Hétérotrophie algale : effets de la gentamycine et de la cycloheximide sur les activités hétérotrophes et photosynthétiques des bacteries et des algues

Afin de tenter de déterminer les parts respectives des activités bactérienne et algale, nous avons testé d'une part, l'action d'une substance antibactérienne (la gentamycine), et d'autre part, l'action d'un inhibiteur métabolique des cellules eucaryotes (la cycloheximide) sur des cultures d'algues et de bactéries et sur des échantillons provenant du milieu naturel et soumis à des filtrations différentielles. Les effets de ces inhibiteurs ont été testés au niveau des activités hétérotrophe et photosynthétique globales, mais également au niveau de l'incorporation des marqueurs radioactifs lors de la synthèse des macromolécules et des composés de faible poids moléculaire.Les résultats obtenus laissent apparaître que l'inhibition de l'activité bactérienne par la gentamyclne est significative mais non complète (pourcentage d'inhibition moyen = 67 %). De plus, l'efficacité de la gentamycine augmente avec la durée d'incubation. Par ailleurs, les effets secondaires de la gentamycine sur les activités hétérotrophe et photosynthétique d'une culture de Melosira italica subsp. subarctica sont acceptables seulement pour des incubations de courte durée (< 4 heures).En revanche, l'emploi de la cycloheximide s'est révélé sans aucun effet significatif sur les activités photosynthétique et hérérotrophe de la culture de Melosira, même après 24 h d'incubation.A partir des échantillons prélevés en milieu naturel, l'emploi de la gentamycine a permis de réduire l'interférence bactérienne dans les mesures d'activité hétérotrophe algale. Enfin, nous avons pu constater que la gentamycine modifie l'allocation des marqueurs radioactifs dans les macromolécules.The ability of many planktonic algae to use particulate and/or dissolved organic carbon directly by phagotrophy or osmotrophy in laboratory cultures is well documented (DROOP, 1974; NEILSON and LEWIN, 1974; HELLEBUST and LEWIN, 1977; BIRD and KALFF, 1986). In axenic cultures, numerous microalgae grow in the dark with micromolar concentrations of diverse organic nutrients as their sole sources of carbon and energy (RIVKIN and PUTT, 1987). However, to demonstrate algal heterotrophy in the field, it is necessary to differentiate between bacterial and algal activities. In the course of this study, we tested the effect of an antibacterial substance (gentamycin) and of a metabolic inhibitor of eukaryotic cells (cycloheximide) on algal and bacterial cultures, and also on lake water samples submitted to differential filtration. The effect of these inhibitors was tested both at the overall heterotrophic and photosynthetic activities level and the level of the incorporation of radiolabeled tracers in macromolecules and low molecular weight compounds.Gentamycin was tested on bacteria and on an axenic culture of the diatom Melosira italica subsp. subarctica, the dominant species of the spring phyto-planktonic bloom of many temperate lakes. Bacterial culture was obtained by filtration of a senescent culture of Melosira through a 0.45 µm pore-size membrane. During exponential growth, gentamycin (40 µg.ml-1) was added to different flasks containing 100 ml of culture, 30 mn after gentamycin addition, Na H14CO3 (12µCi/100 ml) was introduced into the flasks. In each case, two replicates were incubated