Search

Chronic myeloid leukemia (CML) is a myeloproliferative disease that activates multiple signaling pathways, causing cells to produce higher levels of reactive oxygen species (ROS). Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major generator of ROS in leukemia, and marine natural products have shown promising activities for the treatment of hematopoietic malignancies. In the present study, we investigated the effect of the marine microalga

The advancement in next-generation sequencing technologies and the dropping of sequencing costs have seen an increase in the amount of transcriptome data generated each year. These data are of big potential for identifying genes and molecular pathways of interest across a plethora of organisms. However, navigating these resources requires some bioinformatics and evolutionary skills. Here, we describe a protocol of transcriptome data mining for genes of interest, from the creation of a protein database to the inference of phylogenetic trees, which was used for marine protists, but can be used as general pipeline across different taxa.

Multiple myeloma (MM) is a blood cancer that occurs in the plasma cells (PCs), a type of white blood cell. Despite the progress of several current treatments that prolong the overall patient’s survival, most MM cases are incurable. For this reason, many efforts have been undertaken by the scientific community in the search for new treatments. BLENREPTM and Aplidin® are two marine-derived drugs currently in use for MM. In addition, other natural products have been identified from marine organisms, tested for their possible anticancer properties, and are in preclinical or clinical trials for MM, including cytarabine, a compound in use for leukaemia treatment. Between the most successful marine compounds in fighting MM, there are molecules with specific targets, such as the elongation factor 1-alpha 2 and proteasome inhibitors, and compounds conjugated with antibodies that recognise specific cell types and direct the drug to the correct cell target. Active compounds belong to different chemical classes, from cyclic peptides to alkaloids, highlighting the importance of screening the plethora of compounds produced by marine organisms. In this review, we summarise the current state of art of MM therapies focusing on the marine natural product emerging roles.

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.

Melanoma is considered a multifactorial disease etiologically divided into melanomas related to sun exposure and those that are not, but also based on their mutational signatures, anatomic site, and epidemiology. The incidence of melanoma skin cancer has been increasing over the past decades with 132,000 cases occurring globally each year. Marine organisms have been shown to be an excellent source of natural compounds with possible bioactivities for human health applications. In this review, we report marine compounds from micro- and macro-organisms with activities in vitro and in vivo against melanoma, including the compound Marizomib, isolated from a marine bacterium, currently in phase III clinical trials for melanoma. When available, we also report active concentrations, cellular targets and mechanisms of action of the mentioned molecules. In addition, compounds used for UV protection and melanoma prevention from marine sources are discussed. This paper gives an overview of promising marine molecules which can be studied more deeply before clinical trials in the near future.

Microalgae are known to produce a plethora of compounds derived from the primary and secondary metabolism. Different studies have shown that these compounds may have allelopathic, antimicrobial, and antipredator activities. In addition, in vitro and in vivo screenings have shown that several compounds have interesting bioactivities (such as antioxidant, anti-inflammatory, anticancer, and antimicrobial) for the possible prevention and treatment of human pathologies. Additionally, the enzymatic pathways responsible for the synthesis of these compounds, and the targets and mechanisms of their action have also been investigated for a few species. However, further research is necessary for their full exploitation and possible pharmaceutical and other industrial applications. Here, we review the current knowledge on the chemical characteristics, biological activities, mechanism of action, and the enzymes involved in the synthesis of microalgal metabolites with potential benefits for human health.

Aflatoxin B1 (AFB1), the mainly Aspergillus fungi derived mycotoxin, is well known for its carcinogenic effects on liver, and frequently occurs in food supplies, leading to fatal consequences in both farm animals and humans. Poultry, one of the most important segments of agro-industry, has been demonstrated to be extremely sensitive to AFB1 intake, which results in chickens’ low performance, decreased quality of both eggs and meat and a negative economic feedback. Oxidative stress caused by AFB1 plays a crucial role in chickens’ kidney damage by generating lipid peroxidation accompanied by a concomitant increase in the antioxidant enzymes involved in ROS metabolism (NADPH oxidase isoform 4 (NOX4) and its regulatory subunit p47-phox). The aim of the present work was to investigate the benefits of dietary supplementation, in chickens affected by AFB1 mycotoxicosis, using a new Feed additive (FA) containing a mixture of a tri-octahedral Na-smectite with a ligno-cellulose-based material an antioxidant adjuvant. Exposure of AFB1-treated chickens to the feed additive induced a significant down-regulation of both NOX4 and p47-phox genes expression levels. This trend was confirmed by their protein expression, demonstrating the great potential of the FA to counteract oxidative stress. To conclude, these results could open new perspectives in the methods of feeding chickens, using eco-friendly dietary supplements able to reduce AFB1-induced mycotoxicosis and to ameliorate poultry performances.

According to the WHO classification of tumors, more than 150 typologies of hematopoietic and lymphoid tumors exist, and most of them remain incurable diseases that require innovative approaches to improve therapeutic outcome and avoid side effects. Marine organisms represent a reservoir of novel bioactive metabolites, but they are still less studied compared to their terrestrial counterparts. This review is focused on marine natural products with anticancer activity against hematological tumors, highlighting recent advances and possible perspectives. Until now, there are five commercially available marine-derived compounds for the treatment of various hematopoietic cancers (e.g., leukemia and lymphoma), two molecules in clinical trials, and series of compounds and/or extracts from marine micro- and macroorganisms which have shown promising properties. In addition, the mechanisms of action of several active compounds and extracts are still unknown and require further study. The continuous upgrading of omics technologies has also allowed identifying enzymes with possible bioactivity (e.g., l-asparaginase is currently used for the treatment of leukemia) or the enzymes involved in the synthesis of bioactive secondary metabolites which can be the target of heterologous expression and genetic engineering.

Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 &micro, M, 15 &micro, M, and 20 &micro, M. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.

