Polyphosphate (PolyP) for alveolar cleft repair: study protocol for a pilot randomized controlled trial.

OBJECTIVE: Bone grafting is an important surgical procedure to restore missing bone in patients with alveolar cleft lip/palate, aiming to stabilize either sides of the maxillary segments by inducing new bone formation, and in bilateral cleft cases also to stabilize the pre-maxilla. Polyphosphate (PolyP), a physiological polymer composed of orthophosphate units linked together with high-energy phosphate bonds, is a naturally existing compound in platelets which, when complexed with calcium as Ca-polyP microparticles (Ca-polyP MPs), was proven to have osteoinductive properties in preclinical studies. AIM: To evaluate the feasibility, safety, and osteoinductivity of Ca-polyP MPs as a bone-inducing graft material in humans. METHODS: This prospective non-blinded first-in-man clinical pilot study shall consist of 8 alveolar cleft patients of 13 years or older to evaluate the feasibility and safety of Ca-PolyP MPs as a bone-inducing graft material. Patients will receive Ca-polyP graft material only or Ca-polyP in combination with biphasic calcium phosphate (BCP) as a bone substitute carrier. During the trial, the participants will be investigated closely for safety parameters using radiographic imaging, regular blood tests, and physical examinations. After 6 months, a hollow drill will be used to prepare the implantation site to obtain a biopsy. The radiographic imaging will be used for clinical evaluation; the biopsy will be processed for histological/histomorphometric evaluation of bone formation. DISCUSSION: This is the first-in-man study evaluating the safety and feasibility of the polyP as well as the potential regenerative capacity of polyP using an alveolar cleft model. TRIAL REGISTRATION: Indonesian Trial Registry INA-EW74C1N . Registered on 12 June 2020.

Dynamic contrast-enhanced magnetic resonance imaging for monitoring neovascularization during bone regeneration-a randomized in vivo study in rabbits.

OBJECTIVES: Micro-computed tomography (µ-CT) and histology, the current gold standard methods for assessing the formation of new bone and blood vessels, are invasive and/or destructive. With that in mind, a more conservative tool, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), was tested for its accuracy and reproducibility in monitoring neovascularization during bone regeneration. Additionally, the suitability of blood perfusion as a surrogate of the efficacy of osteoplastic materials was evaluated. MATERIALS AND METHODS: Sixteen rabbits were used and equally divided into four groups, according to the time of euthanasia (2, 3, 4, and 6 weeks after surgery). The animals were submitted to two 8-mm craniotomies that were filled with blood or autogenous bone. Neovascularization was assessed in vivo through DCE-MRI, and bone regeneration, ex vivo, through µ-CT and histology. RESULTS: The defects could be consistently identified, and their blood perfusion measured through DCE-MRI, there being statistically significant differences within the blood clot group between 3 and 6 weeks (p = 0.029), and between the former and autogenous bone at six weeks (p = 0.017). Nonetheless, no significant correlations between DCE-MRI findings on neovascularization and µ-CT (r =-0.101, 95% CI [-0.445; 0.268]) or histology (r = 0.305, 95% CI [-0.133; 0.644]) findings on bone regeneration were observed. CONCLUSIONS: These results support the hypothesis that DCE-MRI can be used to monitor neovascularization but contradict the premise that it could predict bone regeneration as well.

Bioinspired self-assembly of tyrosinase-modified silicatein and fluorescent core-shell silica spheres.

Inspired by the intermolecular cross-linking of mussel foot proteins and their adhesive properties, tyrosinase has been used to modify recombinant silicatein. DOPA/DOPAquinone-mediated cross-linking and interfacial interactions enhanced both self-assembly of silicatein building blocks and templating of core-shell silica spheres, resulting in fluorescent biomimetic silicatein-silica hybrid mesofibers.

In vitro degradation of porous PLLA/pearl powder composite scaffolds.

The in vitro degradation behavior of poly-L-lactide (PLLA), PLLA/aragonite pearl powder and PLLA/vaterite pearl powder scaffolds was investigated. The scaffolds were soaked in phosphate buffer solution (PBS) up to 200 days. Scanning electron microscopy (SEM), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC) were used to observe any degradation of the scaffolds. Degradation behaviors such as changes in pH, porosity, bulk density, water absorption, weight loss and mechanical properties were discussed. The results show that a gradual increase of the pH in composite scaffolds can decrease the rate of hydrolysis of PLLA. PLLA/vaterite and PLLA/aragonite scaffolds have a similar degradation behavior but a slower rate of degradation than PLLA.

