Chemical characterization of polysaccharides from Gracilaria gracilis from Bizerte (Tunisia).

Polysaccharides were extracted from Gracilaria gracilis collected from Manzel Jemil Lake in Bizerte Tunisia, with two different solvents (water and NaOH 0.3 M). Different assays were performed on samples (total sugars, neutral sugars, uronic acids, anhydrogalactose, proteins, sulphates, pyruvates), followed by high performance anion-exchange chromatography (HPAEC) to observe the monosaccharide composition, high pressure size exclusion chromatography with multi-angle laser light scattering (HPSEC-MALS) to obtain the molecular mass, Fourier transform infrared spectroscopy (FTIR), and 1D and 2D nuclear magnetic resonance (NMR) to access to structural data. Results have shown that the polysaccharide extracted from Gracilaria gracilis collected from Manzel Jemil Lake in Bizerte Tunisia, is of agar type but with high molecular mass and some original structural features. Hence, the sample was found to contain 9 % of pyruvate groups and is partly sulphated at the C4 of ß-d-galactose and methylated on C2 of anhydro-α-l-galactose. The polymer from G. gracilis from Bizerte thus presents a never described structure that could be interesting for further rheological or biological activities applications.

Spot urine collection: A valid alternative to total urine collection for metabolomic studies in dairy cattle.

Urine is a highly suitable biological matrix for metabolomics studies. Total collection for 24-h periods is the gold standard as it ensures the presence of all metabolites excreted throughout the day. However, in animal studies, it presents limitations related to animal welfare and also due to alterations of the metabolome originating from the use of acid for preventing microbial growth or microbial contamination. In this study, we investigated whether spot urine collection is a practical alternative to total collection for metabolomic studies in lactating cows. For this purpose, we collected urine samples from 4 lactating Holstein cows fed 4 diets in a 4 × 4 Latin square design. Urine was collected for 24 h using a collecting device (i.e., total collection) or collected once per day 4 h after the morning feeding (i.e., spot urine collection). Dietary treatments differed by the amount of nitrogen content (high vs. low) and by the nature of the energy (starch vs. fiber). Urine metabolome was analyzed by 2 untargeted complementary methods, nuclear magnetic resonance and hydrophilic-interaction liquid chromatography (HILIC) coupled to a time-of-flight mass spectrometer, and by 1 targeted method, HILIC-tandem mass spectrometry. Although sampling technique had an effect on the abundance of metabolites detected, spot urine samples were equally capable of showing differences in urine metabolome than samples from total collection. When considering nitrogen levels in the diet, the robustness and precision for discriminating high- and low-nitrogen diets was equally achieved with both sampling techniques. A total of 22 discriminant metabolites associated with the N level of diets were identified from untargeted HILIC coupled to a time-of-flight mass spectrometer (n = 9) and nuclear magnetic resonance (n = 11), and 2 from targeted HILIC-tandem mass spectrometry. Alternatively, starch or fiber in the diet induced less changes in the metabolome that were not clearly discriminated independently of the sampling technique. We concluded that spot urine collection can successfully reveal differences in the urine metabolome elicited by dietary N levels and be used as a substitute of total urinary 24-h collection for metabolomic studies.

Environmental scenarii for the degradation of oxo-polymers.

The fate of oxo-polymers in nature is strongly dependent on environmental conditions, mainly on the intensity and duration of sunshine, which vary with the season and the climate. In this work, we report the effect of different scenarii on the production and the molecular composition of oligomers released from oxo-biodegradable HDPE films. Under our experimental conditions, the duration of accelerated weathering corresponded to a period of 3 months to 3 years of exposure to outside conditions under temperate climate. In addition, the oligomers were extracted in three different solvents: i) water to mimics the natural environment; ii) acetone and chloroform to identify oligomers trapped in the polymer matrix. The combination of high-resolution mass spectrometry and 1H NMR spectroscopy gives an extensive picture of the relative concentrations and the structural compositions of the extracted oligomers in the different tested conditions. In particular, the masses, the number of oxygen and carbon atoms could be determined for up to 2283 molecules. Globally the concentration and the size of oligomers increased with the duration of extraction, the level of aging of the polymer and the use of non-polar solvents. Surprisingly, the presence of highly oxidized molecules in acetone and chloroform extract, suggested an important swelling of HPDE films in these solvents and a better diffusion of these oligomers in the matrix. In nature, the biodegradability of oligomers could result from processes occurring both at the molecular (oxidation) and the macromolecular (diffusion and release) levels.