Background and Aims Type 2 diabetes mellitus is a pathological and chronical condition that induce a rearrangement in carbohydrate metabolism (Kaneto H et al. Antioxid Redox Signal. 2007). Even if the pathological mechanism of DN is not yet clear, NADPH oxidase (NOX) has been demonstrated to be involved in kidney ROS (Reactive Oxygen Species) generation during diabetic state (Palicz A. et al. J Biol Chem. 2001). Therefore, NOX-4, whose p22-phox subunit have shown a pivotal role in diabetes development, has been considered a potential pharmacological target in DN (Gill PS and Wilcox CS. Antioxid Redox Signal. 2006). In previous experiments performed on diabetic rat animal model with DN, we have found a beneficial effect of an extract rich in anthocyanins (ANT), members of flavonoids group (Shih PH. J Agric Food Chem. 2007). The aim of the present work was to investigate the molecular mechanisms involved in the DN and the protective effects of the new extract particularly abundant in ANT, the Red orange and Lemon Extract (RLE). Method Zucker diabetic fatty rats were divided into three groups and treated by gavage daily until 30 weeks of age, as follows: control group (ZF rats) received 1 mL of normal saline; ZDF Group (ZDF rats) received 1mL of normal saline; ZDF + RLE group (ZDF rats) treated with 90 mg/ kg of RLE dissolved in 1mL of normal saline. We examined the gene and protein expression of NOX4 catalytic subunit and its regulatory subunit p22-phox through Real Time PCR and western blot analysis. Results The expression gene levels of NOX4 significantly increased in diabetic rats ZDF, compared to control ZF (***p Conclusion In conclusion, we hypothesize that the protective effect of RLE on the development of DN is related to its ability to inhibit NADPH oxidase activity and opens new perspectives for the treatment of DN.

Diabetic nephropathy (DN) is the primary cause of end-stage renal disease, worldwide, and oxidative stress has been recognized as a key factor in the pathogenesis and progression of DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has the most important contribution to reactive oxygen species generation during the development of DN. Bioactive compound use has emerged as a potential approach to reduce chronic renal failure. Therefore, a red orange and lemon extract (RLE) rich in anthocyanins was chosen in our study, to reduce the toxic renal effects during the development of DN in Zucker diabetic fatty rat (ZDF). RLE effects were examined daily for 24 weeks, through gavage, in ZDF rats treated with RLE (90 mg/kg). At the end of the experiment, ZDF rats treated with RLE showed a reduction of the diabetes-associated up-regulation of both NOX4 and the p47-phox and p22-phox subunits, and restored the BAX/BCL-2 ratio respect to ZDF rats. Furthermore, RLE was able to reduce the oxidative DNA damage measured in urine samples in ZDF rats. This study showed that RLE could prevent the renal damage induced by DN through its capacity to inhibit NOX4 and apoptosis mechanisms. ispartof: JOURNAL OF CLINICAL MEDICINE vol:9 issue:5 ispartof: location:Switzerland status: published

Chlorophyll breakdown products are usually studied for their antioxidant and anti-inflammatory activities. The chlorophyll derivative Pheophorbide a (PPBa) is a photosensitizer that can induce significant anti-proliferative effects in several human cancer cell lines. Cancer is a leading cause of death worldwide, accounting for about 9.6 million deaths, in 2018 alone. Hence, it is crucial to monitor emergent compounds that show significant anticancer activity and advance them into clinical trials. In this review, we analyze the anticancer activity of PPBa with or without photodynamic therapy and also conjugated with or without other chemotherapic drugs, highlighting the capacity of PPBa to overcome multidrug resistance. We also report other activities of PPBa and different pathways that it can activate, showing its possible applications for the treatment of human pathologies.

Diatoms are the most diverse and abundant group of phytoplankton species and represent a huge reservoir of marine natural products with possible application for human health. Several diatoms are known to have anticancer, anti-inflammatory, antioxidant and anti-microbial properties, but the compounds responsible of these activities are often still unknown. The diatom Cylindrotheca closterium showed anti-inflammatory properties inhibiting TNFα release in human monocytic leukemia cells. In this study, we present the full transcriptome of C. closterium, and used an -omic approach to identify transcripts coding enzymes that can be involved in the synthesis/degradation of anti-inflammatory compounds. This approach allowed to identify phosphatidylinositol-3-phosphatase, phosphatidylinositol 3-kinase catalytic subunit type 3, phosphatidylinositol N-acetylglucosaminyltransferase subunit A, monogalactosyldiacylglycerol synthase and violaxanthin de-epoxidase, which are known to be involved in anti-inflammatory compound metabolism. When C. closterium was cultured in silica-starvation conditions, selected as stress condition to potentially trigger the synthesis of bioactive metabolites, anti-inflammatory activity was lost and expression levels of the analyzed transcripts were reduced. These data suggested that the control culturing condition was the most active. This study used for the first time a transcriptomic-guided approach to identify enzymes involved in anti-inflammatory compound metabolism, directing future discoveries of marine natural products in microalgae.

Diatoms are photosynthetic organisms with potential biotechnological applications in the bioremediation sector, having shown the capacity to reduce environmental concentrations of different pollutants. The diatom Cylindrotheca closterium is known to degrade di-n-butyl phthalate (DBP), one of the most abundant phthalate esters in aquatic environments and a known endocrine-disrupting chemical. In this study, we present for the first time the in silico identification of two putative DBP hydrolases (provisionally called DBPH1 and DBPH2) in the transcriptome of C. closterium. We modeled the structure of both DBPH1-2 and their proposed interactions with the substrate to gain insights into their mechanism of action. Finally, we analyzed the expression levels of the two putative hydrolases upon exposure of C. closterium to different concentrations of DBP (5 and 10 mg/l) for 24 and 48 h. The data showed a DBP concentration-dependent increase in expression levels of both dbph1 and 2 genes, further highlighting their potential involvement in phthalates degradation. This is the first identification of phthalate-degrading enzymes in microalgae, providing new insights into the possible use of diatoms in bioremediation strategies targeting phthalates.

