68Ga-Labelled Carbon Nanoparticles for Ventilation PET/CT Imaging: Physical Properties Study and Comparison with Technegas®.

PURPOSE: The use of 68Ga-labelled carbon nanoparticles has been proposed for lung ventilation PET/CT imaging. However, no study has assessed the physical properties of 68Ga-labelled carbon nanoparticles. The aim of this study therefore was to evaluate the shape and size of 68Ga-labelled carbon nanoparticles, and to determine the composition of the aerosol, as opposed to 99mTc-labelled carbon nanoparticles aerosol. PROCEDURES: 99mTc- and 68Ga-labelled carbon nanoparticles, stable gallium carbon nanoparticles, 0.9 % NaCl and 0.1 N HCl-based carbon nanoparticles were produced using an unmodified Technegas® generator, following the usual technique used for clinical Technegas® production. The shape and size of particles were studied by transmission electron microscopy (TEM) after decay of the radioactive samples. The composition of the 68Zn- and 99Tc-labelled carbon nanoparticles aerosols was assessed using scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX) analysis after decay of the 68Ga- and 99mTc-labelled carbon nanoparticles, respectively. RESULTS: On TEM, all samples showed similar shape with hexagonally structured primary particles, agglomerated in clusters. The mean diameters of primary stable gallium carbon nanoparticles, 99Tc- and 68Zn-labelled carbon nanoparticles were 22.4 ± 10 nm, 20.9 ± 7.2 nm and 19.8 ± 11.7 nm, respectively. CONCLUSION: Using Technegas® generator in the usual clinical way, 99mTc- and 68Ga-labelled carbon nanoparticles demonstrated similar shape and diameters in the same size range size. These results support the use of 68Ga-labelled carbon nanoparticles for the assessment of regional lung ventilation function with PET imaging.

Qualitative and quantitative study of the highly specialized lipid tissues of cetaceans using HR-MAS NMR and classical GC.

Cetacean adipose tissues contain an extremely very wide variety of acyl-chains present in triacylglycerols and / or wax esters. In addition, changes in the lipid composition across organs suggest fine stratification. It therefore remains technically challenging to describe precisely the lipid organization of these tissues. In the present study, we used in parallel HR-MAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) and GC (gas-chromatography) to characterize and quantify the lipids and fatty acyl-chains from the blubber and melon of two odontocete species. Both methods generated very similar compositions, but each presented clear advantages. While GC underestimated the amount of short branched fatty acyl-chains, which are specific to cetacean adipose tissues and most probably of primary importance for their functioning, HR-MAS NMR allowed for their exact quantification. Conversely, when HR-MAS NMR could only discriminate a few types of fatty acyl-chain families, GC unambiguously identified and quantified most of them. In addition, this technique allowed for the determination of the wax esters molecular species. Our results further suggest that the stratification of these adipose tissues relies on changes in the triacylglycerol to wax ester ratio and in the fatty acyl composition of triacylglycerols, but not on changes in the wax esters composition. Altogether, our data show that the complementarities of these two approaches result in lipid analyses of unprecedented precision, paving the way for the detailed description of the fatty acyl composition of cetacean adipose tissues and the understanding of their functioning.

The Relationship between NMR Chemical Shifts of Thermally Polarized and Hyperpolarized 89 Y Complexes and Their Solution Structures.

Recently developed dynamic nuclear polarization (DNP) technology offers the potential of increasing the NMR sensitivity of even rare nuclei for biological imaging applications. Hyperpolarized 89 Y is an ideal candidate because of its narrow NMR linewidth, favorable spin quantum number (I=1/2 ), and long longitudinal relaxation times (T1 ). Strong NMR signals were detected in hyperpolarized 89 Y samples of a variety of yttrium complexes. A dataset of 89 Y NMR data composed of 23 complexes with polyaminocarboxylate ligands was obtained using hyperpolarized 89 Y measurements or 1 H,89 Y-HMQC spectroscopy. These data were used to derive an empirical equation that describes the correlation between the 89 Y chemical shift and the chemical structure of the complexes. This empirical correlation serves as a guide for the design of 89 Y sensors. Relativistic (DKH2) DFT calculations were found to predict the experimental 89 Y chemical shifts to a rather good accuracy.

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus.

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response.

Triggering bilayer to inverted-hexagonal nanostructure formation by thiol-ene click chemistry on cationic lipids: consequences on gene transfection.

