Toxicity and inflammatory potential of mineral fibres: The contribute of released soluble metals versus cell contact direct effects.

Asbestos fibres have been considered an environmental hazard for decades. However, little is known about the attempts of circulating immune cells to counteract their toxicity. We addressed the early effects of fibre-released soluble factors (i.e. heavy metals) in naïve immune cells, circulating immediately below the alveolar/endothelial cell layer. By comparison, the direct fibre effects on endotheliocytes were also studied since these cells are known to sustain inflammatory processes. The three mineral fibres analysed showed that mainly chrysotile (CHR) and erionite (ERI) were able to release toxic metals in extracellular media respect to crocidolite (CRO), during the first 24 h. Nevertheless, all three fibres were able to induce oxidative stress and genotoxic damage in indirectly challenged naïve THP-1 monocytes (separated by a membrane). Conversely, only CHR-released metal ions induced apoptosis, NF-κB activation, cytokines and CD163 gene overexpression, indicating a differentiation towards the M0 macrophage phenotype. On the other hand, all three mineral fibres in direct contact with HECV endothelial cells showed cytotoxic, genotoxic and apoptotic effects, cytokines and ICAM-I overexpression, indicating the ability of these cells to promote an inflammatory environment in the lung independently from the type of inhaled fibre. Our study highlights the different cellular responses to mineral fibres resulting from both the nature of the cells and their function, but also from the chemical-physical characteristics of the fibres. In conclusion, CHR represented the main pro-inflammatory trigger, able to recruit and activate circulating naïve monocytes, through its released metals, already in the first 24 h after inhalation.

The influence of cation exchange on the possible mechanism of erionite toxicity: A synchrotron-based micro-X-ray fluorescence study on THP-1-derived macrophages exposed to erionite-Na.

Fibrous erionite is the only zeolite classified as Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Carcinogenesis induced by erionite is thought to involve several factors as biopersistence, the iron role and cation exchange processes. To better understand these mechanisms, a detailed investigation at the micro scale was performed, collecting elemental information on iron and cation release and their distribution in biological systems by synchrotron micro-X-ray fluorescence mapping (SR-micro-XRF) and synchrotron micro-X-ray absorption spectroscopy (SR-micro-XANES) at the TwinMic beamline (Elettra synchrotron) and at the ID21 beamline of the European Synchrotron Radiation Facility (ESRF). By microscopy and chemical mapping, highly detailed maps of the chemical and morphological interaction of biological systems with fibres could be produced. In detail, THP-1 cell line derived macrophages, used as in vitro model, were analysed during erionite-Na phagocytosis at different time intervals, after single dose exposure. For comparison, cellular fluorescent probes were also used to evaluate the intracellular free sodium and calcium concentrations. Synchrotron analyses visualised the spatial distribution of both fibre and mineral particle associated metals during the phagocytosis, describing the mechanism of internalisation of erionite-Na and its accessory mineral phases. The intracellular distribution of metals and other cations was mapped to evaluate metal release, speciation changes and/or cation exchange during phagocytosis. The fluorescent probes complemented microchemical data clarifying, and confirming, the cation distribution observed in the SR-micro-XRF maps. The significant cytoplasmic calcium decrease, and the concomitant sodium increase, after the fibre phagocytosis seemed due to activation of plasma membrane cations exchangers triggered by the internalisation while, surprisingly, the ion-exchange capacity of erionite-Na could play a minor role in the disruption of the two cations intracellular homeostasis. These results help to elucidate the role of cations in the toxicity of erionite-treated THP-1 macrophages and add knowledge to its carcinogenicity process.

Chitosan-Collagen Electrospun Nanofibers Loaded with Curcumin as Wound-Healing Patches.