Silicate modulates the cross-talk between osteoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation.

It has been shown that inorganic monomeric and polymeric silica/silicate, in the presence of the biomineralization cocktail, increases the expression of osteoprotegerin (OPG) in osteogenic SaOS-2 sarcoma cells in vitro. In contrast, silicate does not affect the steady-state gene expression level of the osteoclastogenic ligand receptor activator of NF-κB ligand (RANKL). In turn it can be expected that the concentration ratio of the mediators OPG/RANKL increases in the presence of silicate. In addition, silicate enhances the growth potential of SaOS-2 cells in vitro, while it causes no effect on RAW 264.7 cells within a concentration range of 10-100 µM. Applying a co-cultivation assay system, using SaOS-2 cells and RAW 264.7 cells, it is shown that in the presence of 10 µM silicate the number of RAW 264.7 cells in general, and the number of TRAP(+) RAW 264.7 cells in particular markedly decreases. The SaOS-2 cells retain their capacity of differential gene expression of OPG and RANKL in favor of OPG after exposure to silicate. It is concluded that after exposure of the cells to silicate a factor(s) is released from SaOS-2 cells that causes a significant inhibition of osteoclastogenesis of RAW 264.7 cells. It is assumed that it is an increased secretion of the cytokine OPG that is primarily involved in the reduction of the osteoclastogenesis of the RAW 264.7 cells. It is proposed that silicate might have the potential to stimulate osteogenesis in vivo and perhaps to ameliorate osteoporotic disorders.

Induction of apoptosis in mussel Mytilus galloprovincialis gills by model cytotoxic agents.

Apoptosis signaling pathway was investigated in the marine mussel Mytilus galloprovincialis exposed to various stressors. Analyses were performed in mussels exposed to two major pollutants of the aquatic environment: tributyltin and the water soluble fraction of diesel oil, for 1 h and animals were then maintained in sea water for a recovery period of 6 and 24 h. Apoptosis was evaluated at several levels of the cell signaling cascade by measuring Bcl-xS expression, caspase-3 activity and DNA damage (Fast micromethod(®) and TUNEL techniques). H(2)O(2) was used as a control of apoptosis induction for validation of the assays. Results showed an induction of Bcl-xS expression, a protein implicated in apoptosis, after 1 h exposure to all concentrations of chemicals. Moreover, in the same manner, apoptotic DNA damage was induced with all chemicals tested. Besides, caspase 3 activity was detected after 1 h exposure to low doses of TBT and diesel oil while the high concentrations induced this protein after 6 h. The achieved data were also correlated with our previous study, demonstrating an induction of the mitogen-activated protein kinase (MAPK) activity in the mussel M. galloprovincialis exposed to the same conditions. In conclusion, this study was one of the first characterizing the MAP kinase cell signaling pathway leading to apoptosis in the mussel M. galloprovincialis exposed to chemicals. It showed for the first time that the Bcl-xS protein was present in these mussels as in other species and played a role in apoptosis mediation. Moreover, the main originality of this work was that it showed that two apoptotic pathways might be present in the mussel: a caspase 3-dependent and a caspase 3-independent pathways.

MAP kinase cell signaling pathway as biomarker of environmental pollution in the sponge Suberites domuncula.

In the present study, we analyzed the effects of two major pollutants of the environment, tributyltin (TBT) and water-accommodated fraction (WAF) of diesel oil, on MAP kinase activation, apoptosis induction and DNA damage, in the marine sponge Suberites domuncula. Our results clearly demonstrated a differential activation of the MAPKs depending on the chemicals tested. TBT induced the activation of p38 and JNK while diesel oil enhanced activation of both ERK and p38. The activation of MAPKs was observed after 1 h exposure and 6 and 24 h of recovery in seawater. In addition, DNA fragmentation, assessed by two techniques, the Fast micromethod(®) and the TUNEL assay, was detected after sponges were treated with both chemicals. Moreover, the study of caspase 3/7 activity showed that apoptosis was induced and triggered with all concentrations of TBT but only at high diesel oil concentrations. After TBT exposure, a correlation was observed between JNK activation, caspase 3 activity and DNA damage while p38 activation followed the two latter parameters at high concentrations of diesel oil, suggesting that sponges enhanced a specific apoptotic pathway depending on the xenobiotic tested. This study demonstrated a high signal response by the sponge Suberites domuncula to the tested chemicals. Cell signaling pathway studies may thus be of use in water quality biomonitoring programs.

