Buccal micronucleus cytome assay

The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifestyle factors, genotoxin exposure and genotype on DNA damage, chromosome malsegregation and cell death. The biomarkers measured in this assay have been associated with increased risk of accelerated ageing, cancer and neurodegenerative diseases. This protocol describes one of the current established methods for buccal cell collection using a small-headed toothbrush, the generation of a single-cell suspension, slide preparation using cytocentrifugation, fixation and staining using Feulgen and Light Green for both bright field and fluorescence microscopic analysis. The scoring criteria for micronuclei and other nuclear anomalies are also described in detail. The protocol in its current form takes approximately 4 h to complete from the time of buccal cell collection to the generation of stained slides for microscopic analysis.Philip Thomas, Nina Holland, Claudia Bolognesi, Micheline Kirsch-Volders, Stefano Bonassi, Errol Zeiger, Siegfried Knasmueller & Michael Fenec

Methacrylic Acid Based Polymer Networks with a High Content of Unfunctionalized Nanosilica: Particle Distribution, Swelling, and Rheological Properties

The poor stability and tendency to agglomerate of unfunctionalized nano-SiO2 in the presence of ionic species presents a challenge for preparing poly(methacrylic acid)/nano-SiO2 nanocomposite (NC) hydrogels with desired strength and swelling capability. We proposed a facile and eco-friendly method for the preparation of PMAA/SiO2 NC hydrogels using unfunctionalized silica nanoparticles (NPs) in the form of a suspension. SEM and TEM analyses showed that the NP distribution in the polymer matrix highly depended on the particle concentration. At lower concentrations (up to 13.9 wt %), the NPs were uniformly dispersed as single nanoparticles. With an increase in NP concentration, homogeneously dispersed nanoscale aggregates were formed, while a further increase in the silica concentration led to the formation of homogeneous structures consisting of mutually interacting nanosilica particles coated with PMAA. Swelling experiments confirmed that the silica NPs behaved as adhesive fillers that interacted with PMAA chains, causing the formation of a thin polymer layer strongly adsorbed at the particle interface. The thicknesses of the adsorbed polymer layer, as well as the swelling kinetic parameters, were strongly influenced by nanoparticle size and concentration. Combining nanosilica and PMAA in the form of a soft hydrogel network provided stabilization of the NPs and ensured better mechanical properties of the obtained NC hydrogels compared to pure polymer matrix. The optimal loadings, necessary to ensure the most improved dynamical-mechanical properties, were found in the case of the formation of homogeneously dispersed, nanosized silica aggregates in a PMAA matrix