Composite chitosan-collagen nanofibrous mats embedded with curcumin were prepared via a single-step electrospinning procedure and explored as wound-healing patches with superior biological activity. A mild crosslinking protocol consisting of a short exposure to ammonia vapor and UV radiation was developed to ensure proper stability in physiological-like conditions without affecting the intrinsic biocompatibility of chitosan and collagen. The fabricated composite patches displayed a highly porous, homogeneous nanostructure consisting of fibers with an average diameter of 200 nm, thermal stability up to 200 °C, mechanical features able to ensure protection and support to the new tissues, and water-related properties in the ideal range to allow exudate removal and gas exchange. The release kinetic studies carried out in a simulated physiological environment demonstrated that curcumin release was sustained for 72 h when the mats are crosslinked hence providing prolonged bioactivity reflected by the displayed antioxidant properties. Remarkably, combining chitosan and collagen not only ensures prolonged stability and optimal physical-chemical properties but also allows for better-promoting cell adhesion and proliferation and enhanced anti-bacteriostatic capabilities with the addition of curcumin, owing to its beneficial anti-inflammatory effect, ameliorating the attachment and survival/proliferation rates of keratinocytes and fibroblasts to the fabricated patches.

Photoprotective and Anti-Aging Properties of the Apical Frond Extracts from the Mediterranean Seaweed Ericaria amentacea.

There is a growing interest in using brown algal extracts thanks to the bioactive substances they produce for adaptation to the marine benthic environment. We evaluated the anti-aging and photoprotective properties of two types of extracts (50%-ethanol and DMSO) obtained from different portions, i.e., apices and thalli, of the brown seaweed, Ericaria amentacea. The apices of this alga, which grow and develop reproductive structures during summer when solar radiation is at its peak, were postulated to be rich in antioxidant compounds. We determined the chemical composition and pharmacological effects of their extracts and compared them to the thallus-derived extracts. All the extracts contained polyphenols, flavonoids and antioxidants and showed significant biological activities. The hydroalcoholic apices extracts demonstrated the highest pharmacological potential, likely due to the higher content of meroditerpene molecular species. They blocked toxicity in UV-exposed HaCaT keratinocytes and L929 fibroblasts and abated the oxidative stress and the production of pro-inflammatory cytokines, typically released after sunburns. Furthermore, the extracts showed anti-tyrosinase and anti-hydrolytic skin enzyme activity, counteracting the collagenase and hyaluronidase degrading activities and possibly slowing down the formation of uneven pigmentation and wrinkles in aging skin. In conclusion, the E. amentacea apices derivatives constitute ideal components for counteracting sunburn symptoms and for cosmetic anti-aging lotions.

The Acute Toxicity of Mineral Fibres: A Systematic In Vitro Study Using Different THP-1 Macrophage Phenotypes.

Alveolar macrophages are the first line of defence against detrimental inhaled stimuli. To date, no comparative data have been obtained on the inflammatory response induced by different carcinogenic mineral fibres in the three main macrophage phenotypes: M0 (non-activated), M1 (pro-inflammatory) and M2 (alternatively activated). To gain new insights into the different toxicity mechanisms of carcinogenic mineral fibres, the acute effects of fibrous erionite, crocidolite and chrysotile in the three phenotypes obtained by THP-1 monocyte differentiation were investigated. The three mineral fibres apparently act by different toxicity mechanisms. Crocidolite seems to exert its toxic effects mostly as a result of its biodurability, ROS and cytokine production and DNA damage. Chrysotile, due to its low biodurability, displays toxic effects related to the release of toxic metals and the production of ROS and cytokines. Other mechanisms are involved in explaining the toxicity of biodurable fibrous erionite, which induces lower ROS and toxic metal release but exhibits a cation-exchange capacity able to alter the intracellular homeostasis of important cations. Concerning the differences among the three macrophage phenotypes, similar behaviour in the production of pro-inflammatory mediators was observed. The M2 phenotype, although known as a cell type recruited to mitigate the inflammatory state, in the case of asbestos fibres and erionite, serves to support the process by supplying pro-inflammatory mediators.

Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy.

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 µg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.

