1081. Multi-marker metabarcoding approach to study mesozooplankton at basin scaleSergio Stefanni, David Stanković, Diego Borme, Alessandra De Olazabal, Tea Juretić, Alberto Pallavicini, Valentina Tirelli, 2018, original scientific article Abstract: Zooplankton plays a pivotal role in marine ecosystems and the characterisation of its biodiversity still represents a challenge for marine ecologists. In this study, mesozooplankton composition from 46 samples collected in summer along the western Adriatic Sea, was retrieved by DNA metabarcoding analysis. For the first time, the highly variable fragments of the mtDNA COI and the V9 region of 18S rRNA genes were used in a combined matrix to compile an inventory of mesozooplankton at basin scale. The number of sequences retrieved after quality filtering were 824,148 and 223,273 for COI and 18S (V9), respectively. The taxonomical assignment against reference sequences, using 95% (for COI) and 97% (for 18S) similarity thresholds, recovered 234 taxa. NMDS plots and cluster analysis divided coastal from offshore samples and the most representative species of these clusters were distributed according to the dominant surface current pattern of the Adriatic for the summer period. For selected sampling sites, mesozooplankton species were also identified under a stereo microscope providing insights on the strength and weakness of the two approaches. In addition, DNA metabarcoding was shown to be helpful for the monitoring of non-indigenous marine metazoans and spawning areas of commercial fish species. We defined pros and cons of applying this approach at basin scale and the benefits of combining the datasets from two genetic markers.
Keywords: zooplankton, genetics, biodiversity
Published in DiRROS: 24.07.2024; Views: 313; Downloads: 215
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1082. Cysteine cathepsins B, X and K expression in peri-arteriolar glioblastoma stem cell nichesBarbara Breznik, Clara Limbaeck Stanic, Janko Kos, Mohammed Khurshed, Vashendriya V. V. Hira, Roman Bošnjak, Tamara Lah Turnšek, Cornelis J. F. van Noorden, 2018, original scientific article Abstract: Glioblastoma (GBM) is the most lethal brain tumor also due to malignant and therapy-resistant GBM stem cells (GSCs) that are localized in protecting hypoxic GSC niches. Some members of the cysteine cathepsin family of proteases have been found to be upregulated in GBM. Cathepsin K gene expression is highly elevated in GBM tissue versus normal brain and it has been suggested to regulate GSC migration out of the niches. Here, we investigated the cellular distribution of cathepsins B, X and K in GBM tissue and whether these cathepsins are co-localized in GSC niches. Therefore, we determined expression of these cathepsins in serial paraffin sections of 14 human GBM samples and serial cryostat sections of two samples using immunohistochemistry and metabolic mapping of cathepsin activity using selective fluorogenic substrates. We detected cathepsins B, X and K in peri-arteriolar GSC niches in 9 out of 16 GBM samples, which were defined by co-expression of the GSC marker CD133, the niche marker stromal-derived factor-1α (SDF-1α) and smooth muscle actin as a marker for arterioles. The expression of cathepsin B and X was detected in stromal cells and cancer cells throughout the GBM sections, whereas cathepsin K expression was more restricted to arteriole-rich regions in the GBM sections. Metabolic mapping showed that cathepsin B, but not cathepsin K is active in GSC niches. On the basis of these findings, it is concluded that cathepsins B, X and K have distinct functions in GBM and that cathepsin K is the most likely GSC niche-related cathepsin of the three cathepsins investigated.
Keywords: cysteine cathepsins, glioblastoma stem cells, niches, stroma, proteolytic activity
Published in DiRROS: 24.07.2024; Views: 309; Downloads: 217
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1083. Molecular diversity of ʼCandidatus Phytoplasma maliʼ and ʼCa. P. prunorumʼ in orchards in SloveniaMarina Dermastia, Dorian Dolanc, Petra Mlinar, Nataša Mehle, 2018, original scientific article Abstract: Phytoplasmas from the 16Sr-X apple proliferation (AP) group are quarantine species in Europe and causal agents of the most important diseases of fruit trees within the family Rosaceae, namely apple proliferation, European stone fruit yellows and pear decline. In this study, a detailed insight into the molecular diversity of isolates of two phytoplasmas from the AP group, i. e. ‘Candidatus Phytoplasma mali’ and ‘Ca. P. prunorum’ obtained from different orchards in Slovenia, was estimated by a multilocus sequence typing, based on analysis of the genomic regions of aceF, pnp, secY and imp. With seven and five genotypes defined for ‘Ca. P. mali’ and ‘Ca. P. prunorum’ isolates, respectively, imp was the most variable among the applied markers. On the other hand, pnp was the least variable with three genotypes defined for ‘Ca. P. mali’ isolates and only one for ‘Ca. P. prunorum’ isolates. The presented results complete the survey of the AP group phytoplasma diversity in Slovenia, which has started with the recent analysis of the ‘Ca. P. pyri’. The comparison of results with those from several European countries shows an important genetic diversity of the Slovenian genotypes with some previously unknown. The genotype distribution reflects the geographic position of Slovenia. Additional grafting experiments with apricot trees tolerant to ‘Ca. P. prunorum’ demonstrated that the tolerance status is transmissible. Some possible mechanisms involved in the process are discussed.
