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Sponge Enzymes and Cells for Innovative AppLications

Final Report Summary - SPECIAL (Sponge Enzymes and Cells for Innovative AppLications)

Executive Summary:
The SPECIAL project aimed at delivering breakthrough technologies for the biotechnological production of cellular metabolites and extracellular biomaterials from marine sponges. These include a platform technology to produce secondary metabolites from a wide range of sponge species, a novel in vitro method for the production of biosilica and recombinant technology for the production of marine collagen.

Research on cellular metabolites was based upon our previous finding that non-growing sponges continuously release large amounts of cellular material. Production of biosilica was realized through biosintering, a novel enzymatic process that was recently discovered in siliceous sponges. Research on sponge collagen focused on finding the optimal conditions for expression of the related genes.

Alongside this research, the project has identified and developed new products from sponges, thus realizing the promises of marine biotechnology. Specifically, the project focused on potential anticancer drugs and novel biomedical/industrial applications of biosilica and collagen, hereby taking advantage of the unique physico-chemical properties of these extracellular sponge products.

The consortium unites seven world-class research institutions covering a wide range of marine biotechnology-related disciplines and four knowledge-intensive SMEs that are active in the field of sponge culture, drug development and nanobiotechnology.

The project is clearly reflecting the strategic objectives outlined in the position paper European Marine Strategy (2008); it definitely contributed to enhance marine biotechnology at a multi-disciplinary, European level, while providing new opportunities for the European industry to exploit natural marine resources in a sustainable way. In particular the biotechnological potential of marine sponges, which has for a long time been considered as an eternal promise, is being realized with the findings and outputs from the SPECIAL project.
Project Context and Objectives:
1.2.1 Introduction: a SPECIAL focus on sponges
The sea harbours a rich biodiversity, which provides a plentiful resource of potential new products for society. Yet, after their initial discovery, no progress is made regarding the advancement of many of these marine natural products because biological materials needed for consecutive development cannot be sufficiently supplied. In particular sponges, that are extremely rich in natural products, are considered as a promising biological resource that cannot be fully exploited because they are notoriously difficult to culture. Therefore, sponges are the most logical target for a project that addresses bottlenecks in culture methods for marine organisms. Hence, the main objective of the SPECIAL proposal was to establish sustainable, controlled technologies for the production of sponge materials. Hereby, we distinguish two types of sponge products to which different biotechnological approaches were applied:

1. Intracellularly produced compounds - secondary metabolites that may serve as leads for pharmaceutical drugs. In this proposal, we have focused on potential anticancer compounds. Cancer remains a major problem for human health and many potential cures have been found in marine sponges. To enable the development of these anticancer compounds and other intracellular sponge products, a generic technique to produce small quantities of sponge biomass under controlled conditions is urgently needed. Based upon a recent breakthrough in our understanding of sponge cell proliferation, we established such a platform technology within the SPECIAL project. Such a platform technique can provide the materials necessary to execute the initial steps in drug discovery and development.

2. Extracellularly produced biomaterials such as collagen and biosilica. There is an increasing demand for alternative sources of collagen, which are needed for a wide variety of applications (food, cosmetics, healthcare). Sponge collagen has unique physico-chemical properties and is as such a promising resource, but sponge collagen is not available in large quantities. Sponges have also been noted to produce many unique biosilica structures that provide a magnificent source of inspiration for novel products in various fields of biomimetic application. In contrast to intracellular metabolites, which usually have a very complex biosynthesis, most extracellular products may be produced through (recombinant) enzymatic processes in vitro, thus preventing the need to culture the whole organism. Regarding these in vitro production techniques, SPECIAL has addressed specific bottlenecks related to productivity and controllability. Moreover, we have developed new enzymatic processes to produce biosilica based materials upon the completely novel concept of biosintering. From biosintering, the creation of micro-electronic and nanomechanical/nano-optical elements will become possible as their initial steps have been addressed in the scope of SPECIAL. Unprecedented hybrid polymer/biosilica - based biomedical applications also derived by the joint expertise of the consortium. Improved mariculture protocols specifically optimised for the bulk production of sponge collagen have been concomitantly implemented and different methodologies for the extraction of sponge collagen have been evaluated.

By addressing these topics, SPECIAL have demonstrated that biotechnology-based development is a feasible and sustainable avenue towards the commercialisation of natural products from marine sponges.

1.2.2 Approach: what makes the SPECIAL proposal so special
The SPECIAL project combines discovery and development of sponge-derived products with studies on methods related to the production of these products. Whereas the main emphasis was on production methods, concurrent efforts on discovery and development were deemed as needed to complete the innovation chain and thus fully demonstrate the concept of sustainable biotechnology-based product development.

In SPECIAL, we have developed a generic technology to produce small amounts of biomass of a large variety of sponge species, which were then considered to access the potential of intracellularly produced compounds. The technology is based upon our recent breakthrough finding that, in non-growing sponges, one particular cell type proliferates at amazingly high rates, the excess production of cellular material being released into the environment. To evaluate the applicability of this massive cell release as a platform technique for producing sponge biomass, we have firstly identified the presence of this phenomenon in other sponges species (WP2), in particular among the ones that bear compounds showing anticancer activity (WP6). Hereby, we have initially focused on species with previously identified anticancer compounds that are already under study within the SPECIAL consortium. Concurrently, we have searched for new anticancer compounds and rapid cell proliferation in sponges obtained from habitats that have hitherto been somewhat neglected (WP1). These two parallel activities have allowed us to identify a number of species that show both anticancer activity and rapid cell proliferation. These elective species can serve as targets for consecutive research and development. The percentage of double-positive species also shows the general applicability of the new technique.

Production of extracellular sponge materials have been based upon novel approaches to use enzymes for in vitro biomineralization (WP5). It was recently discovered that siliceous sponges exhibit enzymatic varieties of sintering processes (these are processes whereby a powder is transformed into a bulk material by thermal treatment just below the melting temperature of the main constituent material). Sintering usually needs high temperatures. In sponge spicules, sintering of “biosilica glass” is observed at low temperature and low pressure (“biosintering”). Biosintering is a completely new concept to produce tailor-made siliceous structures that provides a large array of opportunities to generate new products (implemented in WP7).

For controlled production of the other targeted extracellular sponge biomaterial (collagen), two alternative approaches have been followed: i) A recombinant, enzymatic process to produce medical grade collagen under completely controlled conditions (WP4), and ii) Development of mariculture techniques for high collagen content sponge species, thus allowing extraction of collagen without jeopardizing natural populations (WP3). Since considerable efforts have been taken to produce sponge collagen, the project provided as well an excellent opportunity to develop new, collagen-based biomedical applications such as innovative biomaterials acting as scaffolds in bone regeneration approaches and highly innovative hybrid polymer/biosilica - based biomedical applications (WP7).

The approach outlined above is reflected in the following project objectives:

• To collect sponge materials from neglected habitats for consecutive screening for anticancer compounds and new types of collagen (WP1);

• To establish a technique to produce sponge materials through rapid cell proliferation (and release) (WP2);

• To establish suitable mariculture methods for the large-scale production of two species of sponges for further collagen extraction (WP3);

• To proceed towards recombinant production of medical grade sponge collagen (WP4);

• To proceed towards the in vitro production of sponge-based biosilica (WP5);

• To execute anticancer drug development studies using biotechnologically produced sponge metabolites (WP6);

• To develop biomedical applications using biotechnologically produced extracellular sponge materials and novel biosensor approaches (WP7).


The SPECIAL team was excellently suited to execute the proposed work: it united world-class researchers in the fields of sponge culture, sponge enzymes, biomineralization processes and their application, marine bioprospecting, sponge taxonomy, biomaterials and tissue engineering, natural product chemistry and cancer research. The large amount of proprietary innovative know-how embedded in this team was a further added value.

The team was composed of seven research institutions that are leading in their respective fields, supplemented with four knowledge-intensive European SMEs. It included a partner form an International Cooperation Partner Country (ICPC – China) and a partner located in an Outermost European Region (The Azores). These partners both added specific expertise to the consortium that was deemed necessary for a proper execution of the project.

All activities within SPECIAL have been pursued in full compliance with ethical and legal principles of environmental and social sustainability. Sampling, handling, maintenance and experimental operations met national and international regulations related to the use and the conservation of natural resources. Sampling from wild populations was in any case limited to strictly needed quantities and has served only as a starting point for product discovery and culture studies.
All partners, either from EU Member States (including the Outermost European Regions), Associate Countries, or ICPC, have been granted Intellectual Property Rights (IPR) on the project foreground that they have (co)developed. In this way, we have developed routes and technologies for the sustainable exploitation of marine living resources, including those from ICPC.

1.2.3 Outcomes: breakthroughs in sponge biotechnology
The SPECIAL project has delivered a cell proliferation-based platform technique for the production of intracellular compounds and new enzymatic techniques to produce extracellular biomaterials. Both can be regarded as major breakthroughs in sponge biotechnology. As a side strategy for the bulk production of sponge collagen, specific mariculture protocols have been concomitantly implemented. Additionally, the project has also delivered leads for anticancer drugs and new biomedical (and industrial) applications based on silica and collagen. For further implementation of the results after the project, the team is engaged in an endeavour for a new research project (addressing some of the scientific challenges that arose from SPECIAL), while taking advantage of its excellent network of established connections with relevant industrial parties, integrated in an exploitation strategy refined during SPECIAL. Extensive dissemination activities to the scientific community, as well as outreach focusing the laypeople have been an integral part of SPECIAL, contributing to the momentum on marine biotechnology and its potentials, to reinforce the knowledge and innovation capacity of the scientific community on sponge materials and derived applications in particular but also on marine natural products in general, and to increase the awareness of laypeople in science innovations and the wide potential of the Sea.
Project Results:
WP1 - Collection of biological materials – sponges
• Mediterranean Sea
The East Mediterranean (EM) has been sampled by TAU at 10 sites along the Israeli Mediterranean coast as well as one site in the Italian coast. On six occasions samples were collected beyond SCUBA diving depth (once using Trimix and five times using ROV of a private NGO). Five sites along the West Mediterranean (WM) Italian coast have been sampled by GAIA: three sites in the Ligurian Sea (13 sampling times), one site in the Southern Tyrrhenian Sea (2 sampling times), and one site in the Adriatic Sea (1 sampling time). These latter sites cover deep habitats (beyond SCUBA-diving and Trimix-range, sampled four times), caves and a cold seep, sponge-rich environment (“Tegnue di Chioggia”, Adriatic Sea). All samples were photographed in situ, collected and taken to the lab on ice. Here they were frozen after sub-samples (vouchers) were separated and preserved in 95% ethanol for taxonomic (morphological and molecular) identification.
These collections did not retrieve a very high diversity of species (with about 75 species in total), which proved especially true for sampling by SCUBA diving, beyond-SCUBA diving and Trimix. However the target species were found, collected and further treated in the laboratory. Samples (especially of the targeted species) were extracted and separation was done, regarding tasks in other WPs (WP6). Sponges that showed interesting bioactivity, or from which promising microorganisms could be isolated, were recollected. Indeed, besides sponge extracts, 843 bacterial isolates to be examined for their chemical activity were derived from Mediterranean target sponges, cultured and sequenced.
One reason that may explain the relatively low biodiversity sampled is that target sponges were supposed to be large enough to allow further processing, and extraction. This implies that complexes of small encrusting sponges that probably harbour a relatively high biodiversity were largely untapped. To explore for variability into these relatively less diverse sponge collections, one strategy was to sample at different depths, seasons and locations (including “extreme” habitats so far not explored for bioprospecting in the studied sites). Where needed, sampling directed to already collected and new specimens was repeated and extended.
In spite of the relatively low diversity recorded in SCUBA diving, beyond SCUBA diving and Trimix sampling sessions, the newly discovered EM deep reef (sampled by ROV) was surprisingly rich in sponge biodiversity and biomass. This was an encouraging result, as the species residing there are either first records to that part of the Mediteranean (usually known in WM at shallower depths) or species that decades ago went extinct at shallower depth and now have been found in deeper water. The collection in EM that targeted sponges of sizes suitable for chemical extraction, yielded about 50 demosponge species (including 13 from 100 m).



