Biology subjects

Wargnies, M.

Publications and source records attributed to Wargnies, M..

4 recordsLinked to original sources

bioRxiv2025

Quantification of H3.1-nucleosomes using a chemiluminescent immunoassay: a reliable method for neutrophil extracellular trap detection

Neutrophil extracellular traps (NETs) are chromatin-based web-like structures released by activated neutrophils in response to infectious agents. Overproduction or insufficient clearance of NETs contributes to dysfunction of immune response and disease pathogenesis, underlying the importance of early detection and monitoring of NET levels in clinical samples. While existing methods for NETs detection and quantification face limitations, there is a pressing need for a reliable, sensitive, and clinically applicable assay. Since NETs consist of long strains of decondensed chromatin, with nucleosomes as their basic units, we propose circulating H3.1-nucleosomes as biomarkers for NETs detection in clinical plasma samples. In the initial phase of our study, we confirmed the presence of H3.1-nucleosomes by immunofluorescence and immunoprecipitation experiments in two in vitro NET models: neutrophil-like cells differentiated from the HL-60 cell line and primary neutrophils isolated from whole blood, both treated with phorbol 12-myristate 13-acetate to induce NET formation. Subsequently, we developed and analytically validated a chemiluminescent immunoassay for the quantification of circulating H3.1-nucleosomes in plasma. This fully automated assay demonstrates impressive analytical performance in parameters including sensitivity, precision, linearity and reproducibility. Overall, by measuring the H3.1-nucleosome levels in plasma samples from patients suffering from NETs-related diseases compared to healthy donors, we demonstrated the assays potential as a groundbreaking diagnostic tool for disease management.

molecular biology↗

bioRxiv2024

High-Throughput Epigenetic Profiling Immunoassays for Accelerated Disease Research and Clinical Development

Epigenetics, which examines the regulation of genes without modification of the DNA sequence, plays a crucial role in various biological processes and disease mechanisms. Among the different forms of epigenetic modifications, histone post-translational modifications (PTMs) are important for modulating chromatin structure and gene expression. Aberrant levels of histone PTMs are implicated in a wide range of diseases, including cancer, making them promising targets for biomarker discovery and therapeutic intervention. In this context, blood, tissues, or cells serve as valuable resources for epigenetic research and analysis. Traditional methods such as mass spectrometry and western blotting are widely used to study histone PTMs, providing qualitative and (semi)quantitative information. However, these techniques often face limitations that could include throughput and scalability, particularly when applied to clinical samples. To overcome these challenges, we developed and validated 13 Nu.Q(R) immunoassays to detect and quantify specific histone PTM-nucleosomes from K2EDTA plasma samples. Then, we tested these assays on other types of samples, including chromatin extracts from frozen tissues, as well as cell lines and white blood cells Our findings demonstrate that the Nu.Q(R) assays offer high specificity, sensitivity, precision and linearity, making them effective tools for epigenetic profiling. A comparative analysis of HeLa cells using mass spectrometry, Western blot, and Nu.Q(R) immunoassays revealed a consistent histone PTMs signature, further validating the effectiveness of these assays. Additionally, we successfully applied Nu.Q(R) assays across various biological samples, including human tissues from different organs and specific white blood cell subtypes, highlighting their versatility and applicability in diverse biological contexts.

molecular biology↗

bioRxiv2024

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei operates in the gluconeogenic direction in cellulo under in vivo-like conditions

In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis glucose 6- phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We showed here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, (i) fructose-1,6- bisphosphatase (FBPase), the canonical enzyme performing this reaction, (ii) sedoheptulose- 1,7-bisphosphatase (SBPase) and (iii) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyse the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas a 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we showed for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is probably due to the supramolecular organisation of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channelling.

microbiology↗

bioRxiv2019

\"Metabolic contest\", a new way to control carbon source preference

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, allowing to redirect cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism, named \"metabolic contest\", for regulating the use of carbon sources without nutrient sensing and signaling. In contrast to most microorganisms, trypanosomes show a glycerol-to-glucose preference that depends on the combination of three conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-like organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways being both ATP-dependent (glycerol kinase and hexokinase, respectively) and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being ~80-fold higher than the hexokinase activity.

microbiology↗