71. 2D and 3D in vitro assays to quantify the invasive behavior of glioblastoma stem cells in response to SDF-1[alpha]Vashendriya V. V. Hira, Barbara Breznik, Cornelis J. F. van Noorden, Tamara Lah Turnšek, Remco J. Molenaar, 2020, original scientific article Abstract: Invasion is a hallmark of cancer and therefore in vitro invasion assays are important tools in cancer research. We aimed to describe in vitro 2D transwell assays and 3D spheroid assays to quantitatively determine the invasive behavior of glioblastoma stem cells in response to the chemoattractant SDF-1α. Matrigel was used as a matrix in both assays. We demonstrated quantitatively that SDF-1α increased invasive behavior of glioblastoma stem cells in both assays. We conclude that the 2D transwell invasion assay is easy to perform, fast and less complex whereas the more time-consuming 3D spheroid invasion assay is physiologically closer to the in vivo situation.
Keywords: 2D transwell invasion assay, 3D spheroid invasion assay, cancer cell, cellular invasion, glioblastoma stem cells
Published in DiRROS: 22.07.2024; Views: 212; Downloads: 170
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72. CCR5-mediated signaling is involved in invasion of glioblastoma cells in its microenvironmentMetka Novak, Miha Koprivnikar Krajnc, Barbara Hrastar, Barbara Breznik, Bernarda Majc, Mateja Mlinar, Ana Rotter, Andrej Porčnik, Jernej Mlakar, Katja Stare, Richard G. Pestell, Tamara Lah Turnšek, 2020, original scientific article Abstract: Abstract
The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc.
Keywords: CCL5, CCR5, chemokines, glioblastoma, invasion, maraviroc, mesenchymal stem cells
Published in DiRROS: 22.07.2024; Views: 207; Downloads: 43
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73. Ecological patterns of polychaete assemblages associated with the Mediterranean stony coral Cladocora caespitosa (Linnaeus, 1767) : a comparison of sites in two biogeographic zones (Adriatic and Aegean Sea)Valentina Pitacco, Giorgos Chatzigeorgiou, Barbara Mikac, Lovrenc Lipej, 2021, original scientific article Abstract: The Mediterranean stony coral Cladocora caespitosa (Linnaeus, 1767) is a well-known habitat builder, and as such hosts a diversified faunal assemblage. Although polychaetes are one of the most abundant and diverse macrobenthic groups associated with C. caespitosa colonies, our knowledge of their ecological features in this association is still limited. The aim of this paper was to gather and compare the most comprehensive data available on polychaetes associated with C. caespitosa in the Adriatic and the Aegean Seas, and to test for differences between these geographic areas. To this end, differences were tested in terms of: (i) richness and structure of polychaete assemblages; (ii) feeding and functional traits of assemblages; (iii) the main factors influencing those aspects, (iv) the relationship between polychaete assemblages richness and Cladocora colony size, and estimate richness. Differences were observed between the Adriatic and the Aegean Seas, in terms of richness, species composition and relative proportion of the dominant feeding guild (filter feeders most abundant in the Aegean and carnivores in the Adriatic) and motility mode (sessile most abundant in the Aegean and motile in the Adriatic). Conversely, cosmopolitan and Atlanto-Mediterranean species dominated the assemblages in both geographic areas, and the same Species-Area Relation model proved to be effective for richness estimation in both geographic areas.
Keywords: benthic ecology, habitat builder species, polychaeta, Mediterranean Sea
Published in DiRROS: 19.07.2024; Views: 180; Downloads: 71
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74. Feasibility of droplet digital PCR analysis of plasma cell-free DNA from kidney transplant patientsBarbara Jerič Kokelj, Maja Štalekar, Sebastian Vencken, David Dobnik, Polona Kogovšek, Matjaž Stanonik, Miha Arnol, Maja Ravnikar, 2021, original scientific article Abstract: Increasing research demonstrates the potential of donor-derived cell-free DNA (dd-cfDNA) as a biomarker for monitoring the health of various solid organ transplants. Several methods have been proposed for cfDNA analysis, including real-time PCR, digital PCR, and next generation sequencing-based approaches. We sought to revise the droplet digital PCR (ddPCR)-based approach to quantify relative dd-cfDNA in plasma from kidney transplant (KTx) patients using a novel pilot set of assays targeting single nucleotide polymorphisms that have a very high potential to distinguish cfDNA from two individuals. The assays are capable of accurate quantification of down to 0.1% minor allele content when analyzing 165 ng of human DNA. We found no significant differences in the yield of extracted cfDNA using the three different commercial kits tested. More cfDNA was extracted from the plasma of KTx patients than from healthy volunteers, especially early after transplantation. The median level of donor-derived minor alleles in KTx samples was 0.35%. We found that ddPCR using the evaluated assays within specific range is suitable for analysis of KTx patientsʼ plasma but recommend prior genotyping of donor DNA and performing reliable preamplification of cfDNA.
Keywords: kidney transplantation, droplet digital PCR, plasma cell-free DNA, minor allele quantification, assay evaluation, graft health monitoring
Published in DiRROS: 19.07.2024; Views: 239; Downloads: 113
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75. TRIM28 selective nanobody reduces glioblastoma stem cell invasionAndrej Porčnik, Metka Novak, Barbara Breznik, Bernarda Majc, Barbara Hrastar, Neja Šamec, Alja Zottel, Ivana Jovchevska, Miloš Vittori, Ana Rotter, Radovan Komel, Tamara Lah Turnšek, 2021, original scientific article Abstract: Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool.
