ABSTRACT
AIM: The aim of the present study was to assess the alterations in morphology, roughness, and composition of the surfaces of a conventional and a flowable composite attachment engaged with aligners, and to evaluate the release of resin monomers and their derivatives in an aqueous environment. METHODS: Zirconia tooth-arch frames (nâ =â 20) and corresponding thermoformed PET-G aligners with bonded attachments comprising two composite materials (universal-C and flowable-F) were fabricated. The morphological features (stereomicroscopy), roughness (optical profilometry), and surface composition (ATR-FTIR) of the attachments were examined before and after immersion in water. To simulate intraoral use, the aligners were removed and re-seated to the frames four times per day for a 7-day immersion period. After testing, the eluents were analyzed by LC-MS/MS targeting the compounds Bis-GMA, UDMA, 2-HEMA, TEGDMA and BPA and by LC-HRMS for suspect screening of the leached dental material compounds and their degradation products. RESULTS: After testing, abrasion-induced defects were found on attachment surfaces such as scratches, marginal cracks, loss of surface texturing, and fractures. The morphological changes and debonding rate were greater in F. Comparisons (before-after testing) revealed a significantly lower Sc roughness parameter in F. The surface composition of the aligners after testing showed minor changes from the control, with insignificant differences in the degree of Câ =â C conversion, except for few cases with strong evidence of hydrolytic degradation. Targeted analysis results revealed a significant difference in the compounds released between Days 1 and 7 in both materials. Insignificant differences were found when C was compared with F in both timeframes. Several degradation products were detected on Day 7, with a strong reduction in the concentration of the targeted compounds. CONCLUSIONS: The use of aligners affects the surface characteristics and degradation rate of composite attachments in an aqueous environment, releasing monomers, and monomer hydrolysates within 1-week simulated use.
Subject(s)
Composite Resins , Materials Testing , Methacrylates , Surface Properties , Zirconium , Zirconium/chemistry , Composite Resins/chemistry , Methacrylates/chemistry , Polyethylene Glycols/chemistry , Polymethacrylic Acids/chemistry , Polyurethanes/chemistry , Bisphenol A-Glycidyl Methacrylate/chemistry , Dental Materials/chemistry , In Vitro Techniques , Humans , Tooth Movement Techniques/instrumentation , Tooth Movement Techniques/methods , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
BACKGROUND: The aim of the present study was to investigate qualitatively and quantitatively the elution of substances from polyester-urethane (Invisalign™) aligners and resin composite attachments (Tetric EvoFlow) in vivo. METHODS: Patients (n = 11) treated with the aligners and attachments (16 per patient, without other composite restorations) for an average of 20 months, who were planned for attachment removed were enrolled in the study. Patients were instructed to rinse with 50 mL of distilled water upon entry and the rinsing solution was collected (before removal). Then, the attachments were removed with low-speed tungsten carbide burs for adhesive residue removal, a thorough water rinsing was performed immediately after the grinding process to discard grinding particle residues, and subsequently, after a second water-rinsing the solution was collected for analysis (after removal). The rinsing solutions were analyzed for targeted (LC-MS/MS: Bis-GMA, DCDMA, UDMA, BPA) and untargeted (LC-HRMS: screening of leached species and their degradation products) compounds. RESULTS: Targeted analysis revealed a significant reduction in BPA after attachment removal (4 times lower). Bis-GMA, DCDMA, UDMA were below the detection limit before removal but were all detectable after removal with Bis-GMA and UDMA at quantifiable levels. Untargeted analysis reviled the presence of mono-methacrylate transformation products of Bis-GMA (Bis-GMA-M1) and UDMA (UDMA-M1), UDMA without methacrylate moieties (UDMA-M2), and 4-(dimethylamino) benzoic acid (DMAB), the degradation product of the photo-initiator ethyl-4-(dimethylamino) benzoate (EDMAB), all after attachment removal. Several amino acids and endogenous metabolites were also found both before and after removal. CONCLUSIONS: Elevated levels of BPA were traced instantaneously in patients treated with Invisalign™ and flowable resin composite attachments for the testing period. BPA was reduced after attachment removal, but residual monomers and resin degradation products were found after removal. Alternative resin formulations and attachment materials may be utilized to reduce eluents.
