131. Loop-mediated isothermal amplification : rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultryPolona Kogovšek, Jerneja Ambrožič, Alenka Dovč, Tanja Dreo, Hristo Hristov, Uroš Krapež, Maja Ravnikar, Brigita Slavec, Marjetka Lotrič, Jana Žel, Olga Zorman-Rojs, 2018, original scientific article Abstract: Infections with pathogenic Escherichia colican lead to different animal- and human-associated diseases. E. coliinfections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli(APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. colispread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in <35 min, and it can be applied to other important microorganisms to allow the required prompt measures to be taken.
Keywords: APEC virulence genes, isothermal amplification LAMP, on-site detection, colibacillosis
Published in DiRROS: 24.07.2024; Views: 28; Downloads: 6
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132. High-throughput sequencing facilitates characterisation of a ʺforgottenʺ plant virus : the case of a henbane mosaic virus infecting tomatoAnja Pecman, Denis Kutnjak, Nataša Mehle, Magda Tušek-Žnidarič, Ion Gutiérrez-Aguirre, Patricija Pirnat, Ian Adams, Neil Boonham, Maja Ravnikar, 2018, original scientific article Abstract: High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, Potyviridae), which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M (Carlavirus, Betaflexiviridae), Southern tomato virus (Amalgavirus, Amalgamaviridae) and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.
Keywords: henbane mosaic virus, Tomato, high-throughput sequencing, host range analysis, phylogeny, Potyvirus
Published in DiRROS: 24.07.2024; Views: 17; Downloads: 7
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133. Localization patterns of cathepsins K and X and their predictive value in glioblastomaBarbara Breznik, Clara Limbaeck Stanic, Andrej Porčnik, Andrej Blejec, Miha Koprivnikar Krajnc, Roman Bošnjak, Janko Kos, Cornelis J. F. van Noorden, Tamara Lah Turnšek, 2018, original scientific article Abstract: Background
Glioblastoma is a highly aggressive central nervous system neoplasm characterized by extensive infiltration of malignant cells into brain parenchyma, thus preventing complete tumor eradication. Cysteine cathepsins B, S, L and K are involved in cancer progression and are overexpressed in glioblastoma. We report here for the first time that cathepsin X mRNA and protein are also abundantly present in malignant glioma.
Materials and methods
Gene expression of cathepsins K and X was analyzed using publically-available tran-scriptomic datasets and correlated with glioma grade and glioblastoma subtype. Kaplan-Maier survival analysis was performed to evaluate the predictive value of cathepsin K and X mRNA expression. Cathepsin protein expression was localized and semi-quantified in tumor tissues by immunohistochemistry.
Results
Highest gene expression of cathepsins K and X was found in glioblastoma, in particular in the mesenchymal subtype. Overall, high mRNA expression of cathepsin X, but not that of cathepsin K, correlated with poor patients’ survival. Cathepsin K and X proteins were abundantly and heterogeneously expressed in glioblastoma tissue. Immuno-labeling of cathepsins K and X was observed in areas of CD133-positive glioblastoma stem cells, localized around arterioles in their niches that also expressed SDF-1α and CD68. mRNA levels of both cathepsins K and X correlated with mRNA levels of markers of glioblastoma stem cells and their niches.
Conclusions
The presence of both cathepsins in glioblastoma stem cell niche regions indicates their possible role in regulation of glioblastoma stem cell homing in their niches. The clinical relevance of this data needs to be elaborated in further prospective studies.
Keywords: cathepsins, glioblastoma, immunohistochemistry, patient survival, cancer stem cell niches
Published in DiRROS: 24.07.2024; Views: 20; Downloads: 8
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134. A new species of Stenasellus Dollfus, 1897 from Iran, with a key to the western Asian species (Crustacea, Isopoda, Stenasellidae)Valiallah Khalaji-Pirbalouty, Yaser Fatemi, Mohammad Javad Malek-Hosseini, Matjaž Kuntner, 2018, original scientific article Abstract: A new stenasellid isopod is described from Tashan Cave, Khuzestan Province, south-west Iran, belonging to the genus Stenasellus Dollfus, 1897. The first recorded species of Stenasellidae from Iran, Stenasellus tashanicus sp. n., is diagnosed by the presence of antennae with a minute squama bearing paired, long, robust setae; a maxilliped endite with six coupling hooks; and slender appendix masculina with an acute apex. A revised generic diagnosis is provided with a key to the six known western Asian Stenasellus species.
