341. Pregled pojavljanja močvirske uharice Asio flammeus v Sloveniji med letoma 1995 in 2015 ter verjetno gnezdenje v eruptivnem letu 2008Al Vrezec, 2016, izvirni znanstveni članek Povzetek: Between 1995 and 2015 the number of records of Short-eared Owl Asio flammeus in Slovenia increased drastically, especially after 2007, but the species occurred regularly every year since 2002. Before that, the Short-eared Owl was regarded as a very rare migrant in Slovenia. Most of the observations were from wintering and migration periods, and the most important areas for the species in Slovenia were Ljubljansko barje, Lake Cerknica, surroundings of the water reservoir Medvedce and coastal wetlands. In 2008 and 2013 the Short-eared Owl occurred in large numbers, and these years were regarded as irruptive. Flocks of 2 to 8 birds were observed. At Ljubljansko barje, increased numbers of observed Short-eared Owls coincided with a large population of small mammals (species of the genus Apodemus and Microtus) and poor snow cover in 2008, and at least three communal roost sites were found that year. In the irruptive year 2013 there was a greater number of Short-eared Owls observed at the Medvedce water reservoir. On the plain at Lesce near the village of Smokuč an injured second year female was found at the end of March 2008 with a developing brood patch in its initial stage. It is likely that the female attempted to nest, which confirms the status of the species as occasional breeder in Slovenia. The last confirmed breeding in Slovenia was recorded in 1936 at Ljubljansko barje. In addition to local conditions (population of small mammals, snow cover) the frequency of occurrence of the Short-eared Owl in Slovenia is also affected by the population of development in the Boreal region and changes in migratory characteristics of the species in Europe. Therefore, an increase of the number of Short-eared Owls in Slovenia is expected in the future, as well as breeding attempts by this nomadic owl in seasons with high populations of small mammals and green winters, of course, if appropriate meadow habitat is still preserved. Ključne besede: močvirska uharica, pojavljanje, gnezdenje, erupcija, Ljubljansko barje Objavljeno v DiRROS: 26.07.2024; Ogledov: 146; Prenosov: 78
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342. Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraeaKatarina Karničar, Igor Drobnak, Marko Petek, Vasilka Magdevska, Jaka Horvat, Robert Vidmar, Špela Baebler, Ana Rotter, Polona Jamnik, Štefan Fujs, Boris Turk, Marko Fonović, Kristina Gruden, Gregor Kosec, Hrvoje Petković, 2016, izvirni znanstveni članek Povzetek: Background
Omics approaches have significantly increased our understanding of biological systems. However, they have had limited success in explaining the dramatically increased productivity of commercially important natural products by industrial high-producing strains, such as the erythromycin-producing actinomycete Saccharopolyspora erythraea. Further yield increase is of great importance but requires a better understanding of the underlying physiological processes.
Results
To reveal the mechanisms related to erythromycin yield increase, we have undertaken an integrated study of the genomic, transcriptomic, and proteomic differences between the wild type strain NRRL2338 (WT) and the industrial high-producing strain ABE1441 (HP) of S. erythraea at multiple time points of a simulated industrial bioprocess. 165 observed mutations lead to differences in gene expression profiles and protein abundance between the two strains, which were most prominent in the initial stages of erythromycin production. Enzymes involved in erythromycin biosynthesis, metabolism of branched chain amino acids and proteolysis were most strongly upregulated in the HP strain. Interestingly, genes related to TCA cycle and DNA-repair were downregulated. Additionally, comprehensive data analysis uncovered significant correlations in expression profiles of the erythromycin-biosynthetic genes, other biosynthetic gene clusters and previously unidentified putative regulatory genes. Based on this information, we demonstrated that overexpression of several genes involved in amino acid metabolism can contribute to increased yield of erythromycin, confirming the validity of our systems biology approach.