Subject(s)
Composite Resins , Methacrylates , Polyurethanes , Humans , Polyurethanes/chemistry , Composite Resins/chemistry , Female , Male , Methacrylates/chemistry , Saliva/chemistry , Bisphenol A-Glycidyl Methacrylate/chemistry , Adult , Orthodontic Appliances, Removable , Polyesters/chemistry , para-Aminobenzoates/analysis , Young Adult , Adolescent , Tooth Movement Techniques/instrumentation , Tooth Movement Techniques/methods , Tandem Mass Spectrometry , Chromatography, LiquidABSTRACT
Cyanobacteria can reach high densities in eutrophic lakes, which may cause problems due to their potential toxin production. Several methods are in use to prevent, control or mitigate harmful cyanobacterial blooms. Treatment of blooms with low concentrations of hydrogen peroxide (H2O2) is a promising emergency method. However, effects of H2O2 on cyanobacteria, eukaryotic phytoplankton and zooplankton have mainly been studied in controlled cultures and mesocosm experiments, while much less is known about the effectiveness and potential side effects of H2O2 treatments on entire lake ecosystems. In this study, we report on three different lakes in the Netherlands that were treated with average H2O2 concentrations ranging from 2 to 5 mg L-1 to suppress cyanobacterial blooms. Effects on phytoplankton and zooplankton communities, on cyanotoxin concentrations, and on nutrient availability in the lakes were assessed. After every H2O2 treatment, cyanobacteria drastically declined, sometimes by more than 99%, although blooms of Dolichospermum sp., Aphanizomenon sp., and Planktothrix rubescens were more strongly suppressed than a Planktothrix agardhii bloom. Eukaryotic phytoplankton were not significantly affected by the H2O2 additions and had an initial advantage over cyanobacteria after the treatment, when ample nutrients and light were available. In all three lakes, a new cyanobacterial bloom developed within several weeks after the first H2O2 treatment, and in two lakes a second H2O2 treatment was therefore applied to again suppress the cyanobacterial population. Rotifers strongly declined after most H2O2 treatments except when the H2O2 concentration was ≤ 2 mg L-1, whereas cladocerans were only mildly affected and copepods were least impacted by the added H2O2. In response to the treatments, the cyanotoxins microcystins and anabaenopeptins were released from the cells into the water column, but disappeared after a few days. We conclude that lake treatments with low concentrations of H2O2 can be a successful tool to suppress harmful cyanobacterial blooms, but may negatively affect some of the zooplankton taxa in lakes. We advise pre-tests prior to the treatment of lakes to define optimal treatment concentrations that kill the majority of the cyanobacteria and to minimize potential side effects on non-target organisms. In some cases, the pre-tests may discourage treatment of the lake.
Subject(s)
Cyanobacteria , Phytoplankton , Animals , Hydrogen Peroxide , Lakes/microbiology , Zooplankton , Ecosystem , Cyanobacteria/physiologyABSTRACT
Harmful algal blooms events have been reported worldwide and during the last decades are occurred with increasing frequency and intensity due to the climate change and the high inputs of nutrients in freshwaters from anthropogenic activities. During blooms cyanobacteria release in water their toxic secondary metabolites, known as cyanotoxins, along with other bioactive metabolites. Due to the negative impacts of these compounds on aquatic ecosystems and public health, there is an urgent need to detect and identify known and unknown cyanobacterial metabolites in surface waters. In the frame of the present study, a method based on liquid chromatography - high resolution mass spectrometry (LC-HRMS) was developed to investigate the presence of cyanometabolites in bloom samples from Lake Karaoun, Lebanon. Data analysis was performed using Compound Discoverer software with related tools and databases in combination to the CyanoMetDB mass list for detection, identification and structural elucidation of the cyanobacterial metabolites. In the course of this study, 92 cyanometabolites were annotated including 51 cyanotoxins belonging to microcystins, 15 microginins, 10 aeruginosins, 6 cyclamides, 5 anabaenopeptins, a cyanopeptolin, the dipeptides radiosumin B and dehydroradiosumin, the planktoncyclin and a mycosporine-like amino acid. Out of them, 7 new cyanobacterial metabolites, the chlorinated MC-ClYR, [epoxyAdda5]MC-YR, MC-LI, aeruginosin 638, aeruginosin 588, microginin 755C and microginin 727 were discovered. Moreover, the presence of anthropogenic contaminants was recorded indicating the pollution of the lake and emphasizing the need for assessment of the co-occurrence of cyanotoxins, other cyanobacterial metabolites and other compounds hazardous to the environment. Overall, results prove the suitability of the proposed approach for the detection of cyanobacterial metabolites in environmental samples but also highlight the necessity of spectral libraries for these compounds, considering the absence of their reference standards.