An Innovative In Vitro Open-Angle Glaucoma Model (IVOM) Shows Changes Induced by Increased Ocular Pressure and Oxidative Stress.

Primary Open-Angle Glaucoma (POAG) is a neurodegenerative disease, and its clinical outcomes lead to visual field constriction and blindness. POAG's etiology is very complex and its pathogenesis is mainly explained through both mechanical and vascular theories. The trabecular meshwork (TM), the most sensitive tissue of the eye anterior segment to oxidative stress (OS), is the main tissue involved in early-stage POAG, characterized by an increase in pressure. Preclinical assessments of neuroprotective drugs on animal models have not always shown correspondence with human clinical studies. In addition, intra-ocular pressure management after a glaucoma diagnosis does not always prevent blindness. Recently, we have been developing an innovative in vitro 3Dadvanced human trabecular cell model on a millifluidicplatform as a tool to improve glaucoma studies. Herein, we analyze the effects of prolonged increased pressure alone and, in association with OS, on such in vitro platform. Moreover, we verify whethersuch damaged TM triggers apoptosis on neuron-like cells. The preliminary results show that TM cells are less sensitive to pressure elevation than OS, and OS-damaging effects were worsened by the pressure increase. The stressed TM releases harmful signals, which increase apoptosis stimuli on neuron-like cells, suggesting its pivotal role in the glaucoma cascade.

Effect of sodium alginate molecular structure on electrospun membrane cell adhesion.

Alginate-based electrospun nanofibers prepared via electrospinning technique represent a class of materials with promising applications in the biomedical and pharmaceutical industries. However, to date, the effect of alginate molecular mass and block composition on the biological response of such systems remains to some extent unclear. As such, in the present work, three alginates (i.e., M.pyr, L.hyp, A.nod) with different molecular features are employed to prepare nanofibers whose ability to promote cell adhesion is explored by using both skin and bone cell lines. Initially, a preliminary investigation of the raw materials is carried out via rheological and zeta-potential measurements to determine the different grade of polyelectrolyte behaviour of the alginate samples. Specifically, both the molecular mass and block composition are found to be important factors affecting the alginate response, with long chains and a predominance of guluronic moieties leading to a marked polyelectrolyte nature (i.e., lower dependence of the solution viscosity upon the polymer concentration). Subsequently, physically crosslinked alginate nanofibrous mats are first morphologically characterized via both scanning electron and atomic force microscopy, which show a homogenous and defect-free structure, and their biological response is then evaluated. Noticeably, fibroblast and keratinocyte cell lines do not show significant differences in terms of cell adhesion on the three mats (i.e., 30-40% and 10-20% with respect to the seeded cells, respectively), with the formers presenting a greater affinity toward the alginate-based nanofibers. Conversely, both the investigated osteoblast cells are characterized by a distinct behaviour depending on the alginate type. Specifically, polysaccharide samples with an evident polyelectrolyte nature are found to better promote cell viability (i.e., cell adhesion in the range 15-36% with respect to seeded cells) compared to the ones displaying a nearly neutral behaviour (i.e., cell adhesion in the range 5-25% with respect to seeded cells). Therefore, the obtained results, despite being preliminary, suggest that the alginate type (i.e., molecular structure properties) may play a topical role in conditioning the efficiency of healing patches for bone reparation, but it has a negligible effect in the case of skin regeneration.

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.

In vitro toxicity of fibrous glaucophane.

