By exploiting the extremely large effective cross sections (10 217 10 216 cm 2 ͞molecule) available from surface-enhanced Raman scattering (SERS), we achieved the first observation of single molecule Raman scattering. Measured spectra of a single crystal violet molecule in aqueous colloidal silver solution using one second collection time and about 2 3 10 5 W͞cm 2 nonresonant near-infrared excitation show a clear "fingerprint" of its Raman features between 700 and 1700 cm 21. Spectra observed in a time sequence for an average of 0.6 dye molecule in the probed volume exhibited the expected Poisson distribution for actually measuring 0, 1, 2, or 3 molecules.
The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.
Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N6-methyladenosine (m6A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m6A motifs are enriched in circRNAs and a single m6A site is sufficient to drive translation initiation. This m6A-driven translation requires initiation factor eIF4G2 and m6A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m6A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.
Historically, N6-methyladenosine (m6A) has been identified as the most abundant internal modification of messenger RNA (mRNA) in eukaryotes 1. Its mammalian function remained unknown until recently, when it was reported that thousands of mammalian mRNAs and long noncoding RNAs (lncRNAs) show m6A modification 2,3 and that m6A demethylases are required for mammalian energy homeostasis and fertility 4,5. As yet, the identity of m6A methyltransferases (MTase) and the molecular mechanisms regulated by m6A remains unclear. Here, we show that two proteins, the putative m6A MTase, methyltransferase-like 3 (Mettl3) 6, and a related but uncharacterized protein Mettl14, function synergistically to control m6A formation in mammalian cells. Since m6A modification is involved in cell fate determination in yeast 7,8 and embryo development in plant 9,10, we knocked down Mettl3 and Mettl14, respectively, in mouse embryonic stem cells (mESCs). The resulting cells displayed equivalent phenotypes characterized by lack of m6A RNA methylation and lost self-renewal capability. We also observed that a large number of transcripts, including many encoding developmental regulators, showed m6A methylation inversely correlated with mRNA stability and gene expression. Further analysis suggested that some of these effects were mediated through Human antigen R (HuR) and microRNA pathways. Overall our work provides first experimental evidence of mammalian m6A MTases and reveals a previously unknown gene regulatory mechanism operating in mESCs through m6A methylation. This mechanism is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.
We report the establishment of a library of micromolded elastomeric micropost arrays to modulate substrate rigidity independently of effects on adhesive and other material surface properties. We demonstrate that micropost rigidity impacts cell morphology, focal adhesions, cytoskeletal contractility, and stem cell differentiation. Furthermore, early changes in cytoskeletal contractility predicted later stem cell fate decisions at the single cell level.Cell function is regulated primarily by extracellular stimuli, including soluble and adhesive factors that bind to cell surface receptors. Recent evidence suggests that mechanical properties of the extracellular matrix (ECM), particularly rigidity, can also mediate cell signaling, proliferation, differentiation, and migration 1,2 . Culturing cells on hydrogels derived from natural ECM proteins at different densities has dramatic effects on cell adhesion, morphology, and function 3 . However, changing densities of the gels impacts not only mechanical rigidity, but also the amount of ligand, leaving uncertainty as to the relevant contribution of these two matrix properties on the observed cellular response. Synthetic ECM analogs such as polyacrylamide or polyethylene glycol gels, which vary rigidity by modulating the amount of cross-linker, has revealed that substrate rigidity alone can modulate many cellular functions, including stem cell differentiation 4-6 . However, altered cross-linker amount impacts not only bulk mechanics, but also molecular-scale material properties including porosity, surface chemistry, backbone flexibility, and binding properties of immobilized adhesive ligands 7,8 . Consequently, whether cells transduce substrate rigidity at the microscopic scale (eg sensing the rigidity between adhesion sites) or the nanoscopic scale (eg sensing local alterations in receptor-ligand binding characteristics) remains an open question 7,8 . While hydrogels will continue to play a major role in characterizing and controlling cell-material interactions, alternative approaches are necessary to further elucidate the basis by which cells sense changes in substrate rigidity.
Abstract-A widely recognized shortcoming of model predictive control (MPC) is that it can usually only be used in applications with slow dynamics, where the sample time is measured in seconds or minutes. A well-known technique for implementing fast MPC is to compute the entire control law offline, in which case the online controller can be implemented as a lookup table. This method works well for systems with small state and input dimensions (say, no more than five), few constraints, and short time horizons. In this paper, we describe a collection of methods for improving the speed of MPC, using online optimization. These custom methods, which exploit the particular structure of the MPC problem, can compute the control action on the order of 100 times faster than a method that uses a generic optimizer. As an example, our method computes the control actions for a problem with 12 states, 3 controls, and horizon of 30 time steps (which entails solving a quadratic program with 450 variables and 1284 constraints) in around 5 ms, allowing MPC to be carried out at 200 Hz.Index Terms-Model predictive control (MPC), real-time convex optimization.
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