Microalgae are photosynthetic unicellular organisms that can be found in very different environments, both terrestrial and marine, including extreme environments such as cold, hot and high/low salinity [...]

There is an urgent need to discover new drug entities due to the increased incidence of severe diseases as cancer and neurodegenerative pathologies, and reducing efficacy of existing antibiotics. Recently, there is a renewed interest in exploring the marine habitat for new pharmaceuticals also thanks to the advancement in cultivation technologies and in molecular biology techniques. Microorganisms represent a still poorly explored resource for drug discovery. The possibility of obtaining a continuous source of bioactives from marine microorganisms, more amenable to culturing compared to macro-organisms, may be able to meet the challenging demands of pharmaceutical industries. This would enable a more environmentally-friendly approach to drug discovery and overcome the over-utilization of marine resources and the use of destructive collection practices. The importance of the topic is underlined by the number of EU projects funded aimed at improving the exploitation of marine organisms for drug discovery.

Ochratoxin A (OTA) is a mycotoxin which represents an emerging problem for both animal and human health, due to its high presence in feed and foods. Exposure to OTA is associated with oxidative stress-induced nephrotoxicity. Therefore, the identification of new antioxidant or adsorbent substances with protective action constitutes one of the main challenges to reduce the negative effects induced by mycotoxins. For this purpose, we investigated the effect of two innovative feed additives, a bio-organoclay (CHS) and a mixture of a tri-octahedral Na-smectite with a ligno-cellulose based material (MIX) alone or in combination with OTA in kidneys of treated chickens. Real-Time PCR analyses for NADPH oxidase 4 (NOX) and p47-phox were performed to evaluate oxidative stress. Our results demonstrated an increase in NOX and p47-phox levels in OTA-treated chickens. Moreover, CHS, more than MIX, was able to reduce OTA-induced toxicity, restoring NOX levels. Taken together, these findings highlight the potential beneficial role of CHS in reverting OTA-induced nephrotoxicity in chickens and could lead to the production of healthier foods with beneficial consequences for human and animal health.

Due to the unique biodiversity and the physical-chemical properties of their environment, marine microorganisms have evolved defense and signaling compounds that often have no equivalent in terrestrial habitats. The aim of this study was to screen extracts of the dinoflagellate Amphidinium carterae for possible bioactivities (i.e., anticancer, anti-inflammatory, anti-diabetes, antibacterial and antifungal properties) and identify bioactive compounds. Anticancer activity was evaluated on human lung adenocarcinoma (A549), human skin melanoma (A2058), human hepatocellular carcinoma (HepG2), human breast adenocarcinoma (MCF7) and human pancreas carcinoma (MiaPaca-2) cell lines. Antimicrobial activities were evaluated against Gram-positive bacteria (Staphylococcus aureus MRSA and MSSA), Gram-negative bacteria (i.e., Escherichia coli and Klebsiella pneumoniae), Mycobacterium tuberculosis and the fungus Aspergillus fumigatus. The results indicated moderate biological activities against all the cancer cells lines and microorganisms tested. Bioassay-guided fractionation assisted by HRMS analysis allowed the detection of one new and two known amphidinols that are potentially responsible for the antifungal and cytotoxic activities observed. Further isolation, purification and structural elucidation led to a new amphidinol, named amphidinol 22. The planar structure of the new compound was determined by analysis of its HRMS and 1D and 2D NMR spectra. Its biological activity was evaluated, and it displayed both anticancer and antifungal activities.

Microalgae are currently considered one of the most promising resources for biofuel production, aquaculture feedstock and new pharmaceuticals. Among them, green algae of the genus Tetraselmis are extensively studied for their lipid accumulation in nutrient-starvation conditions. In this paper, we present the full-transcriptome of Tetraselmis suecica and differential expression analysis between nitrogen-starved and -repleted conditions (at stationary phase) focusing not only on lipid metabolism but giving new insights on nutrient starvation responses. Transcripts involved in signal transduction pathways, stress and antioxidant responses and solute transport were strongly up-regulated when T. suecica was cultured under nitrogen starvation. On the contrary, transcripts involved in amino acid synthesis, degradation of sugars, secondary metabolite synthesis, as well as photosynthetic activity were down-regulated under the same conditions. Among differentially expressed transcripts, a polyketide synthase and three lipoxygenases (involved in the synthesis of secondary metabolites with antipredator, anticancer and anti-infective activities) were identified, suggesting the potential synthesis of bioactive compounds by this microalga. In addition, the transcript for a putative nitrilase, enzyme used in nitrile bioremediation, is here reported for the first time for T. suecica. These findings give new insights on T. suecica responses to nutrient starvation and on possible biotechnological applications for green algae.

