Formation and Structure of Fluorescent Silver Nanoclusters at Interfacial Binding Sites Facilitating Oligomerization of DNA Hairpins

Fluorescent, DNA‐stabilized silver nanoclusters (DNA‐AgNCs) are applied in a range of applications within nanoscience and nanotechnology. However, their diverse optical properties, mechanism of formation, and aspects of their composition remain unexplored, making the rational design of nanocluster probes challenging. Herein, a synthetic procedure is described for obtaining a high yield of emissive DNA‐AgNCs with a C‐loop hairpin DNA sequence, with subsequent purification by size‐exclusion chromatography (SEC). Through a combination of optical spectroscopy, gel electrophoresis, inductively coupled plasma mass spectrometry (ICP‐MS), and small‐angle X‐ray scattering (SAXS) in conjunction with the systematic study of various DNA sequences, the low‐resolution structure and mechanism of the formation of AgNCs were investigated. Data indicate that fluorescent DNA‐AgNCs self‐assemble by a head‐to‐head binding of two DNA hairpins, bridged by a silver nanocluster, resulting in the modelling of a dimeric structure harboring an Ag₁₂ cluster.     

Adsorption of nicotinic acid on the surface of nanosized hydroxyapatite and structurally modified hydroxyapatite

In the present paper, hydroxyapatite and structurally modified hydroxyapatite were investigated to establish the best material for nicotinic acid adsorption. Structurally modified hydroxyapatite wa prepared by adding sodium silicate in the reaction medium. The influence of silica concentration, presence of small amounts of metal ions, temperature and initial concentrations of nicotinic acid solutions on the adsorption capacity, were studied. Results indicated that structurally modified hydroxyapatite doped with copper adsorbed the highest amount of nicotinic acid. For this material the adsorption capacity was 0.232 mg nicotinic acid / g material, at an initial concentration of 10⁻⁴ M nicotinic acid. For all types of materials, best results were obtained at 15°C. The amount of nicotinic acid adsorbed increases with the decrease in temperature and with the increase in the initial concentration of nicotinic acid. Adsorption kinetics data were modeled using pseudo-first and pseudo-second order models while the interference due to diffusion was analyzed with intraparticle diffusion model. The results indicate that pseudo-second order model best describes the adsorption kinetics data, indicating the formation of chemical bonding.     

Pfropf-Polymerisation von Methylmethacrylat auf Wolle mit dem System von Methylmethacrylat auf Wolle mit dem Redox-System H2O2-Na-Thiosulfat

Ohne Zusammenfassung     

Electrospray low energy CID and MALDI PSD fragmentations of protonated sulfinamide cross-linked peptides

Murine S100A8 (A8) is a major cytoplasmic neutrophil protein and is converted to novel oxidation products containing Cys-epsilon amino-Lys sulfinamide cross-links and Met-sulfoxide by the neutrophil oxidant HOCl. Seven products were separated using RP-HPLC, with electrospray ionization mass spectrometry (ESI-MS) masses after deconvolution of 10,354, 10,388, +/- 1, and 20,707, +/- 3 Da, and all were resistant to reduction by dithiothreitol. The major products with masses of 10,354 Da contained Cys41-Lys34/35 intramolecular cross-links. Additional isomeric products with identical masses (10,354 Da) were isolated and peptide mapping and ESI/MS indicated that Cys41 forms covalent sulfinamide cross-links with either Lys6, Lys76, Lys83, or Lys87 present in A8. Electrospray low energy collisionally induced (CID) spectra of multiply-charged AspN digest peptides with sulfinamide cross-links contained characteristic fragmentations that corresponded to simple cleavage of the nitrogen-sulfur bond with charge retention on either of the fragment ions, allowing conformation of cross-linked peptides. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post source decay spectra of [M + H] + ions of the same sulfinamide-containing cross-linked peptides fragment similarly, but additional facile fragmentation reactions corresponding to formation of a protonated peptide containing de-hydroalanine were attributed to cleavage of the carbon-sulfur bond. In addition, lose of methanesulfenic acid from Met-sulfoxide was observed. A sulfinamide-containing adduct was isolated after incubation of the A8/HOCl reaction mixture with Lys or alpha N-acetyl Lys with masses of 10,500 or 10,542 Da. ESI/MS/MS and MALDI/post decay source (PSD) analysis of A8(32)-(57)-sulfinamide showed the same characteristic fragmentations as those in the sulfinamide cross-linked peptides, confirming the Cys41-Lys sulfinamide cross-link and suggesting that peptide-peptide sulfinamides may all fragment similarly, allowing ready identification of these cross-links in proteins from more complex biological materials.     