Subject(s)
Cyanobacteria , Lakes , Lakes/analysis , Ecosystem , Cyanobacteria/metabolism , Microcystins/analysis , Chromatography, Liquid/methods , Mass Spectrometry , Cyanobacteria ToxinsABSTRACT
In the present study, the photocatalytic performance of exfoliated graphitic carbon nitride (g-C3N4) catalysts, with enhanced properties and response in UV and visible light irradiation, was evaluated for the removal of selected contaminants i.e., diuron, bisphenol A and ethyl paraben. Commercial TiO2 Degussa P25 was also used as a reference photocatalyst. The g-C3N4 catalysts demonstrated good photocatalytic activity which in some cases is comparable to TiO2 Degussa P25 leading to high removal percentages of the studied micropollutants under UV-A light irradiation. In contrast to TiO2 Degussa P25, g-C3N4 catalysts were also able to degrade the studied micropollutants under visible light irradiation. For all the studied g-C3N4 catalysts under both UV-A and visible light irradiation, the overall degradation rate decreases in the order of bisphenol A > diuron > ethyl paraben. Among the studied g-C3N4, the chemically exfoliated catalyst (g-C3N4-CHEM) showed superior photocatalytic activity under UV-A light irradiation due to its enhanced characteristics, such as pore volume and specific surface area and ~ 82.0 % in 6 min, ~75.7 % in 15 min and ~ 96.3 % in 40 min removals were achieved for BPA, DIU and EP, respectively. Under visible light irradiation, the thermally exfoliated catalyst (g-C3N4-THERM) demonstrated the best photocatalytic performance and the degradation ranged from ~29.5 to 59.4 % after 120 min. EPR data revealed that the three g-C3N4 semiconductors generate mainly O2â¢-, whereas TiO2 Degussa P25 generates both HO⢠and O2â¢-, the latter only under UV-A light irradiation. Nevertheless, the indirect formation of HO⢠in the case of g-C3N4 should also be considered. Hydroxylation, oxidation, dealkylation, dechlorination and ring opening were the main degradation pathways. The process proceeded without significant alterations in toxicity levels. Based on the results, heterogeneous photocatalysis using g-C3N4 catalysts is a promising method for the removal of organic micropollutants without the formation of harmful transformation products.
Subject(s)
Diuron , LightABSTRACT
Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography-mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8-13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723.
Subject(s)
Cyanobacteria , Tandem Mass Spectrometry , Humans , Chromatography, Liquid/methods , Workflow , Cyanobacteria/metabolism , Microcystins/chemistry , Oligopeptides/metabolismABSTRACT
AIM: To quantitatively assess the degree of conversion and the water-leaching targeted compound from 3D-printed aligners. MATERIALS AND METHODS: 3D-printed aligners were made of photopolymerized resin (Tera Harz TC85A). The molecular structure and degree of conversion of the set resin were investigated by ATR-FTIR spectroscopy (n = 5). The aligners (n = 10) were immersed in double distilled water for 1 week at 37°C and the eluents were analysed using liquid chromatography/mass spectrometry methods (LC-ESI-MS/MS for urethane dimethacrylate [UDMA] and LC-APCI-MS/MS for bispenol-A [BPA]). RESULTS: The resin was composed of aliphatic vinyl ester-urethane monomers, with acrylate and/or methacrylate functionalization. The degree of conversion was estimated as to 83%. There was no detection of BPA in any of the assessed samples (0.25 µg/l). Quantifiable amounts of UDMA were detected in all the exposed samples, ranging from 29 to 96 µg/l. CONCLUSIONS: Although efficiently polymerized and BPA free, the great variability in the amount of UDMA monomer leached from the examined samples may raise concerns on potential health hazards after repeated intraoral exposure, which is indicated for this class of materials.
