Project description:Cancer cell culture models frequently rely on fetal bovine serum as a source of protein and lipid factors that support cell survival and proliferation; however, serum-containing media imperfectly mimics the in vivo cancer environment. Recent studies suggest that typical serum-containing cell culture conditions can mask cancer dependencies, for example on cholesterol biosynthesis enzymes, that exist in vivo and emerge when cells are cultured in media that provides more realistic levels of lipids. Here we describe a high-throughput screen that identified fenretinide and ivermectin as small molecules whose cytotoxicity is greatly enhanced in lipid-restricted media formulations. Mechanism of action studies indicate that the ivermectin-induced cell death involves oxidative stress, while fenretinide likely targets DEGS1, a lipid desaturase necessary for ceramide synthesis, to induce cell death. Notably, both fenretinide and ivermectin have previously demonstrated in vivo anticancer efficacy despite their low cytotoxicity under typical cell culture conditions. These studies reveal ceramide synthesis as a targetable vulnerability of cancer cells cultured under lipid-restricted conditions and suggest a general screening strategy for identifying additional cancer dependencies masked by culture conditions unrepresentative of the in vivo environment.
Project description:Ferroptosis is an iron-dependent form of cell death driven by biochemical and metabolic alterations resulting in oxidation within the lipid compartment. Calcium is a potent signaling molecule ascribed to diverse cellular processes including migration, neurotransmitter function, and cell death. Here we elucidate a crucial link between calcium homeostasis and ferroptotic cell death through the identification of the tetraspanin MS4A15. Ectopic MS4A15 expression specifically protects against ferroptosis by depleting endoplasmic reticulum stores. In an unexpected connection, prolonged calcium dysregulation stimulates fundamental remodeling to ferroptosis-resistant monounsaturated and plasmalogen lipid species. Application of this discovery revealed that augmenting luminal calcium sensitizes cancer cell lines previously refractory to ferroptosis. This finding provides a unique mechanistic basis for ferroptosis sensitivity and resolves a long-standing query into the role of calcium in oxidative cell death. Manipulating calcium homeostasis offers an unprecedented strategy for overcoming therapy resistance in cancer.
Project description:Time-restricted feeding improves metabolic health independently of dietary macronutrient composition or energy restriction. To understand the mechanisms underpinning the effects of time-restricted feeding, we investigated the metabolic and transcriptomic profile of skeletal muscle and serum samples from 11 overweight/obese men. In muscle, 4-10% of transcripts and 14% of metabolites were periodic, with the amplitude of the metabolites lower after time-restricted feeding. Core clock genes were unaltered by either intervention, while time-restricted feeding induced rhythmicity of genes related to lipid and amino acid transport. In serum, 49-65% of the metabolites had diurnal rhythms across both conditions, with the majority being lipids. Time-restricted feeding shifted the skeletal muscle metabolite profile from predominantly lipids to amino acids. Our results show time-restricted feeding differentially affects the amplitudes and rhythmicity of serum and skeletal muscle metabolites, and regulates the rhythmicity of genes controlling lipid and amino acid transport, without perturbing the core clock.
Project description:The genome-wide transcriptional response of S. cerevisiae cells upon transfer to methionine-restricted media is investigated. DHO strain (BY4742 background) was cultivated batch-wise in SDC media at 30C and 180 rpm up to mid-exponential phase (OD600 ~ 0.6). The cells of the main culture were divided in three aliquots at the mid-exponential phase, centrifuged at 6000 rpm for 5 min, washed twice with deionized and distilled sterile water prior to their transfer to the treatment media. Treatment media comprised of SDC + 0.75% methionine for the methionine-restricted case while SDC is used for the control case. Sample collection for transcriptional profiling was done at the 2nd hour of the transfer. The experiments were carried out in biological triplicates.
Project description:The role of CD1-restricted, lipid-specific T cells in vivo remains to be elucidated. We used microarrays to detail the global program of gene expression accompanied by the activation of CD1-restricted, lipid-specific T cells. Monkeys were immunized with BCG bacteria to evoke CD1-restricted, mycolyl glycolipid-specific T cell responses at high frequency. The PBMCs were stimulated in the presense or absense of mycolyl glycolipid antigen, followed by RNA extraction and hybridization on Affymetrix microarrays.
Project description:RNA-seq of three different Yarrowia lipolytica strains: OKYL029 (control, W29 derived), OKYL049 (high-lipid producing strain), JFYL007 (or Q4, no-lipid producing strain). The strains were grown on DELFT media containing either ammonium sulphate or urea as nitrogen sources. DELFT media had two different carbon-to-nitrogen (C/N) ratios: a nitrogen limiting ratio of 116 and a carbon limiting ratio of 3. The experiments were performed in chemostats, with two different dilution rates of 0,06 and 0,1. Every condition was performed in quadruplicate, and triplicates were selected for RNA-seq.
Project description:Cytokines utilize the transcription factor STAT5 to control cell-specific and universal genes. In general, the magnitude of cell-restricted gene activation greatly exceeds that of universal genes, with a mechanistic explanation yet to be supplied. Genome-wide studies have identified putative STAT5-based mammary-specific enhancers and universal STAT5-controlled regulatory elements, an opportunity to investigate mechanisms underlying their differential response to cytokines. We have now interrogated the integrity and function of both categories of regulatory elements using biological and genetic approaches. During lactation, STAT5 occupies mammary-specific and universal cytokine-responsive elements. Following lactation, prolactin levels decline and STAT5-dependent enhancers at mammary-specific genes are decommissioned with 24 hours while universal regulatory complexes remain intact. These differential sensitivities are linked to STAT5 concentrations and the mammary-specific Stat5 autoregulatory enhancer. In its absence, mammary-specific enhancers, but not universal elements, fail to be fully established. Upon termination of lactation, STAT5 binding to a subset of mammary enhancers is substituted by STAT3. No STAT3 binding was observed at the most sensitive STAT5 enhancers, suggesting that upon hormone withdrawal their chromatin becomes inaccessible. This study, for the first time, provides molecular insight into the differential sensitivities of mammary-specific and universal cytokine-sensing enhancers.