Replicative aging is associated with loss of genetic heterogeneity from extrachromosomal circular DNA in <i>Saccharomyces cerevisiae</i>

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Circular DNA can arise from all parts of eukaryotic chromosomes. In yeast, circular ribosomal DNA (rDNA) accumulates dramatically as cells age, however little is known about the accumulation of other chromosome-derived circles or the contribution of such circles to genetic variation in aged cells. We profiled circular DNA in Saccharomyces cerevisiae populations sampled when young and after extensive aging. Young cells possessed highly diverse circular DNA populations but 94% of the circular DNA were lost after ∼15 divisions, whereas rDNA circles underwent massive accumulation to >95% of circular DNA. Circles present in both young and old cells were characterized by replication origins including circles from unique regions of the genome and repetitive regions: rDNA and telomeric Y' regions. We further observed that circles can have flexible inheritance patterns: [HXT6/7circle] normally segregates to mother cells but in low glucose is present in up to 50% of cells, the majority of which must have inherited this circle from their mother. Interestingly, [HXT6/7circle] cells are eventually replaced by cells carrying stable chromosomal HXT6 HXT6/7 HXT7 amplifications, suggesting circular DNAs are intermediates in chromosomal amplifications. In conclusion, the heterogeneity of circular DNA offers flexibility in adaptation, but this heterogeneity is remarkably diminished with age.ISSN:1362-4962ISSN:0301-561

The Moral of the Tale: Stories, Trust, and Public Engagement with Clinical Ethics via Radio and Theatre.

Trust is frequently discussed with reference to the professional-patient relationship. However, trust is less explored in relation to the ways in which understanding of, and responses to, questions of ethics are discussed by both the "public" and "experts." Public engagement activity in healthcare ethics may invoke "trust" in analysing a moral question or problem but less frequently conceives of trust as integral to "public engagement" itself. This paper explores the relationship between trust and the ways in which questions of healthcare ethics are identified and negotiated by both "experts" and the public. Drawing on two examples from the author's "public engagement" work-a radio programme for the British Broadcasting Corporation and work with a playwright and theatre-the paper interrogates the ways in which "public engagement" is often characterized. The author argues that the common approach to public engagement in questions of ethics is unhelpfully constrained by a systemic disposition which continues to privilege the professional or expert voice at the expense of meaningful exchange and dialogue. By creating space for novel interactions between the "expert" and the "public," authentic engagement is achieved that enables not only the participants to flourish but also contributes to trust itself

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development

Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner, whereas alpha-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h(-1) that influences protein production. Thus, for lower specific growth rates, the alpha-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas for higher specific growth rates, the two strains respond differently to changes in the specific growth rate

Industrial Systems Biology of Saccharomyces cerevisiae Enables Novel Succinic Acid Cell Factory.

Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the alpha-keto-glutarate dehydrogenase catalyzed conversion of alpha-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive cell factory for over-producing different platform chemicals

Analysis of Hypoxia and Hypoxia-Like States through Metabolite Profiling

In diverse organisms, adaptation to low oxygen (hypoxia) is mediated through complex gene expression changes that can, in part, be mimicked by exposure to metals such as cobalt. Although much is known about the transcriptional response to hypoxia and cobalt, little is known about the all-important cell metabolism effects that trigger these responses.Herein we use a low molecular weight metabolome profiling approach to identify classes of metabolites in yeast cells that are altered as a consequence of hypoxia or cobalt exposures. Key findings on metabolites were followed-up by measuring expression of relevant proteins and enzyme activities. We find that both hypoxia and cobalt result in a loss of essential sterols and unsaturated fatty acids, but the basis for these changes are disparate. While hypoxia can affect a variety of enzymatic steps requiring oxygen and heme, cobalt specifically interferes with diiron-oxo enzymatic steps for sterol synthesis and fatty acid desaturation. In addition to diiron-oxo enzymes, cobalt but not hypoxia results in loss of labile 4Fe-4S dehydratases in the mitochondria, but has no effect on homologous 4Fe-4S dehydratases in the cytosol. Most striking, hypoxia but not cobalt affected cellular pools of amino acids. Amino acids such as aromatics were elevated whereas leucine and methionine, essential to the strain used here, dramatically decreased due to hypoxia induced down-regulation of amino acid permeases.These studies underscore the notion that cobalt targets a specific class of iron proteins and provide the first evidence for hypoxia effects on amino acid regulation. This research illustrates the power of metabolite profiling for uncovering new adaptations to environmental stress

Transcriptome and Proteome Exploration to Model Translation Efficiency and Protein Stability in Lactococcus lactis

This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms

Patterns and mechanisms of early Pliocene warmth

About five to four million years ago, in the early Pliocene epoch, Earth had a warm, temperate climate. The gradual cooling that followed led to the establishment of modern temperature patterns, possibly in response to a decrease in atmospheric CO2 concentration, of the order of 100 parts per million, towards preindustrial values. Here we synthesize the available geochemical proxy records of sea surface temperature and show that, compared with that of today, the early Pliocene climate had substantially lower meridional and zonal temperature gradients but similar maximum ocean temperatures. Using an Earth system model, we show that none of the mechanisms currently proposed to explain Pliocene warmth can simultaneously reproduce all three crucial features. We suggest that a combination of several dynamical feedbacks underestimated in the models at present, such as those related to ocean mixing and cloud albedo, may have been responsible for these climate conditions

Genomic Binding Profiling of the Fission Yeast Stress-Activated MAPK Sty1 and the bZIP Transcriptional Activator Atf1 in Response to H2O2

10.1371/journal.pone.0011620PLoS ONE57