No abstract

Background/Aim: Proteomics technologies provide fundamental insights into the high organizational complexity and diversity of the central nervous system. In the present study, high-resolution mass spectrometry (MS) was applied in order to identify whole-proteome content of anatomically distinct and functionally specific mouse brain regions. Materials and Methods: Brains from eight 8-week-old C57BL/6N normal male mice were separated into seven anatomically district regions. The protein content of each region was analyzed by highthroughput nano-liquid chromatography-MS/MS Orbitrap elite technology.

The unicellular eukaryote S. cerevisiae is an invaluable resource for the study of basic eukaryotic cellular and molecular processes. However, due to its small size compared to other eukaryotic organisms the study of subcellular structures is challenging. Expansion microscopy (ExM) holds great potential to study the intracellular architecture of yeast, especially when paired with pan-labelling techniques visualising the full protein content inside cells. ExM allows to increase imaging resolution by physically enlarging a fixed sample that is embedded and cross- linked to a swellable gel followed by isotropic expansion in water. The cell wall present in fungi – including yeast – and Gram-positive bacteria is a resilient structure that resists denaturation and conventional digestion processes usually used in ExM protocols, resulting in uneven expansion. Thus, the digestion of the cell wall while maintaining the structure of the resulting protoplasts are crucial steps to ensure isotropic expansion. For this reason, specific experimental strategies are needed, and only a few protocols are currently available. We have developed a modified ExM protocol for S. cerevisiae, with 4x expansion factor, which allows the visualisation of the ultrastructure of the cells. Here, we describe the experimental procedure in detail, focusing on the most critical steps required to achieve isotropic expansion for ExM of S. cerevisiae.

Cryptococcus spp. are fungal species belonging to Tremellomycetes, Agaricomycotina, Basidiomycota, and several members are responsible for cryptococcosis, one of the most ubiquitous human mycoses. Affecting mainly immune suppressed patients, but also immune competent ones, the members of this genus present a high level of genetic diversity. In this study, two mitochondrial intergenic regions, i.e. nad1-cob and cob-rps3, were tested for the intra- or interspecies discrimination and identification of strains and species of the genus Cryptococcus. Phylogenetic trees were constructed based on individual and concatenated sequences from representative pathogenic strains of the C. neoformans/C. gattii complex, representing serotypes and AFLP genotypes of all newly introduced species of this complex. Using both intergenic regions, as well as the concatenated dataset, the strains clustered in accordance with the new taxonomy. These results suggest that identification of Cryptococcus strains is possible by employing these mitochondrial intergenic regions using PCR amplification as a quick and effective method to elucidate genotypic and taxonomic differences. Thus, these regions may be applicable to a broad range of clinical studies, leading to a rapid recognition of the clinical profiles of patients.