Keywords: apple proliferation, multilocus sequence typing, phytoplasma diversity, Slovenia
Published in DiRROS: 24.07.2024; Views: 270; Downloads: 191
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1084. ITS2 metabarcoding analysis complements lichen mycobiome diversity dataElisa Banchi, David Stanković, Fernando Fernández-Mendoza, Fabrizia Gionechetti, Alberto Pallavicini, Lucia Muggia, 2018, original scientific article Abstract: Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a well-characterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes.
Keywords: ascomycetes, basidiomycetes, endophytes, fungal isolates, ion torrent, ITS1
Published in DiRROS: 24.07.2024; Views: 282; Downloads: 226
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1085. Ecotoxicity of disinfectant benzalkonium chloride and its mixture with antineoplastic drug 5-fluorouracil towards alga Pseudokirchneriella subcapitataTina Eleršek, Maja Ženko, Metka Filipič, 2018, original scientific article Abstract: Background
Benzalkonium chloride (BAC) is one of the most common ingredients of the disinfectants. It is commonly detected in surface and wastewaters where it can interact with the residues of pharmaceuticals that are also common wastewater pollutants. Among the latter, the residues of antineoplastic drugs are of particular concern as recent studies showed that they can induce adverse effect in aquatic organisms at environmentally relevant concentrations.
Methods
Ecotoxicity of BAC as an individual compound and in a binary mixture with an antineoplastic drug 5-fluorouracil (5-FU) was determined towards alga Pseudokirchneriella subcapitata, a representative of primary producers. The toxicity of the BAC+5-FU binary mixture was predicted by the two basic models: concentration addition (CA) and independent action (IA), and compared to the experimentally determined toxicity. Additionally combination index (CI) was calculated to determine the type of interaction.
Results
After 72 h exposure to BAC a concentration dependent growth inhibition of P. subcapitata was observed with an EC50 0.255 mg/L. Comparing the predicted no effect concentration to the measured concentrations in the surface waters indicate that BAC at current applications and occurrence in aquatic environment may affect algal populations. The measured toxicity of the mixture was higher from the predicted and calculated CI confirmed synergistic effect on the inhibition of algal growth, at least at EC50 concentration. The observed synergism may have impact on the overall toxicity of wastewaters, whereas it is less likely for general environments because the concentrations of 5-FU are several orders of magnitude lower from its predicted no effect concentration.
Discussion
These results indicate that combined effects of mixtures of disinfectants and antineoplastic drugs should be considered in particular when dealing with environmental risk assessment as well as the management of municipal and hospital wastewaters.
Keywords: ecotoxicity, benzalkonium chloride, algae, 5-fluorouracil, synergistic inhibition
Published in DiRROS: 24.07.2024; Views: 288; Downloads: 378
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1086. The pattern of 1-aminocyclopropane-1-carboxylate oxidase induction in the tomato leaf petiole abscission zone is independent of expression of the ribonuclease-LX-encoding LeLX geneMarko Chersicola, Aleš Kladnik, Magda Tušek-Žnidarič, Amnon Lers, Marina Dermastia, 2018, original scientific article Abstract: The abscission of tomato leaves occurs in the petiole abscission zone, and its late stage includes two spatially divided processes: cell separation and programmed cell death (PCD). Both of these processes are regulated by ethylene. The last step in ethylene biosynthesis is conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene, which is catalysed by the enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO); however, the location of ACO in the leaf petiole abscission zone is not known. The tomato gene LeLX encodes ribonuclease LX, which is a marker for PCD and is induced by ethylene during abscission, but its association with ACO has not been explored.
In a tomato transgenic line 1-7 with inhibited expression of LeLX showing delayed leaf abscission, the morphology and ultrastructure of the leaf petiole abscission zone was examined. In this zone of the cv.’VF36’ and of a transgenic line 1-7, spatiotemporal differences in expression of LeACO1 and LeACO4 were analysed and ACO protein was detected immunohistochemically.
In comparison to wild-type plants, there were no obvious morphological and ultrastructural features in the abscission zone of plants of a transgenic line 1-7 before and after abscission induction. LeACO1 expression was low before abscission induction, and increased 24 h after induction, although with no apparent spatial pattern. In contrast, LeACO4 was expressed before abscission induction, and its transcript level declined 24 h after induction on the distal side of the abscission zone fracture. In the LeLX-inhibited transgenic line, there were no significant differences in LeACO1 and LeACO4 expression in the petiole abscission zone, in comparison to wild-type plants. In addition, the ACO protein was immunolocalised to the vascular tissues that traverse the petiole abscission zone in plants of wild type and of a transgenic line 1-7; and additionally in the plane of future abscission zone fracture of transgenic-line plants.
The results suggest temporal differential expression of the LeACO genes in tomato leaf petioles and vascular localisation of ACO1 protein. Additionally, the results indicate that expression of LeACO genes is not affected by suppression of the LeLX expression.
Keywords: abscission, 1-aminocyclopropane-1-carboxylate oxidase, ethylene, gene expression, localization, RNase LX
Published in DiRROS: 24.07.2024; Views: 301; Downloads: 168
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1089. Temperature effects explain continental scale distribution of cyanobacterial toxinsEvanthia Mantzouki, Tina Eleršek, 2018, original scientific article Abstract: nsight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains.
Keywords: microcystin, anatoxin, cylindrospermopsin, temperature, direct effects, indirect effects, spatial distribution, European Multi Lake Survey
Published in DiRROS: 24.07.2024; Views: 274; Downloads: 246
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