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1022. Surveillance of human enteric viruses in coastal waters using concentration with methacrylate monolithic supports prior to detection by RT-qPCRJosé Gonçalves, Ion Gutiérrez-Aguirre, Mukundh Narayanan Balasubramanian, Maja Zagorščak, Maja Ravnikar, Valentina Turk, 2018, original scientific article Abstract: This is the first surveillance study using methacrylate monolithic supports to concentrate environmental coastal water samples, prior to molecular target detection by RT-qPCR. Rotaviruses (RoV) and Noroviruses (NoV) were monitored in a polluted area at the Bay of Koper (Gulf of Trieste, Northern Adriatic Sea) and at a nearby bathing area and mussel farm areas.
RoV and NoV are released into the Bay of Koper, with higher rates close to the discharge of the wastewater treatment plant, however, they can be detected at recreational and mussel farming areas. Our results showed that water bodies considered safe based on FC concentrations, can still have low, yet potentially infective, concentrations of human viruses.
Keywords: Rotavirus, Norovirus, faecal coliforms, RT-qPCR, methacrylate monolithic columns, enteric virus concentration
Published in DiRROS: 24.07.2024; Views: 292; Downloads: 160
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1023. NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate hostJean-Marie Volland, Arno Schintlmeister, Helena Zambalos, Siegfried Reipert, Patricija Mozetič, Salvador Espada-Hinojosa, Valentina Turk, Michael Wagner, Monika Bright, 2018, original scientific article Abstract: The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.
Keywords: microbial ecology, symbiosis
Published in DiRROS: 24.07.2024; Views: 257; Downloads: 214
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1024. Cell death is not sufficient for the restriction of potato virus Y spread in hypersensitive response-conferred resistance in potatoTjaša Lukan, Špela Baebler, Maruša Pompe Novak, Katja Guček, Maja Zagorščak, Anna Coll Rius, Kristina Gruden, 2018, original scientific article Abstract: Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1-mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this resistance remains unknown.
Keywords: potato virus Y, salicylic acid, hypersensitive response, programmed cell death, callose deposits, necrotic lesion
Published in DiRROS: 24.07.2024; Views: 264; Downloads: 236
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1025. Plant X-tender : an extension of the AssemblX system for the assembly and expression of multigene constructs in plantsTjaša Lukan, Fabian Machens, Anna Coll Rius, Špela Baebler, Katrin Messerschmidt, Kristina Gruden, 2018, original scientific article Abstract: Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.
Keywords: cloning, plasmid construction, polymerase chain reaction
Published in DiRROS: 24.07.2024; Views: 294; Downloads: 179
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1026. Use of HuH6 and other human-derived hepatoma lines for the detection of genotoxins : a new hope for laboratory animals?Monika Waldherr, Miroslav Mišík, Franziska Ferk, Jana Tomc, Bojana Žegura, Metka Filipič, Wolfgang Mikulits, Sören Mai, Oskar Haas, Wolfgang W. Huber, Elisabeth Haslinger, Siegfried Knasmüller, 2018, original scientific article Abstract: Cell lines which are currently used in genotoxicity tests lack enzymes which activate/detoxify mutagens. Therefore, rodent-derived liver preparations are used which reflect their metabolism in humans only partly; as a consequence misleading results are often obtained. Previous findings suggest that certain liver cell lines express phase I/II enzymes and detect promutagens without activation; however, their use is hampered by different shortcomings. The aim of this study was the identification of a suitable cell line. The sensitivity of twelve hepatic cell lines was investigated in single cell gel electrophoresis assays. Furthermore, characteristics of these lines were studied which are relevant for their use in genotoxicity assays (mitotic activity, p53 status, chromosome number, and stability). Three lines (HuH6, HCC1.2, and HepG2) detected representatives of five classes of promutagens, namely, IQ and PhIP (HAAs), B(a)P (PAH), NDMA (nitrosamine), and AFB1 (aflatoxin), and were sensitive towards reactive oxygen species (ROS). In contrast, the commercially available line HepaRG, postulated to be a surrogate for hepatocytes and an ideal tool for mutagenicity tests, did not detect IQ and was relatively insensitive towards ROS. All other lines failed to detect two or more compounds. HCC1.2 cells have a high and unstable chromosome number and mutated p53, these features distract from its use in routine screening. HepG2 was frequently employed in earlier studies, but pronounced inter-laboratory variations were observed. HuH6 was never used in genotoxicity experiments and is highly promising, it has a stable karyotype and we demonstrated that the results of genotoxicity experiments are reproducible.
