Although the use of genetically modified plants for bioremediation, or the in situ cleaning of contaminated sites, has been known for quite some time, little attention has so far been paid to the production of antibodies in plants and their ex vivo application in selective depletion. Therefore, highly affine and specific antibodies against algal toxins using microcystin as an example will be produced in plants at low cost within this research project. The basis is a monoclonal antibody (Mab 10E7, species: mouse) generated in a former research project. The sequence of the variable domains will be determined, optimized for plants and sub cloned into suitable plant transformation vectors, which already contain constant antibody sequences. In addition, a scFv fragment containing different tag sequences and fusion proteins will be constructed. Leaf-based (tobacco) as well as seed-based (barley) systems will be used.Affinity-purified plant-produced antibodies (plantibodies) will be characterized in detail for their binding properties using microtitre plate-ELISA and surface plasmon resonance (SPR). The monoclonal mouse antibody will be used as reference. To assure cost-efficiency for future applications, roughly purified fractions (sequential pH and temperature treatment followed by filtration) will be tested for the upscaling. Following immobilization of the plantibody fractions on suitable substrates, for instance membranes, porous polymer monoliths or in porous glasses, their application for depletion will be defined using model water samples spiked fortified with microcystins.
Man-made eutrophication of lakes has caused increased frequencies of cyanobacterial blooms, which go along with a reduction in abundance of Daphnia, the major herbivore of the phytoplankton. This reduced abundance of Daphnia is a major cause for the fact that food web manipulations frequently cause only short term improvements of water quality. The most frequently reported toxins of cyanobacterial blooms are microcystins, which are hepatotoxic to mammals and increase mortality in Daphnia. Although it has frequently been shown that Daphnia coexisting with cyanobacteria show increased tolerance to toxic cyanobacteria, the genetic basis of this tolerance is entirely unknown. Here the genetic basis of tolerance to microcystins in Daphnia will be investigated. A microcystin-producing strain and a mutant of that strain, which is deficient in microcystins, will be fed to a microcystin-tolerant and a microcystin-sensitive clone of Daphnia magna. Transcriptome analysis based on RNASeq will be used to identify differentially expressed genes that either differ constitutively in expression among the two Daphnia clones or are differentially expressed upon ingestion of microcystins. Bioinformatic analysis of the data and metabolic mapping will be used to identify key enzymes and genes that mediate tolerance to microcystins. Expression of selected differentially expressed genes will be validated in an independent experiment using qPCR. Further evidences for a mediation of microcystin tolerance by selected differentially expressed genes will be gained by investigating how the concentration of dietary microcystins affect gene expression. This will be achieved by feeding liposomes that are loaded with microcystins to Daphnia. Understanding the molecular mechanisms is indispensable for the understanding of the evolution of microcystin tolerance in Daphnia and is of potentially pivotal importance for the management of lakes with toxic cyanobacterial blooms.
We aim at understanding the occurrence and ecotoxicological effects of selected cyanobacterial toxins and elucidating their uptake and modes of action in fish. The data will contribute to the hazard and risk assessment. Cyanobacteria have gained worldwide importance as their intense growth in eutrophic surface waters. Microcystis and Planktothrix spp. are most often observed to develop high densities in lake water. These phytoplankton species may produce harmful toxins such as cyclic oligopeptides, however, the biological functions of most cyanobacterial oligopeptides are not known. Microcystin-LR (MC-LR) may have a severe impact on aquatic biota, or even lifestock drinking contaminated water. However, in vivo uptake mechanisms and elimination kinetics of microcystins are poorly understood. Human health problems mainly originate from chronic exposure to low microcystin concentrations in poorly treated drinking water, leading to liver cancer. Cyanopeptolins, another class of cyanobacterial oligopeptides are not investigated for their toxicity. To date, there is a lack of understanding of the uptake kinetics, metabolism and effects of these peptide toxins in fish including their molecular effects. The aim of this project is to deepen our understanding on the formation, molecular and cellular effects of these cyanobacterial toxins. Research is promoted by limnologists, environmental toxicologists and chemists focusing on a common research question, thereby investigating multiple aspects including isolation, structural and toxicological characterization, preparation of a series of derivatives to probe the toxicology and mode of action, in particular of cyanopeptolin CP1020 and MC-LR. Uptake through transmembrane transporters, kinetics and molecular and developmental effects will be studied in zebrafish. Following questions will be addressed for a better hazard and risk assessment of these toxins: What toxin patterns exist in P. rubescens, and what are their chemical structures and toxicity? How is the uptake, distribution and elimination kinetics of fluorescent MC-LR and CP1020 in zebrafish? What is the role of uptake transporters (Oatp) and efflux transporters (ABC-transporters) in zebrafish hepatocytes? What are the molecular effects and mechanisms of action of MC-LR and CP1020 on the transcriptome in zebrafish? By tackling these questions and by linking limnology, chemistry and ecotoxicology we will provide a unique multidisciplinary training opportunity across multiple areas.
