De Novo Assembly of the Genome of the Sea Urchin Paracentrotus lividus (Lamarck 1816).

The Mediterranean purple sea urchin Paracentrotus lividus (Lamarck 1816) is a remarkable model system for molecular, evolutionary and cell biology studies, particularly in the field of developmental biology. We sequenced the genome, performed a de novo assembly, and analysed the assembly content. The genome of P. lividus was sequenced using Illumina NextSeq 500 System (Illumina) in a 2 × 150 paired-end format. More than 30,000 open reading frames (ORFs), (more than 8000 are unique), were identified and analysed to provide molecular tools accessible for the scientific community. In particular, several genes involved in complex innate immune responses, oxidative metabolism, signal transduction, and kinome, as well as genes regulating the membrane receptors, were identified in the P. lividus genome. In this way, the employment of the Mediterranean sea urchin for investigations and comparative analyses was empowered, leading to the explanation of cis-regulatory networks and their evolution in a key developmental model occupying an important evolutionary position with respect to vertebrates and humans.

Potential of Polar Lipids Isolated from the Marine Sponge Haliclona (Halichoclona) vansoesti against Melanoma.

Marine sponges represent a good source of natural metabolites for biotechnological applications in the pharmacological, cosmeceutical, and nutraceutical fields. In the present work, we analyzed the biotechnological potential of the alien species Haliclona (Halichoclona) vansoesti de Weerdt, de Kluijver & Gomez, 1999, previously collected in the Mediterranean Sea (Faro Lake, Sicily). The bioactivity and chemical content of this species has never been investigated, and information in the literature on its Caribbean counterpart is scarce. We show that an enriched extract of H. vansoesti induced cell death in human melanoma cells with an IC50 value of 36.36 µg mL-1, by (i) triggering a pro-inflammatory response, (ii) activating extrinsic apoptosis mediated by tumor necrosis factor receptors triggering the mitochondrial apoptosis via the involvement of Bcl-2 proteins and caspase 9, and (iii) inducing a significant reduction in several proteins promoting human angiogenesis. Through orthogonal SPE fractionations, we identified two active sphingoid-based lipid classes, also characterized by nuclear magnetic resonance and mass spectrometry, as the main components of two active fractions. Overall, our findings provide the first evaluation of the anti-cancer potential of polar lipids isolated from the marine sponge H. (Halichoclona) vansoesti, which may lead to new lead compounds with biotechnological applications in the pharmaceutical field.

Ferroptosis precedes apoptosis to facilitate specific death signalling by fatty acids.

Cell death is physiologically induced by specific mediators. However, our power to trigger the process in selected cells is quite limited. The protandric shrimp Hippolyte inermis offers a possible answer. Here, we analyse a de novo transcriptome of shrimp post-larvae fed on diatoms. The sex ratio of diatom-fed shrimps versus shrimps fed on control diets was dramatically altered, demonstrating the disruption of the androgenic gland, and their transcriptome revealed key modifications in gene expression. A wide transcriptomic analysis, validated by real-time qPCR, revealed that ferroptosis represents the primary factor to re-shape the body of this invertebrate, followed by further apoptotic events, and our findings open biotechnological perspectives for controlling the destiny of selected tissues. Ferroptosis was detected here for the first time in a crustacean. In addition, this is the first demonstration of a noticeable effect prompted by an ingested food, deeply impacting the gene networks of a young metazoan, definitely determining its future physiology and sexual differentiation.

Sponges and Their Symbionts as a Source of Valuable Compounds in Cosmeceutical Field.

In the last decades, the marine environment was discovered as a huge reservoir of novel bioactive compounds, useful for medicinal treatments improving human health and well-being. Among several marine organisms exhibiting biotechnological potential, sponges were highlighted as one of the most interesting phyla according to a wide literature describing new molecules every year. Not surprisingly, the first marine drugs approved for medical purposes were isolated from a marine sponge and are now used as anti-cancer and anti-viral agents. In most cases, experimental evidence reported that very often associated and/or symbiotic communities produced these bioactive compounds for a mutual benefit. Nowadays, beauty treatments are formulated taking advantage of the beneficial properties exerted by marine novel compounds. In fact, several biological activities suitable for cosmetic treatments were recorded, such as anti-oxidant, anti-aging, skin whitening, and emulsifying activities, among others. Here, we collected and discussed several scientific contributions reporting the cosmeceutical potential of marine sponge symbionts, which were exclusively represented by fungi and bacteria. Bioactive compounds specifically indicated as products of the sponge metabolism were also included. However, the origin of sponge metabolites is dubious, and the role of the associated biota cannot be excluded, considering that the isolation of symbionts represents a hard challenge due to their uncultivable features.