The health hazard represented by the exposure to asbestos may also concern other minerals with asbestos-like crystal habit. One of these potentially hazardous minerals is fibrous glaucophane. Fibrous glaucophane is a major component of blueschist rocks of California (USA) currently mined for construction purposes. Dust generated by the excavation activities might potentially expose workers and the general public. The aim of this study was to determine whether fibrous glaucophane induces in vitro toxicity effects on lung cells by assessing the biological responses of cultured human pleural mesothelial cells (Met-5A) and THP-1 derived macrophages exposed for 24 h and 48 h to glaucophane fibres. Crocidolite asbestos was tested for comparison. The experimental configuration of the in vitro tests included a cell culture without fibres (i.e., control), cell cultures treated with 50 µg/mL (i.e., 15.6 µg/cm2) of crocidolite fibres and 25-50-100 µg/mL (i.e., 7.8-15.6-31.2 µg/cm2) of glaucophane fibres. Results showed that fibrous glaucophane may induce a decrease in cell viability and an increase in extra-cellular lactate dehydrogenase release in the tested cell cultures in a concentration dependent mode. Moreover, it was found that fibrous glaucophane has a potency to cause oxidative stress. The biological reactivity of fibrous glaucophane confirms that it is a toxic agent and, although it apparently induces lower toxic effects compared to crocidolite, exposure to this fibre may be responsible for the development of lung diseases in exposed unprotected workers and population.

The Remarkable Antioxidant and Anti-Inflammatory Potential of the Extracts of the Brown Alga Cystoseira amentacea var. stricta.

Inflammation and oxidative stress are part of the complex biological responses of body tissues to harmful stimuli. In recent years, due to the increased understanding that oxidative stress is implicated in several diseases, pharmaceutical industries have invested in the research and development of new antioxidant compounds, especially from marine environment sources. Marine seaweeds have shown the presence of many bioactive secondary metabolites, with great potentialities from both the nutraceutical and the biomedical point of view. In this study, 50%-ethanolic and DMSO extracts from the species C. amentacea var. stricta were obtained for the first time from seaweeds collected in the Ligurian Sea (north-western Mediterranean). The bioactive properties of these extracts were then investigated, in terms of quantification of specific antioxidant activities by relevant ROS scavenging spectrophotometric tests, and of anti-inflammatory properties in LPS-stimulated macrophages by evaluation of inhibition of inflammatory cytokines and mediators. The data obtained in this study demonstrate a strong anti-inflammatory effect of both C. amentacea extracts (DMSO and ethanolic). The extracts showed a very low grade of toxicity on RAW 264.7 macrophages and L929 fibroblasts and a plethora of antioxidant and anti-inflammatory effects that were for the first time thoroughly investigated. The two extracts were able to scavenge OH and NO radicals (OH EC50 between 392 and 454 µg/mL; NO EC50 between 546 and 1293 µg/mL), to partially rescue H2O2-induced RAW 264.7 macrophages cell death, to abate intracellular ROS production in H2O2-stimulated macrophages and fibroblasts and to strongly inhibit LPS-induced inflammatory mediators, such as NO production and IL-1α, IL-6, cyclooxygenase-2 and inducible NO synthase gene expression in RAW 264.7 macrophages. These results pave the way, for the future use of C. amentacea metabolites, as an example, as antioxidant food additives in antiaging formulations as well as in cosmetic lenitive lotions for inflamed and/or damaged skin.

Can Polyphenols in Eye Drops Be Useful for Trabecular Protection from Oxidative Damage?

Polyphenols, with anti-oxidant properties, counteract oxidative stress effects. Increasing evidence has found oxidative stressto be the main risk factor for trabecular meshwork (TM) damage, leading to high-tension glaucoma. Topical anti-oxidants could represent a new target for glaucoma treatment. Our aim is to investigate the protective mechanisms on a human TM culture of a patented polyphenol and fatty acid (iTRAB®)formulation in response to oxidative stress using an advanced invitromodel consisting of 3D-human TM cells, embedded in a natural hydrogel, and a milli-scaled multi-organ device model for constantdynamic conditions. The 3D-human TM cells(3D-HTMCs) were treated daily with 500 µM H2O2or 500 µM H2O2and 0.15% iTRAB®(m/v) for 72 h, and molecular differences in the intracellular reactive oxygen species (iROS), state of the cells, activation of the apoptosis pathway and NF-kB and the expression ofinflammatory and fibrotic markers wereanalyzed at different time-points.Concomitant exposure significantly reduced iROS and restored TM viability, iTRAB® having a significant inhibitory effect on the apoptotic pathway, activation of NF-κB, induction of pro-inflammatory (IL-1α, IL-1ß and TNFα) and pro-fibrotic (TGFß) cytokines and the matrix metalloproteinase expressions. It is clear that this specific anti-oxidant provides a valid TM protection, suggesting iTRAB® could be an adjuvant therapy in primary open-angle glaucoma (POAG).

