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Recent studies have shown that the Mediterranean white sea bream Diplodus sargus includes the invasive green alga Caulerpa cylindracea in its diet, with consequent metabolic and enzymatic alterations. As a result of this novel alimentary habit, the bioactive algal red pigment caulerpin has been detected in its tissues. However, this may not be an isolated case: other fish species have also been reported to feed on C. cylindracea, although the possible accumulation of caulerpin in their tissues has not yet been investigated. In this report, we analysed stomach contents and caulerpin levels in the native sparid species Spondyliosoma cantharus, Sarpa salpa, and Diplodus vulgaris, and in the scarid Sparisoma cretense, along with the Lessepsian siganid Siganus luridus. C. cylindracea was found in the stomachs of all but one fish species, the exception being S. cretense, in which prey items could not be determined due to the high degree of digestion. Chemical analysis of fish tissues revealed that only S. cantharus and S. salpa accumulated caulerpin, while no traces of the compound were detected in the other species. Despite intense research efforts on natural products obtained from C. cylindracea, a complete picture of the impacts caused by fish including this alga in their diet has not been elucidated. The identification of caulerpin in other Mediterranean native fish suggests a need for further research in order to assess the possible transfer of such molecules to humans through seafood consumption.

Ecballium elaterium , also known as squirting cucumber, is a plant which is widespread in temperate regions of Europe, Africa and Asia. The plant is considered to be one of the oldest used drugs. In the last decades, E. elaterium has been widely studied as a source of triterpene metabolites named cucurbitacins, often found as glycosylated derivatives, used by the plant as defensive agents. Such metabolites exhibit several biological activities, including cytotoxic, anti-inflammatory, and anti-cancer. Interestingly, the bioactive properties of E. elaterium extracts have been investigated in dozens of studies, especially by testing the apolar fractions, including the essential oils, extracted from leaves and fruits. The purpose of this review is to provide an overview of the chemical profile of different parts of the plants (leaves, flowers, and seeds) analyzing the methods used for structure elucidation and identification of single metabolites. The pharmacological studies on the isolated compounds are also reported, to highlight their potential as good candidates for drug discovery.

We investigated the effects of dietary delivered self-DNA in the model insect Drosophila melanogaster . Self-DNA administration resulted in low but significant lethality in Drosophila larvae and considerably extended the fly developmental time. This was characterized by the abnormal persistence of the larvae in the L2 and L3 stages, which largely accounted for the average 72 h delay observed in pupariation, as compared to controls. In addition, self-DNA exposure affected adult reproduction by markedly reducing both female fecundity and fertility, further demonstrating its impact on Drosophila developmental processes. The effects on the metabolites of D. melanogaster larvae after exposure to self-DNA were studied by NMR, LC-MS, and molecular networking. The results showed that self-DNA feeding reduces the amounts of all metabolites, particularly amino acids and N-acyl amino acids, which are known to act as lipid signal mediators. An increasing amount of phloroglucinol was found after self-DNA exposure and correlated to developmental delay and egg-laying suppression. Pidolate, a known intermediate in the γ-glutamyl cycle, also increased after exposure to self-DNA and correlated to the block of insect oogenesis.

Despite low temperatures, poor nutrient levels and high pressure, microorganisms thrive in deep-sea environments of polar regions. The adaptability to such extreme environments renders deep-sea microorganisms an encouraging source of novel, bioactive secondary metabolites. In this study, we isolated 77 microorganisms collected by a remotely operated vehicle from the seafloor in the Fram Strait, Arctic Ocean (depth of 2454 m). Thirty-two bacteria and six fungal strains that represented the phylogenetic diversity of the isolates were cultured using an One-Strain-Many-Compounds (OSMAC) approach. The crude EtOAc extracts were tested for antimicrobial and anticancer activities. While antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecium was common for many isolates, only two bacteria displayed anticancer activity, and two fungi inhibited the pathogenic yeast Candida albicans . Due to bioactivity against C. albicans and rich chemical diversity based on molecular network-based untargeted metabolomics, Aspergillus versicolor PS108-62 was selected for an in-depth chemical investigation. A chemical work-up of the SPE-fractions of its dichloromethane subextract led to the isolation of a new PKS-NRPS hybrid macrolactone, versicolide A ( 1 ), a new quinazoline (-)-isoversicomide A ( 3 ), as well as three known compounds, burnettramic acid A ( 2 ), cyclopenol ( 4 ) and cyclopenin ( 5 ). Their structures were elucidated by a combination of HRMS, NMR, [α] D , FT-IR spectroscopy and computational approaches. Due to the low amounts obtained, only compounds 2 and 4 could be tested for bioactivity, with 2 inhibiting the growth of C. albicans (IC 50 7.2 µg/mL). These findings highlight, on the one hand, the vast potential of the genus Aspergillus to produce novel chemistry, particularly from underexplored ecological niches such as the Arctic deep sea, and on the other, the importance of untargeted metabolomics for selection of marine extracts for downstream chemical investigations.