The role of association of ions in ionic liquid/molecular solvent mixtures on metal extraction.

Several mixtures of butyl acetate (BA) and an ionic liquid (1-methyl-3-octylpyrrolidinium bis(trifluoromethylsulfonyl)imide, [C1C8Pyrro][NTf2], 1-octyl-pyridinium bis(trifluoromethylsulfonyl)imide, [C8Pyr][NTf2], 1-butyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide, [C1C4Im][NTf2] or 1-octyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide, [C1C8Im][NTf2]) have been characterized by measuring density, viscosity and conductivity, from pure BA to pure ILs at 298.15 K. The ionicity of these mixtures has been determined on the basis of electrical conductivity and NMR spectroscopy. IR spectra of these mixtures were used to examine the interactions between ions. In parallel, Pt(iv) was extracted from the acidic aqueous phase towards mixtures of BA and [C1C8Im][NTf2] over the entire composition range. A drastic modification in the distribution coefficient of Pt(iv) was observed at ca xAB = 0.8. A drop was also observed in the ionicity of the extracting phase (IL + BA) at a similar composition. The variation of the distribution coefficient is ascribed to changes in the interactions in the mixtures, which in turn could induce changes in the extraction mechanism.

Characterization of oxidized oligomers from polyethylene films by mass spectrometry and NMR spectroscopy before and after biodegradation by a Rhodococcus rhodochrous strain.

The objective of this work was to develop a new approach to assess the specificity and the efficiency of biodegradation of oxidized oligomers extracted from aged HDPE polyethylene films and to bring insight on the mechanisms occurring during biodegradation. 1H NMR spectroscopy and LC Orbitrap™ mass spectrometry were combined together with data processing using Kendrick mass defect calculation and Van Krevelen Diagram. We showed that the molecular weight of extracted oligomers was lower than 850 Da with maximum chain length of 55 carbon atoms. The oligomers were divided into 11 classes of molecules with different oxidation state ranging from 0 to 10. All classes included series of chemically related compounds including up to 19 molecules. 95% of the soluble oligomers were assimilated by a strain of Rhodococcus rhodocchrous after 240 days of incubation. Large highly oxidized molecules completely disappeared while the other classes of molecules were still represented. Molecules containing 0-1 oxygen atom were less degraded. A strong shift to smaller molecules (<450 Da, 25 carbon atoms) was observed suggesting that longer molecules disappeared more rapidly than the smaller ones. It opens new perspectives on biodegradation processes as not only intracellular ß-oxidation must be considered but also extracellular mechanisms leading to chain cleavages.

Can the tricyanomethanide anion improve CO2 absorption by acetate-based ionic liquids?

Carbon dioxide absorption by mixtures of two ionic liquids with a common cation-1-butyl-3-methylimidazolium acetate, [C4C1Im][OAc], and 1-butyl-3-methylimidazolium tricyanomethanide, [C4C1Im][C(CN)3]-was determined experimentally at pressures below atmospheric pressure as a function of temperature between 303 K and 343 K, and at 303 K as a function of pressure up to 10 bar. It is observed that the absorption of carbon dioxide decreases with increasing tricyanomethanide anion concentration and with increasing temperature, showing a maximum of 0.4 mole fraction of carbon dioxide in pure [C4C1Im][OAc] at 303 K. At this temperature, the CO2 absorption in the mixtures [C4C1Im][OAc](1-x)[C(CN)3]x is approximately the mole-fraction average of that in the pure ionic liquids. By applying an appropriate thermodynamic treatment, after identification of the species in solution, it was possible to calculate both the equilibrium constant, Keq, and Henry's law constant, KH, in the different mixtures studied thus obtaining an insight into the relative contribution of chemical and physical absorption of the gas. It is shown that chemical sorption proceeds through a 1 : 2 stoichiometry between CO2 and acetate-based ionic liquid. The presence of the C(CN)3- anion does not significantly affect the chemical reaction of the gas with the solvent (Keq = 75 ± 2 at 303 K) but leads to lower Henry's law constants (from KH = 77.8 ± 0.6 bar to KH = 49.5 ± 0.5 bar at 303 K), thus pointing towards larger physical absorption of the gas. The tricyanomethanide anion considerably improves the mass transfer by increasing the fluidity of the absorbent as proven by the larger diffusivities of all the ions when the concentration of the C(CN)3- anion increases in the mixtures.

