Quantification of Toxin Biosynthesis Genes In Cyanobacteria and Dinoflagellates - Genetic Factors as Predictors of Toxin Production in the Environment

Harmful algal blooms (HABs) are events caused by the massive proliferation of microscopic, often photosynthetic organisms that inhabit both fresh and marine waters. Although HABs are essentially a natural phenomenon, they now cause worldwide concern. Recent anthropogenic effects, such as climate change and eutrophication via nutrient runoff, can be seen in their increased prevalence and severity. Cyanobacteria and dinoflagellates are often the causative organisms of HABs. In addition to adverse effects caused by the sheer biomass, certain species produce highly potent toxic compounds: hepatotoxic microcystins are produced exclusively by cyanobacteria and neurotoxic saxitoxins, also known as paralytic shellfish toxins (PSTs), by both cyanobacteria and dinoflagellates. Specific biosynthetic genes in the cyanobacterial genomes direct the production of microcystin and paralytic shellfish toxins. Recently also the first paralytic shellfish toxin gene sequences from dinoflagellate genomes have been elucidated. The public health risks presented by HABs are evident, but the monitoring and prediction of toxic events is challenging. Characterization of the genetic background of toxin biosynthesis, including that of microcystins and paralytic shellfish toxins, has made it possible to develop highly sensitive molecular tools which have shown promise in the monitoring and study of potentially toxic microalgae. In this doctoral work, toxin-specific genes were targeted in the developed PCR and qPCR assays for the detection and quantification of potentially toxic cyanobacteria and dinoflagellates in the environment. The correlation between the copy numbers of the toxin biosynthesis genes and toxin production were investigated to assess whether the developed methods could be used to predict toxin concentrations. The nature of the correlation between gene copy numbers and amount of toxin produced varied depending on the targeted gene and the producing organism. The combined mcyB copy numbers of three potentially microcystin-producing cyanobacterial genera showed significant positive correlation to the observed total toxin production. However, the presence of PST-specific sxtA, sxtG, and sxtB genes of cyanobacterial origin was found to be a poor predictor of toxin production in the studied area. Conversely, the dinoflagellate sxtA4 was a good qualitative indicator of a neurotoxic bloom both in the laboratory and in the field, and population densities reflected well the observed toxin concentrations. In conclusion, although the specificity of each potential targeted toxin biosynthesis gene must be assessed individually during method development, the results obtained in this doctoral study support the use of quantitative PCR -based approaches in the monitoring of toxic cyanobacteria and dinoflagellates.Siirretty Doriast

Rapid quantification of mcyB copy numbers on dry chemistry PCR chips and predictability of microcystin concentrations in freshwater environments

Microcystin-producing cyanobacteria cause serious water quality problems worldwide, which has led to growing pressure for more intensive monitoring. Molecular biology methods that are based on identification and enumeration of biosynthetic genes, such as quantitative PCR, show promise in this respect. To be practical in a wide range of settings, these methods need to be usable also by laboratory personnel who do not have previous experience in PCR setup. Here we present a real-time quantitative mcyB dry chemistry PCR assay capable of identifying the three globally most common microcystin-producing cyanobacterial genera, Anabaena, Microcystis and Planktothrix. It minimizes the amount of liquid handling and avoids direct contact with the PCR reagents at the time of analysis. Large quantities of virtually identical chips can be manufactured, improving the comparability of results. Using the dry chemistry PCR chips, freshwater environmental samples from Finnish and Estonian lakes, rivers and reservoirs were analyzed for mcyB. The chip format was found to be highly suitable for water sample analysis due to its ease-of-use, good sensitivity and amplification efficiency. Significant positive correlation (Spearman&#39;s rank correlation, &rho;&nbsp;&gt;&nbsp;0.66, P&nbsp;&lt;&nbsp;0.001) was observed between combined mcyB copy numbers from Microcystis, Anabaena, Planktothrix and total microcystin concentrations, regardless of the method used to measure the toxins (ELISA or LC&ndash;MS). Positive correlations were observed also for single lakes.</p

Cyanobacterial akinete distribution, viability, and cyanotoxin records in sediment archives from the Northern Baltic Sea

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations, respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only be revived from the near-surface layers of the sediment, corresponding to an estimated age of 1–3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the Baltic Sea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria

