1311. Lethal and sub-lethal effects and modulation of gene expression induced by T kinase inhibitors in zebrafish (Danio Rerio) embryosTina Eleršek, Matjaž Novak, Mateja Mlinar, Igor Virant, Nika Bahor, Karin Leben, Bojana Žegura, Metka Filipič, 2022, original scientific article Abstract: Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted.
Keywords: aquatic toxicity, tyrosine kinase inhibitors, zebrafish embryo toxicity test, gene expression, environmental hazard
Published in DiRROS: 16.07.2024; Views: 306; Downloads: 196
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1312. Hydrodynamic cavitation efficiently inactivates potato virus Y in waterArijana Filipić, Tadeja Lukežič, Katarina Bačnik, Maja Ravnikar, Meta Ješelnik, Tamara Košir, Martin Petkovšek, Mojca Zupanc, Matevž Dular, Ion Gutiérrez-Aguirre, 2022, original scientific article Abstract: Waterborne plant viruses can destroy entire crops, leading not only to high financial losses but also to food shortages. Potato virus Y (PVY) is the most important potato viral pathogen that can also affect other valuable crops. Recently, it has been confirmed that this virus is capable of infecting host plants via water, emphasizing the relevance of using proper strategies to treat recycled water in order to prevent the spread of the infectious agents. Emerging environmentally friendly methods such as hydrodynamic cavitation (HC) provide a great alternative for treating recycled water used for irrigation. In the experiments conducted in this study, laboratory HC based on Venturi constriction with a sample volume of 1 L was used to treat water samples spiked with purified PVY virions. The ability of the virus to infect plants was abolished after 500 HC passes, corresponding to 50 min of treatment under pressure difference of 7 bar. In some cases, shorter treatments of 125 or 250 passes were also sufficient for virus inactivation. The HC treatment disrupted the integrity of viral particles, which also led to a minor damage of viral RNA. Reactive species, including singlet oxygen, hydroxyl radicals, and hydrogen peroxide, were not primarily responsible for PVY inactivation during HC treatment, suggesting that mechanical effects are likely the driving force of virus inactivation. This pioneering study, the first to investigate eukaryotic virus inactivation by HC, will inspire additional research in this field enabling further improvement of HC as a water decontamination technology.
Keywords: hydrodynamic cavitation, potato virus Y, virus inactivation, water decontamination
Published in DiRROS: 16.07.2024; Views: 314; Downloads: 212
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1313. Differential responses of coexisting owls to annual small mammal population fluctuations in temperate mixed forestUrška Ratajc, Martin Breskvar, Sašo Džeroski, Al Vrezec, 2022, original scientific article Abstract: Montane temperate forests in central and southern Europe host diverse small mammal assemblages, but the fluctuations in these assemblages in correlation with owl predators are still poorly explored. The key questions of our study were how coexisting owls responded to different prey fluctuations and whether any particular small mammal species governed predator–prey co-dynamics. We conducted a long-term study (2004–2020) in low-elevation (300–1100 m above sea level) mixed Beech and Silver Fir forest in the northern Dinaric Alps (central Slovenia). Monitoring data on the main small mammal groups – mice Muridae, voles Cricetidae, dormice Gliridae and shrews Soricidae – and three owl species – the Ural Owl Strix uralensis, Tawny Owl Strix aluco and Boreal Owl Aegolius funereus – were collected annually. To find relationships between prey and predator populations, we used two types of supervised machine learning approaches and addressed three predictive modelling tasks of multi-target regression. The dominant species in the small mammal assemblage, the Yellow-necked Mouse Apodemus flavicollis, had a key role in determining predator populations and their breeding performance. We noted higher sensitivity to small mammal fluctuations in boreal zone owl species (Boreal Owl and Ural Owl), which reach their southern distribution limit in the Dinaric Alps, whereas the temperate zone species (Tawny Owl) seemed to be less affected. In years of prey shortage, the Boreal Owl was found to presumably abandon its territories, the Ural Owl suppressed breeding and the Tawny Owl sustained breeding activity by shifting prey selection. Low-elevation forests appeared to be suboptimal habitat for the competitive subordinate Boreal Owl, which may exploit occasional outbreaks of small mammal populations in these habitats even in the presence of larger competitors. Whether low-elevation forests can play a role in maintaining threatened and cold-adapted Boreal Owl populations in central and southern Europe in the face of recent ecosystem changes due to climate and environmental changes remains an open scientific question.
Keywords: sove, mali sesalci, populacijska dinamika, strojno učenje
Published in DiRROS: 16.07.2024; Views: 273; Downloads: 121
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1314. HepG2 spheroids as a biosensor-like cell-based system for (geno)toxicity assessmentMartina Štampar, Sonja Žabkar, Metka Filipič, Bojana Žegura, 2022, original scientific article Abstract: 3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool.
