Exploitation of endogenous protease activity in raw mastitic milk by MALDI-TOF/TOF

The proteic profiling of bovine milk produced by cows with subclinical mastitis was obtained by MALDI mass spectrometry. A simple procedure of chemical fractionation of raw milk was developed, whereby less complex fractions of proteins were obtained prior to mass spectrometric and SDS-PAGE analysis. The profiles of milk proteins thus obtained could allow the identification of either early markers of the acute phase of mastitis or endogenous peptide of innate immune response. The activity of the endogenous proteases in raw milk produced from each quarter of healthy and mastic cows was therefore assayed over 24-, 48-, 96-, and 216-h incubation at 37 degrees C at both physiological and acid pH. Sequence-specific peptides were identified for each fraction by MS/MS experiments, and all tandem mass spectra were evaluated using MASCOT database searching. The results show a specific proteolytic activity of endogenous enzyme toward beta-casein precursor (P02666), alpha-S2-casein (P02663), alpha-S1-casein (P02662), and kappa-casein (P02668).     

Metastatic Prostate Cancer Cells Secrete Methylglyoxal-Derived MG-H1 to Reprogram Human Osteoblasts into a Dedifferentiated,Malignant-like Phenotype: A Possible Novel Player in Prostate Cancer Bone Metastases

Bone metastases from prostate cancer (PCa) result from a complex cross-talk between PCa cells and osteoblasts (OB). Thus, targeting this interplay has become an attractive strategy to interfere with PCa bone dissemination. The agents currently used in clinical trials have proved ineffective, boosting research to identify additional mechanisms that may be involved in this two-directional talk. Here, we investigated whether and how 5-hydro-5-methylimidazolone (MG-H1), a specific methylglyoxal (MG)-derived advanced glycation end product (AGE), was a novel player in the dialogue between PCa and OB to drive PCa bone metastases. Conditioned medium from osteotropic PC3 PCa cells, pre-treated or not with a specific MG scavenger, was administrated to human primary OB and cell morphology, mesenchymal trans-differentiation, pro-osteogenic determinants, PCa-specific molecules, and migration/invasion were studied by phase-contrast microscopy, real-time PCR, western blot and specific assays, respectively. We found that PC3 cells were able to release MG-H1 that, by binding to the receptor for AGEs (RAGE) on OB, reprogrammed them into a less-differentiate phenotype, endowed with some PCa-specific molecular features and malignant properties, in a mechanism involving reactive oxidative species (ROS) production and NF-kB pathway activation. These findings provide novel insights into the mechanisms of PCa osteoblastic metastases and foster in vivo research toward new therapeutic strategies interfering with PCa/OB cross-talk.     

Chemical and physical modifications of electrospun keratin nanofibers induced by heating treatments

In this study, we aimed to examine to study the effects of heating treatments on wool‐derived keratin nanofibers obtained by electrospinning. Interestingly, the keratin nanofibers did not lose their nanofibrous shape at high temperatures. On the contrary, the diameter of keratin nanofibers significantly decreased after heating. Even the chemical structure of the keratin was not significantly altered by heating, as observed by IR spectroscopy, except for a shoulder at 1720 cm^?1 and the amide III band. The spectral feature changes matched the formation of crosslinking amide bonds between protein chains well. Moreover, for various applications, it is essential that nanofibers are stable in water. Heating treatments were carried out at different temperatures and times to assess what heating conditions gave the keratin nanofibers water stability. Finally, no significant changes in the thermal behavior were observed in the samples after heating treatments compared to the untreated keratin nanofibers; this was a sign of negligible or slight degradations of the protein chains. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40532.     

Amino acids as modulators of lipoxygenase oxidation mechanism. The identification and structural characterization of spin adducts intermediates by electron spin resonance and tandem mass spectrometry

Antioxidant activity is displayed by amino acids, such as tryptophan (Trp), tyrosine (Tyr) and histidine (His) in the spontaneous oxidation of linoleic acid (LA). In addition, when Trp was incubated with soybean lipoxygenase (LOX 1) and LA, a modulating effect was observed. The elucidation of the reaction pathways was achieved through the identification, by electron spin resonance (ESR) spectroscopy, of α-[4-pyridyl 1-oxide]-N-t-butyl nitrone (POBN) adducts with the selected amino acids. The latter, when electrosprayed (ESI) were detected in the gas-phase as radical cations. They were structurally characterized by tandem mass spectrometry (MS/MS) through collision-induced dissociation (CID) of the adducts. The kinetic data obtained from selected model systems suggested a reversible radical scavenging activity of tryptophan on the intermediate dienyl radical formed in the lipids lipoxygenase cascade.     