The ramification of cationic amphiphiles on their unsaturated lipid chains is readily achieved by using the thiol-ene click reaction triggering the formation of an inverted hexagonal phase (HII). The new ramified cationic lipids exhibit different bio-activities (transfection, toxicity) including higher transfection efficacies on 16HBE 14o-cell lines.

Rapid identification of osmolytes in tropical microalgae and cyanobacteria by (1)H HR-MAS NMR spectroscopy.

In this study, we report the chemical characterization of 47 tropical microalgae and cyanobacteria by HR-MAS. The generated data confirm the interest of HR-MAS as a rapid screening technique with the major advantage of its easiness. The sample is used as powder of freeze-dried microalgae without any extraction process before acquisition. The spectral fingerprints of strains are then tested as variables for a chemotaxonomy study to discriminate cyanobacteria and dinoflagellates. The individual factor map generated by PCA analysis succeeds in separating the two groups, essentially thanks to the presence of specific carbohydrates. Furthermore, more resolved signals enable to identify many osmolytes. More precisely the characteristics δ of 2-O-alpha-D-glucosylglycerol (GG) are observed in all 21 h-MAS spectra of tropical cyanobacteria. After specific extraction, complementary analysis by 1D and 2D-NMR spectroscopies validates the identification of this osmolyte.

HRMAS NMR Analysis of Algae and Identification of Molecules of Interest via Conventional 1D and 2D NMR: Sample Preparation and Optimization of Experimental Conditions.

Nuclear magnetic resonance (NMR) has become an astounding tool for molecular characterization. Thanks to the development of probes and the increase of magnetic field, NMR has entered the field of biology and facilitated the identification of natural compounds. Indeed, this nondestructive NMR tool makes possible the complete characterization of less and less quantities of material via 1D and 2D sequences on many nuclei (e.g., (1)H, (13)C, (31)P, (15)N). More recently, the development of high-resolution magic-angle spinning (HRMAS) probes have permitted direct analysis of living tissue (e.g., a piece of algae) without prior extraction providing information on both the total content and the ratio of different molecules within the sample; thus HRMAS facilitates a wide range of analyses, such as species differentiation or studies of metabolomics according to various environmental or experimental conditions. This chapter describes the specific sample preparation, based on an algal sample or extract, required for all NMR analyses in order to optimize the NMR response and obtain the most valuable information.

NMR use to quantify phlorotannins: the case of Cystoseira tamariscifolia, a phloroglucinol-producing brown macroalga in Brittany (France).

Among the most renowned natural products from brown algae, phlorotannins are phloroglucinol polymers that have been extensively studied, both for their biotechnological potential and their interest in chemical ecology. The accurate quantification of these compounds is a key point to understand their role as mediators of chemical defense. In recent years, the Folin-Ciocalteu assay has remained a classic protocol for phlorotannin quantification, even though it frequently leads to over-estimations. Furthermore, the quantification of the whole pool of phlorotannins may not be relevant in ecological surveys. In this study, we propose a rapid (1)H qNMR method for the quantification of phlorotannins. We identified phloroglucinol as the main phenolic compound produced by the brown macroalga Cystoseira tamariscifolia. This monomer was detected in vivo using (1)H HR-MAS spectroscopy. We quantified this molecule through (1)H qNMR experiments using TSP as internal standard. The results are discussed by comparison with a standard Folin-Ciocalteu assay performed on purified extracts. The accuracy and simplicity of qNMR makes this method a good candidate as a standard phlorotannin assay.

Structural elucidation, in vitro antioxidant and photoprotective capacities of a purified polyphenolic-enriched fraction from a saltmarsh plant.

In temperate saltmarshes, halophytic plants have to daily protect their internal tissues against sunlight and UV rays. Consequently, they develop adaptive responses such as the synthesis of secondary metabolites, including polyphenols. The present study focused on the biological activities of fractions enriched in polyphenols from Salicornia ramosissima. Three different extracts were obtained by purification processes to concentrate polyphenols: a crude hydroalcoholic extract, and two purified fractions: an ethyl acetate fraction (EAF) and an aqueous fraction. Phenolic and flavonoid contents, antioxidant (DPPH radical-scavenging activity, reducing activity, ß-carotene linoleic acid system and the ORAC method) and sunscreen properties (Sun Protection Factor and UVA-Protection Factor) were assessed by in vitro tests. The purification process was effective in increasing phenolic and flavonoid contents as well as antioxidant and sunscreen capacities of the EAF. The EAF appeared to be a broad spectrum UV absorber. The chemical structure of 10 EAF polyphenols was elucidated using 2D NMR and mass spectrometry spectra. Furthermore, a correlation was observed between phenolic composition and biological activity. These findings are encouraging for the future use of S. ramosissima as a potential source of antioxidant and photoprotectant molecules for industrial applications.