Subject(s)
Composite Resins , Polymethacrylic Acids , Humans , Composite Resins/chemistry , Bisphenol A-Glycidyl Methacrylate/chemistry , Polymethacrylic Acids/chemistry , Tandem Mass Spectrometry , Polyethylene Glycols/chemistry , Methacrylates/chemistry , Polyurethanes/chemistry , Printing, Three-Dimensional , Materials TestingABSTRACT
In the last decades, cyanobacterial harmful algal blooms (CyanoHABs) pose an intensifying ecological threat. Microcystis aeruginosa is a common CyanoHAB species in freshwater ecosystems, with severe toxic effects in a wide range of organisms. In the present paper we examined whether transient and short (48 h) exposure of fish embryos to sublethal levels of M. aeruginosa crude extract (200 mg biomass dw L-1) affects swimming performance at later life stages (end of metamorphosis, ca 12 mm TL, 22,23 days post-fertilization). Pre-exposed metamorphosing larvae presented a significant decrease in swimming performance (9.7 ± 1.6 vs 11.4 ± 1.7 TL s-1 in the control group, p < 0.01), and a significant decrease in the ventricle length-to-depth ratio (1.23 ± 0.15 vs 1.42 ± 0.15 in control fish, p < 0.05). In addition, extract-exposed fish presented significantly elevated rates of vertebral abnormalities (82 ± 13% vs 7 ± 4% in the control group), mainly consisting of the presence of extra neural and haemal processes. No significant differences between groups were detected in survival and growth rates. Results are discussed in respect to the mechanisms that might mediate the detected cyanobacterial effects. This is the first evidence of a direct link between sublethal exposure to M. aeruginosa during the embryonic period and swimming performance at later life-stages. Decreased swimming performance, altered cardiac shape, and elevated vertebral abnormalities in response to early exposure to M. aeruginosa could have significant effects on fish populations in the wild.
Subject(s)
Microcystis , Water Pollutants, Chemical , Animals , Ecosystem , Embryonic Development , Microcystins , Swimming , Water Pollutants, Chemical/toxicity , ZebrafishABSTRACT
Lake Karaoun is the largest artificial lake in Lebanon and serves multiple purposes. Recently, intensive cyanobacterial blooms have been reported in the lake, raising safety and aesthetic concerns related to the presence of cyanotoxins and cyanobacterial taste and odor (T&O) compounds, respectively. Here, we communicate for the first time results from a recent investigation by LC-MS/MS covering multiple cyanotoxins (microcystins (MCs), anatoxin-a, cylindrospermopsin, nodularin) in water and fish collected between 2019 and 2020. Eleven MCs were identified reaching concentrations of 211 and 199 µg/L for MC-LR and MC-YR, respectively. Cylindrospermopsin, anatoxin-a and nodularin were not detected. The determination of the total MCs was also carried out by ELISA and Protein Phosphatase Inhibition Assay yielding comparable results. Molecular detection of cyanobacteria (16S rRNA) and biosynthetic genes of toxins were carried out by qPCR. Untargeted screening analysis by GC-MS showed the presence of T&O compounds, such as ß-cyclocitral, ß-ionone, nonanal and dimethylsulfides that contribute to unpleasant odors in water. The determination of volatile organic compounds (VOCs) showed the presence of anthropogenic pollutants, mostly dichloromethane and toluene. The findings are important to develop future monitoring schemes in order to assess the risks from cyanobacterial blooms with regard to the lake's ecosystem and its uses.
