Project description:Purpose: To gain additional insights to candidate factors promoting longevity in Caloric Restricted Conditioned Media (CRCM), we performed transcriptomics analysis (RNA-seq) on BY4741 yeast cells grown in non-restricted SC media supplemented with CRCM, Non-restricted Conditioned Media (NRCM), or water as a control with a goal of identifying physiological responses that could be linked to specific metabolites. Methods: RNA profiles of NR (SC, 2% glucose) treated cells supplemented with water, 2% CRCM, or 2% NRCM at log phase, 24, and 96 hours post-inoculation. These samples were conducted in triplicate. Following inoculation, we harvested cells at log phase, 24 hrs (late diauxic shift), and 96 hrs (stationary phase).Total RNA was isolated using the hot acid phenol method and then processed into Illumina DNA sequencing libraries. Briefly, total RNA was treated with DNase I for 10 min at 37°C and then measured for concentration and quality with an Agilent Bioanalyzer. PolyA mRNA selection was performed on 5µg of the DNase-treated total RNA with the NEBNext Poly(A) mRNA magnetic isolation module (E7490). DNA sequencing libraries were then generated with the NEBNExt Ultra Directional RNA library Prep kit for Illumina (E7420). Libraries were sequenced on an Illumina NextSeq 500 by the UVA Genome Analysis and Technology Core. Results: Sequencing reads were mapped to the sacCer3 genome using bowtie2 with default settings. We preprocessed sequencing data from the UVA genomics core and analyzed differential gene expression in R using DESeq2. We found that In log phase cells, there were no significantly upregulated or downregulated genes in the CRCM or NRCM samples as compared to the water supplemented control (FDR <0.05). At the 24 hr timepoint, CRCM samples diverged from the NRCM and water supplemented control samples, and showed many more differentially regulated genes than the NRCM-treated samples. At the 96 hr timepoint, NRCM samples also became more clearly separated from the control in a PCA plot , though there were still a large number of genes exclusively altered in the CRCM samples. Furthermore, the YCL064C (CHA1) gene clearly stood out as being the most significantly upregulated in conditioned media supplementation. Conclusions: RNAseq data led us to explore YCL064C (CHA1) gene clearly stood out as being the most significantly upregulated in the conditioned media experiments. CHA1 encodes a predominantly mitochondrial L-serine (L-threonine) deaminase that catabolizes these amino acids. RNA-seq based transcriptome analysis allowed us to explore L-serine contribution to longevity phenotype we observed with CRCM.

Project description:Studies using yeast have advanced our understanding of both replicative and chronological aging, leading to the discovery of longevity genes that have homologues in higher eukaryotes. Chronological lifespan in yeast is conventionally defined as the lifespan of a non-dividing cell. To date, this parameter has only been estimated under calorically restricted (CR) conditions, mimicked by starvation. Since post-mitotic cells in higher eukaryotes are rarely calorically-restricted, we sought to develop an alternative experimental system where non-dividing yeast would age chronologically, in the presence of excess nutrients. We report here on a system wherein alginate-encapsulated yeast are packed in a pH- and temperature-controlled bioreactor, then continuously fed non-limiting substrate for extended periods of time. We present demographic, physiological and genomic evidence indicating that after ~120 hrs, immobilized cells cease dividing, remain metabolically very active and retain >95% viability for periods of 17 days. Over the same time interval, starved planktonic cells, cultured using the same media, and also controlled for temperature and pH, retained < 1 % viability in both aerobic and anaerobic cultures,. Unlike planktonic yeast, continuously-fed immobilized cells hyper-accumulate glycogen. FACS analysis of SYTOX green-stained yeast confirms that immobilized cells completely arrest within 5 days of culture, and unlike starving planktonic cells, remain free thereafter of replicative stress and are non-apoptotic. This unusual state is supported by a global gene expression profile that is stable over time, repeatable across replicate experiments, and altogether distinct from planktonic cells cultured in the presence and absence of limiting nutrients. DNA expression profiling, performed here for the very first time on immobilized cells, reveals that glycolytic genes and their trans-acting regulatory elements are upregulated, as are genes involved in remodeling the cell wall and resisting stress; by contrast, many genes that promote cell cycle progression and carry out oxidative metabolism are repressed. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously upregulated in the immobilized state, suggesting that nutrient-sensing pathways may play a role in cell viability and longevity when yeast are immobilized and placed in prolonged culture under calorically-unrestricted conditions. The cell cycle arrest in the immobilized state is mediated by RIM 15. Over the time-course of our experiments, well-fed, non-diving immobilized cells do not appear to age. Keywords: comparison of growth states and a time course of immobilized cells Nine growth conditions or time points with two to four replicates each

Project description:Studies using yeast have advanced our understanding of both replicative and chronological aging, leading to the discovery of longevity genes that have homologues in higher eukaryotes. Chronological lifespan in yeast is conventionally defined as the lifespan of a non-dividing cell. To date, this parameter has only been estimated under calorically restricted (CR) conditions, mimicked by starvation. Since post-mitotic cells in higher eukaryotes are rarely calorically-restricted, we sought to develop an alternative experimental system where non-dividing yeast would age chronologically, in the presence of excess nutrients. We report here on a system wherein alginate-encapsulated yeast are packed in a pH- and temperature-controlled bioreactor, then continuously fed non-limiting substrate for extended periods of time. We present demographic, physiological and genomic evidence indicating that after ~120 hrs, immobilized cells cease dividing, remain metabolically very active and retain >95% viability for periods of 17 days. Over the same time interval, starved planktonic cells, cultured using the same media, and also controlled for temperature and pH, retained < 1 % viability in both aerobic and anaerobic cultures,. Unlike planktonic yeast, continuously-fed immobilized cells hyper-accumulate glycogen. FACS analysis of SYTOX green-stained yeast confirms that immobilized cells completely arrest within 5 days of culture, and unlike starving planktonic cells, remain free thereafter of replicative stress and are non-apoptotic. This unusual state is supported by a global gene expression profile that is stable over time, repeatable across replicate experiments, and altogether distinct from planktonic cells cultured in the presence and absence of limiting nutrients. DNA expression profiling, performed here for the very first time on immobilized cells, reveals that glycolytic genes and their trans-acting regulatory elements are upregulated, as are genes involved in remodeling the cell wall and resisting stress; by contrast, many genes that promote cell cycle progression and carry out oxidative metabolism are repressed. Stress resistance transcription factor MSN4 and its upstream effector RIM15 are conspicuously upregulated in the immobilized state, suggesting that nutrient-sensing pathways may play a role in cell viability and longevity when yeast are immobilized and placed in prolonged culture under calorically-unrestricted conditions. The cell cycle arrest in the immobilized state is mediated by RIM 15. Over the time-course of our experiments, well-fed, non-diving immobilized cells do not appear to age. Keywords: comparison of growth states and a time course of immobilized cells

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A cell-nonautonomous mechanism of yeast chronological aging regulated by caloric restriction and one-carbon metabolism.

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