681. Inactivation of pepper mild mottle virus in water by cold atmospheric plasmaArijana Filipić, David Dobnik, Magda Tušek-Žnidarič, Bojana Žegura, Alja Štern, Gregor Primc, Miran Mozetič, Maja Ravnikar, Jana Žel, Ion Gutiérrez-Aguirre, 2021, izvirni znanstveni članek Povzetek: Water scarcity is one of the greatest threats for human survival and quality of life, and this is increasingly contributing to the risk of human, animal and plant infections due to waterborne viruses. Viruses are transmitted through polluted water, where they can survive and cause infections even at low concentrations. Plant viruses from the genus Tobamovirus are highly mechanically transmissible, and cause considerable damage to important crops, such as tomato. The release of infective tobamoviruses into environmental waters has been reported, with the consequent risk for arid regions, where these waters are used for irrigation. Virus inactivation in water is thus very important and cold atmospheric plasma (CAP) is emerging in this field as an efficient, safe, and sustainable alternative to classic waterborne virus inactivation methods. In the present study we evaluated CAP-mediated inactivation of pepper mild mottle virus (PMMoV) in water samples. PMMoV is a very resilient water-transmissible tobamovirus that can survive transit through the human digestive tract. The efficiency of PMMoV inactivation was characterized for infectivity and virion integrity, and at the genome level, using test plant infectivity assays, transmission electron microscopy, and molecular methods, respectively. Additionally, the safety of CAP treatment was determined by testing the cytotoxic and genotoxic properties of CAP-treated water on the HepG2 cell line. 5-min treatment with CAP was sufficient to inactivate PMMoV without introducing any cytotoxic or genotoxic effects in the in-vitro cell model system. These data on inactivation of such stable waterborne virus, PMMoV, will encourage further examination of CAP as an alternative for treatment of potable and irrigation waters, and even for other water sources, with emphasis on inactivation of various viruses including enteric viruses. Ključne besede: enteric viruses, pepper mild mottle virus, virus inactivation, water decontamination, cold atmospheric plasma Objavljeno v DiRROS: 19.07.2024; Ogledov: 149; Prenosov: 122
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682. Cytotoxic and genotoxic effects of cyanobacterial and algal extracts-microcystin and retinoic acid contentMichal Bittner, Alja Štern, Marie Smutna, Klara Hilscherova, Bojana Žegura, 2021, izvirni znanstveni članek Povzetek: In the last decade, it has become evident that complex mixtures of cyanobacterial bioactive substances, simultaneously present in blooms, often exert adverse effects that are different from those of pure cyanotoxins, and awareness has been raised on the importance of studying complex mixtures and chemical interactions. We aimed to investigate cytotoxic and genotoxic effects of complex extracts from laboratory cultures of cyanobacterial species from different orders (Cylindrospermopsis raciborskii, Aphanizomenon gracile, Microcystis aeruginosa, M. viridis, M. ichtyoblabe, Planktothrix agardhii, Limnothrix redekei) and algae (Desmodesmus quadricauda), and examine possible relationships between the observed effects and toxin and retinoic acid (RA) content in the extracts. The cytotoxic and genotoxic effects of the extracts were studied in the human hepatocellular carcinoma HepG2 cell line, using the MTT assay, and the comet and cytokinesis-block micronucleus (cytome) assays, respectively. Liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) was used to detect toxins (microcystins (MC-LR, MC-RR, MC-YR) and cylindrospermopsin) and RAs (ATRA and 9cis-RA) in the extracts. Six out of eight extracts were cytotoxic (0.04–2 mgDM/mL), and five induced DNA strand breaks at non-cytotoxic concentrations (0.2–2 mgDM/mL). The extracts with genotoxic activity also had the highest content of RAs and there was a linear association between RA content and genotoxicity, indicating their possible involvement; however further research is needed to identify and confirm the compounds involved and to elucidate possible genotoxic effects of RAs. Ključne besede: cyanobacteria, algae, extracts, complex mixtures, genotoxicity, cytotoxicity, retinoic acids, microcystins, cyanotoxins, chemical analysis Objavljeno v DiRROS: 19.07.2024; Ogledov: 156; Prenosov: 132
