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

Long non-coding RNAs defining major subtypes of B cell precursor acute lymphoblastic leukemia

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a novel class of RNA due to its diverse mechanism in cancer development and progression. However, the role and expression pattern of lncRNAs in molecular subtypes of B cell acute lymphoblastic leukemia (BCP-ALL) have not yet been investigated. Here, we assess to what extent lncRNA expression and DNA methylation is driving the progression of relapsed BCP-ALL subtypes and we determine if the expression and DNA methylation profile of lncRNAs correlates with established BCP-ALL subtypes. METHODS: We performed RNA sequencing and DNA methylation (Illumina Infinium microarray) of 40 diagnosis and 42 relapse samples from 45 BCP-ALL patients in a German cohort and quantified lncRNA expression. Unsupervised clustering was applied to ascertain and confirm that the lncRNA-based classification of the BCP-ALL molecular subtypes is present in both our cohort and an independent validation cohort of 47 patients. A differential expression and differential methylation analysis was applied to determine the subtype-specific, relapse-specific, and differentially methylated lncRNAs. Potential functions of subtype-specific lncRNAs were determined by using co-expression-based analysis on nearby (cis) and distally (trans) located protein-coding genes. RESULTS: Using an integrative Bioinformatics analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific and 942 relapse-specific lncRNAs and the methylation profile of three subtypes of BCP-ALL. The 1235 subtype-specific lncRNA signature represented a similar classification of the molecular subtypes of BCP-ALL in the independent validation cohort. We identified a strong correlation between the DUX4-specific lncRNAs and genes involved in the activation of TGF-β and Hippo signaling pathways. Similarly, Ph-like-specific lncRNAs were correlated with genes involved in the activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we found 23 promoter methylated lncRNAs epigenetically facilitating their expression levels. CONCLUSION: Here, we describe a set of subtype-specific and relapse-specific lncRNAs from three major BCP-ALL subtypes and define their potential functions and epigenetic regulation. The subtype-specific lncRNAs are reproducible and can effectively stratify BCP-ALL subtypes. Our data uncover the diverse mechanism of action of lncRNAs in BCP-ALL subtypes defining which lncRNAs are involved in the pathogenesis of disease and are relevant for the stratification of BCP-ALL subtypes

Long non-coding RNAs defining major subtypes of B cell precursor acute lymphoblastic leukemia

Recent studies implicated that long non-coding RNAs (lncRNAs) may play a role in the progression and development of acute lymphoblastic leukemia, however, this role is not yet clear. In order to unravel the role of lncRNAs associated with B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) subtypes, we performed transcriptome sequencing and DNA methylation array across 82 BCP-ALL samples from three molecular subtypes (DUX4, Ph-like, and Near Haploid or High Hyperdiploidy). Unsupervised clustering of BCP-ALL samples on the basis of their lncRNAs on transcriptome and DNA methylation profiles revealed robust clusters separating three molecular subtypes. Using extensive computational analysis, we developed a comprehensive catalog of 1235 aberrantly dysregulated BCP-ALL subtype-specific lncRNAs with altered expression and methylation patterns from three subtypes of BCP-ALL. By analyzing the co-expression of subtype-specific lncRNAs and protein-coding genes, we inferred key molecular processes in BCP-ALL subtypes. A strong correlation was identified between the DUX4 specific lncRNAs and activation of TGF-β and Hippo signaling pathways. Similarly, Ph-like specific lncRNAs were correlated with genes involved in activation of PI3K-AKT, mTOR, and JAK-STAT signaling pathways. Interestingly, the relapse-specific differentially expressed lncRNAs correlated with the activation of metabolic and signaling pathways. Finally, we showed a set of epigenetically altered lncRNAs facilitating the expression of tumor genes located at their cis location. Overall, our study provides a comprehensive set of novel subtype and relapse-specific lncRNAs in BCP-ALL. Our findings suggest a wide range of molecular pathways are associated with lncRNAs in BCP-ALL subtypes and provide a foundation for functional investigations that could lead to new therapeutic approaches

C-KIT Signaling Depends on Microphthalmia-Associated Transcription Factor for Effects on Cell Proliferation

