DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.

Emerging evidence has linked the gut microbiome to human obesity. We performed a metagenome-wide association study and serum metabolomics profiling in a cohort of lean and obese, young, Chinese individuals. We identified obesity-associated gut microbial species linked to changes in circulating metabolites. The abundance of Bacteroides thetaiotaomicron, a glutamate-fermenting commensal, was markedly decreased in obese individuals and was inversely correlated with serum glutamate concentration. Consistently, gavage with B. thetaiotaomicron reduced plasma glutamate concentration and alleviated diet-induced body-weight gain and adiposity in mice. Furthermore, weight-loss intervention by bariatric surgery partially reversed obesity-associated microbial and metabolic alterations in obese individuals, including the decreased abundance of B. thetaiotaomicron and the elevated serum glutamate concentration. Our findings identify previously unknown links between intestinal microbiota alterations, circulating amino acids and obesity, suggesting that it may be possible to intervene in obesity by targeting the gut microbiota.

BackgroundWe recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing.Methodology/Principal FindingsWe used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1–2 nucleotides.ConclusionsWe have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.

We developed a general method to detect cellular small molecule-RNA conjugates that does not rely on the reactivity of the small molecule, revealing NAD-linked RNA in E. coli and S. venezuelae. Subsequent characterization shows NAD is a 5’ modification of RNA, cannot be installed in vitro through aberrant transcriptional initiation, is only found among smaller cellular RNAs, and is present at a surprisingly high abundance of ~3000 copies per cell.

DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection of a pilot library of 65 macrocycles. In this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. Integrating these four developments, we executed the translation of 13 824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNAtemplated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.

Compared with the rapidly expanding set of known biological roles for RNA, the known chemical diversity of cellular RNA has remained limited primarily to canonical RNA, 3 -aminoacylated tRNAs, nucleobase-modified RNAs, and 5 -capped mRNAs in eukaryotes. We developed two methods to detect in a broad manner chemically labile cellular small molecule-RNA conjugates. The methods were validated by the detection of known tRNA and rRNA modifications. The first method analyzes small molecules cleaved from RNA by base or nucleophile treatment. Application to Escherichia coli and Streptomyces venezuelae RNA revealed an RNAlinked hydroxyfuranone or succinyl ester group, in addition to a number of other putative small molecule-RNA conjugates not previously reported. The second method analyzes nuclease-generated mononucleotides before and after treatment with base or nucleophile and also revealed a number of new putative small molecule-RNA conjugates, including 3 -dephospho-CoA and its succinyl-, acetyl-, and methylmalonyl-thioester derivatives. Subsequent experiments established that these CoA species are attached to E. coli and S. venezuelae RNA at the 5 terminus. CoA-linked RNA cannot be generated through aberrant transcriptional initiation by E. coli RNA polymerase in vitro, and CoA-linked RNA in E. coli is only found among smaller (Շ200 nucleotide) RNAs that have yet to be identified. These results provide examples of small molecule-RNA conjugates and suggest that the chemical diversity of cellular RNA may be greater than previously understood. mass spectrometry ͉ RNA modifications ͉ coenzyme A O ver the past few decades, RNA has emerged as much more than an intermediary in biology's central dogma. Ribozymes (1), riboswitches (2), microRNAs (miRNAs) (3), small interfering RNAs (siRNAs) (4), Piwi-interacting RNAs (piRNAs) (5), small nuclear RNAs (snRNAs) (6), CRISPR sRNAs (7), RNA transcriptional regulators (8), and long noncoding RNAs (9, 10) are all examples of RNAs that are thought to play a wide range of catalytic, regulatory, or defensive roles in the cell. Models of early biotic systems have proposed even broader roles for RNA, including the possibility that RNA-tethered molecules participated in RNA-templated chemical reactions as an early form of metabolism (11)(12)(13)(14)(15)(16).In contrast with these newer insights into its functional diversity, the known chemical diversity of natural RNA has remained limited primarily to canonical polyribonucleotides, 3Ј-aminoacylated tRNAs (17), modified nucleobases in a variety of RNAs (18), and 5Ј-capped mRNAs in eukaryotes (19)(20). This disparity between functional and chemical diversity, coupled with the powerful functional properties of synthetic small molecule-nucleic acid conjugates (21-24) led us to speculate that small molecule-RNA conjugates beyond those previously described may exist in modern cells as evolutionary fossils or even as novel RNAs with functions enabled by their modifications.To begin to explore this possibility, we have developed and implemented a ...