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683. First restoration experiment for Gongolaria barbata in Slovenian coastal waters : What can go wrong?Martina Orlando-Bonaca, Valentina Pitacco, Petra Slavinec, Milijan Šiško, Tihomir Makovec, Annalisa Falace, 2021, izvirni znanstveni članek Povzetek: The global decline of brown algal forests along rocky coasts is causing an exceptional biodiversity loss. Regardless of conservation efforts, different techniques have been developed for large-scale restoration strategies in the Mediterranean Sea. In this study we tested ex situ pilot restoration of Gongolaria barbata (=Treptacantha barbata) for the first time in Slovenian coastal waters. Healthy apical fronds of the species were collected and the development of recruits on clay tiles was followed under laboratory conditions for 20 days. Despite the experimental difficulties experienced, especially due to the lack of antibiotics to prevent the growth of the biofilm, G. barbata recruits were outplanted in the sea on two concrete plates with 48 tiles each, protected by purpose-built cages to avoid grazing by herbivorous fish. The high survival rate of juveniles after four months in the field (89% of the tiles on the plate that was constantly protected) suggests that outplanting G. barbata is an operable approach for restoration efforts in the northern Adriatic Sea. Our first experiment in Slovenian coastal waters provides new information for the optimization of the best practices during the laboratory cultivation and addresses the early steps of restoration and introduction of young thalli in the natural environment. Objavljeno v DiRROS: 19.07.2024; Ogledov: 160; Prenosov: 106
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684. Molecular systematics of the long-snouted deep water dogfish (Centrophoridae, Deania) with implications for identification, taxonomy, and conservationSergio Stefanni, Diana Catarino, Pedro A. Ribeiro, Mafalda Freitas, Gui Menezes, Francis Neat, David Stanković, 2021, izvirni znanstveni članek Povzetek: According to the most recent taxonomical revision, the deep-sea dogfish genus Deania encompasses four species. Three of them, D. calcea, D. profundorum, and D. hystricosa, occur in the North Atlantic. Whilst D. profundorum can be identified by the presence of a subcaudal keel, the other two species are not easily visually distinguished. Uncertainties over identification raises concerns over stock units and whether management plans are adequate. In this study we compared onboard visual identification of Deania specimens, with morphological inspection of skin denticles under stereo microscope and with independent molecular taxonomical assignment using two molecular markers. Particular emphasis was paid to specimens identified as D. calcea and D. hystricosa in the NE Atlantic where these species potentially occur sympatrically and may be easily confused. In the past the species have been discriminated on the basis of the size of skin denticles (skin roughness), but our study showed that the crown length of skin denticles covaries with size (and sex), irrespective of species, and therefore this is not a reliable morphological character and should not be used to discriminate between the two species. Phylogenetic analyses did not indicate that D. hystricosa to be a distinct lineage from D. calcea. Interestingly, however four individuals (specimens from: UK, Azores Is., Madeira Is. and Seine seamount) formed a well-defined sub-clade nested within the D. calcea clade, possibly a signature of a past vicariance event or a result of coalescent stochasticity. Ključne besede: deep-sea sharks, fishery by-catch, phylogeny, Atlantic Ocean, deep-sea dogfish, skin denticles Objavljeno v DiRROS: 19.07.2024; Ogledov: 161; Prenosov: 96
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685. On the potential of ensemble forecasting for the prediction of meteotsunamis in the Balearic Islands : sensitivity to atmospheric model parameterizationsBaptiste Mourre, A. Santana, A. Buils, L. Gautreau, Matjaž Ličer, Agusti Jansá, B. Casas, B. Amengual, Joaquín Tintoré, 2021, izvirni znanstveni članek Povzetek: This study investigates the potential of ensemble forecasting using full realistic high-resolution nested atmosphere–ocean models for the prediction of meteotsunamis in Ciutadella (Menorca, Spain). The sensitivity of model results to the parameterizations of the atmospheric model is assessed considering the ten most significant recent meteotsunami events for which observations are available. Different schemes adapted to high-resolution Weather Research and Forecasting model simulations were used for the representation of cumulus, microphysics, planetary boundary layer and longwave and shortwave radiations. Results indicate a large spread of the ensemble simulations in terms of the final magnitude of the meteotsunamis. While the modeling system is shown to be able to realistically trigger tsunamigenic atmospheric disturbances in more than 90% of the situations, the small-scale characteristics of these disturbances are significantly modified with the change of parameterizations, leading to significant differences in the magnitude of the simulated sea-level response. No preferred set of parameterizations can be identified that leads to either the largest or the most realistic magnitudes in the majority of situations. Instead, the performance of a given set of parameterizations is found to change with the meteotsunami event under consideration. Importantly, the observed magnitude of the extreme sea-level oscillations lies within the range of a nine-member ensemble in 70% of the cases. This ensemble approach would then allow to generate a realistic range of possibilities in the majority of events, thus improving the realism of meteotsunami predictions compared to single deterministic forecasts. Ključne besede: meteotsunamis prediction, atmosphere, ocean modeling, ensemble forecasting, atmospheric model parameterizations Objavljeno v DiRROS: 19.07.2024; Ogledov: 140; Prenosov: 126
