11. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detectionDavid Dobnik, Dejan Štebih, Andrej Blejec, Dany Morisset, Jana Žel, 2016, original scientific article Abstract: The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.
Keywords: digital PCR, DNA targets, GMO detection
Published in DiRROS: 25.07.2024; Views: 156; Downloads: 96
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12. Solanum venturii, a suitable model system for virus-induced gene silencing studies in potato reveals StMKK6 as an important player in plant immunityDavid Dobnik, Ana Lazar, Tjaša Stare, Kristina Gruden, Vivianne G. A. A. Vleeshouwers, Jana Žel, 2016, original scientific article Abstract: Background
Virus-induced gene silencing (VIGS) is an optimal tool for functional analysis of genes in plants, as the viral vector spreads throughout the plant and causes reduced expression of selected gene over the whole plant. Potato (Solanum tuberosum) is one of the most important food crops, therefore studies performing functional analysis of its genes are very important. However, the majority of potato cultivars used in laboratory experimental setups are not well amenable to available VIGS systems, thus other model plants from Solanaceae family are used (usually Nicotiana benthamiana). Wild potato relatives can be a better choice for potato model, but their potential in this field was yet not fully explored. This manuscript presents the set-up of VIGS, based on Tobacco rattle virus (TRV) in wild potato relatives for functional studies in potato–virus interactions.
Results
Five different potato cultivars, usually used in our lab, did not respond to silencing of phytoene desaturase (PDS) gene with TRV-based vector. Thus screening of a large set of wild potato relatives (different Solanum species and their clones) for their susceptibility to VIGS was performed by silencing PDS gene. We identified several responsive species and further tested susceptibility of these genotypes to potato virus Y (PVY) strain NTN and N. In some species we observed that the presence of empty TRV vector restricted the movement of PVY. Fluorescently tagged PVYN-GFP spread systemically in only five of tested wild potato relatives. Based on the results, Solanum venturii (VNT366-2) was selected as the most suitable system for functional analysis of genes involved in potato–PVY interaction. The system was tested by silencing two different plant immune signalling-related kinases, StWIPK and StMKK6. Silencing of StMKK6 enabled faster spreading of the virus throughout the plant, while silencing of WIPK had no effect on spreading of the virus.
Conclusions
The system employing S. venturii (VNT366-2) and PVYN-GFP is a suitable method for fast and simple functional analysis of genes involved in potato–PVY interactions. Additionally, a set of identified VIGS responsive species of wild potato relatives could serve as a tool for general studies of potato gene function.
Keywords: potato, virus-induced gene silencing, VIGS, potato virus Y, PVY, Solanum venturii, StWIPK, StMKK6, TRV
Published in DiRROS: 25.07.2024; Views: 141; Downloads: 136
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14. Identification of proteins associated with clinical and pathological features of proliferative diabetic retinopathy in vitreous and fibrovascular membranesIngeborg Klaassen, Ewout W. de Vries, Ilse M.C. Vogels, Antoine H. C. van Kampen, Machteld I. Bosscha, David H. W. Steel, Cornelis J. F. van Noorden, Sarit Y Lesnik-Oberstein, Reinier O. Schlingemann, 2017, original scientific article Abstract: Purpose
To identify the protein profiles in vitreous associated with retinal fibrosis, angiogenesis, and neurite formation in epiretinal fibrovascular membranes (FVMs) in patients with proliferative diabetic retinopathy (PDR).
Methods
Vitreous samples of 5 non-diabetic control patients with vitreous debris and 7 patients with PDR membranes were screened for 507 preselected proteins using the semi-quantitative RayBio® L-series 507 antibody array. From this array, 60 proteins were selected for a custom quantitative antibody array (Raybiotech, Human Quantibody® array), analyzing 7 control patients, 8 PDR patients with FVMs, and 5 PDR patients without FVMs. Additionally, mRNA levels of proteins of interest were measured in 10 PDR membranes and 11 idiopathic membranes and in retinal tissues and cells to identify possible sources of protein production.
Results
Of the 507 proteins screened, 21 were found to be significantly elevated in PDR patients, including neurogenic and angiogenic factors such as neuregulin 1 (NRG1), nerve growth factor receptor (NGFR), placental growth factor (PlGF) and platelet derived growth factor (PDGF). Angiopoietin-2 (Ang2) concentrations were strongly correlated to the degree of fibrosis and the presence of FVMs in patients with PDR. Protein correlation analysis showed PDGF to be extensively co-regulated with other proteins, including thrombospondin-1 and Ang2. mRNA levels of glial-derived and brain/derived neurotrophic factor (GDNF and BDNF) were elevated in PDR membranes. These results were validated in a second study of 52 vitreous samples of 32 PDR patients and 20 control patients.
