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.

A scheme enabling the complete sampling of multidimensional NMR domains within a single continuous acquisition is introduced and exemplified. Provided that an analyte's signal is sufficiently strong, the acquisition time of multidimensional NMR experiments can thus be shortened by orders of magnitude. This could enable the characterization of transient events such as proteins folding, 2D NMR experiments on samples being chromatographed, bring the duration of higher dimensional experiments (e.g., 4D NMR) into the lifetime of most proteins under physiological conditions, and facilitate the incorporation of spectroscopic 2D sequences into in vivo imaging investigations. The protocol is compatible with existing multidimensional pulse sequences and can be implemented by using conventional hardware; its performance is exemplified here with a variety of homonuclear 2D NMR acquisitions. F ew analytical techniques in science match in either breadth or depth the impact achieved by nuclear magnetic resonance (NMR) (1). After establishing itself as a tool for the characterization of organic molecules (2), the use of NMR has spread throughout the decades, reaching into areas as diverse as pharmaceutics, metabolic studies, structural biology, solid state chemistry, condensed matter physics, rheology, medical diagnosis (where it is known as MRI), and more recently neurobiology (1-8). In addition to assuming in all these disciplines a position of preeminence, the principles of NMR have served as paradigm to other physical methods that also rely on the interaction between radiation and matter (9, 10). Lying at the core of the expansion of NMR are powerful protocols developed over the years for characterizing the nuclear spin environment; in particular, the Fourier transform (FT) and the multidimensional method of NMR analysis (11-13). Contemporary NMR applications in all the scientific disciplines mentioned above rely routinely on Fourier and multidimensional spectroscopies for their implementation.Unidimensional Fourier spectroscopy brings the so-called multiplex advantage into NMR, enabling a shortening in the data-scanning time by orders of magnitude and bringing about a concomitant increase in the signal-to-noise ratio (S͞N) achievable per unit time. FT-NMR methods, the principles of which eventually extended to many other forms of spectroscopy (14), encode the complete spectral distribution I() being sought by acquiring a single time-domain transient S(t), from which I() is reconstructed via a discrete version of the FT calculation I() ϭ (1͞2)͐ 0 AT S(t)eϪit dt. By contrast, the goal of multidimensional spectroscopy is not just to measure but to correlate NMR frequencies over several spectral dimensions. For instance in 2D NMR, the scheme (13)provides a time-domain set S(t 1 , t 2 ), from which correlations between frequencies during the evolution and acquisition periods can be extracted asdt 1 dt 2 . Collecting the 2D S(t 1 , t 2 ) NMR data set over sufficiently large regions of the time domain is usually carried out by a ...

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Two-dimensional nuclear magnetic resonance (2D NMR) provides one of the foremost contemporary tools available for the elucidation of molecular structure, function, and dynamics. Execution of a 2D NMR experiment generally involves scanning a series of time-domain signals S(t(2)), as a function of a t(1) time variable which undergoes parametric incrementation throughout independent experiments. Very recently, we proposed and demonstrated a general approach whereby this serial mode of data acquisition is parallelized, enabling the acquisition of complete bidimensional NMR data sets via the recording of a single transient. The present paper discusses in more detail various conceptual and experimental aspects of this novel 2D NMR methodology. The basic principles of the approach are reviewed, various homo- and heteronuclear NMR applications are illustrated, and the main features and artifacts affecting the method are derived. Extensions to higher-dimensional experiments are also briefly noted.

The genus Solanum comprises three food crops (potato, tomato, and eggplant), which are consumed on daily basis worldwide and also producers of notorious anti-nutritional steroidal glycoalkaloids (SGAs). Hydroxylated SGAs (i.e. leptinines) serve as precursors for leptines that act as defenses against Colorado Potato Beetle (Leptinotarsa decemlineata Say), an important pest of potato worldwide. However, SGA hydroxylating enzymes remain unknown. Here, we discover that 2-OXOGLUTARATE-DEPENDENT-DIOXYGENASE (2-ODD) enzymes catalyze SGA-hydroxylation across various Solanum species. In contrast to cultivated potato, Solanum chacoense, a widespread wild potato species, has evolved a 2-ODD enzyme leading to the formation of leptinines. Furthermore, we find a related 2-ODD in tomato that catalyzes the hydroxylation of the bitter α-tomatine to hydroxytomatine, the first committed step in the chemical shift towards downstream ripening-associated non-bitter SGAs (e.g. esculeoside A). This 2-ODD enzyme prevents bitterness in ripe tomato fruit consumed today which otherwise would remain unpleasant in taste and more toxic.