Cell Reactivity to Different Silica

The interaction between mineral structures and living beings is increasingly attracting the interest of research. The formation of skeletons, geomicrobiology, the study of the origin of life, soil biology, benthos biology, human and mammalian diseases generated by the inhalation of dust and biomaterials are some examples of scientific areas where the topic has a relevance. In this chapter we focus on cell reactivity to siliceous rocks and to the various forms of silicon dioxide, in particular. The examples here reported carefully review howsuch minerals may strongly affect different living beings, from simple ones to humans. The biomineralogy concept is explained, focusing on the effects of rocks on cell growth and development. The toxic action of silicon dioxide in mammalian lungs is the oldest evidence of crystalline silica bioactivity. More recently, we could demonstrate that crystalline silica has a deep impact on cell biology throughout the whole animal kingdom. One of the most illustrative case studies is the marine sponge Chondrosia reniformis, which has the amazing ability to incorporate and etch crystalline silica releasing dissolved silicates in the medium. This specific and selective action is due to the chemical reaction of ascorbic acid with quartz surfaces. One consequence of this is an increased production of collagen. The discovery of this mechanism opened the door to a new understanding of silica toxicity for animal cells and mammalian cells in particular. The presence of silica in sea water and substrates also affects processes like the settlement of larvae and the growth of diatoms. The following sections review all such aspects

Recombinant production of hydroxylated collagen polypeptides derived from the Demospongia C. reniformis: a new biotechnological source of marine collagen

Collagen from marine sources is a very attractive alternative to bovine collagen. In biomedicine, several applications of collagens derived from sponges have been described, many of which from the demospongia Chondrosia reniformis. The production of these molecules in recombinant form would allow to obtain well-defined molecular types and to limit the expensive procedures of sample recovery and purification. Here we report the realization of a yeast strain able to produce hydroxylated collagen polypeptides derived from C. reniformis. First the cDNAs coding for the \uf061 and \uf062\uf020 subunits of the sponge enzyme prolyl-4-hydroxylase (P\uf034\uf048\uf029 were identified by PCR approach, then they were cloned in pPink and in pPIC6\uf061 vectors respectively and finally they were stably integrated in the genome of the methanotrophic yeast Pichia pastoris. The strain with the highest P4H activity was then transformed with a third expression vector (pPICZ) containing the coding region of a sponge collagen polypeptide in frame with an Histidine-TAG at the 3\u2019-end. Recombinant protein expression was then induced and collagen polypeptides were purified by affinity chromatography and analysed by SDS-PAGE followed by Mass Spectrometry, in order to confirm the sequence and the presence of hydroxy-proline residues. The biocompatibility of the recombinant proteins was evaluated by MTT test in human neonatal fibroblasts and furthermore, tissue culture plates were treated with 10 \uf06dg/ml recombinant protein solution to test its cell adhesion properties. In conclusion, a first recombinant hydroxylated marine collagen polypeptide was produced in the Pichia system, resulting biocompatible and suggesting its use as porcine gelatin substitute for cell culture. Research supported by EU (FP7 grant agreement n: 266033 SPonge Enzyme End Cell for Innovative AppLication-SPECIAL

Silica-induced fibrosis: An ancient response from the early metazoans

Exposure to crystalline silica particles causes silicosis, an occupational disease leading to an overproduction of collagen in the lung. The first step of this pathology is characterized by the release of inflammatory mediators. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine directly involved in silica-induced pulmonary fibrosis. The marine demosponge Chondrosia reniformis is able to incorporate silica grains and partially dissolve the crystalline forms apparently without toxic effects. In the present work, C. reniformis tissue explants were treated with fine quartz dust and the expression level of fibrogenic genes was assayed by qPCR, demonstrating an overexpression of a fibrillar and a non-fibrillar collagen and of prolyl-4-hydroxylase enzyme. The deposition of new collagen could also be documented in quartztreated sponge explants. Furthermore, TNF pro-inflammatory cytokine overexpression and involvement in silica-induced sponge collagen biosynthesis was demonstrated in quartz-treated explants as compared with controls by means of specific TNF inhibitors affecting the fibrogenic gene response. As no documentable detrimental effect was observed in treated explants, we conclude that the C. reniformis unique quartz engulfment and erosion is physiological and beneficial to the animal, leading to new collagen synthesis and strengthening of the body stiffness. Thus, we put forward the hypothesis that an ancient physiological behaviour from the lowest of the Metazoa, persisting through evolution via the samemolecularmediators such as TNF, may have become the cause of disease in the specialized tissues of higher animals such as mammals