This review is an overview on marine bioactive peptides with promising activities for the development of alternative drugs to fight human pathologies. In particular, we focus on potentially prolific producers of peptides in microorganisms, including sponge-associated bacteria and marine photoautotrophs such as microalgae and cyanobacteria. Microorganisms are still poorly explored for drug discovery, even if they are highly metabolically plastic and potentially amenable to culturing. This offers the possibility of obtaining a continuous source of bioactive compounds to satisfy the challenging demands of pharmaceutical industries. This review targets peptides because of the variety of potent biological activities demonstrated by these molecules, including antiviral, antimicrobial, antifungal, antioxidant, anticoagulant, antihypertensive, anticancer, antidiabetic, antiobesity, and calcium-binding bioactivities. Several of these peptides have already gained recognition as effective drug agents in recent years. We also focus on cutting-edge omic approaches for the discovery of novel compounds for pharmacological applications. With rapid depletion of natural resources, omic technologies may be the solution to efficiently produce a vast variety of novel peptides with unique pharmacological potential.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Diatoms are unicellular algae playing a key role as photosynthetic organisms in the world’s ocean food webs. The chemical ecology of planktonic diatoms is well documented, but few studies have reported on the effects of benthic diatoms on their consumers, also due to difficulties in the collection, quantification and massive culturing of benthic species. Here for the first time we investigate the effects of feeding on two abundantly occurring benthic diatoms, Nanofrustulum shiloi and Cylindrotheca closterium, isolated from the leaves of the seagrass Posidonia oceanica, on the sea urchin Paracentrotus lividus. Adult P. lividus were fed for one month on diets of either one of the two diatoms and on the green alga Ulva rigida, used as a feeding control. By combining morphological, metabolomic and de novo transcriptomic approaches, we demonstrate toxigenic effect on embryos generated by females fed with these benthic diatoms. Furthermore, chemical analysis reveal the presence of polyunsaturated aldehydes only for N. shiloi, and a high production of other oxylipins (cytotoxic compounds on their grazers and on cancer cell lines) for both diatoms, including some additional peaks not correlated to the canonic oxylipins commonly observed in planktonic diatoms. These findings open new perspectives in the study of diatom secondary metabolites influencing their grazers.

This review is an overview on marine bioactive peptides with promising activities for the development of alternative drugs to fight human pathologies. In particular, we focus on potentially prolific producers of peptides in microorganisms, including sponge-associated bacteria and marine photoautotrophs such as microalgae and cyanobacteria. Microorganisms are still poorly explored for drug discovery, even if they are highly metabolically plastic and potentially amenable to culturing. This offers the possibility of obtaining a continuous source of bioactive compounds to satisfy the challenging demands of pharmaceutical industries. This review targets peptides because of the variety of potent biological activities demonstrated by these molecules, including antiviral, antimicrobial, antifungal, antioxidant, anticoagulant, antihypertensive, anticancer, antidiabetic, antiobesity, and calcium-binding bioactivities. Several of these peptides have already gained recognition as effective drug agents in recent years. We also focus on cutting-edge omic approaches for the discovery of novel compounds for pharmacological applications. With rapid depletion of natural resources, omic technologies may be the solution to efficiently produce a vast variety of novel peptides with unique pharmacological potential.

With the goal of identifying neuroactive secondary metabolites from microalgae, a microscale in vivo zebrafish bioassay for antiseizure activity was used to evaluate bioactivities of the diatom Skeletonema marinoi, which was recently revealed as being a promising source of drug-like small molecules. A freeze-dried culture of S. marinoi was extracted by solvents with increasing polarities (hexane, dichloromethane, methanol and water) and these extracts were screened for anticonvulsant activity using a larval zebrafish epilepsy model with seizures induced by the GABAA antagonist pentylenetetrazole. The methanolic extract of S. marinoi exhibited significant anticonvulsant activity and was chosen for bioassay-guided fractionation, which associated the bioactivity with minor constituents. The key anticonvulsant constituent was identified as the nucleoside inosine, a well-known adenosine receptor agonist with previously reported antiseizure activities in mice and rat epilepsy models, but not reported to date as a bioactive constituent of microalgae. In addition, a UHPLC-HRMS metabolite profiling was used for dereplication of the other constituents of S. marinoi. Structures of the isolated compounds were elucidated by nuclear magnetic resonance and high-resolution spectrometry. These results highlight the potential of zebrafish-based screening and bioassay-guided fractionation to identify neuroactive marine natural products.

Several EU projects funded during the FP7 programme and currently under the topic ‘Blue Growth’ of Horizon 2020 aim at improving the exploitation of marine organisms for several biotechnological applications (e.g. pharmaceutical, nutraceutical, cosmeceutical, bioenergy and other industrial applications). Here we focus only on those related to marine drug discovery such as BAMMBO, BlueGenics, GIAVAP, LIPOYEASTS, MaCuMBA, MAMBA, MAREX, MarineBiotech, PharmaSea, PolyModE, SeaBioTech, SUNBIOPATH, EMBRIC, INMARE, NoMorFilm and TASCMAR. We describe the major achievements in this field in terms of improved sampling in extreme environments, optimization in cultivation methods with the creation of ad hoc systems and bioreactors/raceway ponds, speeding up the production of a specific product by genetically modifying microorganisms and sequencing genomes/transcriptomes of marine species in order to identify enzymes of industrial interest or gene clusters responsible for the synthesis of bioactives. Several enzymes and new metabolites have been found allowing for important discoveries and new products for development in different market sectors: health, personal care, cosmetics and nutrition. We also discuss the urgent need to find new funding opportunities in H2020 that will allow candidate lead compounds identified by these projects to advance to preclinical testing in order to assess chemical, biological and toxicological properties. Overcoming these difficulties would allow marine drug discovery to reach its full potential, thereby supporting the development of blue biotechnology in Europe.

Marine microalgae are considered a potentially new and valuable source of biologically active compounds for applications in several biotechnology sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. Considering the increasing rate of antibiotic-resistance bacteria and infections by fungi, 46 microalgae have been screened in this study for possible antibacterial and antifungal activities. Two different extraction methods have been used in order to increase the probability of finding positive hits. In particular, we screened microalgae in both control and nutrient stress conditions. We also tested different strains for 7 species in order to study potentially different bioactivities due to strain diversity. Results showed that extracts of two diatoms, Skeletonema costatum and Chaetoceros pseudocurvisetus, had anti-tuberculosis activity and were active only when cultured in the control and phosphate-starvation conditions, while the nitrogen starvation condition showed no activity. In addition, we tested both the organic and water extracts and found that only the organic extracts for both diatoms were active. The organic extracts of these two diatom species were not toxic on normal human cell lines.