Rapid geographical differentiation of the European spread brown macroalga Sargassum muticum using HRMAS NMR and Fourier-Transform Infrared spectroscopy.

Two recent techniques based on chemical footprinting analysis, HRMAS NMR and FTIR spectroscopy, were tested on a brown macroalgal model. These powerful and easily-to-use techniques allowed us to discriminate Sargassum muticum specimens collected in five different countries along Atlantic coasts, from Portugal to Norway. HRMAS NMR and FTIR permitted the obtaining of an overview of metabolites produced by the alga. Based on spectra analysis, results allowed us to successfully group the samples according to their geographical origin. HRMAS NMR and FTIR spectroscopy respectively point out the relation between the geographical localization and the chemical composition and demonstrated macromolecules variations regarding to environmental stress. Then, our results are discussed in regard of the powerful of these techniques together with the variability of the main molecules produced by Sargassum muticum along the Atlantic coasts.

Chemical and enzymatic fractionation of cell walls from Fucales: insights into the structure of the extracellular matrix of brown algae.

BACKGROUND AND AIMS: Brown algae are photosynthetic multicellular marine organisms evolutionarily distant from land plants, with a distinctive cell wall. They feature carbohydrates shared with plants (cellulose), animals (fucose-containing sulfated polysaccharides, FCSPs) or bacteria (alginates). How these components are organized into a three-dimensional extracellular matrix (ECM) still remains unclear. Recent molecular analysis of the corresponding biosynthetic routes points toward a complex evolutionary history that shaped the ECM structure in brown algae. METHODS: Exhaustive sequential extractions and composition analyses of cell wall material from various brown algae of the order Fucales were performed. Dedicated enzymatic degradations were used to release and identify cell wall partners. This approach was complemented by systematic chromatographic analysis to study polymer interlinks further. An additional structural assessment of the sulfated fucan extracted from Himanthalia elongata was made. KEY RESULTS: The data indicate that FCSPs are tightly associated with proteins and cellulose within the walls. Alginates are associated with most phenolic compounds. The sulfated fucans from H. elongata were shown to have a regular α-(1→3) backbone structure, while an alternating α-(1→3), (1→4) structure has been described in some brown algae from the order Fucales. CONCLUSIONS: The data provide a global snapshot of the cell wall architecture in brown algae, and contribute to the understanding of the structure-function relationships of the main cell wall components. Enzymatic cross-linking of alginates by phenols may regulate the strengthening of the wall, and sulfated polysaccharides may play a key role in the adaptation to osmotic stress. The emergence and evolution of ECM components is further discussed in relation to the evolution of multicellularity in brown algae.

Isolation of turbinaric acid as a chemomarker of Turbinaria conoides (J. Agardh) Kützing from South Pacific Islands.

Several species of the genus Turbinaria coexist along the coasts of islands in the Indian and Pacific Oceans. Among these brown algae, Turbinaria ornata and T. conoides are sister species that are difficult to differentiate using exclusively morphological characters. Based on in vivo nuclear magnetic resonance and chromatographic techniques, i.e., liquid and gas chromatography-mass spectrometry analysis, combined with phylogenetic data, we successfully identified turbinaric acid in T. conoides samples from several Indian and Pacific Ocean islands. This nonvariable discriminant molecule was only identified in T. conoides specimens, but not in the two allied species T. ornata and T. decurrens. Results are discussed with regard to turbinaric acid as an interesting chemomarker isolated from T. conoides and the rapid discrimination of Turbinaria specimens using chemical assays.

Biosynthesis and characterization of polyhydroxyalkanoates by Pseudomonas guezennei from alkanoates and glucose.