in the light and two in the dark for 0.5, 1, 2, 4, 6, 8, 12 and 24 hours. After incubation, aliquots were collected on a filtration membrane (0.45 µm) and the radioactivity was measured using an LKB liquid scintillation counter to estimate photosynthetic activity. Incorporation of inorganic carbon into macromolecules was measured using the procedure described by LANCELOT and MATHOT (1985), which allows the separation of lipids, polysaccharides, proteins and low molecular weight compounds (e.g. amino acids, organic acids and monosaccharides) by virtue of their relative solubilities in different extraction solvents :- lipids were extracted with a 2/1 (v/v) chloroform-methanol mixture;- low molecular weight compounds were extracted with hot ethanol;- proteins were precipated with TCA at elevated temperature; this also separated them from polysaccharides. Nucleic acids were recovered with the polysaccharides fraction. Results are given as a percentage of total radioactivity. The same method, as that described for photosynthetic incorporation, was used to measure incorporation of glucose 3H (19 nmoles.l-1) and for the allocation of radioactive marker in macromolecules. To assess the effect of cycloheximide, the same procedure as that described for gentamycin was used. However, due to the lack of references, three cycloheximide concentrations were tested = 50, 100 and 150 µg.ml-1, and aliquots were collected on a 0.2 µm pore-size membrane.Field samples were taken in Lake Pavin, an oligomesotrophic French lake, during the sedimentation of Melosira italica subsp. subarctica, the prevailing species of the spring bloom. The effects of gentamycin on photosynthetic and heterotrophic incorporations were tested with the came procedure as that described for cultures. However, radioactivity was measured for different size tractions : 0.2-0.45 µm, 0.45-5 µm and 5-160 µm, after incubations for 0.5, 1, 3, 6, 12 and 24 hours. Phytoplanktonic cells were counted on a Wild M40 inverted microscope and bacterial enumeration was realized in epifluorescence microscopy after staining with acridine orange (HOBBIE el al., 1977).Results show that gentamycin used at 40 µg.ml-1 inhibits significantly but not completely the activity of the bacterial culture (mean inhibition percent = 67 %). Gentamycin efficiency increases with incubation time, the inhibition reaching 81 % after 24 h. At the same time, the secondary effects of gentamycin on heterotrophic and photosynthetic activities of Melosira italica subsp. subarctica in culture were only tolerable with short incubation times (< 4 h), when the percents of inhibition were respectively 13.6 and 12.2%. On the other band, cycloheximide produced no significant effect on photosynthetic and heterotrophic activities of Melosira italica in culture, the percent of inhibition always remaining below 6.5 %.The use of gentamycin in natural samples reduced bacterial interference with algal heterotrophic activily measurements. The percent of inhibition caused by gentamycin was high (< 76 %) In the small-size fraction where the bacterial biomass predominate on the phytoplanktonic one.Lastly, in all samples, we could demonstrate that gentamycin modifies the allocation of inorganic carbon and radioactivity in macromolecules. The incorporation in proteins is significantly reduced essentially to benefit of the incorporation in low molecular weight compounds