Assessment of Common Cyanotoxins in Cyanobacteria of Biological Loess Crusts

Cyanotoxins are a diverse group of bioactive compounds produced by cyanobacteria that have adverse effects on human and animal health. While the phenomenon of cyanotoxin production in aquatic environments is well studied, research on cyanotoxins in terrestrial environments, where cyanobacteria abundantly occur in biocrusts, is still in its infancy. Here, we investigated the potential cyanotoxin production in cyanobacteria-dominated biological loess crusts (BLCs) from three different regions (China, Iran, and Serbia) and in cyanobacterial cultures isolated from the BLCs. The presence of cyanotoxins microcystins, cylindrospermopsin, saxitoxins, and beta-N-methylamino-L-alanine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, while the presence of cyanotoxin-encoding genes (mcyE, cyrJ, sxtA, sxtG, sxtS, and anaC) was investigated by polymerase chain reaction (PCR) method. We could not detect any of the targeted cyanotoxins in the biocrusts or the cyanobacterial cultures, nor could we amplify any cyanotoxin-encoding genes in the cyanobacterial strains. The results are discussed in terms of the biological role of cyanotoxins, the application of cyanobacteria in land restoration programs, and the use of cyanotoxins as biosignatures of cyanobacterial populations in loess research. The article highlights the need to extend the field of research on cyanobacteria and cyanotoxin production to terrestrial environments

Cyanobacterial Akinete Distribution, Viability, and Cyanotoxin Records in Sediment Archives from the Northern Baltic Sea

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection  of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only berevived from the near-surface layers of the sediment, corresponding to an estimated age of 1-3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the BalticSea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria.</p

Tarkistuslistan käyttökokemuksia keskusleikkausosastolla : Kvantitatiivinen kyselytutkimus Oulun yliopistollisen sairaalan keskusleikkausosaston sairaanhoitajille

Anestesiologeista, kirurgeista, hoitajista ja potilasturvallisuusasiantuntijoista koostuva kansainvälinen työryhmä on laatinut leikkaustiimin tarkistuslistan parantamaan yhteistyötä ja kommunikaatiota perioperatiivisessa hoitotyössä. Tarkistuslistalla pyritään huomioimaan potilasturvallisuutta vaarantavat asiat ennen anestesian aloitusta, ennen toimenpiteen aloitusta ja ennen potilaan leikkaussalista poistumista. Tarkistuslistan on tutkitusti todistettu vähentävän leikkauskuolleisuutta ja muita komplikaatioita. Tutkimuksen tarkoituksena oli kuvailla Oulun yliopistollisen sairaalan keskusleikkausosaston sairaanhoitajien kokemuksia ja näkemyksiä tarkistuslistan käytöstä sekä pyrkiä selvittämään mahdollisia käyttöön liittyviä ongelmia ja sairaanhoitajien kehittämisideoita. Tavoitteena on tarjota keskusleikkausosastolle mahdollisuus kehittää tarkistuslistaa ja tarvittaessa kouluttaa henkilökuntaa tarkistuslistan käyttämisessä sekä tuoda esille tarkistuslistan merkitys potilasturvallisuutta edistävänä tekijänä. Opinnäytetyön tilaajana toimi Oulun yliopistollisen sairaalan keskusleikkausosasto. Opinnäytetyö oli määrällinen tutkimus, johon kuului myös kaksi avointa kysymystä. Aineisto kerättiin syyskuussa 2012 verkkokyselyllä Webropol-alustalla, jonne vietiin keskusleikkausosaston tarkistuslistaa hyödyntäen laadittu mittari. Kysely välitettiin 176 sairaanhoitajalle, joista 56 vastasi kyselyyn. Vastausprosentti oli 31,8 %. Aineisto analysoitiin Webropol-alustalla laadittujen taulukoiden avulla. Avointen kysymysten kohdalla käytettiin induktiivista sisällönanalyysia. Tulosten perusteella sairaanhoitajat kokivat tarkistuslistan potilasturvallisuutta parantavaksi tekijäksi. Tarkistuslistaa ei koettu liian työlääksi käyttää ja sitä hyödynnetäänkin hyvin perioperatiivisessa hoitotyössä. Sairaanhoitajat kokivat tarkistuslistan käytön myös parantavan yhteistyötä leikkaus- ja anestesiatiimin välillä. Tuloksissa oli havaittavissa eroja leikkaus- ja anestesiapuolen sekä pehmytkudoskirurgian toimialueen ja tukielin- ja neurokirurgian toimialueen välillä esimerkiksi siinä, kenelle tarkistuslistan lukeminen kuuluu ja kuinka potilaan perussairaudet ja anestesialaitteisto varmistetaan ennen anestesian aloitusta. Tutkimustuloksia voidaan hyödyntää keskusleikkausosastolla perioperatiivisen hoitotyön potilasturvallisuuden kehittämiseen. Tarkistuslistaa voidaan kehittää sitä päivittäin käyttävien sairaanhoitajien kokemusten perusteella toimivammaksi ja tarkoitustaan vastaavammaksi eri tilanteissa. Tuloksia ei voi yleistää koskemaan muita leikkausosastoja, sillä tulokset ovat sidoksissa tutkittavaan ympäristöön ja tarkistuslistaan, joka on muokattu keskusleikkausosaston tarpeisiin sopivaksi.World Health Organization has developed a Surgical Safety Checklist to improve the cooperation among the operating room staff and the safety of surgical care. The Checklist has been found to be an effective way to ensure patient safety before, during and after surgery. The commissioner of this study was Surgical Department of Oulu University Hospital. In Surgical Department an edited version of WHO’s Checklist has been used for a couple of years. There are no earlier studies about the user experience of a Surgical Safety Checklist in perioperative nursing in the Surgical Department of Oulu University Hospital. The purposes of this study were to describe nurses’ user experiences of a Surgical Safety Checklist in Surgical Department, to find out possible problems in the use of Checklist and also to find out nurses’ development proposals. This study was mainly quantitative, but it also contained two open-ended questions. The data were collected with a questionnaire on the Internet. The questionnaire was made on the basis of the Surgical Safety Checklist used in Surgical Department. 56 of 176 nurses answered the survey. Quantitative data were analyzed using the tablets made by Webropol Online Survey and Analysis Software. The open-ended questions were content analysed. The study revealed that nurses regarded the Surgical Safety Checklist as a way to improve patient safety and a tool of cooperation between anaesthetic and theatre nurses. It is not guaranteed that the Checklist is a part of every operation but still nurses did not find it too laborious to go through. There were some differences between theatre and anaesthetic nurses, between those who worked in orthopaedic and gastroenterological operating theatres and between men and women. Differences were not very significant. The results are not generalizable to nurses in other hospitals or in other surgical departments. The thesis will offer Surgical Department a chance to develop the Checklist, to teach the staff to use the Checklist properly and to highlight its meaning for patient safety

