Quantitative proteomic analysis of bacterial enzymes released in cheese during ripening

Due to increasingly available bacterial genomes in databases, proteomic tools have recently been used to screen proteins expressed by micro-organisms in food in order to better understand their metabolism in situ. While the main objective is the systematic identification of proteins, the next step will be to bridge the gap between identification and quantification of these proteins. For that purpose, a new mass spectrometry-based approach was applied, using isobaric tagging reagent for quantitative proteomic analysis (iTRAQ), which are amine specific and yield labelled peptides identical in mass. Experimental Swiss-type cheeses were manufactured from microfiltered milk using Streptococcus thermophilus ITG ST20 and Lactobacillus helveticus ITG LH1 as lactic acid starters. At three ripening times (7, 20 and 69 days), cheese aqueous phases were extracted and enriched in bacterial proteins by fractionation. Each sample, standardised in protein amount prior to proteomic analyses, was: i) analysed by 2D-electrophoresis for qualitative analysis and ii) submitted to trypsinolysis, and labelled with specific iTRAQ tag, one per ripening time. The three labelled samples were mixed together and analysed by nano-LC coupled on-line with ESI-QTOF mass spectrometer. Thirty proteins, both from bacterial or bovine origin, were identified and efficiently quantified. The free bacterial proteins detected were enzymes from the central carbon metabolism as well as stress proteins. Depending on the protein considered, the quantity of these proteins in the cheese aqueous extract increased from 2.5 to 20 fold in concentration from day 7 to day 69 of ripening.     

12-O-tetradecanoylphorbol-13-acetate inhibits aminophospholipid translocase activity and modifies the lateral motions of fluorescent phospholipid analogs in the plasma membrane of bovine aortic endothelial cells

Interstitial cystitis (IC) is a relatively uncommon and enigmatic disorder characterized by pain in the bladder and pelvic region, typically accompanied by urinary urgency and frequency. Fibromyalgia is a more common disorder, with the prominent symptoms being diffuse musculoskeletal pain and fatigue, and it has been well established that there is substantial clinical overlap between fibromyalgia and chronic fatigue syndrome (CFS). Although genitourinary and musculoskeletal symptoms predominate in IC and fibromyalgia respectively, both disorders share a number of features, including similar demographics, "allied conditions" (e.g. irritable bowel syndrome, headaches, etc.), natural history, aggravating factors, and efficacious therapy. We hypothesized that there was substantial clinical overlap between fibromyalgia and IC, and examined cohorts of individuals with these two disorders in parallel, to compare the spectrum of symptomatology. Sixty fibromyalgia patients, 30 IC patients, and 30 age-matched healthy controls were questioned regarding current symptomatology. A dolorimeter examination was also performed in the three groups to assess peripheral nociception. We found that the frequency of current symptoms was very similar for the fibromyalgia and IC groups. Both the fibromyalgia and IC patients displayed increased pain sensitivity when compared to healthy individuals, at both tender and control points. These data suggest that IC and fibromyalgia have significant overlap in symptomatology, and that IC patients display diffusely increased peripheral nociception, as is seen in fibromyalgia. Although central mechanisms have been suspected to contribute to the pathogenesis of fibromyalgia for some time, we speculate that these same types of mechanisms may be operative in IC, which has traditionally been felt to be a bladder disorder.     

Rheology of cellulose nanofibrils and silver nanowires for the development of screen-printed antibacterial surfaces

Journal of Materials Science - TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl)-oxidized cellulose nanofibrils (T-CNF) and silver nanowires (Ag NWs) were formulated as active inks. Their rheological...     

Independent control of sol molar mass and gel content in acrylate polymer/latexes

Poly n-butylacrylate latexes are commonly used as the base ingredient in the formulation of pressure sensitive adhesives, PSA. In the typical starved semi-batch process carried out to produce this latex and due to the non-linear nature of the kinetics, chain transfer to polymer plus bimolecular termination by combination, polymer networks are produced. The molar mass distribution of these polymer latexes is characterized by a soluble and an insoluble fraction, so-called gel. Both fractions strongly affect the adhesion properties, but unfortunately the independent control of these properties is a difficult task, that has not been solved yet. In this work, the concentrations of chain transfer agent and cross-linker were used in an attempt to exercise control over the molar mass of the soluble part and the amount of gel polymer. It was found that by simultaneously manipulating both variables it was possible to modify the gel content of the polymer without completely sacrificing the sol molar mass. The adhesion properties, tack and resistance to shear, measured on films cast from the latex demonstrated that a good PSA can be obtained by properly balancing the amount of CTA and cross-linker in the formulation.     

Velocity-map imaging at low extraction fields

We present a velocity-map imaging (VMI) setup for photoelectron imaging that utilizes low electric extraction fields. This avoids any complications that could arise from electrostatic interactions between the extraction field and the molecular properties that are probed and has a minimal effect on the trajectory of ions in ion beam experiments. By using an attractive potential supplied to the detector, and keeping the electrodes at ground (zero) potential, we show that fringe fields between the VMI arrangement and the vacuum chamber can be eliminated, which is important in experiments on ions.     

Assessing the macroporous structure of monolithic columns by transmission electron microscopy

A set of monolithic stationary phases representing a broad span of monomers and porogens have been characterized directly in their capillary chromatographic format by computational assessment of their pore structure from transmission electron micrographs obtained after in situ embedment of the monoliths in contrast resin, followed by dissolution of the fused-silica tubing, further encasement of the resin-embedded monolith, and microtomy. This technique has been compared to mercury intrusion, a more conventional technique for macroporosity estimation. Supplementing the embedding resin by lead methacrylate gave a negative staining, and the resulting micrographs showed a good contrast between the polymeric monoliths and the embedding resin that allowed studies on the pore formation and polymer development. The technique was also applied to a commercial monolithic silica column.     

p-n Semiconductor membrane for electrically tunable ion current rectification and filtering

We show that a semiconductor membrane made of two thin layers of opposite (n- and p-) doping can perform electrically tunable ion current rectification and filtering in a nanopore. Our model is based on the solution of the 3D Poisson equation for the electrostatic potential in a double-cone nanopore combined with a transport model. It predicts that, for appropriate biasing of the membrane-electrolyte system, transitions from ohmic behavior to sharp rectification with vanishing leakage current are achievable. Furthermore, ion current rectifying and filtering regimes of the nanopore correspond to different charge states in the p-n membrane, which can be tuned with appropriate biasing of the n- and p- layers.     

Regulation of the Expression,Oligomerisation and Signaling of the Inhibitory Receptor CLEC12A by Cysteine Residues in the Stalk Region

CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor’s expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.     

Dielectric properties of Al V2O5 Al thin film sandwich structures

Vanadium pentoxide thin films were prepared by the electron beam evaporation technique onto Corning 7059 glass substrates kept at a temperature of T_s=423 K. The dielectric properties of Al V₂O₅ Al thin film sandwich structures were studied in the frequency range 0.1–100 kHz and in the temperature range 125–450 K. Both the dielectric constant and the dielectric loss factor were found to depend on frequency and temperature. The activation energy obtained for the dielectric relaxation process was about 0.36 eV.