• Red Sea
TAU sampling in the Red sea has been done at one site. All samples were frozen after sub-samples (vouchers) were separated for taxonomical (morphological and molecular) identification. The collection work focused on recollecting four species, which in anti-cancer tests showed promising results. From Red Sea targeted sponge species 1153 bacterial isolates were cultured (and sequenced) to be examined for their chemical activity.
The ROV purchased by Porifarma arrived to Israel on February 2012 to be employed for samples collection in the Red Sea. For assembling the ROV and to operate it PF & TAU assigned people and Seamor (the company that built the custom made ROV) sent one of their personnel to assist in the initial stages of operating the ROV, This ROV failed to work even after repeated trials of repairs by the manufacturer. Therefore another ROV was rented and used for the research.

• Azores
Sampling surveys were conducted by SCUBA diving in several localities along both the North (Mosteiros, Porto das Capelas, Baía de São Vicente, Porto da Ribeirinha) and South coasts (Ilhéu de São Roque, Caloura) of São Miguel Island between 10-25 m depth. Encountered morphotypes were scored as to their relative abundance in each site as well as to their general size. Specimens were photographed in situ and a small fragment of each morphotype was collected for taxonomic assignment. After taxonomic identification a second round of dives was performed and the most abundant species were re-collected to be supplied to the other partners. In the lab small sub-samples (preserved in 96% ethanol) were kept as vouchers for taxonomic identification. These are deposited at the Zoological Collections of the Biology Department of the University of the Azores (DBUAc.Por). The remainder samples were stored at -80º C for 1 week and freeze-dried during 2 days (Labconco freezone 2.5).
A total of 71 specimens were collected in the course of the sampling campaigns performed in São Miguel Island. Of these, specimens of Thymosia guernei (Chondrosida, Chondrillidae) were supplied fresh to UMINHO for collagen extraction whereas 23 specimens were freeze-dried and supplied to TAU for chemical extraction.
An ROV sampling campaign was set-up in São Miguel Island in June 2012 as a joint activity between UAzores and PF partner. However, after several diving attempts major technical problems forced the campaign to an end. No samples from deeper areas were thus collected.
• Caribbean and Zanzibar
Sampling in the Caribbean was made by TAU and PF. Some samples whose extracts proved to be of interest in WP6 were recollected (10 samples of 4 species). All samples were frozen after sub-samples (vouchers) were separated for taxonomical (morphological and molecular) identification.
On March 2013 a small group went to Zanzibar in order to collect four sponge species that have shown promissing results in anti-cancer tests evaluation. Overall 31 species have been collected which included the desired Theonella conica, Haliclona debilis and Haliclona bawiana that were retirieved, while a fourth species apparently underwent local extinction due to sea urchins population explosion.

• Morphological and molecular identification of sponge specimens
In this Task we aimed at implementing a DNA-assisted taxonomic system by integrating both morphological and molecular methods for species identification. For such purpose, data regarding samples collection, external and internal morphological characters as well as molecular barcodes were generated and integrated in a database built for the project in Filemaker Pro. Two batches of samples were received from GAIA for identification. The first batch contained 63 specimens that were morphologically identified as belonging to 27 taxa. The second batch contained 96 specimens that were assigned to 38 taxa. Not all specimens could be identified down to species level and some may in fact constitute new undescribed species. A subset of the generated data, including morphological descriptions and COI sequences, was submitted to the Sponge Barcoding Project (SBP - http://www.spongebarcoding.org/). A total of 38 species (3 Calcarea, 2 Homoscleromorpha and 33 Demospongiae) were identified from the material collected in São Miguel Island. In addition, 9 species were identified from the material collected in Curaçao by PF. GAIA sent a total of 159 specimens collected in the Mediterranean for identification. These were assigned 44 taxa distributed among 11 orders. In the East Mediterranean, Red Sea, and Zanzibar 129 sponge species have been collected (TAU) during the course of the reserch. Morphological and molecular means were used to identify species.


WP2 – Sponge Proliferation Application
WP2 of the SPECIAL projects aims at developing a generic production tool for sponge materials, based on the proliferation capacity of sponges, recently discovered by our research group. This Sponge Proliferation Application (SPA) revolves around rapid turnover (balance between cell proliferation and loss) of sponge cells. First, we demonstrate that this process is commonly occurring in a wide variety of sponge species from different ecosystems. Secondly, we studied different growth mechanisms (steady state, somatic growth, wound healing and regeneration and degeneration) using a combination of histology, immunohistochemistry and cell kinetic assessments to develop a diagnostic tool to determine the different physiological ‘states’ sponges are in. Thirdly, we built recirculation freshwater and artificial seawater facilities to test different biotic (food and trace elements) and abiotic (temperature, light, salinity, water flow) parameters on the physiological state of our model species. The results obtained from project SPECIAL initiates us to control and manipulate these physiological states in future studies, to ultimately be able to control a high output of sponge biomass for biotechnological purposes.

• Proliferation
Eight model species were used for the steady-state (rapid cell turnover without increase in sponge biomass) proliferation assay were: Halisarca caerulea, Chondrilla caribensis, Scopalina ruetzleri, Clanthria venosa, Haliclona vansoesti and Monanchora arbuscula (Caribbean reef); Mycale microsigmatosa (Caribbean mangrove); Chondrosia reniformis (Mediterranean). A high level of cell proliferation, mainly in the filter cells (i.e. choanocytes) was found in seven out of eight sponge species and may be termed ‘proliferative’.

Most (six out of eight species) showed a consistent proliferation of 16 – 19 % after 6 h, whereas the Caribbean Mangrove species M. Microsigmatosa showed a ‘high’ choanocyte proliferation of >70 %. The Caribbean reef species H. vansoesti was the only species showing a wide range of choanocyte proliferation, from as low as 2.8 % to as high as 73 % and was termed ‘variable’. The clear histological difference in cell proliferation between sponge individuals might point to different growth mechanisms (steady state versus growth or degeneration), suggesting that proliferation may be influenced by environmental and/or intrinsic stimuli.

• Detritus production (cell loss)
All species studied (tropical reef) produce significant amounts of detritus. Production of detritus (percentage mg DW detritus per mg DW tissue) seems to be stable between specimen of the same species and within specimen. Between sponge species there is a high variety in detritus production per dry weight sponge. Preliminary data show that there are basically two types of detritus producers:
• ‘High-Production-Sponges’ (HPS): Halisarca caerulea, Clathria venosa and Monanchora arbuscula (Production ranges between 13 and 49 % of their biomass);
• ‘Low-Production-Sponges’(LPS): Scopalina ruetzleri, Chondrilla caribensis and Haliclona vansoesti (production <12% of their biomass; sponges between 5 – 20 mm of thickness).
There is a clear difference in production rates (mg DW detritus per specimen) between day and night, sometimes up to two- fourfold increase during the night. Detritus production is affected during wound healing and regeneration as compared to steady state. For our model sponge species Halisarca caerulea, we found a decrease in detritus production after inflicting a wound and a subsequent recovery in production within 6 days.
Fatty acids were found in all the detritus samples. All samples contained very long-chained fatty acids, linking the detritus to its sponge source. There are no apparent differences in fatty acid profiles between the different times of collection (fresh versus old detritus).
We conclude that the combination of cell turnover and histology provides a powerful, novel tool to determine sponge growth ‘states’. Moreover, the collection of detritus may provide an additional, fast diagnostic tool to determine the health of a sponge under ex situ conditions.

• Development of SPA production platform
The first goal is to determine to what extent the rapid cell proliferation is maintained in sponges under controlled and semi-controlled aquarium conditions. Porifarma applied its proprietary knowledge on designing regimes for maintenance of sponges in captivity to create conditions that will result in maintaining sponges in a controlled physiological (growth) state. Regime design will include optimization of hydrodynamic conditions, food quality and quantity, trace element composition (in particular iron), temperature, salinity and light. Two 1500 L climate controlled artificial seawater facilities are constructed at the University of Amsterdam. The two system were designed as a tropical system with a water temperature of 26 °C and a (temperate) Mediterranean system with a seasonal temperature regimes from 16 °C (winter) to 22 °C (summer). Live sponges (Halisarca caerulea, Scopalina ruetzleri, Haliclona vansoesti, and Mycale microsigmatosa) have been collected in Curacao and transported successfully to the systems.

In addition to our seawater model species, we located the freshwater sponge species Ephydatia fluviatilis from the Amsterdam canals, and added this cosmopolitan species as a future model. Freshwater sponges are easily maintained and provide excellent models for cell kinetic studies. We additionally (to the marine systems) constructed two 3000 L freshwater aquaria systems. The freshwater sponges are currently kept successfully for 15 months to date.

We now, for the first time, successfully implemented our approach of cell turnover and histology on a controlled ex situ aquaculture. The assessment of biological activity of the sponge-derived detritus finalizes the development of SPA in this first stage. Future studies will therefore focus on the manipulation of different growth states of sponges to further control and influence a sufficient and continuous supply of sponge biomass for science and application.