Two Benthic Diatoms, Nanofrustulum shiloi and Striatella unipunctata, Encapsulated in Alginate Beads, Influence the Reproductive Efficiency of Paracentrotus lividus by Modulating the Gene Expression.

Physiological effects of algal metabolites is a key step for the isolation of interesting bioactive compounds. Invertebrate grazers may be fed on live diatoms or dried, pelletized, and added to compound feeds. Any method may reveal some shortcomings, due to the leaking of wound-activated compounds in the water prior to ingestion. For this reason, encapsulation may represent an important step of bioassay-guided fractionation, because it may assure timely preservation of the active compounds. Here we test the effects of the inclusion in alginate (biocompatible and non-toxic delivery system) matrices to produce beads containing two benthic diatoms for sea urchin Paracentrotus lividus feeding. In particular, we compared the effects of a diatom whose influence on P. lividus was known (Nanofrustulum shiloi) and those of a diatom suspected to be harmful to marine invertebrates, because it is often present in blooms (Striatella unipunctata). Dried N. shiloi and S. unipunctata were offered for one month after encapsulation in alginate hydrogel beads and the larvae produced by sea urchins were checked for viability and malformations. The results indicated that N. shiloi, already known for its toxigenic effects on sea urchin larvae, fully conserved its activity after inclusion in alginate beads. On the whole, benthic diatoms affected the embryogenesis of P. lividus, altering the expression of several genes involved in stress response, development, skeletogenesis and detoxification processes. Interactomic analysis suggested that both diatoms activated a similar stress response pathway, through the up-regulation of hsp60, hsp70, NF-κB, 14-3-3 ε and MDR1 genes. This research also demonstrates that the inclusion in alginate beads may represent a feasible technique to isolate diatom-derived bioactive compounds.

Bioactivity Screening of Antarctic Sponges Reveals Anticancer Activity and Potential Cell Death via Ferroptosis by Mycalols.

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemimycale topsenti, and Hemigellius pilosus. Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.

A Metataxonomic Approach Reveals Diversified Bacterial Communities in Antarctic Sponges.

Marine sponges commonly host a repertoire of bacterial-associated organisms, which significantly contribute to their health and survival by producing several anti-predatory molecules. Many of these compounds are produced by sponge-associated bacteria and represent an incredible source of novel bioactive metabolites with biotechnological relevance. Although most investigations are focused on tropical and temperate species, to date, few studies have described the composition of microbiota hosted by Antarctic sponges and the secondary metabolites that they produce. The investigation was conducted on four sponges collected from two different sites in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018. Collected species were characterized as Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemigellius pilosus and Microxina sarai by morphological analysis of spicules and amplification of four molecular markers. Metataxonomic analysis of these four Antarctic sponges revealed a considerable abundance of Amplicon Sequence Variants (ASVs) belonging to the phyla Proteobacteria, Bacteroidetes, Actinobacteria and Verrucomicrobia. In particular, M. (Oxymycale) acerata, displayed several genera of great interest, such as Endozoicomonas, Rubritalea, Ulvibacter, Fulvivirga and Colwellia. On the other hand, the sponges H. pilosus and H. (Rhizoniera) dancoi hosted bacteria belonging to the genera Pseudhongella, Roseobacter and Bdellovibrio, whereas M. sarai was the sole species showing some strains affiliated to the genus Polaribacter. Considering that most of the bacteria identified in the present study are known to produce valuable secondary metabolites, the four Antarctic sponges could be proposed as potential tools for the discovery of novel pharmacologically active compounds.

PAHs and PCBs Affect Functionally Intercorrelated Genes in the Sea Urchin Paracentrotus lividus Embryos.

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) represent the most common pollutants in the marine sediments. Previous investigations demonstrated short-term sublethal effects of sediments polluted with both contaminants on the sea urchin Paracentrotus lividus after 2 months of exposure in mesocosms. In particular, morphological malformations observed in P. lividus embryos deriving from adults exposed to PAHs and PCBs were explained at molecular levels by de novo transcriptome assembly and real-time qPCR, leading to the identification of several differentially expressed genes involved in key physiological processes. Here, we extensively explored the genes involved in the response of the sea urchin P. lividus to PAHs and PCBs. Firstly, 25 new genes were identified and interactomic analysis revealed that they were functionally connected among them and to several genes previously defined as molecular targets of response to the two pollutants under analysis. The expression levels of these 25 genes were followed by Real Time qPCR, showing that almost all genes analyzed were affected by PAHs and PCBs. These findings represent an important further step in defining the impacts of slight concentrations of such contaminants on sea urchins and, more in general, on marine biota, increasing our knowledge of molecular targets involved in responses to environmental stressors.