Poriferan survivin exhibits a conserved regulatory role in the interconnected pathways of cell cycle and apoptosis.

Survivin orchestrates intracellular pathways during cell division and apoptosis. Its central function as mitotic regulator and inhibitor of cell death has major implications for tumor cell proliferation. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution a complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulator, a survivin-like protein (SURVL) of one of the earliest-branching metazoan taxa was identified and functionally characterized. SURVL of the sponge Suberites domuncula shares considerable similarities with its metazoan homologs, ranging from conserved exon/intron structure to presence of protein-interaction domains. Whereas sponge tissue shows a low steady-state level, SURVL expression was significantly upregulated in rapidly proliferating primmorph cells. In addition, challenge of tissue and primmorphs with heavy metal or lipopeptide stimulated SURVL expression, concurrent with the expression of a newly discovered caspase. Complementary functional analyses in transfected HEK-293 cells revealed that heterologous expression of a SURVL-EFGP fusion not only promotes proliferation but also enhances resistance to cadmium-induced cell death. Taken together, these results suggest both a deep evolutionary conserved dual role of survivin and an equally conserved central position in the interconnected pathways of cell cycle and apoptosis.

Activation of MAP kinase signaling pathway in the mussel Mytilus galloprovincialis as biomarker of environmental pollution.

Stimulation of MAP kinase signal transduction pathway by various stressful stimuli was investigated in the marine bivalve Mytilus galloprovincialis. Analyses were performed in animals exposed in laboratory to selected pollutants and in mussels collected in winter and summer along the eastern Adriatic coast (Croatia). Effects of oxidative stress, induced by tributyltin, hydrogen peroxide and water soluble fraction of diesel fuel on the activation/phosphorylation of the three Mitogen-Activated Protein Kinases (MAPKs) p38, JNK and ERK using a newly developed ELISA procedure were evaluated. MAP kinase activation was analyzed 1h after exposure of mussels to chemical agents, and after recovery periods of 6 and 24h. Our results clearly indicated that pollutants generated different patterns of induction of the MAPK phosphorylation. Indeed, only pp38 and pJNK were activated with 11, 33 and 100 microg/L TBT, reaching a maximum activation after 6h in seawater following treatment of mussels with 11 microg/L TBT. Treatment with 0.074 and 0.222 mM H2O2 enhanced activation of both p38 and ERK. These two kinases were activated after 1h exposure, followed by a diminution after 6h of recovery in seawater and a reactivation after 24h. The levels of phosphorylated P38 and JNK were increased after mussel exposure with 7.5, 15 and 30% of water soluble fraction of diesel oil. P38 was activated concentration dependently at 1h exposure. Additionally, field study pointed out seasonal differences in MAP kinases activation as mussels collected during summer had a higher enzyme activation state than in winter, as well as sampling site differences which could be correlated to the industrial/tourism activity and environmental stresses (salinity). All the results converge towards MAP kinase signaling pathway being induced by various pollutants in M. galloprovincialis. This signaling cascade should be considered as a possible biomarker of environmental stress and pollution.

Luciferase a light source for the silica-based optical waveguides (spicules) in the demosponge Suberites domuncula.

Two classes of sponges (animal phylum Porifera) possess a siliceous skeleton which is composed of spicules. Studying the optical fiber-mechanical properties of large spicules from hexactinellid sponges (> 5 cm) it was demonstrated that they are effective light-collecting optical fibers. Here, we report that the demosponge Suberites domuncula is provided with a biosensor system composed of the (organic) light producing luciferase and the (inorganic) light transducing silica spicules. The light transmission feature of these smaller spicules (200 microm) has been demonstrated and the ability of sponge tissue to generate light has been proven. Screening for a luciferase gene in S. domuncula was successful; the recombinant luciferase was prepared and shown to be bioactive. The luciferase protein is abundantly present in the close neighborhood of the spicules. The expression of the luciferase gene is under the control of light.

