Project description:Larvae were reared on standard diet until early third instar, at which time they were washed and transferred to standard diet lacking yeast. The animals remained on this diet until four days after emergence, when one group of adults was switched back to standard diet containing yeast (group Y) while another remained on the diet lacking yeast (group NY). Flies from both groups were killed every hour for the next twelve hours, creating 24 samples across the two treatments. In addition, four samples of flies were killed just before the start of the time course and used as baseline replicates for the no yeast (NY) and yeast (Y) treatments. Baseline replicates were temporally ordered as noted for change-point analysis. No yeast (NY) treatment samples at hours four and eight did not yield microarray data due to insufficient RNA. Total RNA was extracted from whole animals using Trizol (Invitrogen). Sample processing and microarray hybridization/scanning were performed at the Brown University Center for Genetics and Genomics according to Affymetrix protocol. Change-point Analysis Results Table: Results of running change-point analysis on dChip normalized data. Normalized data was transformed into yeast (Y):no yeast (NY) signal ratios, and change-point analysis was performed by GeneTrace on these ratios (see publication for more information on change-point analysis). Raw Data, Not Normalized Table: Raw data (not normalized). Image files were analyzed by Affymetrix Microarray Suite (MAS) 5.0 with no normalization and no scaling. Signal abundance measurements, present [P], marginal [M], or absent [A] calls, and detection p-values reported were all produced by MAS 5.0. Note that there is no data for no yeast (NY) treatment samples at hours 4 and 8 due to insufficient RNA yield. Keywords = insulin, diet, nutrition Keywords: other
Project description:Barth syndrome (BTHS) is a rare X-linked recessively inherited disorder caused by variants in the TAFAZZIN gene. The pathogenic variants lead to impaired conversion of monolysocardiolipin (MLCL) into mature phospholipid cardiolipin (CL). The accumulation of MLCL and mature CL deficiency is a diagnostic marker for BTHS. The clinical spectrum includes cardiomyopathy, skeletal myopathy, neutropenia, and delays in growth. In severely affected BTHS patients, the cardiac phenotype is early onset, heterogeneous and unpredictable. Ultimately, these patients may require a cardiac transplant early in their life. Unfortunately, the pathophysiological mechanisms of BTHS are poorly understood, and treatment options for BTHS remain symptomatic. In this study, we analysed heart samples from five paediatric male BTHS individuals (5 month-15 years old) and compared them to tissues from 24 non-failing donors (19-71 years old) using a newly developed integrated omics method that combines metabolomics, lipidomics and proteomics using a single sample. This comprehensive analysis confirms expected changes in established diagnostic markers such as CL and MLCL, as well as severe and pleiotropic alterations in mitochondrial phenotype and metabolic output, a substrate shift in energy metabolism, and an elevation of heart-failure markers. It also reveals striking interindividual differences between BTHS individuals. Combined, we describe a powerful analytical tool for the in-depth analysis of metabolic disorders and a solid foundation for the understanding of BTHS disease phenotypes in cardiac tissues.
Project description:For yeast cells, tolerance to high levels of ethanol is vital both in their natural environment and in industrially relevant conditions. We recently genotyped experimentally evolved yeast strains adapted to high levels of ethanol and identified mutations linked to ethanol tolerance. In this study, by integrating genomic sequencing data with quantitative proteomics profiles from six evolved strains (data set identifier PXD006631) and construction of protein interaction networks, we elucidate exactly how the genotype and phenotype are related at the molecular level. Our multi-omics approach points to the rewiring of numerous metabolic pathways affected by genomic and proteomic level changes, from energy-producing and lipid pathways to differential regulation of transposons and proteins involved in cell cycle progression. One of the key differences is found in the energy-producing metabolism, where the ancestral yeast strain responds to ethanol by switching to respiration and employing the mitochondrial electron transport chain. In contrast, the ethanol-adapted strains appear to have returned back to energy production mainly via glycolysis and ethanol fermentation, as supported by genomic and proteomic level changes. This work is relevant for synthetic biology where systems need to function under stressful conditions, as well as for industry and in cancer biology, where it is important to understand how the genotype relates to the phenotype.
Project description:We identified a molecule in a synthetic lethal screen with ira2Δ in yeast called Y100. Y100 targets ira2Δ deficient yeast and inhibits NF1-deficient tumor cells. Y100 disrupts proteostasis, metabolic homeostasis, and induces the formation of mitochondrial superoxide in NF1 deficient cancer cells. Here, we examined the transcriptional response following treatment with Y100 or a vehicle control.
Project description:The yeast Saccharomyces cerevisiae has long been used to produce alcohol from glucose and other sugars. While much is known about glucose metabolism, relatively little is known about the receptors and signaling pathways that indicate glucose availability. Here, we compare the two glucose receptor systems in S. cerevisiae. The first is a heterodimer of transporter-like proteins (transceptors), while the second is a seven-transmembrane receptor coupled to a large G protein (Gpa2) that acts in coordination with two small G proteins (Ras1 and Ras2). Through comprehensive measurements of glucose-dependent transcription and metabolism, we demonstrate that the two receptor systems have distinct roles in glucose signaling: the G-protein-coupled receptor directs carbohydrate and energy metabolism, while the transceptors regulate ancillary processes such as ribosome, amino acids, cofactor and vitamin metabolism. The large G-protein transmits the signal from its cognate receptor, while the small G-protein Ras2 (but not Ras1) integrates responses from both receptor pathways. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.
