1071. Accurate quantification and characterization of adeno-associated viral vectorsDavid Dobnik, Polona Kogovšek, Tjaša Jakomin, Nejc Košir, Magda Tušek-Žnidarič, Maja Leskovec, Stephen M. Kaminsky, Janet Mostrom, Hyunmi Lee, Maja Ravnikar, 2019, original scientific article Abstract: One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.
Keywords: absolute quantification, AAV, gene therapy, electron microscopy, digital PCR, real-time PCR
Published in DiRROS: 23.07.2024; Views: 323; Downloads: 160
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1072. Genomic evidence of recombination in the basidiomycete Wallemia mellicolaSun Xiaohuan, Cene Gostinčar, Fang Chao, Janja Zajc, Hou Yong, Song Zewei, Nina Gunde-Cimerman, 2019, original scientific article Abstract: One of the most commonly encountered species in the small basidiomycetous sub-phylum Wallemiomycotina is Wallemia mellicola, a xerotolerant fungus with a widespread distribution. To investigate the population characteristics of the species, whole genomes of twenty-five strains were sequenced. Apart from identification of four strains of clonal origin, the distances between the genomes failed to reflect either the isolation habitat of the strains or their geographical origin. Strains from different parts of the world appeared to represent a relatively homogenous and widespread population. The lack of concordance between individual gene phylogenies and the decay of linkage disequilibrium indicated that W. mellicola is at least occasionally recombining. Two versions of a putative mating-type locus have been found in all sequenced genomes, each present in approximately half of the strains. W. mellicola thus appears to be capable of (sexual) recombination and shows no signs of allopatric speciation or specialization to specific habitats.
Keywords: population genomics, halotolerance, xerotolerance, basidiomycete, allergenic fungus, recombination
Published in DiRROS: 23.07.2024; Views: 283; Downloads: 189
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1073. Digital PCR as an effective tool for GMO quantification in complex matricesAlexandra Bogožalec Košir, Tina Demšar, Dejan Štebih, Jana Žel, Mojca Milavec, 2019, original scientific article Abstract: The increased use of genetically modified organisms (GMOs) is accompanied by increased complexity of the matrices that contain GMOs. The most common DNA-based approach for GMO detection and quantification is real-time quantitative polymerase chain reaction (qPCR). However, as qPCR is sensitive to inhibitors and relies on standard curves for quantification, it has limited application in GMO quantification for complex matrices. To overcome this hurdle in DNA quantification, we present droplet digital PCR (ddPCR) assays that were designed to target ‘Roundup Ready’ soybean and the soybean reference gene. Three ddPCR assays were transferred from qPCR to QX100/QX200 ddPCR platforms and characterised. Together, the fitness-for-purpose study on four real-life samples and the use of a chamber-based PCR system, showed that dPCR has great potential to improve such measurements in GMO testing and monitoring of food authenticity.
Keywords: genetically modified organisms, digital PCR, GMO quantification, complex matrices
Published in DiRROS: 23.07.2024; Views: 260; Downloads: 163
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1074. Poročilo o preskusu št.: LVG 2024-074 : vzorec št. 2024/00385Nikica Ogris, Špela Hočevar, Patricija Podkrajšek, Barbara Piškur, 2024, expertise, arbitration decision Keywords: varstvo gozdov, morfološke analize, Fusarium circinatum, Pinus, borov smolasti rak, PCR
Published in DiRROS: 23.07.2024; Views: 267; Downloads: 0
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1075. Cold atmospheric plasma as a novel method for inactivation of potato virus Y in water samplesArijana Filipić, Gregor Primc, Rok Zaplotnik, Nataša Mehle, Ion Gutiérrez-Aguirre, Maja Ravnikar, Miran Mozetič, Jana Žel, David Dobnik, 2019, original scientific article Abstract: While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for inactivation of viruses in water used in closed irrigation systems. We measured the plasma-mediated viral RNA degradation as well as the plasma-induced loss of viral infectivity using potato virus Y as a model virus due to its confirmed water transmissibility and economic as well as biological importance. We showed that only 1 min of plasma treatment is sufficient for successful inactivation of viruses in water samples with either high or low organic background. The plasma-mediated inactivation was efficient even at markedly higher virus concentrations than those expected in irrigation waters. Obtained results point to reactive oxygen species as the main mode of viral inactivation. Our laboratory-scale experiments confirm for the first time that plasma has an excellent potential as the eukaryotic virus inactivation tool for water sources and could thus provide a cost-effective solution for irrigation mediated plant virus transmission. The outstanding inactivation efficiency demonstrated by plasma treatments in water samples offers further expansions of its application to other water sources such as reused wastewater or contaminated drinking waters, as well as other plant, animal, and human waterborne viruses, ultimately leading to the prevention of water scarcity and numerous human, animal, and plant infections worldwide.
Keywords: cold atmospheric plasma, potato virus Y, virus inactivation, water decontamination
Published in DiRROS: 23.07.2024; Views: 248; Downloads: 184
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1076. Poročilo o preskusu št.: LVG 2024-072 : vzorec št. 2024/00313Tine Hauptman, Špela Hočevar, Barbara Piškur, 2024, expertise, arbitration decision Keywords: varstvo gozdov, morfološke analize, program preiskav, Geosmithia morbida, bolezen tisočerih rakov, vektorji, rtPCR
Published in DiRROS: 23.07.2024; Views: 282; Downloads: 0
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1079. Poročilo o preskusu št.: LVG 2024-063 : vzorec št. 2024/00185Tine Hauptman, Špela Hočevar, Barbara Piškur, 2024, expertise, arbitration decision Keywords: varstvo gozdov, morfološke analize, program preiskav, Geosmithia morbida, bolezen tisočerih rakov, vektorji, rtPCR
Published in DiRROS: 23.07.2024; Views: 256; Downloads: 0
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1080. Who cares about ocean acidification in the Plasticene?Rachel Tiller, Francisco Arenas, Charles Galdies, Francisco Leitão, Alenka Malej, Beatriz Martinez Romera, Cosimo Solidoro, Robert Stojanov, Valentina Turk, Roberta Guerra, 2019, original scientific article Abstract: Plastics is all the rage, and mitigating marine litter is topping the agenda for nations pushing issues such as ocean acidification, or even climate change, away from the public consciousness. We are personally directly affected by plastics and charismatic megafauna is dying from it, and it is something that appears to be doable. So, who cares about the issue of ocean acidification anymore? We all should. The challenge is dual in the fact that is both invisible to the naked eye and therefore not felt like a pressing issue to the public, thereby not reaching the top of the agenda of policy makers; but also that it is framed in the climate change narrative of fear - whereby it instills in a fight-or-flight response in the public, resulting in their avoidance of the issue because they feel they are unable to take action that have results. In this article, we argue that the effective global environmental governance of ocean acidification, though critical to address, mitigate against and adapt to, is hindered by the both this lack of perception of urgency in the general public, fueled by a lack of media coverage, as well as a fight-or-flight response resulting from fear. We compare this to the more media friendly and plastics problem that is tangible and manageable. We report on a media plots of plastics and ocean acidification coverage over time and argue that the issue needs to be detangled from climate change and framed as its own issue to reach the agenda at a global level, making it manageable to assess and even care about for policy makers and the public alike?
Keywords: oceans, acidification, plastics
Published in DiRROS: 23.07.2024; Views: 291; Downloads: 140
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