Tailoring the properties of acetate-based ionic liquids using the tricyanomethanide anion.

The equilibrium and transport properties of mixtures of two ionic liquids - [C4C1Im][OAc] and [C4C1Im][C(CN)3] - were determined and interpreted at the molecular level using vibration spectroscopy, NMR and molecular dynamics simulation. The non-ideality of the mixtures [C4C1Im][OAc](1-x)[C(CN)3]x was characterized by V(E) = +0.28 cm(3) mol(-1) (293 K, x = 0.65) and H(E) = -2.2 kJ mol(-1) for x = 0.5. These values could be explained by a rearrangement of the hydrogen-bond network of the mixture that favours the interaction of the acetate anion with the imidazolium cation at position C2. The dynamic properties of the mixture are also dramatically influenced by the composition with a decrease of the viscosity and an increase of self-diffusion coefficients of the ions when the amount of tricyanomethanide anion increases in the mixture.

Antioxidant activities of a polyglucuronic acid sodium salt obtained from TEMPO-mediated oxidation of xanthan.

A xanthouronic acid sodium salt called xanthouronan was produced from xanthan by regioselective oxidation with NaOCl/NaBr using 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO) as catalyst. The efficiency of the one pot TEMPO-mediated oxidation was confirmed by HPAEC-PAD, (13)C NMR, and FT-IR. The oxidation degree was close to 98% and the mass yield of this new polyglucuronic acid was higher than 90% (w/w). The macromolecular characterization of xanthouronan using SEC-MALLS showed a molecular size reduced by a third due to the oxidation treatment and the degree of polymerization (DP) of the xanthouronan form was about 665. The evaluation of the enzymatic degradation of this C-6 carboxylated xanthan by various polysaccharide hydrolases and one polysaccharide lyase showed its high resistant to biodegradation. The antioxidant activity of xanthouronan was also tested by using the 2,2'-diphenyl-1-picrylhydrazyle (DPPH) and hydroxyl radical procedures. At 1 g/L, xanthouronan presented 75% of the ascorbic acid antioxidant activity.

In vivo 31P and 13C NMR investigations of Rhodococcus rhodochrous metabolism and behaviour during biotransformation processes.

AIMS: The strain Rhodococcus rhodochrous OBT18 was isolated from a water treatment plant used to decontaminate industrial effluents containing benzothiazole derivatives. Aims of the work are to study the central metabolism of this strain and more specifically its behaviour during biodegradation of 2-aminobenzothiazole. METHODS AND RESULTS: In vivo(13)C and (31)P NMR experiments showed that this strain contains storage compounds such as polyphosphates, glycogen and trehalose and produces biosurfactants containing trehalose as sugar unit. Trehalose can be synthesized after reversion of the glycolytic pathway. In vivo(31)P NMR experiments showed that energy metabolism markers such as the intracellular pH and the ATP concentration did not change during biotransformation processes when R. rhodochrous was exposed to potentially toxic compounds including iron complexes and (* )OH radicals. Also R. rhodochrous recovers the normal values of ATP and pH after anoxia/reoxygenation cycle very quickly. CONCLUSIONS: Rhodococcus rhodochrous carbon and energy metabolism is well adapted to different stresses and consequently to live in the environment where conditions are constantly changing. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study can be used to understand the behaviour of this bacterium in natural environments but also in water treatment plants where iron and UV light are present.

Sarcolemma phospholipid structure investigated by immunogold electron microscopy and (31)P NMR spectroscopy with lanthanide ions.

The biological functions of plasma membranes depend greatly on the biophysical properties resulting from protein and phospholipid structure. We investigated the phospholipid structure of the normal sarcolemma membrane, which is known to be highly dysfunctional in myopathies. Combining electron microscopy and (31)P nuclear magnetic resonance (NMR) spectroscopy on isolated sarcolemma vesicles, we find that (i) the sarcolemma vesicles maintain the in-vivo cellular sidedness, (ii) the phospholipid mobility is close to that observed in model membranes (similar lateral diffusion coefficients and spin-lattice T(1) relaxation times). Using broad-band and magic angle spinning (31)P NMR spectroscopy with lanthanide ions (Pr(3+)), it is possible to quantify the distribution of phospholipids between internal and external membrane layers, showing that the trans-bilayer distribution is highly asymmetrical.

