenic biosilica via the surface-immobilized enzyme silicatein, protein adsorption, surface morphology and wettability were assessed. Finally, the cultivation of osteoblastic SaOS-2 cells on the surface-functionalized CHS-g-PCL was followed by analyses of cell viability, mineral deposition and alkaline phosphatase activity. These characterizations revealed a composite that combines the versatile properties of CHS-g-PCL with the osteogenic activity of the silicatein/biosilica coating and, hence, represents an innovative alternative to conventionally used CHS/PCL composites for biomedical applications, where stable bone-material interfaces are required.

Cellular effects of bacterial N-3-Oxo-dodecanoyl-L-Homoserine lactone on the sponge Suberites domuncula (Olivi, 1792): insights into an intimate inter-kingdom dialogue.

Sponges and bacteria have lived together in complex consortia for 700 million years. As filter feeders, sponges prey on bacteria. Nevertheless, some bacteria are associated with sponges in symbiotic relationships. To enable this association, sponges and bacteria are likely to have developed molecular communication systems. These may include molecules such as N-acyl-L-homoserine lactones, produced by Gram-negative bacteria also within sponges. In this study, we examined the role of N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL) on the expression of immune and apoptotic genes of the host sponge Suberites domuncula. This molecule seemed to inhibit the sponge innate immune system through a decrease of the expression of genes coding for proteins sensing the bacterial membrane: a Toll-Like Receptor and a Toll-like Receptor Associated Factor 6 and for an anti-bacterial perforin-like molecule. The expression of the pro-apoptotic caspase-like 3/7 gene decreased as well, whereas the level of mRNA of anti-apoptotic genes Bcl-2 Homolog Proteins did not change. Then, we demonstrated the differential expression of proteins in presence of this 3-oxo-C12-HSL using 3D sponge cell cultures. Proteins involved in the first steps of the endocytosis process were highlighted using the 2D electrophoresis protein separation and the MALDI-TOF/TOF protein characterization: α and ß subunits of the lysosomal ATPase, a cognin, cofilins-related proteins and cytoskeleton proteins actin, α tubulin and α actinin. The genetic expression of some of these proteins was subsequently followed. We propose that the 3-oxo-C12-HSL may participate in the tolerance of the sponge apoptotic and immune systems towards the presence of bacteria. Besides, the sponge may sense the 3-oxo-C12-HSL as a molecular evidence of the bacterial presence and/or density in order to regulate the populations of symbiotic bacteria in the sponge. This study is the first report of a bacterial secreted molecule acting on sponge cells and regulating the symbiotic relationship.

The silicatein propeptide acts as inhibitor/modulator of self-organization during spicule axial filament formation.

Silicateins are crucial enzymes that are involved in formation of the inorganic biosilica scaffold of the spicular skeleton of siliceous sponges. We show that silicatein acquires its structure-guiding and enzymatically active state by processing of silicatein from pro-silicatein to the mature enzyme. A recombinant propeptide (PROP) of silicatein from the siliceous demosponge Suberites domuncula was prepared, and antibodies were raised against the peptide. In sponge tissue, these antibodies reacted with both surface structures and the central region of the spicules. Using phage display expression, spicule-binding 12-mer peptides were identified that are rich in histidine residues. In the predicted tertiary structure of PROP, these histidine residues are only present in the α-helical region. The recombinant PROP was found to inhibit self-assembly of silicatein molecules. By light scattering, it was shown that, in the presence of 4 m urea, the recombinant silicatein is obtained in the mono/oligomeric form with a hydrodynamic radius of 4 nm, while lower urea concentrations promote self-aggregation and assembly of the protein. Finally, it is shown that the enzymatic activity of silicatein is abolished by PROP in silicatein samples that predominantly contain mono- or oligomeric silicatein particles, but the enzyme is not affected if present in the filamentous aggregated form. It is concluded that the functions of silicatein, acting as a structural template for its biosilica product and as an enzyme, are modulated and controlled by its propeptide.

Deep metazoan phylogeny: when different genes tell different stories.