Conclusions
Our comprehensive omics approach, carried out in industrially relevant conditions, enabled the identification of key pathways affecting erythromycin yield and suggests strategies for rapid increase in the production of secondary metabolites in industrial environment. Ključne besede: aktinomicete, Saccharopolyspora erythraea, sekundarni metaboliti, antibiotiki, eritromicin, biosinteza, metabolno inženirstvo, proteomika Objavljeno v DiRROS: 25.07.2024; Ogledov: 177; Prenosov: 132
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343. Determination of the activity signature of key carbohydrate metabolism enzymes in phenolic-rich grapevine tissuesElizabeth Dunn Covington, Thomas Roitsch, Marina Dermastia, 2016, izvirni znanstveni članek Povzetek: Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenolsand polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and inter-fere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabo-lism, while based on our recently published one for quantitative measurement of activities using coupled spectrophoto-metric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grape-vine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteriašCandidatusPhytoplasma solani’, known to alter carbohydrate metabolism in grapevine. The described adaptations maybe useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inhe-rently high levels of phenolic compounds. Ključne besede: AGPase, carbohydrates, invertases, sucrose synthase, panel of enzyme activity assays, phytoplasma Objavljeno v DiRROS: 25.07.2024; Ogledov: 168; Prenosov: 98
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344. DNAqua-Net : developing new genetic tools for bioassessment and monitoring of aquatic ecosystems in EuropeFlorian Leese, Tina Eleršek, Cene Fišer, Ana Rotter, Bojana Žegura, Irena Maček, 2016, izvirni znanstveni članek Povzetek: The protection, preservation and restoration of aquatic ecosystems and their functions are of global importance. For European states it became legally binding mainly through the EU-Water Framework Directive (WFD). In order to assess the ecological status of a given water body, aquatic biodiversity data are obtained and compared to a reference water body. The quantified mismatch obtained determines the extent of potential management actions. The current approach to biodiversity assessment is based on morpho-taxonomy. This approach has many drawbacks such as being time consuming, limited in temporal and spatial resolution, and error-prone due to the varying individual taxonomic expertise of the analysts. Novel genomic tools can overcome many of the aforementioned problems and could complement or even replace traditional bioassessment. Yet, a plethora of approaches are independently developed in different institutions, thereby hampering any concerted routine application. The goal of this Action is to nucleate a group of researchers across disciplines with the task to identify gold-standard genomic tools and novel eco-genomic indices for routine application in biodiversity assessments of European fresh- and marine water bodies. Furthermore, DNAqua-Net will provide a platform for training of the next generation of European researchers preparing them for the new technologies. Jointly with water managers, politicians, and other stakeholders, the group will develop a conceptual framework for the standard application of eco-genomic tools as part of legally binding assessments. Ključne besede: aquatic ecosystems, biodiversity, monitoring, genomic tools Objavljeno v DiRROS: 25.07.2024; Ogledov: 157; Prenosov: 75
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345. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detectionDavid Dobnik, Dejan Štebih, Andrej Blejec, Dany Morisset, Jana Žel, 2016, izvirni znanstveni članek Povzetek: The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets. Ključne besede: digital PCR, DNA targets, GMO detection Objavljeno v DiRROS: 25.07.2024; Ogledov: 192; Prenosov: 116
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346. Enhanced detection of pathogenic enteric viruses in coastal marine environment by concentration using methacrylate monolithic chromatographic supports paired with quantitative PCRMukundh Narayanan Balasubramanian, Nejc Rački, José Gonçalves, Katarina Kovač, Magda Tušek-Žnidarič, Valentina Turk, Maja Ravnikar, Ion Gutiérrez-Aguirre, 2016, izvirni znanstveni članek Povzetek: Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10–100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM® C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods. Ključne besede: rotavirus, norovirus, seawater, fecal contamination, qPCR, sewage Objavljeno v DiRROS: 25.07.2024; Ogledov: 157; Prenosov: 87
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347. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosisAlison S. Devonshire, Jernej Pavšič, Mojca Milavec, Jana Žel, 2016, izvirni znanstveni članek Povzetek: Background
Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring.
Methods
To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories.
Results
dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude.
Conclusions
TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB. Ključne besede: digital PCR, diagnostics Objavljeno v DiRROS: 25.07.2024; Ogledov: 167; Prenosov: 102
