A mussel tissue certified reference material for multiple phycotoxins. Part 3: homogeneity and stability

A candidate certified reference material (CRM) for multiple shellfish toxins (domoic acid, okadaic acid and dinophysistoxins, pectenotoxins, yessotoxin, azaspiracids and spirolides) has been prepared as a freeze-dried powder from mussel tissues (Mytilus edulis). Along with the certified values, the most important characteristics for a reference material to be fit-for-purpose are homogeneity and stability. Acceptable between-bottle homogeneity was found for this CRM. Within-bottle homogeneity was assessed using domoic acid, and it was shown that repeated subsampling of the CRM can be performed precisely down to 0.35 g. Both short- and long-term stability studies carried out under isochronous conditions demonstrated excellent stability of the various toxins present in the material. While degradation of some analytes was observed at +60°C in short-term studies, it was determined that shipping at ambient temperature is adequate. No instability was detected in long-term stability studies, and it was shown that the material can be held at +18°C safely for up to 1 year. To guarantee stability of the CRM over its lifetime the stock will be maintained at −20°C. The results of the homogeneity and stability testing show that CRM–FDMT1 is appropriate for its intended use in quality assurance and quality control of shellfish toxin analysis methods.     

Tridentate ionic hydrogen-bonding interactions of the 5-fluorocytosine cationic dimer and other 5-fluorocytosine analogues characterized by IRMPD spectroscopy and electronic structure calculations

Ionic hydrogen-bonding interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, along with the cationic (proton-bound) dimer have been characterized by infrared multiple-photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD action spectra, in combination with calculated spectra and relative energetics, indicate that it is most probable that predominantly a single isomer exists in each experiment. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest-energy isomer convincingly matches the experimental spectrum. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) involving a tridentate ionic hydrogen-bonding interaction. The three sites of intermolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significant calculated binding energy of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, suggests that a tridentate-proton-bound dimer was observed predominantly in the experiments. Comparison of the calculated relative Gibbs free energies (298 K) for this species and several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV.     

Modification of poly(styrene-co-divinylbenzene) membrane by grafting of salicylic acid via a ketone bridge

Chelating solid phase extraction is a method particularly adapted to the recovery of metallic ions in polluted waters. The principle consists in the partitioning of the analyte between the solution and a solid sorbent grafted by a ligand. Aromatic organic crosslinked copolymers, which are commercially available, permit to consider numerous ways of linking the ligand to the sorbent. In this way, the grafting of salicylic acid (SA) via a ketone bridge on Amberlite^® XAD-4 resin has been previously achieved with success. Then the same grafting on Empore^™ SDB-XC membrane was also considered. The yield obtained is 33%. The determination of complexing capacities highlights the fact that about 20% of the grafted complexing sites could efficiently retain metallic ions. For the first time, a ligand has been covalently linked to Empore^™ SDB-XC membrane.     

Chloroform sorption in nanoporous crystalline and amorphous phases of syndiotactic polystyrene

The transport of chloroform in films of atactic polystyrene and of semicrystalline syndiotactic polystyrene in its nanoporous form (δ‐form) has been investigated by gravimetric analysis. Experimental tests have been performed at 35, 49, and 56 °C and at several vapor pressures ranging from 0.5 to 100 Torr. Nonequilibrium lattice fluid prediction of the amorphous sorption behavior was used to enucleate the sorption contribution of the crystalline nanoporous phase from semicrystalline samples. Sorption behavior of the crystalline phase has been interpreted on the basis of Langmuir equation. Moreover, the chloroform sorption at low activities in the crystalline nanoporous phase has been predicted by using Grand Canonical Monte Carlo molecular simulations. Isosteric heats of sorption were also experimentally evaluated for the crystalline phase, and compared with the corresponding prediction of molecular simulation. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 8–15, 2008     

Approaching the proteoglycome: molecular interactions of proteoglycans and their functional output

[Image: see text] Through their diverse core protein modules and glycan/glycosaminoglycan moieties, proteoglycans may engage in numerous cellular and molecular interactions which are dispensable during embryogenesis, are essential for the maintenance of a healthy state and are prone to modulation in pathological conditions. Proteoglycan interactions may involve binding to other structural components of the ECM, to cell surface receptors, to membrane-associated components, and to soluble signaling molecules, which through this interaction may become entrapped in the ECM or sequestered at the cell surface. Understanding of these multiple interplays is therefore of paramount importance and requires a detailed mapping through what we define as the proteoglycome.     

C2C12 myoblast sensitivity to different apoptotic chemical triggers

Apoptosis is a form of cell death crucial for normal development and tissue homeostasis. Its typical features include chromatin changes, nuclear breakdown, plasma membrane blebbing and splitting of cellular content into apoptotic bodies, that progressively undergo phagocytosis.Apoptosis is considered essential for skeletal muscle development, where defective cells are deleted during differentiation. In addition, it plays a relevant role in several muscle myopathies, as well as in denervation and disuse.The aim of this study was to evaluate muscle cell sensitivity to different apoptotic triggers, acting through different mechanisms of action. Chemical agents, active against distinct intracellular targets, such as mitochondrial respiratory chain and DNA, have been chosen to better highlight cell death mechanisms. To induce apoptosis, C2C12 myoblasts have been exposed to H₂O₂, staurosporine, cisplatin and etoposide, at different doses and incubation times, and they have been analysed by flow cytometry, scanning and transmission electron microscopy.Flow cytometry analysis revealed a certain subdiploid peak after all treatments. The best apoptotic effect was observable, as confirmed at reverted microscope, at minimum doses and after the major exposure time.At ultrastructural level programmed cell death has been observed. Characteristic chromatin condensation and margination, as well as apoptotic bodies, frequently appeared, even if in the presence of secondary necrosis; surface blebs were also observed during scanning microscopic observation.In particular, exposure to H₂O₂ or staurosporine showed the largest number of myoblasts in late apoptotic stages and in secondary necrosis. Cisplatin treatments revealed few early apoptotic cells. The analysis of etoposide-induced apoptosis was in agreement with data obtained from flow cytometry, indicating a significant increase of apoptotic cell number.These results suggest that all conditions are able to induce apoptosis in C2C12 myoblasts, which occurs, considering trigger mechanisms of action, mostly following the mitochondrial pathway, if not excluding that due to DNA damage. Therefore, mitochondria permeability alteration is an important step in skeletal muscle programmed cell death. This last conclusion seems to have a significant relevance in understanding the mechanisms involved in muscle disorders, denervation and chronic muscle disuse, conditions frequently characterized by a decline in mitochondrial content and by an increase of mitochondrial apoptosis susceptibility.     

Ethane-bridged zinc porphyrin dimers in Langmuir-Shäfer thin films: structural and spectroscopic properties

This work reports on the structural and spectroscopic properties of ethane-bridged Zn porphyrin dimers (1) in Langmuir-Sch?fer (LS) thin films by combining scanning force microscopy (SFM) with film balance, UV-vis absorption, fluorescence, and nanosecond laser flash photolysis measurements. Results show that depending on the surface pressure the Langmuir films of pure 1 can be arranged in two different condensed phases, whereas SFM of the LS films shows characteristic fractal networks constituted by nanoscopic aggregates. The spectral findings agree with a picture in which 1 is apparently present in the anti conformation but aggregated in a sort of H-type structure whose optical features resemble those of the syn conformer. This type of structure is not responsive to light stimuli. By diluting 1 in arachidic acid the porphyrin aggregation is significantly minimized with 1 exhibiting almost exclusively the anti conformation. As a result the LS films become photoresponsive, showing fluorescence emission and triplet-triplet transient absorption.