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Povzetek: Expanding international pet trade has emerged as one of the main introduction pathways of aquatic invasive species, with ornamental crayfish species commonly available on the EU and global markets. Besides most frequently studied crayfish pathogens, such as Aphanomyces astaci and white spot syndrome virus (WSSV), ornamental crayfish carry associated microbial communities, which may potentially lead to the emergence of known or even novel diseases following intentional or unintentional release of animals into the wild. This is especially problematic in the case of viruses, which represent an important, yet considerably understudied, group of crayfish pathogens. Here we analyzed viromes of hepatopancreas tissue of four crayfish species acquired in the international pet trade in Europe (Procambarus clarkii, Procambarus alleni, Cherax holthuisi, and Cherax quadricarinatus) using a high throughput sequencing based metagenomic approach. Seven different known viruses were identified, which were previously either directly associated with crayfish (WSSV, Cherax quadricarinatus reovirus, chequa iflavirus, athtab bunya-like virus) or with hosts from subphylum Crustacea or invertebrates associated with freshwater environment (Shahe ispoda virus 5, Dicistroviridae sp.). Additional sequences represented 8 potential novel and divergent RNA viruses, most similar to sequences belonging to members of Picornavirales, Elliovirales, Reovirales, Hepelivirales, Tolivirales and Ghabrivirales orders. We discuss our findings in relation to their phylogenetic relationships, geographical origins, and putative pathogenicity implications. The results highlight the need for further research into the risks related to disease emergence associated with the pet trade.
Ključne besede: virome, RNA viruses, ornamental crayfish, pet-trade, high throughput sequencing, ecology, molecular biology, pathology, virology, environmental risk assessment
Objavljeno v DiRROS: 23.05.2025; Ogledov: 1351; Prenosov: 1070
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Povzetek: Anatoxins (ATX) are globally occurring toxins produced by some species of cyanobacteria in aquatic habitats. They can cause acute poisoning in animals, leading to muscle paralysis and respiratory failure, and might also pose a long-term health risk to humans. Thanks to advances in molecular methods and genomic knowledge, it is now possible to rapidly detect and quantify the genes associated with cyanotoxin production for most major groups of cyanotoxins except ATX. The aim of this study was to develop and validate a new quantitative PCR (qPCR) assay for general detection of all potential ATX producers in the environment. After specificity testing in silico and in vitro with 16 cyanobacterial strains (endpoint PCR, amplicon sequencing and qPCR), two assays targeting the anaC gene were thoroughly validated for linearity, amplification efficiency, sensitivity, dynamic range, inter-assay and intra-assay variability, and the influence of background DNA. The assays were then applied to 144 environmental samples of plankton and biofilm from lakes and rivers whose ATX content had previously been measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Amplification efficiency of the two designed assays was between 94% and 103%, and the limits of quantification and detection were up to, but mostly below, 322 and 32 cells/mL, respectively. Both assays showed better or equal specificity in cyanobacterial cultures than currently available PCR assays and were able to predict the presence of ATX detected by LC-MS/MS in most environmental samples (83 % in plankton and 52–62% in biofilm). A higher number of discrepancies between qPCR and LC-MS/MS results in biofilm than in plankton samples indicates limited knowledge and sparse genomic data on benthic cyanobacteria. These assays are the first published general qPCR assays targeting all ATX producers and could provide water managers with a rapid and cost-effective risk assessment to better protect human and animal health.
Ključne besede: anatoxins (ATX), cyanobacteria, qPCR assay, anaC gene, LC-MS/MS, environmental microbiology, environmental science, toxicology, molecular biology
Objavljeno v DiRROS: 17.12.2024; Ogledov: 1361; Prenosov: 745
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Povzetek: Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant%pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.
Ključne besede: plant diseases, potato, molecular biology
Objavljeno v DiRROS: 02.08.2024; Ogledov: 1629; Prenosov: 1006
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Povzetek: The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.
Ključne besede: biotechnology, molecular biology
Objavljeno v DiRROS: 25.07.2024; Ogledov: 1366; Prenosov: 916
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Povzetek: PaintOmics is a web server for the integrative analysis and visualisation of multi-omics datasets using biological pathway maps. PaintOmics 4 has several notable updates that improve and extend analyses. Three pathway databases are now supported: KEGG, Reactome and MapMan, providing more comprehensive pathway knowledge for animals and plants. New metabolite analysis methods fill gaps in traditional pathway-based enrichment methods. The metabolite hub analysis selects compounds with a high number of significant genes in their neighbouring network, suggesting regulation by gene expression changes. The metabolite class activity analysis tests the hypothesis that a metabolic class has a higher-than-expected proportion of significant elements, indicating that these compounds are regulated in the experiment. Finally, PaintOmics 4 includes a regulatory omics module to analyse the contribution of trans-regulatory layers (microRNA and transcription factors, RNA-binding proteins) to regulate pathways. We show the performance of PaintOmics 4 on both mouse and plant data to highlight how these new analysis features provide novel insights into regulatory biology.
Ključne besede: PaintOmics 4, web tools, datasets, analysis methods, molecular biology
Objavljeno v DiRROS: 17.07.2024; Ogledov: 1658; Prenosov: 1326
Celotno besedilo (3,53 MB)
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