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97. A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteriaMaša Jablonska, Tina Eleršek, 2024, izvirni znanstveni članek 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: 39; Prenosov: 16
 Celotno besedilo (1,86 MB)
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100. Amino acid sequence encodes protein abundance shaped by protein stability at reduced synthesis costFilip Buric, Sandra Viknander, Xiaozhi Fu, Oliver Lemke, Oriol Gracia Carmona, Jan Zrimec, Lukasz Szyrwiel, Michael Mülleder, Markus Ralser, Aleksej Zelezniak, 2025, izvirni znanstveni članek Povzetek: Understanding what drives protein abundance is essential to biology, medicine, and biotechnology. Driven by evolutionary selection, an amino acid sequence is tailored to meet the required abundance of a proteome, underscoring the intricate relationship between sequence and functional demand. Yet, the specific role of amino acid sequences in determining proteome abundance remains elusive. Here we show that the amino acid sequence alone encodes over half of protein abundance variation across all domains of life, ranging from bacteria to mouse and human. With an attempt to go beyond predictions, we trained a manageable-size Transformer model to interpret latent factors predictive of protein abundances. Intuitively, the model's attention focused on the protein's structural features linked to stability and metabolic costs related to protein synthesis. To probe these relationships, we introduce MGEM (Mutation Guided by an Embedded Manifold), a methodology for guiding protein abundance through sequence modifications. We find that mutations which increase predicted abundance have significantly altered protein polarity and hydrophobicity, underscoring a connection between protein structural features and abundance. Through molecular dynamics simulations we revealed that abundance-enhancing mutations possibly contribute to protein thermostability by increasing rigidity, which occurs at a lower synthesis cost.
Ključne besede: molecular biology, biotechnology, bioinformatics, deep learning, gene expression, synthetic biology, protein abundance, amino acid sequence, evolutionary selection, transformer model, MGEM (Mutation guided by an embedded manifold)
Objavljeno v DiRROS: 17.12.2024; Ogledov: 34; Prenosov: 14
Celotno besedilo (13,19 MB)
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