Conclusions
This exploratory study reveals protein networks that potentially contribute to neurite outgrowth, angiogenesis and fibrosis in the formation of fibrovascular membranes in PDR. We identified a possible role of Ang2 in fibrosis and the formation of FVMs, and of the neurotrophic factors NRG1, PDGF and GDNF in neurite growth that occurs in all FVMs in PDR.
Published in DiRROS: 25.07.2024; Views: 113; Downloads: 132
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15. Salicylic acid perturbs sRNA-gibberellin regulatory network in immune response of potato to Potato virus Y infectionMaja Križnik, Marko Petek, David Dobnik, Živa Ramšak, Špela Baebler, Stephan Pollmann, Jan F. Kreuze, Jana Žel, Kristina Gruden, 2017, original scientific article Abstract: Potato virus Y is the most economically important potato viral pathogen. We aimed at unraveling the roles of small RNAs (sRNAs) in the complex immune signaling network controlling the establishment of tolerant response of potato cv. Désirée to the virus. We constructed a sRNA regulatory network connecting sRNAs and their targets to link sRNA level responses to physiological processes. We discovered an interesting novel sRNAs-gibberellin regulatory circuit being activated as early as 3 days post inoculation (dpi) before viral multiplication can be detected. Two endogenous sRNAs, miR167 and phasiRNA931 were predicted to regulate gibberellin biosynthesis genes GA20-oxidase and GA3-oxidase. The increased expression of phasiRNA931 was also reflected in decreased levels of GA3-oxidase transcripts. Moreover, decreased concentration of gibberellin confirmed this regulation. The functional relation between lower activity of gibberellin signaling and reduced disease severity was previously confirmed in Arabidopsis-virus interaction using knockout mutants. We further showed that this regulation is salicylic acid-dependent as the response of sRNA network was attenuated in salicylic acid-depleted transgenic counterpart NahG-Désirée expressing severe disease symptoms. Besides downregulation of gibberellin signaling, regulation of immune receptor transcripts by miR6022 as well as upregulation of miR164, miR167, miR169, miR171, miR319, miR390, and miR393 in tolerant Désirée, revealed striking similarities to responses observed in mutualistic symbiotic interactions. The intertwining of different regulatory networks revealed, shows how developmental signaling, disease symptom development, and stress signaling can be balanced.
Keywords: gibberellin, miRNA/siRNA, plant immunity, potato, Potato virus Y, salicylic acid, symbiosis, tolerance
Published in DiRROS: 25.07.2024; Views: 132; Downloads: 85
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16. 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: 149; Downloads: 102
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17. 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: 132; Downloads: 62
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18. Abbreviated 13C-mixed triglyceride breath test for detection of pancreatic exocrine insufficiency performs equally as standard 5-hour test in patients after gastrectomy performed for gastric cancerDarko Siuka, Kristina Kumer, Borut Štabuc, David Štubljar, David Drobne, Rado Janša, 2022, original scientific article Keywords: abbreviated 13C-mixed triglyceride breath test, pancreatic exocrine insufficiency, gastrectomy, faecal elastase, gastric cancer
Published in DiRROS: 25.07.2024; Views: 232; Downloads: 47
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19. Zoantharians (Hexacorallia: Zoantharia) associated with cold-water corals in the Azores Region: new species and associations in the deep seaMarina Carreiro-Silva, Oscar Ocaňa, David Stanković, ĺris Sampaio, Filipe M. Porteiro, Marie-Claire Fabri, Sergio Stefanni, 2017, original scientific article Abstract: Zoantharians are a group of cnidarians that are often found in association with marine invertebrates, including corals, in shallow and deep-sea environments. However, little is known about deep-sea zoantharian taxonomy, specificity and nature of their associations with their coral hosts. In this study, analyses of molecular data (mtDNA COI, 16S, and 12S rDNA) coupled with ecological and morphological characteristics were used to examine zoantharian specimens associated with cold-water corals (CWC) at depths between 110 and 800 m from seamounts and island slopes in the Azores region. The zoantharians examined were found living in association with stylasterids, antipatharians and octocorals. From the collected specimens, four new species were