The effects of climate change are likely to be dependent on local settings. Nonetheless, the compounded effects of global and regional stressors remain poorly understood. Here, we used CO2 vents to assess how the effects of ocean acidification on the seagrass, Posidonia oceanica, and the associated epiphytic community can be modified by enhanced nutrient loading. P. oceanica at ambient and low pH sites was exposed to three nutrient levels for 16 months. The response of P. oceanica to experimental conditions was assessed by combining analyses of gene expression, plant growth, photosynthetic pigments and epiphyte loading. At low pH, nutrient addition fostered plant growth and the synthesis of photosynthetic pigments. Overexpression of nitrogen transporter genes following nutrient additions at low pH suggests enhanced nutrient uptake by the plant. In addition, enhanced nutrient levels reduced the expression of selected antioxidant genes in plants exposed to low pH and increased epiphyte cover at both ambient and low pH. Our results show that the effects of ocean acidification on P. oceanica depend upon local nutrient concentration. More generally, our findings suggest that taking into account local environmental settings will be crucial to advance our understanding of the effects of global stressors on marine systems.

Dinoflagellates are phytoplanktonic organisms found in both freshwater and marine habitats. They are often studied because related to harmful algal blooms but they are also known to produce bioactive compounds for the treatment of human pathologies. The aim of this study was to sequence the full transcriptome of the dinoflagellate Amphidinium carterae in both nitrogen-starved and -replete culturing conditions (1) to evaluate the response to nitrogen starvation at the transcriptional level, (2) to look for possible polyketide synthases (PKSs) in the studied clone (genes that may be involved in the synthesis of bioactive compounds), (3) if present, to evaluate if nutrient starvation can influence PKS expression, (4) to look for other possible enzymes of biotechnological interest and (5) to test strain cytotoxicity on human cell lines. Results showed an increase in nitrogen metabolism and stress response in nitrogen-starved cells and confirmed the presence of a type I β-ketosynthase. In addition, L-asparaginase (used for the treatment of Leukemia and for acrylamide reduction in food industries) and cellulase (useful for biofuel production and other industrial applications) have been identified for the first time in this species, giving new insights into possible biotechnological applications of dinoflagellates.

The seagrass Zostera marina is an important marine ecosystem engineer, greatly influencing oxygen and carbon fluctuations in temperate coastal areas. Although photosynthetically driven gas fluxes a ...

Ocean acidification is a major threat for marine life but seagrasses are expected to benefit from high CO2. In situ (long-term) and transplanted (short-term) plant incubations of the seagrass Cymodocea nodosa were performed near and away the influence of volcanic CO2 vents at Vulcano Island to test the hypothesis of beneficial effects of CO2 on plant productivity. We relate, for the first time, the expression of photosynthetic, antioxidant and metal detoxification-related genes to net plant productivity (NPP). Results revealed a consistent pattern between gene expression and productivity indicating water origin as the main source of variability. However, the hypothesised beneficial effect of high CO2 around vents was not supported. We observed a consistent long- and short-term pattern of gene down-regulation and 2.5-fold NPP decrease in plants incubated in water from the vents and a generalized up-regulation and NPP increase in plants from the vent site incubated with water from the Reference site. Contrastingly, NPP of specimens experimentally exposed to a CO2 range significantly correlated with CO2 availability. The down-regulation of metal-related genes in C. nodosa leaves exposed to water from the venting site suggests that other factors than heavy metals, may be at play at Vulcano confounding the CO2 effects.

Here we present the results of a multiple organizational level analysis conceived to identify acclimative/adaptive strategies exhibited by the seagrass Posidonia oceanica to the daily fluctuations in the light environment, at contrasting depths. We assessed changes in photophysiological parameters, leaf respiration, pigments, and protein and mRNA expression levels. The results show that the diel oscillations of P. oceanica photophysiological and respiratory responses were related to transcripts and proteins expression of the genes involved in those processes and that there was a response asynchrony between shallow and deep plants probably caused by the strong differences in the light environment. The photochemical pathway of energy use was more effective in shallow plants due to higher light availability, but these plants needed more investment in photoprotection and photorepair, requiring higher translation and protein synthesis than deep plants. The genetic differentiation between deep and shallow stands suggests the existence of locally adapted genotypes to contrasting light environments. The depth-specific diel rhythms of photosynthetic and respiratory processes, from molecular to physiological levels, must be considered in the management and conservation of these key coastal ecosystems. Portuguese funds from FCT - Foundation for Science and Technology [UID/Multi/04326/2013]; SZN PhD fellowship via the Open University; ESF COST Action Seagrass Productivity: From Genes to Ecosystem Management [ES0906] info:eu-repo/semantics/publishedVersion

Effects of the toxic Gulf of Mexico dinoflagellate Karenia brevis on its copepod grazers are equivocal, in that this dinoflagellate is ingested by various copepods, but may be toxic to, and/or nutritionally inadequate for grazers. Recent studies have shown that when the Mediterranean copepod Calanus helgolandicus fed on K. brevis, egg production rates and egg viability declined steadily over time, reaching almost zero levels after 5 days of feeding ( Turner et al., 2012 ). The K. brevis strain used in that study was chemically analyzed and shown not to contain any brevetoxins, but may have contained other harmful metabolites. Here we present gene expression analyses by reverse transcription-quantitative PCR (RT-qPCR) in C. helgolandicus after different exposure times (3, 5 and 8 days) to the same non-brevetoxin-containing K. brevis strain examined previously. The analyzed genes have a primary role in generic stress responses, defense systems (e.g. aldehyde, free fatty acid and free radical detoxification) and apoptosis regulation in other organisms. After 3 days of feeding on K. brevis, gene expression levels in copepods did not show significant changes. However, after 5 days of exposure, expression of stress-response genes was significantly reduced. After prolonged exposure for 8 days, expression of genes for 5 of 6 aldehyde dehydrogenases was strongly reduced, but expression of other genes was not coherently reduced, or increased. Microscopic observations revealed that after only a single day of feeding on K. brevis, freshly-spawned eggs were morphologically similar to those produced by copepods feeding on control diets, but after 3 days of feeding on K. brevis, eggs exhibited altered membrane cell morphology and apoptotic features such as granulation and degeneration of the cytoplasm matrix, compared to the unaltered eggs produced by copepods feeding on control diets. The changes in gene expression levels in copepods feeding on K. brevis were similar to those noted previously for copepods feeding upon diatoms that contained oxylipins, which interfered with copepod reproductive success. Changes in gene expression in copepods feeding upon toxic algal diets may offer important early warning signals of copepod stress responses to these diets.