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686. 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, izvirni znanstveni članek Povzetek: 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. Objavljeno v DiRROS: 19.07.2024; Ogledov: 270; Prenosov: 97
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687. Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicalsMartina Štampar, Helle Frandsen, Adelina Rogowska-Wrzesinska, Krzysztof Wrzesinski, Metka Filipič, Bojana Žegura, 2021, izvirni znanstveni članek Povzetek: The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations. Ključne besede: in vitro 3D cell model, 21-day old spheroids, cytotoxic, genotoxic, gene expression Objavljeno v DiRROS: 19.07.2024; Ogledov: 125; Prenosov: 160
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688. Production of volatile moth sex pheromones in transgenic Nicotiana benthamiana plantsRubèn Mateos Fernández, Elena Moreno Gimenéz, Silvia Gianoglio, Alfredo Quijano-Rubio, Jose Gavaldá-García, Lucía Estellés, Alba Rubert, José Luis Rambla, Marta Vazquez-Vilar, Estefanía Huet, Asunción Fernández-del-Carmen, Ana Espinosa-Ruiz, Mojca Juteršek, Sandra Vacas, Ismael Navarro Fuertes, Vicente Navarro-Llopis, Jaime Primo, Diego Orzaez, 2021, izvirni znanstveni članek Povzetek: Plant-based bioproduction of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here, we describe the engineering of transgenic plants producing (Z)-11-hexadecenol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them into Nicotiana benthamiana plants. The constructs contained the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera fatty acyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, the severity of which correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH, but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered that harboured an intact pheromone pathway and which produced moderate levels of Z11-16OAc (11.8 μg g-1 FW) and high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone biodispenser strategies and establish a roadmap for biotechnological improvements. Objavljeno v DiRROS: 19.07.2024; Ogledov: 287; Prenosov: 137
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689. A mini-TGA protein modulates gene expression through heterogeneous association with transcription factorsŠpela Tomaž, Marko Petek, Tjaša Lukan, Karmen Pogačar, Katja Stare, Erica Teixeira Prates, Daniel A. Jacobson, Jan Zrimec, Gregor Bajc, Matej Butala, Maruša Pompe Novak, Ajda Taler-Verčič, Aleksandra Usenik, Dušan Turk, Anna Coll Rius, Kristina Gruden, 2022, izvirni znanstveni članek Povzetek: TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families. Objavljeno v DiRROS: 18.07.2024; Ogledov: 195; Prenosov: 91
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690. Hyperthermia as a potential cornerstone of effective multimodality treatment with radiotherapy, cisplatin and PARP inhibitor in IDH1-mutated cancer cellsMohammed Khurshed, Elia Prades-Sagarra, Sarah Al Saleh, Peter Sminia, Johanna W Wilmink, Remco J. Molenaar, Hans Crezee, Cornelis J. F. van Noorden, 2022, izvirni znanstveni članek Povzetek: Mutations in the isocitrate dehydrogenase 1 (IDH1MUT) gene occur in various types of malignancies, including ~60% of chondrosarcomas, ~30% of intrahepatic cholangiocarcinomas and >80% of low-grade gliomas. IDH1MUT are causal in the development and progression of these types of cancer due to neomorphic production of the oncometabolite D-2-hydroxyglutarate (D-2HG). Intracellular accumulation of D-2HG has been implicated in suppressing homologous recombination and renders IDH1MUT cancer cells sensitive to DNA-repair-inhibiting agents, such as poly-(adenosine 5′-diphosphate–ribose) polymerase inhibitors (PARPi). Hyperthermia increases the efficacy of DNA-damaging therapies such as radiotherapy and platinum-based chemotherapy, mainly by inhibition of DNA repair. In the current study, we investigated the additional effects of hyperthermia (42 °C for 1 h) in the treatment of IDH1MUT HCT116 colon cancer cells and hyperthermia1080 chondrosarcoma cancer cells in combination with radiation, cisplatin and/or a PARPi on clonogenic cell survival, cell cycle distribution and the induction and repair of DNA double-strand breaks. We found that hyperthermia in combination with radiation or cisplatin induces an increase in double-strand breaks and cell death, up to 10-fold in IDH1MUT cancer cells compared to IDH1 wild-type cells. This vulnerability was abolished by the IDH1MUT inhibitor AGI-5198 and was further increased by the PARPi. In conclusion, our study shows that IDH1MUT cancer cells are sensitized to hyperthermia in combination with irradiation or cisplatin and a PARPi. Therefore, hyperthermia may be an efficacious sensitizer to cytotoxic therapies in tumors where the clinical application of hyperthermia is feasible, such as IDH1MUT chondrosarcoma of the extremities. Ključne besede: isocitrate dehydrogenase, PARP, hyperthermia, D‐2‐hydroxyglutarate, radiotherapy, cisplatin Objavljeno v DiRROS: 18.07.2024; Ogledov: 195; Prenosov: 153
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