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Ključne besede: varstvo gozdov, morfološke analize, Geosmithia morbida, bolezen tisočerih rakov, qPCR, Juglans, program preiskav
Objavljeno v DiRROS: 30.08.2024; Ogledov: 182; Prenosov: 0
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Povzetek: Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer—PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins—mcyE, saxitoxins—sxtA, cylindrospermopsins—cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.
Ključne besede: extreme environments, cylindrospermopsin, sulphidic springs, caves, qPCR, PC-IGS
Objavljeno v DiRROS: 05.08.2024; Ogledov: 234; Prenosov: 152
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Povzetek: 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.
Ključne besede: CHO cell line, stable recombinant protein production, gene expression, RT-qPCR, DNA microarray, mMarker genes
Objavljeno v DiRROS: 29.07.2024; Ogledov: 269; Prenosov: 287
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Povzetek: Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10–100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM® C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods.
Ključne besede: rotavirus, norovirus, seawater, fecal contamination, qPCR, sewage
Objavljeno v DiRROS: 25.07.2024; Ogledov: 248; Prenosov: 132
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Povzetek: In this study, we investigated the importance of plant cover for secondary succession and soil fungal community development in remediated substrates after EDTA washing of metal-contaminated soils. The abundance of the total fungal community, determined by ITS fungal marker genes (Internal Transcribed Spacer region), and root colonisation by arbuscular mycorrhizal (AM) fungi were monitored in two types of soil material (calcareous and acidic) sown with perennial ryegrass (Lolium perenne L.) and without plant cover (bulk soil). Four months after the start of the experiment, the abundance of ITS genes in the soil clearly showed that the presence of plants was the main factor affecting the total fungal community, which increased in the rhizosphere soil in most treatments, while it remained at a low level in the bulk soil (without plants). Interestingly, the addition of environmental inoculum, i.e., rhizosphere soil from a semi-natural meadow, did not have a positive effect on the abundance of the total fungal community. While fungal ITS genes were detected in soils at the end of the first growing season, arbuscular mycorrhizal (AM) structures were scarce in Lolium roots in all treatments throughout the first season. However, in the second season, more than a year after the start of the experiment, AM fungal colonisation was detected in Lolium roots in virtually all treatments, with the frequency of colonised root length ranging from 30% to >75% in some treatments, the latter also in remediated soil. This study demonstrates the importance of plants and rhizosphere in the development and secondary succession of fungal communities in soil, which has important implications for the revitalisation of remediated soils and regenerative agriculture.
Ključne besede: heavy metals, arbuscular mycorrhiza, remediation, revitalisation, secondary succession, biodiversity, qPCR, toxic metals pollution
Objavljeno v DiRROS: 19.09.2022; Ogledov: 950; Prenosov: 462
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