Ikääntyneiden kotiravitsemuksen kartoittaminen

Opinnäytetyön tavoitteena oli tuottaa työkalu ikääntyneiden kotiravitsemuksen arvioimista varten ja opinnäytetyö on tehty Lohjan kaupungin tilauksesta. Opinnäytetyössä tuotettu työ kalu on suunniteltu Lohjan kaupungin omaishoitajakoordinaattoreiden käyttöä varten. Työka lun tyyliksi valittiin tarkistuslista, jonka on tarkoitus helpottaa omaishoitajakoordinaattorin tehtävää arvioitaessa ikääntyneiden kotiravitsemusta. Tarkistuslistan haluttiin olevan helposti ymmärrettävä, helppokäyttöinen sekä nopea käyttöinen, johon ei menisi liikaa aikaa. Opinnäytetyö toteutettiin kehittämistyönä, joka sisälsi kaksi erilaista vaihetta. Ensimmäisenä vaiheena oli nykytilanteiden kartoittaminen haastatteluilla sekä teoriatietoon perehtyminen. Opinnäytetyön teoria osassa käydään läpi ravitsemukseen sekä omaishoitajuuteen liittyvää teoriatietoa ja opinnäytetyön toteutusosuudessa kerrotaan tarkemmin haastatteluista ja niissä esiin nousseista asioista. Toisena vaiheena oli työkalun luominen sekä sen arvioiminen. Opinnäytetyössä tuotettu työkalun kysymykset luotiin teoriatiedon sekä omaishoitajakoordi naattoreiden haastatteluiden pohjalta. Opinnäytetyössä käytettiin tutkimuksiin sekä näyttöön perustuvaan tietoon pohjautuvia lähteitä. Opinnäytetyössä tuotetun tuotoksen arviointiin osallistui omaishoitajakoordinaattorit sekä opinnäytetyön tilaajan yhteyshenkilö, ja heidän antaman palautteen pohjalta alkuperäiseen tarkistuslistaan tehtiin muutoksia, jotta se olisi heidän toiveidensa mukainen