Haplotyping of putative microRNA-binding sites in the SNAP-25 gene

Synaptosomal-associated protein 25 (SNAP-25) plays a crucial role in exocitosis. Single nucleotide polymorphisms (rs3746544 and rs1051312) in the 3' un-translated region of the SNAP-25 gene have been described to be in association with attention-deficit hyperactivity disorder. As the disease affects millions of children world-wide, understanding the genetic background of attention-deficit hyperactivity disorder is of crucial importance. Efficient and reliable PCR-RFLP protocols were elaborated for the genotyping of the rs3746544 and rs1051312 SNPs employing a high-throughput capillary electrophoresis method for fragment analysis. A novel real-time PCR-based technique was used applying sequence specific TaqMan probes to haplotype the two SNPs, and the G-C haplotype could not be detected in a large Caucasian population (N=1376). These findings have been confirmed by molecular biology tools as well as by the PHASE Bayesian computational approach. In silico analyses have suggested that the two SNPs might alter microRNA binding and thus have an effect on SNAP-25 production. We have demonstrated that this biological information can be revealed only by direct haplotype analysis emphasizing the importance of our novel molecular haplotye analysis protocol. Results of the study of the two SNPs might shed light on the association of SNAP-25 variants and pathological phenotypes at the molecular level.     

Identification of noncovalent dimeric complexes of the recombinant murine S100 protein CP10 by electrospray ionization mass spectrometry and chemical cross-linking

CP10 is a chemotactic S100 protein expressed by murine myeloid cells. A specific noncovalently linked dimeric complex of recombinant Ala₄₃ CP10 was identified after electrospray ionization mass spectrometry using a nondenaturing solvent of 5-mM ammonium acetate (pH 6. 5) and source temperature of 50°C. With a low cone voltage (75 V), major ions were observed at ～2075, 2305, and 2613 Da, which were attributed to partially desolvated multiply charged noncovalently linked dimeric species (+10, +9, and +8 charge states, respectively). Deconvolution produced a broad peak centered around 20750 Da corresponding to the approximate mass of dimeric recombinant Ala₄₃ CP10. Increasing the cone voltage, and collisionally activating the complex, gradually reduced the intensity of these dimeric ions, with a concomitant increase in higher and lower charge state monomeric ions. The intensities of these dimeric ions were greatly reduced in spectra recorded with a source temperature of 140°C and cone voltage of 75 V, indicating a thermally unstable noncovalent complex. Similar spectra were obtained using recombinant CP10. Specific noncovalent S100 dimeric complexes were confirmed by chemically cross-linking recombinant Ala₄₃ CP10 or CP10 with bis (sulfosuccinimidyl) suberate, followed by SDS/PAGE. The dominant silver-stained and CP10-immunoreactive component migrated at 20,000 suggesting that the complex represents the major isoform in solution.     

Preparation and characterization of poly(acrylic acid)-based nanoparticles

The present investigation describes the synthesis and characterization of nanoparticles based on poly(acrylic acid) (PAA) intramolecularly cross-linked with diamine, 2,2′-(ethylenedioxy)bis(ethylamine), using water-soluble carbodiimide. The aqueous colloid dispersions of nanoparticles were clear or mildly opalescent depending on the ratio of cross-linking, pH of the solution, and the molecular weight of PAA, finding consistent with values of transmittance between 3% and 99%. The structure was determined by nuclear magnetic resonance spectroscopy, and the particle size was identified by dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. It was found that particle size depends on the pH, and at a given pH, it was caused by the ratio of cross-linking and the molecular weight of PAA. Particle size measured by TEM varied in the range of 20 and 80 nm. In the swollen state, the average size of the particles measured by DLS was in the range of 35–160 nm.     

Analytical methods for detection of gluten in food--method developments in support of food labeling legislation

The current essential therapy of celiac disease is a strict adherence to a gluten-free diet. Besides food products that are naturally gluten-free, "very low gluten" and "gluten-free" bakery products have become available. The availability of immunochemical and other analytical methods to determine gluten markers in foods is of utmost importance to ensure the well being of gluten-sensitive individuals. The aim of this review was to evaluate if currently available methodologies are suitable to meet the requirements of food labeling standards for individual gluten source declaration, in order to achieve policy objectives. Codex Alimentarius and European Union (EU) legislation and gluten detection methodologies applicable at present have been summarized and compared. In 2009, the European Commission issued Regulation No. 41/2009 concerning the composition and labeling of foodstuffs suitable for people intolerant to gluten. This review constitutes a basis to investigate the possibility to develop a proteomic-based method for the specific detection of gluten-containing cereals in food products, especially at or around the limits specified in EU legislation.