RESUMEN
Research in cancer nanotechnology is entering its third decade, and the need to study interactions between nanomaterials and cells remains urgent. Heterogeneity of nanoparticle uptake by different cells and subcellular compartments represent the greatest obstacles to a full understanding of the entire spectrum of nanomaterials' effects. In this work, we used flow cytometry to evaluate changes in cell cycle associated with non-targeted nanocomposite uptake by individual cells and cell populations. Analogous single cell and cell population changes in nanocomposite uptake were explored by X-ray fluorescence microscopy (XFM). Very few nanoparticles are visible by optical imaging without labeling, but labeling increases nanoparticle complexity and the risk of modified cellular uptake. XFM can be used to evaluate heterogeneity of nanocomposite uptake by directly imaging the metal atoms present in the metal-oxide nanocomposites under investigation. While XFM mapping has been performed iteratively in 2D with the same sample at different resolutions, this study is the first example of serial tomographic imaging at two different resolutions. A cluster of cells exposed to non-targeted nanocomposites was imaged with a micron-sized beam in 3D. Next, the sample was sectioned for immunohistochemistry as well as a high resolution "zoomed in" X-ray fluorescence (XRF) tomography with 80 nm beam spot size. Multiscale XRF tomography will revolutionize our ability to explore cell-to-cell differences in nanomaterial uptake.
RESUMEN
Loss of heterozygosity (LOH) is observed during vegetative growth and reproduction of diploid genotypes through mitotic crossovers, aneuploidy caused by nondisjunction, and gene conversion. We aimed to test the role that LOH plays during adaptation of two highly heterozygous Saccharomyces cerevisiae genotypes to multiple environments over a short time span in the laboratory. We hypothesized that adaptation would be observed through parallel LOH events across replicate populations. Using genome resequencing of 70 clones, we found that LOH was widespread with 5.2 LOH events per clone after â¼500 generations. The most common mode of LOH was gene conversion (51%) followed by crossing over consistent with either break-induced replication or double Holliday junction resolution. There was no evidence that LOH involved nondisjunction of whole chromosomes. We observed parallel LOH in both an environment-specific and environment-independent manner. LOH largely involved recombining existing variation between the parental genotypes, but also was observed after de novo, presumably beneficial, mutations occurred in the presence of canavanine, a toxic analog of arginine. One highly parallel LOH event involved the ENA salt efflux pump locus on chromosome IV, which showed repeated LOH to the allele from the European parent, an allele originally derived by introgression from S. paradoxus Using CRISPR-engineered LOH we showed that the fitness advantage provided by this single LOH event was 27%. Overall, we found extensive evidence that LOH could be adaptive and is likely to be a greater source of initial variation than de novo mutation for rapid evolution of diploid genotypes.