Variations à court terme des compartiments planctoniques d'un lac humique du Bouclier canadien

Les variations spatio-temporelles à court terme des compartiments planctoniques ont été étudiées simultanément du 30 juillet au 5 août 1986 dans un lac humique du Bouclier canadien. L'abondance du bactérioplancton fluctue de 1,4 à 1,7.106 cell. ml.-1 (coloration DAPI) ou de 2,7 à 7,7.106 cell. ml-1; (coloration Acridine Orange). La production du bactérioplancton estimée par incorporation de 3H méthyl thimidine, varie de 4 à 24.106 cell. l-1 h-1. Les valeurs d'activité hétérotrophe potentielle bactérienne estimée par assimilation de 14C glucose, s'échelonnent de 0,007 à 0,065 µg C.l-1. h-1. La biomasse pigmentaire (chlore. a et pheopigments) varie de 6,8 à 21,7 mg.m-3 . La production primaire est très faible (max. : 10 mg C. m-3 .h-1; 20 mg C.m-2 .h-1 ) et décroît très rapidement avec la profondeur (25 % à 82 % dans le premier mètre). Le microzooplancton (Rotifères, Bosmina, nauplies) représente plus de 90 % du peuplement zooplanctonique et les taux de broutage global du macrozooplancton sont faibles (25 % j-1). Les compartiments hétérotrophes prédominent dans le métabolisme du lac par rapport au compartiment autotrophe. Les patrons de variation spatiale reflètent la stratification verticale des compartiments et des processus autotrophes et hétérotrophes. Les maxima de photosynthèse, d'ATP et de production bactérienne se situent dans les eaux épilimnétiques tandis que les maxima d'abondance du bactérioptancton et des pigments se rencontrent dans l'hypolimnion. Le zooplancton est plus dense et broute d'avantage dans la strate 1-3 m. Ces variations spatiales semblent influencer l'activité hétérotrophe potentielle du bactérioptancton. Nos résultats montrent aussi des variations temporelles à court terme de la production primaire, de l'assimilation hétérotrophe et du broutage du macrozooplancton. Cette étude préliminaire met en lumière la nécessité de tenir compte des variations à court terme lors des études des relations trophodynamiques dans les écosystèmes planctoniques.Short term spatial and temporal variations in planktonic compartments were studied simultaneously, from July 30th to August 5th, 1986, in a humic lake on the Canadian Shield. Abundance of bacterioplankton ranged from 1,4 to 1,7 106 cell. ml-1 (DAP1) or from 2,7 to 7,7 106 cell. ml-1 (Acridine Orange). Bacterial production, as measured by incorporation of 3H methyl-thimidine, was estimated at 4-24 106 cells. l-1. h-1 and potential heterotrophic bacterial activity ranged from 0,007 to 0,065 µg C.l-1. h-1, as estimated by 14C glucose incorporation. Pigments biomass (chloro. a and phaeopigments) varied from 6,8 to 21,7 mg m-3. Primary production was low (max. : 10 mg C.m-3. h-1; 20 mg C.m-2. h-1) and decreased rapidly with depth (25-82 % in 1 m depth). Microzooplankton (rotifera, Bosmina, nauplii) accounted for more than 90 % of total numbers and macrozooplankton global grazing rates were low (25 % d-1). The heterotrophic compartments play a greater rate in the lake metabolism than the autotrophic compartment. Spatial variations reflect the stratification of the heterotrophic and autotrophic organisms and processes with depth. Maximum levels in primary production, ATP and bacterial production occur in epilimnetic waters, while maxima in bacterial numbers and algal pigments occur in the hypolimnion. Zooplancton density and macrozooplankton grazing rates were higher in the 1-3 m strata. These spatial patterns seem to influence the vertical profiles of the bacterial potential heterotrophic activity. Our results also show short term temporal variations in primary production, potential heterotrophic activity of bacterioplankton and macrozooplankton grazing rates. This preliminary study stresses the importance of short term variations in the assessment of the trophodynamics of the planktonic food wed

Localization of a 64-kDa phosphoprotein in the lumen between the outer and inner envelopes of pea chloroplasts

The identification and localization of a marker protein for the intermembrane space between the outer and inner chloroplast envelopes is described. This 64-kDa protein is very rapidly labeled by [γ-32P]ATP at very low (30 nM) ATP concentrations and the phosphoryl group exhibits a high turnover rate. It was possible to establish the presence of the 64-kDa protein in this plastid compartment by using different chloroplast envelope separation and isolation techniques. In addition comparison of labeling kinetics by intact and hypotonically lysed pea chloroplasts support the localization of the 64-kDa protein in the intermembrane space. The 64-kDa protein was present and could be labeled in mixed envelope membranes isolated from hypotonically lysed plastids. Mixed envelope membranes incorporated high amounts of 32P from [γ-32P]ATP into the 64-kDa protein, whereas separated outer and inner envelope membranes did not show significant phosphorylation of this protein. Water/Triton X-114 phase partitioning demonstrated that the 64-kDa protein is a hydrophilic polypeptide. These findings suggest that the 64-kDa protein is a soluble protein trapped in the space between the inner and outer envelope membranes. After sonication of mixed envelope membranes, the 64-kDa protein was no longer present in the membrane fraction, but could be found in the supernatant after a 110000 × g centrifugation

