In Vivo-In Vitro Comparative Toxicology of Cadmium Sulphide Quantum Dots in the Model Organism Saccharomyces cerevisiae.

The aim of this work was to use the yeast Saccharomyces cerevisiae as a tool for toxicogenomic studies of Engineered Nanomaterials (ENMs) risk assessment, in particular focusing on cadmium based quantum dots (CdS QDs). This model has been exploited for its peculiar features: a short replication time, growth on both fermentable and oxidizable carbon sources, and for the contextual availability of genome wide information in the form of genetic maps, DNA microarray, and collections of barcoded mutants. The comparison of the whole genome analysis with the microarray experiments (99.9% coverage) and with the phenotypic analysis of 4688 barcoded haploid mutants (80.2% coverage), shed light on the genes involved in the response to CdS QDs, both in vivo and in vitro. The results have clarified the mechanisms involved in the exposure to CdS QDs, and whether these ENMs and Cd2+ exploited different pathways of response, in particular related to oxidative stress and to the maintenance of mitochondrial integrity and function. Saccharomyces cerevisiae remains a versatile and robust alternative for organismal toxicological studies, with a high level of heuristic insights into the toxicology of more complex eukaryotes, including mammals.

Tau monoclonal antibody generation based on humanized yeast models: impact on Tau oligomerization and diagnostics.

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr(18). For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.

Mucin biopolymers prevent bacterial aggregation by retaining cells in the free-swimming state.

Many species of bacteria form surface-attached communities known as biofilms. Surrounded in secreted polymers, these aggregates are difficult both to prevent and eradicate, posing problems for medicine and industry. Humans play host to hundreds of trillions of microbes that live adjacent to our epithelia, and we are typically able to prevent harmful colonization. Mucus, the hydrogel overlying all wet epithelia in the body, can prevent bacterial contact with the underlying tissue. The digestive tract, for example, is lined by a firmly adherent mucus layer that is typically devoid of bacteria, followed by a second, loosely adherent layer that contains numerous bacteria. Here, we investigate the role of mucus as a principle arena for host-microbe interactions. Using defined in vitro assays, we found that mucin biopolymers, the main functional constituents of mucus, promote the motility of planktonic bacteria and prevent their adhesion to underlying surfaces. The deletion of motility genes, however, allows Pseudomonas aeruginosa to overcome the dispersive effects of mucus and form suspended antibiotic-resistant flocs, which mirror the clustered morphology of immotile natural isolates found in the cystic fibrosis lung mucus. Mucus may offer new strategies to target bacterial virulence, such as the design of antibiofilm coatings for implants.

The splicing mutant of the human tumor suppressor protein DFNA5 induces programmed cell death when expressed in the yeast Saccharomyces cerevisiae.

DFNA5 was first identified as a gene responsible for autosomal dominant deafness. Different mutations were found, but they all resulted in exon 8 skipping during splicing and premature termination of the protein. Later, it became clear that the protein also has a tumor suppression function and that it can induce apoptosis. Epigenetic silencing of the DFNA5 gene is associated with different types of cancers, including gastric and colorectal cancers as well as breast tumors. We introduced the wild-type and mutant DFNA5 allele in the yeast Saccharomyces cerevisiae. The expression of the wild-type protein was well tolerated by the yeast cells, although the protein was subject of degradation and often deposited in distinct foci when cells entered the diauxic shift. In contrast, cells had problems to cope with mutant DFNA5 and despite an apparent compensatory reduction in expression levels, the mutant protein still triggered a marked growth defect, which in part can be ascribed to its interaction with mitochondria. Consistently, cells with mutant DFNA5 displayed significantly increased levels of ROS and signs of programmed cell death. The latter occurred independently of the yeast caspase, Mca1, but involved the mitochondrial fission protein, Fis1, the voltage-dependent anion channel protein, Por1 and the mitochondrial adenine nucleotide translocators, Aac1 and Aac3. Recent data proposed DFNA5 toxicity to be associated to a globular domain encoded by exon 2-6. We confirmed these data by showing that expression of solely this domain confers a strong growth phenotype. In addition, we identified a point mutant in this domain that completely abrogated its cytotoxicity in yeast as well as human Human Embryonic Kidney 293T cells (HEK293T). Combined, our data underscore that the yeast system offers a valuable tool to further dissect the apoptotic properties of DFNA5.