Identification, Purification and Molecular Characterization of Chondrosin, a New Protein with Anti-tumoral Activity from the Marine Sponge Chondrosia Reniformis Nardo 1847.

: Chondrosia reniformis is a common marine demosponge showing many peculiarities, lacking silica spicules and with a body entirely formed by a dense collagenous matrix. In this paper, we have described the identification of a new cytotoxic protein (chondrosin) with selective activity against specific tumor cell lines, from C. reniformis, collected from the Liguria Sea. Chondrosin was extracted and purified using a salting out approach and molecular weight size exclusion chromatography. The cytotoxic fractions were then characterized by two-dimensional gel electrophoresis and mass spectrometry analysis and matched the results with C. reniformis transcriptome database. The procedure allowed for identifying a full-length cDNA encoding for a 199-amino acids (aa) polypeptide, with a signal peptide of 21 amino acids. The mature protein has a theoretical molecular weight of 19611.12 and an IP of 5.11. Cell toxicity assays showed a selective action against some tumor cell lines (RAW 264.7 murine leukemia cells in particular). Cell death was determined by extracellular calcium intake, followed by cytoplasmic reactive oxygen species overproduction. The in silico modelling of chondrosin showed a high structural homology with the N-terminal region of the ryanodine receptor/channel and a short identity with defensin. The results are discussed suggesting a possible specific interaction of chondrosin with the Cav 1.3 ion voltage calcium channel expressed on the target cell membranes.

Corrigendum to An advanced in vitro model to assess glaucoma onset.

In this manuscript, which appeared in ALTEX 37, 265-274 (doi: 10.14573/altex.1909262), the affiliation of Stefania Vernazza should read: Stefania Vernazza 5# 5 IRCCS-Fondazione Bietti, Rome, Italy and the address for correspondence should read: Stefania Vernazza, PhD, IRCCS, Fondazione Bietti via Livenza 3, 00198 Rome, Italy (stefania.vernazza@yahoo.it).

An advanced in vitro model to assess glaucoma onset.

Glaucoma is the second leading cause of blindness worldwide. Currently, glaucoma treatments aim to lower intraocular pressure by decreasing aqueous humor production or increasing aqueous humor outflow through pharmacological approaches or trabeculectomy. The lack of an effective cure requires new therapeutic strategies. We compared the bio­logical responses of a three-dimensional trabecular meshwork model with or without perfusion bioreactor technology to better understand the early molecular changes induced by prolonged oxidative stress conditions induced by repeated daily peroxide exposure. We used standard 3D cultures of trabecular meshwork cells in Matrigel cultured under either static and dynamic conditions for one week. We studied changes in F-actin expression and organization in the cells, cellular metabolic activity, proinflammatory gene expression, expression of pro- and anti-apoptotic proteins, PARP-1 cleavage, and NFκB activation in the model. We demonstrate that the dynamic conditions improve the adaptive behavior of 3D trabecular meshwork cultures to chronic oxidative stress via offsetting pathway activation.

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 ± 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 × 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.

2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study.

A physiologically relevant in vitro human-based model could be the 'gold standard' to clarify the pathological steps involved in glaucoma onset. In this regard, human 3D cultures may represent an excellent starting point to achieve this goal. Indeed, the 3D matrix allows to re-create the in vivo-like tissue architecture, maintaining its functionality and cellular behaviour, compared to the 2D model. Thus, we propose a comparison between the 2D and 3D in vitro models of human trabecular meshwork cells in terms of cellular responses after chronic stress exposure. Our results showed that 3D-cells are more sensitive to intracellular reactive oxidative specie production induced by hydrogen peroxide treatment, compared to 2D cultures. Additionally, in 3D cultures a more accurate regulation of the apoptosis trigger and cell adaptation mechanisms was detected than in 2D models. In line with these findings, the 3D-HTMC model shows the ability to better mimic the in vivo cell behaviour in adaptive responses to chronic oxidative stress than 2D.

