Folding structures of the c-MYC I-motifs.

<p>Schematic drawing of the folding structures of the c-MYC I-motifs formed in the C11T sequence (A), the C20T sequence (B), and the two equilibrating conformations in the C11/20/23T sequence (C). The C⁺-C base pairs are shown in white boxes. The dashed boxes indicate the possible C⁺-C base pairs that are in dynamic equilibrium and thus show weaker and broader C⁺-C imino peaks. (cytosine  =  yellow sphere, adenine  =  green sphere, thymine  =  blue sphere.)</p

Variable pH 1D ¹H NMR of C11/14/20/23T c-MYC I-motif.

<p>Cytosine- and thymine- imino regions of 1D ¹H NMR spectra of C11/14/20/23T at various pHs at 7°C.</p

Imino proton assignments of C11T and C20T c-MYC I-motif.

<p>Imino proton assignments of C11T (A) and C20T (B) using 1D ¹⁵N-filtered experiments on site-specific 6% ¹⁵N-labeled oligonucleotides. Each site-specifically labeled cytosine is shown above its corresponding spectrum. The assignment of all cytosine imino protons is shown above the 1D spectra of the corresponding sequence. All samples are prepared at pH 5.5. NMR experiments were performed at 7°C except for the C7-labeled C20T which was performed at 1°C.</p

Variable temperature 1D ¹H NMR of c-MYC I-motif sequences.

<p>Imino proton regions of variable temperature 1D ¹H NMR spectra of C11T, C20T, C11/20/23T, and C11/14/20/23T at pH 5.5.</p

The Major G‑Quadruplex Formed in the Human Platelet-Derived Growth Factor Receptor β Promoter Adopts a Novel Broken-Strand Structure in K⁺ Solution

Overexpression of platelet-derived growth factor receptor β (PDGFR-β) has been associated with cancers and vascular and fibrotic disorders. PDGFR-β has become an attractive target for the treatment of cancers and fibrotic disorders. DNA G-quadruplexes formed in the GC-rich nuclease hypersensitivity element of the human PDGFR-β gene promoter have been found to inhibit PDGFR-β transcriptional activity. Here we determined the major G-quadruplex formed in the PDGFR-β promoter. Instead of using four continuous runs with three or more guanines, this G-quadruplex adopts a novel folding with a broken G-strand to form a primarily parallel-stranded intramolecular structure with three 1 nucleotide (nt) double-chain-reversal loops and one additional lateral loop. The novel folding of the PDGFR-β promoter G-quadruplex emphasizes the robustness of parallel-stranded structural motifs with a 1 nt loop. Considering recent progress on G-quadruplexes formed in gene-promoter sequences, we suggest the 1 nt looped G_<i>i</i>NG_<i>j</i> motif may have been evolutionarily selected to serve as a stable foundation upon which the promoter G-quadruplexes can build. The novel folding of the PDGFR-β promoter G-quadruplex may be attractive for small-molecule drugs that specifically target this secondary structure and modulate PDGFR-β gene expression

P–N Cooperative Borane Activation and Catalytic Hydroboration by a Distorted Phosphorous Triamide Platform

Studies of the stoichiometric and catalytic reactivity of a geometrically constrained phosphorous triamide <b>1</b> with pinacolborane (HBpin) are reported. The addition of HBpin to phosphorous triamide <b>1</b> results in cleavage of the B–H bond of pinacolborane through addition across the electrophilic phosphorus and nucleophilic N-methylanilide sites in a cooperative fashion. The kinetics of this process of were investigated by NMR spectroscopy, with the determined overall second-order empirical rate law given by ν = −<i>k</i>[<b>1</b>]­[HBpin], where <i>k</i> = 4.76 × 10^–5 M^–1 s^–1 at 25 °C. The B–H bond activation process produces P-hydrido-1,3,2-diazaphospholene intermediate <b>2</b>, which exhibits hydridic reactivity capable of reacting with imines to give phosphorous triamide intermediates, as confirmed by independent synthesis. These phosphorous triamide intermediates are typically short lived, evolving with elimination of the N-borylamine product of imine hydroboration with regeneration of the deformed phosphorous triamide <b>1</b>. The kinetics of this latter process are shown to be first-order, indicative of a unimolecular mechanism. Consequently, catalytic hydroboration of a variety of imine substrates can be realized with <b>1</b> as the catalyst and HBpin as the terminal reagent. A mechanistic proposal implicating a P–N cooperative mechanism for catalysis that incorporates the various independently verified stoichiometric steps is presented, and a comparison to related phosphorus-based systems is offered

Antioxidant Drug Tempol Promotes Functional Metabolic Changes in the Gut Microbiota