WP3 – Sponge mariculture
Within WP3 of the SPECIAL project, we aimed to develop culture protocols for three Mediterranean sponge species, Chondrilla nucula, Chondrosia reniformis and Axinella damicornis in order to supply raw materials for marine collagen production. The main aim of the work within WP3 was to develop cost-effective and reliable mariculture techniques for these sponges, for application in commercial aquaculture.
An additional aim of the work package was to develop sponge mariculture technology that is suited for inclusion as an extractive component in Integrated MultiTrophic Aquaculture, taking advantage of the efficient filter feeding capacities of the sponges. To test the idea, explants of Chondrosia reniformis were grown in a pristine area and in an area polluted by open-cage fish farming. It was anticipated that the sponges would benefit from the organic waste produced by the fish and that the environment would benefit from the filtering activities of the sponges. For the latter aspect it is essential, however, that pumping activity is maintained under polluted conditions. Hence, both the growth of sponges and their pumping activity was measured at both locations.
A. damicornis was found unsuitable for open sea mariculture. C. reniformis was grown on PVC plates, whereby applying an angle of 90 or more (i.e. vertical or overhanging) was found to be most successful. Culture efficiency was not satisfactory and needs further optimization. The main issue that remains to be resolved is the loss of explants; explants glued to PVC plates and other supports tend to drop off from their supports. The explants that remain attached usually show reasonable growth, in particular during the first 6 months after explantation.

By applying vertical plates, we were also capable of growing C. reniformis in areas polluted by fish farms, which hitherto failed due to smothering of the explants by fish farm sediments. The explants survived and grew even better than at a pristine location. Despite the higher growth rates, it was found that the sponges at the polluted site had a twofold lower volumetric pumping velocity, indicating that pumping activity is controlled by food availability. Average annual growth at the fish farm site (140%) is too low, however, for commercial culture of C. reniformis for large scale production of food grade collagen. When applied properly, the technique may be useful and feasible to produce cosmetic grade or medical grade collagen, which has a higher added value. It should be taken into consideration that production volumes needed for these applications of collagen may be much lower than for food grade collagen. Hence, sustainable harvesting of natural populations of this abundantly occurring species may provide a competitive alternative.
The work on C. nucula was most successful; this species could be grown effectively on concrete supports. The cultures were vulnerable to losses due to stormy weather, but by applying protective measures during the initial stage, the risk for loss of explants could be minimized. Growth rates up to 1200% in 481 days were obtained for individual explants, the average annual growth rate being between 300 and 500%. There was a strong seasonal effect on growth, being highest in the period between May and December. Repetitive cloning of cultured specimen of C. nucula was also successful, the F1 generation of explants showing the same growth pattern as the F0 parent sponges. This makes the species suitable for broodstock optimization through genotypic selection.

When considering the culture of C. nucula as an alternative source of collagen to C. reniformis, the following three issues must be taken into consideration:
1. Collagen content in C. nucula is lower than in C. reniformis (<20% of the wet weight in C. nucula vs 40-50% of the wet weight in C. reniformis);
2. Collagen content in C. nucula is highly variable throughout the season, hence, optimization of the timing of harvesting should be elaborated;
3. In contrast to C. reniformis, C. nucula contains siliceous spicules, which may obfuscate the production of food grade collagen from this species.

Given the three considerations above, we conclude that despite its better potential for culture, culture of C. nucula for large-scale production of collagen needs a few further optimization steps. The current method is suitable for the production of cosmetic grade and medical grade collagen.


WP4 – Enzymatic production of collagen
The aim of WP4 was the biochemistry and molecular biology characterization of collagen derived from the marine sponge C. reniformis in order to improve the biomass production of this compound and produce marine collagen in recombinant form.

• Molecular characterization of one of the most represented collagen genes in the sponge and histological analysis of its distribution in the sponge body.
Using PCR approach, a full length cDNA coding for a non-fibrillar collagen was identified and successively its tissue distribution analysis was performed using in situ hybridisation and qPCR. The non-fibrillar collagen cDNA is 2,563 nucleotides long. It contains an open reading frame of 2,229 nucleotides, coding for a protein of 743 amino acids. The putative translation product has an estimated Mr of 72.12 kDa and can be divided in several domains. The expression patterns of the non-fibrillar collagen gene studied in C. reniformis by in situ hybridization reveals an high to very high concentrations of mRNA d in the cortex (apical and basal), while the medullar part of the sponge was virtually devoid of collagen hybridization signals. Quantitative PCR analysis confirmed these results.

• Molecular characterization of the prolyl hydroxilases genes
Both subunit alpha and beta (PDI) of the prolyl-4-hydroxylase gene in C. reniformis were identified using the same approach. The C. reniformis prolyl-4-hydroxylase cDNA (P4Hach) is 1,789 nucleotides long. It contains an open reading frame of 1,599 nucleotides, coding for a protein of 533 amino acids. The putative translation product of P4Hach has an estimated Mr of 61.27 kDa. The C. reniformis protein disulfiide isomerase cDNA (PDIch) is 1,798 nucleotides long. It contains a an open reading frame of 1,578 nucleotides, coding for a protein of 526 amino acids. The putative translation product of PDIch has an estimated Mr of 58.75 kDa. The identification of these two last sequences represents a key step for the realization of the recombinant collagen.

• Transcriptome analysis of the sponge model
The transcriptome analysis of this sponge was performed using high quality total RNA from a healthy and fleshly collected C. reniformis. The assembling of the 665,421 reads obtained from the 454 pyrosequencing of the normalized cDNA, gave 19,678 trasncripts (isotig) belonging to 13,900 isogroups. A specific research for collagen transcript gave 117 isotig related to collagen, 8 were related to fibrillar collagen types and 109 to non-fibrillar types, 43 of which were connected to vertebrate type type IV collagen.

• Establishment of a suitable experimental model of C. reniformis
For the biochemistry studies of collagen, initially three different models were considered i) animal model, ii) fragmorph model and iii) cellular model. The encouraging results obtained with the fragmorph model and the contextual challenge in obtaining sponge cells from C. reniformis did not advise for the prosecution in this last direction. The animal and fragmorphs models have been used mainly. C. reniformis was easy to maintain in aquaria for experimental purposes. Soluble collagen could be extracted from the sponge body and tested for suitability in cell culture and tissue engineering. Masson’s trichrome staining was used to observe the general organization of the sponge and to identify some cell types and Picro-sirius red staining, through polarized light, evidenced the orientation and size of collagen fibres. Chondrosia reniformis resulted also a good model for the in vitro cultivation of sponge fragments. The species were indeed easy to keep in aquaria and showed good recovery and regeneration after fragmentation. The regeneration process of the 50-80 mm3 fragments lasted several days and resulted in a rounded or ovoid body shape. The use of fragmorphs produced from a single animal increases the quality of replicates and reduces the environmental impact of animal collection for experimental purposes. Fragmorph models were used to evaluate the main agonists stimulating collagen synthesis. The collagen production was assayed as non fibrillar collagen transcript level using quantitative PCR. The collagen mRNA levels were analyzed in C. reniformis fragmorphs following their treatment with soluble silicates and with vitamin C, the obtained data indicate that these compounds can up regulate the collagen gene expression. Fragmorph samples were also treated with different kind of silica dust, biogenic silica as diatomaceus heart and grounded sponge silica spicules or mineral dust as grounded silica sand or quartz dust (MinUSil ,a well characterized quartz dust from US Silica Company). The obtained data indicated that diatomaceous heart can significantly improve the collagen gene expression, the sand quartz can also positively regulate the COLch gene expression although to a lesser extent than the diatomaceous earth. No positive effects on collagen expression were observed using grounded sponge spicules. At the end of the treatment, the external epithelium was partially restored under each condition, but the morphology of the respective fragmorphs was significantly different. The samples treated with quartz dust were larger than those treated either with minced spicules that were much more rounded. The difference may be due to the latter models trying to minimize their surface/volume ratio hence exposing the least surface to the powder.

• Proteomic analysis of extracellular matrix protein
Nowadays, the use of mass spectrometry is considered the best approach to structurally characterize proteins and their post-translational modifications (PTMs). The most common procedure exploited for this purpose is called “bottom up” and consists in the proteolytic digestion of the macromolecule with specific enzymes followed by their separation and characterization. In this project, the structural analysis was preceded by the extraction of collagen from the sponge chondrosia reniformis. The collagen was extracted from the sponge using different protocols set up in the first year of activity. These methods lead to obtain two main fractions, soluble and insoluble. Initially was studied the soluble fraction of native collagen. The sample was denatured, reduced and alkylated. To perform the enzymatic digestion the denatured proteins need to be previously separated from the reaction mixture; commonly this step is performed using the gel filtration chromatography. The purified material was digested with trypsin and the resulting peptides were analyzed by high performance liquid chromatography coupled with a mass spectrometer equipped with an electrospray ion source (HPLC-ESI_MS). In spite of the different ionic strength altermatively used during the extraction of these soluble samples (1M NaCl, pH 7.5 or 0,1M NaCl, pH 7.4 in 50 mM Tris-HCl), the HPLC-MS analysis returned identical results (with differences only in the relative abundances of the different signals). As resulting from MS analysis some peaks were obtained; unfortunately, three signals (rt=57.2 57.9 and 44.4 minutes) showed m/z 288.29 m/z 288.35 and m/z 381.13 due to human keratins. This is not totally unexpected, as all the sponge samples, from fishing to final laboratory treatment, were handled by different people. All the other peaks have ratios included in the 250-900 m/z range. It is possible to include these signals in two major categories. On the one hand, some of these signals show features of different hypothetical fragments (m/z 300.18 332.16 331.23 269.14) of classical fibrillar collagens, containing typical residues (glycine-x-y, where x and y could be proline, hydroxyproline, lysine and hydroxylisine). On the other hand, some signals (572.350 603.360 850.410) correspond, as unspecific cleavages, to peptides belonging to fibrillar collagens from other marine sponges (Clathria prolifera) found performing an in silico on-line analysis (findpep, expasy.org) with an accuracy of about 16 ppm.Since the soluble extracted material didn’t provide suitable results, the attention was focused on the insoluble preparation subsequently dried or resuspended in a denaturing medium. After specific digestion of this fraction with trypsin, the peptides obtained were analyzed by HPLC-MS using the electrospray ionization coupled, from time to time, with analyzers at different resolutions. Working on the insoluble preparation, previous results got in full scan at low resolution mode were very useful to identify the presence of some significative peptides expected from the cDNA sequence relative to the non fibrillar collagen here cloned. After this previous screening performed with an ion-trap instrument the samples were analyzed by high resolution instrumentats to both identify PTMs and to define the sequence of candidates fragments using different approaches (peptide mass fingerprint and “de novo” sequence). In particular, after the digestion conducted in solution or after SDS gel separation, we were able to certainly confirm the presence of three peptides deduced by the in silico digestion of the non-fibrillar collagen cloned. These results were confirmed through high resolution (HR) tandem mass spectrometry also allowing to structurally characterize other five expected. With the same approach, conducted using two different HR mass spectrometers, these data were validated and extended obtaining the coverage of more than 50% of the expected sequence. Moreover some possible oxidation sites were identified and validated by HPLC analysis. With analogous experiments, some hydroxylation sites were identified. These modifications are characteristic of collagen that can, in this way, generate some cross-links between the helixes. This indicates that some portion of the sequence could be present, in the native form, in the classical triple helix fold expected for the collagen.

