The stunting effect of an oxylipins-containing macroalgae extract on sea urchin reproduction and neuroblastoma cells viability.

Different bioactive molecules extracted from macroalgae, including oxylipins, showed interesting potentials in different applications, from healthcare to biomaterial manufacturing and environmental remediation. Thus far, no studies reported the effects of oxylipins-containing macroalgae extracts on embryo development of marine invertebrates and on neuroblastoma cancer cells. Here, the effects of an oxylipins-containing extract from Ericaria brachycarpa, a canopy-forming brown algae, were investigated on the development of Arbacia lixula sea urchin embryos and on SH-SY5Y neuroblastoma cells viability. Embryos and cells were exposed to concentrations covering a full 0-100% dose-response curve, with doses ranging from 0 to 40 µg mL-1 for embryos and from 0 to 200 µg mL-1 for cells. These natural marine toxins caused a dose-dependent decrease of normal embryos development and of neuroblastoma cells viability. Toxicity was higher for exposures starting from the gastrula embryonal stage if compared to the zygote and pluteus stages, with an EC50 significantly lower by 33 and 68%, respectively. Embryos exposed to low doses showed a general delay in development with a decrease in the ability to calcify, while higher doses caused 100% block of embryo growth. Exposure of SH-SY5Y neuroblastoma cells to 40 µg mL-1 for 72 h caused 78% mortality, while no effect was observed on their neuronal-like cells derivatives, suggesting a selective targeting of proliferating cells. Western Blot experiments on both model systems displayed the modulation of different molecular markers (HSP60, HSP90, LC3, p62, CHOP and cleaved caspase-7), showing altered stress response and enhanced autophagy and apoptosis, confirmed by increased fragmented DNA in apoptotic nuclei. Our study gives new insights into the molecular strategies that marine invertebrates use when responding to their environmental natural toxins and suggests the E. brachycarpa's extract as a potential source for the development of innovative, environmentally friendly products with larvicide and antineoplastic activity.

Environmental DNA: The First Snapshot of the Vertebrate Biodiversity in Three Sicilian Lakes.

Freshwater ecosystems play a key role in global diversity and are subject to a series of anthropic impacts, often leading to biodiversity loss. The organisms inhabiting these sites continuously release DNA into the environment through cells, excrement, gametes and/or decomposing matter; thus, evaluation of this eDNA could revolutionize the monitoring of biodiversity. In this study, environmental DNA metabarcoding was used for the first time in three Sicilian lakes: Lake Poma, Piana degli Albanesi Lake and Lake Scanzano. Results obtained provide the first snapshot of vertebrate biodiversity in these three lakes, where little is known, to provide valuable information useful for creating a baseline of knowledge regarding the biodiversity in these three lakes. Another important result was the detection of marine species, most likely due to some kind of anthropogenic contamination. Environmental DNA is a useful tool to evaluate both biodiversity and the ecological status of the environment; it has the potential to complement traditional methods, and the use of both approaches may offer a more comprehensive understanding of the ecosystem.

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRT-PCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis.

In Vitro Cytotoxic Effect of Aqueous Extracts from Leaves and Rhizomes of the Seagrass Posidonia oceanica (L.) Delile on HepG2 Liver Cancer Cells: Focus on Autophagy and Apoptosis.

Aqueous extracts from Posidonia oceanica's green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose-response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 µg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizome-derived preparation. The underlying death-promoting mechanisms identified involved the down-regulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer properties.

New Bioactive Peptides from the Mediterranean Seagrass Posidonia oceanica (L.) Delile and Their Impact on Antimicrobial Activity and Apoptosis of Human Cancer Cells.

