Biology subjects

Kovacs, G. M.

Publications and source records attributed to Kovacs, G. M..

6 recordsLinked to original sources

bioRxiv2025

Petasol Exhibits Rapid and Efficient Epithelial Transport in Epithelial Barrier Models

The epithelial permeability of petasol, a sesquiterpene derivative with reported antiviral properties, remains largely unexplored despite its potential as a therapeutic candidate. Here, we systematically characterize the transport of petasol across epithelial barriers using a combination of in vitro transwell models, automated millifluidic sampling, mathematical modeling, optical- and HPLC-mass spectroscopy. Vero E6 and Caco-2 cell layers were employed as epithelial barrier models, with transepithelial electrical resistance (TEER) measurements confirming the formation of functionally intact epithelial layers. Mathematical modeling of passive diffusion and cellular uptake allowed estimation of key transport parameters, including permeability coefficients and intracellular sequestration rates. Trans-barrier transport of petasol was compared to that of nitazoxanide, chloroquine, and FITC-dextran. Our results reveal that petasol exhibits efficient and rapid transport across both epithelial cell types without substantial intracellular accumulation or metabolic degradation, suggesting favorable properties for systemic delivery.

pharmacology and toxicology↗

bioRxiv2025

Characterizing Solute Transport Across Cell Layers: Artifact Correction and Parameter Extraction from a Simplified Three-Compartment Model

Quantifying solute transport across epithelial cell layers grown on transwell inserts is a common approach in early-stage drug development to estimate pharmacokinetic properties such as absorption and bioavailability. To increase throughput and reduce variability, these assays are increasingly automated, including the use of robotic or microfluidic systems for time-resolved sampling. However, both automated and manual sampling can introduce systematic artifacts, such as residual volume retention and surface adsorption, that distort concentration time series and affect downstream analysis. To fully realize the potential precision of automated measurements, we propose a mathematical correction to account for sampling artifacts; then to fit the corrected data to a three-compartment model that captures membrane diffusion, cellular sequestration, and metabolic loss. The method is demonstrated on datasets from transwell epithelial barrier transport assays. We suggest that the considered three-compartment model yields mechanistically more meaningful parameters than the conventional apparent permeability (Papp) measure. The proposed approach thus enables more accurate characterization of analyte interactions with the barrier cell layer, supporting better-informed assessments of compound behavior in in vitro transport systems.

pharmacology and toxicology↗

bioRxiv2025

Multi-locus phylogenetic network analysis of Ampelomyces mycoparasites isolated from diverse powdery mildews in Australia and the generation of two de novo genome assemblies

The interactions between powdery mildews (Ascomycota, Erysiphaceae), obligate biotrophic pathogens of many plants, and pycnidial fungi belonging to the genus Ampelomyces, are classic examples of specific mycoparasitic relationships. These interactions are common and finely tuned tritrophic relationships amongst host plants, powdery mildews, and Ampelomyces mycoparasites wherever these organisms co-occur in the field. Selected Ampelomyces strains have already been developed as biocontrol agents of powdery mildew infections of some crops. In Australia, their study has received little attention so far. Only a single Ampelomyces strain, included in a whole-genome sequencing (WGS) project, was known from this continent. Here, we report the isolation of 20 more Ampelomyces strains from eight powdery mildew species in Australia. Multi-locus phylogenetic network analyses of all the 21 Australian Ampelomyces strains carried out in combination with 32 reference strains from overseas revealed that the Australian strains belonged to four molecular taxonomic units (MOTUs). All those MOTUs were delimited earlier based on Ampelomyces strains isolated in Europe, North America, and elsewhere. Analyses indicated possible recombination events amongst phylogenetically different Ampelomyces strains. Based on the phylogenetic analyses, two Australian strains belonging to different MOTUs were selected for WGS. Long-read (minION) and short-read (Illumina) technologies were used to provide genome assemblies with high completeness. Both assemblies reported here had bipartite structure, i.e., consisted of AT-rich, gene-sparse regions interspersed with GC-balanced, gene-rich regions. These new high-quality assemblies and evidence-based annotations are important resources for future analyses of mycoparasitic interactions to disentangle molecular mechanisms underlying mycoparasitism, possible new biocontrol applications, and natural tritrophic relationships.