Recent studies have identified the important role of the gut microbiota in the pathogenesis and progression of obesity and related metabolic disorders. The antioxidant tempol was shown to prevent or reduce weight gain and modulate the gut microbiota community in mice; however, the mechanism by which tempol modulates weight gain/loss with respect to the host and gut microbiota has not been clearly established. Here we show that tempol (0, 1, 10, and 50 mg/kg p.o. for 5 days) decreased cecal bacterial fermentation and increased fecal energy excretion in a dose-dependent manner. Liver ¹H NMR-based metabolomics identified a dose-dependent decrease in glycogen and glucose, enhanced glucogenic and ketogenic activity (tyrosine and phenylalanine), and increased activation of the glycolysis pathway. Serum ¹H NMR-based metabolomics indicated that tempol promotes enhanced glucose catabolism. Hepatic gene expression was significantly altered as demonstrated by an increase in <i>Pepck</i> and <i>G6pase</i> and a decrease in <i>Hnf4a</i>, <i>ChREBP</i>, <i>Fabp1</i>, and <i>Cd36</i> mRNAs. No significant change in the liver and serum metabolomic profiles was observed in germ-free mice, thus establishing a significant role for the gut microbiota in mediating the beneficial metabolic effects of tempol. These results demonstrate that tempol modulates the gut microbial community and its function, resulting in reduced host energy availability and a significant shift in liver metabolism toward a more catabolic state

Orthogonal Comparison of GC–MS and ¹H NMR Spectroscopy for Short Chain Fatty Acid Quantitation

Short chain fatty acids (SCFAs) are important regulators of host physiology and metabolism and may contribute to obesity and associated metabolic diseases. Interest in SCFAs has increased in part due to the recognized importance of how production of SCFAs by the microbiota may signal to the host. Therefore, reliable, reproducible, and affordable methods for SCFA profiling are required for accurate identification and quantitation. In the current study, four different methods for SCFA (acetic acid, propionic acid, and butyric acid) extraction and quantitation were compared using two independent platforms including gas chromatography coupled with mass spectrometry (GC–MS) and ¹H nuclear magnetic resonance (NMR) spectroscopy. Sensitivity, recovery, repeatability, matrix effect, and validation using mouse fecal samples were determined across all methods. The GC–MS propyl esterification method exhibited superior sensitivity for acetic acid and butyric acid measurement (LOD < 0.01 μg mL^–1, LOQ < 0.1 μg mL^–1) and recovery accuracy (99.4%–108.3% recovery rate for 100 μg mL^–1 SCFA mixed standard spike in and 97.8%–101.8% recovery rate for 250 μg mL^–1 SCFAs mixed standard spike in). NMR methods by either quantitation relative to an internal standard or quantitation using a calibration curve yielded better repeatability and minimal matrix effects compared to GC–MS methods. All methods generated good calibration curve linearity (<i>R</i>² > 0.99) and comparable measurement of fecal SCFA concentration. Lastly, these methods were used to quantitate fecal SCFAs obtained from conventionally raised (CONV-R) and germ free (GF) mice. Results from global metabolomic analysis of feces generated by ¹H NMR and bomb calorimetry were used to further validate these approaches

Metabolomics Reveals that Aryl Hydrocarbon Receptor Activation by Environmental Chemicals Induces Systemic Metabolic Dysfunction in Mice

Environmental exposure to dioxins and dioxin-like compounds poses a significant health risk for human health. Developing a better understanding of the mechanisms of toxicity through activation of the aryl hydrocarbon receptor (AHR) is likely to improve the reliability of risk assessment. In this study, the AHR-dependent metabolic response of mice exposed to 2,3,7,8-tetrachlorodibenzofuran (TCDF) was assessed using global ¹H nuclear magnetic resonance (NMR)-based metabolomics and targeted metabolite profiling of extracts obtained from serum and liver. ¹H NMR analyses revealed that TCDF exposure suppressed gluconeogenesis and glycogenolysis, stimulated lipogenesis, and triggered inflammatory gene expression in an <i>Ahr</i>-dependent manner. Targeted analyses using gas chromatography coupled with mass spectrometry showed TCDF treatment altered the ratio of unsaturated/saturated fatty acids. Consistent with this observation, an increase in hepatic expression of <i>stearoyl coenzyme A desaturase 1</i> was observed. In addition, TCDF exposure resulted in inhibition of <i>de novo</i> fatty acid biosynthesis manifested by down-regulation of acetyl-CoA, malonyl-CoA, and palmitoyl-CoA metabolites and related mRNA levels. In contrast, no significant changes in the levels of glucose and lipid were observed in serum and liver obtained from <i>Ahr</i>-null mice following TCDF treatment, thus strongly supporting the important role of the AHR in mediating the metabolic effects seen following TCDF exposure

MOESM1 of Reversing methanogenesis to capture methane for liquid biofuel precursors

Additional file 1. This file consists of four supplemental tables and six supplemental figures. Table S1 lists the components in the HS medium used to grow ANME-1 Mcr-producing M. acetivorans on methane and 0.1 mM or 10 mM FeCl3. Table S2 shows the fold changes of differentially expressed genes in M. acetivorans/pES1-MATmcr3 grown on methane and 0.1 mM FeCl3, in comparison to the same strain grown on methanol. Table S3 lists the strains and plasmids, and Table S4 lists the oligonucleotides, used in this study. Figure S1 shows the three promoters used to express ANME-1 mcrBGA genes. Figure S2 shows the detected McrA-FLAG in M. acetivorans/pES1-MATmcr3-flag grown on methane after five days. Figure S3 shows the detection of ANME-1 mcrA after 30 days of growth on methane. Figure S4 shows the GC/MS spectra of culture supernatants used to identify acetate from H13CO. Figure S5 shows the flux through the various reactions in the methanogenesis pathway of M. acetivorans estimated by 13C-metabolic flux analysis using 13C-labeled bicarbonate as the input tracer. Figure S6 shows a simplified methanogenesis pathway from CO2 and CH3OH of M. acetivorans