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1042. Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leavesNataša Mehle, Polona Kogovšek, Nejc Rački, Tjaša Jakomin, Ion Gutiérrez-Aguirre, Petra Kramberger, Maja Ravnikar, 2017, original scientific article Abstract: Waterborne and seedborne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT-qPCR results with sequencing of RT-PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds.
Keywords: PSTVd, PepMV, seeds, water, concentration, loop-mediated isothermal amplification
Published in DiRROS: 24.07.2024; Views: 355; Downloads: 163
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1044. 1-aminocyclopropane-1-carboxylate oxidase induction in tomato flower pedicel phloem and abscission related processes are differentially sensitive to ethyleneMarko Chersicola, Aleš Kladnik, Magda Tušek-Žnidarič, Tanja Mrak, Kristina Gruden, Marina Dermastia, 2017, original scientific article Abstract: Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.
Keywords: abscission, ACO, cell separation, ethylene, laser microdissection, programmed cell death, tomato, ultrastructure
Published in DiRROS: 24.07.2024; Views: 265; Downloads: 320
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1047. Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell linesBarbara Breznik, Helena Motaln, Miloš Vittori, Ana Rotter, Tamara Lah Turnšek, 2017, original scientific article Abstract: Glioblastoma multiforme are an aggressive form of brain tumors that are characterized by distinct invasion of single glioblastoma cells, which infiltrate the brain parenchyma. This appears to be stimulated by the communication between cancer and stromal cells. Mesenchymal stem cells (MSCs) are part of the glioblastoma microenvironment, and their ‘cross-talk’ with glioblastoma cells is still poorly understood. Here, we examined the effects of bone marrow-derived MSCs on two different established glioblastoma cell lines U87 and U373. We focused on mutual effects of direct MSC/glioblastoma contact on cellular invasion in three-dimensional invasion assays in vitro and in a zebrafish embryo model in vivo. This is the first demonstration of glioblastoma cell-type-specific responses to MSCs in direct glioblastoma co-cultures, where MSCs inhibited the invasion of U87 cells and enhanced the invasion of U373. Inversely, direct cross-talk between MSCs and both of glioblastoma cell lines enhanced MSC motility. MSC-enhanced invasion of U373 cells was assisted by overexpression of proteases cathepsin B, calpain1, uPA/uPAR, MMP-2, -9 and -14, and increased activities of some of these proteases, as determined by the effects of their selective inhibitors on invasion. In contrast, these proteases had no effect on U87 cell invasion under MSC co-culturing. Finally, we identified differentially expressed genes, in U87 and U373 cells that could explain different response of these cell lines to MSCs. In conclusion, we demonstrated that MSC/glioblastoma cross-talk is different in the two glioblastoma cell phenotypes, which contributes to tumor heterogeneity.
Keywords: glioblastoma multiforme, proteases, mesenchymal stem cells, tumor heterogeneity, zebrafish model
Published in DiRROS: 24.07.2024; Views: 304; Downloads: 203
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1049. Pridobivanje in uporaba ekstraktivov iz lesa in drevesne skorjePrimož Oven, Ida Poljanšek, Urša Osolnik, Viljem Vek, 2024, original scientific article Abstract: Ekstraktivi so nizkomolekularne spojine, ki so v vseh rastlinskih tkivih, tudi v lesu in drevesni skorji. V drevesu imajo pomembne ekološke in fiziološke funkcije, obenem pa predstavljajo bioosnovane proizvode z visoko dodano vrednostjo, ki jih je mogoče pridobivati tudi iz lesa slabše kakovosti in biomasnih ostankov gozdnih lesnih verig. V prispevku bomo ekstraktive razvrstili, pojasnili, kakšna je kakovostna in količinska spremenljivost po posameznih tkivih izbranih drevesnih vrst, predstavili bomo načine pridobivanja in področja uporabe. Ocenjujemo, da je pridobivanje ekstraktivov dokaj enostaven tehnološki postopek, ki bi bil v obliki manjše biorafinerije izvedljiv v lokalnih okoljih, kjer so na voljo zadostne količine lesne biomase.
Keywords: les, skorja, ekstraktivi, kaskadna raba, biorafinacija
Published in DiRROS: 24.07.2024; Views: 313; Downloads: 80
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1050. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNAJernej Pavšič, Alison S. Devonshire, Andrej Blejec, Carole A. Foy, Fran Van Heuverswyn, Gerwyn M. Jones, Heinz Schimmel, Jana Žel, Jim F. Huggett, Nicholas Redshaw, Maria Karczmarczyk, Erkan Mozioglu, Sema Akyürek, Müslüm Akgöz, Mojca Milavec, 2017, original scientific article Abstract: Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.
Keywords: digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials
Published in DiRROS: 24.07.2024; Views: 359; Downloads: 210
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