• Transformation of a yeast strain with C. reniformis genes
The enzymatic production of recombinant marine collagen was developed following the experimental design described in literature. The P. pastoris strains used for this project are PichiaPink TM (Life technologies). They are ade 2 auxotrophs, unable to grow in the absence of adenine due to full deletion of the ADE 2 gene. Once the full length cDNA sequence coding for the alpha and beta subunits of C. reniformis prolyl-hydroxylase had been obtained, the expression vectors pPinkHC\α, pPinkLC\α and αPIC6B\PDI were successfully prepared. Linearized pPinkHC or pPinkLC and αPIC6B were used for the co-trasformation reaction of four different PichiaPink strains, a wild type and three different protease defective genes strains. Overall eight different yeast strains were prepared. After a their first selection in agar pates without adenine and with blasticidin antibiotic and a standard PCR screening in order to confirm the presence of both expression cassettes in the yeast genome, a clone for each eight prepared strains a quantitative PCR was performed for a relative gene copy number assay. The induction of the recombinant polypeptides was carry on at 30°C in presence of 0.5% of methanol for 60 hours and finally the yeast cell were recovered and lysate with glass beads. The prolyl-hydroxylase activity was assayed on the post-mitochondrial cell lysate using a standard method based on radioactive compound incorporation. A first comparison among the four PichiaPink strains was performed using the high-copy pPink version. The data obtained indicated that the strain 2 had the best prolyl-hydroxylase activity. Successively the comparison between the high-copy pPink version and low copy version using the strain 2 revealed that the latter was the best. Finally a time course expression analysis was conducted using the strain 2 with the low copy version of pPink vector (L2) indicating that 48 hours were the best expression time. Once identified the P.pastoris strain with the best prolyl-hydroxylase activity (L2), it was made competent and transformed with linearized pPICZ\Colch in order to obtain the Colch strain. After a first selection on zeocin® agar plates and a conventional PCR screening for to detect the presence of all three recombinant genes in yeast genome, eight clones of Colch strain were isolated and a quantitative PCR was performed for relative gene copy number assay. The methanol induction of the expression of the three recombinant polypeptides was carried using the clone with the highest collagen gene copies (clone 4). The quantitative analysis of the transcripts was obtained by qPCR approach on a negative control, on L2 strain and on Colch4 strain. The SDS analysis of recombinant polypeptide was performed on supernatant cell lysate of L2 and Colch4 strains. The region corresponding to the 70 kD was excise from the acrylamide gel for the MS analysis.

• MS analysis of recombinant collagen produced by the yeast strain.
The sample obtained from the cell culture supernatant as from the cell lysate of transformed pichia pastoris yeast was processed similarly to the native stuff. However, due to the complexity of the matrix, the analysis were conducted only digesting the protein after their separation by SDS-PAGE electrophoresis. In this way, comparing the material coming from the transformed cells with the untreated one, some bands were identified, in gel digested with trypsin and subsequently analyzed as previously described. A lot of peptides relative to the expression product obtained through the transformation of the yeast were identified; all the fragments are consistent with those expected by the “in silico” digestion of the deduced non-fibrillar collagen sequence and many of these peptides are the same found in the sponge and so its possible to be sure that the transformed yeast is able to produce the expected collagenic polipeptide.


WP5 - Biosintering for biosilica production
Sponge primmorphs (3D cell culture system) from the demosponge Suberites domuncula have been optimized with respect to the production of biosilica. UMC could demonstrate that in primmorphs inorganic trace elements, e.g. manganese ions, can affect the morphology of the spicules formed. The spatio-temporal expression of the principle genes/proteins involved in formation and maturation/hardening of biosilica, such as silicatein, silintaphin-1, and aquaporin, were investigated using primmorphs in qRT-PCR, Western and/or in situ hybridization experiments. The morphology and composition of the spicules formed in primmorphs under various incubation conditions were analyzed by SEM and EDX techniques. Based on the results, optimized media conditions for spicule formation in primmorph cultures have been designed. The synthesis of new spicules involves the formation of organic cylinder-like structures surrounding the spicules in the extraspicular space that contain silicatein and a calcium-dependent lectin. Immunogold electron microscopy revealed that these organic cylinders are formed only in the presence of both silicate and retinoic acid. The organic cylinders allow the radial apposition of new silica layers during the growth of the spicules. The two key enzymes of the retinoid pathway, the β-carotene dioxygenase and the retinal dehydrogenase have been cloned from S. domuncula. In addition, UMC could demonstrate that retinoic acid causes a strong up-regulation of the expression of the gene encoding the bone morphogenetic protein-1 (BMP-1) in S. domuncula primmorphs.

Besides silintaphin-1, UMC and NRCGA-CAGS investigated a second silicatein interactor, silintaphin-2, that had been identified by conventional yeast two-hybrid library screening and a novel solid-phase pull-down assay. The expression of silintaphin-2 was studied in primmorphs. UMC and NRCGA-CAGS could demonstrate that the three main components of the organic cylinder formed during spicule formation are synthesized in sclerocytes (silicatein and silintaphin-2) and archaeocytes (galectin). The expression of the genes encoding silicatein and silintaphin-2 is under the control of silicate. Collagen is synthesized by the lophocytes. Its synthesis is regulated by myotrophin, which is released from sclerocytes. Silintaphin-2 has been shown to be formed by proteolytic cleavage from a longer-sized precursor, presilintaphin-2. This reaction is mediated by BMP-1. The formation of BMP-1 is regulated by retinoic acid that is formed from β-carotene that originates from chromocytes. The expression levels of silicatein and silintaphin-2, which are strongly increased in the presence of silicate, are not affected by retinoic acid.
Applying this primmorph system, UMC and NRCGA-CAGS showed that during the initial phase of spicule synthesis nanofibrils with a diameter of around 10 nm are formed that comprise bundles between 10 and 20 nanofibrils. These nanofibrillar bundles become embedded into the Si-rich matrix.
One essential task for the use of primmorphs for fabrication of biosilica structures and structures of other metal oxides that has been solved is the hardening of the biosilica structures. Primmorphs grown in the presence of Mn-sulfate were shown to form spicules that comprise instead of a smooth surface a rough surface which is decorated with irregular biosilica deposits. qRT-PCR experiments revealed a differential expression of silicatein and aquaporin transcripts in the Mn-treated primmorphs versus non-treated primmorphs. The expression of the gene encoding aquaporin-8 was found to be drastically down-regulated in primmorphs treated with Mn-sulfate, while the expression of silicatein gene remained almost unimpaired.
In addition, UMC and NRCGA-CAGS investigated and elucidated the mechanism by which primmorphs form hard biosilica structures from the initially formed soft biosilica material and sinter the hardened silica material to 3-dimensional hierarchical structures. Our results revealed that the protein component of the biosilica spicules has not only biocatalytic (silicatein) and structure-directing activity (silintaphin-1) but also facilitates the “biosintering” process of the silica nanospheres formed by silicatein. This process is most evident during maturation of demosponge spicules which fuse completely, in contrast to the lamellae of the hexactinellid spicules. In vitro experiments revealed that bio-sintering is most likely driven by silicatein which remains within the biosilica matrix after silica formation.
The newly formed spicules contain relatively high concentrations of sodium and potassium as revealed by ICP-AES and EDX analysis. About 80% of these alkali metals are removed during maturation of the spicules. This process is accompanied by the removal of water. The progress in the silicatein-driven polycondensation process can be explained by the exchange of sodium and potassium ions from the biosilica product formed by silicatein to the negatively charged aspartic acid and glutamic acid residues of the silintaphin-2 molecule. UMC and NRCGA-CAGS could demonstrate that silintaphin-2 comprises anionic amino acid clusters in the N-terminal region of the protein, which are rich in Asp and Glu. A peptide corresponding to this region significantly enhanced the silicatein-driven biosilicification process.
In addition, UMC and NRCGA-CAGS could demonstrate, using the primmorph system, that the formation of cellular processes is involved in the axial growth of spicules. The spicule formation is accompanied by the formation of evaginations of the spicule-forming cells (sclerocytes) into the axial canal of the growing and elongating spicules. Further UMC and NRCGA-CAGS could show that, around a cell extension protruding into the axial canal, silicatein molecules are released from storage vesicles (silicasomes) into the space between the cell membrane and the inner surface of the silica mantel that surrounds the axial canal, and catalyze biosilica deposition at the inner surface.
Primmorphs turned out to be a suitable model to study the biosintering process of biosilica. Using high resolution methods the fusion zones during the biosintering process have been analyzed by NRCGA-CAGS. Based on our data we concluded that the water molecules that are released during the polycondensation reaction are removed from the site of biosilica formation by importing them into cells that surround the spicules. These cells migrate and transport the water further away, allowing a more rapid adjustment of the water equilibrium. NRCGA-CAGS and UMC could demonstrate that aquaporin gene is involved in the syneresis process of biosilica formed by silicatein. The removal of water does not only accelerate the rate of biosilica formation but also the formation of a more compact biosilica product. As a result, the diameter of the biosilica mantel decreases during spicule formation. The shrinkage of the spicule could also be corroborated by theoretic calculations.
Several strategies to modify the morphology of spicular nanostructures generated in primmorph cultures have been developed. In addition, UMC succeeded to develop methods for bioencapsulation of cells in a biosilica shell. The bioencapsulation of enzymes or whole cells has gained increasing interest in biotechnology and biomedicine. Based on the fact that silicatein allows the production of silica under mild, physiological conditions, the technique of silicatein-mediated biosilica synthesis can even be applied for the production of silica-encapsulated bacterial biosensors. UMC could show that the heterologous expression of silicatein on the surface of bacteria transformed with a silicatein cDNA can be applied for the synthesis of porous biocompatible silica shells around the bacteria. The advantage of such biosilica shells is an easier handling of the bacteria and an increase in their mechanical and chemical stability.
The glass-fibre-like sponge spicules act as optical fibres that transmit light with high efficiency. Based on these properties, first steps were undertaken to use the recombinant biosilica forming enzymes to fabricate optical components under physiological processing conditions. Applying the silicatein-mediated bio-polycondensation and biosilica molding technology, we succeeded to achieve a controlled fabrication of silica layers with electrically-insulating properties. This new bio-inspired technique has the advantage compared to current techniques that the enzymatic biosilica synthesis can be performed at ambient temperatures (room temperature), neutral pH and silicate concentrations below 1 mM, while industri¬ally applied processes require high temperatures above the glass transition temperature.


