Aims: Identification of the mycobiota associated to the marine echinoderm Holothuria poli and investigation of cytotoxic and pro-osteogenic potential of isolated strains. Methods and results: Fungal strains were isolated from the animal's bodywall, intestine and faeces. The species identification was based on DNA barcoding and morphophysiological observations. Forty-seven species were identified, all are Ascomycota and mainly belonging to Aspergillus and Penicillium genera. Sixteen strains were grown on three media for chemical extraction. Cytotoxic activity was tested on a hepatic cancer cell line (HepG2), the cells viability was evaluated after treatment using a resazurin based assay (AlamarBlue). Pro-osteogenic activity was tested on human Mesenchymal stem cell, differentiation was measured as the alkaline phosphatase production through reaction with p-nitrophenylphosphate or as the cells ability to mineralize calcium using a colorimetric kit (StanBio). Cytotoxic activity was recorded for four fungal species while five of 48 extracts highlighted bioactivity towards human mesenchymal stem cells. Conclusions: The presence of relevant animal-associated mycobiota was observed in H. poli and selected strains showed cytotoxic potential and proosteogenic activity. Significance and Impact of the Study: Our work represents the first report of a Mediterranean Sea cucumber mycobiota and highlights the isolates potential to synthetize compounds of pharmaceutical interest for regenerative medicine.

Fungi populate deep Oceans in extreme habitats characterized by high hydrostatic pressure, low temperature and absence of sunlight. Marine fungi are potential major contributors to biogeochemical events, critical for marine communities and food web equilibrium under climate change conditions and a valuable source of novel extremozymes and small molecules. Despite their ecophysiological and biotechnological relevance, fungal deep-sea biodiversity has not yet been thoroughly characterized. In this study, we describe the culturable mycobiota associated with the deepest margin of the European Western Continental Shelf: sediments sampled at the Porcupine Bank and deep-water corals and sponges sampled in the Whittard Canyon. Eighty-seven strains were isolated, belonging to 43 taxa and mainly Ascomycota. Ten species and four genera were detected for the first time in the marine environment and a possible new species of Arachnomyces was isolated from sediments. The genera Cladosporium and Penicillium were the most frequent and detected on both substrates, followed by Candida and Emericellopsis. Our results showed two different fungal communities: sediment-associated taxa which were predominantly saprotrophic and animalassociated taxa which were predominantly symbiotic. This survey supports selective fungal biodiversity in the deep North Atlantic, encouraging further mycological studies on cold water coral gardens, often overexploited marine habitats.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

Chronic diseases characterized by bone and cartilage loss are associated with a reduced ability of progenitor cells to regenerate new tissues in an inflammatory environment. A promising strategy to treat such diseases is based on tissue repair mediated by human mesenchymal stem cells (hMSCs), but therapeutic outcomes are hindered by the absence of small molecules to efficiently modulate cell behavior. Here, we applied a high-throughput drug screening technology to bioprospect a large library of extracts from Irish deep-sea organisms to induce hMSC differentiation toward musculoskeletal lineages and reduce inflammation of activated macrophages. The library included extracts from deep-sea corals, sponges and filamentous fungi representing a novel source of compounds for the targeted bioactivity. A validated hit rate of 3.4% was recorded from the invertebrate library, with cold water sea pens (octocoral order Pennatulacea), such as Kophobelemnon sp. and Anthoptilum sp., showing the most promising results in influencing stem cell differentiation toward osteogenic and chondrogenic lineages. Extracts obtained from deep-sea fungi showed no effects on stem cell differentiation, but a 6.8% hit rate in reducing the inflammation of activated macrophages. Our results demonstrate the potential of deep-sea organisms to synthetize pro-differentiation and immunomodulatory compounds that may represent potential drug development candidates to treat chronic musculoskeletal diseases.