In the past few years extracellular vesicles called exosomes have gained huge interest of scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is accurate size characterization and quantification of exosomes because of the lack of reliable characterization techniques. In this work, the emphasis was focused on a method development to size-separate, characterize, and quantify small amounts of exosomes by asymmetrical-flow field-flow fractionation (AF4) technique coupled to a multidetection system (UV and MALS). Batch DLS (dynamic light-scattering) and NTA (nanoparticle tracking analysis) analyses of unfractionated exosomes were also conducted to evaluate their shape and internal structure, as well as their number density. The results show significant influence of cross-flow conditions and channel thickness on fractionation quality of exosomes, whereas the focusing time has less impact. The AF4/UV-MALS and DLS results display the presence of two particles subpopulations, that is, the larger exosomes and the smaller vesicle-like particles, which coeluted in AF4 together with impurities in early eluting peak. Compared to DLS and AF4-MALS results, NTA somewhat overestimates the size and the number density for larger exosome population, but it discriminates the smaller particle population.

Molecular studies of salt tolerance of eukaryotic microorganisms have until recently been limited to the baker's yeast Saccharomyces cerevisiae and a few other moderately halotolerant yeast. Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. H. werneckii is unique in its adaptability to fluctuations in salt concentrations, as it can grow without NaCl as well as in the presence of up to 5 M NaCl. On the other hand, W. ichthyophaga requires at least 1.5 M NaCl for growth, but also grows in up to 5 M NaCl. Our studies have revealed the novel and intricate molecular mechanisms used by these fungi to combat high salt concentrations, which differ in many aspects between the extremely halotolerant H. werneckii and the halophilic W. ichthyophaga. Specifically, the high osmolarity glycerol signaling pathway that is important for sensing and responding to increased salt concentrations is here compared between H. werneckii and W. ichthyophaga. In both of these fungi, the key signaling components are conserved, but there are structural and regulation differences between these pathways in H. werneckii and W. ichthyophaga. We also address differences that have been revealed from analysis of their newly sequenced genomes. The most striking characteristics associated with H. werneckii are the large genetic redundancy, the expansion of genes encoding metal cation transporters, and a relatively recent whole genome duplication. In contrast, the genome of W. ichthyophaga is very compact, as only 4884 protein-coding genes are predicted, which cover almost three quarters of the sequence. Importantly, there has been a significant increase in their hydrophobins, cell-wall proteins that have multiple cellular functions.

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HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S. cerevisiae, Hog1 is phosphorylated already at low osmolyte concentrations, and regulates expression of a similar set of genes independent of osmolyte type. To understand how HwHog1 kinases activity is regulated in H. werneckii, we studied HwHog1A/B activation in vivo, by following phosphorylation of HwHog1A/B in H. werneckii exposed to various osmolytes, and in vitro, by measuring kinase activities of recombinant HwHog1A, HwHog1B and Hog1ΔC. To this end, highly pure and soluble recombinant Hog1 homologues were isolated from insect cells. Our results demonstrate that HwHog1A/B are, in general, transiently phosphorylated in cells shocked with ≥3 M osmolyte, yet constitutive phosphorylation is observed at extreme NaCl and KCl concentrations. Importantly, phosphorylation profiles differ depending on the osmolyte type. Additionally, phosphorylated recombinant HwHog1A/B show lower specific kinase activities compared to Hog1ΔC. In summary, HOG pathway MAPKs in the extremely halotolerant H. werneckii show unique characteristics compared to S. cerevisiae homologues. The reported findings contribute to defining the key determinants of H. werneckii osmotolerance, which is important for its potential transfer to economically relevant microorganisms and crops.