Marine cyanobacteria compounds with anticancer properties: a review on the implication of apoptosis

Marine cyanobacteria have been considered a rich source of secondary metabolites with potential biotechnological applications, namely in the pharmacological field. Chemically diverse compounds were found to induce cytoxicity, anti-inflammatory and antibacterial activities. The potential of marine cyanobacteria as anticancer agents has however been the most explored and, besides cytotoxicity in tumor cell lines, several compounds have emerged as templates for the development of new anticancer drugs. The mechanisms implicated in the cytotoxicity of marine cyanobacteria compounds in tumor cell lines are still largely overlooked but several studies point to an implication in apoptosis. This association has been related to several apoptotic indicators such as cell cycle arrest, mitochondrial dysfunctions and oxidative damage, alterations in caspase cascade, alterations in specific proteins levels and alterations in the membrane sodium dynamics. In the present paper a compilation of the described marine cyanobacterial compounds with potential anticancer properties is presented and a review on the implication of apoptosis as the mechanism of cell death is discussed.

Are known cyanotoxins involved in the toxicity of picoplanktonic and filamentous north atlantic marine cyanobacteria?

Eight marine cyanobacteria strains of the genera Cyanobium, Leptolyngbya, Oscillatoria, Phormidium, and Synechococcus were isolated from rocky beaches along the Atlantic Portuguese central coast and tested for ecotoxicity. Strains were identified by morphological characteristics and by the amplification and sequentiation of the 16S rDNA. Bioactivity of dichloromethane, methanol and aqueous extracts was assessed by the Artemia salina bioassay. Peptide toxin production was screened by matrix assisted laser desorption/ionization time of flight mass spectrometry. Molecular analysis of the genes involved in the production of known cyanotoxins such as microcystins, nodularins and cylindrospermopsin was also performed. Strains were toxic to the brine shrimp A. salina nauplii with aqueous extracts being more toxic than the organic ones. Although mass spectrometry analysis did not reveal the production of microcystins or other known toxic peptides, a positive result for the presence of mcyE gene was found in one Leptolyngbya strain and one Oscillatoria strain. The extensive brine shrimp mortality points to the involvement of other unknown toxins, and the presence of a fragment of genes involved in the cyanotoxin production highlight the potential risk of cyanobacteria occurrence on the Atlantic coast.

Antimicrobial and cytotoxic assessment of marine cyanobacteria - synechocystis and synechococcus

Aqueous extracts and organic solvent extracts of isolated marine cyanobacteria strains were tested for antimicrobial activity against a fungus, Gram-positive and Gram-negative bacteria and for cytotoxic activity against primary rat hepatocytes and HL-60 cells. Antimicrobial activity was based on the agar diffusion assay. Cytotoxic activity was measured by apoptotic cell death scored by cell surface evaluation and nuclear morphology. A high percentage of apoptotic cells were observed for HL-60 cells when treated with cyanobacterial organic extracts. Slight apoptotic effects were observed in primary rat hepatocytes when exposed to aqueous cyanobacterial extracts. Nine cyanobacteria strains were found to have antibiotic activity against two Gram-positive bacteria, Clavibacter michiganensis subsp. insidiosum and Cellulomonas uda. No inhibitory effects were found against the fungus Candida albicans and Gram-negative bacteria. Marine Synechocystis and Synechococcus extracts induce apoptosis in eukaryotic cells and cause inhibition of Gram-positive bacteria. The different activity in different extracts suggests different compounds with different polarities.