A new,very sensitive method of assessment of ptaquiloside,the major bracken carcinogen in the milk of farm animals

We describe a new method to detect trace levels of ptaquiloside (Pta), a major carcinogen of bracken fern in biological samples such as milk from farm animals. The method involves the absorption of analyte on carbograph followed by elution with solvents mixtures. The unstable analyte is then converted into Br-Pt (II), which is specific for Pta, as it is not a natural decay product of the glycoside in aqueous media. An internal standard, the Br-pterosine-d₂, prepared in our laboratories has been used. Detection and quantification are possible with gas chromatography/mass spectrometry (GC/MS) in single ion monitoring mode (SIM). The detectable amount is in the range of ppb. The method allowed us to detect Pta not only in the milk from bracken fern-poisoned cattle but also, for the first time, in the milk from healthy farm animals such as sheep, goat, horse, and donkey mares.     

Biological control of peach fungal pathogens by commercial products and indigenous yeasts

The potential use of the commercial biocontrol products Serenade (Bacillus subtilis QST-713) and Trichodex (Trichoderma harzianum Rifai strain T39) to inhibit the postharvest pathogenic molds Penicillium crustosum and Mucor circinelloides was investigated. Both products exhibited antagonistic activity in vitro against the pathogens, reducing their growth at different levels. In addition, epiphytic yeasts isolated from peaches were identified as Candida maltosa, Pichia fermentans, and Pichia kluyveri by PCR-restriction fragment length polymorphism of internal transcribed spacer regions and screened for antagonistic activity against the same molds. The efficacy of biocontrol in vitro was dependent on the concentration of the yeast cells. Optimal yeast concentrations were above 10(7) CFU ml(-1). However, C. maltosa and P. fermentans were more effective than P. kluyveri in inhibiting molds. The exclusion of antifungal metabolite production and direct competition for nutrients or space with the pathogens was proposed as the mechanism of biocontrol. Application of biocontrol agents directly on artificially wounded peach fruits significantly reduced the incidence of mold rot during storage at 20 degrees C.     

S100B Protein as a Therapeutic Target in Multiple Sclerosis: The S100B Inhibitor Arundic Acid Protects from Chronic Experimental Autoimmune Encephalomyelitis

S100B is an astrocytic protein behaving at high concentration as a damage-associated molecular pattern molecule. A direct correlation between the increased amount of S100B and inflammatory processes has been demonstrated, and in particular, the inhibitor of S100B activity pentamidine has been shown to ameliorate clinical scores and neuropathologic-biomolecular parameters in the relapsing-remitting experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. This study investigates the effect of arundic acid (AA), a known inhibitor of astrocytic S100B synthesis, in the chronic experimental autoimmune encephalomyelitis, which is another mouse model of multiple sclerosis usually studied. By the daily evaluation of clinical scores and neuropathologic-molecular analysis performed in the spinal cord, we observed that the AA-treated group showed lower severity compared to the vehicle-treated mice, particularly in the early phase of disease onset. We also observed a significant reduction of astrocytosis, demyelination, immune infiltrates, proinflammatory cytokines expression and enzymatic oxidative reactivity in the AA-treated group. Overall, our results reinforce the involvement of S100B in the development of animal models of multiple sclerosis and propose AA targeting the S100B protein as a focused potential drug to be considered for multiple sclerosis treatment.     

Model checking fault tolerant systems

This paper proposes a modelling approach suitable for formalizing fault tolerant systems, taking into account different fault scenarios. Verification of the properties of such systems is then performed using model checking. A general framework for the formal specification and verification of fault tolerant systems is defined starting from these principles, and experience with its application to two case studies is then presented. Copyright © 2002 John Wiley & Sons, Ltd.     

Oleuropein expression in olive oils produced from drupes stoned in a spring pitting apparatus (SPIA)

A new device for the preparation of stoned olive oils, called SPIA, has been developed, aiming at fulfilling the targets of: (i) employing a less powered engine, (ii) reducing the size of the machine and (iii) reaching a good efficiency in terms of oil yields. Oleuropein expression was used as a biochemical parameter to distinguish stoned oils from oils produced by conventional milling systems. In vitro experiments performed by exposing oleuropein to pit enzymes, showed an exponential decay of the substrate.