Thiazole orange-induced c-di-GMP quadruplex formation facilitates a simple fluorescent detection of this ubiquitous biofilm regulating molecule

Recently, there has been an explosion of research activities in the cyclic dinucleotides field. Cyclic dinucleotides, such as c-di-GMP and c-di-AMP, have been shown to regulate bacterial virulence and biofilm formation. c-di-GMP can exist in different aggregate forms, and it has been demonstrated that the polymorphism of c-di-GMP is influenced by the nature of cation that is present in solution. In previous work, polymorphism of c-di-GMP could only be demonstrated at hundreds of micromolar concentrations of the dinucleotide, and it has been a matter of debate if polymorphism of c-di-GMP exists under in vivo conditions. In this Article, we demonstrate that c-di-GMP can form G-quadruplexes at low micromolar concentrations when aromatic molecules such as thiazole orange template the quadruplex formation. We then use this property of aromatic molecule-induced G-quadruplex formation of c-di-GMP to design a thiazole orange-based fluorescent detection of this important signaling molecule. We determine, using this thiazole orange assay on a crude bacterial cell lysate, that WspR D70E (a constitutively activated diguanylate cyclase) is functional in vivo when overexpressed in E. Coli . The intracellular concentration of c-di-GMP in an E. Coli cell that is overexpressed with WspR D70E is very high and can reach 2.92 mM.     

Model for the surface reconstruction phase transition of clean and hydrogen-adsorbed W(001) surfaces: Low-coverage solution

We summarize the theoretical indications and experimental evidence justifying a shortrange structural model for the clean and H-adsorbed W(001) surface reconstruction phase transition. A detailed study of the effect of the adsorbate treated as a lattice gas within the context of such a model is presented. The results, though based on mean field theory, are accurate in the low-cover-age region within the context of the model and succeed in reproducing the low-coverage features of the phase diagram, thus enabling a determination of some of the interaction constants in the model. The energy difference between distortions along the 〈11〉 and 〈10〉 directions is 3.0 × 10^?3 eV per atom. The force exerted on a surface W atom by an adsorbed H atom in an adjacent bridge site is $\text{0.04}\text{eV}\text{u}{}_0$, where u₀ is the magnitude of the clean surface W atom displacement at low temperature.     

Precise mass measurement of a double-stranded 500 base-pair (309 kDa) polymerase chain reaction product by negative ion electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) has been used to determine the mass of a double-stranded 500 base-pair (bp) polymerase chain reaction (PCR) product with an average theoretical mass of the blunt-ended (i.e. unadenylated) species of 308 859.35 Da. The PCR product was generated from the linearized bacteriophage Lambda genome which is a double-stranded template. Utilization of ethanol precipitation in tandem with a rapid microdialysis step to purify and desalt the PCR product was crucial to obtain a precise mass measurement. The PCR product (0.8 pmol/μL) was electrosprayed from a solution containing 75% acetonitrile, 25 mM piperidine, and 25 mM imidazole and was infused at a rate of 200 nL/min. The average molecular mass and the corresponding precision were determined using the charge-states ranging from 172 to 235 net negative charges. The experimental mass and corresponding precision (reported as the 95% confidence interval of the mean) was 309 406 +/- 27 Da (87 ppm). The mass accuracy was compromised due to the fact that the PCR generates multiple products when using Taq polymerase due to the non-template directed 3'-adenylation. This results in a mixture of three PCR products with nearly identical mass (i.e. blunt-ended, mono-adenylated and di-adenylated) with unknown relative abundances that were not resolved in the spectrum. Thus, the experimental mass will be a weighted average of the three species which, under our experimental conditions, reflects a nearly equal concentration of the mono- and di-adenylated species. This report demonstrates that precise mass measurements of PCR products up to 309 kDa (500 bp) can be routinely obtained by ESI-FTICR requiring low femtomole amounts. Copyright 1999 John Wiley & Sons, Ltd.     

Competition between direct and concerted movements in surface diffusion with application to the Au(110) surface

Recent calculations of the energetic barriers for diffusion of Au atoms on the Au(110) surface have shown that for moves perpendicular to the close-packed atomic rows, concerted, or multi-atom movements are energetically favorable over single-atom movements for the same displacement. Entropic hindrances, however are expected to limit concerted moves. In the present paper this competition is investigated via simulation techniques based on model potentials that reproduce the relevant energtic and entropic features of the Au(110) situation. Apparently non-Arrhenius behavior is found for the concerted motions. A new scheme, which avoids the necessity of long simulation runs to determine the “hop” rate at several temperatures, for determining entropic barriers directly is proposed and tested. Though this method is applied in the present context to a simplified model for the interatomic potentials, it can be straightforwardly extended to realistic treatments of those interactions.     

Cysteine adducts of human haemoglobin measured by isoelectric focusing in polyacrylamide gels with a non-linear pH gradient

The in vitro formation of adducts from human haemoglobin formed by alkylation with methyl-methanesulphonate, dimethyl sulphate and iodoacetamide was determined with isoelectric focusing in ultra-thin polyacrylamide gels with a non-linear pH gradient. The most important adduct seen in the gels was identified as HbA alkylated at the beta-93 cysteine. Influences of the chemical nature of the alkylating agents and of the biological environment are discussed. The method is suggested to be applicable to monitoring the biological effects of low, long-term exposure to mixtures of alkylating agents or of exposure to unknown compounds.