Moringa oleifera is a traditional food crop widespread in Asiatic, African, and South American continents. The plant, able to grow in harsh conditions, shows a high nutritional value and medicinal potential evidencing cardioprotective, anti-inflammatory, antioxidant, and antimicrobial properties. The purpose of this study was the phytochemical analysis of M. oleifera and the identification of the antimicrobial compounds by combining a chemical approach with in vitro tests. The metabolite profile of M. oleifera polar and apolar extracts of leaves and seeds were investigated by using Nuclear Magnetic Resonance spectroscopy and Gas Chromatography-Mass Spectrometry. The antimicrobial activity of all of the obtained extract was evaluated against four bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Salmonella enterica). The chemical analysis provided a wide set of metabolites that were identified and quantified. Moreover, apolar extracts from seeds showed a significant concentration-dependent antimicrobial activity against S. aureus and S. epidermidis, (4 mg/mL reduced the viability up to 50%) that was associated to the content of specific fatty acids. Our results remarked the advantages of an integrated approach for the identification of plant metabolites and its use in association with biological tests to recognize the compounds responsible for bioactivity without compounds purification.

Lettuce plants were grown in a greenhouse affected by the fungal pathogen Fusarium oxysporum to test the effects on plant metabolomics by different organic treatments. Three foliar application treatments were applied: a commercial compost tea made of aerobically fermented plant organic matter, a pure lyophilized microalga Artrospira platensis , commonly named spirulina, and the same microalga previously exposed during its culture to a natural uptake from medium enriched with F. oxysporum fragmented DNA (NAT). The experiment is the first attempt to observe in field conditions, the use and effects of a natural microbial library as a carrier of pathogenic fungal DNA for disease control. Untargeted NMR metabolomics and chemometrics showed that foliar organic application significantly reduced fumaric and formic acids, aromatic amino acids, and nucleosides, while increasing ethanolamine. A strong decrease in phenolic acids and an increase in citric acid and glutamine were specifically observed in the NAT treatment. It is noteworthy that the exposure of a known biostimulant microalga to fungal DNA in its culture medium was sufficient to induce detectable changes in the metabolomic profiles of the fertilized plants. These findings deserve further investigation to assess the potential relevance of the presented approach in the field of crop biostimulation and biocontrol of plant pathogens.

Seaweed endophytic (algicolous) fungi are talented producers of bioactive natural products. We have previously isolated two strains of the endophytic fungus, Pyrenochaetopsis sp. FVE-001 and FVE-087, from the thalli of the brown alga Fucus vesiculosus . Initial chemical studies yielded four new decalinoylspirotetramic acid derivatives with antimelanoma activity, namely pyrenosetins A-C ( 1 - 3 ) from Pyrenochaetopsis sp. strain FVE-001, and pyrenosetin D ( 4 ) from strain FVE-087. In this study, we applied a comparative metabolomics study employing HRMS/MS based feature-based molecular networking (FB MN) on both Pyrenochaetopsis strains. A higher chemical capacity in production of decalin derivatives was observed in Pyrenochaetopsis sp. FVE-087. Notably, several decalins showed different retention times despite the same MS data and MS/MS fragmentation pattern with the previously isolated pyrenosetins, indicating they may be their stereoisomers. FB MN-based targeted isolation studies coupled with antimelanoma activity testing on the strain FVE-087 afforded two new stereoisomers, pyrenosetins E ( 5 ) and F ( 6 ). Extensive NMR spectroscopy including DFT computational studies, HR-ESIMS, and Mosher's ester method were used in the structure elucidation of compounds 5 and 6 . The 3' R ,5' R stereochemistry determined for compound 6 was identical to that previously reported for pyrenosetin C ( 3 ), whose stereochemistry was revised as 3' S ,5' R in this study. Pyrenosetin E ( 5 ) inhibited the growth of human malignant melanoma cells (A-375) with an IC 50 value of 40.9 μM, while 6 was inactive. This study points out significant variations in the chemical repertoire of two closely related fungal strains and the versatility of FB MN in identification and targeted isolation of stereoisomers. It also confirms that the little-known fungal genus Pyrenochaetopsis is a prolific source of complex decalinoylspirotetramic acid derivatives.

