Cyanobacteria (“blue-green algae”) are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called “harmful algal blooms”, particularly in freshwater systems, a number of these metabolites have been associated - as “toxins”, or commonly “cyanotoxins” - with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.

Nodularia spumigena é uma espécie cianobacteriana conhecida como produtora da hepatotoxina nodularina. Essa cianotoxina é uma potente e irreversível inibidora de proteínas fosfatases da família serina/treonina (PP1 e PP2A) de células eucarióticas e é uma promotora tumoral e suspeita carcinogéna. Além da nodularina, a N. spumigena também é produtora de outros peptídeos não ribossômicos, tais como espumiginas, aeruginosinas e anabaenopeptinas. O primeiro relato de N. spumigena formadora de florações no Brasil ocorreu em 2011 em tanques de produção de camarões no Rio Grande, RS, e estimulou o interesse na obtenção de informações sobre o seu genoma e potencial biossíntético. Dessa forma, a objetivo deste estudo foi avaliar os aspectos genômicos e funcionais da linhagem Nodularia spumigena CENA596 isolada de um tanque de produção de camarões de Rio Grande. Para isso, uma cultura da linhagem N. spumigena CENA596 foi submetida a um tratamento com hipoclorito de sódio (2%) para eliminação de contaminantes e o DNA extraído das células tratadas foi sequenciado na plataforma MiSeq e analisado com ferramentas genômicas. O sequenciamento e a montagem do seu genoma originaram 291 sequências contíguas com percentual GC de 41,19 e tamanho total de 5.189.679 pb. A análise filogenética baseada na sequência do gene que codifica o 16S rRNA agrupou a linhagem CENA596 com outras de N. spumigena da Austrália e América do Norte. Na árvore filogenômica construída com as sequências concatenadas de 31 proteínas, a linhagem brasileira CENA596 agrupou-se com valor de reamostragem de 100% com a N. spumigena CCY9414 originária do mar Báltico. As análises comparativas entre os genomas dessas duas linhagens indicaram um grande número de genes compartilhados, os quais estão relacionados principalmente ao metabolismo primário das células. Por outro lado, foram encontrados genes específicos para cada uma delas que estão envolvidos em respostas celulares a estresses oxidativos, patógenos e antibióticos. A mineração do genoma da N. spumigena CENA596 revelou 13 agrupamentos gênicos hipoteticamente relacionados à síntese de metabólitos secundários, a maioria dos quais mostrou similaridade significativa com agrupamentos conhecidos. As análises químicas confirmaram a produção de duas variantes de nodularina, espumigina, namalida, aeruginosina e aminoácidos tipo micosporina, e uma variante de geosmina. A linhagem brasileira N. spumigena CENA596 mostrou-se capaz de produzir uma variedade significante de moléculas bioativas e seu genoma revelou-se ser consideravelmente conservado em relação ao genoma da linhagem CCY9414, a qual é conhecida por causar grandes florações tóxicas no Mar Báltico / Nodularia spumigena is a cyanobacterial species known as a producer of the hepatotoxin nodularin. This cyanotoxin is a potent and irreversible inhibitor of eukaryotic cell serine/threonine protein phosphatases (PP1 and PP2A) and is a tumor promoter and suspected carcinogen. In addition to nodularin, N. spumigena is also produces other non-ribosomal peptides, such as spumigins, aeruginosines and anabaenopeptins. The first report of bloom-forming N. spumigena in Brazil occurred in 2011 in shrimp production ponds, Rio Grande, RS, and stimulated interest in obtaining information on its genome and biosynthetic potential. Thus, the objective of this study was to evaluate the genomic and functional aspects of the strain N. spumigena CENA596 isolated from a shrimp production pond of the Rio Grande. For this, a culture of the strain N. spumigena CENA596 was submitted to a treatment with sodium hypochlorite (2%) to eliminate contaminants and the DNA extracted from treated cells was sequenced in a platform MiSeq and analyzed with genomic tools. Genome sequencing and assembly resulted in 291 contiguous sequences with GC percentage of 41.19 and total size of 5,187,679 bp. Phylogenetic analysis based on the gene sequence encoding the 16S rRNA grouped the strain CENA596 with other N. spumigena from Australia and North America. In the phylogenomic tree constructed with the concatenated sequences of 31 proteins, the Brazilian strain CENA596 grouped with a bootstrap value of 100% with the N. spumigena CCY9414 originating from the Baltic sea. Comparative analyses between the genomes of these two strains indicated a large number of shared genes, which are mainly related to the primary metabolism of the cells. Otherwise, genes specific for each of the two strains were identified as involved in cellular responses to oxidative stress, pathogens and antibiotics. Genome mining revealed 13 gene clusters hypothetically related to the synthesis of secondary metabolites, most of which showed significant similarity to known clusters. Chemical analyses confirmed the production of two variants of nodularin, spumigin, namalide, aeruginosin and mycosporine-like amino acid, and one variant of geosmin. The Brazilian strain N. spumigena CENA596 was able to produce a significant variety of bioactive molecules and its genome revealed to be considerably conserved in relation to the genome of the strain CCY9414, which is known to cause large toxic blooms in the Baltic Sea

No description available.