identified: (1) Epizoanthus martinsae sp. n. associated with the antipatharian Leiopathes sp.; (2) Parazoanthus aliceae sp. n. associated with the stylasterid Errina dabneyi (Pourtalès, 1871); (3) Zibrowius alberti sp. n. associated with octocorals of the family Primnoidae [Paracalyptrophora josephinae (Lindström, 1877)] and the family Plexauridae (Dentomuricea aff. meteor Grasshoff, 1977); (4) Hurlizoanthus hirondelleae sp. n. associated with the primnoid octocoral Candidella imbricata (Johnson, 1862). In addition, based on newly collected material, morphological and molecular data and phylogenic reconstruction, the zoantharian Isozoanthus primnoidus Carreiro-Silva, Braga-Henriques, Sampaio, de Matos, Porteiro & Ocaña, 2011, associated with the primnoid octocoral Callogorgia verticillata (Pallas, 1766), was reclassified as Zibrowius primnoidus comb. nov. The zoantharians, Z. primnoidus comb. nov., Z. alberti sp. n., and H. hirondelleae sp. n. associated with octocorals showed evidence of a parasitic relationship, where the zoantharian progressively eliminates gorgonian tissue and uses the gorgonian axis for structure and support, and coral sclerites for protection. In contrast, the zoantharian P. aliceae sp. n. associated with the stylasterid E. dabneyi and the zoantharian E. martinsae sp. n. associated with the antipatharian Leiopathes sp., appear to use the coral host only as support with no visible damage to the host. The monophyly of octocoral-associated zoantharians suggests that substrate specificity is tightly linked to the evolution of zoantharians.
Keywords: antipatharians, octocorals, Azores, gorgonians, molecular taxonomy, phylogeny, stylasterids
Published in DiRROS: 24.07.2024; Views: 134; Downloads: 104
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20. Testing the influence of sediment granulometry on heterotrophic respiration with a new laboratory flow-through systemNataša Mori, Barbara Debeljak, David Kocman, Tatjana Simčič, 2017, original scientific article Abstract: Purpose
Increased sedimentation due to land use intensification is increasingly affecting carbon processing in streams and rivers around the globe. This study describes the design of a laboratory-scale flow-through incubation system as a tool for the rapid estimation of sediment respiration. The measurements were compared with those obtained using an in situ closed chamber respiration method. The influence of sediment size on respiration rates was also investigated.
Materials and methods
Measurements were conducted on a pre-alpine gravel-bed river sediment separated into the following grain size fractions: > 60 mm (14.3%), 60–5 mm (60.2%), 5–2 mm (13.7%), 2–0.063 mm (11.1%) and <0.063 mm (0.6%). Concurrently, in situ and laboratory measurements were carried out on a naturally heterogeneous sediment. In situ respiration was determined in closed chambers as O2 consumption over time, while in the laboratory, respiration was determined using flow-through respiration chambers. Oxygen concentrations were measured using a fibre-optic oxygen meter positioned at the inflow and outflow from the chamber.
Results and discussion
The mean respiration rates within naturally mixed riverbed sediments were 1.27 ± 0.3 mg O2 dm−3 h−1 (n = 4) and 0.77 ± 0.1 mg O2 dm−3 h−1 (n = 3) for the flow-through chamber system and closed chamber system, respectively. Respiration rates were statistically significantly higher in the flow-through chamber system (t test, p < 0.05), indicating that closed chamber measurements underestimated the oxygen consumption within riverbed sediments. Sediment grain size was found to significantly affect respiration rates in both systems (ANOVA, p < 0.001) with the fine sediment fraction (particle size <0.063 mm) having the highest respiration rate (rflow-through = 51 ± 23 mg O2 dm−3 h−1). The smallest fractions (2–0.063 and <0.063 mm), which represent approximately 12% of total sediment volume, contributed 60% of total respiration.
Conclusions
The study demonstrated that flow-through respiration chambers more accurately estimate the respiration rate within riverbed sediments than in situ closed chambers, since the former experiment imitates the natural conditions where continuous interstitial flow occurs in the sediments. We also demonstrated that fine sediments (<5 mm) substantially contribute to heterotrophic respiration in the studied gravel-bed river.
Keywords: carbon fluxes, freshwaters, geomorphology, hyporheic zone, respiration, sediments
Published in DiRROS: 24.07.2024; Views: 123; Downloads: 85
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