Project description:Centromeres ensure accurate chromosome segregation, yet their DNA evolves rapidly across eukaryotes, leaving the origin of new centromere architectures unresolved. In the brewer’s yeast Saccharomyces cerevisiae (order Saccharomycetales), compact, genetically defined “point” centromeres replaced large, repeat-rich, epigenetically specified centromeres, but how this transition occurred has been unclear. Competing models have proposed either descent with modification from ancestral epigenetic centromeres or acquisition from selfish plasmid DNA. Here we map and characterize centromeres in the sister order Saccharomycodales and identify evolutionarily related “proto-point” centromeres that bridge repeat-rich and point centromeres. Proto-point centromeres contain a single centromeric nucleosome positioned over an AT-rich core, but retain relaxed organization and sequence variability in flanking cis-elements. In two species, including Saccharomycodes ludwigii, proto-point centromeres are embedded within clusters of Ty5 long terminal repeat (LTR) retrotransposons, and their core CDEII and flanking motifs share sequence similarity to Ty5 LTR sequence. Comparative genomics, synteny, and phylogenetic analyses across multiple yeast orders show that Ty5-cluster centromeres are ancient genomic features and support a model in which proto-point and point centromeres evolved by co-option of Ty5 LTR sequences in an ancestor with retrotransposon-rich centromeres, rather than by horizontal transfer from the 2-µm plasmid. These results indicate that yeast point centromeres are direct descendants of retrotransposons and illustrate how transposable elements can be repurposed to create genetically encoded centromeres.
Project description:Systems biology is increasingly being applied in nanosafety research for observing and predicting the biological perturbations inflicted by exposure to nanoparticles (NPs). In the present study, we used a combined transcriptomics and proteomics approach to assess the responses of human monocytic cells to Au-NPs of two different sizes with three different surface functional groups, i.e., alkyl ammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated Au-NPs. Cytotoxicity screening using THP-1 cells revealed a pronounced cytotoxicity for the ammonium-terminated Au-NPs, while no cell death was seen after exposure to the carboxylated or PEG-modified Au-NPs. Moreover, Au-NR3+ NPs, but not the Au-COOH NPs, were found to trigger dose-dependent lethality in vivo in the model organism, Caenorhabditis elegans. RNA sequencing combined with mass spectrometry-based proteomics predicted that the ammonium-modified Au-NPs elicited mitochondrial dysfunction. The latter results were validated by using an array of assays to monitor mitochondrial function. Au-NR3+ NPs were localized in mitochondria of THP-1 cells. Moreover, the cationic Au-NPs triggered autophagy in macrophage-like RFP-GFP-LC3 reporter cells, and cell death was aggravated upon inhibition of autophagy. Taken together, these studies have disclosed mitochondria-dependent effects of cationic Au-NPs resulting in the rapid demise of the cells.
Project description:Peptides present in growth media are essential for nitrogen nutrition and optimal growth of lactic acid bacteria. In addition, according to their amino acid composition, they can also directly or indirectly play regulatory roles and influence global metabolism. This is especially relevant during the propagation phase to produce high cell counts of active lactic acid bacteria used as starters in the dairy industry. In the present work, we aimed at investigating how the respective compositions of two different yeast extracts, with a specific focus on peptide content, influenced Streptococcus thermophilus metabolism during growth under pH-controlled conditions. In addition to free amino acid quantification, we used a multi-omics approach (peptidomics, proteomics, and transcriptomics) to identify peptides initially present in the two culture media and to follow S. thermophilus gene expression and bacterial protein production during growth. The free amino acid and peptide compositions of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar levels of growth of S. thermophilus and led to equivalent final biomasses. However, transcriptomics and proteomics showed differential gene expression and protein production in several S. thermophilus metabolic pathways, especially amino acid, citrate, urease, purine, and pyrimidine metabolisms. The probable role of the regulator CodY is discussed in this context. Moreover, we observed significant differences in the production of regulators and of a quorum sensing regulatory system. The possible roles of yeast extract peptides on the modulation of the quorum sensing system expression are evaluated.IMPORTANCE Improving the performance and industrial robustness of bacteria used in fermentations and food industry remains a challenge. We showed here that two Streptococcus thermophilus fermentations, performed with the same strain in media that differ only by their yeast extract compositions and, more especially, their peptide contents, led to similar growth kinetics and final biomasses, but several genes and proteins were differentially expressed/produced. In other words, subtle variations in peptide composition of the growth medium can finely tune the metabolism status of the starter. Our work, therefore, suggests that acting on growth medium components and especially on their peptide content, we could modulate bacterial metabolism and produce bacteria differently programmed for further purposes. This might have applications for preparing active starter cultures.
Project description:In this study, we present the first genome-wide recombination map for mitochondrial DNA in yeast. We also assess the impact of the genetic background and of several gene deletions on the recombination profiles.