The main objective of this study was the application and comparison of different assays in assessing toxicity of cyanobacterial samples, and also characterizing toxicity of the field samples. Therefore, toxicity of purified microcystin-LR (MC-LR) and cyanobacterial samples collected from the Hartbeespoort (HBP) Dam (winter and summer seasons of 2005/2006) and Kruger National Park (KNP) were investigated and compared using the ELISA, mouse bioassay, catfish primary hepatocytes (in vitro assay) and protein phosphatase inhibition (PPi) assays. During sampling in the summer season at the HBP Dam, the dam surface was covered with a thick-green layer of cyanobacterial scum and a foul smell coming from the water surface was always present. Only blue-green streaks of cyanobacteria covered the dam surface during the winter season. All HBP Dam samples (winter and summer samples) and KNP samples (Nhlanhanzwani Dam, Mpanama Dam and Sunset Dam) were dominated by Microcystis aeruginosa with the exception of Makhohlola Dam samples which were found to have no cyanobacteria. The World Health Organization (WHO) has proposed a guideline value for human use of 1.0 µg/L (0.001 mg/L) for MC-LR, the most common microcystin (MC) variant, in drinking water (WHO 1998), whereas 2 000 Microcystis cells/mL have been recommended as the limit of cyanobacteria in drinking water for animals (DWAF 1996). Cyanotoxin concentrations exceeding the prescribed guideline value were detected in all HBP Dam samples (ELISA results ranging between 3.67 to 86.08 mg/L; PPi results ranging between 2.99 to 54.90 mg/L) and KNP samples (ELISA results ranging between 0.1 to 49.41 mg/L; PPi results ranging between 0.006 to 10.95 mg/L) using both the ELISA and PPi assays. In the current study, a dose of about 175 µg/kg of purified MC-LR was demonstrated to be lethal in male CD-1 SPF mice. The HBP Dam summer samples and Nhlanganzwani Dam samples were the only cyanobacterial samples that resulted in death (acute toxicity) of mice. In order to be able to investigate further the in vivo effects of cyanotoxins, transmission electron microscopy (TEM) was used to complement results obtained from the in vivo assay. Ultrastructural changes of varying degree were observed in livers of mice exposed to both the HBP Dam winter and summer samples. Early stages of hepatocyte to hepatocyte disassociation, slight vesiculation of endoplasmic reticulum (ER) and swollen mitochondria were the most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam winter samples. The most significant ultrastructural changes produced in mouse hepatocyte tissues by the HBP Dam summer samples were massive hepatic haemorrhage indicated by the appearance of erythrocytes between hepatocytes and the extensive vesiculation of ER. This is the first time that the African sharptooth catfish primary hepatocyte model has been used to assess the hepatotoxicity of purified MC-LR and cyanotoxin-containing water samples. In this study, the toxicity of cyanobacterial samples and purified MC-LR to cause hepatotoxicity in mice was confirmed in vitro using the catfish primary cell line. A comparison among the cyanobacterial samples using EC50 showed the following hepatotoxicity trend in the catfish primary cell line: HBP Dam summer samples > Nhlanganzwani Dam samples > HBP Dam winter samples > Mpanama Dam samples > Sunset Dam samples > Makhohlola Dam samples. The HBP Dam samples were the most hepatotoxic and Makhohlola Dam samples were the least hepatotoxic. The EC50 for purified MC-LR using the catfish primary hepatocytes was about 91 nM. A statistical comparison of the assays used in this study (i.e. ELISA, PPi, mouse test and cytotoxicity [catfish primary hepatocyte] assays) was performed based on the Kappa coefficient (K). An almost perfect agreement (K > 0.80) was observed between the mouse test and cytotoxicity assay; mouse test and ELISA; cytotoxicity assay and ELISA; and ELISA and PPi assay. In conclusion, field samples collected during the summer season were found to have very high levels of toxins and a higher degree of toxicity when compared to the winter samples. The cytotoxicity assay using African sharptooth catfish (Clarias gariepinus) primary hepatocytes has been shown for the first time to produce results similar to those observed when using the mouse bioassay in assessing cyanobacterial toxicity. Therefore, this primary cell line may be used as a potential alternative to the mouse assay in toxicity testing of cyanotoxins. Three KNP dams (Nhlanganzwani Dam, Mpanama Dam and Sunset Dam) investigated in this study were found to contain Microcystis aeruginosa. All four KNP dams (Nhlanganzwani Dam, Mpanama Dam, Makhohlola Dam and Sunset Dam) had cyanotoxin levels above the prescribed guideline value, which is of concern and warrants further investigations to the effects on wildlife in the park. Future studies will include use of High Performance Liquid Chromatography (HPLC) to investigate the toxin profile of the field samples in order to fully describe the different classes/or types of toxins present in the samples. More validation studies that could give a more comprehensive understanding about the sensitivity of the catfish primary cell line for microcystins will also be undertaken. / Dissertation (MSc (Paraclinical Studies))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

