1001. ALF : a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysisAlexandra Bogožalec Košir, Alfred J. Arulandhu, Marleen Voorhuijzen, Hongmei Xiao, Rico Hagelaar, Martijn Staats, Adalberto Costessi, Jana Žel, Esther Kok, Jeroen P. van Dijk, 2017, original scientific article Abstract: The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.
Keywords: biotechnology, molecular biology
Published in DiRROS: 25.07.2024; Views: 242; Downloads: 211
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1002. 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, original scientific article Abstract: 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).
Keywords: next generation sequencing, small RNA, ribosomal RNA depleted total RNA, detection, plant viruses, plant viroids
Published in DiRROS: 25.07.2024; Views: 279; Downloads: 181
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1003. Newly isolated bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae families have variable effects on putative novel Dickeya spp.Špela Alič, Tina Mikuletič, Magda Tušek-Žnidarič, Maja Ravnikar, Nejc Rački, Matjaž Peterka, Tanja Dreo, 2017, original scientific article Abstract: Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20∘C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents.
Keywords: bacteriophages, genome sequencing, resistance development, convective interaction media monolith chromatography
Published in DiRROS: 25.07.2024; Views: 306; Downloads: 217
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1004. Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCRAlexandra Bogožalec Košir, Carla Divieto, Jernej Pavšič, Stefano Pavarelli, David Dobnik, Tanja Dreo, Roberto Bellotti, Maria Paola Sassi, Jana Žel, 2017, original scientific article Abstract: Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Keywords: droplet digital PCR, droplet volume, DNA quantification, optical microscopy imaging
Published in DiRROS: 25.07.2024; Views: 303; Downloads: 235
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1005. 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, original scientific article Abstract: 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.
Keywords: cancer, cell cycle, DNA damage, intravital imaging, RNA interference, theranostics
Published in DiRROS: 25.07.2024; Views: 303; Downloads: 208
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1006. Novel, male-produced aggregation pheromone of the cerambycid beetle Rosalia alpina, a priority species of European conservation concernAlenka Žunič Kosi, Yunfan Zou, Michal Hoskovec, Al Vrezec, Nataša Stritih Peljhan, Jocelyn G. Millar, 2017, original scientific article Abstract: Several recent studies have demonstrated the great potential for exploiting semiochemicals in ecology and conservation studies. The cerambycid beetle Rosalia alpina represents one of the flagship species of saproxylic insect biodiversity in Europe. In recent years its populations appear to have declined substantially, and its range has shrunk considerably as a result of forest management and urbanization. Here, we collected volatile chemicals released by males and females of R. alpina. Analyses of the resulting extracts revealed the presence of a single male-specific compound, identified as a novel alkylated pyrone structure. In field bioassays in Slovenia, traps baited with the synthesized pyrone captured both sexes of R. alpina, indicating that the pyrone functions as an aggregation pheromone. Our results represent the first example of a new structural class of pheromones within the Cerambycidae, and demonstrate that pheromone-baited traps can provide a useful tool for sampling R. alpina. This tool could be particularly useful in the ongoing development of conservation strategies for the iconic but endangered Alpine longicorn.
Keywords: pheromones, beetles, plants, endangered species, conservation science
Published in DiRROS: 25.07.2024; Views: 282; Downloads: 374
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1007. Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean linesAlexandra Bogožalec Košir, Bjørn Spilsberg, Arne Holst-Jensen, Jana Žel, David Dobnik, 2017, original scientific article Abstract: Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.
Keywords: droplet digital PCR, genetically modified organisms, soybean
Published in DiRROS: 25.07.2024; Views: 279; Downloads: 155
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1008. Impact of the COVID-19 epidemic on cancer burden and cancer care in Slovenia : a follow-up studyTina Žagar, Sonja Tomšič, Vesna Zadnik, Nika Bric, Mojca Birk, Blaž Vurzer, Ana Mihor, Katarina Lokar, Irena Oblak, 2022, original scientific article Abstract: In Slovenia, cancer care services were exempt from government decrees for COVID-19 containment. Nevertheless, cancer control can be impacted also by access to other health services and changes in health-seeking behaviour. In this follow up study, we explored changes in cancer burden and cancer care beyond the first months after the onset of the COVID-19 epidemic.Materials and methods. We analysed routinely collected data for the period January 2019 through July 2022 from three sources: (1) pathohistological and clinical practice cancer notifications from two major cancer centres in Ljubljana and Maribor (source: Slovenian Cancer Registry); (2) referrals issued for oncological services (source: e-referral system); and (3) outpatient appointments and diagnostic imaging performed (source: administrative data of the Institute of Oncology Ljubljana – IOL). Additionally, changes in certain clinical and demographic characteristics in patients diagnosed and treated during the epidemic were analysed using the Hospital-Based Cancer Registry of the IOL (period 2015–2021).Results. After a drop in referrals to follow-up cancer appointments in April 2020, in June-August 2020, there was an increase in referrals, but it did not make-up for the drop in the first wave; the numbers in 2021 and 2022 were even lower than 2020. Referrals to first cancer care appointments and genetic testing and counselling increased in 2021 compared to 2019 and in 2022 increased further by more than a quarter. First and follow-up outpatient appointments and cancer diagnostic imaging at the IOL dropped after the onset of the epidemic in March 2020 but were as high as expected according to 2019 baseline already in 2021. Some deficits remain for follow-up outpatients’ appointments in surgical and radiotherapy departments. There were more CT, MRI and PET scans performed during the COVID-19 period than before. New cancer diagnoses dropped in all observed years 2020, 2021 and until July 2022 by 6%, 3% and 8%, respectively, varying substantially by cancer type. The largest drop was seen in the 50−64 age group (almost 14% in 2020 and 16% in 2021), while for patients older than 80 years, the numbers were above expected according to the 2015–2019 average (4% in 2020, 8% in 2021).Conclusions. Our results show a varying effect of COVID-19 epidemic in Slovenia for different types of cancers and at different stages on the patient care pathway – it is probably a mixture of changes in health-seeking behaviour and systemic changes due to modifications in healthcare organisation on account of COVID-19. A general drop in new cancer cases reflects disruptions in the pre-diagnostic phase and could have profound long-term consequences on cancer burden indicators.
Keywords: cancer, covid-19, delay in diagnosis
Published in DiRROS: 25.07.2024; Views: 308; Downloads: 191
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