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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: 275; Downloads: 195
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Abstract: Colorado potato beetle (CPB) is an agricultural pest of solanaceous crops, notorious for its rapid resistance development to chemical pesticides. Foliar spraying of dsRNA formulations is a promising innovative technology providing highly specific and environmentally acceptable option for CPB management. We designed dsRNA to silence CPB mesh gene (dsMESH) and performed laboratory feeding trials to assess impacts on beetle survival and development. We compared the effectiveness of in vivo and in vitro produced dsRNA in a series of laboratory experiments. We additionally performed a field trial in which the efficacy of dsRNA sprayed onto potato foliage was compared to a spinosad-based insecticide. We showed that dsMESH ingestion consistently and significantly impaired larval growth and decreased larval survival in laboratory feeding experiments. In vivo produced dsRNA performed similarly as in vitro synthesized dsRNA in laboratory settings. In the field trial, dsMESH was as effective in controlling CPB larvae as a commercial spinosad insecticide, its activity was however slower. We discuss limitations and benefits of a potential dsMESH-based CPB management strategy and list some important RNAi based CPB research topics, which will have to be addressed in future.
Keywords: RNA interference, RNA interference feeding, ds RNA, gene silencing, RNAi pest control, survival analysis, field trial
Published in DiRROS: 22.07.2024; Views: 262; Downloads: 107
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Abstract: Fish farm-originating organic matter can modify the ecological processes in a benthic ecosystem. This was investigated in the sediments of the northern Adriatic Sea by measuring δ13C signature of nematodes, harpacticoids, and sedimentary organic matter, and by assessing pore water nutrients and bacterial composition. In a mesocosm experiment, 13C-labeled diatoms were added on top of sediment cores and 13C enrichment was measured as a proxy of diatom uptake by meiofauna. The δ13C signatures were depleted under fish farming cages compared to the reference site, as observed for sedimentary organic matter (−24.4‰ vs. −21.8‰), for nematodes (−22.5‰ vs. −17.7‰), and for harpacticoids (−25.3‰ vs. −20.8‰). The direct consumption of fish feed (−22.2‰) was not traced in meiofauna taxa. Nematodes from the farm site likely reflect a diet comprising sedimentary organic matter, as they were enriched by 2‰ relative to the sedimentary organic matter. The nematodes from the reference site were enriched by 4.2‰ relative to the sedimentary organic matter, which implies that they rely on more enriched food sources, like diatoms, which was confirmed by their uptake of 13C-labeled diatoms. The nematode assemblage incorporated more diatom 13C than harpacticoids, making them more important players in the carbon flux from diatoms to higher trophic levels at the reference site. Harpacticoids from the reference site were enriched by 1.1‰ compared to sedimentary organic matter, implying that this was their primary food source. Harpacticoids from the farm site were depleted by 0.9‰ relative to the sedimentary organic matter, indicating they were influenced by a very depleted food source like bacteria. Harpacticoids from both the cage and reference sites consumed 13C-labeled diatoms, which implies their diet might span a broad δ13C range, from bacteria to diatoms. Pore water nutrients with high dissolved inorganic carbon, phosphate, and ammonium concentration indicated an elevated microbial degradation of organic compounds under the fish farm. The denaturing gradient gel electrophoresis analysis showed a 70% similarity between sediment bacteria communities from the fish farm and reference site. The study demonstrated that fish farm-originating organic matter enters the meiofauna food chain, and that nematodes and harpacticoids use different food sources under the fish farm and at the reference site.
Published in DiRROS: 22.07.2024; Views: 239; Downloads: 163
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Abstract: Senescence has been studied since a long time by theoreticians in ecology and evolution, but empirical support in natural population has only recently been accumulating. One of the current challenges is the investigation of senescence of multiple fitness components and the study of differences between sexes. Until now, studies have been more frequently conducted on females than on males and rather in long-lived than in short-lived species. To reach a more fundamental understanding of the evolution of senescence, it is critical to investigate age-specific survival and reproduction performance in both sexes and in a large range of species with contrasting life histories. In this study, we present results on patterns of age-specific and sex-specific variation in survival and reproduction in the whinchat Saxicola rubetra, a short-lived passerine. We compiled individual-based long-term datasets from seven populations that were jointly analyzed within a Bayesian modeling framework. We found evidence for senescence in survival with a continuous decline after the age of 1 year, but no evidence of reproductive senescence. Furthermore, we found no clear evidence for sex effects on these patterns. We discuss these results in light of previous studies documenting senescence in short-lived birds. We note that most of them have been conducted in populations breeding in nest boxes, and we question the potential effect of the nest boxes on the shape of age-reproductive trajectories.
Keywords: actuarial senescence, age-specific demographic rate, aging, Saxicola rubetra, whinchat
Published in DiRROS: 22.07.2024; Views: 242; Downloads: 138
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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: 268; Downloads: 81
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