No description available.

The cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) now appears to be a cause of Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). Its production by cyanobacteria throughout the world combined with multiple mechanisms of BMAA neurotoxicity, particularly to vulnerable subpopulations of motor neurons, has significantly increased interest in investigating exposure to this non-protein amino acid as a possible risk factor for other forms of neurodegenerative illness. We here provide a brief overview of BMAA studies and provide an introduction to this collection of scientific manuscripts in this special issue on BMAA.

<p>U doktorskoj disertaciji je rađena analiza produkcije i procena stabilnosti sekundarnih metabolita cijanobakterija. U analizi pigmenata cijanobakterija kao biomarkera u paleoklimatskoj rekonstrukciji poslužio je nov model analize-AMMI model, koji pruža značajan doprinos odabiru odgovarajućih biomarkera u paleoklimatiskoj rekonstrukciji. Analizom produkcije MOSA i MOMA 15 vodenih i zemlji&scaron;nih cijanobakterija NSCCC sposobnost produkcije MOSA je uočena kod 8, a MOMA kod svih sojeva. Kultivacija u različitim pH vrednostima podloge i različitim temperaturnim uslovima nije pokazala znatan uticaj na produkciju MOSA. Produkcija MOMA je bila izraženija u baznoj sredini (pH 9.0) i na vi&scaron;im temperaturama (30-35 &deg;C). UV svetlost se pokazala kao najznačajniji faktor i inicirala je najveću produkciju MOSA i MOMA kod svih sojeva. Veća koncentracija azota u podlozi nije uticala na povećanje produkcije MOSA kod većine analiziranih sojeva, dok je znatno uticala na povećanje produkcije MOMA kod svih analiziranih sojeva.<br />Analizom produkcije pigmenata 19 lesnih cijanobakterija NSCCC prisustvo fikobilina i MOSA je uočeno u svim analiziranim kulturama, dok sposobnost produkcije MOMA nije uočena u dve kulture lesnih cijanobakterija NSCCC. Tokom posmatranog vremenskog perioda uočena je razgradnja ukupnih fikobilina u kontrolnim uslovima rasta, dok za isti vremenski period nije do&scaron;lo do degradacije MOSA i MOMA u kontrolnim uslovima.<br />Procenom stabilnosti MOSA i MOMA delovanjem abiotičkih faktora (različitih pH i temperaturnih vrednosti podloge, različitog vremena izlaganja UV svetlosti) utvrđeno je da su MOSA i MOMA pokazali izraženu stabilnost na testirane abiotičke faktore. Procenom stabilnosti pigmenata nakon delovanja biotičkih faktora primetna je intenzivnija razgradnja ukupnih fikobilina posmatrano u zavisnosti od vremena, do postizanja potpune degradacije u pojednim kulturama lesnih cijanobakterija, dok je uočeno da su MOSA i MOMA pokazali stabilnu strukturu i u testu biodegradabilnosti nije do&scaron;lo do njihove degradacije.<br />Analizom prisustva MOSA i MOMA u lesnom sedimentu i biolo&scaron;kim lesnim pokoricama njihovo prisustvo je uočeno u svim analiziranim uzorcima. Takođe, kori&scaron;ćenjem LC-MS(/MS) metode utvrđeno je prisustvo scitonemina u 10 terestričnih kultura NSCCC.<br />Analizom toksičnosti i produkcije mikrocistina, cilindrospermopsina i saksitoksina lesnih cijanobakterija NSCCC dobijeni su negativni rezultati. Razvojem novih metoda za detekciju cijanotoksina u terestričnim ekosistemima potrebno je proveriti dobijene rezultate. Procenom stabilnosti mikrocistina referentnog soja Microcystis aeruginosa PCC 7806, uočena je njegova izrazita stabilnost tokom vremenskog perioda od 96 h u kontrolnim uslovima i delovanjem tri bakterijska soja.<br />Shodno dobijenim rezultatima, UV za&scaron;titni pigmenti su mnogo podesniji za paleoklimatsku rekonstrukciju od fikobilina s obzirom da molekuli MOSA i MOMA imaju postojaniju strukturu i da nisu degradirani tokom posmatranih stresnih uslova. Usled nemogućnosti detekcije cijanotoksina lesnih cijanobakterija, i pored izražene stabilnosti mikrocistina referentnog soja, cijanotoksini se ne mogu smatrati biomarkerima cijanobakterija u geolo&scaron;kim istraživanjima.<br />Postavljanje BLOCDUST teorije i otkriće stabilnosti MOSA i MOMA i njihove upotrebe kao pouzdanih biomarkera u paleoklimatskoj rekonstrukciji predstavlja osnovu za mnoga buduća istraživanja od neprocenjivog naučnog značaja, pogotovo u paleoklimatskoj rekonstrukciji lesa. Predloženi scenario se može smatrati osnovom u paleoklimatskoj rekonstrukciji.