Brown alga Bifurcaria bifurcata is an extraordinarily rich source of linear (acylic) diterpenes with enormous structural diversity. As part of our interest into secondary metabolites of the Irish seaweeds, here we report four new acyclic diterpenes ( 1 - 4 ) and seven known terpenoids ( 5 - 11 ) from the CHCl 3 extract of B. bifurcata . The planar structures of the new metabolites were elucidated by means of 1D and 2D NMR, HRMS, and FT-IR spectroscopy. Since linear diterpenes are highly flexible compounds, the assignment of their stereochemistry by conventional methods, e.g., NOESY NMR, is difficult. Therefore, we employed extensive quantum-mechanical prediction of NMR chemical shifts and optical rotation analyses to identify the relative and absolute configurations of the new compounds 1 - 4 . Several compounds moderately inhibited the human breast cancer cell line (MDA-MB-231) with IC 50 values ranging from 10.0 to 33.5 μg/mL. This study not only demonstrates the vast capacity of the Irish B. bifurcata to produce highly oxygenated linear diterpenoids, but also highlights the potential of new methodologies for assignment of their stereogenic centers.

A diketopiperazine has been purified from a culture filtrate of the endophytic fungus Paraphaeosphaeria sporulosa , isolated from healthy tissues of strawberry plants in a survey of microbes as sources of anti-bacterial metabolites. Its structure has been determined by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analyses and was found to be identical to cyclo (L-Pro-L-Phe) purified from species of other fungal genera. This secondary metabolite has been selected following bioguided-assay fractionation against two strains of Salmonella enterica , the causal agent of bovine gastroenteritis. The diketopiperazine cyclo (L-Pro-L-Phe), isolated for the first time from Paraphaeosphaeria species, showed minimum inhibitory concentration (MIC) values of 71.3 and 78.6 μg/mL against the two S. enterica strains. This finding may be significant in limiting the use of synthetic antibiotics in animal husbandry and reducing the emergence of bacterial multidrug resistance. Further in vivo experiments of P. sporulosa diketopiperazines are important for the future application of these metabolites.

The fungal genus Pyrenochaetopsis is commonly found in soil, terrestrial, and marine environments, however, has received little attention as a source of bioactive secondary metabolites so far. In a recent work, we reported the isolation and characterization of three new anticancer decalinoyltetramic acid derivatives, pyrenosetins A-C, from the Baltic Fucus vesiculosus -derived endophytic fungus Pyrenochaetopsis sp. FVE-001. Herein we report a new pentacyclic decalinoylspirotetramic acid derivative, pyrenosetin D ( 1 ), along with two known decalin derivatives wakodecalines A ( 2 ) and B ( 3 ) from another endophytic strain Pyrenochaetopsis FVE-087 isolated from the same seaweed and showed anticancer activity in initial screenings. The chemical structures of the purified compounds were elucidated by comprehensive analysis of HR-ESIMS, FT-IR, [a] D , 1D and 2D NMR data coupled with DFT calculations of NMR parameters and optical rotation. Compounds 1-3 were evaluated for their anticancer and toxic potentials against the human malignant melanoma cell line (A-375) and the non-cancerous keratinocyte cell line (HaCaT). Pyrenosetin D ( 1 ) showed toxicity towards both A-375 and HaCaT cells with IC 50 values of 77.5 and 39.3 μM, respectively, while 2 and 3 were inactive. This is the third chemical study performed on the fungal genus Pyrenochaetopsis and the first report of a pentacyclic decalin ring system from the fungal genus Pyrenochaetopsis .

The saponin composition of a specimen of black sea cucumber, Holothuria atra collected in the Persian Gulf was studied by a combined approach including LC-MS/MS, Molecular Networking, pure compound isolation, and NMR spectroscopy. The saponin composition of Holothuria atra turned out to be more complex than previously reported. The most abundant saponins in the extract ( 1 ⁻ 4 ) were isolated and characterized by 1D- and 2D-NMR experiments. Compound 1 was identified as a new triterpene glycoside saponin, holothurin A5. The side chain of the new saponin 1 , unprecedented among triterpene glycosides, is characterized by an electrophilic enone function, which can undergo slow water or methanol addition under neutral conditions. The cytotoxic activity of compounds 1 ⁻ 4 , evaluated on the human cervix carcinoma HeLa cell line, was remarkable, with IC 50 values ranging from 1.2 to 2.5 µg/mL.