Removal of Cd(II), Zn(II) and Pb(II) from aqueous solutions by brown marine macro algae: kinetic modelling

Specific marine macro algae species abundant at the Portuguese coast (Laminaria hyperborea, Bifurcaria bifurcata, Sargassum muticum and Fucus spiralis) were shown to be effective for removing toxic metals (Cd(II), Zn(II) and Pb(II)) from aqueous solutions. The initial metal concentrations in solution were about 75–100 mg L−1. The observed biosorption capacities for cadmium, zinc and lead ions were in the ranges of 23.9–39.5, 18.6–32.0 and 32.3–50.4 mg g−1, respectively. Kinetic studies revealed that the metal uptake rate was rather fast, with 75% of the total amount occurring in the first 10 min for all algal species. Experimental data were well fitted by a pseudo-second order rate equation. The contribution of internal diffusion mechanism was significant only to the initial biosorption stage. Results indicate that all the studied macro algae species can provide an efficient and cost-effective technology for eliminating heavy metals from industrial effluents.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

Exploring bioactive properties of marine cyanobacteria Isolated from the Portuguese coast: high potential as a source of anticancer compounds

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria.

Antitumor activity of hierridin B, a cyanobacterial secondary metabolite found in both filamentous and unicellular marine strains

Cyanobacteria are widely recognized as a valuable source of bioactive metabolites. The majority of such compounds have been isolated from so-called complex cyanobacteria, such as filamentous or colonial forms, which usually display a larger number of biosynthetic gene clusters in their genomes, when compared to free-living unicellular forms. Nevertheless, picocyanobacteria are also known to have potential to produce bioactive natural products. Here, we report the isolation of hierridin B from the marine picocyanobacterium Cyanobium sp. LEGE 06113. This compound had previously been isolated from the filamentous epiphytic cyanobacterium Phormidium ectocarpi SAG 60.90, and had been shown to possess antiplasmodial activity. A phylogenetic analysis of the 16S rRNA gene from both strains confirmed that these cyanobacteria derive from different evolutionary lineages. We further investigated the biological activity of hierridin B, and tested its cytotoxicity towards a panel of human cancer cell lines; it showed selective cytotoxicity towards HT-29 colon adenocarcinoma cells.

Bisphenol A migration from plastic materials: direct insight of ecotoxicity in Daphnia magna

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) whose migration from food packaging is recognized worldwide. However, the real overall food contamination and related consequences are yet largely unknown. Among humans, children’s exposure to BPA has been emphasized because of the immaturity of their biological systems. The main aim of this study was to assess the reproductive impact of BPA leached from commercially available plastic containers used or related to child nutrition, performing ecotoxicological tests using the biomonitoring species Daphnia magna. Acute and chronic tests, as well as single and multigenerational tests were done. Migration of BPA from several baby bottles and other plastic containers evaluated by GC-MS indicated that a broader range of foodstuff may be contaminated when packed in plastics. Ecotoxicological test results performed using defined concentrations of BPA were in agreement with literature, although a precocious maturity of daphnids was detected at 3.0 mg/L. Curiously, an increased reproductive output (neonates per female) was observed when daphnids were bred in the polycarbonate (PC) containers (145.1±4.3 % to 264.7±3.8 %), both in single as in multigenerational tests, in comparison with the negative control group (100.3±1.6 %). A strong correlated dose-dependent ecotoxicological effect was observed, providing evidence that BPA leached from plastic food packaging materials act as functional estrogen in vivo at very low concentrations. In contrast, neonate production by daphnids cultured in polypropylene and non-PC bottles was slightly but not significantly enhanced (92.5±2.0 % to 118.8±1.8 %). Multigenerational tests also revealed magnification of the adverse effects, not only on fecundity but also on mortality, which represents a worrying trend for organisms that are chronically exposed to xenoestrogens for many generations. Two plausible explanations for the observed results could be given: a non-monotonic dose–response relationship or a mixture toxicity effect.

Drilling of fibre reinforced plastic laminates

The use of fibre reinforced plastics – FRP’s – in structures is under a considerable increase. Advantages of their use are related with their low weight, high strength and stiffness. The improvement of the dynamic characteristics has been profitable for aeronautics, automobile, railway, naval and sporting goods industries. Drilling is a widely used machining technique as it is needed to assemble parts in a structure. This is a unique machining process, characterized by the existence of two different mechanisms: extrusion by the drill chisel edge and cutting by the rotating cutting lips. Drilling raises particular problems that can reduce mechanical and fatigue strength of the parts. In this work, quasi-isotropic hybrid laminates with 25% of carbon fibre reinforced plies and 4 mm thickness are produced, tested and drilled. Three different drill geometries are compared. Results considered are the interlaminar fracture toughness in Mode I – GIc –, thrust force during drilling and delamination extent after drilling. A bearing test is performed to evaluate tool influence on the load carrying capacity of the plate. Results consider the influence of drill geometry on delamination. A correlation linking plate damage to bearing test results is presented.