The demand for new molecules to counter bacterial resistance to antibiotics and tumor cell resistance is increasingly pressing. The Mediterranean seagrass Posidonia oceanica is considered a promising source of new bioactive molecules. Polypeptide-enriched fractions of rhizomes and green leaves of the seagrass were tested against Gram-positive (e.g., Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Escherichia coli), as well as towards the yeast Candida albicans. The aforementioned extracts showed indicative MIC values, ranging from 1.61 µg/mL to 7.5 µg/mL, against the selected pathogens. Peptide fractions were further analyzed through a high-resolution mass spectrometry and database search, which identified nine novel peptides. Some discovered peptides and their derivatives were chemically synthesized and tested in vitro. The assays identified two synthetic peptides, derived from green leaves and rhizomes of P. oceanica, which revealed interesting antibiofilm activity towards S. aureus, E. coli, and P. aeruginosa (BIC50 equal to 17.7 µg/mL and 70.7 µg/mL). In addition, the natural and derivative peptides were also tested for potential cytotoxic and apoptosis-promoting effects on HepG2 cells, derived from human hepatocellular carcinomas. One natural and two synthetic peptides were proven to be effective against the "in vitro" liver cancer cell model. These novel peptides could be considered a good chemical platform for developing potential therapeutics.

HPLC/HRMS and GC/MS for Triacylglycerols Characterization of Tuna Fish Oils Obtained from Green Extraction.

BACKGROUND: Fish oil is one of the most common lipidic substances that is consumed as a dietary supplement. The high omega-3 fatty acid content in fish oil is responsible for its numerous health benefits. Fish species such as mackerel, herring, tuna, and salmon are particularly rich in these lipids, which contain two essential omega-3 fatty acids, known as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). OBJECTIVES: Due to the scarcity of information in the literature, this study aimed to conduct a qualitative and quantitative characterization of triglycerides (TAGs) in crude tuna fish oil using HPLC/HRMS. Fatty acid (FA) determination was also performed using GC/MS. The tuna fish oils analyzed were produced using a green, low-temperature process from the remnants of fish production, avoiding the use of any extraction solvents. RESULTS: The analyses led to the tentative identification and semi-quantitation of 81 TAGs. In silico saponification and comparison with fatty acid methyl ester results helped to confirm the identified TAGs and their quantities. The study found that the produced oil is rich in EPA, DHA, and erucic acid, while the negligible isomerization of fatty acids to trans-derivatives was observed.

Transcriptomic and Bioinformatic Analyses Identifying a Central Mif-Cop9-Nf-kB Signaling Network in Innate Immunity Response of Ciona robusta.

The Ascidian C. robusta is a powerful model for studying innate immunity. LPS induction activates inflammatory-like reactions in the pharynx and the expression of several innate immune genes in granulocyte hemocytes such as cytokines, for instance, macrophage migration inhibitory factors (CrMifs). This leads to intracellular signaling involving the Nf-kB signaling cascade that triggers downstream pro-inflammatory gene expression. In mammals, the COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) complex also results in the activation of the NF-kB pathway. It is a highly conserved complex in vertebrates, mainly engaged in proteasome degradation which is essential for maintaining processes such as cell cycle, DNA repair, and differentiation. In the present study, we used bioinformatics and in-silico analyses combined with an in-vivo LPS exposure strategy, next-generation sequencing (NGS), and qRT-PCR to elucidate molecules and the temporal dynamics of Mif cytokines, Csn signaling components, and the Nf-κB signaling pathway in C. robusta. A qRT-PCR analysis of immune genes selected from transcriptome data revealed a biphasic activation of the inflammatory response. A phylogenetic and STRING analysis indicated an evolutionarily conserved functional link between the Mif-Csn-Nf-kB axis in ascidian C. robusta during LPS-mediated inflammation response, finely regulated by non-coding molecules such as microRNAs (miRNAs).

Controlled uptake of PFOA in adult specimens of Paracentrotus lividus and evaluation of gene expression in their gonads and embryos.

Perfluorooctanoic acid (PFOA) has been largely used in the manufacturing industry but a few years ago it turned out to be a dangerous pollutant which is now of concern for terrestrial and aquatic environments. Here, we investigated the bioaccumulation of PFOA in the sea urchin Paracentrotus lividus after exposure to different concentrations of the pollutant for 28 days. We observed rapid uptake of PFOA in the coelomic fluid collected weekly during the exposure period and high bioaccumulation in gonads at the end of the experiment. Interestingly, animals were also able to fast depurate when relocated to a clean environment. In addition, to assess the effect of PFOA on sea urchins' physiological pathways, we analysed the expression profile of some marker genes both in the gonads and in the embryos obtained from parents exposed to PFOA. Our results suggest that PFOA is a persistent, bioaccumulative compound that adversely affects the health of the exposed organisms and their offspring by causing significant changes in the expression of some key target genes and the occurrence of developmental anomalies in the embryos.