First identification of a fatal fungal infection of the marine sponge Chondrosia reniformis by Aspergillus tubingensis.

Sponges are considered promising sources of biomolecules for both pharmaceutical and cosmetic interests as well as for the production of biomaterials suitable for tissue engineering and regenerative medicine. Accordingly, the ability to grow sponges in captivity and in healthy conditions to increase their biomass is a required goal for the development of sponge aquaculture systems. To date, little information is available about the pathogenicity of fungi associated with sponges. In our study, we identified an infection in freshly collected specimens of Chondrosia reniformis (Porifera, Demospongiae) and determined that the fungus Aspergillus tubingensis was the pathogen responsible. This is the first description of a natural infection of C. reniformis by A. tubingensis. Despite raising an inflammatory response by means of an increase in tumour necrosis factor (TNF) mRNA, the infected C. reniformis specimens were not able to control the fungal infection, leading to rotting in 15 d. Characterization of this infection shows that a widely distributed fungus can represent a potential hazard to sponge aquaculture industries and how, especially in stressed or compromised marine environments, this fungus could represent a fatal opportunistic pathogen.

Insights into the evolution of metazoan regenerative mechanisms: roles of TGF superfamily members in tissue regeneration of the marine sponge Chondrosia reniformis.

Tissue repair is an adaptive and widespread metazoan response. It is characterised by different cellular mechanisms and complex signalling networks that involve numerous growth factors and cytokines. In higher animals, transforming growth factor-ß (TGF-ß) signalling plays a fundamental role in wound healing. In order to evaluate the involvement of TGF superfamily members in lower invertebrate tissue regeneration, sequences for putative TGF ligands and receptors were isolated from the transcriptome of the marine sponge Chondrosia reniformis We identified seven transcripts that coded for TGF superfamily ligands and three for TGF superfamily receptors. Phylogenetically, C. reniformis TGF ligands were not grouped into any TGF superfamily clades and thus presumably evolved independently, whereas the TGF receptors clustered in the Type I receptor group. We performed gene expression profiling of these transcripts in sponge regenerating tissue explants. Data showed that three ligands (TGF1, TGF3 and TGF6) were mainly expressed during early regeneration and seemed to be involved in stem cell maintenance, whereas two others (TGF4 and TGF5) were strongly upregulated during late regeneration and thus were considered pro-differentiating factors. The presence of a strong TGF inhibitor, SB431542, blocked the restoration of the exopinacoderm layer in the sponge explants, confirming the functional involvement of the TGF pathway in tissue regeneration in these early evolved animals.

Simulated microgravity induces nuclear translocation of Bax and BCL-2 in glial cultured C6 cells.

Alterations in the control of apoptotic processes were observed in cells during space flight or under simulated microgravity, the latter obtained with the 3D-Random Positioning Machine (3D-RPM). Usually the proteins Bax and Bcl-2, act as pro- or anti-apoptotic regulators. Here we investigated the effects of simulated microgravity obtained by the 3D-RPM on cell viability, localization and expression of Bax and Bcl-2 in cultures of glial cancerous cells. We observed for the first time a transient cytoplasmic/nuclear translocation of Bax and Bcl-2 triggered by changing gravity vector. Bax translocates into the nucleus after 1 h, is present simultaneously in the cytoplasm after 6 h and comes back to the cytoplasm after 24 h. Bcl-2 translocate into the nucleus only after 6 h and comes back to the cytoplasm after 24 h. Physiological meaning, on the regulation of apoptotic event and possible applicative outcomes of such finding are discussed.