New recycling approaches for thermoset polymeric composite wastes – an experimental study on polyester based concrete materials filled with fibre reinforced plastic recyclates

In this study, a new waste management solution for thermoset glass fibre reinforced polymer (GFRP) based products was assessed. Mechanical recycling approach, with reduction of GFRP waste to powdered and fibrous materials was applied, and the prospective added-value of obtained recyclates was experimentally investigated as raw material for polyester based mortars. Different GFRP waste admixed mortar formulations were analyzed varying the content, between 4% up to 12% in weight, of GFRP powder and fibre mix waste. The effect of incorporation of a silane coupling agent was also assessed. Design of experiments and data treatment was accomplished through implementation of full factorial design and analysis of variance ANOVA. Added value of potential recycling solution was assessed by means of flexural and compressive loading capacity of GFRP waste admixed mortars with regard to unmodified polymer mortars. The key findings of this study showed a viable technological option for improving the quality of polyester based mortars and highlight a potential cost-effective waste management solution for thermoset composite materials in the production of sustainable concrete-polymer based products.

Experimental study on polyester based concretes filled with glass fibre reinforced plastic recyclates – a contribution to composite materials sustainability

The development and applications of thermoset polymeric composites, namely fibre reinforced plastics (FRP), have shifted in the last decades more and more into the mass market [1]. Despite of all advantages associated to FRP based products, the increasing production and consume also lead to an increasing amount of FRP wastes, either end-of-lifecycle products, or scrap and by-products generated by the manufacturing process itself. Whereas thermoplastic FRPs can be easily recycled, by remelting and remoulding, recyclability of thermosetting FRPs constitutes a more difficult task due to cross-linked nature of resin matrix. To date, most of the thermoset based FRP waste is being incinerated or landfilled, leading to negative environmental impacts and supplementary added costs to FRP producers and suppliers. This actual framework is putting increasing pressure on the industry to address the options available for FRP waste management, being an important driver for applied research undertaken cost efficient recycling methods. [1-2]. In spite of this, research on recycling solutions for thermoset composites is still at an elementary stage. Thermal and/or chemical recycling processes, with partial fibre recovering, have been investigated mostly for carbon fibre reinforced plastics (CFRP) due to inherent value of carbon fibre reinforcement; whereas for glass fibre reinforced plastics (GFRP), mechanical recycling, by means of milling and grinding processes, has been considered a more viable recycling method [1-2]. Though, at the moment, few solutions in the reuse of mechanically-recycled GFRP composites into valueadded products are being explored. Aiming filling this gap, in this study, a new waste management solution for thermoset GFRP based products was assessed. The mechanical recycling approach, with reduction of GFRP waste to powdered and fibrous materials was applied, and the potential added value of obtained recyclates was experimentally investigated as raw material for polyester based mortars. The use of a cementless concrete as host material for GFRP recyclates, instead of a conventional Portland cement based concrete, presents an important asset in avoiding the eventual incompatibility problems arisen from alkalis silica reaction between glass fibres and cementious binder matrix. Additionally, due to hermetic nature of resin binder, polymer based concretes present greater ability for incorporating recycled waste products [3]. Under this scope, different GFRP waste admixed polymer mortar (PM) formulations were analyzed varying the size grading and content of GFRP powder and fibre mix waste. Added value of potential recycling solution was assessed by means of flexural and compressive loading capacities of modified mortars with regard to waste-free polymer mortars.

