1. Analysis of glioblastoma patients' plasma revealed the presence of microRNAs with a prognostic impact on survival and those of viral originKlemen Zupančič, Helena Motaln, Miomir Knežević, Urška Verbovšek, Marjan Koršič, Tamara Lah Turnšek, Primož Rožman, Matjaž Jeras, Matjaž Hren, Kristina Gruden, Andrej Blejec, Matija Veber, Ana Herman, Andrej Porčnik, Vid Podpečan, 2015, izvirni znanstveni članek Povzetek: Background
Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.
Experimental Procedures
MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits.
Results
We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients’ survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.
Conclusion
The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients
Ključne besede: microRNAs, glioblastoma multiforme, biomarkers, RNA extraction, viral disease diagnosis
Objavljeno v DiRROS: 26.07.2024; Ogledov: 19; Prenosov: 6
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2. RNA-dependent RNA polymerase 1 in potato (Solanum tuberosum) and its relationship to other plant RNA-dependent RNA polymerasesLydia J. R. Hunter, Samuel F. Brockington, Alex M. Murphy, Adrienne E. Pate, Kristina Gruden, Stuart A. MacFarlane, Peter Palukaitis, John P. Carr, 2016, izvirni znanstveni članek Povzetek: Cellular RNA-dependent RNA polymerases (RDRs) catalyze synthesis of double-stranded RNAs that can serve to initiate or amplify RNA silencing. Arabidopsis thaliana has six RDR genes; RDRs 1, 2 and 6 have roles in anti-viral RNA silencing. RDR6 is constitutively expressed but RDR1 expression is elevated following plant treatment with defensive phytohormones. RDR1 also contributes to basal virus resistance. RDR1 has been studied in several species including A. thaliana, tobacco (Nicotiana tabacum), N. benthamiana, N. attenuata and tomato (Solanum lycopersicum) but not to our knowledge in potato (S. tuberosum). StRDR1 was identified and shown to be salicylic acid-responsive. StRDR1 transcript accumulation decreased in transgenic potato plants constitutively expressing a hairpin construct and these plants were challenged with three viruses: potato virus Y, potato virus X, and tobacco mosaic virus. Suppression of StRDR1 gene expression did not increase the susceptibility of potato to these viruses. Phylogenetic analysis of RDR genes present in potato and in a range of other plant species identified a new RDR gene family, not present in potato and found only in Rosids (but apparently lost in the Rosid A. thaliana) for which we propose the name RDR7.
Ključne besede: RNA polymerases, RNA, potato
Objavljeno v DiRROS: 25.07.2024; Ogledov: 15; Prenosov: 4
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3. Next generation sequencing for detection and discovery of plant viruses and viroids : comparison of two approachesAnja Pecman, Denis Kutnjak, Ion Gutiérrez-Aguirre, Ian Adams, Adrian Fox, Neil Boonham, Maja Ravnikar, 2017, izvirni znanstveni članek Povzetek: Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).
Ključne besede: next generation sequencing, small RNA, ribosomal RNA depleted total RNA, detection, plant viruses, plant viroids
Objavljeno v DiRROS: 25.07.2024; Ogledov: 17; Prenosov: 7
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4. RECQ1 helicase silencing decreases the tumour growth rate of U87 glioblastoma cell xenografts in zebrafish embryosMiloš Vittori, Barbara Breznik, Katja Hrovat, Saša Kenig, Tamara Lah Turnšek, 2017, izvirni znanstveni članek Povzetek: RECQ1 helicase has multiple roles in DNA replication, including restoration of the replication fork and DNA repair, and plays an important role in tumour progression. Its expression is highly elevated in glioblastoma as compared to healthy brain tissue. We studied the effects of small hairpin RNA (shRNA)-induced silencing of RECQ1 helicase on the increase in cell number and the invasion of U87 glioblastoma cells. RECQ1 silencing reduced the rate of increase in the number of U87 cells by 30%. This corresponded with a 40% reduction of the percentage of cells in the G2 phase of the cell cycle, and an accumulation of cells in the G1 phase. These effects were confirmed in vivo, in the brain of zebrafish (Danio rerio) embryos, by implanting DsRed-labelled RECQ1 helicase-silenced and control U87 cells. The growth of resulting tumours was quantified by monitoring the increase in xenograft fluorescence intensity during a three-day period with fluorescence microscopy. The reduced rate of tumour growth, by approximately 30% in RECQ1 helicase-silenced cells, was in line with in vitro measurements of the increase in cell number upon RECQ1 helicase silencing. However, RECQ1 helicase silencing did not affect invasive behaviour of U87 cells in the zebrafish brain. This is the first in vivo confirmation that RECQ1 helicase is a promising molecular target in the treatment of glioblastoma.