Although the chemical warfare between invasive and native species has become a central problem in invasion biology, the molecular mechanisms by which bioactive metabolites from invasive pests influence local communities remain poorly characterized. This study demonstrates that the alkaloid caulerpin (CAU)-a bioactive component of the green alga Caulerpa cylindracea that has invaded the entire Mediterranean basin-is an agonist of peroxisome proliferator-activated receptors (PPARs). Our interdisciplinary study started with the in silico prediction of the ligand-protein interaction, which was then validated by in vivo, ex vivo and in vitro assays. On the basis of these results, we candidate CAU as a causal factor of the metabolic and behavioural disorders observed in Diplodus sargus , a native edible fish of high ecological and commercial relevance, feeding on C. cylindracea . Moreover, given the considerable interest in PPAR activators for the treatment of relevant human diseases, our findings are also discussed in terms of a possible nutraceutical/pharmacological valorisation of the invasive algal biomasses, supporting an innovative strategy for conserving biodiversity as an alternative to unrealistic campaigns for the eradication of invasive pests.

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels, and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (α-GalCer), a lipid antigen-stimulating invariant natural killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/α-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo , without inducing iNKT anergy. Moreover, co-delivery of α-GalCer and a MHC class I restricted tumor-associated antigenic determinant to antigen-presenting cells via bacteriophages strongly boosted adaptive CD8 + T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free α-GalCer and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe, inexpensive, and versatile tool.

Among marine organisms, sponges are the richest sources of pharmacologically-active compounds. Stemming from a previous lead discovery program that gathered a comprehensive library of organic extracts of marine sponges from the off-shore region of Portugal, crude extracts of Erylus cf . deficiens collected in the Gorringe Bank (Atlantic Ocean) were tested in the innovative high throughput screening (HTS) assay for inhibitors of indoleamine 2,3-dioxygenase (IDO) and showed activity. Bioassay guided fractionation of the dichloromethane extract led to the isolation of four new glycolipids, named erylusamide A - D . The structures of the isolated compounds were established by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and chemical derivatization. The metabolites shared a pentasaccharide moiety constituted by unusual highly acetylated ᴅ-glucose moieties as well as ᴅ-xylose and ᴅ-galactose. The aglycones were unprecedented long chain dihydroxyketo amides. Erylusamides A , B and D differ in the length of the hydrocarbon chain, while erylusamide C is a structural isomer of erylusamide B ., Competing Interests: The authors declare no conflict of interest.

The glycerophosphoinositols constitute a class of biologically active lipid-derived mediators whose intracellular levels are modulated during physiological and pathological cell processes. Comprehensive assessment of the role of these compounds expands beyond the cellular biology of lipids and includes rapid and unambiguous measurement in cells and tissues. Here we describe a sensitive and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative analysis of the most abundant among these phosphoinositide derivatives in mammalian cells, the glycerophosphoinositol (GroPIns). The method has been developed in mouse Raw 264.7 macrophages with limits of quantitation at 3 ng/ml. Validation on the same cell line showed excellent response in terms of linear dynamic range (from 3 to 3,000 ng/ml), intra-day and inter-day precision (coefficient of variation ≤ 7.10%) and accuracy (between 98.1 and 109.0%) in the range 10-320 ng/ml. As proof of concept, a simplified analytical platform based on this method and external calibration was also tested on four stimulated and unstimulated cell lines, including Raw 264.7 macrophages, Jurkat T-cells, A375MM melanoma cells and rat basophilic leukemia RBL-2H3 cells. The results indicate a wide variation in GroPIns levels among different cell lines and stimulation conditions, although the measurements were always in line with the literature. No significant matrix effects were observed thus indicating that the here proposed method can be of general use for similar determinations in cells of different origin.

The glycerophosphodiester phosphodiesterases are evolutionarily conserved proteins that have been linked to several patho/physiological functions, comprising bacterial pathogenicity and mammalian cell proliferation or differentiation. The bacterial enzymes do not show preferential substrate selectivities among the glycerophosphodiesters, and they are mainly dedicated to glycerophosphodiester hydrolysis, producing glycerophosphate and alcohols as the building blocks that are required for bacterial biosynthetic pathways. In some cases, this enzymatic activity has been demonstrated to contribute to bacterial pathogenicity, such as with Hemophilus influenzae. Mammalian glyerophosphodiesterases have high substrate specificities, even if the number of potential physiological substrates is continuously increasing. Some of these mammalian enzymes have been directly linked to cell differentiation, such as GDE2, which triggers motor neuron differentiation, and GDE3, the enzymatic activity of which is necessary and sufficient to induce osteoblast differentiation. Instead, GDE5 has been shown to inhibit skeletal muscle development independent of its enzymatic activity., (© 2013 FEBS.)