41. Quantitative analysis of food and feed samples with droplet digital PCRDany Morisset, Dejan Štebih, Mojca Milavec, Kristina Gruden, Jana Žel, 2013, izvirni znanstveni članek Povzetek: In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.
Ključne besede: droplet digital PCR, ddPCR, genetically modified organisms
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42. A "crossomics" study analysing variability of different components in peripheral blood of health caucasoid individualsKristina Gruden, Matjaž Hren, Ana Herman, Andrej Blejec, Tanja Albrecht, Joachim Selbig, Chris Bauer, Jochannes Schuchardt, Michal Or-Guil, Klemen Zupančič, Urban Švajger, Borut Štabuc, Alojz Ihan, Andreja Nataša Kopitar, Maja Ravnikar, Miomir Knežević, Primož Rožman, Matjaž Jeras, 2012, izvirni znanstveni članek Povzetek: Background: Different immunotherapy approaches for the treatment of cancer andautoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. Methodology/Principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex ććcrossomicsćć analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4+T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. Conclusions/Significance: Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.
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43. Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamianaQing Liu, Mohammad Majdi, Katarina Cankar, Miriam Goedbloed, Tatsiana Charnikhova, Francel W. A. Verstappen, Ric de Vos, Jules Beekwilder, Sander van der Krol, Harro Bouwmeester, 2011, izvirni znanstveni članek Povzetek: The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase(GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here weshow that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resultedin a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g-1 FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.
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44. Erwinia amylovora novel plasmid pEI70 : complete sequence, biogeography, and role in aggressiveness in the fire blight phytopathogenPablo Llop, J. Cabrefiga, T. Smits, Tanja Dreo, Silvia Barbé, Joanna Pulawska, Alain Bultreys, Jochen Blom, Brion Duffy, Emilio Montesinos, María M. López, 2011, izvirni znanstveni članek Povzetek: Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.
Ključne besede: plant diseases, bacteria, plamid
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45. 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, izvirni znanstveni članek Povzetek: 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.
Ključne besede: plant viruses, plant diseases
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46. Characterization of GM events by insert knowledge adapted re-sequencing approachesLitao Yang, Congmao Wang, Arne Holst-Jensen, Dany Morisset, Yongjun Lin, Dabing Zhang, 2013, izvirni znanstveni članek Povzetek: Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events.
Ključne besede: molecular engineering in plants, DNA recombination, plant molecular biology, next-generation sequencing
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47. [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, izvirni znanstveni članek Povzetek: 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.
Ključne besede: plant biotechnology, plant-virus interaction, potato virus Y, agroinfiltration, beta-1, 3-glucanase
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48. Clinical and structural features of sperm head vacuoles in men included in the in vitro fertilization programmeNina Fekonja, Jasna Štrus, Magda Tušek-Žnidarič, Katja Knez, Eda Vrtačnik-Bokal, Ivan Verdenik, Irma Virant-Klun, 2014, izvirni znanstveni članek Povzetek: The human sperm head vacuoles and their role in male infertility are still poorly understood. The aim of this study was to identify the clinical and ultrastructural features of human sperm head vacuoles in men included in the in vitro fertilization programme: men with normal (normozoospermia) and impaired sperm morphology (teratozoospermia). The sperm samples were observed under 6000-time magnification using motile sperm organelle morphology examination (MSOME). The proportion of sperm with head vacuoles was evaluated and related to the outcome of in vitro fertilization. The sperm of men with impaired sperm morphology was characterized by a higher proportion of sperm head vacuoles. The sperm head vacuoles were related to impaired semen quality (sperm concentration, motility, and morphology) but were not influenced by male factors (semen volume, height, age, weight, or body mass index). Moreover, sperm head vacuoles were related to impaired fertilization rate merely after classical in vitro fertilization (IVF), while there was no relation to pregnancy. In a subgroup of men, the sperm was fixed and observed by transmission electron microscopy (TEM). The ultrastructural study revealed that sperm head vacuoles are large nuclear indentations of various sizes and positions, packed with membranous material organized in membrane whorls (MW).
Ključne besede: human sperm head vacuoles, male infertility
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49. Environmental footprint of external thermal insulation composite systems with different insulation typesTajda Potrč Obrecht, Katja Malovrh Rebec, Friderik Knez, Roman Kunič, Andraž Legat, 2016, objavljeni znanstveni prispevek na konferenci Povzetek: In terms of upcoming energy directive for Nearly Zero Energy Houses (nZEB), we are very much focused on building skin and its properties. Not only thermal characteristics and design, but also durability and environmental aspects should play a role, when deciding on which system will be implemented. External Thermal Insulation Composite Systems or ETICS are generally made of adhesive, insulation, render with mesh reinforcement, primer and finish coat. In the following case study we have presented a life cycle assessment (LCA) study of three ETICS with different types of insulation: expanded polystyrene (EPS), mineral wool and wood fiber board insulation. The study complies to the standard EN 15804:2012. It was conducted in the program Gabi using the Gabi Professional 2012 Database. The scope of the study is covering the production phase (raw material supply, transport to the factory, manufacturing). We have compared the functional unit of 1 sqm of the ETICS system with U-value 0.27 W/m2K taking into account different environmental impact categories. In the calculation the characterization factors proposed by Centre of Environmental Science (CML) at Leiden University were used. The comparison of ETICS shows the important impact of the insulation type used. Also there are some differences in the amount of other ETICS components applied, since changing the type of insulation affects the environmental footprint of the ETICS.
Ključne besede: ETICS, life cycle assessment, nearly zero energy house, insulition types
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50. An international comparability study on quantification of mRNA gene expression ratios: CCQM-P103.1Alison S. Devonshire, Tina Demšar, Jana Žel, Andrej Blejec, Mojca Milavec, 2016, izvirni znanstveni članek Povzetek: Measurement of RNA can be used to study and monitor a range of infectious and non-communicable diseases, with profiling of multiple gene expression mRNA transcripts being increasingly applied to cancer stratification and prognosis. An international comparison study (Consultative Committee for Amount of Substance (CCQM)-P103.1) was performed in order to evaluate the comparability of measurements of RNA copy number ratio for multiple gene targets between two samples. Six exogenous synthetic targets comprising of External RNA Control Consortium (ERCC) standards were measured alongside transcripts for three endogenous gene targets present in the background of human cell line RNA. The study was carried out under the auspices of the Nucleic Acids (formerly Bioanalysis) Working Group of the CCQM. It was coordinated by LGC (United Kingdom) with the support of National Institute of Standards and Technology (USA) and results were submitted from thirteen National Metrology Institutes and Designated Institutes. The majority of laboratories performed RNA measurements using RT-qPCR, with datasets also being submitted by two laboratories based on reverse transcription digital polymerase chain reaction and one laboratory using a next-generation sequencing method. In RT-qPCR analysis, the RNA copy number ratios between the two samples were quantified using either a standard curve or a relative quantification approach. In general, good agreement was observed between the reported results of ERCC RNA copy number ratio measurements. Measurements of the RNA copy number ratios for endogenous genes between the two samples were also consistent between the majority of laboratories. Some differences in the reported values and confidence intervals (‘measurement uncertainties’) were noted which may be attributable to choice of measurement method or quantification approach. This highlights the need for standardised practices for the calculation of fold change ratios and uncertainties in the area of gene expression profiling.
Ključne besede: RNA copy number ratio, RT-qPCR, gene expression, normalisation, standardisation, molecular diagnostic, transcriptomics, cancer, diagnostics, biomarker identification and validation
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