The seagrass Zostera marina is an important marine ecosystem engineer, greatly influencing oxygen and carbon fluctuations in temperate coastal areas. Although photosynthetically driven gas fluxes are well studied, the impact of the plant’s mitochondrial respiration on overall CO2 and O2 fluxes in marine vegetated areas is not yet understood. Likewise, the gene expression in relation to the respiratory pathway has not been well analyzed in seagrasses. This study uses a combined approach, studying respiratory oxygen consumption rates in darkness simultaneously with changes in gene expression, with the aim of examining how respiratory oxygen consumption fluctuates on a diel basis. Measurements were first made in a field study where samples were taken directly from the ocean to the laboratory for estimations of respiratory rates. This was followed by a laboratory study where measurements of respiration and expression of genes known to be involved in mitochondrial respiration were conducted for 5 days under light conditions mimicking natural summer light (i.e., 15 h of light and 9 h of darkness), followed by 3 days of constant darkness to detect the presence of a potential circadian clock. In the field study, there was a clear diel variation in respiratory oxygen consumption with the highest rates in the late evening and at night (0.766 and 0.869 µmol O2 m−2 s−1, respectively). These repetitive diel patterns were not seen in the laboratory, where water conditions (temperature, pH, and oxygen) showed minor fluctuations and only light varied. The gene expression analysis did not give clear evidence on drivers behind the respiratory fluxes; however, expression levels of the selected genes generally increased when the seagrass was kept in constant darkness. While light may influence mitochondrial respiratory fluxes, it appears that other environmental factors (e.g., temperature, pH, or oxygen) could be of significance too. As seagrasses substantially alter the proportions of both oxygen and inorganic carbon in the water column and respiration is a great driver of these alterations, we propose that acknowledging the presence of respiratory fluctuations in nature should be considered when estimating coastal carbon budgets. As dark respiration in field at midnight was approximately doubled from that of midday, great over-, or underestimations of the respiratory carbon dioxide release from seagrasses could be made if values are just obtained at one specific time point and considered constant. Electronic supplementary material The online version of this article (doi:10.1007/s00227-017-3168-z) contains supplementary material, which is available to authorized users.

Published version. Source at https://doi.org/10.3389/fmars.2016.00068 Marine microalgae are considered a potentially new and valuable source of biologically active molecules for applications in the food industry as well as in the pharmaceutical, nutraceutical, and cosmetic sectors. They can be easily cultured, have short generation times and enable an environmentally-friendly approach to drug discovery by overcoming problems associated with the over-utilization of marine resources and the use of destructive collection practices. In this study, 21 diatoms, 7 dinoflagellates, and 4 flagellate species were grown in three different culturing conditions and the corresponding extracts were tested for possible antioxidant, anti-inflammatory, anticancer, anti-diabetes, antibacterial, and anti-biofilm activities. In addition, for three diatoms we also tested two different clones to disclose diversity in clone bioactivity. Six diatom species displayed specific anti-inflammatory, anticancer (blocking human melanoma cell proliferation), and anti-biofilm (against the bacteria Staphylococcus epidermidis) activities whereas, none of the other microalgae were bioactive against the conditions tested for. Furthermore, none of the 6 diatom species tested were toxic on normal human cells. Culturing conditions (i.e., nutrient starvation conditions) greatly influenced bioactivity of the majority of the clones/species tested. This study denotes the potential of diatoms as sources of promising bioactives for the treatment of human pathologies.

The stability on the expression level of putative reference genes (RGs) for reverse transcriptase-quantitative PCR (RT-qPCR) was tested in the seagrass Posidonia oceanica for plants collected in different environmental conditions (at different depths and in different salinity and pH). Besides adult plants, seedlings were also used for pH and salinity experiments. The putative RGs encompassed the most frequently used in other species to normalize RT-qPCR in a range of experimental conditions. Assignment of the best RGs has been performed with three Excel-based applets, BestKeeper, geNorm and NormFinder, and the results showed that best RGs may change depending on the experimental conditions tested, plant age and on the software utilized. Preferred sets of RGs are proposed for each of the conditions and a general procedure concerning their selection is discussed. In addition, expression levels of four genes involved in plant response to light, salinity and pH variation have also been evaluated.

In this study we develop gene expression tools in Calanus helgolandicus to study the effects of toxic diatom diets on copepod fitness and survival. We demonstrate that when adult females are fed on the control dinoflagellate Prorocentrum minimum and the flagellate Rhodomonas baltica, which are not known to produce toxic oxylipins, there are no significant changes in alpha- and beta-tubulin (microtubule subunits) gene expression levels. By contrast, the oxylipin-producing diatom Skeletonema marinoi influences tubulin expression levels which were markedly down-regulated. We scored a panel of putative reference genes (ACT, EFA, GAPDH, 18S, S7, S20, ATPs, UBI and IST) and found that two (S20 and S7) were highly stable in the tested conditions and can be used for further experiments. To normalize reverse transcription-quantitative real time polymerase chain reaction (RT-qPCR) data we also used the third best reference gene, GAPDH, considering the possibility that the two ribosomal proteins could be co-regulated. This pilot study will pave the way for further investigations on which genes are affected by diatom diets and clarify when and if a stress response or a detoxification mechanism becomes visible in C. helgolandicus. Moreover, the RT-qPCR analysis presented here may also be useful to study the effects of other diets and/or environmental factors such as salinity, temperature, pollution and other toxic compounds, on gene expression levels in this copepod species.