WP6 – Antitumour drug discovery and development
Partners TAU, UNIGE and UAzores have collected sponges of different species and from different locations. From freshly collected and freeze-dried sponges partners TAU and UNIGE made crude extracts by homogenization, extraction and evaporation.

• Sponge extracts showing tumour specific antitumour activity
A large number of crude extracts has been screened for antitumour activity (partner KI, UNIGE and ATR) and active extracts have been separated sequentially by different chromatographic methods by partners TAU and UNIGE. About 230 different fractions were tested and those fractions that showed activity in tumour cell bioassays were further separated with HPLC. Final purification of active compound, achieved by semi- preparative normal phase HPLC column and NMR determination of structure revealed structure of 12 compounds so far.
Prior to the start of the SPECIAL project, KI had screened 30 different extracts obtained from TAU and one of them, obtained from a sponge from Florida Cribrochalina vasculum, was found to possess anti-tumour activity in tumour cells of different origin (Non small cell lung cancer (NSCLC), breast cancer (BC) and Acute Myeloid Leukaemia (AML)). This extract was selected for further purification. In parallel to the identification of active compounds from the extract, partner KI could demonstrate apoptosis-activation of caspase-3 as one mechanism of action for anti-tumour effect. Despite the fact that that this extract showed clear, cell death-inducing capacity in tumour cells normal cell toxicity was evident. Further testing was terminated as there was no therapeutic window allowing mechanistic studies of important pathways or in vivo evaluation in mice tumour models. Nevertheless, it illustrate the importance of simultaneously testing of extracts on tumour and normal cells early on when analyzing sponge compounds to exclude nonspecific cytotoxic effects.
The same Florida-derived sponge C. vasculum was recollected and extract were made for identifying compounds with antitumour activity. Importantly although from the same sponge this extract did not contain the compounds isolated from the original extract illustrating the complexity of sponge-associated tumour research. HPLC purification of this extract revealed a new set of acetylene containing compounds some of which showed tumour cell selective toxicity in small and non small lung cancer cells (NSCLC/SCLC) as well as in ovarian tumour cell lines. Some of the compounds isolated from parental extract have been isolated and described previously to possess anti-tumour activity while others possess a structure previously not described. Importantly, partner KI demonstrated that these compounds are not toxic to Caco-2 (representing intestine toxicity in ADMET) and to BEAS-2B (bronchial epithelium cells) whereas in HepG2 which commonly is used for liver toxicity assessment in vitro of chemical compounds, these C. vasculum compounds were found indeed to be toxic. The interpretation of this finding is that since HepG2 in fact are tumour cells they may not be an appropriate model to address liver toxicity in response to sponge compounds aimed for anti-tumour purposes. All in all a tumour therapeutic window was established which will enable further development of these compounds for an anti-tumour drug. To further assess the activity of C. vasculum compounds TAU prepared synthetic analogs of compounds which showed tumour specific toxicity and on which mechanism characterization is ongoing.

Partner KI has screened additional collection of 96 extracts collected by partner TAU from different sponges (e.g. Iotrochota biotulata, Adocia atra, Theonela conica) and at different locations (Mediterranean, Red Sea, Caribbean, Indian Ocean) (supplementary Table 1) 16 of them were found to decrease tumour cell viability by more than 90% yet some of which also reduced viability of normal fibroblasts. Based on these screening results, biomaterial supply and previous described isolated compounds partners TAU and KI decided to focus on the extracts from sponges Theonella conica, Haliclona debilis, Iotrochota birotulata and Svenzea zeai and from Solitary tunicate. However, further fractionation, purification and testing of sponge extract from Svenzea zeai, Haliclona debilis and Iotrochota birotulata revealed no tumour specific activity in the LC tumour cells examined. The Theonella conica fractionated extract showed tumour specific activity in one fraction, which reduced cell viability of multiple LC tumour cell lines yet did not influence cell survival of normal cells. Further purification revealed one fraction with tumour specific toxicity in lung and ovarian cancer cells without influencing fibroblast and bronchial epithelial cells survival indicating a tumour specific therapeutic window. The isolation of pure compound is on the way and its mechanism of action will be worked on. Partners TAU and KI worked further on solitary tunicate extracts collected at Bahamas and Florida. At present fractions with tumour specific activity is at hand but the active compounds remains to be revealed.
70 different extracts isolated from multiple sponge species were collected at different localizations by partner GAIA and UAzores and extracted by partner TAU. These extracts have undergone profiling for antitumour activity by partner KI using NSCLC and SCLC cells and normal diploid lung fibroblasts WI-38. Further purification revealed no tumour specific toxicity and at present partners TAU and KI decided due to time limitations of the project to terminate further work on these fractions
It was also shown that compounds from sponges could also inhibit growth of tumour progenitor stem cells (also called tumour initiating cells (TICs)) of NSCLC, previously shown to be resistant to conventional treatment. In summary, activity of all tested extracts (C. vasculum, T. conica, solitary tunicate, Reniera sarai, Iricinia) against TICs demonstrated similar or even better activity than in bulk NSCLC cells. Partner KI can therefore conclude that sponge-derived compounds likely are promising for CT-treatment resistant tumour cells as well.
Partner UNIGE have evaluated the cytotoxicity of two novel diadenosine homodinucleotides adenine, diadenosine 5’ analogues P18 and P24 which were generated by ADP-ribosyl cyclases (ADPRC) extracted from Axinella polypoides. P18 and P24 were toxic in micromolar and submicromolar concentrations respectively on different human tumour cell lines (erythroleukemia cells: TF1, NSCLC, BC cells) and patient derived AML-and CLL cells. Importantly, mononuclear cells isolated from healthy donors were not affected indicating tumour selectively. The plan ahead for P18, P24 extract is to extend screening into NSCLC and SCLC cells as well as to look for vivo effect in NSCLC xenografts in SCID mice and delineate mechanism further. Partner UNIGE has also isolated and purified high molecular weight extracts from the marine sponge Chondrosia reniformis. Extract showed cytotoxic effect in NSCLC and breast cancer cells. Further work aims to isolate the active compounds from these fractions using HPLC and further characterize of anti-tumour mechanisms.
Partner KI also worked on a synthetic analog of poly-APS- compound isolated from sponge Reniera sarai and analyzed its anti-tumour activity in NSCLC cells. In a recently submitted publication, partner KI demonstrate that APS8 triggers apoptotic signaling activation in NSCLC tumour cells by blocking nicotinic acetylcholine receptors (nAChRs), a molecular target shown to be overexpressed and active at least in sub fraction of LC cases. Further work aims to understand if APS8 also might be a therapy for small cell lung carcinoma or if APS8 can be used in combination with different chemotherapies.

• Sponge extracts inducing dysfunctional mitochondrial signalling
Partner UNIGE characterized the effect of P24, P18 (from A. polypoides) and of the C.reniformis sponge extract on mitochondrial function, in particular on the proton gradient (ΔΨm) and on the permeability transition pore (PTP). Results showed loss of mitochondrial potential with P24 and C. reniformis extract but not with P18. Partner UNIGE can demonstrate that P24 activates the purinergic receptor/channel P2X7 leading to influx of extracellular Ca2+ and loss of the mitochondrial proton gradient, which subsequently triggers cellular apoptosis whereas P18 behaves as a P2X7 antagonist and has no such effects.

• Sponge extract showing induction of senescence in PTEN deficient tumour cells
Senescence is an irreversible cell growth arrest that blocks the proliferation of cancer cells and triggers tumour clearance. Given that tumours frequently have decreased level of PTEN, partner ATR has focused on extracts that act in PTEN deficient tumour cells and induces senescence. Partner ATR has developed an screening assay platform (PICS assay) which enables identification of senescence-inducing specific compounds by using PTEN deficient and proficient mouse embryo fibroblasts (MEFs) from genetically modified mice (Pten-/-) (8). Within the SPECIAL project Partner ATR obtained 150 different extract from partner TAU and an additional 47 extracts from GAIA from different sponges or their associated microbes i.e. bacteria or fungi. Partner ATR can demonstrate that 36 show a statistically significant effect on cell growth of Pten-/- MEFs, and 6 of them were also able to induce a significant growth arrest in the same cells whereas one extract showed pro-senescence activity in the Pten-/- but not Pten wt MEFSs. This pro-senescence candidate extract NF13EtAc (c24) is from a fungus extracted from a sponge belonging to the family of the Ircinia (Supplementary Table 2). Partner TAU has made purification of fractions which is to be s re-tested in the PICS assay with the aim to identify a pure pro-senescence compound to be tested for effects on prostate cancer stem cells and also in vivo models of Pten null prostate conditional mouse model (Ptenpc-/-).

• Sponge compounds inducing tumour specific pro-apoptotic signaling
For two of the C. vasculum compounds partner KI also investigated which signaling pathways are responsible for their anti-tumour activity. Both compounds induced apoptotic nuclear morphology in NSCLC tumour cells, caused activation of mitochondria-mediated apoptotic signaling as reflected by activation of pro-apoptotic Bcl-2 proteins Bak and Bax, triggered depolarization of mitochondria and release of cytochrome c with subsequent induction of caspase-3 activation and execution of an apoptotic phenotype. Importantly, all these apoptotic features were not evident in diploid fibroblasts WI-38 illustrating a tumour-specific apoptotic action of these C. vasculum compounds. To further understand mechanism of action gene expression alterations in response to C. vasculum compound in tumour and normal cells were compared using Affymetrix gene array. A large set of genes was significantly down regulated in tumour cells whereas in normal lung fibroblasts no genes were altered. Computerized pathway analysis (IPA and Gene set enrichment) suggested cell cycle regulation to be one mechanism of action, which indeed was confirmed in cell cycle distribution analysis of tumour cells where a G2/M accumulation was evident. Gene expression data also indicated inhibitory effect on growth factor receptor regulated kinases e.g. Akt and ERK phosphorylation g by C. vasculum compounds and increased JNK activation. All in all this suggest a tumour-specific mechanism involving switch of pro-survival signaling to pro-death possibly via Bad phosphorylation to Bak/Bax activation and mitochondria-mediated apoptosis.

