1. In silico determination of novel SARS-CoV-2 envelope protein ion channel inhibitorsNina Kobe, Lennart Dreisewerd, Matic Pavlin, Polona Kogovšek, Črtomir Podlipnik, Uroš Grošelj, Miha Lukšič, 2025, original scientific article Abstract: The SARS-CoV-2 envelope protein (2-EPRO), a viroporin crucial for viral pathogenesis, is a promising target for antiviral drug development as it is highly conserved and functionally important. Although it is a promising therapeutic target for the treatment of COVID-19, it has often been overlooked in previous studies. In this study, a high-throughput virtual screening of nearly one billion compounds was performed, followed by rigorous filtering and re-docking. Eight best-scoring and chemically versatile lead candidates were identified. In molecular dynamics simulations, three of these ligands showed stable protein-ligand complexes occupying the 2-EPRO channel pore. Among these, ZINC001799167680 (L3) and ZINC001081252239 (L2) exhibited the strongest binding a˙inity, with key interactions at residues ASN15, THR11 and GLU8 identified by Molecular Mechanics Poisson-Boltzmann Surface Area analysis. All ligands were compared with the known inhibitor rimantadine and showed stronger binding to the protein. These in silico results highlight the potential of focusing on the 2-EPRO ion channel in the development of novel COVID-19 therapeutics and pave the way for further in vitro and in vivo studies.
Keywords: SARS-CoV-2 envelope protein, viroporin, ion channel, channel blockers, molecular dynamics simulations, free energy calculations, drug discovery
Published in DiRROS: 05.09.2025; Views: 268; Downloads: 109
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2. Test performance study on qPCR assays for detection of Phyllosticta citricarpaTjaša Jakomin, Janja Zajc Žunič, Polona Kogovšek, 2025, original scientific article Abstract: Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on TEF region, for detecting P. citricarpa through a collaborative test performance study (TPS). DNA from the isolates of Phyllosticta spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related Phyllosticta species, while Pc-TEF1 showed higher specificity for P. citricarpa included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of P. citricarpa, contributing to the effective management of CBS disease in citrus production and trade.
Keywords: test performance study, Phyllosticta citricarpa, real time PCR, TEF1, biotechnology
Published in DiRROS: 07.05.2025; Views: 575; Downloads: 525
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3. Dynamics of responses in compatible potato - potato virus Y interaction are modulated by salicylic acidŠpela Baebler, Katja Stare, Maja Kovač, Andrej Blejec, Nina Prezelj, Tjaša Stare, Polona Kogovšek, Maruša Pompe Novak, S. Rosahl, Maja Ravnikar, Kristina Gruden, 2011, original scientific article Abstract: To investigate the dynamics of the potato – Potato virus Y (PVY) compatible interaction in relation to salicylic acid - controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVYNTN, the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid - analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.
Keywords: plant viruses, plant diseases
Published in DiRROS: 04.03.2025; Views: 531; Downloads: 335
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4. [Beta]-1,3-glucanase class III promotes spread of PVY[sup]NTN and improves in planta protein productionDavid Dobnik, Špela Baebler, Polona Kogovšek, Maruša Pompe Novak, Dejan Štebih, Gabriela Panter, Nikolaja Janež, Dany Morisset, Jana Žel, Kristina Gruden, 2013, original scientific article Abstract: Glucanases are enzymes regulating the size exclusion limit and permeability of plasmodesmata and play a role in biotic stress. In plant genomes, they are encoded as relatively large gene families divided into four classes. Most studies of plant virus interactions have focused on glucanases from classes I and II. In our study, we have evaluated the role of the β-1,3-glucanase class III (Glu-III) gene in the potato–potato virus YNTN (PVYNTN) interaction and implemented the findings to plant biotechnology application. Potato cultivars Désirée and Santé, which are tolerant and extremely resistant to PVYNTN, respectively, were stably transformed with Agrobacterium tumefaciens harbouring constructs for Glu-III overexpression. Localization of Glu-III protein in patches within the cell wall was determined by tagging the Glu-III protein with green fluorescent protein. Transgenic and non-transgenic plants were challenged with PVYNTN and its multiplication and spreading was followed. Differences in viral spread were observed between transgenic lines overexpressing Glu-III and non-transgenic lines, with stronger and faster viral spread in transgenic Désirée, and some multiplication in transgenic Santé. In addition, the ability of Glu-III to improve in planta protein production after agroinfiltration was tested. The results have shown that Glu-III overexpression enables faster spreading of vectors between cells and better protein production, which could be beneficial in improving in planta protein production system using viral vectors.
Keywords: plant biotechnology, plant-virus interaction, potato virus Y, agroinfiltration, beta-1, 3-glucanase
Published in DiRROS: 04.03.2025; Views: 665; Downloads: 787
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5. Bacterial filtration efficiency of different masksTamara Košir, Katja Fric, Arijana Filipić, Polona Kogovšek, 2022, original scientific article Abstract: Face coverings, such as surgical masks and respirators, have an important role in preventing bacterial and viral transmission, especially during a global pandemic like COVID-19. Therefore, to secure their availability, new manufacturers and the use of novel materials must be encouraged. However, masks and their materials must first be properly tested for safety and efficiency, as required by the relevant standard, valid in a specific region. All standards prescribe determination of the bacterial filtration efficiency (BFE) of masks. In this study, we report the establishment of a test method for the BFE of face masks in accordance with European standard EN 14683:2019, by which we tested 52 samples, each composed of 3 to 5 subsamples, of surgical and cloth masks, respirators, filters, and mask materials. Forty-seven out of the 52 samples reached a BFE above 75 %. Of these, 16 samples had a BFE of 75 % to 95 %, 3 had a BFE of 95 % to 98 %, while 28 reached a filtration efficiency above 98 %. Our findings show that all tested samples provided some level of protection, most of which met the requirements for the national or European market.