Molecular phylogenetic analyses have produced a plethora of controversial hypotheses regarding the patterns of diversification of non-bilaterian animals. To unravel the causes for the patterns of extreme inconsistencies at the base of the metazoan tree of life, we constructed a novel supermatrix containing 122 genes, enriched with non-bilaterian taxa. Comparative analyses of this supermatrix and its two non-overlapping multi-gene partitions (including ribosomal and non-ribosomal genes) revealed conflicting phylogenetic signals. We show that the levels of saturation and long branch attraction artifacts in the two partitions correlate with gene sampling. The ribosomal gene partition exhibits significantly lower saturation levels than the non-ribosomal one. Additional systematic errors derive from significant variations in amino acid substitution patterns among the metazoan lineages that violate the stationarity assumption of evolutionary models frequently used to reconstruct phylogenies. By modifying gene sampling and the taxonomic composition of the outgroup, we were able to construct three different yet well-supported phylogenies. These results show that the accuracy of phylogenetic inference may be substantially improved by selecting genes that evolve slowly across the Metazoa and applying more realistic substitution models. Additional sequence-independent genomic markers are also necessary to assess the validity of the phylogenetic hypotheses.

Biogenic inorganic polysilicates (biosilica): formation and biomedical applications.

The siliceous sponges, the demosponges and hexactinellid glass sponges, are unique in their ability to form biosilica structures with complex architectures through an enzyme-catalyzed mechanism. The biosilica skeleton of these sponges with its hierarchically structure and exceptional opto-mechanical properties has turned out to be an excellent model for the design of biomimetic nanomaterials with novel property combinations. In addition, biosilica shows morphogenetic activity that offers novel applications in the field of bone tissue engineering and repair. In recent years, much progress has been achieved towards the understanding of the principal enzymes, the silicateins that form the sponge skeletal elements, the spicules, and their self-assembling and structure-guiding properties. The discovery of the silicatein-interacting, scaffolding proteins provided new insights in the mechanism of spiculogenesis. The now available toolbox of enzymes and proteins that are involved in biosilica formation and the biosilica material synthesized by them are of great interest for a variety of applications from nanobiotechnology to nanomedicine.

Osteogenic potential of a biosilica-coated P(UDMA-co-MPS) copolymer.

A P(UDMA-co-MPS) copolymer was surface-functionalized through the polycondensation activity of the enzyme silicatein. The resulting biosilica coating significantly enhanced mineralization of osteoblastic cells, thereby revealing its osteogenic potential. Consequently, the functionalized copolymer may be explored as an alternative to conventionally used acrylics in applications where stable bone-material interfaces are required.

Dual effect of inorganic polymeric phosphate/polyphosphate on osteoblasts and osteoclasts in vitro.

Inorganic polymeric phosphate/polyphosphate (polyP) is a natural polymer existing in both pro- and eukaryotic systems. In the present study the effect of polyP as well as of polyP supplied in a stoichiometric ratio of 2 m polyP:1 m CaCl2 [polyP (Ca(2+) complex)] on the osteoblast-like SaOS-2 cells and the osteoclast-like RAW 264.7 cells was determined. Both polymers are non-toxic for these cells up to a concentration of 100 µm. In contrast to polyP, polyP (Ca(2+) complex) significantly induced hydroxyapatite formation at a concentration > 10 µm, as documented by alizarin red S staining and scanning electron microscopic (SEM) inspection. Furthermore, polyP (Ca(2+) complex) triggered in SaOS-2 cells transcription of BMP2 (bone morphogenetic protein 2), a cytokine involved in maturation of hydroxyapatite-forming cells. An additional activity of polyP (Ca(2+) complex) is described by showing that this polymer impairs osteoclastogenesis. At concentrations > 10 µm polyP (Ca(2+) complex) slows down the progression of RAW 264.7 cells to functional osteoclasts, as measured by the expression of TRAP (tartrate-resistant acid phosphatase). Finally, it is shown that 10-100 µm polyP (Ca(2+) complex) inhibited phosphorylation of IκBα by the respective kinase in RAW 264.7 cells. We concluded that polyP (Ca(2+) complex) displays a dual effect on bone metabolizing cells. It promotes hydroxyapatite formation in SaOS-2 cells (osteoblasts) and impairs maturation of the osteoclast-related RAW 264.7 cells.