Keywords: Stenasellidae, Tashan Cave (Iran)
Published in DiRROS: 24.07.2024; Views: 31; Downloads: 10
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135. DiNAR: revealing hidden patterns of plant signalling dynamics using Diferential Network Analysis in RMaja Zagorščak, Andrej Blejec, Živa Ramšak, Marko Petek, Tjaša Stare, Kristina Gruden, 2018, original scientific article Abstract: Background
Progress in high-throughput molecular methods accompanied by more complex experimental designs demands novel data visualisation solutions. To specifically answer the question which parts of the specifical biological system are responding in particular perturbation, integrative approach in which experimental data are superimposed on a prior knowledge network is shown to be advantageous.
Results
We have developed DiNAR, Differential Network Analysis in R, a user-friendly application with dynamic visualisation that integrates multiple condition high-throughput data and extensive biological prior knowledge. Implemented differential network approach and embedded network analysis allow users to analyse condition-specific responses in the context of topology of interest (e.g. immune signalling network) and extract knowledge concerning patterns of signalling dynamics (i.e. rewiring in network structure between two or more biological conditions). We validated the usability of software on the Arabidopsis thaliana and Solanum tuberosum datasets, but it is set to handle any biological instances.
Conclusions
DiNAR facilitates detection of network-rewiring events, gene prioritisation for future experimental design and allows capturing dynamics of complex biological system. The fully cross-platform Shiny App is hosted and freely available at https://nib-si.shinyapps.io/DiNAR. The most recent version of the source code is available at https://github.com/NIB-SI/DiNAR/ with a DOI 10.5281/zenodo.1230523 of the archived version in Zenodo.
Keywords: biological networks, clustering, gene expression, time series, dynamic network analysis, dynamic data visualisation, web application, multi-conditional datasets, background knowledge
Published in DiRROS: 24.07.2024; Views: 29; Downloads: 10
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138. Assessment of hyperbaric oxygenation treatment response in parotid glands by T2 mapping following radiotherapy for head and neck tumoursJernej Vidmar, Ksenija Cankar, Maja Grošelj, Žarko Finderle, Igor Serša, 2022, original scientific article Abstract: Background: The study was designed to evaluate the influence of hyperbaric oxygenation therapy (HBOT) on the parotid gland in patients following radiotherapy for head and neck tumours. Patients and methods: HBOT response was monitored by 3T magnetic resonance imaging (MRI) using T 2 mapping and subsequent measurement of mean T 2 and T 2 variability as well as by salivary tests (salivary flow, buffer capacity, and pH). Eighteen patients previously treated with irradiation doses between 50 and 80 Gy as well as 18 healthy gender and age matched controls were enrolled. MRI was performed prior to HBOT (40.2 ± 20 months after radiotherapy) and after 20 daily HBOT at 2.5 ATA (absolute atmosphere). Each HBOT consisted of breathing 100% oxygen for 90 minutes. Results: Significant differences in mean T 2 prior to HBOT were observed between the ipsilateral irradiated (121 ± 20 ms), contralateral parotids (107 ± 21) and control group (96 ± 12 ms). A positive correlation in patients between T 2 variability and irradiation dose was detected in contralateral parotids before HBOT (R = 0.489, p = 0.0287). In addition, negative correlations were observed between mean T 2 in the ipsilateral as well as the contralateral gland and salivary flow before and after HBOT. Negative correlations between mean T 2, T 2 variability and pH of unstimulated saliva were also observed in the sides of parotid before and after HBOT. Conclusions: The study confirmed that T 2 mapping had a potential for monitoring the differences between irradiated and normal parotid glands. It could also be useful in the assessment of the glandular tissue response to HBOT.
Keywords: MRI, T2 mapping, hyperbaric oxygenation therapy, head and neck tumours
Published in DiRROS: 24.07.2024; Views: 12; Downloads: 4
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139. 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: 15; Downloads: 5
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140. 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: 15; Downloads: 5
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