[Silicateins identification of the freshwater sponge L. baicalensis].

Siliceous spicules of the freshwater Baikal sponge Lubomirskia baicalensis contain several proteins including silicateins. Existences of four different genes of silicatein alpha (alpha1, alpha2, alpha3, alpha4) which are related to silicatein alpha from the sea sponges were found when cDNA library analysis was made. The intron-exon structure of the full-size silicatein alpha1 gene was determined. This gene has total length of 1988 bp and includes 6 introns (1007 bp) and 7 exons (981 bp). With use of mass-spectrometric analysis of the spicule proteins tryptic digest, two silicateins alpha were authentically found.

Changing distribution of group A rotavirus G-types and genetic analysis of G9 circulating in Japan.

A total of 1,797 fecal specimens from infants and children with acute gastroenteritis in Japan from July 2000 to June 2003 were tested for group A rotavirus by ELISA, RT-PCR, RNA-PAGE and latex agglutination methods. Of these, 439 were found to be positive for group A rotavirus and this presented 24.4%. In 2000-2001, G1 was the most prevalent (45.5%) followed by G2 (32.5%), G3 (12.3%), G9 (5.9%) and G4 (2.6%). However, G2 was found predominant with 40% in the following year (2001-2002). Interestingly, G9 had a rapid increase of infection up to 17.8%. In 2002-2003, G3 dominated over other G-types with 34%. Another interesting feature of the study was the demonstration of great genetic diversity among G9 strains in Japan. Worth of note was the first prevalence pattern of rotavirus G-types with an increase of G2, G3 as well as G9 and a decrease of G1 during the 20 year-survey of rotavirus infection in Japan.

Monitoring chemical and physical stress using sea urchin immune cells.

Coelomocytes are the cells freely circulating in the body fluid contained in echinoderm coelom and constitute the defence system, which, in response to injuries, host invasion, and adverse conditions, is capable of chemotaxis, phagocytosis, and production of cytotoxic metabolites. Red and colourless amoebocytes, petaloid and philopodial phagocytes, and vibratile cells are the cell types that, in different proportions, constitute the mixed coelomocyte cell population found in sea urchins. Advances in cellular and molecular biology have made it possible to identify a number of specific proteins expressed in coelomocytes under resting conditions or when activated by experimentally induced stress. Only recently, coelomocytes have been used for pollution studies with the aim of introducing a new biosensor for detection of stress at both cellular and molecular levels, as sentinel of sea health. In this chapter, we briefly review the important features of these valuable cells and describe studies on their use in the laboratory and in the field for the assessment of chemical and physical pollution of the sea.

DNA damage and developmental defects after exposure to UV and heavy metals in sea urchin cells and embryos compared to other invertebrates.

The depletion of the stratospheric ozone layer and the resulting increase in hazardous ultraviolet-B (UV-B) radiation reaching the Earth are of major concern not only for terrestrial but also for aquatic organisms. UV-B is able to penetrate clear water to ecologically significant depths. This chapter deals with the effects of UV radiation on DNA integrity in marine benthic organisms, in particular sea urchins in comparison to other marine invertebrates (sponges and corals). These animals cannot escape the damaging effects of UV-B radiation and may be additionally exposed to pollution from natural or anthropogenic sources. Besides eggs and larvae that lack a protective epidermal layer and are particularly prone to the damaging effects of UV radiation, coelomocytes from the sea urchin Paracentrotus lividus were used as a "cellular sensor" to analyse the effects on DNA caused by UV-B, heavy metals (cadmium), and their combined actions. From our data we conclude that sea urchin coelomocytes as well as cells from other marine invertebrates are useful bioindicators of UV-B and heavy metal stress, responding to these stressors with different extents of DNA damage.

New natural products from the sponge-derived fungus Aspergillus niger.