Subject(s)
Cyanobacteria Toxins/analysis , Lakes/chemistry , Water Pollutants, Chemical/analysis , Animals , Carps , Cyanobacteria/chemistry , Cyanobacteria/genetics , Cyanobacteria Toxins/genetics , Environmental Monitoring/methods , Eutrophication , Fresh Water/chemistry , Lakes/microbiology , Lebanon , Odorants , RNA, Ribosomal, 16S , Taste , Volatile Organic Compounds/analysisABSTRACT
Cyanotoxins (CTs) produced by cyanobacteria in surface freshwater are a major threat for public health and aquatic ecosystems. Cyanobacteria can also produce a wide variety of other understudied bioactive metabolites such as oligopeptides microginins (MGs), aeruginosins (AERs), aeruginosamides (AEGs) and anabaenopeptins (APs). This study reports on the co-occurrence of CTs and cyanopeptides (CPs) in Lake Vegoritis, Greece and presents their variant-specific profiles obtained during 3-years of monitoring (2018-2020). Fifteen CTs (cylindrospermopsin (CYN), anatoxin (ATX), nodularin (NOD), and 12 microcystins (MCs)) and ten CPs (3 APs, 4 MGs, 2 AERs and aeruginosamide (AEG A)) were targeted using an extended and validated LC-MS/MS protocol for the simultaneous determination of multi-class CTs and CPs. Results showed the presence of MCs (MC-LR, MC-RR, MC-YR, dmMC-LR, dmMC-RR, MC-HtyR, and MC-HilR) and CYN at concentrations of <1 µg/L, with MC-LR (79%) and CYN (71%) being the most frequently occurring. Anabaenopeptins B (AP B) and F (AP F) were detected in almost all samples and microginin T1 (MG T1) was the most abundant CP, reaching 47.0 µg/L. This is the first report of the co-occurrence of CTs and CPs in Lake Vegoritis, which is used for irrigation, fishing and recreational activities. The findings support the need for further investigations of the occurrence of CTs and the less studied cyanobacterial metabolites in lakes, to promote risk assessment with relevance to human exposure.
Subject(s)
Bacterial Toxins/analysis , Cyanobacteria , Peptides/analysis , Water Pollutants/analysis , Chlorophyll A/analysis , Environmental Monitoring , Greece , Lakes/analysis , Lakes/microbiologyABSTRACT
Cyanobacteria are photosynthetic microorganisms that are able to produce a large number of secondary metabolites. In freshwaters, under favorable conditions, they can rapidly multiply, forming blooms, and can release their toxic/bioactive metabolites in water. Among them, anabaenopeptins (APs) are a less studied class of cyclic bioactive cyanopeptides. The occurrence and structural variety of APs in cyanobacterial blooms and cultured strains from Greek freshwaters were investigated. Cyanobacterial extracts were analyzed with LC-qTRAP MS/MS using information-dependent acquisition in enhanced ion product mode in order to obtain the fragmentation mass spectra of APs. Thirteen APs were detected, and their possible structures were annotated based on the elucidation of fragmentation spectra, including three novel ones. APs were present in the majority of bloom samples (91%) collected from nine Greek lakes during different time periods. A large variety of APs was observed, with up to eight congeners co-occurring in the same sample. AP F (87%), Oscillamide Y (87%) and AP B (65%) were the most frequently detected congeners. Thirty cyanobacterial strain cultures were also analyzed. APs were only detected in one strain (Microcystis ichtyoblabe). The results contribute to a better understanding of APs produced by freshwater cyanobacteria and expand the range of structurally characterized APs.
Subject(s)
Bacterial Toxins/isolation & purification , Cyanobacteria/chemistry , Fresh Water/microbiology , Peptides, Cyclic/isolation & purification , Greece , Microcystis/chemistryABSTRACT
Microginins are the less common class of bioactive linear cyanobacterial peptides. Recently, an investigation for their presence in cyanobacteria from Greek freshwaters and strain cultures was carried out. The present dataset is related to the research article "New microginins from cyanobacteria of Greek freshwaters" [1]. Cyanobacterial biomass from bloom samples and cultured strains were extracted with aqueous methanol. Extracts were analysed by liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometer (LC-qTRAP MS/MS) in information dependent acquisition (IDA) mode. Enhanced ion product (EIP) mode was applied for the collection of ion fragmentation spectra. Identification of microginins was based on the characteristic fragment ions of the unique microginin amino acid 3-amino-2-hydroxy-decanoic acid (Ahda) and its modified forms. The analysis of fragmentation spectra revealed 51 microginin structures, including 36 new variants. This article provides the dataset of fragmentation mass spectra of the microginins detected in cyanobacteria from Greek freshwaters. As this class of cyanopeptides is produced by cyanobacteria from different geographical regions, the aim of this dataset is to enable the identification of microginins in future studies and therefore to contribute to a better evaluation of their presence in freshwater bodies worldwide.