Keywords: hepatic cell lines, p53, comet assay, genotoxicity
Published in DiRROS: 24.07.2024; Views: 286; Downloads: 189
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1027. Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCRDavid Dobnik, Tina Demšar, Ingrid Huber, Lars Gerdes, Sylvia Broeders, Nancy Roosens, Frédéric Debode, Gilbert Berben, Jana Žel, 2018, original scientific article Abstract: Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection.
Keywords: digital PCR, droplet digital PCR, absolute quantification, reference materials, GMO quantification
Published in DiRROS: 24.07.2024; Views: 396; Downloads: 155
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1028. Uncertainty of modelled flow regime for flow-ecological assessment in Southern EuropeOlga Vigiak, Stefanie Lutz, Angeliki Mentzafou, Gabriele Chiogna, Tuo Ye, Bruno Majone, Hylke Beck, Ad de Roo, Anna Malagó, Faycal Bouraoui, Rohini Kumar, Luis Samaniego, Ralf Merz, Christos Gamvroudis, Nikolaos Skoulikidis, Nikolaos P. Nikolaidis, Alberto Bellin, Vicenç Acuňa, Nataša Mori, Ralf Ludwig, Alberto Pistocchi, 2018, original scientific article Abstract: Sustainable water basin management requires characterization of flow regime in river networks impacted by anthropogenic pressures. Flow regime in ungauged catchments under current, future, or natural conditions can be assessed with hydrological models. Developing hydrological models is, however, resource demanding such that decision makers might revert to models that have been developed for other purposes and are made available to them (‘off-the-shelf’ models). In this study, the impact of epistemic uncertainty of flow regime indicators on flow-ecological assessment was assessed at selected stations with drainage areas ranging from about 400 to almost 90,000 km2 in four South European basins (Adige, Ebro, Evrotas and Sava). For each basin, at least two models were employed. Models differed in structure, data input, spatio-temporal resolution, and calibration strategy, reflecting the variety of conditions and purposes for which they were initially developed. The uncertainty of modelled flow regime was assessed by comparing the modelled hydrologic indicators of magnitude, timing, duration, frequency and rate of change to those obtained from observed flow. The results showed that modelled flow magnitude indicators at medium and high flows were generally reliable, whereas indicators for flow timing, duration, and rate of change were affected by large uncertainties, with correlation coefficients mostly below 0.50. These findings mirror uncertainty in flow regime indicators assessed with other methods, including from measured streamflow. The large indicator uncertainty may significantly affect assessment of ecological status in freshwater systems, particularly in ungauged catchments. Finally, flow-ecological assessments proved very sensitive to reference flow regime (i.e., without anthropogenic pressures). Model simulations could not adequately capture flow regime in the reference sites comprised in this study. The lack of reliable reference conditions may seriously hamper flow-ecological assessments. This study shows the pressing need for improving assessment of natural flow regime at pan-European scale.
Keywords: flow regime, ecohydrological assessment, SWAT, mHM, lisflood, HYPERstream
Published in DiRROS: 24.07.2024; Views: 326; Downloads: 173
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1029. Statistical modeling of long-term grapevine response to "Candidatus Phytoplasma solani" infection in the fieldAna Rotter, Petra Nikolić, Neža Turnšek, Polona Kogovšek, Andrej Blejec, Kristina Gruden, Marina Dermastia, 2018, original scientific article Abstract: Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.
Keywords: plant diseases, plant pathology
Published in DiRROS: 24.07.2024; Views: 471; Downloads: 246
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