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348. Dispersal similarly shapes both population genetics and community patterns in the marine realmGuillem Chust, Ernesto Villarino, Anne Chenuil, Xabier Irigoien, Nihayet Bizsel, Antonio Bode, Serena Fonda Umani, Patricija Mozetič, Ángel Borja, 2016, izvirni znanstveni članek Povzetek: Dispersal plays a key role to connect populations and, if limited, is one of the main processes to maintain and generate regional biodiversity. According to neutral theories of molecular evolution and biodiversity, dispersal limitation of propagules and population stochasticity are integral to shaping both genetic and community structure. We conducted a parallel analysis of biological connectivity at genetic and community levels in marine groups with different dispersal traits. We compiled large data sets of population genetic structure (98 benthic macroinvertebrate and 35 planktonic species) and biogeographic data (2193 benthic macroinvertebrate and 734 planktonic species). We estimated dispersal distances from population genetic data (i.e., FST vs. geographic distance) and from %-diversity at the community level. Dispersal distances ranked the biological groups in the same order at both genetic and community levels, as predicted by organism dispersal ability and seascape connectivity: macrozoobenthic species without dispersing larvae, followed by macrozoobenthic species with dispersing larvae and plankton (phyto- and zooplankton). This ranking order is associated with constraints to the movement of macrozoobenthos within the seabed compared with the pelagic habitat. We showed that dispersal limitation similarly determines the connectivity degree of communities and populations, supporting the predictions of neutral theories in marine biodiversity patterns. Ključne besede: sea, phytoplankton, dispersal, distribution, ocean sampling, Atlantic Ocean, Gulf of Trieste Objavljeno v DiRROS: 25.07.2024; Ogledov: 136; Prenosov: 130
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349. Solanum venturii, a suitable model system for virus-induced gene silencing studies in potato reveals StMKK6 as an important player in plant immunityDavid Dobnik, Ana Lazar, Tjaša Stare, Kristina Gruden, Vivianne G. A. A. Vleeshouwers, Jana Žel, 2016, izvirni znanstveni članek Povzetek: Background
Virus-induced gene silencing (VIGS) is an optimal tool for functional analysis of genes in plants, as the viral vector spreads throughout the plant and causes reduced expression of selected gene over the whole plant. Potato (Solanum tuberosum) is one of the most important food crops, therefore studies performing functional analysis of its genes are very important. However, the majority of potato cultivars used in laboratory experimental setups are not well amenable to available VIGS systems, thus other model plants from Solanaceae family are used (usually Nicotiana benthamiana). Wild potato relatives can be a better choice for potato model, but their potential in this field was yet not fully explored. This manuscript presents the set-up of VIGS, based on Tobacco rattle virus (TRV) in wild potato relatives for functional studies in potato–virus interactions.
Results
Five different potato cultivars, usually used in our lab, did not respond to silencing of phytoene desaturase (PDS) gene with TRV-based vector. Thus screening of a large set of wild potato relatives (different Solanum species and their clones) for their susceptibility to VIGS was performed by silencing PDS gene. We identified several responsive species and further tested susceptibility of these genotypes to potato virus Y (PVY) strain NTN and N. In some species we observed that the presence of empty TRV vector restricted the movement of PVY. Fluorescently tagged PVYN-GFP spread systemically in only five of tested wild potato relatives. Based on the results, Solanum venturii (VNT366-2) was selected as the most suitable system for functional analysis of genes involved in potato–PVY interaction. The system was tested by silencing two different plant immune signalling-related kinases, StWIPK and StMKK6. Silencing of StMKK6 enabled faster spreading of the virus throughout the plant, while silencing of WIPK had no effect on spreading of the virus.
Conclusions
The system employing S. venturii (VNT366-2) and PVYN-GFP is a suitable method for fast and simple functional analysis of genes involved in potato–PVY interactions. Additionally, a set of identified VIGS responsive species of wild potato relatives could serve as a tool for general studies of potato gene function. Ključne besede: potato, virus-induced gene silencing, VIGS, potato virus Y, PVY, Solanum venturii, StWIPK, StMKK6, TRV Objavljeno v DiRROS: 25.07.2024; Ogledov: 164; Prenosov: 152
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350. Metabolic consequences of infection of grapevine (Vitis vinifera L.) cv. ʺModra frankinjaʺ with flavescence dorée phytoplasmaNina Prezelj, Elizabeth Covington, Thomas Roitsch, Kristina Gruden, Lena Fragner, Wolfram Weckwerth, Marko Chersicola, Maja Vodopivec, Marina Dermastia, 2016, izvirni znanstveni članek Povzetek: Flavescence dorée, caused by the quarantine phytoplasma FDp, represents the most devastating of the grapevine yellows diseases in Europe. In an integrated study we have explored the FDp–grapevine interaction in infected grapevines of cv. “Modra frankinja” under natural conditions in the vineyard. In FDp-infected leaf vein-enriched tissues, the seasonal transcriptional profiles of 14 genes selected from various metabolic pathways showed an FDp-specific plant response compared to other grapevine yellows and uncovered a new association of the SWEET17a vacuolar transporter of fructose with pathogens. Non-targeted metabolome analysis from leaf vein-enriched tissues identified 22 significantly changed compounds with increased levels during infection. Several metabolites corroborated the gene expression study. Detailed investigation of the dynamics of carbohydrate metabolism revealed significant accumulation of sucrose and starch in the mesophyll of FDp-infected leaves, as well as significant up-regulation of genes involved in their biosynthesis. In addition, infected leaves had high activities of ADP-glucose pyrophosphorylase and, more significantly, sucrose synthase. The data support the conclusion that FDp infection inhibits phloem transport, resulting in accumulation of carbohydrates and secondary metabolites that provoke a source-sink transition and defense response status. Ključne besede: fructose utilization, gene expression, invertase, metabolome, soluble sugars, starch, sucrose synthase, SWEET, VvDMR6 Objavljeno v DiRROS: 25.07.2024; Ogledov: 178; Prenosov: 167
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