Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency.

SummaryThe red colour of tomato (Lycopersicon esculentum) fruits is provided by the carotenoid pigment lycopene whose concentration increases dramatically during the ripening process. A single dominant gene, Del, in the tomato mutant Delta changes the fruit colour to orange as a result of accumulation of δ-carotene at the expense of lycopene. The cDNA for lycopene ε-cyclase (CrtL-e), which converts lycopene to δ-carotene, was cloned from tomato. The primary structure of CRTL-E is 71% identical to the homologous polypeptide from Arabidopsis and 36% identical to the tomato lycopene β-cyclase, CRTL-B. The CrtL-e gene was mapped to a single locus on chromosome 12 of the tomato linkage map. This locus co-segregated with the Del gene. In the wild-type tomato, the transcript level of CrtL-e decreases at the 'breaker' stage of ripening to a nondetectable level in the ripe fruit. In contrast, it increases approximatley 30-fold during fruit ripening in the Delta plants. The Delta mutation does not affect carotenoid composition nor the mRNA level of CrtL-e in leaves and flowers. These results strongly suggest that the mutation Del is an allele of the gene for ε-cyclase. Together with previous data, our results indicate that the primary mechanism that controls lycopene accumulation in tomato fruits is based on the differential regulation of expression of carotenoid biosynthesis genes. During fruit development, the mRNA levels for the lycopene-producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase, while the mRNA levels of the genes for the lycopene β-and ε-cyclases, which convert lycopene to either β-or δ-carotene, respectively, decline and completely disappear.

Microfluidic organ-on-a-chip technology aims to replace animal toxicity testing, but thus far has demonstrated few advantages over traditional methods. Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity, as well as pluripotency and disease processes. However, current methods to evaluate mitochondrial activity still rely on end-point assays, resulting in limited kinetic and prognostic information. Here, we present a liveron-chip device capable of maintaining human tissue for over a month in vitro under physiological conditions. Mitochondrial respiration was monitored in real time using two-frequency phase modulation of tissue-embedded phosphorescent microprobes. A computer-controlled microfluidic switchboard allowed contiguous electrochemical measurements of glucose and lactate, providing real-time analysis of minute shifts from oxidative phosphorylation to anaerobic glycolysis, an early indication of mitochondrial stress. We quantify the dynamics of cellular adaptation to mitochondrial damage and the resulting redistribution of ATP production during rotenone-induced mitochondrial dysfunction and troglitazone (Rezulin)-induced mitochondrial stress. We show troglitazone shifts metabolic fluxes at concentrations previously regarded as safe, suggesting a mechanism for its observed idiosyncratic effect. Our microfluidic platform reveals the dynamics and strategies of cellular adaptation to mitochondrial damage, a unique advantage of organ-on-chip technology.microfluidics | liver tissue engineering | toxicology | organ-on-a-chip

To navigate O2 gradients, C. elegans can modulate turning rates and speed of movement. Aerotaxis can be reprogrammed by experience or engineered artificially. We propose a model in which prolonged activation of the AQR, PQR, and URX neurons by low O2 switches on previously inactive O2 sensors. This enables aerotaxis to low O2 environments and may encode a "memory" of previous cultivation in low O2.

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