microbiology↗

bioRxiv2019

High throughput competitive fluorescence polarization assay reveals functionalredundancy in the S100 protein family

The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To assess their interactome, high-throughput, systematic analysis is indispensable, which allows one to get not only qualitative but quantitative insight into their protein-protein interactions (PPIs). We have chosen an unbiased assay, fluorescence polarization (FP) that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified (orphan). Our results demonstrate that in vitro FP assays are highly suitable for quantitative ligand binding studies of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family.\n\nAuthor summaryFunctional similarity among a protein family can be essential in order to understand proteomic data, to find biomarkers, or in inhibitor design. Proteins with similar functions can compensate the loss-of-function of the others, their expression can co-vary under pathological conditions, and simultaneous targeting can lead to better results in the clinics. To investigate this property one can use sequence-based approaches. However, this path can be difficult. In the case of the vertebrate specific, evolutionary young, S100 family, phylogenetic approaches lead to ambiguous results. To overcome this problem, we applied a high-throughput biochemical approach to experimentally measure the binding affinities of a large number of S100 interactions. We performed unbiased fluorescence polarization assay, involving the complete human S100ome (20 paralogs) and 13 known interaction partners. We used this measured 20x13 (260) protein-protein interaction array to reveal the functional relationships within the family. Our work provide a general framework for studies focusing on phenotype-based domain classification.

biochemistry↗

bioRxiv2019

The genome of Auriculariopsis ampla sheds light on fruiting body development and wood-decay of bark-inhabiting fungi

The Agaricomycetes are fruiting body forming fungi that produce some of the most efficient enzyme systems to degrade woody plant materials. Despite decades-long interest in the ecological and functional diversity of wood-decay types and in fruiting body development, the evolution of the genetic repertoires of both traits are incompletely known. Here, we sequenced and analyzed the genome of Auriculariopsis ampla, a close relative of the model species Schizophyllum commune. Comparative analyses of wood-decay genes in these and other 29 Agaricomycetes species revealed that the gene family composition of A. ampla and S. commune are transitional between that of white rot species and less efficient wood-degraders (brown rot, ectomycorrhizal). Rich repertoires of suberinase and tannase genes were found in both species, with tannases generally restricted to species that preferentially colonize bark-covered wood. Analyses of fruiting body transcriptomes in both A. ampla and S. commune highlighted a high rate of divergence of developmental gene expression. Several genes with conserved developmental expression were found, nevertheless, including 9 new transcription factors as well as small secreted proteins, some of which may serve as fruiting body-specific effector molecules. Taken together, the genome sequence and developmental transcriptome of Auriculariopsis ampla has highlighted novel aspects of wood-decay diversity and of fruiting body development in mushroom-forming fungi.

evolutionary biology↗

bioRxiv2019

GFP transformation sheds more light on a widespread mycoparasitic interaction

Powdery mildews (PMs), ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic PMs. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed the green fluorescent protein (GFP), to (i) improve the visualization of the PM-Ampelomyces interaction; and (ii) decipher the environmental fate of Ampelomyces before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse PM, leaf and soil samples containing GFP transformants were examined with fluorescence microscopy compared to brightfield and DIC optics. We showed for the first time that Ampelomyces can persist up to 21 days on PM-free host plant surfaces, where it can attack PM structures as soon as these appear after this period. As a saprobe in decomposing, PM-infected leaves on the ground, and also in autoclaved soil, Ampelomyces developed new hyphae, but did not sporulate. These results indicate that Ampelomyces occupies a niche in the phyllosphere where it acts primarily as a mycoparasite of PMs. Our work has established a framework for a molecular genetic toolbox for Ampelomyces using ATMT.

microbiology↗