ABSTRACT
Cyanobacteria can form extensive blooms in water with concurrent production and release of a large number of chemically diverse and bioactive metabolites, including hazardous toxins. Significant number of the metabolites belongs to non-ribosomal peptides, with unique residues, unusual structures and great potential for biotechnological application. The biosynthetic pathways of the peptides generate tens of variants, but only part of them has been identified. Microginins are an understudied class of cyanobacterial linear peptides with a characteristic decanoic acid derivative amino acid residue in their structure. In this study, cyanobacterial blooms and isolated strains from Greek lakes were analyzed for the presence of microginins by liquid chromatography coupled to hybrid triple quadrupole/linear ion trap mass spectrometer (LC-qTRAP MS/MS). Microginin structures were elucidated based on the obtained fragmentation spectra. A large number of microginins occurred in blooms of Greek freshwaters and the most frequently detected were Microginin FR1 (70% of samples), Microginin T1 (52%), Microginin 565B (52%), Microginin T2 (43%), and Microginin 565A (43%). Additionally, nine cyanobacterial strains i.e. Nostoc oryzae, Synechococcus sp., Microcystis aeruginosa, Microcystis viridis, and five Microcystis sp., were found to produce microginins. Thirty-six new microginin structures were characterized out of fifty-one totally detected variants. This is the first time that such a diversity of microginins is reported to be present in water bodies. Results clearly demonstrate the great metabolomic potential of cyanobacteria that inhabit Greek freshwaters and significantly expand the knowledge of cyanobacterial secondary metabolites with regards to the class of microginins.
Subject(s)
Cyanobacteria/metabolism , Water Pollution , Chromatography, Liquid , Environmental Monitoring , Greece , Lakes/analysis , Metabolomics , Microcystis/metabolism , Peptides , Tandem Mass Spectrometry , Water MicrobiologyABSTRACT
Marine cyanobacteria are considered a prolific source of bioactive natural products with a range of biotechnological and pharmacological applications. However, data on the production of natural compounds from sponge-associated cyanobacteria are scarce. This study aimed to assess the potential of sponge-associated cyanobacteria strains representing different taxonomic groups for the production of bioactive compounds and the biological activity of their extracts. Phylogenetic analysis of sponge-associated cyanobacteria and screening for the presence of genes encoding non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) were performed. Methanol extracts of the sponge-associated strains were analyzed for cyanotoxin production and tested for antioxidant activity and cytotoxic activity against several human cancer cell lines and pathogenic bacteria. PKS were detected in all sponge-associated strains examined, indicating the metabolic potential of the isolates. PKS genes were more ubiquitous than NRPS genes. Cyanotoxins (i.e., cylindrospermopsin, anatoxin-a, nodularin, and microcystins) were not detected in any of the sponge-associated cyanobacterial strains. Strains belonging to Leptothoe, Pseudanabaena, and Synechococcus were found to have activity mainly against Staphylococcus aureus. In addition, sponge-associated Leptothoe strains (TAU-MAC 0915, 1015, 1115, and 1215) were found to be highly cytotoxic and in most cases more effective against human cancer cell lines than against normal cells. Extracts with the most promising bioactivity deserve further investigation in order to isolate and identify the bioactive molecule(s).
Subject(s)
Anti-Bacterial Agents/toxicity , Antineoplastic Agents/toxicity , Bacterial Toxins/toxicity , Cyanobacteria/metabolism , Porifera/microbiology , Animals , Anti-Bacterial Agents/metabolism , Antineoplastic Agents/metabolism , Bacterial Toxins/metabolism , Bioprospecting , Cell Line, Tumor , Cell Survival/drug effects , Escherichia coli/drug effects , Humans , Pseudomonas aeruginosa/drug effects , Secondary Metabolism , Staphylococcus aureus/drug effectsABSTRACT
Cyanobacteria are a diverse group of photosynthetic Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against a broad spectrum of organisms and cell lines. In this study, 29 strains isolated from freshwaters in Greece were classified using a polyphasic approach and assigned to Chroococcales, Synechococcales, and Nostocales, representing 11 genera and 17 taxa. There were good agreements between 16S ribosomal RNA (rRNA)-cpcBA-internal genetic spacer (IGS) characterization and morphological features, except for the Jaaginema-Limnothrix group which appears intermixed and needs further elucidation. Methanol extracts of the strains were analyzed for cyanotoxin production and tested against pathogenic bacteria species and several cancer cell lines. We report for the first time a Nostoc oryzae strain isolated from rice fields capable of producing microcystins (MCs) and a Chlorogloeopsis fritschii strain isolated from the plankton of a lake, suggesting that this species may also occur in freshwater temperate habitats. Strains with very high or identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activities. Extracts from Synechococcus cf. nidulans showed the most potent antibacterial activity against Staphylococcus aureus, whereas Jaaginema sp. strains exhibited potent cytotoxic activities against human colorectal adenocarcinoma and hepatocellular carcinoma cells. Jaaginema Thessaloniki Aristotle University Microalgae and Cyanobacteria (TAU-MAC) 0110 and 0210 strains caused pronounced changes in the actin network and triggered the formation of numerous lipid droplets in hepatocellular carcinoma and green monkey kidney cells, suggesting oxidative stress and/or mitochondrial damage leading to apoptosis.
