Caractères biogéochimiques de la matière organique dans la colonne d'eau et les sédiments d'un écosystème saumâtre: l'étang de Thau - Variations saisonnières

Le long de la côte méditerranéenne française du Golfe du Lion, l'étang de Thau présente des caractères assez particuliers. Il est parfois soumis à des conditions anoxiques appelées "malaigues" qui résultent de l'accumulation de matières organiques durant la période chaude liée au développement des macrophytes. Ces dépôts organiques associés aux biomasses résultant des activités conchylicoles et aux apports extérieurs contribuent en cours d'année aux échanges biogéochimiques entre la colonne d'eau et les dépôts.Dans ce même milieu, l'analyse de la distribution et de la nature de la matière organique par des méthodes fines comme la chromatographie liquide haute performance ou la pyrolyse a permis de préciser son origine et son évolution dans la colonne d'eau et les dépôts. Durant les quatre saisons, les particularités de la matière organique ont donc été analysées en terme d'accumulation, de dégradation et de conservation. L'été constitue une période de production et de dégradation. L'automne est principalement caractérisé par des processus dégradatifs et des apports terrigènes (composés phénoliques). L'hiver correspond à une période de relative stabilité de la matière organique consécutive aux conditions froides. Le printemps enfin représente une période de reprise de l'activité biologique produisant une matière organique fraîche riche en sucres.Sous les tables conchylicoles on observe un accroissement de la matière organique dans la colonne d'eau et les dépôts. Mais les processus actifs de dégradation réduisent considérablement la quantité de matière organique déposée. Les résultats de ces mécanismes varient selon les stations sous table et hors table.Dans les dépôts les résultats de la dégradation dans la colonne d'eau amènent à une décroissance des composés biodégradables et à un accroissemenet des composés résistants comme les phénols et les hydrocarbures aromatiques. Ces processus de minéralisation s'accroissent vers la profondeur dans les dépôts au profit du pôle aromatique.Les relations entre les nutriments et la matière organique qui constitue à la fois leur source et leur puits se marquent bien sous les tables conchylicoles où les sels nutritifs s'accumulent en surface.The Thau lagoon along the French Mediterranean coast of the Gulf of Lions has unusual characteristics. It is sometimes subjected to anoxic conditions, known as "malaigues", which result from the accumulation of organic matter during the warmer period. Throughout the year this organic deposition, associated oyster farming and terrigenous inputs, contributes to biogeochemical exchanges between the water column and the underlying deposits. In this same environment, high-resolution analytical techniques (HPLC ; PY-GC-MS) were used to analyze the distribution and nature of the organic matter and to determine its origin and behaviour in the water column and sediments.Total suspended matter (TSM) was determined by filtration of water samples pumped up from different levels of the water column and filtered onto glass fiber filters (GF/F grade) previously heated at 450 °C for 4 hours. Particulate organic carbon (POC) was determined on the same samples with a Leco CS 125 analyzer after removal of inorganic carbonates by treatment with a H2 SO4 (2N) solution. Dissolved organic carbon (DOC) was determined on the filtrates using a Shimadzu TOC 5000 analyzer. The determination of polysaccharides in the TSM was achieved by a colorimetric method involving a H2 SO4 (3N) solution and anthrone reagent (Gallali 1972).Phenolic compounds were determined by high performance liquid chromatography (HPLC) after cupric oxide alkaline oxidation of TSM samples. The oxidized samples were acidified (HCl, 2N) and subjected to liquid-liquid extraction with ethyl acetate (Hartley & Buchan 1973; Hedges & Ertel 1982). The limit of detection is 10-4 g and the precision of the method is about 2% for each compound. Separation and quantification of phenolic monomers was carried out by HPLC (Hartley & Buchan, 1973 ; Serve et al., 1983). Of a total of 28 identified products, eleven represent the monomers constituting lignin and are taken into account according to