Fractionation of the EtOAc extract of a static culture of Aspergillus niger isolated from the Mediterranean sponge Axinella damicornis yielded eight secondary metabolites, out of which seven compounds (2-8) proved to be new natural products, whereas one was identified as the known fungal pigment cycloleucomelone (1). The new compounds included the 3,3'-bicoumarin bicoumanigrin (2), the structurally unusual 4-benzyl-1H-pyridin-6-one derivatives aspernigrins A and B (3 and 4), and pyranonigrins A-D (5-8), the latter featuring a novel pyrano[3,2-b]pyrrole skeleton hitherto unprecedented in nature. All structures were elucidated on the basis of extensive one- and two-dimensional NMR spectroscopic studies ((1)H, (13)C, COSY, HMQC, HMBC, NOE difference spectra) and mass spectral analysis. For the two chiral molecules 4 and 5, the absolute configurations were established by quantum chemical calculations of their circular dichroism (CD) spectra. In each case, two independent methods, i.e., a molecular dynamics approach taking into consideration the molecular flexibility, and a conformational analysis followed by Boltzmann weighting of the single CD spectra calculated for the conformers thus obtained, led to identical results without the need of any empirical comparison of chiroptical data reported for reference compounds. Bicoumanigrin (2) showed moderate cytotoxicity against human cancer cell lines in vitro. In addition, aspernigrin B (4) was found to display a strong neuroprotective effect against glutamic acid.

Stress-70 proteins in marine mussel Mytilus galloprovincialis as biomarkers of environmental pollution: a field study.

In the present work we have investigated levels of stress-70 proteins in the gills of mussel Mytilus galloprovincialis collected seasonally from subtidal rocky shores at 6 different sites of the Rovinj coastal area (Northern Adriatic, Croatia). 1-D analysis (SDS-PAGE) using monoclonal mouse antibodies anti-HSP70 detected two bands of stress-70 proteins, 70 and 72 kDa constitutively present during the year. 2-D analysis (IEF+SDS-PAGE) proved that the antibodies used detected HSP70 (pI 5.7-5.9) and HSP72 (pI 5.5-5.6). The quantification of stress-70 proteins was possible using 200 ng of external HSP70 protein standard included on every blot. Maximal levels of HSP72 and HSP70 were observed in mussels in summer (September), and minimal levels in winter (December), and only HSP70 showed significant correlation with the sea temperature (r=+0.822, p<0.05). Acclimatization of mussels to a different lower salinity under experimental conditions proved that small changes in sea salinity (Delta=2 psu) could not cause significant stress-70 proteins induction. Results indicated that there are significant differences in HSP70 and HSP72 content in mussels from the control site (S-1) and mussels from other sampling sites with urban and industrial pollution. The usefulness of stress-70 proteins as biomarkers of environmental pollution is discussed.

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula.

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B cells and mutations in the BTK gene cause X-linked agammaglobulinemia. Kinases from the Tec family are not present in Caenorhabditis elegans and, until now, they were found only in insects and higher animal taxa. Our finding implies that the BTK/TEC genes are of a very ancient origin.

The chemokine networks in sponges: potential roles in morphogenesis, immunity and stem cell formation.

Porifera (sponges) are now well accepted as the phylum which branched off first from the common ancestor of all metazoans, the Urmetazoa. The transition to the Metazoa became possible because during this phase, cell-cell as well as cell-matrix adhesion molecules evolved which allowed the formation of a colonial stage of animals. The next prerequisite for the evolution to the Urmetazoa was the establishment of an effective immune system which, flanked by apoptosis, allowed the formation of a first level of individuation. In sponges (with the model Suberites domuncula and Geodia cydonium), the main mediators of the immune responses are the chemokines. Since sponges lack a vascular system and consequently blood cells (in the narrow sense), we have used the term chemokines (in a broad sense) to highlight that the complex network of intercellular mediators initiates besides differentiation processes also cell movement. In the present review, the cDNAs encoding the following chemokines were described and the roles of their deduced proteins during self-self and nonself recognition outlined: the allograft inflammatory factor, the glutathione peroxidase, the endothelial-monocyte-activating polypeptide, the pre-B-cell colony-enhancing factor and the myotrophin as well as an enzyme, the (2-5)A synthetase, which is involved in cytokine response in vertebrates. A further step required to reach the evolutionary step of the integrated stage of the Urmetazoa was the acquisition of a stem cell system. In this review, first markers for stem cells (mesenchymal stem cell-like protein) as well as for chemokines involved in the maintenance of stem cells (noggin and glia maturation factor) are described at the molecular level, and a first functional analysis is approached. Taken together, it is outlined that the chemokine network was essential for the establishment of metazoans, which evolved approximately 600 to 800 million years ago.