Subject(s)
Bacterial Toxins/analysis , Cyanobacteria/isolation & purification , Animals , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Biodiversity , Cell Line , Cell Survival/drug effects , Chlorocebus aethiops , Complex Mixtures/pharmacology , Cyanobacteria/classification , Cyanobacteria/genetics , Fresh Water/microbiology , Greece , Humans , Microalgae/classification , Microalgae/genetics , Microalgae/isolation & purification , Phylogeny , RNA, Ribosomal, 16SABSTRACT
Increased rates of reactive oxygen/nitrogen species (ROS/RNS) are involved in almost all cancer types, associated with tumor development and progression, causing damage to biomolecules such as proteins, nucleic acids and membrane lipids, in different biological compartments. We used a human tumor xenograft mouse model to evaluate for the first time in parallel the remodeling of fatty acid moieties in erythrocyte membrane phospholipids and the level of ROS-induced DNA lesions in liver and kidney tissues. Using liquid chromatography tandem mass spectrometry the 5'R and 5'S diastereoisomers of 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine, together with 8-oxo-7,8-dihydro-2'-deoxyadenosine, were determined in mice at young (4- and 5-weeks) and old (17-weeks) ages and compared with control SCID mice without tumor implantation. Tumor-bearing mice showed a higher level of ROS-damaged nucleosides in genomic DNA as the age and tumor progress, compared to controls (1.07-1.53-fold in liver and 1.1-1.4-fold in kidney, respectively). The parallel fatty acid profile of erythrocyte membranes showed a profound lipid remodeling during tumor and age progression consisting of PUFA consumption and SFA enrichment (ca 28% and 58%, respectively, in late stage tumor-bearing mice), markers of enhanced oxidative and proliferative processes, respectively. Membrane lipid remodeling and ROS-induced DNA lesions may be combined to afford an integrated scenario of cancer progression and ageing, reinforcing a holistic vision among molecular markers rather than the biomarker identification in a single compartment.
ABSTRACT
Cyanobacterial diversity associated with sponges remains underestimated, though it is of great scientific interest in order to understand the ecology and evolutionary history of the symbiotic relationships between the two groups. Of the filamentous cyanobacteria, the genus Leptolyngbya is the most frequently found in association with sponges as well as the largest and obviously polyphyletic group. In this study, five Leptolyngbya-like sponge-associated isolates were investigated using a combination of molecular, chemical, and morphological approach and revealed a novel marine genus herein designated Leptothoe gen. nov. In addition, three new species of Leptothoe, Le. sithoniana, Le. kymatousa, and Le. spongobia, are described based on a suite of distinct characters compared to other marine Leptolyngbyaceae species/strains. The three new species, hosted by four sponge species, showed different degrees of host specificity. Leptothoe sithoniana and Le. kymatousa hosted by the sponges Petrosia ficiformis and Chondrilla nucula, respectively, seem to be more specialized than Le. spongobia, which was hosted by the sponges Dysidea avara and Acanthella acuta. All three species contained nitrogen-fixing genes and may contribute to the nitrogen budget of sponges. Leptothoe spongobia TAU-MAC 1115 isolated from Acanthella acuta was shown to produce microcystin-RR indicating that microcystin production among marine cyanobacteria could be more widespread than previously determined.