</p> / <p>This PhD thesis analyzed the production and stability of cyanobacterial secondary metabolites. The results describe the effects of pH, temperature and light source combined with the effects of medium nitrogen content on the production of the MOSA and MOMA compounds of aquatic and soil cyanobacterial strains through AMMI model. The application of the AMMI model represents a significant contribution to the selection of appropriate biomarkers in the paleoclimatic reconstructions because it reveals the increased production of certain secondary metabolites in certain environmental conditions. MOSA compounds were observed in 8 out of 15 soil and aquatic cyanobacterial strains, while MOMA compounds were found in all 15 strains. Results show that exposure to UV light induced a higher synthesis of both pigments. The production of the MOSA compounds was clearly increased by UV irradiation and other treatments did not show a significant impact on its production. The production of MOMA compounds was increased by several stress factors including pH (pH 9.0), temperature (30-35 &deg;C), nitrogen content and UV irradiation. A higher concentration of nitrogen in the medium did not influence the increase in the production of MOSA compounds in most of the analyzed strains, while it significantly influenced the increase in the production of MOMA in all analyzed strains.<br />By analyzing the production of pigments in 19 loess cultures, phycobilins and MOSA were present in all examined loess isolates, while presence of MOMA was not detected in two samples from China. In control conditions, it was observed degradation of phycobilins depending on time, but MOSA and MOMA showed a stable structure.<br />Stability assessment of the MOSA and MOMA by the treatment with abiotic factors (different pH and temperature values of the medium, different time of exposure to UV light) revealed their pronounced stability on tested abiotic factors. Considering unstable structure of phycobilins in the presence of bacterial strains, phycobilins cannot be considered as biomarkers in loess studies. Detected results indicate that MOSA and MOMA have stable core structures resistant to bacterial strains, which makes them potentially good biomarkers for paleoclimatic reconstruction.<br />Moreover, the presence of MOSA and MOMA compounds was confirmed in loess sediment samples and BLC. Also, the LC-MS(/MS) method revealed the presence of scytonemin in 10 terrestrial cyanobacterial cultures.<br />Testing of the toxicity of loess cyanobacterial cultures and their ability to produce microcystins, cylindrospermopsin and saxitoxins, negative results were obtained. The development of new methods for detection of cyanotoxins in terrestrial ecosystems is necessary in order to revise obtained results. By assessing the stability of microcystins of the reference strain of Microcystis aeruginosa PCC 7806, its pronounced stability was observed over a 96 h in control conditions and in the presence of three bacterial strains.<br />Due to MOSA and MOMA narrow environment and organism specificity, as well as its structural stability, these metabolites are designated with a strong potential to be used as a cyanobacterial biomarker in paleoclimatic research. Due to the inability to detect cyanotoxins in loess cyanobacteria, despite the pronounced stability of the microcystin of the reference strain, cyanotoxins cannot be considered as adequate biomarkers of cyanobacteria in geological research.<br />The production of the MOSA and MOMA compounds across examined stress conditions, and further, their presence in loess samples and BLCs indicate the potential of these compounds to be regarded as biomarkers in paleoclimatic research of lacustrine/marine and loess sediments. Setting up the BLOCDUST theory and determining stability of MOSA and MOMA compounds and their aplication as a convinient biomarkers of cyanobacteria in paleoclimatic reconstruction provides the foundation for many future research of invaluable scientific significance, especially in the paleoclimatic reconstruction of loess. The proposed scenario can be considered as one of the basic model for paleoclimatic reconstruction.</p>

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