Hedges & Parker (1976), Hedges & Mann (1979) and Hedges & Ertel (1982). The products of oxidative hydrolysis of lignin belong to the following three series : 4-hydroxybenzyl "H" (p-hydroxybenzoic acid, p-hydroxybenzaldehyde, p-hydroxyacetophenone), 3-methoxy-4-hydroxybenzylic "V" (Vanillyl) and 3,5-methoxy-4-hydroxybenzylic "S" (Syringyl). Each of these three series presents an alkyl side chain with 1, 2 or 3 carbon atoms. The compounds in C6-C1 can be acids or aldehydes, those in C6-C2 are ketones and those in C6-C3 are acids. The latter, having a phenylpropenic structure, belong to the Cinnamyl "C" series (ferulic acid, p-coumaric acid). Separation of phenols was carried out on a Merck analytical column (250 mm long x 4 mm in diameter) with a Lichrosorb reversed phase C18 stationary phase of 5 µm granulometry, equipped with a precolumn (40 mm long) containing the same phase. Elution was achieved with ternary eluents (water, acetonitrile, acetic acid), in a high pressure binary gradient (Charrière 1991). The eluted products were determined qualitatively, by comparison of their retention times with those of commercial products (detection in UV at 275 nm), after a co-injection if necessary, and quantitatively by an internal standard method (phloroglucinol : 1,3,5-benzenetriol and p-anisic acid : p-methoxybenzoic acid).Analysis of the major classes of organic compounds was carried out by coupled pyrolysis - gas chromatography - mass spectrometry. A CDS 1000 pyrolysis probe was directly fitted with a Perkin-Elmer 8700 gas chromatograph (GC) equipped with a TR-WAX capillary column (length: 30 m, diameter: 0.32 mm, phase thickness: 0.50 µm). Pyrolysis temperature was 700 °C for 10 s and the column temperature was programmed from 60°C to 240 °C at a rate of 6 °C/min according to Puigbo et al. (1989). Pyrolysis fragments were identified by coupling the GC to a HP 5989 mass spectrometer. Twenty three major peaks were selected on the pyrochromatograms and each selected compound was expressed as a percentage of the sum of the surface of these 23 peaks Pyrolysis products were grouped into five main families, each of them including similar molecules or closely related chemical structures: aromatic hydrocarbons, nitrogenous compounds, sugars, phenols and amino sugars.The survey of all these parameters showed some characteristic differences over the four seasons. Summer appears as a period when the biological production reaches maximum levels in the water column. At that time, organic matter is stratified with high levels of accumulation in the deeper layers. DOC is also abundant throughout the water column and organic compounds belonging to the class of sugars decrease according to depth. Autumn corresponds to Mediterranean storms and typical rainfalls. Terrestrial inputs increase in this season and degradative processes affect the organic matter that was produced in large quantities in the summer by the autotrophic organisms of the lagoon. DOC is recycled and reflects the degradation of autochthonous organic material. Winter, with reduced TSM levels related to low terrestrial inputs, is characterized by a homogenization of the water column and a weak biological activity. Lignin-derived phenols are abundant and correspond to a period of low biological activity. In contrast, in the spring the biological activity recovers, as indicated by the high sugar content of the DOC and by a homogenization of the water column.Under the oyster beds, an increase of organic matter is observed in the water column as well as in the sediments. However, the active degradation processes in summer and autumn reduce considerably the amount of the settling organic matter. The results of these processes are variable according to whether the stations are under or outside of the oyster beds. Degradation in the water column leads to a decrease of biodegradable compounds in the sediments and an increase in resistant compounds like phenols and aromatic hydrocarbons. These mineralization processes increase with depth in deposits, as reflected by higher proportions of aromatic compounds. The relationship between nutrients and organic matter, the latter constituting both their source and their sink, appears in sediments under oyster beds, where the inorganic nutrients accumulate at the surface