• In vivo effect of sponge compound in (Ptenpc-/-) mice
Partners TAU, KI and ATR selected synthetic compound from sponge C. vasculum for in vivo testing in PTEN-driven prostate cancer mice model. To determine the maximum tolerable dose of compound to be used for in vivo trial in Ptenpc-/- mice in vivo toxicity evaluation was first performed in wt C57Bl6 mice without tumours. At the highest doses 1g/kg and 100mg/kg a tendency to leucopenia and lymphopenia was evident. During studies of subacute toxicity (25mg/kg repeatedly for 5 days) revealed increased levels of creatinine and cholesterol. The increase in creatinine level should be investigated by prolonging the period of subacute administration. The hematological analysis showed no other statistically significant differences in any of analyzed parameters upon the subacute molecule administration. However, DMSO that was used as a solvent shows significant side effect in mice indicating limitation of high doses use in in vivo testing. Anti-tumour activity was evaluated in a Pten null prostate conditional (Ptenpc-/-) that partner ATR established. In this model both Pten alleles are specifically inactivated in the prostate causing tumour growth. C. vasculum compound was administered at dose of 50mg/kg every other day for 4 weeks to the mice. No response was evident in terms of tumour size and histology. There was a massive inflammatory response in abdominal cavity in treated mice and, although less intense, in control mice as well. This indicates that DMSO is not a proper solvent to use in in vivo experiments and a different vehicle such as ethyl acetate is preferred. In terms of the lack of tumour response to treatment this may be due to inadequate compound uptake in tumour or that these particular tumour cells with inactivation of Pten may not be responsive. Tumour xenografts established from NSCLC or ovarian cancer cells, which responded in vitro to C. vasculum compound will be tested in the next round of animal experiments.


WP7 - Development of biomedical and industrial applications
The work developed in WP7 was dedicated to the development of sponge-derived biomaterials for further application in different sectors, with particular emphasis in tissue engineering and regenerative medicine (collagen and hybrid polymer-biosilica materials) and sensors technology (encapsulation of primmorphs). In this regard, it aimed:
- the development of sponge collagen based structures to be applied in tissue engineering strategies;
- the development of a hybrid polymer/biosilica biomedical system for tissue engineering approaches;
- the scale up of primmorph cultures towards the development of nano-biofactories;
- the development of a prototype of a biosilica encapsulated primmorph stress sensor.

• Development of collagen based structures towards tissue engineering
Marine sponges are well known for the biosynthesis of several toxic compounds and this particularity was actually addressed in WP6 when evaluating possible antitumoral leads produced intracellularly in marine sponges. Thus, when considering the use of other sponge materials in applications in which cytotoxicity can be an issue, such as tissue engineering, this behaviour must be accessed. During SPECIAL it was described that the available procedures to the extraction of collagen from marine sponges, based in acetic acid solution or in basic solution in the presence of a concentrated chaotropic agent, result in both cases in highly cytotoxic collagen solutions. However, it was demonstrated to be possible to form structures with the extracted collagen from those solutions without retaining the toxic compounds.
The sponge collagen structures developed in SPECIAL were collagen coatings and collagen membranes. Such structures were considered interesting in the context of skin tissue engineering and its effect on the behaviour of specific cell lines have been addressed particularly in the collagen coatings.
Regarding the collagn membranes, two challenges were faced in SPECIAL. By one side, it was hard to achieve significant amounts of sponge collagen and the method achieving higher extraction yields results in a precipitated collagen found extremelly difficult to solubilize. On the other side, once in solution, there was a need of collagen processing to achieve membrane stable in aqueous medium to support cell culture. Both challenges have been successfully overcomed. Different combinations of solvent solutions have been tested and an effective method has been established for the solubilization of extracted sponge collagen. The resulting solutions were afterwards submitted to a procedure of freeze-drying together with cross-linking with the common agent EDC/NHS, which rendered structures stable in PBS.

By analysing the sponges’ morphology, it was interesting to find amazing porous structures, mainly composed by collagen, ceramics (depending on the species, calcium carbonates or biosilica) and sponge cells. The collagenous structures was then as inspiration for the development of nature made scaffolds, in a truly biomimetic approach. It was possible to calculate a porosity of about 70%, with interconnected pores with an average diameter of about 200 µm, deemed quite interesting for bone tissue engineering.
In this perspective, the mechanical properties of several sponge species have been addressed, as well as its water uptake upon incubation in PBS solution, exhibiting quite different responses depending on the sponge species, thus covering a wide range of behaviours.
Despite the promising observations made up to this moment, two challenges arose: the presence of toxic compounds, which needed to be efficiently removed to allow the use of such structures in biomedical applications, and the presence of sponge cells, which would certainly lead to a immune reaction upon implantation of such construct in the human body, for which they also needed to be removed. The SPECIAL team was then dedicated to evaluate strategies of removing both compounds and cells and it successfully proposes a simple procedure to jointly address both challenges. It was thus possible to produce nature made scaffolds from marine sponges, in which osteoblast-like cells were successfully cultured, thus illustrating their potential for bone tissue regeneration.

• Transfection of primmorph cultures and further up-scale
Sponge primmorphs are difficult to transfect. Conventional techniques such as using calcium phosphate, liposomes, or others are not successful but the SPECIAL team has been able to develop an innovative method for transfecting primmorphs using a microporator. The efficacy of this method has been demonstrated by transfection of primmorphs of the demosponge S. domuncula, using the pipet-type electroporation device.
Moreover, it has been possible to expand those cultures up to 4-liter bioreactors.
With the developed technology, it is now possible to transfect primmorphs allowing the expression of selected cDNAs of biotechnological interest in these cell aggregates. Moreover, it is also promoted the production of biosintered nanostructures.

• Fabrication of a biosilica encapsulated primmorph stress responsive biosensor
The OWLS technique allows in situ and label-free studies of surface processes at molecular level. It is based on the measurement of the resonance angle of polarized laser light which is diffracted by a grating and incoupled into a thin waveguide layer. The mass of the bound molecules or cells can be calculated from the change of the resonance angle. The OWLS technique can also be applied for the detection and discrimination of living and damaged bacterial cells. The formation of a biosilica shell around E. coli transformed with the silicatein gene has been used to develop a bacterial biosensor by immobilizing the bacterial cells at mild conditions in the presence of TEOS on the surface of the SiO2-containing sensor chips. Applying this mild immobilization technique a stable E. coli layer could be created on the sensor surface. This bacterial biosensor has been successfully used as an effective tool for the detection of various stressors (e.g. hydrogen peroxide), environmental pollutants (pesticides, e.g. carbofuran) and antibiotics (e.g. chloramphenicol) in real time measurement.


• Hybrid polymer/biosilica – based biomedical system for bone tissue engineering
In the second reporting period, UMC-Mainz and NTM could show that biosilica also displays morphogenetic activity on cells after embedding in a Na-alginate-based hydrogel. SaOS-2 cells embedded into the hydrogel showed an increased growth and an increased formation of hydroxyapatite nodules after exposure to mineralization activation cocktail if silica was present in the hydrogel. Moreover, in the silica-containing hydrogels an enhanced expression of the gene encoding BMP-2, as well as a higher expression rate of the genes encoding collagen type I and carbonic anhydrase, an enzyme involved in bone formation/dissolution was found. The effect of silica from the hybrid material on the expression of other marker genes of bone formation (besides BMP-2 and collagen type I) was also studied. The results revealed that silica present in the hydrogel enhances the expression of collagen type V, as well as of osteopontin and osteonectin genes. Osteopontin and osteonectin are glycoproteins involved in the mineralization process by binding to hydroxyapatite and collagen. Moreover, it was concluded that silica causes its morphogenetic effect via BMP-2 rather than the BMP-2-independent RUNX2 pathway.
Based on these findings silica-containing alginate hydrogels have been proposed to be suitable as a morphogenetically active matrix for 3D cell printing.

• Bioactive bioceramics from marine sponges
The potential of bioceramics from different sponges for novel biomedical applications, in particular through bioactivity, was evaluated. Sponge bioceramics have been obtained by calcination of sponge samples, in which all organic and toxic compounds existent in marine sponges are removed. Petrosia ficidormis exhibited a stable 3D biosilica porous structure; Agelas oroides and Chondrosia reniformis bioceramics were recovered as powder, being the former composed by silicates with incorporation of calcium and magnesium and the latter corresponding to exogenous siliceous spicules and sand grains incorporated by the sponge. The bioactivity of these bioceramics was assessed by the formation of a bone-like apatite layer upon immersion in a simulated body fluid, following the methodology of Kokubo and co-workers. The formation of the typical cauliflower-like crystals of hydroxyapatite was observed on the surfaces of Agelas oroides and Chondrosia reniformis bioceramics. The bioceramics of Petrosia ficidormis didn’t exhibited inherent bioactivity, which needs to me stimulated following surface chemistry functionalization processes. Moreover, the non-cytotoxic behaviour of Chondrosia reniformis and Agelas oroides ceramics suggests its use on bone tissue engineering strategies.
Potential Impact:
Brief address of potential impact of SPECIAL project
Marine organisms have provided a large proportion of the bioactive natural products reported over the last decades. Since biomedical and biotechnology industries are continually searching for new functional materials aimed for health related applications, such role is expecting to be more and more significant in the future.
Being sessile aquatic organisms, sponges have developed an incredibly diverse chemical arsenal to fight external threats (predators, pathogens, competitors), which place them, among the many phyla found in the oceans, as the most promising avenue for marine (blue) biotechnology.
In this context, SPECIAL project had the ambition of discover and develop sponge derived products that may result in valuable applications for EU industries in several sectors, with particular incidence in the biomedical field. In more detail, focus was given in anticancer compounds and biomedical applications based in collagen and biosilica structures, resulting in non-pharmaceutical anticancer products, sponge collagen systems and bioceramics for further in-vitro and in-vivo testing of tissue regeneration and a stress biosensor prototype.
With this, the project is expected to have a strong impact in European Marine Research and related fields in two ways:
• significant contribution to show the great potential in producing biomass of marine organisms with known bioactive properties, thus allowing the design of sustainable production pathways, but avoiding destructive in situ harvesting processes;
• identification of possible applications of products derived from produced biological materials that will enable strong synergies between marine related industries with other economic fields, thus showing the great interest in exploring sea as a background for the needed breakthroughs to kick-off European competitiveness in the scientific, technological and economic arenas.