Bioactive Molecules from the Innate Immunity of Ascidians and Innovative Methods of Drug Discovery: A Computational Approach Based on Artificial Intelligence.

The study of bioactive molecules of marine origin has created an important bridge between biological knowledge and its applications in biotechnology and biomedicine. Current studies in different research fields, such as biomedicine, aim to discover marine molecules characterized by biological activities that can be used to produce potential drugs for human use. In recent decades, increasing attention has been paid to a particular group of marine invertebrates, the Ascidians, as they are a source of bioactive products. We describe omics data and computational methods relevant to identifying the mechanisms and processes of innate immunity underlying the biosynthesis of bioactive molecules, focusing on innovative computational approaches based on Artificial Intelligence. Since there is increasing attention on finding new solutions for a sustainable supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of marine invertebrates' innate immunity.

Natural Anticancer Peptides from Marine Animal Species: Evidence from In Vitro Cell Model Systems.

Anticancer peptides are short and structurally heterogeneous aminoacidic chains, which display selective cytotoxicity mostly against tumor cells, but not healthy cells, based on their different cell surface properties. Their anti-tumoral activity is carried out through interference with intracellular homeostasis, such as plasmalemma integrity, cell cycle control, enzymatic activities and mitochondrial functions, ultimately acting as angiogenesis-, drug resistance- and metastasis-inhibiting agents, immune stimulators, differentiation inducers and necrosis or extrinsic/intrinsic apoptosis promoters. The marine environment features an ever-growing level of biodiversity, and seas and oceans are poorly exploited mines in terms of natural products of biomedical interest. Adaptation processes to extreme and competitive environmental conditions led marine species to produce unique metabolites as a chemical strategy to allow inter-individual signalization and ensure survival against predators, infectious agents or UV radiation. These natural metabolites have found broad use in various applications in healthcare management, due to their anticancer, anti-angiogenic, anti-inflammatory and regeneration abilities. The aim of this review is to pick selected studies that report on the isolation of marine animal-derived peptides and the identification of their anticancer activity in in vitro cultures of cancer cells, and list them with respect to the taxonomical hierarchy of the source organism.

A Novel Peptide with Antifungal Activity from Red Swamp Crayfish Procambarus clarkii.

The defense system of freshwater crayfish Procambarus clarkii as a diversified source of bioactive molecules with antimicrobial properties was studied. Antimicrobial activity of two polypeptide-enriched extracts obtained from hemocytes and hemolymph of P. clarkii were assessed against Gram positive (Staphylococcus aureus, Enterococcus faecalis) and Gram negative (Pseudomonas aeruginosa, Escherichia coli) bacteria and toward the yeast Candida albicans. The two peptide fractions showed interesting MIC values (ranging from 11 to 700 µg/mL) against all tested pathogens. Polypeptide-enriched extracts were further investigated using a high-resolution mass spectrometry and database search and 14 novel peptides were identified. Some peptides and their derivatives were chemically synthesized and tested in vitro against the bacterial and yeast pathogens. The analysis identified a synthetic derivative peptide, which showed an interesting antifungal (MIC and MFC equal to 31.2 µg/mL and 62.5 µg/mL, respectively) and antibiofilm (BIC50 equal to 23.2 µg/mL) activities against Candida albicans and a low toxicity in human cells.

Green Extraction of Polyphenols from Waste Bentonite to Produce Functional Antioxidant Excipients for Cosmetic and Pharmaceutical Purposes: A Waste-to-Market Approach.

In an ever-growing perspective of circular economy, the development of conscious, sustainable and environmental-friendly strategies to recycle the waste products is the key point. The scope of this work was to validate the waste bentonite from the grape processing industries as a precious matrix to extract polyphenols by applying a waste-to-market approach aimed at producing novel functional excipients. The waste bentonite was recovered after the fining process and opportunely pre-treated. Subsequently, both the freeze dried and the so-called "wet" bentonites were subjected to maceration. PEG200 and Propylene Glycol were selected as solvents due to their ability to dissolve polyphenols and their wide use in the cosmetic/pharmaceutical field. The extracts were evaluated in terms of yield, density, pH after water-dilution, total phenolic (Folin-Ciocalteu) and protein (Bradford) contents, antioxidant power (DPPH), amount of some representative polyphenols (HPLC-DAD), cytocompatibility and stability. Both solvents validated the bentonite as a valuable source of polyphenols and led to colored fluids characterized by an acidic pH after water-dilution. The best extract was obtained from the wet bentonite with PEG200 and highlighted the highest phenolic content and consequently the strongest antioxidant activity. Additionally, it displayed proliferative properties and resulted almost stable over time. Hence, it might be directly used as polyphenols-enriched functional novel raw material for cosmetic and pharmaceutics purposes.