19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI)

We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with ?-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 ?M

Hydrodynamic gene delivery in human skin using a hollow microneedle device

Microneedle devices have been proposed as a minimally invasive delivery system for the intradermal administration of nucleic acids, both plasmid DNA (pDNA) and siRNA, to treat localised disease or provide vaccination. Different microneedle types and application methods have been investigated in the laboratory, but limited and irreproducible levels of gene expression have proven to be significant challenges to pre-clinical to clinical progression. This study is the first to explore the potential of a hollow microneedle device for the delivery and subsequent expression of pDNA in human skin. The regulatory approved MicronJet600® (MicronJet hereafter) device was used to deliver reporter plasmids (pCMVβ and pEGFP-N1) into viable excised human skin. Exogenous gene expression was subsequently detected at multiple locations that were distant from the injection site but within the confines of the bleb created by the intradermal bolus. The observed levels of gene expression in the tissue are at least comparable to that achieved by the most invasive microneedle application methods e.g. lateral application of a microneedle. Gene expression was predominantly located in the epidermis, although also evident in the papillary dermis. Optical coherence tomography permitted real time visualisation of the sub-surface skin architecture and, unlike a conventional intradermal injection, MicronJet administration of a 50 μL bolus appears to create multiple superficial microdisruptions in the papillary dermis and epidermis. These were co-localised with expression of the pCMVβ reporter plasmid. We have therefore shown, for the first time, that a hollow microneedle device can facilitate efficient and reproducible gene expression of exogenous naked pDNA in human skin using volumes that are considered to be standard for intradermal administration, and postulate a hydrodynamic effect as the mechanism of gene delivery

Elucidation of tonic and activated B-cell receptor signaling in Burkitt's lymphoma provides insights into regulation of cell survival.

Burkitt's lymphoma (BL) is a highly proliferative B-cell neoplasm and is treated with intensive chemotherapy that, because of its toxicity, is often not suitable for the elderly or for patients with endemic BL in developing countries. BL cell survival relies on signals transduced by B-cell antigen receptors (BCRs). However, tonic as well as activated BCR signaling networks and their relevance for targeted therapies in BL remain elusive. We have systematically characterized and compared tonic and activated BCR signaling in BL by quantitative phosphoproteomics to identify novel BCR effectors and potential drug targets. We identified and quantified ∼16,000 phospho-sites in BL cells. Among these sites, 909 were related to tonic BCR signaling, whereas 984 phospho-sites were regulated upon BCR engagement. The majority of the identified BCR signaling effectors have not been described in the context of B cells or lymphomas yet. Most of these newly identified BCR effectors are predicted to be involved in the regulation of kinases, transcription, and cytoskeleton dynamics. Although tonic and activated BCR signaling shared a considerable number of effector proteins, we identified distinct phosphorylation events in tonic BCR signaling. We investigated the functional relevance of some newly identified BCR effectors and show that ACTN4 and ARFGEF2, which have been described as regulators of membrane-trafficking and cytoskeleton-related processes, respectively, are crucial for BL cell survival. Thus, this study provides a comprehensive dataset for tonic and activated BCR signaling and identifies effector proteins that may be relevant for BL cell survival and thus may help to develop new BL treatments

高温環境下における筋労作時並びに安静時の発汗反応に及ぼす運動鍛錬の影響

博士（医学）神戸大

Class IA PI3Kinase Regulatory Subunit, p85α, Mediates Mast Cell Development through Regulation of Growth and Survival Related Genes

Stem cell factor (SCF) mediated KIT receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from KIT are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI3Kinase (PI3K) including p85α, p85β, p50α, and p55α. While it is known that PI3K plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including growth, survival and cycling are not known. We show that loss of p85α impairs the growth, survival and cycling of mast cell progenitors (MCp). To delineate the molecular mechanism (s) by which p85α regulates mast cell growth, survival and cycling, we performed microarray analyses to compare the gene expression profile of MCps derived from WT and p85α-deficient mice in response to SCF stimulation. We identified 151 unique genes exhibiting altered expression in p85α-deficient cells in response to SCF stimulation compared to WT cells. Functional categorization based on DAVID bioinformatics tool and Ingenuity Pathway Analysis (IPA) software relates the altered genes due to lack of p85α to transcription, cell cycle, cell survival, cell adhesion, cell differentiation, and signal transduction. Our results suggest that p85α is involved in mast cell development through regulation of expression of growth, survival and cell cycle related genes

Integrated In Silico Analysis of Pathway Designs for Synthetic Photo-Electro-Autotrophy