There is a large potential for gaining more value from these sponge based products. These products, resulting from sponges’ rich and diversified chemistry, are required by many fields of knowledge and industries, including pharmaceutics and the biomedical field. Possible applications include bioactive agents (for instance as drugs), biomaterials and medical devices and for cancer research. These traditional and yet innovative uses of sponge-based materials could potentially generate significant additional revenue. In this context, SPECIAL project aimed to take sponge valorisation and exploitation a large step further. By focussing in very high added value fields like biomedical and biotechnology, SPECIAL project explored (and is exploring) not only incremental R&D pathways to establish sustainable and controlled technologies for the production of sponge materials, but also exploit in a non-disruptive manner novel applications for these materials, with a strong emphasis in the pharmaceutical field and regenerative medicine.

SPECIAL addresses, hence, the main issues raised in the call text by:
• Generating new products for society through marine biotechnology, thus contributing both to the quality of life and to the competitiveness of several sectors of the European industry mentioned in the call text. The needs for new products are high, in particular in the fields of cancer and drug-resistant infections, which are ranked second and fourth in the league table of the leading causes for death, respectively. Hence, the economic potential of these products is large: for example, more than 40% of the top-selling pharmaceuticals on the market today are derived from naturally occurring substances. Nevertheless, most of these products come from terrestrial and microbial resources, while only few natural compounds derived from marine animals and plants have been introduced on the market, which is in contrast to the huge biochemical potential of these living resources. SPECIAL provided the concerted, integrated approach needed to efficiently exploit these natural resources.
• Increasing the potential of European marine biotechnology by creating durable public-private collaboration. The European added value of this project lies in its huge potential to integrate the currently fragmented marine biotechnological activities in Europe and beyond (ICPC participation).
• Promoting sustainability by implementing routes for sustainable exploitation of marine natural resources. In this way, the current constraints to the full development of Blue Biotechnology (i.e. the “supply problem” and the related environmental and ethical considerations) were properly addressed, by the delivery of several new and innovative approaches that may be taken up by the wide scientific and industrial community for further implementation on different targets.

Production of biological materials for consecutive research steps was primarily done by using sustainable biotechnological methods, such as aquaculture, high output platforms and recombinant technology. The implementation of these actions established a sustainable way of exploitation of marine natural resources. Moreover, the set up of these raw materials and derived products may open new possibilities for biomedical and pharmaceutical industries, as well as for microelectronics (nanotechnology). New compounds with new bioactivities can be found, with clear application in the development of new drugs. The porposal of new biomaterials will contribute to the advancement of tissue engineering, pursuing to overcome the limitations of the current therapies in regenerative medicine. Even the application of sponge-derived materials in microelectronics and nanotechnology received added light from the outcomes of SPECIAL project, by demonstrating the exceptional ability of sponges and sponge cell cultures (primmorphs) of biosintering to generate biosintered metal oxide nanostructures with tailored opto-electro-mechanical properties.

From an academic point of view, the results of the SPECIAL project will additionally impact on several fundamental and applied sciences, namely with new alternatives for valorisation of marine based products, improved production techniques through enzymatic processes, increase the available compounds with bioactivity and increased confidence in engineering and designing natural and bioinspired materials. Moreover, the generation of state-of-the-art knowledge will benefit the undergraduate students training in the enrolled high education institutions, contributing to the formation of highly qualified human resources.

To accomplish such a challenging project, a consortium has been set, combining the expertise of a wide range of SMEs and some of the best research institutions in the fields relevant for this project. Overcoming some European fragmentation, the high complementarity between partners resulted in a synergy that one believe is being capable to contribute to enhance European industry competitiveness when facing with USA or Japan’s leading companies. Due to their extensive networks, the industrial partners in SPECIAL are excellent intermediates to create durable alliance between science and industry in the field of marine biotechnology. This interaction was promoted by targeted dissemination activities and within SPECIAL Open days.

The successful establishment of sustainable and controlled technologies for the production of sponge materials and applicative development of sponge based products in this project will have a large societal (health and human resources), environmental, economic and market impacts. Many of these will occur through the indirect actions of the project:
• Health (Societal):
- Improvement of patients´ quality of life in the long term due to the introduction of novel product pipelines;
- Increase in patient recovery rate;
- Reduction in the duration of treatments.
• Human Resources (Societal):
- Retention of highly qualified human resources in Europe;
- Development of multi-disciplinary R&D professionals;
- Contribute to professional and scientific mobility.
• Environment:
- Design of sustainable production pathways, avoiding destructive in situ harvesting processes;
- Sustainable exploitation of marine natural resources;
• Economy / Market:
- Novel high added value products;
- Transfer of specialized technology into the industry and market;
- Improved European SME competitiveness.

Dissemination activities and Exploitation of results
Project SPECIAL has developed a complex and successful Dissemination and Outreach strategy, aimed at delivering messages (about its scientific results and the potential of such results, the sustainable use of natural resources, the various facets of research life as well as the importance of EU-supported collaborative actions) to a diversified audience consisting of peers, university students, decision makers, industrial actors, pupils/children and adults. As for adults, these include both the so-called “general public” and a more specific segment corresponding to SCUBA divers. Indeed, diving and science go hand in hand. While diving, people from the most diverse layers of society have a unique experience of nature that often leads to increased curiosity, understanding and awareness. More important, with a few million recreational divers worldwide of different age, gender and socio-economic background, diving relies on an impressive network of multipliers that can effectively pass the acquired knowledge to their non-diving entourages. SPECIAL relates very well to the experiences of SCUBA divers, which makes them an ideal target (and tool) for outreach.
To achieve in its ambitious plan, SPECIAL developed a strategy based on the use of effective and cross-linked multipliers (old and new media), plus direct contact via meetings, lectures, Open days and fairs. We can calculate that at least 74.000 LinkedIn users operating in the fields addressed by SPECIAL, SCUBA diving and/or science communication (LinkedIn groups) received news from the project.

In terms of audience that has been engaged, outcomes may be summarized as below. Further details on means and media will be given in the subsequent sections.
- Peers. SPECIAL has been largely promoted to peers via direct contacts and contacts mediated by mailing lists (including scientific societies and associations), social networks and the customary channels for scientific research (papers, workshops, conferences etc). Peers have made use of the website, of the SPECIAL publications and have taken active part in the Open days.
- University students. They have been reached via mailing lists, direct contacts within the University and at the SPECIAL Open days. Other resources available to University students include the SPECIAL publications, the website (especially in the outreach section) and the SPECIAL YouTube channel. Much of the video material produced covers aspects of interest to this share of the audience.
- Decision makers. Policy/decision makers and administrators have been engaged at various levels, ranging from local administration (e.g. Guimarães city hall and other local administrations from Minho and Northern Portugal Region, UMINHO; Alassio Municipality, GAIA) to the highermost positions (e.g. President’s Advisor for Maritime Affairs and the President of the Portuguese Scientific Funding Agency, UMINHO; President Shimon Perez, TAU; President Wen Jibao, UMC), focusing on the promotion of the SPECIAL activities and of Blue Biotechnology. In many of these occasions, a multiplier effect towards the general public was obtained thanks to the associated media coverage.
- Industry. The SPECIAL Consortium possesses a strong background of relationship with the industrial sector, which is the baseline for the work of the CEC. Additionally, promotion of the project to further prospective partners has occurred via channels offered by the Internet, i.e. social media and mailing lists, and by the participation of SPECIAL partners to relevant events at the national and international levels.
- School students, teens, children. Most of the activities targeting this share of the audience are linked to Open days/events and videos, although website visits to specific sections indicate children may contribute to a small but relevant fraction of the website audience.
- Adults. A plan was laid down focusing on two main groups: divers and the remaining general public. While the general audience as such has been targeted by press releases and by most of the Open days, divers have a specific interest in marine-related topics and are therefore an ideal subgroup to produce a multiplier effect. SPECIAL has tackled this specific segment of audience by entering dedicated blogs, magazines and groups in social media, besides producing outreach and video material for the website and its YouTube channel.


• SPECIAL Website
The SPECIAL Website was intended with the double aim to support external (i.e. towards the public) and internal (i.e. within the Consortium) dissemination. Focus has been mostly on, but not limited to, activities and outcomes of SPECIAL. The SPECIAL website was reachable through the address www.project-special.eu and two e-mail addresses were identified for internal and external communication with the project: info@project-special.eu (leading to the Coordinator’s e-mail addresses) appears on the website homepage, open to the public, and partners@project-special.eu (leading to the list of contacts included in the Consortium Agreement). The website has been constantly updated and, after an initial phase when visits were monitored approximately twice per week and then twice per month – around day 1 and day 15, the visits count has been performed on a monthly basis during the last 12 months.

At the end of November 2013 (end of project) the SPECIAL website consists of approximately 210 pages. Indeed, besides the usual sections Home page, Overview, People, Resources (featuring a list of the SPECIAL publications, with a direct link to them; access to the SPECIAL newsletter also occurs via this section), Visuals (photo and videos, the latter via a specifically designated YouTube channel), News, Links (including EC background documentation) and Events, an entirely new section dedicated to outreach has been developed starting in April 2013. This includes a brief overview of sponges, then leading to: i) a section focusing on some of the most common sponges encountered in the Mediterranean and Red Seas; ii) a section focusing on sponge biotechnology, in a lay-man language; iii) a section dedicated to striking stories about sponges, with links to videos and material from elsewhere in the web. Additionnaly, to make the best use of the non-sensitive data contained in the SPECIAL sponge database, the consortium decided to feed them into the Sponge Barcoding Project, a high-profile, international endeavour that will ensure long-term sustainability of and open-access to the knowledge. The SPECIAL website (both in the news and in the outreach sections) and consortium will ensure appropriate promotion of the event once the required technical and administrative steps have been finalised.
At the end of November 2013 the website has received nearly 490,000 visits. Altogether, the news pages have been visited over 400,000 times. The photo gallery area has been visited over 6,000 times. The gallery dedicated to the Sponge Bob event that featured also SPECIAL has been visited ca. 1,650 times since its creation at the end of June 2012. The latter is an important achievement as the audience interested likely consists of children. The posters and keynotes presented at the Genoa Open day have been visited around 7,700 times. Finally, and in spite of its recent creation, the section dedicated to outreach has been visited around 4,000 times. According to Google statistics, a large fraction of visitors are aged below 34, thus confirming the appeal to the young generations as well as to the young researchers’ community. The geographical distribution of visitors’ origin is evenly distributed across the globe. Approximately 93% of visits from social networks come via LinkedIn, where promotion is carried out in dozens of groups including Biotech/Blue Biotech, Science Communication and diving environments.
Most of the success of the SPECIAL promotional strategy is due to the extensive and cross-linked use of new media, such as social networks, mailing lists and blogs. Specifically for the latter, two articles targeting SCUBA divers appeared respectively on ScubaPortal (http://www.scubaportal.it/chondrilla-nucula.html) and on the official PADI blog (http://www.padi.com/blog/2013/11/14/what-can-you-do-with-a-sponge/). PADI is the largest SCUBA diving certifying agency in the world, covering ca. 60% of a market estimated in several million active divers. This is a very important achievement especially aimed at drawing divers’ attention to sponges via the SPECIAL outreach section, and from here to Blue Biotech and EU-funded research. An interview to Martina Milanese, with many comments on and links to SPECIAL, is available on ScubaPath (http://mydivejob.com/).