Seagrasses colonized the sea on at least three independent occasions to form the basis of one of the most productive and widespread coastal ecosystems on the planet. Here we report the genome of Zostera marina (L.), the first, to our knowledge, marine angiosperm to be fully sequenced. This reveals unique insights into the genomic losses and gains involved in achieving the structural and physiological adaptations required for its marine lifestyle, arguably the most severe habitat shift ever accomplished by flowering plants. Key angiosperm innovations that were lost include the entire repertoire of stomatal genes, genes involved in the synthesis of terpenoids and ethylene signalling, and genes for ultraviolet protection and phytochromes for far-red sensing. Seagrasses have also regained functions enabling them to adjust to full salinity. Their cell walls contain all of the polysaccharides typical of land plants, but also contain polyanionic, low-methylated pectins and sulfated galactans, a feature shared with the cell walls of all macroalgae and that is important for ion homoeostasis, nutrient uptake and O2/CO2 exchange through leaf epidermal cells. The Z. marina genome resource will markedly advance a wide range of functional ecological studies from adaptation of marine ecosystems under climate warming, to unravelling the mechanisms of osmoregulation under high salinities that may further inform our understanding of the evolution of salt tolerance in crop plants.

Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy to higher trophic levels. However, these unicellular organisms produce secondary metabolites deriving from the oxidation of fatty acids, collectively termed oxylipins, with negative effects on predators, such as copepods, that feed on them (e.g. reduction in survival, egg production and hatching success) and, indirectly, on higher trophic levels. Here, a multidisciplinary study (oxylipin measurements, copepod fitness, gene expression analyses, chlorophyll distribution, phytoplankton composition, physico-chemical characteristics) was carried out at the end of the spring diatom bloom in April 2011 in the Northern Adriatic Sea (Mediterranean Sea) in order to deeply investigate copepod diatom interactions, chemical communication and response pathways. The results show that the transect with the lowest phytoplankton abundance had the lowest copepod egg production and hatching success, but the highest oxylipin concentrations. In addition, copepods in both the analyzed transects showed increased expression levels of key stress-related genes (e.g. heat-shock proteins, catalase, glutathione S-transferase, aldehyde dehydrogenase) compared to control laboratory conditions where copepods were fed with the dinoflagellate Prorocentrum minimum which does not produce any oxylipins. New oxylipins that have never been reported before for microalgae are described for the first time, giving new insights into the complex nature of plant-animal signaling and communication pathways at sea. This is also the first study providing insights on the copepod response during a diatom bloom at the molecular level. (C) 2016 Elsevier B.V. All rights reserved.

Background The dense phytoplankton blooms that characterize productive regions and seasons in the oceans are dominated, from high to low latitudes and from coast line to open ocean, by comparatively few, often cosmopolitan species of diatoms. These key dominant species may undergo dramatic changes due to global climate change. Results In order to identify molecular stress-indicators for the ubiquitous diatom species Skeletonema marinoi, we tested stress-related genes in different environmental conditions (i.e. nutrient starvation/depletion, CO2-enrichment and combined effects of these stressors) using RT-qPCR. The data show that these stressors impact algal growth rate, inducing early aging and profound changes in expression levels of the genes of interest. Conclusions Most analyzed genes (e.g. antioxidant-related and aldehyde dehydrogenases) were strongly down-regulated which may indicate a strategy to avoid unnecessary over-investment in their respective proteins. By contrast, key genes were activated (e.g. HSPs, GOX) which may allow the diatom species to better cope with adverse conditions. We propose the use of this panel of genes as early bio-indicators of environmental stress factors in a changing ocean. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1574-5) contains supplementary material, which is available to authorized users.

Studies on stress genes are fundamental to understand how marine organisms maintain or re-estabilish a normal metabolism in face of physical or chemical disturbances. Aquatic organisms are in fact constantly exposed to environmental stimuli and natural and/or dissolved anthropogenic variables/compounds, including both physical (e.g. cold, heat, salinity and pH) and chemical (e.g. heavy metals, hydrocarbons and other pollutants) stressors. Human activities have intensified in coastal area, increasing the number of stressors that act simultaneously over natural systems (e.g. ocean acidification and eutrophication). In this study, Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) was used to characterize metabolic processes at the cellular level in response to natural CO2-enrichment and artificial nutrient-enrichment in proximity of a volcanic vent located in the Ischia island (Gulf of Naples, Tyrrhenian Sea). We evaluated the differential expression of selected stress genes in the seagrass Posidonia oceanica collected in a control site and in the vicinity of the CO2 vents. In each location, plants experienced three different nutrient concentrations: natural (without adding any nutrient), low- and high- enrichments. Results show that nutrient addition mainly induced an over-expression of genes codifying for antioxidant proteins, in sites not influenced by CO2-enrichment. In particular, we observed an increase in the activity of glutathione synthase, responsible of the synthesis of the antioxidant protein glutathione. In addition, we also observed the up-regulation of glutathione peroxidase, catalase, ascorbate reductase and cythocrome P450. When analysing the effects of nutrients in the acidified site, trends in expression changes were similar, but expression levels were notably lower. Interestingly, the over-expression of the above mentioned genes was always higher at low nutrient exposure, while other antioxidant enzymes (i.e. glutathione S-transferase and glutathione reductase) were more activated in high nutrient conditions. The difference in response between acidified and control site and in different nutrient conditions seems to derive from the combined affect of multiple stressors, in a way that still remains obscure. Effects of different stressors should be disentangled in order to identify stress-specific genes as early indicators of stressful conditions at sea and during laboratory experiments.