Ključne besede: cancer, cell cycle, DNA damage, intravital imaging, RNA interference, theranostics
Objavljeno v DiRROS: 25.07.2024; Ogledov: 26; Prenosov: 13
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5. Potato virus Y infection alters small RNA metabolism and immune response in tomatoMaria I. Prigigallo, Maja Križnik, Domenico De Paola, Domenico Catalano, Kristina Gruden, Mariella M. Finetti-Sialer, Fabrizio Cillo, 2019, izvirni znanstveni članek Povzetek: Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype.
Ključne besede: RNA silencing, plant defense response, Potato virus Y, molecular plant-virus interactions, microRNA, secondary small interfering RNA, small RNA sequencing, phasiRNA
Objavljeno v DiRROS: 24.07.2024; Ogledov: 18; Prenosov: 4
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6. Structural basis for the multitasking nature of the potato virus Y coat proteinAndreja Kežar, Luka Kavčič, Martin Pólak, Jiři Nováček, Ion Gutiérrez-Aguirre, Magda Tušek-Žnidarič, Anna Coll Rius, Katja Stare, Kristina Gruden, Maja Ravnikar, David Pahovnik, Ema Žagar, Franci Merzel, Gregor Anderluh, Marjetka Podobnik, 2019, izvirni znanstveni članek Povzetek: Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
Ključne besede: plant pathogens, potato virus Y, viral RNA
Objavljeno v DiRROS: 23.07.2024; Ogledov: 41; Prenosov: 14
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7. Scaphoideus titanus forecasting and management : quo vadis?Elena Gonella, Giovanni Benelli, Nathalie Arricau-Bouvery, Domenico Bosco, Meta Virant-Doberlet, 2024, pregledni znanstveni članek Povzetek: Scaphoideus titanus Ball (Hemiptera: Cicadellidae) is a major pest for European viticulture due to its high efficiency in the transmission of one of the most destructive pathogens for grapevine, namely flavescence dorée phytoplasmas. Although it plays a major role in spreading this disease, S. titanus is part of a complex epidemiological cycle involving several alternative vectors with variable relevance for phytoplasma spread. Here we provide an updated review on S. titanus monitoring and modelling, as well as the available tools for management of this pest and for limiting phytoplasma transmission and, thus, also spread. Insecticide-based control is examined; additional emphasis is placed on innovative and low-impact control approaches, such as vibrational mating disruption, biocontrol, and methods to reduce vector competence. We also discuss the main emerging challenges to the implementation of effective and sustainable control programs against S. titanus.
Ključne besede: epidemiology, pest monitoring, modelling, Integrated Pest Management, vibrational mating disruption, biological control, plant resistance elicitors, RNA-interference, symbiotic control, entomology, insects, plant protection
Objavljeno v DiRROS: 23.07.2024; Ogledov: 18; Prenosov: 13
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8. Validating the potential of double-stranded RNA targeting Colorado potato beetle mesh gene in laboratory and field trialsMarko Petek, Anna Coll Rius, Rok Ferenc, Jaka Razinger, Kristina Gruden, 2020, izvirni znanstveni članek Povzetek: 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.
Ključne besede: RNA interference, RNA interference feeding, ds RNA, gene silencing, RNAi pest control, survival analysis, field trial
Objavljeno v DiRROS: 22.07.2024; Ogledov: 29; Prenosov: 8
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9. Virome analysis of signal crayfish (Pacifastacus leniusculus) along its invasion range reveals diverse and divergent RNA virusesKatarina Bačnik, Denis Kutnjak, Silvija Černi, Ana Bielen, Sandra Hudina, 2021, izvirni znanstveni članek Povzetek: Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.
Ključne besede: signal crayfish virome, RNA viruses, invasive alien species, invasion range, high-throughput sequencing
Objavljeno v DiRROS: 19.07.2024; Ogledov: 50; Prenosov: 24
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10. Looking beyond virus detection in RNA sequencing data : lessons learned from a community-based effort to detect cellular plant pathogens and pestsAnnelies Haegeman, Yoika Foucart, Kris De Jonghe, Thomas Goedefroit, Maher Al Rwahnih, Neil Boonham, Thierry Candresse, Yahya Gaafar, Oscar Hurtado-Gonzales, Zala Kogej Zwitter, Denis Kutnjak, Janja Lamovšek, Irena Mavrič Pleško, 2023, izvirni znanstveni članek Povzetek: High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.
Ključne besede: plant viruses, plant virus detection, plant virology, high-throughput sequencing, RNA sequencing, plant tissues, plant pathogen, diagnostics, high-throughput sequencing, metagenomics, metatranscriptomics
Objavljeno v DiRROS: 12.07.2024; Ogledov: 87; Prenosov: 29
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