Circulating adrenomedullin estimates survival and reversibility of organ failure in sepsis: the prospective observational multinational Adrenomedullin and Outcome in Sepsis and Septic Shock-1 (AdrenOSS-1) study

Background: Adrenomedullin (ADM) regulates vascular tone and endothelial permeability during sepsis. Levels of circulating biologically active ADM (bio-ADM) show an inverse relationship with blood pressure and a direct relationship with vasopressor requirement. In the present prospective observational multinational Adrenomedullin and Outcome in Sepsis and Septic Shock 1 (, AdrenOSS-1) study, we assessed relationships between circulating bio-ADM during the initial intensive care unit (ICU) stay and short-term outcome in order to eventually design a biomarker-guided randomized controlled trial. Methods: AdrenOSS-1 was a prospective observational multinational study. The primary outcome was 28-day mortality. Secondary outcomes included organ failure as defined by Sequential Organ Failure Assessment (SOFA) score, organ support with focus on vasopressor/inotropic use, and need for renal replacement therapy. AdrenOSS-1 included 583 patients admitted to the ICU with sepsis or septic shock. Results: Circulating bio-ADM levels were measured upon admission and at day 2. Median bio-ADM concentration upon admission was 80.5 pg/ml [IQR 41.5-148.1 pg/ml]. Initial SOFA score was 7 [IQR 5-10], and 28-day mortality was 22%. We found marked associations between bio-ADM upon admission and 28-day mortality (unadjusted standardized HR 2.3 [CI 1.9-2.9]; adjusted HR 1.6 [CI 1.1-2.5]) and between bio-ADM levels and SOFA score (p &lt; 0.0001). Need of vasopressor/inotrope, renal replacement therapy, and positive fluid balance were more prevalent in patients with a bio-ADM &gt; 70 pg/ml upon admission than in those with bio-ADM ≤ 70 pg/ml. In patients with bio-ADM &gt; 70 pg/ml upon admission, decrease in bio-ADM below 70 pg/ml at day 2 was associated with recovery of organ function at day 7 and better 28-day outcome (9.5% mortality). By contrast, persistently elevated bio-ADM at day 2 was associated with prolonged organ dysfunction and high 28-day mortality (38.1% mortality, HR 4.9, 95% CI 2.5-9.8). Conclusions: AdrenOSS-1 shows that early levels and rapid changes in bio-ADM estimate short-term outcome in sepsis and septic shock. These data are the backbone of the design of the biomarker-guided AdrenOSS-2 trial. Trial registration: ClinicalTrials.gov, NCT02393781. Registered on March 19, 2015

New engineering approach for the development and demonstration of a multi-purpose platform for the Blue Growth Economy

Aquaculture is currently the fastest growing food sector in the world and the open oceans are seen as one of the most likely areas for large scale expansion [1], [2], [3]. The global demand for seafood is continuing to rise sharply, driven by both population growth and increased per capita consumption, whilst wild capture fisheries are constrained in their potential to produce more seafood. A recently funded EC project, the Blue Growth Farm BGF (GA n. 774426, 1st June 2018 ÷ 30th September 2021) aims at contributing to this world need with an original solution. The Blue Growth Farm proposes an efficient, cost competitive and environmentally friendly multi purpose offshore farm concept based on a modular floating structure, moored to the seabed, meeting requirements of efficiency, cost-competitiveness and environmental friendless, where automated aquaculture and renewable energy production systems are integrated and engineered for profitable applications in the open sea. In the present paper, the overall engineering approach developed to carry out the research work is presented, described and justified. Different technical and scientific challenges are addressed through an integrated industrial engineering design approach, where all disciplines are tuned to achieve the Blue Growth Farm main targets, represented by: i) guaranteeing expected nominal fish production thanks to advanced automation and remote control capabilities; ii) minimizing the pollution introduced at marine ecosystem level when exploiting the marine natural resources, whilst increasing the social acceptance and users community agreement; iii) maximizing the electricity production in the Blue Growth Farm potential installation area ecosystem to provide energy supply to the on board electrical equipment and to dispatch the extra produced electric energy to the land network. Preliminary engineering design results are promising to demonstrate effective increase of safety and efficiency by reducing on board human effort and consequently risks at offshore, thus to make commercial scale open ocean farming a reality