Plants developed several mechanisms to sense light and to cope with its natural diel and seasonal fluctuation. Many species use an endogenous clock to predict signals from the environment and to anticipate circadian light change. Changes in circadian clock genes allow plants to synchronize their physiology with local light and temperature conditions and to adapt to changes in light regimes occurring across environmental gradient (e.g. across latitudes). Understanding the genetic and physiological basis of plants response to the seasonal and daily variation in environmental conditions, could allow to predict the effects on plant physiology of environmental perturbations that could happen in the future, due to anthropogenic pressure and climatic changes. Most of the recently identified genes, associated with endogenous clock in plants, are themselves circadian regulated, with expression peaks phased to occur at a specific time of the day. Here we explore daily variations in the expression of clock genes in the marine angiosperm Posidonia oceanica along its bathymetric distribution, where plants are exposed to a natural cline of light and temperature. To do that, we measured genes expression (by RT-qPCR) of photoreceptors and components of the plant clock at two target depths (5 and 25 meter depth) and at six time points during the day. Sampling was performed in a continuous meadow located in the Bay of Calvi, Corsica (thanks to the ESF Cost Action 0906). We discuss the effects of the influence of the distinct light environments on the peak expression occurring at a specific time of the day between the two depths. Further, we discuss the potential adaptive relevance of these results on P. oceanica fitness and survival.

Seagrass meadows are among the most productive ecosystems, with Posidonia oceanica being the most important species along the Mediterranean coastline. This species forms extensive mono-specific meadows that are extremely sensitive to medium-high levels of disturbance and are being threatened by fast environmental changes caused by global warming and increasing human activities. The impact can either reflect in higher turbidity along the water column and in increased UV radiation, making the light availability one of the most important factors affecting P. oceanica distribution. Plants developed mechanisms of adaptations at multiple levels to track and cope with fluctuations and changes in the light environment. At molecular level, the modulation of gene expression in response to environmental changes allows plants to optimize the utilization of light energy for growth and to prevent damages due to its excess. To detect the relevant molecular adaptation strategies evolved by P. oceanica and to assess the plasticity showed in the acclimation under different light regimes, we are employing studies both in natural and controlled conditions. Here, we describe the differences in photo acclimation of plants living along the bathymetric cline observed in field and in a common garden experiment in mesocosms, after the exposition to contrasting light regimes. Using a transcriptional approach (both RT -qPCR and RNA-seq) coupled with a physiological one, we are also testing potential divergences existing among populations and individuals related to light sensitivity. These data should supply new insights for the management of seagrasses ecosystems, for the development of most successful transplantation strategies and ultimately for conservation of biodiversity of these precious ecosystems.

Submarine volcanic vents are being used as natural laboratories to assess the effects of increased ocean acidity and carbon dioxide (CO2) concentration on marine organisms and communities. However, in the vicinity of volcanic vents other factors in addition to CO2, which is the main gaseous component of the emissions, may directly or indirectly confound the biota responses to high CO2. Here we used for the first time the expression of antioxidant and stress-related genes of the seagrass Posidonia oceanica to assess the stress levels of the species. Our hypothesis is that unknown factors are causing metabolic stress that may confound the putative effects attributed to CO2 enrichment only. We analyzed the expression of 35 antioxidant and stress-related genes of P. oceanica in the vicinity of submerged volcanic vents located in the islands of Ischia and Panarea, Italy, and compared them with those from control sites away from the influence of vents. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was used to characterize gene expression patterns. Fifty-one percent of genes analyzed showed significant expression changes. Metal detoxification genes were mostly down-regulated in relation to controls at both Ischia and Panarea, indicating that P. oceanica does not increase the synthesis of heavy metal detoxification proteins in response to the environmental conditions present at the two vents. The up-regulation of genes involved in the free radical detoxification response (e.g., CAPX, SODCP and GR) indicates that, in contrast with Ischia, P. oceanica at the Panarea site faces stressors that result in the production of reactive oxygen species, triggering antioxidant responses. In addition, heat shock proteins were also activated at Panarea and not at Ischia. These proteins are activated to adjust stress-accumulated misfolded proteins and prevent their aggregation as a response to some stressors, not necessarily high temperature. This is the first study analyzing the expression of target genes in marine plants living near natural CO2 vents. Our results call for contention to the general claim of seagrasses as "winners" in a high-CO2 world, based on observations near volcanic vents. Careful consideration of factors that are at play in natural vents sites other than CO2 and acidification is required. This study also constitutes a first step for using stress-related genes as indicators of environmental pressures in a changing ocean.

Diatoms are a hugely diverse microalgal class, which possesses unique biological features and complex metabolic pathways and may activate sophisticated mechanisms to respond to environmental changes. Abiotic stress factors may limit growth rate of diatoms, but may also trigger intracellular signaling pathways that cause cells to undergo programmed cell death (PCD). Here we investigate the gene expression of different target genes related to cell death, namely programmed cell death 4 (PDCD4), tumor susceptibility gene 101 (TSG101), developmental and cell death (DCD) domain, death specific protein (DSP) and metacaspase (MC), using RT-qPCR in the cosmopolitan coastal centric diatom species Skeletonema marinoi, which contributes significantly to phytoplankton blooms in temperate waters. To this end, we undertook a detailed study of the best reference genes to analyze gene expression in S. marinoi under different experimental conditions (i.e. in different growth phases or under silica starvation). Results showed that DSP gene expression had a clear and constant increase along the S. marinoi growth curve reaching its maximum during the senescent phase. On the contrary, PDCD4, DCD, TSG101 and MC did not show any significant variation. These findings indicate that the DSP gene is a possible PCD marker induced by aging in this diatom species. In contrast, levels of DSP transcripts induced by silica starvation were relatively low compared to those induced by cell aging suggesting differential activation and/or regulation of the PCD machinery in response to different stressful conditions. Our study also expands the list of reference genes available for the diatom S. marinoi for normalization of RT-qPCR data of cells cultivated under different growth phases or under silica starvation.