Im Rahmen des geplanten Vorhabens soll gemeinsam mit israelischen Arbeitsgruppen eine neuartige Methode entwickelt und getestet werden, die die Kontrolle und Eliminierung von cyanobakteriellen Blüten ermöglicht. Cyanobakterielle Massenentwicklungen, die man zunehmend in Seen aller Breitengrade beobachten kann, stellen ein großes Problem sowohl für die Ökosysteme und die Trinkwassergewinnung dar. Im geplanten Vorhaben soll erstmalig ein natürlicher Faktor des Dinoflagellaten Peridinium getestet werden. Spezifische Projektziele sind 1) Die Identifizierung und chemische Strukturaufklärung des Peridiniumfaktors; 2) Die Aufklärung der Wirkungsweise des Peridiniumfaktors; 3) Untersuchungen zur Wirkung des Faktors auf Toxinexpression und Flotierungsverhalten von Microcystis; 4) Die Entwicklung eines Labor-Screeningsystems für den Peridiniumfaktor; 5) Die Entwicklung einer Methode zur Herstellung des Faktors; 6) Die Etablierung eines Sensorsystems für Microcystin und 7) Testung der Methode in Freilandexperimenten. Die im Rahmen des Projektes etablierte Technologie kann zur Verbesserung der Bade- und Trinkwasserqualität in Gewässern eingesetzt werden.
Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains. Quelle: https://www.mdpi.com
The impact of environmental stimuli on the productionof the widespread cyanobacterial hepatotoxinmicrocystin (MC) is under debate. Whereas transcriptionalstudies of the biosynthetic genes suggest aclear influence of light conditions on toxin productionthe data for the metabolite itself are inconsistent andhighly strain-specific. Here, we have reassessed theMC content by using two immunological detectiontechniques that allow a parallel quantification of MCin the methanolic extracts and the residual pellet fractionthat contains high molecular weight proteins. Ourresults show a significant proportion of MC in theprotein bound fraction in strains of Microcystis andPlanktothrix and of the related toxin nodularin (NOD)in Nodularia. Moreover, we could show a very strongincrease of MC after high light illumination in theprotein fraction contributing to a significant overallincrease in MC production under these conditionsthat is not seen in extracts analysed by LC-MS andELISA. The fact that a considerable portion of MC isneglected with current analysis techniques was alsoconfirmed for selected field samples. Immunofluorescencestudies suggest strain-specific differences inthe amount of MC conjugate formation.Quelle: Environmental Microbiology (2013) 15(6), 1810-1820
Gesamtziel des Projektes ist die Erarbeitung einer interdisziplinär angelegten, wissenschaftlichen Konzeption zur Reduzierung der Eutrophierung des 760m2 großen Chaosees auf ein Maß, welches die Trinkwassernutzung ohne aufwändige Aufbereitung erlaubt. Zur Nutzung des Seewassers als Rohwasser soll die Reduktion der durch Algenblüte hervorgerufenen Microcystine durch die Verwendung der 'grünen Leber' auf eine unschädliche Konzentration erreicht werden. Das Vorhaben steht in direktem Zusammenhang mit der nachhaltigen Entwicklung der Wasserressourcen. Wissenschaftliche Arbeitsziele von Experimenten sind, robuste biologische Verfahren zur Aufbereitung des Seewassers als geeignetes Rohwasser für die ortsübliche Trinkwasseraufbereitung bereit zu stellen, um damit die Konzentration der Microcystine auf ein unschädliches Maß zu reduzieren. In diesem Verbund haben wir erstmals die Möglichkeit, im Freiland (Enclosures) diese Laborexperimente zu verifizieren und auf die Freilandsituation zu übertragen. Die Experimente zum Toxinabbau sind für viele Wasserwerke und für die Volksgesundheit von immenser Bedeutung, zumal auch eine Reihe von Stauseen Eutrophierungsprobleme haben.
Lake Naivasha, an important inland water ecosystem and a crucial freshwater resource in the Great African Rift Valley, has displayed clear signals of degradation inrecent decades. We studied the phytoplankton composition and biomass levels in the period 2001-2013 and noted a progressive increase in the occurrence of potentially toxic cyanobacteria. Analyses for the presence of cyanotoxins such as microcystins (MC), cylindrospermopsin (CYN) and anatoxin-a (ATX-a) were carried out on samples collected in 2008-2013. Among the cyanotoxins tested, low concentrations of MC were detected in the lake. This is the first record of the occurrence of MC in Lake Naivasha. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Lake Naivasha were carried out to establish the taxa of the dominant species. Amplification of the aminotrasferase (AMT) domain responsible for cyanotoxin production confirmed the presence of the mcyE gene belonging to the microcystin synthesis gene cluster in field samples containing Microcystis and Planktothrix species. These findings suggest that toxin producing cyanobacteria could become a threat to users of this over- exploited tropical lake in the near future.Quelle: 2013 Elsevier B.V. All rights reserved.
'- Bestimmung von Algentoxinen aus Blaualgen in Gewaessern; - Bewertung der Konzentrationen an Blaualgentoxinen im Rahmen der Badegewaesserhygiene und Erarbeitung von Grundlagen fuer Empfehlungen zur Nutzung der Gewaesser als Badegewaesser; - Erarbeitung der Grundlagen fuer einen ,Screeningtest' auf Algentoxine im Rahmen der Badegewaesseruntersuchungen und der Ableitung von Warnwerten; - Klaerung der Mobilitaet von Algentoxinen im Wasserkreislauf und Bedeutung im Rahmen der Trinkwassergewinnung.
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