371. Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brainMiloš Vittori, Barbara Breznik, Tajda Gredar, Katja Hrovat, Lilijana Bizjak-Mali, Tamara Lah Turnšek, 2016, izvirni znanstveni članek Povzetek: Background
An attractive approach in the study of human cancers is the use of transparent zebrafish (Danio rerio) embryos, which enable the visualization of cancer progression in a living animal.
Materials and methods
We implanted mixtures of fluorescently labeled glioblastoma (GBM) cells and bonemarrow-derived mesenchymal stem cells (MSCs) into zebrafish embryos to study the cellular pathways of their invasion and the interactions between these cells in vivo.
Results
By developing and applying a carbocyanine-dye-compatible clearing protocol for observation of cells in deep tissues, we showed that U87 and U373 GBM cells rapidly aggregated into tumor masses in the ventricles and midbrain hemispheres of the zebrafish embryo brain, and invaded the central nervous system, often using the ventricular system and the central canal of the spinal cord. However, the GBM cells did not leave the central nervous system. With co-injection of differentially labeled cultured GBM cells and MSCs, the implanted cells formed mixed tumor masses in the brain. We observed tight associations between GBM cells and MSCs, and possible cell-fusion events. GBM cells and MSCs used similar invasion routes in the central nervous system.
Conclusions
This simple model can be used to study the molecular pathways of cellular processes in GBM cell invasion, and their interactions with various types of stromal cells in double or triple cell co-cultures, to design anti-GBM cell therapies that use MSCs as vectors. Ključne besede: brain tumors, tumor microenvironment, animal models, xenotransplantation Objavljeno v DiRROS: 25.07.2024; Ogledov: 154; Prenosov: 154
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372. Phytoplankton across Tropical and Subtropical Regions of the Atlantic, Indian and Pacific oceansMarta Estrada, Maximino Delgado, Dolores Blasco, Mikel Latasa, Ana Maria Cabello, Verónica Benitez-Barrios, Eugenio Fraile-Nuez, Patricija Mozetič, Monteserrat Vidal, 2016, izvirni znanstveni članek Povzetek: We examine the large-scale distribution patterns of the nano- and microphytoplankton collected from 145 oceanic stations, at 3 m depth, the 20% light level and the depth of the sub-surface chlorophyll maximum, during the Malaspina-2010 Expedition (December 2010-July 2011), which covered 15 biogeographical provinces across the Atlantic, Indian and Pacific oceans, between 35°N and 40°S. In general, the water column was stratified, the surface layers were nutrient-poor and the nano- and microplankton (hereafter phytoplankton, for simplicity, although it included also heterotrophic protists) community was dominated by dinoflagellates, other flagellates and coccolithophores, while the contribution of diatoms was only important in zones with shallow nutriclines such as the equatorial upwelling regions. We applied a principal component analysis to the correlation matrix among the abundances (after logarithmic transform) of the 76 most frequent taxa to synthesize the information contained in the phytoplankton data set. The main trends of variability identified consisted of: 1) A contrast between the community composition of the upper and the lower parts of the euphotic zone, expressed respectively by positive or negative scores of the first principal component, which was positively correlated with taxa such as the dinoflagellates Oxytoxum minutum and Scrippsiella spp., and the coccolithophores Discosphaera tubifera and Syracosphaera pulchra (HOL and HET), and negatively correlated with taxa like Ophia-ster hydroideus (coccolithophore) and several diatoms, 2) a general abundance gradient between phytoplankton-rich regions with high abundances of dinoflagellate, coccolitho-phore and ciliate taxa, and phytoplankton-poor regions (second principal component), 3) differences in dominant phytoplankton and ciliate taxa among the Atlantic, the Indian and the Pacific oceans (third principal component) and 4) the occurrence of a diatom-dominated assemblage (the fourth principal component assemblage), including several pennate taxa, Planktoniella sol, Hemiaulus hauckii and Pseudo-nitzschia spp., in the divergence regions. Ključne besede: sea, phytoplankton, research programmes, ocean expeditions, Malaspina 2010, distribution, ocean sampling, Atlantic Ocean, Pacific, Indian Ocean Objavljeno v DiRROS: 25.07.2024; Ogledov: 278; Prenosov: 209
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373. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerasesLydia J. R. Hunter, Samuel F. Brockington, Alex M. Murphy, Adrienne E. Pate, Kristina Gruden, Stuart A. MacFarlane, Peter Palukaitis, John P. Carr, 2016, izvirni znanstveni članek Povzetek: Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7. Ključne besede: RNA polymerases, RNA, potato Objavljeno v DiRROS: 25.07.2024; Ogledov: 251; Prenosov: 171
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374. Primary metabolism, phenylpropanoids and antioxidant pathways are regulated in potato as a response to Potato virus Y infectionPolona Kogovšek, Maruša Pompe Novak, Marko Petek, Lena Fragner, Wolfram Weckwerth, Kristina Gruden, 2016, izvirni znanstveni članek Povzetek: Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus—plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed. Objavljeno v DiRROS: 25.07.2024; Ogledov: 178; Prenosov: 174
