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Povzetek: Anatoxins (ATX) are globally occurring toxins produced by some species of cyanobacteria in aquatic habitats. They can cause acute poisoning in animals, leading to muscle paralysis and respiratory failure, and might also pose a long-term health risk to humans. Thanks to advances in molecular methods and genomic knowledge, it is now possible to rapidly detect and quantify the genes associated with cyanotoxin production for most major groups of cyanotoxins except ATX. The aim of this study was to develop and validate a new quantitative PCR (qPCR) assay for general detection of all potential ATX producers in the environment. After specificity testing in silico and in vitro with 16 cyanobacterial strains (endpoint PCR, amplicon sequencing and qPCR), two assays targeting the anaC gene were thoroughly validated for linearity, amplification efficiency, sensitivity, dynamic range, inter-assay and intra-assay variability, and the influence of background DNA. The assays were then applied to 144 environmental samples of plankton and biofilm from lakes and rivers whose ATX content had previously been measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Amplification efficiency of the two designed assays was between 94% and 103%, and the limits of quantification and detection were up to, but mostly below, 322 and 32 cells/mL, respectively. Both assays showed better or equal specificity in cyanobacterial cultures than currently available PCR assays and were able to predict the presence of ATX detected by LC-MS/MS in most environmental samples (83 % in plankton and 52–62% in biofilm). A higher number of discrepancies between qPCR and LC-MS/MS results in biofilm than in plankton samples indicates limited knowledge and sparse genomic data on benthic cyanobacteria. These assays are the first published general qPCR assays targeting all ATX producers and could provide water managers with a rapid and cost-effective risk assessment to better protect human and animal health.
Ključne besede: anatoxins (ATX), cyanobacteria, qPCR assay, anaC gene, LC-MS/MS, environmental microbiology, environmental science, toxicology, molecular biology
Objavljeno v DiRROS: 17.12.2024; Ogledov: 448; Prenosov: 235
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Povzetek: The Annexe 17 of the Final Report of the project LIFE Stop CyanoBloom, presenting the “Report on economic justification of the project” is composed of 14 chapters. The first part of the report gives a general overview of the cyanobacteria and their blooms, and species and toxicity of cyanobacterial toxins. With a literature review, economic consequences of cyanobacterial blooms on human health, commercial fishery, tourism and recreation, and lake monitoring and management have been evaluated. The chapter on phytoplankton and bacterioplankton monitoring options presents the importance of the phytoplankton for the lake ecology and evaluation of the water quality status. Traditional phytoplankton monitoring approaches are compared to the new monitoring options applied in the project. The chapter in-lake cyanobacterial control options give a broad review of available in-lake rehabilitation and remediation options, which can be compared with the electrochemical oxidation technique presented in the following chapter and applied in the project. According to the results obtained during the pilot testing activities, a cost evaluation of the approach has been performed and presented in the report. The last chapter gathers open issues and proposals for the further work in this field.
Ključne besede: cyanobacteria, cyanotoxins, health risk
Objavljeno v DiRROS: 05.09.2024; Ogledov: 652; Prenosov: 460
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Povzetek: The systemic toxicity of cyclic peptides produced by cyanobacteria (CCPs) is not yet completely understood. Apart from the most known damages to the liver and kidneys, symptoms of their neurotoxicity have also been reported. Hepatotoxic CCPs, like microcystins, as well as non-hepatotoxic anabaenopeptins and planktopeptins, all exhibit cytotoxic and cytostatic effects on mammalian cells. However, responses of different cell types to CCPs depend on their specific modes of interaction with cell membranes. This study demonstrates that non-hepatotoxic planktopeptin BL1125 and anabaenopeptins B and F, at concentrations up to 10 µM, affect normal and tumor human astrocytes (NHA and U87-GM) in vitro by their almost immediate insertion into the lipid monolayer. Like microcystin-LR (up to 1 µM), they inhibit Ser/Thr phosphatases and reorganize cytoskeletal elements, with modest effects on their gene expression. Based on the observed effects on intermediate filaments and intermediate filament linkage elements, their direct or indirect influence on tubulin cytoskeletons via post-translational modifications, we conclude that the basic mechanism of CCP toxicities is the induction of inter- and intracellular communication failure. The assessed inhibitory activity on Ser/Thr phosphatases is also crucial since the signal transduction cascades are modulated by phosphorylation/dephosphorylation processes.