Multiple Roles of Diatom-Derived Oxylipins within Marine Environments and Their Potential Biotechnological Applications.

The chemical ecology of marine diatoms has been the subject of several studies in the last decades, due to the discovery of oxylipins with multiple simultaneous functions including roles in chemical defence (antipredator, allelopathic and antibacterial compounds) and/or cell-to-cell signalling. Diatoms represent a fundamental compartment of marine ecosystems because they contribute to about 45% of global primary production even if they represent only 1% of the Earth's photosynthetic biomass. The discovery that they produce several toxic metabolites deriving from the oxidation of polyunsaturated fatty acids, known as oxylipins, has changed our perspectives about secondary metabolites shaping plant-plant and plant-animal interactions in the oceans. More recently, their possible biotechnological potential has been evaluated, with promising results on their potential as anticancer compounds. Here, we focus on some recent findings in this field obtained in the last decade, investigating the role of diatom oxylipins in cell-to-cell communication and their negative impact on marine biota. Moreover, we also explore and discuss the possible biotechnological applications of diatom oxylipins.

Genome Mining as New Challenge in Natural Products Discovery.

Drug discovery is based on bioactivity screening of natural sources, traditionally represented by bacteria fungi and plants. Bioactive natural products and their secondary metabolites have represented the main source for new therapeutic agents, used as drug leads for new antibiotics and anticancer agents. After the discovery of the first biosynthetic genes in the last decades, the researchers had in their hands the tool to understand the biosynthetic logic and genetic basis leading to the production of these compounds. Furthermore, in the genomic era, in which the number of available genomes is increasing, genome mining joined to synthetic biology are offering a significant help in drug discovery. In the present review we discuss the importance of genome mining and synthetic biology approaches to identify new natural products, also underlining considering the possible advantages and disadvantages of this technique. Moreover, we debate the associated techniques that can be applied following to genome mining for validation of data. Finally, we review on the literature describing all novel natural drugs isolated from bacteria, fungi, and other living organisms, not only from the marine environment, by a genome-mining approach, focusing on the literature available in the last ten years.

Lipoxygenase Pathways in Diatoms: Occurrence and Correlation with Grazer Toxicity in Four Benthic Species.

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

Combined Effects of Diatom-Derived Oxylipins on the Sea Urchin Paracentrotus lividus.

Oxylipins are diatom-derived secondary metabolites, deriving from the oxidation of polyunsatured fatty acids that are released from cell membranes after cell damage or senescence of these single-celled algae. Previous results revealed harmful toxic effects of polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) on sea urchin Paracentrotus lividus embryonic development by testing individual compounds and mixtures of the same chemical group. Here, we investigated the combined effects of these compounds on sea urchin development at the morphological and molecular level for the first time. Our results demonstrated that oxylipin mixtures had stronger effects on sea urchin embryos compared with individual compounds, confirming that PUAs induce malformations and HEPEs cause developmental delay. This harmful effect was also confirmed by molecular analysis. Twelve new genes, involved in stress response and embryonic developmental processes, were isolated from the sea urchin P. lividus; these genes were found to be functionally interconnected with 11 genes already identified as a stress response of P. lividus embryos to single oxylipins. The expression levels of most of the analyzed genes targeted by oxylipin mixtures were involved in stress, skeletogenesis, development/differentiation, and detoxification processes. This work has important ecological implications, considering that PUAs and HEPEs represent the most abundant oxylipins in bloom-forming diatoms, opening new perspectives in understanding the molecular pathways activated by sea urchins exposed to diatom oxylipins.

Molecular and Morphological Toxicity of Diatom-Derived Hydroxyacid Mixtures to Sea Urchin Paracentrotus lividus Embryos.