Genome-wide methylation profiling and copy number analysis in atypical fibroxanthomas and pleomorphic dermal sarcomas indicate a similar molecular phenotype

Background: Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are lesions of the skin with overlapping histologic features and unspecific molecular traits. PDS behaves aggressive compared to AFX. Thus, a precise delineation, although challenging in some instances, is relevant. Methods: We examined the value of DNA-methylation profiling and copy number analysis for separating these tumors. DNA-methylation data were generated from 17 AFX and 15 PDS using the Illumina EPIC array. These were compared with DNA-methylation data generated from 196 tumors encompassing potential histologic mimics like cutaneous squamous carcinomas (cSCC; n = 19), basal cell carcinomas (n = 10), melanoma metastases originating from the skin (n = 11), leiomyosarcomas (n = 11), angiosarcomas of the skin and soft tissue (n = 11), malignant peripheral nerve sheath tumors (n = 19), dermatofibrosarcomas protuberans (n = 13), extraskeletal myxoid chondrosarcomas (n = 9), myxoid liposarcomas (n = 14), schwannomas (n = 10), neurofibromas (n = 21), alveolar (n = 19) and embryonal (n = 17) rhabdomyosarcomas as well as undifferentiated pleomorphic sarcomas (n = 12). Results: DNA-methylation profiling did not separate AFX from PDS. The DNA-methylation profiles of the other cases, however, were distinct from AFX/PDS. They reliably assigned to subtype-specific DNA-methylation clusters, although overlap occurred between some AFX/PDS and cSCC. Copy number profiling revealed alterations in a similar frequency and distribution between AFX and PDS. They involved losses of 9p (22/32) and 13q (25/32). Gains frequently involved 8q (8/32). Notably, a homozygous deletion of CDKN2A was more frequent in PDS (6/15) than in AFX (2/17), whereas amplifications were non-recurrent and overall rare (5/32). Conclusions: Our findings support the concept that AFX and PDS belong to a common tumor spectrum. We could demonstrate the diagnostic value of DNA-methylation profiling to delineating AFX/PDS from potential mimics. However, the assessment of certain histologic features remains crucial for separating PDS from AFX

Sarcoma classification by DNA methylation profiling

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications

Forest biodiversity, ecosystem functioning and the provision of ecosystem services

Forests are critical habitats for biodiversity and they are also essential for the provision of a wide range of ecosystem services that are important to human well-being. There is increasing evidence that biodiversity contributes to forest ecosystem functioning and the provision of ecosystem services. Here we provide a review of forest ecosystem services including biomass production, habitat provisioning services, pollination, seed dispersal, resistance to wind storms, fire regulation and mitigation, pest regulation of native and invading insects, carbon sequestration, and cultural ecosystem services, in relation to forest type, structure and diversity. We also consider relationships between forest biodiversity and multifunctionality, and trade-offs among ecosystem services. We compare the concepts of ecosystem processes, functions and services to clarify their definitions. Our review of published studies indicates a lack of empirical studies that establish quantitative and causal relationships between forest biodiversity and many important ecosystem services. The literature is highly skewed; studies on provisioning of nutrition and energy, and on cultural services, delivered by mixed-species forests are under-represented. Planted forests offer ample opportunity for optimising their composition and diversity because replanting after harvesting is a recurring process. Planting mixed-species forests should be given more consideration as they are likely to provide a wider range of ecosystem services within the forest and for adjacent land uses. This review also serves as the introduction to this special issue of Biodiversity and Conservation on various aspects of forest biodiversity and ecosystem services