Ključne besede: astrocytes, cyclic cyanobacterial peptides, cytoskeletal organization, Ser/Thr phosphatases, systemic toxicity, cyanobacteria
Objavljeno v DiRROS: 28.08.2024; Ogledov: 760; Prenosov: 536
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Povzetek: Sensors based on in vivo measurements of photosynthetic pigments fluorescence enable real-time phytoplankton monitoring with high spatial and temporal resolution. A combination of chlorophyll a (CHL) and phycocyanin (PC) fluorescence sensors was used for phytoplankton quantification and differentiation in two small water bodies, Koseze Pond and pond in Hotinja vas. The high correlation of CHL and PC fluorescence signals with biovolume was confirmed during the two-year monitoring in anatural pond environment in spite of a seasonal succession of the phytoplankton. Additionally, disturbances of the sensors were investigated. Water bodies containing predominantly algae yielded false positive signals of the PC sensor, which reached up to 1% of the intensity of the CHL signal. Similarly, underestimated counts of cyanobacteria measured with CHL fluorescence sensor can be adjusted using PC fluorescence sensor.
Ključne besede: small water bodies, algae, cyanobacteria, fluorescence sensors, biovolume
Objavljeno v DiRROS: 01.08.2024; Ogledov: 631; Prenosov: 358
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Povzetek: Background Developing sustainable autotrophic cell factories depends heavily on the availability of robust and well-characterized biological parts. For cyanobacteria, these still lag behind the more advanced E. coli toolkit. In the course of previous protein expression experiments with cyanobacteria, we encountered inconveniences in working with currently available RSF1010-based shuttle plasmids, particularly due to their low biosafety and low yields of recombinant proteins. We also recognized some drawbacks of the commonly used fluorescent reporters, as quantification can be affected by the intrinsic fluorescence of cyanobacteria. To overcome these drawbacks, we envisioned a new chimeric vector and an alternative reporter that could be used in cyanobacterial synthetic biology and tested them in the model cyanobacterium Synechocystis sp. PCC 6803. Methods We designed the pMJc01 shuttle plasmid based on the broad host range RSFmob-I replicon. Standard cloning techniques were used for vector construction following the RFC10 synthetic biology standard. The behavior of pMJC01 was tested with selected regulatory elements in E. coli and Synechocystis sp. PCC 6803 for the biosynthesis of the established GFP reporter and of a new reporter protein, cystatin. Cystatin activity was assayed using papain as a cognate target. Results With the new vector we observed a significantly higher GFP expression in E. coli and Synechocystis sp. PCC 6803 compared to the commonly used RSF1010-based pPMQAK1. Cystatin, a cysteine protease inhibitor, was successfully expressed with the new vector in both E. coli and Synechocystis sp. PCC 6803. Its expression levels allowed quantification comparable to the standardly used fluorescent reporter GFPmut3b. An important advantage of the new vector is its improved biosafety due to the absence of plasmid regions encoding conjugative transfer components. The broadhost range vector pMJc01 could find application in synthetic biology and biotechnology of cyanobacteria due to its relatively small size, stability and ease of use. In addition, cystatin could be a useful reporter in all cell systems that do not contain papain-type proteases and inhibitors, such as cyanobacteria, and provides an alternative to fluorescent reporters or complements them.
Ključne besede: bacteria, cyanobacteria, synthetic biology, proteolytic cleavage, cystatin
Objavljeno v DiRROS: 19.07.2024; Ogledov: 678; Prenosov: 595
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