The European Pine Marten Martes martes (Linnaeus, 1758) Is Autochthonous in Sicily and Constitutes a Well-Characterised Major Phylogroup within the Species (Carnivora, Mustelidae).

No molecular data are currently available for the Sicilian populations of the European pine marten Martes martes, thus preventing any sound inference about its native or non-native status on the island, as well as the local phylogeography of the species. In order to investigate these issues, we sequenced two mtDNA markers in road-killed specimens collected in Sicily. Both markers consistently demonstrated the existence of a well-characterised Sicilian clade of the species, which is endemic to the island and constitutes the sister group of a clade including the Mediterranean and Central-North European major phylogroups of the European pine marten. Such evidence supports the autochthony of Martes martes in Sicily and points to a natural Pleistocene colonisation of the island followed by isolation. The occurrence of a, to date undetected, major phylogroup of the species in Sicily calls for the dedicated monitoring of the Sicilian populations of the species in order to preserve this evolutionarily significant unit.

Cytotoxic capability and the associated proteomic profile of cell-free coelomic fluid extracts from the edible sea cucumber Holothuria tubulosa on HepG2 liver cancer cells.

Hepatocellular carcinoma (HCC) is an aggressive cancer histotype and one of the most common types of cancer worldwide. The identification of compounds that might intervene to restrain neoplastic cell growth appears imperative due to its elevated overall mortality. The marine environment represents a reservoir rich in bioactive compounds in terms of primary and secondary metabolites produced by aquatic animals, mainly invertebrates. In the present study, we determined whether the water-soluble cell-free extract of the coelomic fluid (CFE) of the edible sea cucumber Holothuria tubulosa could play an anti-HCC role in vitro by analyzing the viability and locomotory behavior, cell cycle distribution, apoptosis and autophagy modulation, mitochondrial function and cell redox state of HepG2 HCC cells. We showed that CFE causes an early block in the cell cycle at the G2/M phase, which is coupled to oxidative stress promotion, autophagosome depletion and mitochondrial dysfunction ultimately leading to apoptotic death. We also performed a proteomic analysis of CFE identifying a number of proteins that are seemingly responsible for anti-cancer effects. In conclusion, H. tubulosa's CFE merits further investigation to develop novel promising anti-HCC prevention and/or treatment agents and also beneficial supplements for formulation of functional foods and food packaging material.

Does Bacillus thuringiensis Affect the Stress and Immune Responses of Rhynchophorus ferrugineus Larvae, Females, and Males in the Same Way?

Bacillus thuringiensis (Bt) is considered a potentially useful entomopathogen against red palm weevil (RPW) Rhynchophorus ferrugineus. We compared the effects of Bt on mature larvae, females, and males. The pathogenicity of Bt was evaluated, estimating: Median Lethal Dose (LD50), Median Lethal Time (LT50), Total Hemocyte Count (THC), and Differential Hemocyte Counts (DHC), and the expression of the stress protein Heat Shock Protein 70 (Hsp 70) in hemocytes and the brain. Mortality exhibited a positive trend with the dosage and duration of exposure to Bt. Larvae were more susceptible than adults, and the LD50 of females was almost double the value of that of the larvae. LT50 value was higher for females than for males and larvae. Treatment with sub-lethal doses of Bt induced a decrease in THC in larvae, females, and males. In treated larvae, plasmatocytes decreased, while oenocytes and spherulocytes increased. In treated females, all types of hemocytes decreased, while in males the number of plasmatocytes decreased and granulocytes increased. We also registered the stress response directly on hemocytes showing that, already at 3 h after eating Bt, the expression of the stress protein Hsp 70 was modulated. This effect was also observed in brain tissue at 6 h after treatment. The results confirm that Bt treatment induces a pathogenic state in larvae and adults of both sexes, with effects after only a few hours from ingestion; however, the effects are different in magnitude and in type of target.