Alginate-Based Electrospun Membranes Containing ZnO Nanoparticles as Potential Wound Healing Patches: Biological, Mechanical, and Physicochemical Characterization

In the present work, alginate-based mats with and without ZnO nanoparticles were prepared via an electrospinning technique and subjected to a washing-cross-linking process to obtain highly stable products characterized by thin and homogeneous nanofibers with a diameter of 100 \ub1 30 nm. Using a commercial collagen product as control, the biological response of the prepared mats was carefully evaluated with particular attention paid to the influence of the used cross-linking agent (Ca2+, Sr2+, or Ba2+ ions) and to the presence of nanofillers. Fibroblast and keratinocyte cultures successfully proved the safety of the prepared alginate-based mats, whereas ZnO nanoparticles were found to provide strong antibacteriostatic and antibacterial properties; above all, the strontium- and barium-cross-linked samples showed performances in terms of cell adhesion and growth very similar to those of the commercial collagen membrane despite them showing a significantly lower protein adsorption. Moreover, the mechanical and water-related properties of the strontium-cross-linked mats embedding ZnO nanoparticles were proven to be similar to those of human skin (i.e., Young modulus of 470 MPa and water vapor permeability of 3.8 7 10-12 g/m Pa s), thus proving the ability of the prepared mats to be able to endure considerable stress, maintaining at the same time the fundamental ability to remove exudates. Taking into account the obtained results, the proposed alginate-based products could lead to harmless and affordable surgical patches and wound dressing membranes with a simpler and safer production procedure than the commonly employed animal collagen-derived systems

Molecular cloning, characterization and expression analysis of a prolyl 4-hydroxylase from the marine sponge Chondrosia reniformis

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, \u3b1 and \u3b2. Here, we report the cDNA cloning, characterization, and expression analysis of the \u3b1 and \u3b2 subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the \u3b1 subunit is 533 residues long with an Mr of 59.14 kDa, while the \u3b2 subunit counts 526 residues with an Mr of 58.75 kDa. Phylogenetic analyses showed that \u3b1P4H and \u3b2P4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an \u3b12\u3b22 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that \u3b1P4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the \u3b1P4H levels, whereas ascorbic acid strongly upregulated the \u3b1P4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of \u3b1P4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organism

Modified peptide nucleic acids are internalized in mouse macrophages RAW 264.7 and inhibit inducible nitric oxide synthase

AbstractOverexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3–25-fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43–44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression

Effect of Crosslinking Type on the Physical-Chemical Properties and Biocompatibility of Chitosan-Based Electrospun Membranes

Chitosan nanofibrous membranes are prepared via an electrospinning technique and explored as potential wound healing patches. In particular, the effect of a physical or chemical crosslinking treatment on the mat morphological, mechanical, water-related, and biological properties is deeply evaluated. The use of phosphate ions (i.e., physical crosslinking) allows us to obtain smooth and highly homogenous nanofibers with an average size of 190 nm, whereas the use of ethylene glycol diglycidyl ether (i.e., chemical crosslinking) leads to rougher, partially coalesced, and bigger nanofibers with an average dimension of 270 nm. Additionally, the physically crosslinked mats show enhanced mechanical performances, as well as greater water vapour permeability and hydrophilicity, with respect to the chemically crosslinked ones. Above all, cell adhesion and cytotoxicity experiments demonstrate that the use of phosphate ions as crosslinkers significantly improves the capability of chitosan mats to promote cell viability owing to their higher biocompatibility. Moreover, tuneable drug delivery properties are achieved for the physically crosslinked mats by a simple post-processing impregnation methodology, thereby indicating the possibility to enrich the prepared membranes with unique features. The results prove that the proposed approach may lead to the preparation of cheap, biocompatible, and efficient chitosan-based nanofibers for biomedical and pharmaceutical applications

Optimised solid phase synthesis of a cystine-linked peptide-PNA chimera

A solid phase synthesis method for a cystine-linked peptide-PNA chimera starting from different modified protectedcystine monomers was developed. This strategy dramatically improved the final yield and the specificity of disulphide bondformation in this kind of oligomers, which are traditionally obtained by liquid phase coupling via oxidation of the two terminalcysteines