Mutations in KRAS oncogene are recognized biomarkers that predict lack of response to anti- epidermal growth factor receptor (EGFR) antibody therapies. However, some patients with KRAS wild-type tumors still do not respond, so other downstream mutations in BRAF, PIK3CA and NRAS should be investigated. Herein we used direct sequencing to analyze mutation status for 676 patients in KRAS (codons 12, 13 and 61), BRAF (exon 11 and exon 15), PIK3CA (exon 9 and exon 20) and NRAS (codons12, 13 and 61). Clinicopathological characteristics associations were analyzed together with overall survival (OS) of metastatic colorectal cancer patients (mCRC). We found 35.9% (242/674) tumors harbored a KRAS mutation, 6.96% (47/675) harbored a BRAF mutation, 9.9% (62/625) harbored a PIK3CA mutation and 4.19% (26/621) harbored a NRAS mutation. KRAS mutation coexisted with BRAF, PIK3CA and NRAS mutation, PIK3CA exon9 mutation appeared more frequently in KRAS mutant tumors (P = 0.027) while NRAS mutation almost existed in KRAS wild-types (P<0.001). Female patients and older group harbored a higher KRAS mutation (P = 0.018 and P = 0.031, respectively); BRAF (V600E) mutation showed a higher frequency in colon cancer and poor differentiation tumors (P = 0.020 and P = 0.030, respectively); proximal tumors appeared a higher PIK3CA mutation (P<0.001) and distant metastatic tumors shared a higher NRAS mutation (P = 0.010). However, in this study no significant result was found between OS and gene mutation in mCRC group. To our knowledge, the first large-scale retrospective study on comprehensive genetic profile which associated with anti-EGFR MoAbs treatment selection in East Asian CRC population, appeared a specific genotype distribution picture, and the results provided a better understanding between clinicopathological characteristics and gene mutations in CRC patients.

The residual stress and structural properties of tungsten thin films prepared by magnetron sputtering as a function of sputtering-gas pressure are reported. The films were analyzed in situ by a cantilever beam technique, and ex situ by x-ray diffraction, cross-sectional transmission electron microscopy (TEM), x-ray photoelectron spectroscopy, electron energy-loss spectrometry, and energy-filtered electron diffraction. It is found that the residual stress, microstructure, and surface morphology are clearly correlated. The film stresses, determined in real time during the film formation, depend strongly on the argon pressure and change from highly compressive to highly tensile in a relatively narrow pressure range of 12–26 mTorr. For pressures exceeding ∼60 mTorr, the stress in the film is nearly zero. It is also found that the nonequilibrium A15 W structure is responsible for the observed tensile stress, whereas the stable bcc W or a mixture of bcc W and A15 W are in compression. Cross-sectional TEM evidence indicates that the compressively stressed films contain a dense microstructure without any columns, while the films having tensile stress have a very columnar microstructure. High sputtering-gas pressure conditions yield dendritic-like film growth, resulting in complete relaxation of the residual tensile stresses. Structural details of the A15 W and amorphous W phases were also investigated at the atomic level using energy-filtered electron diffraction with reduced radial distribution function G(r) analysis. By comparing the experimental and simulated G(r) distributions, the A15 W structure is determined to be composed of ordered and stacking faulted W3W structures and the amorphous W has a disordered structure of W3O. The effect of oxygen in stabilizing the A15 phase found is explained on the basis of structural and thermodynamic stability.