ABSTRACT: Gene array experiment to identify genes that are upregulated by saturating creatine levels in vitro and potentially inhibiting creatine transporter (CrT) activity
Project description:We sought to investigate the mechanisms of CrT modulation by saturating extracellular Cr. For this purpose, we used an in vitro system of 3T3 fibroblasts stably overexpressing CrT and followed a global gene array approach to test the hypothesis that saturating intracellular [Cr] increases the expression of candidate molecules that inhibit CrT activity. In this way, we would elucidate mechanisms of redox regulation in cases of fluctuating energy demands. 3T3-CrT cells were grown on 6-well plates to 70% confluency. n=3 replicate wells were treated with 5mM creatine monohydrate in growth media and n=3 wells remained untreated (controls).
Project description:We sought to investigate the mechanisms of CrT modulation by saturating extracellular Cr. For this purpose, we used an in vitro system of 3T3 fibroblasts stably overexpressing CrT and followed a global gene array approach to test the hypothesis that saturating intracellular [Cr] increases the expression of candidate molecules that inhibit CrT activity. In this way, we would elucidate mechanisms of redox regulation in cases of fluctuating energy demands.
Project description:Next-generation proteomics of human brain organoids derived from individuals with creatine transporter deficiency in order to assess their characteristics as models of the pathophysiology.
Project description:Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, the creatine deficiency syndrome (CDS) is manifested by impaired metabolism, transport, and distribution of creatine throughout the body leading to severe forms of mental disabilities. One of the common cause of CDS is mutations that impact the function of the creatine transporter (SLC6A8). This study characterized 30 missense variants of SLC6A8, which include 15 variants of unknown significance and two which were not reported before. These variants were expressed in HEK293 cells, and their subcellular localization and transport activity was assessed. Further, for the first time the interactome of SLC6A8 WT was characterized by quantitative interaction proteomics. Computational methods were then used to first assess the impact of mutations upon the thermodynamic stability of SLC6A8. This was expanded by modeling the impact of mutations upon the inward and outward facing transporter conformation. Next, identified SLC6A8 protein interactions binary complexes were modelled and used to characterize the impact of variants upon the thermodynamic stability of the complexes. This was followed up, by performing for a subset of the variants the interaction proteomics analysis to study changes upon the identified SLC6A8 WT interactome.
Project description:Expression profiling of vastus lateralis muscle of young, healthy, non-obese men supplemented with creatine monohydrate vs. placebo for 10 days. Results identify transcriptional pathways activated in skeletal muscles as a result of acute creatine monohydrate supplementation. Keywords: Supplementation response.
Project description:The metabolic dependencies of cancer cells have significant potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is a novel urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high creatine riboside levels in cancer patients, as well as its implications for the treatment of these aggressive cancers remain unclear. By integrating RNA sequencing and whole exome sequencing of non-small cell lung cancer tissues we sought to understand the source of creatine riboside and the tumor biology associated with this novel metabolite. Utilising multiple lung and liver cancer cohorts, next generation sequencing data was combined with global metabolomics analysis of human tumors, and matched liquid biopsies together with functional studies to reveal that dysregulation of the mitochondrial urea cycle and nucleotide imbalance are associated with high creatine riboside levels and are indicators of poor prognosis. Creatine riboside-high cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of creatine riboside not only as a poor prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve cancer patient survival.
Project description:To determine the miRNAs potentially responsible for the premature-senescence mediated gene expression regulation in human adipose-derived mesenchymal stem cells, we performed illumine bead microarray analyses on parental hMSCs, and globally depleted miRNAs by inhibiting DGCR8, an essential component of miRNA biogenesis. DGCR8 knockdown in hMSCs resulted in severe proliferation defects with senescence-associated changes including markedly increased reactive oxygen species (ROS) levels. In gene expression profiling, thousand two hundred and forty seven transcripts were upregulated and 1,136 genes were downregulated in Dgcr8 knockdown cells compared to control cells (p < 0.05, Fisher’s exact test).