Keywords: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks
Published in DiRROS: 16.07.2024; Views: 312; Downloads: 180
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1315. An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1Samreen Falak, Rainer Macdonald, Eloise J. Busby, Denise M. O'Sullivan, Mojca Milavec, Annabell Plauth, Martin Kammel, Heinz Zeichhardt, Hans-Peter Grunert, Andreas Kummrow, Jim F. Huggett, 2022, original scientific article Abstract: Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.
Published in DiRROS: 16.07.2024; Views: 266; Downloads: 145
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1316. The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four yearsMojca Milavec, Jernej Pavšič, Alexandra Bogožalec Košir, Gerwyn M. Jones, Denise M. O'Sullivan, Alison S. Devonshire, Fran Van Heuverswyn, Maria Karczmarczyk, Jannika Neeb, Annabell Plauth, Philippe Corbisier, Heinz Schimmel, Andreas Kummrow, Jörg Neukammer, Carole A. Foy, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Jim F. Huggett, 2022, original scientific article Published in DiRROS: 15.07.2024; Views: 371; Downloads: 148
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1318. Diferential roles of eNOS in late efects ofVEGF‑A on hyperpermeability in diferent types of endothelial cellsEsmeralda K. Bosma, Shahan Darwesh, Yasmin I. Habani, Maxime Cammeraat, Paola Serrano Martinez, Mathilda E. van Breest Smallenburg, JiaY. Zheng, Ilse M.C. Vogels, Cornelis J. F. van Noorden, Reinier O. Schlingemann, Ingeborg Klaassen, 2023, original scientific article Abstract: Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular
pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute
efects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how
eNOS regulates late efects of VEGF-A-induced hyperpermeability. Here we show that VEGF-A induces
hyperpermeability via eNOS-dependent and eNOS-independent mechanisms at 2 days after VEGF-A
stimulation. Silencing of expression of the eNOS gene (NOS3) reduced VEGF-A-induced permeability
for dextran (70 kDa) and 766 Da-tracer in human dermal microvascular ECs (HDMVECs), but not in
human retinal microvascular ECs (HRECs) and human umbilical vein ECs (HUVECs). However, silencing
of NOS3 expression in HRECs increased permeability to dextran, BSA and 766 Da-tracer in the absence
of VEGF-A stimulation, suggesting a barrier-protective function of eNOS. We also investigated how
silencing of NOS3 expression regulates the expression of permeability-related transcripts, and
found that NOS3 silencing downregulates the expression of PLVAP, a molecule associated with
trans-endothelial transport via caveolae, in HDMVECs and HUVECs, but not in HRECs. Our fndings
underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS
therein, and demonstrate that diferent pathways are activated depending on the EC phenotype.
Keywords: endocytosis, RNAi, hyperpermeability
Published in DiRROS: 15.07.2024; Views: 319; Downloads: 210
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1319. The function of mating plugs in the spider Neriene emphana: mating strategy or sperm protection?Shuang Tian, He Jiang, Yongjia Zhan, Qingqing Wu, Matjaž Kuntner, Lihong Tu, 2023, original scientific article Abstract: Introduction: It is generally thought that mating plugs, where present, impede or reduce the possibilities of female subsequent mating. Behavioral studies on numerous spiders, where mating plugs are common, have generally supported this function. However, mating plugs in spiders could plausibly serve other functions as well. Namely, the structure of entelegyne spermathecae—the morphology of most spiders—could require a mechanism that would prevent sperm from leakage, desiccation, and backflow. Although the form and function of mating plugs in several spider species imply their potential adaptation for sperm protection, this function has never been empirically tested. Methods: Here, we test whether mating plugs in the sheet-web spider Neriene emphana serve as a sperm protective device by investigating its genital morphology, its copulation process, and the precise formation of its amorphous mating plugs. Results: This species constructs secretion plugs through male-female cooperation. Additionally, we found sperm plugs to be formed as a side product of sperm transfer, as well as an intermediate type of secretion plugs. These plug materials are transferred in different mating stages as documented by variations in the rhythm of male palpal application during copulation. We showed that complete copulations always resulted in formation of secretion plugs at spermathecal entrances via laborious deposition of male materials. Discussion: While our findings do not reject that secretion plugs in N. emphana prevent females from subsequent mating, we suggest that they must have evolved to provide sperm protection.
Keywords: entelegyne spermatheca, mating behavior, mating strategy, sperm protection mechanism, mating plug, secretion plug, sperm plug
Published in DiRROS: 15.07.2024; Views: 261; Downloads: 168
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1320. Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomatoBeatriz Romero-Rodríguez, Marko Petek, Chen Jiao, Maja Križnik, Maja Zagorščak, Zhangjun Fei, Eduardo Rodríguez Bejarano, Kristina Gruden, Araceli G. Castillo, 2023, original scientific article Abstract: Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.
Keywords: geminivirus, TYLCV, tomato, transcriptome, miRNA
Published in DiRROS: 15.07.2024; Views: 288; Downloads: 187
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