The strong advances in synthetic biology enable the engineering of novel functions and complex biological features in unprecedented ways, such as implementing synthetic autotrophic metabolism into heterotrophic hosts. A key challenge for the sustainable production of fuels and chemicals entails the engineering of synthetic autotrophic organisms that can effectively and efficiently fix carbon dioxide by using sustainable energy sources. This challenge involves the integration of carbon fixation and energy uptake systems. A variety of carbon fixation pathways and several types of photosystems and other energy uptake systems can be chosen and, potentially, modularly combined to design synthetic autotrophic metabolism. Prior to implementation, these designs can be evaluated by the combination of several computational pathway analysis techniques. Here we present a systematic, integrated in silico analysis of photo-electro-autotrophic pathway designs, consisting of natural and synthetic carbon fixation pathways, a proton-pumping rhodopsin photosystem for ATP regeneration and an electron uptake pathway. We integrated Flux Balance Analysis of the heterotrophic chassis Escherichia coli with kinetic pathway analysis and thermodynamic pathway analysis (Max-min Driving Force). The photo-electro-autotrophic designs are predicted to have a limited potential for anaerobic, autotrophic growth of E. coli, given the relatively low ATP regenerating capacity of the proton pumping rhodopsin photosystems and the high ATP maintenance of E. coli. If these factors can be tackled, our analysis indicates the highest growth potential for the natural reductive tricarboxylic acid cycle and the synthetic pyruvate synthase-pyruvate carboxylate - glyoxylate bicycle. Both carbon fixation cycles are very ATP efficient, while maintaining fast kinetics, which also results in relatively low estimated protein costs for these pathways. Furthermore, the synthetic bicycles are highly thermodynamic favorable under conditions analysed. However, the most important challenge identified for improving photo-electro-autotrophic growth is increasing the proton-pumping rate of the rhodopsin photosystems, allowing for higher ATP regeneration. Alternatively, other designs of autotrophy may be considered, therefore the herein presented integrated modeling approach allows synthetic biologists to evaluate and compare complex pathway designs before experimental implementation.Peer reviewe

Feasibility of Azacitidine Added to Standard Chemotherapy in Older Patients with Acute Myeloid Leukemia — A Randomised SAL Pilot Study

Introduction: Older patients with acute myeloid leukemia (AML) experience short survival despite intensive chemotherapy. Azacitidine has promising activity in patients with low proliferating AML. The aim of this dose-finding part of this trial was to evaluate feasibility and safety of azacitidine combined with a cytarabine- and daunorubicin-based chemotherapy in older patients with AML. Trial Design: Prospective, randomised, open, phase II trial with parallel group design and fixed sample size. Patients and Methods: Patients aged 61 years or older, with untreated acute myeloid leukemia with a leukocyte count of ,20,000/ml at the time of study entry and adequate organ function were eligible. Patients were randomised to receive azacitidine either 37.5 (dose level 1) or 75 mg/sqm (dose level 2) for five days before each cycle of induction (7+3 cytarabine plus daunorubicine) and consolidation (intermediate-dose cytarabine) therapy. Dose-limiting toxicity was the primary endpoint. Results: Six patients each were randomised into each dose level and evaluable for analysis. No dose-limiting toxicity occurred in either dose level. Nine serious adverse events occurred in five patients (three in the 37.5 mg, two in the 75 mg arm) with two fatal outcomes. Two patients at the 37.5 mg/sqm dose level and four patients at the 75 mg/sqm level achieved a complete remission after induction therapy. Median overall survival was 266 days and median event-free survival 215 days after a median follow up of 616 days. Conclusions: The combination of azacitidine 75 mg/sqm with standard induction therapy is feasible in older patients with AML and was selected as an investigational arm in the randomised controlled part of this phase-II study, which is currently halted due to an increased cardiac toxicity observed in the experimental arm. Trial Registration: This trial is registered at clinical trials.gov (identifier: NCT00915252)

Reflections on integrating bioinformatics into the undergraduate curriculum:The Lancaster experience

Bioinformatics is an essential discipline for biologists. It also has a reputation of being difficult for those without a strong quantitative and computer science background. At Lancaster University, we have developed modules for the integration of bioinformatics skills training into our undergraduate biology degree portfolio. This article describes those modules, situating them in the context of the accumulated quarter century of literature on bioinformatics education. The constant evolution of bioinformatics as a discipline is emphasized, drawing attention to the continual necessity to revise and upgrade those skills being taught, even at undergraduate level. Our overarching aim is to equip students both with a portfolio of skills in the currently most essential bioinformatics tools and with the confidence to continue their own bioinformatics skills development at postgraduate or professional level