The biosynthesis of medium chain length poly(3-hydroxyalkanoates) mcl PHAs by Pseudomonas guezennei using glucose, sodium octanoate, and 10-undecenoic acid as sole or mixed carbon sources was investigated. Chemical composition of polyesters was analyzed by GCMS and NMR. The copolyester produced by P. guezennei from glucose mainly consisted of 3-hydroxyoctanoate and 3-hydroxydecanoate, and the presence of 3-hydroxydodec-5-enoate was demonstrated. Using sodium octanoate as the sole nutrient, the microorganism produced a poly(3-hydroxyoctanoate) (PHO) polymer containing up to 94 mol% 3-hydroxyoctanoate. Biosynthesis of poly[(3-hydroxyoctanoate)-co-(3-hydroxyundecenoate)] (PHOU) copolymers bearing terminal reactive double bonds on its side chains with unsaturation degree ranging from 8.8% to 78.2% was obtained by tuning the ratio of sodium octanoate/10-undecenoic acid in the medium. Thermal analysis indicated semi-crystalline polymers with melting temperatures (T(m)) ranging from 46 to 55°C, fusion enthalpy (ΔH) comprised between 3 and 35 J/g and glass transition temperature (T(g)) from -36 to -44°C, except for the highly amorphous 78.2% unsaturated PHOU with a low T(g) (-50°C). Molecular weights determined by GPC ranged from 119000 and 530000 g/mol. The biosynthesis of natural polyesters with controlled ratio of vinyl-terminated side chains is of great interest for further chemical modifications.

Development of a three-steps derivatization assay for the localization of double bond in monounsaturated monomers of poly-beta-hydroxyalkanoates by GC-MS.

A new gas chromatography-mass spectrometry (GC-MS) method for the localization of double bond in monounsaturated 3-hydroxyalkenoic acids monomers has been developed. A three steps derivation assay was used including a methanolysis, then acetylation and dimethyldisulfide (DMDS) addition to alkene groups. Electron impact GC-MS analysis of such derivatives offers characteristic fragments allowing the unambiguous determination of double bond position in side chain. This novel method is well-suited for the routine analysis of poly-beta-hydroxyalkanoates (PHAs), and was used to characterize monounsaturated monomers in both 3-hydroxyalkenoic acids standards as well as in mcl-PHAs and poly(3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) produced by bacterial strain Pseudomonas guezennei from glucose or a mixture of sodium octanoate plus 10-undecenoic acid, respectively.

LC/ESI-MS n and 1H HR-MAS NMR analytical methods as useful taxonomical tools within the genus Cystoseira C. Agardh (Fucales; Phaeophyceae).

Species of the genus Cystoseira are particularly hard to discriminate, due to the complexity of their morphology, which can be influenced by their phenological state and ecological parameters. Our study emphasized on the relevance of two kinds of analytical tools, (1) LC/ESI-MS(n) and (2) (1)H HR-MAS NMR, also called in vivo NMR, to identify Cystoseira specimens at the specific level and discuss their taxonomy. For these analyses, samples were collected at several locations in Brittany (France), where Cystoseira baccata, C. foeniculacea, C. humilis, C. nodicaulis and C. tamariscifolia were previously reported. To validate our chemical procedure, the sequence of the ITS2 has been obtained for each species to investigate their phylogenetic relationships at a molecular level. Our study highlighted the consistency of the two physico-chemical methods, compared to "classical" molecular approach, in studying taxonomy within the genus Cystoseira. Especially, LC/ESI-MS(n) and phylogenetic analyses converged into the discrimination of two taxonomical groups among the 5 species. The occurrence of some specific signals in the (1)H HR-MAS NMR spectra and/or some characteristic chemical compounds during LC/ESI-MS(n) analysis could be regarded as discriminating factors. LC/ESI-MS(n) and (1)H HR-MAS NMR turned out to be two relevant and innovative techniques to discriminate taxonomically this complex genus.

Improved enantioselective synthesis of (-)-linderol A: hindered rotation about aryl-Csp(3) bond.

An improved enantioselective total synthesis of (-)-linderol A has been achieved via a five-step reaction with a 21% overall yield, starting from phloroacetophenone and (-)-alpha-phellandrene, two commercially available reagents. In the diastereoselective epoxidation step, the analysis of the two endocyclic epoxide intermediates reveals a hindered sp(2)-sp(3) rotation, which results in rotational diastereoisomers.

Whole-cell spectroscopy is a convenient tool to assist molecular identification of cultivatable marine bacteria and to investigate their adaptive metabolism.

Recent developments in whole-cell spectroscopic methods allow rapid characterization of microorganisms of interest to human health, but have yet to be widely applied to marine microbiological studies. In this study of bacteria associated with the kelp Laminaria digitata, we have isolated 18 epiphytic bacterial strains from several thalli, sequenced their 16S rDNA, built corresponding phylogenetic trees, and characterized them using spectroscopic methods. Molecular taxonomy revealed Gram(+)Actinobacteria and Gram(-)Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. Twelve marine reference strains (Gram(+)Firmicutes, and Gram(-)Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes) were treated accordingly. Whole-cell MALDI-TOF MS spectral profiles of 29 of the 30 strains were built into a database against which 16 replicate spectra of each strain were compared and categorized into groups. The proton HR-MAS NMR stack plots allowed visual delineation into taxonomic groups according to their most common peaks, in agreement with identifiable compounds from corresponding D(2)O solution spectra. With both methods, these groups corresponded to taxa identified by 16S rDNA sequences, MALDI-TOF MS being more discriminative than HR-MAS NMR. Culture age did not influence the spectral signatures in both approaches. Most cells grown under minimal conditions (VNSS medium) afforded HR-MAS NMR profiles markedly different to those grown in enriched conditions (ZoBell medium), indicating different adaptive metabolic responses between the two media. Spectral signatures obtained under strictly controlled conditions can be used as rapid and reliable tools for taxonomic purposes and as markers of physiological status.