Oxylipins such as polyunsaturated aldehydes (PUAs) and hydroxyacids (HEPEs) are signaling molecules derived from the oxidation of polyunsaturated fatty acids. They are common in diatoms that constitute a major group of microalgae in freshwater and oceanic ecosystems. Although HEPEs represent the most common oxylipins produced by diatoms, little information is available on their effects on marine invertebrates, and most of the information has been obtained by testing individual HEPEs. Our previous studies reported that four hydroxyacids, i.e., 5-, 9-, 11-, and 15-HEPE, were able to induce malformations and a marked developmental delay in sea urchin Paracentrotus lividus embryos, which had not been reported for other oxylipins. Here, we tested a mixture of 5-, 9-, 11-, and 15-HEPE at different concentrations for the first time. The results showed that mixtures of HEPEs have synergistic effects that are much more severe compared to those of individual HEPEs: The HEPE mixtures induced malformations in sea urchin embryos at lower concentrations. Increasing HEPE mixture concentrations induced a marked increase in the number of delayed embryos, until all embryos were delayed at the highest concentration tested. At the molecular level, the HEPE mixtures induced variations in the expression of 50 genes involved in different functional processes, mainly down-regulating these genes at the earliest stages of embryonic development. These findings are ecologically significant, considering that during diatom blooms, sea urchins could accumulate HEPEs in concentrations comparable to those tested in the present study.

UPLC⁻MS/MS Identification of Sterol Sulfates in Marine Diatoms.

Diatoms are unicellular eukaryotic organisms that play a key ecological and biogeochemical role in oceans as major primary producers. Recently, these microalgae have also attracted interest as a promising source of functional products with widespread relevance. Progress in the knowledge of cell and molecular biology of diatoms is envisaged as a key step to understanding regulation of their life cycle in marine environments as well as facilitating their full and profitable exploitation by biotechnological platforms. Recently, we identified sterol sulfates (StS) as regulatory molecules of cell death in the diatom Skeletonema marinoi. As these compounds may have a general role in diatom physiology and chemical signals in aquatic systems, we investigated a suitable tool for their analysis in laboratory and field samples. Herein, we describe a sensitive, fast, and efficient ultra performance liquid chromatography⁻mass spectrometry (UPLC⁻MS) method for qualitative and quantitative analysis of StS from crude extract of diatoms and other microalgae. The method was applied to 13 different strains of our collection of marine protists. This first study suggested a species-specific distribution of StS and identified the sulfated derivatives of 24-methylene cholesterol and 24-methyl cholesterol as the most common members in diatoms.

Marine Sponges and Bacteria as Challenging Sources of Enzyme Inhibitors for Pharmacological Applications.

Enzymes play key roles in different cellular processes, for example, in signal transduction, cell differentiation and proliferation, metabolic processes, DNA damage repair, apoptosis, and response to stress. A deregulation of enzymes has been considered one of the first causes of several diseases, including cancers. In the last several years, enzyme inhibitors, being good candidates as drugs in the pathogenic processes, have received an increasing amount of attention for their potential application in pharmacology. The marine environment is considered a challenging source of enzyme inhibitors for pharmacological applications. In this review, we report on secondary metabolites with enzyme inhibitory activity, focusing our attention on marine sponges and bacteria as promising sources. In the case of sponges, we only reported the kinase inhibitors, because this class was the most representative isolated so far from these marine organisms.

New inter-correlated genes targeted by diatom-derived polyunsaturated aldehydes in the sea urchin Paracentrotus lividus.

The marine environment is continually subjected to the action of stressors (including natural toxins), which represent a constant danger for benthic communities. In the present work using network analysis we identified ten genes on the basis of associated functions (FOXA, FoxG, GFI-1, nodal, JNK, OneCut/Hnf6, TAK1, tcf4, TCF7, VEGF) in the sea urchin Paracentrotus lividus, having key roles in different processes, such as embryonic development and asymmetry, cell fate specification, cell differentiation and morphogenesis, and skeletogenesis. These genes are correlated with three HUB genes, Foxo, Jun and HIF1A. Real Time qPCR revealed that during sea urchin embryonic development the expression levels of these genes were modulated by three diatom-derived polyunsaturated aldehydes (PUAs), decadienal, heptadienal and octadienal. Our findings show how changes in gene expression levels may be used as an early indicator of stressful conditions in the marine environment. The identification of key genes and the molecular pathways in which they are involved represents a fundamental tool in understanding how marine organisms try to afford protection against toxicants, to avoid deleterious consequences and irreversible damages. The genes identified in this work as targets for PUAs can be considered as possible biomarkers to detect exposure to different environmental pollutants.

Evaluating the Effects of an Organic Extract from the Mediterranean Sponge Geodia cydonium on Human Breast Cancer Cell Lines.