Haemolymphatic Parameters in Two Aquaculture Crustacean Species Cherax destructor (Clark, 1836) and Cherax quadricarinatus (Von Martens, 1868).

In the last few years, there has been a notable development in the breeding of freshwater shrimp (astaciculture), which involved various species and in particular, the two Australian Parastacidae species, Cherax destructor and Cherax quadricarinatus. Information about the haemolymphatic parameters of these two species is fragmentary, and filling these gaps becomes important given their importance in aquaculture. Cellular and biochemical parameters were analyzed in both species to create a reference baseline for these parameters to identify the state of welfare or suffering of these animals. The results showed that the total haemocyte count, haemocyte subpopulations, enzymatic activities and pH are similar between the two species, while total protein and osmolality are higher in C. destructor than C. quadricarinatus. Knowledge of these parameters could assist in evaluating the good health status of these species kept in aquaculture facilities.

The potential of antimicrobial peptides isolated from freshwater crayfish species in new drug development: A review.

The much-publicised increased resistance of pathogenic bacteria to conventional antibiotics has focused research effort on the characterization of new antimicrobial drugs. In this context, antimicrobial peptides (AMPs) extracted from animals are considered a promising alternative to conventional antibiotics. In recent years, freshwater crayfish species have emerged as an important source of bioactive compounds. In fact, these invertebrates rely on an innate immune system based on cellular responses and on the production of important effectors in the haemolymph, such as AMPs, which are produced and stored in granules in haemocytes and released after stimulation. These effectors are active against both Gram-positive and Gram-negative bacteria. In this review, we summarise the recent progress on AMPs isolated from the several species of freshwater crayfish and their prospects for future pharmaceutical applications to combat infectious agents.

Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta.

Human Drug Pollution in the Aquatic System: The Biochemical Responses of Danio rerio Adults.

To date, drug pollution in aquatic systems is an urgent issue, and Danio rerio is a model organism to study the toxicological effects of environmental pollutants. The scientific literature has analyzed the effect of human drug pollution on the biochemical responses in the tissues of D. rerio adults. However, the information is still scarce and conflicting, making it difficult to understand its real impact. The scientific studies are not consistent with each other and, until now, no one has grouped their results to create a baseline of knowledge of the possible impacts. In this review, the analysis of literature data highlights that the effects of drugs on adult zebrafishes depend on various factors, such as the tissue analyzed, the drug concentration and the sex of the individuals. Furthermore, the most influenced biochemical responses concern enzymes (e.g., antioxidants and hydrolase enzymes) and total protein and hormonal levels. Pinpointing the situation to date would improve the understanding of the chronic effects of human drug pollution, helping both to reduce it in the aquatic systems and then to draw up regulations to control this type of pollution.

Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring.

The first determination of presence and biodistribution of PFOA in ninety specimens of sea urchin Paracentrotus lividus from two differently contaminated sites along Palermo's coastline (Sicily) is reported. Analyses were performed on the sea urchins' coelomic fluids, coelomocytes, gonads or mixed organs, as well as on seawater and Posidonia oceanica leaves samples from the collection sites. PFOA concentration ranged between 1 and 13 ng/L in seawater and between 0 and 794 ng/g in P. oceanica. The analyses carried out on individuals of P. lividus from the least polluted site (A) showed PFOA median values equal to 0 in all the matrices (coelomic fluid, coelomocytes and gonads). Conversely, individuals collected from the most polluted site (B) showed median PFOA concentrations of 21 ng/g in coelomic fluid, 153 ng/g in coelomocytes, and 195 ng/g in gonads. Calculated bioconcentration factors of log10BCF > 3.7 confirmed the very bioaccumulative nature of PFOA. Significant correlations were found between the PFOA concentration of the coelomic fluid versus the total PFOA concentration of the entire sea urchin. PERMANOVA (p = 0.001) end Welch's t-test (p < 0.001) analyses showed a difference between specimens collected from the two sites highlighting the potential application of P. lividus as sentinel species for PFOA biomonitoring.