Impact of the salt stress on the photosynthetic carbon flux and 13C-label distribution within floridoside and digeneaside in Solieria chordalis.

The flux of photosynthetic carbon used in the synthesis of low-molecular weight carbohydrates (digeneaside and floridoside) was investigated by (13)C and (1)H NMR spectroscopy in samples of the red seaweed, Solieria chordalis, incubated at different salinities (22, 34 and 50psu). Carbohydrates were labelled, by pulse-chase, with the stable isotope (13)C from NaH(13)CO(3). In vivo NMR analyses carried out with a cryogenic probe optimised for (13)C detection were performed directly on the living algal tissues to evidence the labelling of the carbohydrates with neither preliminary extraction nor purification step(s). The isotopic enrichment of each compound was determined by high-resolution (1)H and (13)C NMR spectroscopy. These analyses evidenced different orientations of the flux of the photosynthetic carbon in the algae according to the salt stress. At normal and low salinities, the photosynthetic carbon flux was responsible of 70% and 67% of the floridoside synthetized during the pulse period, respectively, whereas it was only of 30% in the thalli exposed to the high salinity, meaning a biosynthesis of high floridoside amount from endogen source leading to the osmotic regulation. Under normal and hyper-osmotic conditions, the stock of floridoside was used for cellular needs during the chase period, whereas it was not under hypo-osmotic conditions. The characterization of isotopomers composition of floridoside and digeneaside and the analysis of adjacent (13)C-labelling gives much more details on the effects of salinity on the metabolic pathways leading to the synthesis or the degradation of these molecules. High turnover of floridoside was evidenced at normal salinity during the chase period and products issued from the degradation of floridoside would not be used for the novo biosynthesis. That suggests that synthesis and degradation of floridoside may be realized in two different cellular compartments. The presence of more numerous (13)C-(13)C blocks in the carbon skeleton of the molecules from the up salt stressed thalli than in those from no salt stressed algae, concomitant with a slower degree of isotopic enrichment of the molecule, provided evidence that the two metabolic pathways (endogen and photosynthetic) may not share the precursor molecules involved in the floridoside synthesis and that these two routes may be totally separate until the constitution of floridoside molecule.

Composition and distribution of carrabiose moieties in hybrid kappa-/iota-carrageenans using carrageenases.

Hybrid kappa/iota-carrageeans extracted from Gigartina skottsbergii, Chondracanthus chamissoï, and Chondrus crispus were incubated with Pseudoalteromonas carrageenovora kappa-carrageenase and Alteromonas fortis iota-carrageenase. The degradation products as well as the resistant fraction were fully characterized by chromatography, NMR, and mass spectrometry. The low percentage of degradation observed after treatment by the iota-carrageenase suggests that long segments of iota-carrabiose or block of iota-carrageenan are low in abundance. The degradation products of the kappa-carrageenase allow discriminating three modes of kappa-carrabiose distribution: blocks of kappa-carrabiose, kappa-carrabiose rich fraction containing iota-carrabiose units probably randomly distributed, and iota-carrabiose rich fraction which corresponds to the resistant fraction. The proportions of each fraction were related to the botanical origin as well as the place of growth of the seaweed.

Separation of floridoside and isofloridosides by HPLC and complete (1)H and (13)C NMR spectral assignments for D-isofloridoside.

Isofloridosides (1-O-alpha-D-galactopyranosylglycerol) and floridoside (2-O-alpha-D-galactopyranosylglycerol) were extracted from the red alga Porphyra umbilicalis (Linné) Kützing (Bangiales, Rhodophyta). Their separation was achieved by HPLC (NH(2) P50 column) after successive purification of the crude extract by ion-exchange chromatography and HPLC (Sugar-Pak TM1 column). 1D and 2D NMR spectroscopy experiments allowed to completely assign the (1)H and (13)C spectra of D-isofloridoside.