The importance of cholesterol in maintenance of P-glycoprotein activity and its membrane perturbing influence.

In tumour cell lines that display multidrug resistance, expression of P-glycoprotein (P-gp) alters many aspects of biomembrane organization in addition to its well-characterized drug transport activity. We have developed a reconstitution system to directly investigate the effect of purified P-gp on the biophysical properties of lipid bilayers. Using a mixed detergent system it was possible to efficiently reconstitute P-gp at lipid:protein ratios as low as 2.5 (w/w) by removal of detergent using adsorption to SM-2 BioBeads. P-gp was able to alter many biophysical parameters associated with lipid organization within bilayers. For example, the changes in overall fluidity and excimer formation by lipid analogues indicate modified packing organization of bilayer constituents. Surprisingly, given its role in conferring drug resistance, P-gp insertion into bilayers also caused significantly increased permeability to aqueous compounds, also reflecting a modified phospholipid environment. Translocation of various phospholipid species between leaflets of the bilayer was increased in the presence of P-gp; however, the effect was not dependent on ATP hydrolysis by the protein. Physiological concentrations of cholesterol modified P-gp function and the degree to which it perturbed bilayer organization. The basal ATPase activity of P-gp was increased in a dose-dependent fashion by the incorporation of cholesterol in PC:PE liposomes. In addition, the degree to which the modulator verapamil was able to stimulate this basal ATPase activity was reduced by the presence of cholesterol in proteoliposomes. However, the potency of verapamil was unaltered, suggesting a specific effect, not simply caused by lower drug penetration into the cholesterol containing bilayers. In summary, P-gp is able to cause perturbation in the organization of bilayer constituents. Cholesterol imparted "stability" to this perturbation of bilayer organization by P-gp and moreover this led to altered protein function.

Formation of unilamellar vesicles by repetitive freeze-thaw cycles: characterization by electron microscopy and 31P-nuclear magnetic resonance.

It has been reported that repetitive freeze-thaw cycles of aqueous suspensions of dioleoylphosphatidylcholine form vesicles with a diameter smaller than 200 nm. We have applied the same treatment to a series of phospholipid suspensions with particular emphasis on dioleoylphosphatidylcholine/dioleoylphosphatidic acid (DOPC/DOPA) mixtures. Freeze-fracture electron microscopy revealed that these unsaturated lipids form unilamellar vesicles after 10 cycles of freeze-thawing. Both electron microscopy and broad-band 31P NMR spectra indicated a disparity of the vesicle sizes with a highest frequency for small unilamellar vesicles (diameters < or =30 nm) and a population of larger vesicles with a frequency decreasing exponentially as the diameter increases. From 31P NMR investigations we inferred that the average diameter of DOPC/DOPA vesicles calculated on the basis of an exponential size distribution was of the order of 100 nm after 10 freeze-thaw cycles and only 60 nm after 50 cycles. Fragmentation by repeated freeze-thawing does not have the same efficiency for all lipid mixtures. As found already by others, fragmentation into small vesicles requires the presence of salt and does not take place in pure water. Repetitive freeze-thawing is also efficient to fragment large unilamellar vesicles obtained by filtration. If applied to sonicated DOPC vesicles, freeze-thawing treatment causes fusion of sonicated unilamellar vesicles into larger vesicles only in pure water. These experiments show the usefulness of NMR as a complementary technique to electron microscopy for size determination of lipid vesicles. The applicability of the freeze-thaw technique to different lipid mixtures confirms that this procedure is a simple way to obtain unilamellar vesicles.

Solid-state NMR for the study of membrane systems: the use of anisotropic interactions.

The use of solid-state nuclear magnetic resonance (NMR) as a tool to determine the structure of membrane molecules is reviewed with a particular emphasis on techniques that provide information on orientation or order. Experiments reported here have been performed in membranes, rather than in micelles or organic solvents. Several ways to prepare and handle the samples are discussed, like sample orientation and magic-angle spinning (MAS). Results concerning lipids, membrane peptides and proteins are included, as well as a discussion regarding the potential of such methods and their pitfalls.