Picocyanobacteria From a Clade Of Marine Cyanobium Revealed Bioactive Potential Against Microalgae, Bacteria, And Marine

The production of bioactive compounds either toxic or with pharmacological applications by cyanobacteria is well established. However, picoplanktonic forms within this group of organisms have rarely been studied in this context. In this study, the toxicological potential of picocyanobacteria from a clade of marine Cyanobium strains isolated from the Portuguese coast was examined using different biological models. First, strains were identified by applying morphological and molecular approaches and cultured under lab conditions. A crude extract and three fractions reflecting a preliminary segregation of lipophilic metabolites were tested for toxicity with the marine microalga Nannochloropsis sp., the bacteria Pseudomonas sp., the brine shrimp Artemia salina, and fertilized eggs of the sea urchin Paracentrotus lividus. No significant apparent adverse effects were noted against Artemia salina. However, significant adverse effects were found in all other assays, with an inhibition of Nannochloropsis sp. and Pseudomonas sp. growth and marked reduction in Paracentrotus lividus larvae length. The results obtained indicated that Cyanobium genus may serve as a potential source of interesting bioactive compounds and emphasize the importance of also studying smaller picoplanktonic fractions of marine cyanobacteria.

Toxicity assessment of crude and partially purified extracts of marine Synechocystis and Synechococcus cyanobacterial strains in marine invertebrates

Among the Cyanoprokaryota, the genera Synechocystis and Synechococcus have rarely been studied with respect to potential toxicity. This is particularly true with marine environments where studies about the toxicity of cyanobacteria are restricted to filamentous forms at the warmer temperate and tropical regions and also to filamentous forms at cold seas such as the Baltic Sea. In this study, we describe the effects of cyanobacterial strains of the Synechocystis and Synechococcus genera isolated from the marine coast of Portugal, on marine invertebrates. Crude and partially purified extracts at a concentration of 100 mg/ml of freeze-dried material of the marine strains were tested for acute toxicity in nauplii of the brine shrimp Artemia salina, in the rotifer Brachionus plicatillis and in embryos of the sea urchin Paracentrotus lividus and the mussel Mytilus galloprovincialis. The cyanobacterial extracts, especially the crude extract, had an impact on A. salina nauplii. No significant toxic effects were registered against the rotifer. A negative impact of all strains was recorded on the embryonic development of the sea urchin, with toxic effects resulting in an inhibition of embryogenesis or development of smaller larvae. To the mussel embryos, the effects of cyanobacterial extracts resulted in a complete inhibition of embryogenesis. The results of all assays indicate that Synechocystis and Synechococcus marine strains contained toxic compounds to marine invertebrates.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Haemoglobin smart plastic antibody material tailored with charged binding sites on silica nanoparticles: its application as an ionophore in potentiometric transduction

This work uses surface imprinting to design a novel smart plastic antibodymaterial (SPAM) for Haemoglobin (Hb). Charged binding sites are described here for the first time to tailor plastic antibody nanostructures for a large size protein such as Hb. Its application to design small, portable and low cost potentiometric devices is presented. The SPAM material was obtained by linking Hb to silica nanoparticles and allowing its ionic interaction with charged vinyl monomers. A neutral polymeric matrix was created around these and the imprinted protein removed. Additional materials were designed in parallel acting as a control: a neutral imprinted material (NSPAM), obtained by removing the charged monomers from the procedure, and the Non-Imprinted (NI) versions of SPAM and NSPAM by removing the template. SEM analysis confirmed the surface modification of the silica nanoparticles. All materials were mixed with PVC/plasticizer and applied as selective membranes in potentiometric transduction. Electromotive force (emf) variations were detected only for selective membranes having a lipophilic anionic additive in the membrane. The presence of Hb inside these membranes was evident and confirmed by FTIR, optical microscopy and Raman spectroscopy. The best performance was found for SPAM-based selective membranes with an anionic lipophilic additive, at pH 5. The limits of detection were 43.8 mg mL 1 and linear responses were obtained down to 83.8 mg mL 1, with an average cationic slope of +40 mV per decade. Good selectivity was also observed against other coexisting biomolecules. The analytical application was conducted successfully, showing accurate and precise results.