• SPECIAL Video series
Sixteen videos have been uploaded in the SPECIAL YouTube video channel (http://www.youtube.com/user/projectSPECIALeu).

The SPECIAL YouTube channel has been visited ca. 2,300 times. As for the SPECIAL website, approximately an equal number of males and females have watched the SPECIAL videos. In this case, however, the age range is skewed upwards, with a quite regular distribution in the age bracket 35-64.

• SPECIAL Open days
SPECIAL Open days have been intended as one-day events aiming to: i) disseminate SPECIAL activities and results; ii) engage with the audience about S&T; iii) offer high-level lectures about S&T, science ethics and other issues; iv) strengthen the relationship between Industry and Academia. Planned as an additional day at the end of selected project meetings, events have been typically hosted by the institution of the partner in charge of the project meeting itself, albeit different arrangements have also occurred on a case by case basis.

The first SPECIAL Open day was organised in Jerusalem, on October 17th 2011, during one of the intermediate days of the Succot festival. Once a year, the Residence of the President of Israel can be visited by the public. In this day, exhibit highlighting Israel’s achievements in agriculture, science, technology and welfare are on display both inside and in the grounds; designed for the whole family. The SPECIAL team in TAU has taken advantage of this event to present the project and Blue Biotechnology.
Approximately one thousand people joined the event, including the Israeli President Mr. Shimon Peres in person, as well as the Minister of Science and Technology and the Minister of Agriculture.

Another SPECIAL Open day took place in Genoa on October 28th 2011. It came at the end of 2nd Progress Meeting and was jointly organised by GAIA and UNIGE. It was accepted to be part of the programme of the 2011 Genoa Science Festival, an internationally recognised festival collating a wide range of events aiming at the promotion of science and scientific thinking to a broad audience, and committed to engage with society at large in a bidirectional dialogue about science (http://www.festivalscienza.it/site/home.html). The Open day consisted in three key-notes in Blue Biotechnology, by Prof. Rui Reis (UMINHO), Prof. Werner Müller (UMC-Mainz) and Dr. Andrea Alimonti (ATR), followed by oral and poster presentations by young researchers from the project (http://www.project-special.eu/joomla/index.php?option=com_content&view=article&id=90&Itemid=68).
Approximately 200 people attended the SPECIAL Open day in Genoa testifying a high level of satisfaction according to the evaluation questionnaires received.

A third SPECIAL Open day was organised by TAU after the international workshop on “Symbiosis of lower invertebrates with microorganisms” held in Eilat, Israel (by the Red Sea) on February 26 – March 2, 2012. This Open day attracted ca. 70 people, between students, professors and entrepreneurs interested in Blue Biotechnology, and was the ideal floor to meet between different sectors and field, discussing about further scientific and applied opportunities.

A fourth SPECIAL Open day was organised in Genoa thanks to the collaboration with Aquarium of Genoa and Nickleodeon. These had already designed a SpongeBob-dedicated, long-term joint activity. The event provided also for the opportunity to discuss about the future of sponge farming in Liguria, when SPECIAL is concluded, counting with the participation of the Municipality of Alassio (the main location for GAIA’s mariculture activities), the Center for Environmental Education (CEA) from the Municipality of Imperia (with interests in sponge farming for potential environmental applications) and Reef Check Italia (part of a NGO dedicated to monitoring and protecting the reefs worldwide).
A plan named Sponge Friends was presented and was well received by the two Municipalities. Nickelodeon agreed on endeavouring to support fund-rising activities that relate to the Sponge Friends plan. Sponge farming, the SPECIAL project and the plan for Sponge Friends (a plan were therefore presented to an audience of approximately 200 children during the 2012 World Ocean Day, at the Aquarium of Genoa.

The fifth SPECIAL Open day took place in Tel Aviv at the end of the 24M Project Meeting, consisting in two seminars, by Prof. Rui Reis (UMINHO) and by Dr. Ronald Osinga (PF), attracting around 40 people, including students and staff at TAU, and stimulated a lively discussion about the future perspectives of Blue Biotechnology, and the career opportunities it may offer.

The sixth SPECIAL Open day was actually an Open night event associated to a one-week photo exhibition in Alassio (Italy). This event, called “Alassio, un mare Speciale“ (Alassio, a special sea) was organised on July 22nd-30th 2013. The SPECIAL Open night took place on July 24th 2013 in the town’s main square, aiming to promote the activities of SPECIAL and the sustainable sea-based activities. The core of the Open night consisted in the display of underwater images and videos, with live comments from the biologists (namely Dr. Martina Milanese, from GAIA) and followed by a question-and-answer debate with the public, together with a photo exhibition. The SPECIAL Open night and photo exhibition attracted around 100 and 400 people, respectively, mostly of tourists (especially families with children) and locals (generally elderly ones), whom in some cases could not find a seat but eventhough attended standing for the whole duration of the event.

Project SPECIAL was also represented in other events, namely:
• Mainz German “City of Science 2011“, represented by UMC, NRCGA-CAGS and NTM;
• Expo 2012 in Yeosu, Korea (“The Living Ocean and Coast”), represented by UMC-Mainz, NRCGA-CAGS and NTM (May to August of 2012);
• Final Conference of the Coordination and Support Action Marine Biotech, designed as starting point for the launching of an ERA-NET on Marine Biotechnology, in which Project Coordinator Prof. Rui L. Reis presented SPECIAL as a good example of cooperation between academia and industry (March 2013);
• PYMWIMIC Impact Days (impact investors meeting), represented by PF (April 2013);
• School TV, education program for biology at high schools in The Netherlands, represented by PF (May 2013);
• Lobby lunch meeting with a Member of the European Parliament (Ulrike Rodust), discussing the potential of Blue Biotech with other players (academia, industry and policy makers), represented by Tiago H. Silva (UMINHO) (January 2014).

• SPECIAL Publications
SPECIAL has worked on several types of publications, which include: over 30 manuscripts in peer-reviewed scientific journals, around 60 abstracts in conference proceedings, ca 20 press releases in printed and on-line media (such as newspapers, magazines, blogs) and 3 patents.

• Exploitation
Some of the results obtained under the scope of SPECIAL activities have high potential for being further exploited in industrial and commercial context. The enrolled partners are fully aware of its high potential and different ways to explored them are being considered. In this regard, some know-how can be kept inside the institution for further exploitation through services and/or consulting. The protection of intellectual property through patenting is also being considered (and already carried out in some cases, as listed above). In such way, those products and technologies could be further licensed to other companies or explored by the SMEs participants in SPECIAL consortium, thus expecting to have a positive impact in European industrial sector and economy. In order to assure this connection to the industrial sector, some bilateral meetings have taken place between partners of SPECIAL and other companies to discuss the main outcomes of the institutions and possibilities to explore them in industrial and commercial context. Examples are the contacts between UMINHO and STEMMATTERS (Portugal) and an multinational biomedical company, as well as between UMC-Mainz, NTM and Microvacuum Ltd (Hungary).
At this moment, the main products and technologies from SPECIAL that can be envisaged as highly valuable are the following:
- Collection of sponges with identification, which might be interesting for other research purposes;
- Mariculture techniques, tested with two species and eventually applicable to other species, which can thus be exploited by licencing the technology (after patenting) or by selling services or training;
- Production Platform SPA, for the production of sponge biomass in controlled conditions, relevant with findings on valuable compounds from such sponges;
- Yeast strain producing recombinant marine collagen, as a valuable alternative to extracted sponge collagen as well as to other collagens from different sources, depending on the application;
- Sponge primmorph cultures for biosilica production, with potential relevancy to research purposes and to biotechnological and pharmaceutical companies;
- Biofabrication of 3D micro-electronic networks and nanomechanical / nano-optical elements by primmorphs, with relevance in sensor technology;
- biosilica encapsulated primmorph stress responsive biosensor;
- Systems for tissue regeneration, namely sponge collagen structures, natural made scaffolds derived from sponges’ own structure, sponge bioceramics and hybrid collagen/biosilica systems; with relevancy for biomedical companies.
With the support of the CEC, each partner prepared exploitation sheets of identified technologies and products developed within SPECIAL, with information on planned way of exploitation and potential end-users.
Moreover, leaflets with the main technical outputs of the project, with higher potential impact and relevance for external institutions where prepared as support for further meetings fostering exploitation of SPECIAL results. The first use of such material was during the European Forum for Industrial Biotechnology & the Biobased Industry – EFIB 2013, where SPECIAL was represented in a booth by Prof. Marco Giovine (UNIGE) and Dr. Tiago H. Silva (UMINHO). It was an occasion to share knowledge with other biotech endeavors, namely processing plant managers, biotech services providers and biotech clusters, aiming to launch the basis for future collaborative efforts regarding the further exploitation of specific SPECIAL results. In this perspective, opportunities arose to explore scale-up approaches for the production of SPECIAL compounds and materials, to evaluate alternative production processes with incorporation of enzymatic treatments, as well as to contact with private companies aiming to appraise the future market exploitation of those products and/or applications derived from them, after joint activities. The abovementioned leaflets were, in fact, used as a sort of business cards, providing the main contacts of the enrolled scientists together with a brief overview of technical developments (technology, product, service).
List of Websites:
Further details can be found in the project website (http://www.project-special.eu/) or by contacting SPECIAL project coordinator, Prof. Rui L. Reis:

Prof. Rui L. Reis
3B´s Research Group - University of Minho
AvePark, Zona Industrial da Gandra, S. Cláudio do Barco
4806-909 Caldas das Taipas
Guimarães - Portugal

Tel: +351-253-510900
e-mail: rgreis@dep.uminho.pt
url: http://www.3bs.uminho.pt