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Abstract: Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.
Keywords: extraction methods, genetically modified organisms, GMO, decision- making system (biology), GMO testing, cetyltrimethylammonium bromide, CTAB
Published in DiRROS: 29.07.2024; Views: 12; Downloads: 8
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Abstract: The Indo-Pacific egg-carrying copepod Pseudodiaptomus marinus Sato, 1913 was recorded for the first time in the Port of Koper, Slovenia (Gulf of Trieste) in February 2015. This is the fourth finding of this species in the Mediterranean Sea, and the third for the Adriatic Sea. A rather high abundance of 73 ind. m-3 was recorded, which is considerably higher than previous findings in the Adriatic. Maritime transport is presumed to be the main cause of its introduction, primarily through ballast water release. Repeat sampling in May confirmed the presence of this copepod, indicating the possibility that the species has established a stable population in the Port of Koper.
Keywords: copepods, Adriatic Sea, mesozooplankton, ballast waters
Published in DiRROS: 29.07.2024; Views: 21; Downloads: 8
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Abstract: Background Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage. Results The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development. Conclusions The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development.
Keywords: CHO cell line, stable recombinant protein production, gene expression, RT-qPCR, DNA microarray, mMarker genes
Published in DiRROS: 29.07.2024; Views: 15; Downloads: 7
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Abstract: Glioblastoma multiforme is the most lethal of brain cancer, and it comprises a heterogeneous mixture of functionally distinct cancer cells that affect tumor progression. We examined the U87, U251, and U373 malignant cell lines as in vitro models to determine the impact of cellular cross-talk on their phenotypic alterations in co-cultures. These cells were also studied at the transcriptome level, to define the mechanisms of their observed mutually affected genomic stability, proliferation, invasion and resistance to temozolomide. This is the first direct demonstration of the neural and mesenchymal molecular fingerprints of U87 and U373 cells, respectively. U87-cell conditioned medium lowered the genomic stability of U373 (U251) cells, without affecting cell proliferation. In contrast, upon exposure of U87 cells to U373 (U251) conditioned medium, U87 cells showed increased genomic stability, decreased proliferation rates and increased invasion, due to a plethora of produced cytokines identified in the co-culture media. This cross talk altered the expression 264 genes in U87 cells that are associated with proliferation, inflammation, migration, and adhesion, and 221 genes in U373 cells that are associated with apoptosis, the cell cycle, cell differentiation and migration. Indirect and direct co-culturing of U87 and U373 cells showed mutually opposite effects on temozolomide resistance. In conclusion, definition of transcriptional alterations of distinct glioblastoma cells upon co-culturing provides better understanding of the mechanisms of glioblastoma heterogeneity, which will provide the basis for more informed glioma treatment in the future.
Keywords: glioblastoma heterogeneity, U87 cells, temozolomide resistance, cellular cross-talk, transcriptomics
Published in DiRROS: 29.07.2024; Views: 20; Downloads: 5
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Abstract: In 2014, 162 bird species were recorded during the bird ringing activities in Slovenia. Of 155 species, 62,275 birds were ringed, and 107 recoveries of birds ringed in Slovenia and found abroad, 148 foreign recoveries in Slovenia and 1395 local recoveries were recorded. The most frequently ringed species were Blackcap Sylvia atricapilla and Great Tit Parus major. As far as ringed nestlings are concerned, Great Tits and Barn Swalllows Hirundo rustica predominated. Considering the recoveries ringed of found birds abroad, the commonest were Black-headed Gulls Chroicocephalus ridibundus and Mute Swans Cygnus olor. The farthest recovery was a Barn Swallow Hirundo rustica found in the Democratic Republic of Congo (5171 km away). Among the more interesting finds was also the so far southernmost recovery of a Sand Martin Riparia riparia found in Israel. Let us also mention the first recovery of a Corncrake Crex crex, which bred and was ringed in 2013 at Planinsko polje (central Slovenia) and was found in the 2014 breeding season in the Czech Republic. Among rare species, two Little Buntings Emberiza pusilla were caught and ringed. After nine years, the Roller Coracias garrulus bred again in Slovenia in 2014 and its nestlings were ringed. The paper also brings the description of the migration route of the first African migrant, the Black Stork Ciconia nigra, marked with a GPS/GSM telemetric device, which migrated across the Adriatic Sea, Sicily and Sahara to Nigeria.
Keywords: obročkanje, telemetrija, črna štorklja, najdbe (biologija)
Published in DiRROS: 29.07.2024; Views: 19; Downloads: 9
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