521. Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cellsNeža Podergajs, Helena Motaln, Uroš Rajčević, Urška Verbovšek, Marjan Koršič, Nina Obad, Heidi Espedal, Miloš Vittori, Christel Herold-Mende, Hrvoje Miletic, Rolf Bjerkvig, Tamara Lah Turnšek, 2016, original scientific article Abstract: The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment. Keywords: biomarker, CD9, glioblastoma stem cells, neural stem cells, tetraspanin Published in DiRROS: 26.07.2024; Views: 210; Downloads: 132
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522. Velikost populacije prepelice Coturnix coturnix na Ljubljanskem barju se je v dvajsetih letih zmanjšala za polovico, morda pa še za (bistveno?) večDavorin Tome, Al Vrezec, Špela Ambrožič Ergaver, Andrej Kapla, 2016, original scientific article Abstract: In 2015, we used the same method as in the 1989-1996 period to count calling males of the Common Quail Coturnix coturnix in selected 1x1 km squares of Ljubljansko barje (central Slovenia). We counted 39 males, which is 87% less compared to the survey 20 years ago. Quails were found in 56% fewer squares. Since Quail populations are known to fluctuate greatly between years, we think that the most realistic long-term estimate for population decline would be somewhere between 50 to 90%. We detected that within only one week males stopped calling in some squares, while in others they began calling although they had not been detected there before, indicating possible relocations. This dynamics should be considered when designing a population monitoring protocol for the Quail. Keywords: travniki, upad populacije, prepelica, Ljubljansko barje, monitoring Published in DiRROS: 26.07.2024; Views: 632; Downloads: 95
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523. Obročkanje ptic v Sloveniji leta 2015 in pojav velikih krivokljunov Loxia pytyopsittacusAl Vrezec, Dare Fekonja, 2016, original scientific article Abstract: In 2015, 170 bird species were recorded during bird ringing activities in Slovenia. We ringed 73,371 birds belonging to 162 species, there were 132 foreign recoveries of birds ringed in Slovenia, 120 recoveries of birds ringed abroad and found in Slovenia, as well as 1964 local recoveries. The most frequently ringed species were Blackcap Sylvia atricapilla and Great Tit Parus major. In ringed nestlings, Great Tits and Tree Sparrows Passer montanus predominated. In 2015, the first preliminary ringing of Scops Owls Otus scops during migration took place, resulting in the highest number of Scops Owls ringed so far. Concerning recoveries of birds ringed in Slovenia and later recorded abroad and birds ringed abroad and later recorded in Slovenia, the commonest were Mute Swans Cygnus olor and Black-headed Gulls Chroicocephalus ridibundus. The longest-distance recovery concerned a Reed Bunting Emberiza schoeniculus found in Sweden (2,144 km away). Among the interesting finds were also the first finds of ringed Pygmy Cormorants Microcarbo pygmeus so far from breeding sites in Hungary. Among rare species, Yellow-browed Warbler Phylloscopus inornatus, Spanish Sparrow Passer hispaniolensis and a pair of Parrot Crossbills Loxia pytyopsittacus were caught and ringed, the latter for the very first time in Slovenia after more than 100 years. An overview of records of the Parrot Crossbill is given herein, as well as analysis of irruptive years of the Red Crossbills Loxia curvirostra between 1980 and 2015, when the probability of boreal Crossbill species occurrence is the highest. According to the ringers’ data, the irruptive years of Red Crossbills in Slovenia were 1984, 1985, 2007, 2008, 2010, 2011 and 2012. The article points to the probability that Parrot Crossbills have been overlooked in the past, since larger specimens of Crossbills were ringed mostly in irruptive years, but no specific bill measurements important for distinguishing between Parrot and Red Crossbill had been taken. Keywords: obročkanje, najdbe, Slovenija, 2015, veliki krivokljun Published in DiRROS: 26.07.2024; Views: 213; Downloads: 99
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524. Pregled pojavljanja močvirske uharice Asio flammeus v Sloveniji med letoma 1995 in 2015 ter verjetno gnezdenje v eruptivnem letu 2008Al Vrezec, 2016, original scientific article Abstract: 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. Keywords: močvirska uharica, pojavljanje, gnezdenje, erupcija, Ljubljansko barje Published in DiRROS: 26.07.2024; Views: 167; Downloads: 92
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525. 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, original scientific article Abstract: 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. Keywords: aktinomicete, Saccharopolyspora erythraea, sekundarni metaboliti, antibiotiki, eritromicin, biosinteza, metabolno inženirstvo, proteomika Published in DiRROS: 25.07.2024; Views: 208; Downloads: 152
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526. Determination of the activity signature of key carbohydrate metabolism enzymes in phenolic-rich grapevine tissuesElizabeth Dunn Covington, Thomas Roitsch, Marina Dermastia, 2016, original scientific article Abstract: 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. Keywords: AGPase, carbohydrates, invertases, sucrose synthase, panel of enzyme activity assays, phytoplasma Published in DiRROS: 25.07.2024; Views: 189; Downloads: 116
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527. 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, original scientific article Abstract: 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. Keywords: aquatic ecosystems, biodiversity, monitoring, genomic tools Published in DiRROS: 25.07.2024; Views: 188; Downloads: 92
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528. 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, original scientific article Abstract: 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. Keywords: digital PCR, DNA targets, GMO detection Published in DiRROS: 25.07.2024; Views: 228; Downloads: 130
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529. 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, original scientific article Abstract: 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. Keywords: rotavirus, norovirus, seawater, fecal contamination, qPCR, sewage Published in DiRROS: 25.07.2024; Views: 206; Downloads: 114
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530. 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, original scientific article Abstract: 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. Keywords: digital PCR, diagnostics Published in DiRROS: 25.07.2024; Views: 200; Downloads: 115
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