Marine sponges are an excellent source of bioactive secondary metabolites for pharmacological applications. In the present study, we evaluated the chemistry, cytotoxicity and metabolomics of an organic extract from the Mediterranean marine sponge Geodia cydonium, collected in coastal waters of the Gulf of Naples. We identified an active fraction able to block proliferation of breast cancer cell lines MCF-7, MDA-MB231, and MDA-MB468 and to induce cellular apoptosis, whereas it was inactive on normal breast cells (MCF-10A). Metabolomic studies showed that this active fraction was able to interfere with amino acid metabolism, as well as to modulate glycolysis and glycosphingolipid metabolic pathways. In addition, the evaluation of the cytokinome profile on the polar fractions of three treated breast cancer cell lines (compared to untreated cells) demonstrated that this fraction induced a slight anti-inflammatory effect. Finally, the chemical entities present in this fraction were analyzed by liquid chromatography high resolution mass spectrometry combined with molecular networking.

Blue-Print Autophagy: Potential for Cancer Treatment.

The marine environment represents a very rich source of biologically active compounds with pharmacological applications. This is due to its chemical richness, which is claiming considerable attention from the health science communities. In this review we give a general overview on the marine natural products involved in stimulation and inhibition of autophagy (a type of programmed cell death) linked to pharmacological and pathological conditions. Autophagy represents a complex multistep cellular process, wherein a double membrane vesicle (the autophagosome) captures organelles and proteins and delivers them to the lysosome. This natural and destructive mechanism allows the cells to degrade and recycle its cellular components, such as amino acids, monosaccharides, and lipids. Autophagy is an important mechanism used by cells to clear pathogenic organism and deal with stresses. Therefore, it has also been implicated in several diseases, predominantly in cancer. In fact, pharmacological stimulation or inhibition of autophagy have been proposed as approaches to develop new therapeutic treatments of cancers. In conclusion, this blue-print autophagy (so defined because it is induced and/or inhibited by marine natural products) represents a new strategy for the future of biomedicine and of biotechnology in cancer treatment.

Polysaccharides from the Marine Environment with Pharmacological, Cosmeceutical and Nutraceutical Potential.

Carbohydrates, also called saccharides, are molecules composed of carbon, hydrogen, and oxygen. They are the most abundant biomolecules and essential components of many natural products and have attracted the attention of researchers because of their numerous human health benefits. Among carbohydrates the polysaccharides represent some of the most abundant bioactive substances in marine organisms. In fact, many marine macro- and microorganisms are good resources of carbohydrates with diverse applications due to their biofunctional properties. By acting on cell proliferation and cycle, and by modulating different metabolic pathways, marine polysaccharides (including mainly chitin, chitosan, fucoidan, carrageenan and alginate) also have numerous pharmaceutical activities, such as antioxidative, antibacterial, antiviral, immuno-stimulatory, anticoagulant and anticancer effects. Moreover, these polysaccharides have many general beneficial effects for human health, and have therefore been developed into potential cosmeceuticals and nutraceuticals. In this review we describe current advances in the development of marine polysaccharides for nutraceutical, cosmeceutical and pharmacological applications. Research in this field is opening new doors for harnessing the potential of marine natural products.

Molecular response to toxic diatom-derived aldehydes in the sea urchin Paracentrotus lividus.

Diatoms are dominant photosynthetic organisms in the world's oceans and represent a major food source for zooplankton and benthic filter-feeders. However, their beneficial role in sustaining marine food webs has been challenged after the discovery that they produce secondary metabolites, such as polyunsaturated aldehydes (PUAs), which negatively affect the reproductive success of many invertebrates. Here, we report the effects of two common diatom PUAs, heptadienal and octadienal, which have never been tested before at the molecular level, using the sea urchin, Paracentrotus lividus, as a model organism. We show that both PUAs are able to induce teratogenesis (i.e., malformations), as already reported for decadienal, the better-studied PUA of this group. Moreover, post-recovery experiments show that embryos can recover after treatment with all three PUAs, indicating that negative effects depend both on PUA concentrations and the exposure time of the embryos to these metabolites. We also identify the time range during which PUAs exert the greatest effect on sea urchin embryogenesis. Finally, we report the expression levels of thirty one genes (having a key role in a broad range of functional responses, such as stress, development, differentiation, skeletogenesis and detoxification processes) in order to identify the common targets affected by PUAs and their correlation with morphological abnormalities. This study opens new perspectives for understanding how marine organisms afford protection from environmental toxicants through an integrated network of genes.