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105. A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteriaMaša Jablonska, Tina Eleršek, 2024, original scientific article Abstract: 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.
Keywords: anatoxins (ATX), cyanobacteria, qPCR assay, anaC gene, LC-MS/MS, environmental microbiology, environmental science, toxicology, molecular biology
Published in DiRROS: 17.12.2024; Views: 67; Downloads: 28
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108. 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, original scientific article Abstract: 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.
Keywords: 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)
Published in DiRROS: 17.12.2024; Views: 74; Downloads: 29
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109. Numerical analysis of knudsen number of helium flow through gas-focused liquid sheet micro-nozzleKrištof Kovačič, Saša Bajt, Božidar Šarler, 2024, original scientific article Abstract: This work aims to verify whether the continuum mechanics assumption holds for the numerical simulation of a typical sample delivery system in serial femtosecond crystallography (SFX). Knudsen numbers were calculated based on the numerical simulation results of helium flow through the gas-focused liquid sheet nozzle into the vacuum chamber, representing the upper limit of Knudsen number for such systems. The analysed flow is considered steady, compressible, and laminar. The numerical results are mesh-independent, with a Grid Convergence Index significantly lower than 1% for global and local analysis. This study is based on an improved definition of the numerical Knudsen number: a combination of the cell Knudsen number and the physical Knudsen number. In the analysis, no-slip boundary and low-pressure boundary slip conditions are compared. No significant differences are observed. This study justifies using computational fluid dynamics (CFD) analysis for SFX sample delivery systems based on the assumption of continuum mechanics.
Keywords: Knudsen number, compressible hypersonic flow, vacuum, liquid sheet nozzle, sample delivery system, CFD
Published in DiRROS: 17.12.2024; Views: 78; Downloads: 35
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110. Phase-field formulated meshless simulation of axisymmetric Rayleigh-Taylor instability problemKhush Bakhat Rana, Boštjan Mavrič, Rizwan Zahoor, Božidar Šarler, 2024, original scientific article Abstract: A formulation of the immiscible Newtonian two-liquid system with different densities and influenced by gravity is based on the Phase-Field Method (PFM) approach. The solution of the related governing coupled Navier-Stokes (NS) and Cahn-Hillard (CH) equations is structured by the meshless Diffuse Approximate Method (DAM) and Pressure Implicit with Splitting of Operators (PISO). The variable density is involved in all the terms. The related moving boundary problem is handled through single-domain, irregular, fixed node arrangement in Cartesian and axisymmetric coordinates. The meshless DAM uses weighted least squares approximation on overlapping subdomains, polynomial shape functions of second-order and Gaussian weights. This solution procedure has improved stability compared to Chorin's pressure-velocity coupling, previously used in meshless solutions of related problems. The Rayleigh-Taylor instability problem simulations are performed for an Atwood number of 0.76. The DAM parameters (shape parameter of the Gaussian weight function and number of nodes in a local subdomain) are the same as in the authors’ previous studies on single-phase flows. The simulations did not need any upwinding in the range of the simulations. The results compare well with the mesh-based finite volume method studies performed with the open-source code Gerris, Open-source Field Operation and Manipulation (OpenFOAM®) code and previously existing results.
Keywords: two-phase flow, phase-field, two-dimensionality, axisymmetry, diffuse approximate method, PISO
Published in DiRROS: 17.12.2024; Views: 63; Downloads: 31
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