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375. Improved protective effect of umbilical cord stem cell transplantation on cisplatin-induced kidney injury in mice pretreated with antithymocyte globulinŽeljka Večerić-Haler, Andreja Erman, Anton Cerar, Helena Motaln, Katja Kološa, Tamara Lah Turnšek, Snežna Sodin-Šemrl, Katja Lakota, Katjuša Mrak Poljšak, Špela Škrajnar, Simona Kranjc Brezar, Miha Arnol, Martina Perše, 2016, izvirni znanstveni članek Povzetek: Mesenchymal stem cells (MSCs) are recognised as a promising tool to improve renal recovery in experimental models of cisplatin-induced acute kidney injury. However, these preclinical studies were performed on severely immunodeficient animals. Here, we investigated whether human umbilical cord derived MSC treatment could equally ameliorate acute kidney injury induced by cisplatin and prolong survival in mice with a normal immune system and those with a suppressed immune system by polyclonal antithymocyte globulin (ATG). We demonstrated that ATG pretreatment, when followed by MSC transplantation, significantly improved injured renal function parameters, as evidenced by decreased blood urea nitrogen and serum creatinine concentration, as well as improved renal morphology. This tissue restoration was also supported by increased survival of mice. The beneficial effects of ATG were associated with reduced level of inflammatory protein serum amyloid A3 and induced antioxidative expression of superoxide dismutase-1 (SOD-1), glutathione peroxidase (GPx), and hem oxygenase-1 (HO-1). Infused MSCs became localised predominantly in peritubular areas and acted to reduce renal cell death. In conclusion, these results show that ATG diminished in situ inflammation and oxidative stress associated with cisplatin-induced acute kidney injury, the effects that may provide more favourable microenvironment for MSC action, with consequential synergistic improvements in renal injury and animal survival as compared to MSC treatment alone. Ključne besede: mesenchymal stem cells, nephrotoxicity Objavljeno v DiRROS: 25.07.2024; Ogledov: 167; Prenosov: 166
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376. Use of hydrodynamic cavitation in (waste)water treatmentMatevž Dular, Tjaša Griessler Bulc, Ion Gutiérrez-Aguirre, Ester Heath, Tina Kosjek, Aleksandra Krivograd-Klemenčič, Martina Oder, Martin Petkovšek, Nejc Rački, Maja Ravnikar, Andrej Šarc, Brane Širok, Mojca Zupanc, Miha Žitnik, Boris Kompare, 2016, izvirni znanstveni članek Povzetek: The use of acoustic cavitation for water and wastewater treatment (cleaning) is a well known procedure. Yet, the use of hydrodynamic cavitation as a sole technique or in combination with other techniques such as ultrasound has only recently been suggested and employed.
In the first part of this paper a general overview of techniques that employ hydrodynamic cavitation for cleaning of water and wastewater is presented.
In the second part of the paper the focus is on our own most recent work using hydrodynamic cavitation for removal of pharmaceuticals (clofibric acid, ibuprofen, ketoprofen, naproxen, diclofenac, carbamazepine), toxic cyanobacteria (Microcystis aeruginosa), green microalgae (Chlorella vulgaris), bacteria (Legionella pneumophila) and viruses (Rotavirus) from water and wastewater.
As will be shown, hydrodynamic cavitation, like acoustic, can manifest itself in many different forms each having its own distinctive properties and mechanisms. This was until now neglected, which eventually led to poor performance of the technique. We will show that a different type of hydrodynamic cavitation (different removal mechanism) is required for successful removal of different pollutants.
The path to use hydrodynamic cavitation as a routine water cleaning method is still long, but recent results have already shown great potential for optimisation, which could lead to a low energy tool for water and wastewater cleaning. Ključne besede: wastewater, cleaning, hydrodynamic cavitation, pharmaceuticals, cyanobacteria, microalgae, viruses, Legionella bacteria Objavljeno v DiRROS: 25.07.2024; Ogledov: 140; Prenosov: 76
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377. Assessment of the real-time PCR and different digital PCR platforms for DNA quantificationJernej Pavšič, Jana Žel, Mojca Milavec, 2016, izvirni znanstveni članek Povzetek: Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method. Ključne besede: real-time PCR, molecular diagnostics, human cytomegalovirus, DNA quantification, digital PCR Objavljeno v DiRROS: 25.07.2024; Ogledov: 179; Prenosov: 121
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378. Digital PCR for direct quantification of viruses without DNA extractionJernej Pavšič, Jana Žel, Mojca Milavec, 2016, izvirni znanstveni članek Povzetek: DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification. Ključne besede: molecular diagnostics, direct quantification, viruses, virus reference materials, human cytomegalovirus, digital PCR Objavljeno v DiRROS: 25.07.2024; Ogledov: 168; Prenosov: 141
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379. Assessment of short-term effect of platelet-rich plasma treatment of tendinosis using texture analysis of ultrasound imagesKarlo Pintarić, Vladka Salapura, Žiga Snoj, Andrej Vovk, Mojca Božič, Jernej Vidmar, 2023, izvirni znanstveni članek Ključne besede: texture analysis, ultrasonography, tendinosis Objavljeno v DiRROS: 25.07.2024; Ogledov: 157; Prenosov: 69
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