Keywords: bacterial filtration efficiency, face coverings, masks, respirators, Andersen Cascade Impactor, EN 14683:2019
Published in DiRROS: 26.02.2025; Views: 548; Downloads: 456
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6. Extracellular vesicle-bound DNA in urine is indicative of kidney allograft injuryIvana Sedej, Maja Štalekar, Magda Tušek-Žnidarič, Katja Goričar, Nika Kojc, Polona Kogovšek, Vita Dolžan, Miha Arnol, Metka Lenassi, 2022, original scientific article Abstract: Extracellular vesicle-bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer-unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well-characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC-based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell-free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non-rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof-of-principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated.
Keywords: DNA, donor-derived DNA, extracellular vesicles
Published in DiRROS: 26.02.2025; Views: 581; Downloads: 480
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7. Antiviral respiratory masks with plasma-functionalized polypropylene textiles for optimal adsorption of antiviral substanceMark Zver, David Dobnik, Rok Zaplotnik, Miran Mozetič, Alenka Vesel, Arijana Filipić, Polona Kogovšek, Katja Fric, Alja Štern, Gregor Primc, 2024, original scientific article Abstract: During the COVID-19 pandemic, face masks were the first line of defense against the spread of infection. However, infectious viruses may remain on medical textiles, potentially serving as an additional source of infection. Due to their chemical inertness, many textiles cannot be enhanced with antiviral functionalities. Through treatment with low-pressure gaseous plasma, we have activated the surface of a medical-grade melt-blown, non-woven polypropylene textile so that it can absorb sodium dodecyl sulfate, an antimicrobial surfactant. Within two hours of contact time, the functionalized textile has been able to inactivate over 7 log10 PFU mL−1 of bacteriophage phi6, a surrogate of enveloped viruses such as SARS-CoV-2, and it has retained its antiviral properties for over 100 days. The functionalized material has not disrupted facial mask filtration efficiency or breathability. In addition, the in vitro biocompatibility testing in accordance with ISO 10993-5 for testing of medical devices has demonstrated that the selected formulation causes no adverse effects on the mouse fibroblast cell line L-929. With the treatment processes that have been completed within seconds, the method seems to have great potential to produce antiviral textiles against future outbreaks.
Keywords: surgical face masks, plasma functionalization, antiviral materials, virus filtration, breathability
Published in DiRROS: 07.10.2024; Views: 868; Downloads: 738
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8. Molecular screening for cyanobacteria and their cyanotoxin potential in diverse habitatsMaša Jablonska, Tina Eleršek, Polona Kogovšek, Sara Skok, Andreea Oarga-Mulec, Janez Mulec, 2024, original scientific article Abstract: Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer—PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins—mcyE, saxitoxins—sxtA, cylindrospermopsins—cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.
Keywords: extreme environments, cylindrospermopsin, sulphidic springs, caves, qPCR, PC-IGS
Published in DiRROS: 05.08.2024; Views: 981; Downloads: 612
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9. Potato Virus Y infection hinders potato defence response and renders plants more vulnerable to Colorado potato beetle attackMarko Petek, Ana Rotter, Polona Kogovšek, Špela Baebler, Axel Mithöfer, Kristina Gruden, 2014, original scientific article Abstract: In the field, plants are challenged by more than one biotic stressor at the same time. In this study, the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa decemlineata Say; CPB) and Potato virus YNTN (PVYNTN) were investigated through analyses of gene expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile compounds. CPB larval growth was enhanced when reared on secondary PVYNTN-infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVYNTN-infected plants, ethylene signalling pathway induction and induction of auxin response transcription factors were attenuated, while no differences were observed in jasmonic acid (JA) signalling pathway. Similarly to rearing on virus-infected plants, CPB larvae gained more weight when reared on plants silenced in JA receptor gene (coi1). Although herbivore-induced defence mechanism is regulated predominantly by JA, response in coi1-silenced plants only partially corresponded to the one observed in PVYNTN-infected plants, confirming the role of other plant hormones in modulating this response. The release of β-barbatene and benzyl alcohol was different in healthy and PVYNTN-infected plants before CPB larvae infestation, implicating the importance of PVYNTN infection in plant communication with its environment. This was reflected in gene expression profiles of neighbouring plants showing different degree of defence response. This study thus contributes to our understanding of plant responses in agro-ecosystems.
Keywords: insect midgut transcriptional response, gene expression, plant defence, volatile organic compounds, potato
Published in DiRROS: 02.08.2024; Views: 1145; Downloads: 552
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10. LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevinePolona Kogovšek, Jennifer Hodgetts, J. Hall, Nina Prezelj, Petra Nikolić, Nataša Mehle, Rok Lenarčič, Ana Rotter, M. Dickinson, Neil Boonham, Marina Dermastia, Maja Ravnikar, 2015, original scientific article Abstract: In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.
Keywords: flavescence dorée, homogenization, loop-mediated isothermal amplification, on-site application, validation
Published in DiRROS: 26.07.2024; Views: 1026; Downloads: 785
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