Published in DiRROS: 19.07.2024; Views: 174; Downloads: 158
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76. Metagenomic characterization of parental and production CHO cell lines for detection of adventitious virusesKatarina Bačnik, Denis Kutnjak, Barbara Jerič Kokelj, Nika Tuta, Tan Lončar, Matjaž Vogelsang, Maja Ravnikar, 2021, original scientific article Abstract: Viral contamination is a major concern for biological products. Therefore, virus testing of raw materials and cells is essential for the safety of the final product. We used high-throughput sequencing to detect viral-like sequences in selected CHO cell lines. Our aim was to test various approaches of sample preparation, to establish a pipeline for metagenomic analysis and to characterize standard viral metagenome of production and parental CHO cell lines. The comparison of the metagenomics composition of the differently prepared samples showed that among four tested approaches sequencing of ribosomal RNA depleted total RNA is the most promising approach. The metagenomics investigation of one production and three parental CHO cell lines of diverse origin did not indicate the presence of adventitious viral agents in the investigated samples. The study revealed an expected background of virus-like nucleic acids in the samples, which originate from remains of expression vectors, endogenized viral elements and residuals of bacteriophages.
Published in DiRROS: 19.07.2024; Views: 363; Downloads: 131
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77. The cytotoxic effects of cannabidiol and cannabigerol on glioblastoma stem cells may mostly involve GPR55 and TRPV1 signallingTamara Lah Turnšek, Bernarda Majc, Metka Novak, Ajda Sušnik, Barbara Breznik, Andrej Porčnik, Roman Bošnjak, Aleksander Sadikov, Marta Malavolta, Selma Halilčević, Jernej Mlakar, Roby Zomer, 2022, original scientific article Abstract: Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60–70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy.
Keywords: glioblastoma, glioma, cannabigerol, cannabidiol, cannabinoid receptors, stem cells
Published in DiRROS: 18.07.2024; Views: 216; Downloads: 154
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78. Poročilo o preskusu št.: LVG 2024-067 : vzorec št. 2024/00318Nikica Ogris, Špela Hočevar, Zina Devetak, Barbara Piškur, 2024, expertise, arbitration decision Keywords: varstvo gozdov, morfološke analize, program preiskav, Fusarium circinatum, Pinus, borov smolasti rak, PCR
Published in DiRROS: 17.07.2024; Views: 216; Downloads: 0
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79. What did we achieve with VALITEST an EU project on validation in plant pest diagnostics?Charlotte Trontin, Barbara Agstner, Denise Altenbach, Géraldine Anthoine, Hanna Baginska, Ian Brittain, A Chabirand, Anne-Marie Chappé, P. Dahlin, Tanja Dreo, C. Freye-Minks, Camilo Gianinazzi, Catherine Harrison, Glyn Jones, Marco Stefan Kaiser, Marta Luigi, Sébastien Massart, Nataša Mehle, Monica Mezzalama, Hanna Mouaziz, Maja Ravnikar, Tom Raaymakers, Jean-Philippe Renvoise, Mathieu Rolland, Marta Santos, Sam Seddas, René van der Vlugt, Ana Vučurović, Françoise Petter, 2022, original scientific article Keywords: plant pest diagnostics, validation, test performance study, high-throughput, sequencing, reference material, training
Published in DiRROS: 17.07.2024; Views: 232; Downloads: 100
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80. Synthetic cannabinoid WIN 55,212–2 inhibits growth and induces cell death of oral and pancreatic stem-like/poorly differentiated tumor cellsMeng-Wei Ko, Barbara Breznik, Emanuela Senjor, Anahid Jewett, 2022, original scientific article Abstract: We report here that synthetic cannabinoid WIN 55,212–2 inhibits tumor cell proliferation and induces cell death of oral and pancreatic tumor cells, and the effect is much more pronounced on stem-like/poorly differentiated OSCSCs and MP2 cells when compared to well-differentiated OSCCs, and PL-12 tumor cells. In addition, WIN 55,212-2 decreases cell surface expression of CD44, CD54, MHC class I and PD-L1 on oral and pancreatic tumor cells with the exception of PD-L1 expression on well-differentiated PL-12 pancreatic tumor cells which exhibits an increase in the expression rather than a decrease. Overall, we demonstrate that WIN 55,212-2 has an increased targeting activity against cancer stem cells/poorly differentiated oral and pancreatic tumor cells when compared to well-differentiated tumor cells, and furthermore, such differences in function do not correlate with the levels of CB1 and CB2 receptor expression on tumor cells, suggesting it's function either through post-receptor mediated activation and/or yet-to-be identified novel receptors. Intraperitoneal (IP) delivery of WIN 55-212-2 in humanized BLT mice is found to impart an activating potential for NK cells demonstrating increased NK cell mediated cytotoxicity and secretion of IFN-γ in our preliminary experiments. These results not only suggest a direct targeting of CSCs/poorly differentiated tumors by WIN 55-212-2 but also by indirect targeting of such tumors through the activation and increased functions of NK cells.
Keywords: cancer stem cells, cannabinoids, cell death, cancer biology, genetic toxicology
Published in DiRROS: 17.07.2024; Views: 205; Downloads: 137
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