Origin, Regulation, and Fitness Effect of Chromosomal Rearrangements in the Yeast Saccharomyces cerevisiae.

Chromosomal rearrangements comprise unbalanced structural variations resulting in gain or loss of DNA copy numbers, as well as balanced events including translocation and inversion that are copy number neutral, both of which contribute to phenotypic evolution in organisms. The exquisite genetic assay and gene editing tools available for the model organism Saccharomyces cerevisiae facilitate deep exploration of the mechanisms underlying chromosomal rearrangements. We discuss here the pathways and influential factors of chromosomal rearrangements in S. cerevisiae. Several methods have been developed to generate on-demand chromosomal rearrangements and map the breakpoints of rearrangement events. Finally, we highlight the contributions of chromosomal rearrangements to drive phenotypic evolution in various S. cerevisiae strains. Given the evolutionary conservation of DNA replication and recombination in organisms, the knowledge gathered in the small genome of yeast can be extended to the genomes of higher eukaryotes.

Genome-wide mapping of spontaneous genetic alterations in diploid yeast cells.

Genomic alterations including single-base mutations, deletions and duplications, translocations, mitotic recombination events, and chromosome aneuploidy generate genetic diversity. We examined the rates of all of these genetic changes in a diploid strain of Saccharomyces cerevisiae by whole-genome sequencing of many independent isolates (n = 93) subcloned about 100 times in unstressed growth conditions. The most common alterations were point mutations and small (<100 bp) insertion/deletions (n = 1,337) and mitotic recombination events (n = 1,215). The diploid cells of most eukaryotes are heterozygous for many single-nucleotide polymorphisms (SNPs). During mitotic cell divisions, recombination can produce derivatives of these cells that have become homozygous for the polymorphisms, termed loss-of-heterozygosity (LOH) events. LOH events can change the phenotype of the cells and contribute to tumor formation in humans. We observed two types of LOH events: interstitial events (conversions) resulting in a short LOH tract (usually less than 15 kb) and terminal events (mostly cross-overs) in which the LOH tract extends to the end of the chromosome. These two types of LOH events had different distributions, suggesting that they may have initiated by different mechanisms. Based on our results, we present a method of calculating the probability of an LOH event for individual SNPs located throughout the genome. We also identified several hotspots for chromosomal rearrangements (large deletions and duplications). Our results provide insights into the relative importance of different types of genetic alterations produced during vegetative growth.

Inhibition of hepatic cells pyroptosis attenuates CLP-induced acute liver injury.

Pyroptosis is a programmed cell death associated with caspase-1 and accompanied by the secretion of a large number of pro-inflammatory cytokines. In the acute stage of sepsis, the release of several pro-inflammatory cytokines aggravates hepatic cell death, and acute liver injury is aggravated with the progress of the disease, resulting in acute liver failure with a very high mortality rate. The present study investigated the effect of inhibiting hepatic cell pyroptosis on the septic acute liver injury. Septic acute liver injury mice model was established by cecal ligation and puncture (CLP model). The liver tissues were assessed for inflammatory infiltration by HE, serum concentrations of ALT, AST, IL-1ß, and IL-18 were examined by ELISA, hepatic cell pyroptosis was determined by flow cytometry, and expressions of caspase-1 and NLRP3 were assessed by Western blot. CLP-induced acute liver injury was distinct at 24 h post-operation, with the highest hepatic cell pyroptosis rate. The pyroptosis rate and liver injury indexes were positively correlated. Western blot showed that the expressions of pyroptosis-related proteins, caspase-1, and NLRP3, were increased. Normal mouse hepatic cells were cultured in vitro and LPS+ATP introduced to establish the cell model of septic acute liver injury. The expressions of caspase-1, NLRP3, IL-1ß, and IL-18 in LPS+ATP group were significantly higher than the control group by Western blot and ELISA. The inhibitors of NLRP3 (Glyburide) and caspase-1 (AC-YVAD-CMK) alone or in combination were used to pre-treat the hepatic cells, which revealed that the pyroptosis rate was decreased and the cell damage alleviated. The in vivo assay in rats showed that post inhibitor treatment, the 10-days survival was significantly improved and the liver damage reduced. Therefore, inhibiting the hepatic cell pyroptosis could alleviate CLP-induced acute liver injury, providing a novel treatment target for septic acute liver injury.

TLR4-dependent internalization of CX3CR1 aggravates sepsis-induced immunoparalysis.

Sepsis, the most severe manifestation of infection, poses a major challenge to health-care systems around the world. Limited ability to clean and remove the pathogen renders difficulty in septic patients to recover from the phase of immunoparalysis. The present study found the vital role of CX3CR1 internalization on sepsis-induced immunoparalysis. A mouse model with cecal ligation and puncture (CLP) and cell model with lipopolysaccharides (LPS) were employed to explore the relationship between CX3CR1 internalization and septic immunoparalysis. Immunoparalysis model in mice was established 4 days after CLP with significantly decreased proinflammatory cytokines. Flow cytometry analysis found a decreased surface expression of CX3CR1 during immunoparalysis, which was associated with reduced mRNA level and increased internalization of CX3CR1. G-protein coupled receptor kinase 2 (GRK2) and ß-arrestin2 were significantly increased during septic immunoparalysis and involved in the internalization of CX3CR1. TLR4-/- or TLR4 inhibitor-treated macrophages exhibited an inhibited expression of GRK2 and ß-arrestin2, along with reduced internalization of CX3CR1. Moreover, the knockdown of GRK2 and ß-arrestin2 inhibited the internalization of CX3CR1 and led to a higher response on the second hit, which was associated with an increased activation of NF-κB. The critical association between internalization of CX3CR1 and immunosuppression in sepsis may provide a novel reference for clinical therapeutics.