Subject(s)
Cyanobacteria , Phylogeny , RNA, Ribosomal, 16S , SymbiosisABSTRACT
Toxic cyanobacteria occur in Greek surface water bodies. However, studies on the occurrence of cyanotoxins (CTs) are often limited to mainly microcystins (MCs), with use of screening methods, such as ELISA, that are not conclusive of the chemical structure of the CT variants and can be subject to false positive results. A multi-lake survey in Greece (14 lakes) was conducted in water and biomass, targeted to a wide range of multi-class CTs including MCs, nodularin-R (NOD), cylindrospermopsin (CYN), anatoxin-a (ANA-a) and saxitoxins (STXs), using multi-class/variant LC-MS/MS analytical workflows, achieving sensitive detection, definitive identification and accurate quantitation. A wide variety of CTs (CYN, ANA-a, STX, neoSTX, dmMC-RR, MC-RR, MC-YR, MC-HtyR, dm3MC-LR, MC-LR, MC-HilR, MC-WR, MC-LA, MC-LY, MC-LW and MC-LF), were detected, with MCs being the most commonly occurring. In biomass, MC-RR was the most abundant toxin, reaching 754 ng mg-1 dw, followed by MC-LR (458 ng mg-1 dw). CYN and ANA-a were detected for the first time in the biomass of Greek lakes at low concentrations and STXs in lakes Trichonis, Vistonis and Petron. The abundance and diversity of CTs were also evaluated in relation to recreational health risks, in a case study with a proven history of MCs (Lake Kastoria).
Subject(s)
Bacterial Toxins/analysis , Bacterial Toxins/classification , Cyanobacteria/metabolism , Lakes/chemistry , Lakes/microbiology , Chromatography, Liquid , Cyanobacteria/growth & development , Greece , Tandem Mass SpectrometryABSTRACT
The cyanobacterium Cylindrospermopsis raciborskii represents a challenge for researchers and it is extensively studied for its toxicity and invasive behaviour, which is presumably enhanced by global warming. Biogeography studies indicate a tropical origin for this species, with Greece considered as the expansion route of C. raciborskii in Europe. The widening of its geographic distribution and the isolation of strains showing high optimum growth temperature underline its ecological heterogeneity, suggesting the existence of different ecotypes. The dominance of species like C. raciborskii along with their ecotoxicology and potential human risk related problems, render the establishment of a clear phylogeography model essential. In the context of the present study, the characterization of Cylindrospermopsis raciborskii TAU-MAC 1414 strain, isolated from Lake Karla, with respect to its phylogeography and toxic potential, is attempted. Our research provides new insights on the origin of C. raciborskii in the Mediterranean region; C. raciborskii expanded in Mediterranean from North America, whilst the rest of the European strains may originate from Asia and Australia. Microcystin synthetase genes, phylogenetic closely related with Microcystis strains, were also present in C. raciborskii TAU-MAC 1414. We were unable to unambiguously confirm the presence of MC-LR, using LC-MS/MS. Our results are shedding light on the expansion and distribution of C. raciborskii, whilst they pose further questions on the toxic capacity of this species.
Subject(s)
Cylindrospermopsis/classification , Phylogeography , Cylindrospermopsis/genetics , Greece , Likelihood Functions , RNA, Ribosomal, 16S/chemistryABSTRACT
Under ongoing climate change and increasing anthropogenic activity, which continuously challenge ecosystem resilience, an in-depth understanding of ecological processes is urgently needed. Lakes, as providers of numerous ecosystem services, face multiple stressors that threaten their functioning. Harmful cyanobacterial blooms are a persistent problem resulting from nutrient pollution and climate-change induced stressors, like poor transparency, increased water temperature and enhanced stratification. Consistency in data collection and analysis methods is necessary to achieve fully comparable datasets and for statistical validity, avoiding issues linked to disparate data sources. The European Multi Lake Survey (EMLS) in summer 2015 was an initiative among scientists from 27 countries to collect and analyse lake physical, chemical and biological variables in a fully standardized manner. This database includes in-situ lake variables along with nutrient, pigment and cyanotoxin data of 369 lakes in Europe, which were centrally analysed in dedicated laboratories. Publishing the EMLS methods and dataset might inspire similar initiatives to study across large geographic areas that will contribute to better understanding lake responses in a changing environment.