Formation of a micropatterned titania photocatalyst by microcontact printed silicatein on gold surfaces.

The enzyme silicatein has been bioengineered to carry a thiol-bearing Au-affinity tag (Cys-tag) for direct immobilization on gold carriers in shortest time without the need for prior surface functionalization. Through microcontact printing, defined silicatein micropatterns were created on gold surfaces, facilitating the subsequent enzymatically controlled synthesis of photocatalytically active TiO(2).

Nocturnin in the demosponge Suberites domuncula: a potential circadian clock protein controlling glycogenin synthesis in sponges.

Sponges are filter feeders that consume a large amount of energy to allow a controlled filtration of water through their aquiferous canal systems. It has been shown that primmorphs, three-dimensional cell aggregates prepared from the demosponge Suberites domuncula and cultured in vitro, change their morphology depending on the light supply. Upon exposure to light, primmorphs show a faster and stronger increase in DNA, protein and glycogen content compared with primmorphs that remain in the dark. The sponge genome contains nocturnin, a light/dark-controlled clock gene, the protein of which shares a high sequence similarity with the related molecule of higher metazoans. The sponge nocturnin protein was found showing a poly(A)-specific 3'-exoribonuclease activity. In addition, the cDNA of the glycogenin gene was identified for subsequent expression studies. Antibodies against nocturnin were raised and used in parallel with the cDNA to determine the regional expression of nocturnin in intact sponge specimens; the highest expression of nocturnin was seen in the epithelial layer around the aquiferous canals. Quantitative PCR analyses revealed that primmorphs after transfer from light to dark show a 10-fold increased expression in the nocturnin gene. In contrast, the expression level of glycogenin decreases in the dark by 3-4-fold. Exposure of primmorphs to light causes a decrease in nocturnin transcripts and a concurrent increase in glycogenin transcripts. It was concluded that sponges are provided with the molecular circadian clock protein nocturnin that is highly expressed in the dark where it controls the stability of a key metabolic enzyme, glycogenin.

Biosilica: Molecular Biology, Biochemistry and Function in Demosponges as well as its Applied Aspects for Tissue Engineering.

Biomineralization, biosilicification in particular (i.e. the formation of biogenic silica, SiO(2)), has become an exciting source of inspiration for the development of novel bionic approaches following 'nature as model'. Siliceous sponges are unique among silica-forming organisms in their ability to catalyze silica formation using a specific enzyme termed silicatein. In this study, we review the present state of knowledge on silicatein-mediated 'biosilica' formation in marine demosponges, the involvement of further molecules in silica metabolism and their potential applications in nano-biotechnology and bio-medicine. While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. Only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e. biosilicification), hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometres to metres. This peculiar phenomenon has been comprehensively studied in recent years, and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1/silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nano-structured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) with promising results.

Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form.

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.

Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2)) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+)-depletion condition (1 mM CaCl(2)). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast).

Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation).

Bio-silica represents the main mineral component of the sponge skeletal elements (siliceous spicules), while bio-polyphosphate (bio-polyP), a multifunctional polymer existing in microorganisms and animals acts, among others, as reinforcement for pores in cell membranes. These natural inorganic bio-polymers, which can be readily prepared, either by recombinant enzymes (bio-silica and bio-polyP) or chemically (polyP), are promising materials/substances for the amelioration and/or treatment of human bone diseases and dysfunctions. It has been demonstrated that bio-silica causes in vitro a differential effect on the expression of the genes OPG and RANKL, encoding two mediators that control the tuned interaction of the anabolic (osteoblasts) and catabolic (osteoclasts) pathways in human bone cells. Since bio-silica and bio-polyP also induce the expression of the key mediator BMP2 which directs the differentiation of bone-forming progenitor cells to mature osteoblasts and in parallel inhibits the function of osteoclasts, they are promising candidates for treatment of osteoporosis.