Assessment of Common Cyanotoxins in Cyanobacteria of Biological Loess Crusts

Cyanotoxins are a diverse group of bioactive compounds produced by cyanobacteria that have adverse effects on human and animal health. While the phenomenon of cyanotoxin production in aquatic environments is well studied, research on cyanotoxins in terrestrial environments, where cyanobacteria abundantly occur in biocrusts, is still in its infancy. Here, we investigated the potential cyanotoxin production in cyanobacteria-dominated biological loess crusts (BLCs) from three different regions (China, Iran, and Serbia) and in cyanobacterial cultures isolated from the BLCs. The presence of cyanotoxins microcystins, cylindrospermopsin, saxitoxins, and β-N-methylamino-L-alanine was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, while the presence of cyanotoxin-encoding genes (mcyE, cyrJ, sxtA, sxtG, sxtS, and anaC) was investigated by polymerase chain reaction (PCR) method. We could not detect any of the targeted cyanotoxins in the biocrusts or the cyanobacterial cultures, nor could we amplify any cyanotoxin-encoding genes in the cyanobacterial strains. The results are discussed in terms of the biological role of cyanotoxins, the application of cyanobacteria in land restoration programs, and the use of cyanotoxins as biosignatures of cyanobacterial populations in loess research. The article highlights the need to extend the field of research on cyanobacteria and cyanotoxin production to terrestrial environments

Distributions of three <i>Alexandrium</i> species and their toxins across a salinity gradient suggest an increasing impact of GDA producing <i>A. pseudogonyaulax</i> in shallow brackish waters of Northern Europe

Blooms of Alexandrium spp. are a well-known phenomenon in Northern European waters. While A. tamarense/catenella, and A. pseudogonyaulax have been reported from marine waters, high densities of A. ostenfeldii are mainly observed at lower salinities in North Sea estuaries and the Baltic Sea, suggesting salinity as a driver of Alexandrium species composition and toxin distribution. To investigate this relationship, an oceanographic expedition through a natural salinity gradient was conducted in June 2016 along the coasts of Denmark. Besides hydrographic data, phytoplankton and sediment samples were collected for analyses of Alexandrium spp. cell and cyst abundances, for toxin measurement and cell isolation. Plankton data revealed the predominance of A. pseudogonyaulax at all transect stations while A. ostenfeldii and A. catenella generally contributed a minor fraction to the Alexandrium community. High abundances of A. pseudogonyaulax in the shallow enclosed Limfjord were accompanied by high amounts of goniodomin A (GDA). This toxin was also detected at low abundances along with A. pseudogonyaulax in the North Sea and the Kattegat. Genetic and morphological characterization of established strains showed high similarity of the Northern European population to distant geographic populations. Despite low cell abundances of A. ostenfeldii, different profiles of cycloimines were measured in the North Sea and in the Limfjord. This field survey revealed that salinity alone does not determine Alexandrium species and toxin distribution, but emphasizes the importance of habitat conditions such as proximity to seed banks, shelter, and high nutrient concentrations. The results show that A. pseudogonyaulax has become a prominent member of the Alexandrium spp. community over the past decade in the study area. Analyses of long term monitoring data from the Limfjord confirmed a recent shift to A. pseudogonyaulax dominance. Cyst and toxin records of the species in Kiel Bight suggest a spreading potential into the brackish Baltic Sea, which might lead to an expansion of blooms under future climate conditions

Protected freshwater ecosystem with incessant cyanobacterial blooming awaiting a resolution

© 2019 by the authors. For 50 years persistent cyanobacterial blooms have been observed in Lake Ludos (Serbia), a wetland area of international significance listed as a Ramsar site. Cyanobacteria and cyanotoxins can affect many organisms, including valuable flora and fauna, such as rare and endangered bird species living or visiting the lake. The aim was to carry out monitoring, estimate the current status of the lake, and discuss potential resolutions. Results obtained showed: (a) the poor chemical state of the lake; (b) the presence of potentially toxic (genera Dolichospermum, Microcystis, Planktothrix, Chroococcus, Oscillatoria, Woronichinia and dominant species Limnothrix redekei and Pseudanabaena limnetica) and invasive cyanobacterial species Raphidiopsis raciborskii; (c) the detection of microcystin (MC) and saxitoxin (STX) coding genes in biomass samples; (d) the detection of several microcystin variants (MC-LR, MC-dmLR, MC-RR, MC-dmRR, MC-LF) in water samples; (e) histopathological alterations in fish liver, kidney and gills. The potential health risk to all organisms in the ecosystem and the ecosystem itself is thus still real and present. Although there is still no resolution in sight, urgent remediation measures are needed to alleviate the incessant cyanobacterial problem in Lake Ludos to break this ecosystem out of the perpetual state of limbo in which it has been trapped for quite some time