The development of melanocytes is regulated by the tyrosine kinase receptor c-KIT and the basic-helix-loop-helix-leucine zipper transcription factor Mitf. These essential melanocyte survival regulators are also well known oncogenic factors in malignant melanoma. Despite their importance, not much is known about the regulatory mechanisms and signaling pathways involved. In this study, we therefore sought to identify the signaling pathways and mechanisms involved in c-KIT mediated regulation of Mitf. We report that c-KIT stimulation leads to the activation of Mitf specifically through the c-KIT phosphorylation sites Y721 (PI3 kinase binding site), Y568 and Y570 (Src binding site). Our study not only confirms the involvement of Ras-Erk signaling pathway in the activation of Mitf, but also establishes that Src kinase binding to Y568 and Y570 of c-KIT is required. Using specific inhibitors we observe and verify that c-KIT induced activation of Mitf is dependent on PI3-, Akt-, Src-, p38- or Mek kinases. Moreover, the proliferative effect of c-KIT is dependent on Mitf in HEK293T cells. In contrast, c-KIT Y568F and Y721F mutants are less effective in driving cell proliferation, compared to wild type c-KIT. Our results reveal novel mechanisms by which c-KIT signaling regulates Mitf, with implications for understanding both melanocyte development and melanoma

Hoxa9 and Meis1 Cooperatively Induce Addiction to Syk Signaling by Suppressing miR-146a in Acute Myeloid Leukemia

The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia..O. and T. Berg (BE 4198/1-1 and BE 4198/2-1) are supported by the Deutsche Forschungsgemeinschaft (DFG). K.S. is supported by a Leukemia and Lymphoma Society Scholar Award and by the National Cancer Institute (R01 CA140292). F.C. is supported by an EMBO long-term fellowship (1305-2015 and Marie Curie ActionsLTFCOFUND2013/GA-2013-609409). F.K. was supported by grants from Deutsche Krebshilfe (grant 109420; Max-Eder program), fellowship 2010/04 by the European Hematology Association, and by the DFG (SFB 1074, project A5). A.R. was supported by the DFG (SFB 1074, project A5) and the gender equality program by the DFG (SFB 1074, project Z2), a fellowship from the Canadian Institutes of Health Research, and the Baustein Startförderung Program of the Medical Faculty, Ulm University. Work in the Department of Haematology in Cambridge is supported by Bloodwise (grant ref. 13003), the Wellcome Trust (grant ref. 104710/Z/14/Z), the Medical Research Council (MC_PC_12009), the Kay Kendall Leukemia Fund (KKL952), the Cambridge NIHR Biomedical Research Center (NF-BR-0412-10321), the Cambridge Experimental Cancer Medicine Centre itself receives funding from NIHR (NF-EC-0412-10442), the Leukemia and Lymphoma Society of America (grant ref. 07037), and core support grants from the Wellcome Trust (100140/Z/12/Z and 097922/Z/11/Z) and MRC (MC_PC_12009)

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph?+?ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10−3 and 36/67 (53%) and 53/67 (79%) at 10−4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL

Characteristics and outcome of patients with low-/intermediate-risk acute promyelocytic leukemia treated with arsenic trioxide - an international collaborative study

The aim of this study was to characterize a large series of 154 patients with acute promyelocytic leukemia (APL; median age, 53 years; range, 18-90 years) and evaluate real-life outcome after up-front treatment with arsenic trioxide (ATO) and alltrans retinoic acid (ATRA). All patients were included in the prospective NAPOLEON registry (NCT02192619) between 2013 and 2019. APL was de novo in 91% (n=140) and therapy-related in 9% (n=14); 13% (n=20) were older than 70 years. At diagnosis bleeding/hemorrhage was present in 38% and thrombosis in 3%. Complete remission was achieved in 152 patients (99%), whereas two patients (1%) experienced induction death within 18 days after start of therapy. With a median follow-up of 1.99 years (95%-CI, 1.61-2.30 years) 1-year and 2-years overall survival (OS) rates were 97% (95%-CI, 94-100%) and 95% (95%-CI, 91-99%), respectively. Age above 70 years was associated with a significantly shorter OS (P<0.001) as compared to younger patients. So far no relapses were observed. Six patients (4%) died in CR after in median 0.95 years after diagnosis (range, 0.18-2.38 years). Our data confirm the efficiency and durability of ATO/ATRA in the primary management of adult low-/ intermediate-risk APL patients in the real life setting, irrespective of age