Ions from Solution to the Gas Phase: A Molecular Dynamics Simulation of the Structural Evolution of Substance P during Desolvation of Charged Nanodroplets Generated by Electrospray Ionization

Molecular dynamics (MD) simulations are used to model changes in the conformational preferences of a model peptide during the transition from a hydrated environment (charged nanodroplet generated by electrospray ionization) to the solvent-free peptide ion. The charged droplet consists of ∼2400 water molecules, 22 hydronium ions, and 10 chloride and contains a single Substance P (SP) [SP + 3H]³⁺ ion (SP³⁺; amino acid sequence RPKPQQFFGLM-NH₂). Initially, droplet shrinkage involves a combination of solvent evaporation and ejection of excess charge, primarily hydronium ions. Further droplet shrinkage leads to a series of fission events, which includes the loss of some Cl^– ions. SP³⁺ ions adapt to the smaller size droplet through small conformational changes that result in coiling of the hydrophobic C-terminus of the peptide on or near the droplet surface, intramolecular interactions involving the hydrophilic N-terminus of the peptide, and water-mediated interactions between the SP³⁺ ion and H₃O⁺ and Cl^<b>–</b> ions. Calculated collision cross sections (CCS) for SP³⁺ ions at various stages of desolvation are consistent with the results obtained from cryogenic ion mobility-mass spectrometry (cryo-IM-MS) measurements. Specifically, early in the decay of the charged droplet SP³⁺ ions favor an extended conformation, whereas a compact conformer is favored during the final stages of dehydration

Dnmt2-deficiency affects the P-TEFb complex.

<p>(A) Cdk9 expression in Dnmt2-deficient and control ES cells was analyzed by quantitative RT-PCR (left) and Western blot assays (right). (B) Cdk9 expression in Dnmt2-deficient and control hearts was investigated by quantitative RT-PCR (left) (<i>n</i> = 5) and Western blot assays (right). (C) Quantitative RT-PCR for Ctip expression in the hearts of Dnmt2^-/- and Dnmt2^+/+ mice (<i>n</i> = 6). Northern blot assay (D) and quantitative RT-PCR (E) for Rn7sk (7SK) expression in the hearts of Dnmt2^-/- and Dnmt2^+/+ mice. (F) RNA immunoprecipitation using a 5-methyl Cytidine antibody with RNA extracted from Dnmt2^-/- and Dnmt2^+/+ hearts, followed by RT-PCR for Rn7sk (<i>n</i> = 6). Note that Rn7sk is significantly less methylated in Dnmt2^-/- hearts. (G) PTEF-b immunoprecipitation using an antibody against Cdk9 on lysates from Dnmt2- mutant and control ES cells followed by RT-PCR for Rn7sk (<i>n</i> = 3) and subsequent quantification. An anti Cyp2 antibody served as negative control. Note that less Rn7sk is associated to the P-TEFb complex in Dnmt2-deficient cells.</p

Dnmt2-deficiency is associated with increased phosphorylation of RNA polymerase II.

<p>Representative Western Blots for RNA polymerase II (RNA Pol II, upper panels) and quantification of the ratio of phosphorylated to non-phosphorylated RNA Pol II (lower panels) in mouse hearts (A) and ES cells (B) with knockout of Dnmt2 and controls. β-actin was used as a standard. Data are mean ± SEM. *p < 0.05.</p

Primers for quantitative RT-PCR analysis.

<p>Primers for quantitative RT-PCR analysis.</p

Schematic representation of Dnmt2-mediated RNA polymerase II transcriptional activity in cardiac growth.

<p>Storage of inactive protein in a complex with 7SK RNA and inhibitory proteins, release of active kinase (P-TEFb) in Dnmt2 knock out conditions. The phosphorylation of a C-terminal domain of the polymerase by P-TEFb allows elongation of the transcripts. RNAPII CTD phosphorylation increases mRNA and protein expression, which mediates cardiac growth in Dnmt2-deficient mice.</p

Efficient Carbene and Carbyne Formation in Molybdenum(0) and Tungsten(0) Dinitrogen Complexes

Zerovalent group 6 dinitrogen phosphine complexes react with 3,3-diphenylcyclopropene to give vinylcarbyne hydride complexes that have been characterized crystallographically. Computational studies indicate that the observed products resulted from the α-migration and rearrangement of an initially formed vinylcarbene

Development and Application of a Low-Volume Flow System for Solution-State <i>in Vivo</i> NMR

<i>In vivo</i> nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful technique, since it allows samples to be analyzed in their natural, unaltered state, criteria paramount for living organisms. In this study, a novel continuous low-volume flow system, suitable for <i>in vivo</i> NMR metabolomics studies, is demonstrated. The system allows improved locking, shimming, and water suppression, as well as allowing the use of trace amounts of expensive toxic contaminants or low volumes of precious natural environmental samples as stressors. The use of a double pump design with a sump slurry pump return allows algal food suspensions to be continually supplied without the need for filters, eliminating the possibility of clogging and leaks. Using the flow system, the living organism can be kept alive without stress indefinitely. To evaluate the feasibility and applicability of the flow system, changes in the metabolite profile of ¹³C enriched <i>Daphnia magna</i> over a 24-h period are compared when feeding laboratory food vs exposing them to a natural algal bloom sample. Clear metabolic changes are observed over a range of metabolites including carbohydrates, lipids, amino acids, and a nucleotide demonstrating <i>in vivo</i> NMR as a powerful tool to monitor environmental stress. The particular bloom used here was low in microcystins, and the metabolic stress impacts are consistent with the bloom being a poor food source forcing the <i>Daphnia</i> to utilize their own energy reserves