A vitamin-C-derived DNA modification catalysed by an algal TET homologue.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.

TiO2 nanoparticles cause mitochondrial dysfunction, activate inflammatory responses, and attenuate phagocytosis in macrophages: A proteomic and metabolomic insight.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in food and cosmetics but the health impact of human exposure remains poorly defined. Emerging evidence suggests that TiO2 NPs may elicit immune responses by acting on macrophages. Our proteomic study showed that treatment of macrophages with TiO2 NPs led to significant re-organization of cell membrane and activation of inflammation. These observations were further corroborated with transmission electron microscopy (TEM) experiments, which demonstrated that TiO2 NPs were trapped inside of multi-vesicular bodies (MVB) through endocytotic pathways. TiO2 NP caused significant mitochondrial dysfunction by increasing levels of mitochondrial reactive oxygen species (ROS), decreasing ATP generation, and decreasing metabolic flux in tricarboxylic acid (TCA) cycle from 13C-labelled glutamine using GC-MS-based metabolic flux analysis. Further lipidomic analysis showed that TiO2 NPs significantly decreased levels of cardiolipins, an important class of mitochondrial phospholipids for maintaining proper function of electron transport chains. Furthermore, TiO2 NP exposure activates inflammatory responses by increasing mRNA levels of TNF-α, iNOS, and COX-2. Consistently, our targeted metabolomic analysis showed significantly increased production of COX-2 metabolites including PGD2, PGE2, and 15d-PGJ2. In addition, TiO2 NP also caused significant attenuation of phagocytotic function of macrophages. In summary, our studies utilizing multiple powerful omic techniques suggest that human exposure of TiO2 NPs may have profound impact on macrophage function through activating inflammatory responses and causing mitochondrial dysfunction without physical presence in mitochondria.

Discovering a critical transition state from nonalcoholic hepatosteatosis to nonalcoholic steatohepatitis by lipidomics and dynamical network biomarkers.

Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for type 2 diabetes and metabolic syndrome. However, accurately differentiating nonalcoholic steatohepatitis (NASH) from hepatosteatosis remains a clinical challenge. We identified a critical transition stage (termed pre-NASH) during the progression from hepatosteatosis to NASH in a mouse model of high fat-induced NAFLD, using lipidomics and a mathematical model termed dynamic network biomarkers (DNB). Different from the conventional biomarker approach based on the abundance of molecular expressions, the DNB model exploits collective fluctuations and correlations of different metabolites at a network level. We found that the correlations between the blood and liver lipid species drastically decreased after the transition from steatosis to NASH, which may account for the current difficulty in differentiating NASH from steatosis based on blood lipids. Furthermore, most DNB members in the blood circulation, especially for triacylglycerol (TAG), are also identified in the liver during the disease progression, suggesting a potential clinical application of DNB to diagnose NASH based on blood lipids. We further identified metabolic pathways responsible for this transition. Our study suggests that the transition from steatosis to NASH is not smooth and the existence of pre-NASH may be partially responsible for the current clinical limitations to diagnose NASH. If validated in humans, our study will open a new avenue to reliably diagnose pre-NASH and achieve early intervention of NAFLD.