Production and characterization of a trehalolipid biosurfactant produced by the novel marine bacteriumRhodococcussp., strain PML026

AIMS: The aim of this study was to evaluate biosurfactant production by a novel marine Rhodococcus sp., strain PML026 and characterize the chemical nature and properties of the biosurfactant. METHODS AND RESULTS: A novel marine bacterium (Rhodococcus species; strain PML026) was shown to produce biosurfactant in the presence of hydrophobic substrate (sunflower oil). Biosurfactant production (identified as a trehalolipid) was monitored in whole-batch cultures (oil layer and aqueous phase), aqueous phase (no oil layer) and filtered (0·2mum) aqueous phase (no oil or cells; extracellular) and was shown to be closely associated with growth/biomass production. Extracellular trehalolipid levels increased postonset of stationary growth phase. Purified trehalolipid was able to reduce the surface tension of water to 29mN m(-1) at Critical Micellar Concentration (CMC) of c. 250mgl(-1) and produced emulsions that were stable to a wide range of conditions (pH 2-10, temperatures of 20-100°C and NaCl concentrations of 5-25% w/v). Separate chemical analyses of the intact trehalolipid and its constituents demonstrated the compound was in fact a mixture of homologues (>1180MW) consisting of a trehalose moiety esterified to a series of straight chain and hydroxylated fatty acids. CONCLUSIONS: The trehalolipid biosurfactant produced by the novel marine strain Rhodococcus sp. PML026 was characterized and exhibited high surfactant activity under a wide range of conditions. SIGNIFICANCE AND IMPACT OF STUDY: Strain PML026 of Rhodococcus sp. is a potential candidate for bioremediation or biosurfactant production for various applications.

Feeding and overwintering of Antarctic krill across its major habitats: The role of sea ice cover, water depth, and phytoplankton abundance

Antarctic krill (Euphausia superba) were sampled in contrasting habitats: a seasonally ice-covered deep ocean (Lazarev Sea), ice-free shelves at their northern range (South Georgia) and the Antarctic Peninsula (Bransfield Strait), and shelf and oceanic sites in the Scotia Sea. Across 92 stations, representing a year-round average, the food volume in krill stomachs comprised 71 +/- 29% algae, 17 +/- 21% protozoans, and 12 +/- 25% metazoans. Fatty acid trophic markers showed that copepods were consistently part of krill diet, not a switch food. In open waters, both diatom and copepod consumption increased with phytoplankton abundance. Under sea ice, ingestion of diatoms became rare, whereas feeding on copepods remained constant. During winter, larvae contained high but variable proportions of diatom markers, whereas in postlarvae the role of copepods increased with krill body length. Overwintering differed according to habitat. Krill from South Georgia had lower lipid stores than those from the Bransfield Strait or Lazarev Sea. Feeding effort was much reduced in Lazarev Sea krill, whereas most individuals from the Bransfield Strait and South Georgia contained phytoplankton and seabed detritus in their stomachs. Their retention of essential body reserves indicates that krill experienced most winter hardship in the Lazarev Sea, followed by South Georgia and then Bransfield Strait. This was reflected in the delayed development from juveniles to adults in the Lazarev Sea. Circumpolar comparisons of length frequencies suggest that krill growth conditions are more favorable in the southwest Atlantic than in the Lazarev Sea or off East Antarctica because of longer phytoplankton bloom periods and rewarding access to benthic food.

Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m-2s-1) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry of bacteriochlorophylls from Chlorobiaceae: characteristic fragmentations

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry/mass spectrometry (APCI-LC/MS/MS) has been applied to the study of bacteriochlorophylls c, d, and e of phototrophic prokaryotes. Cultures of Chlorobiaceae containing bacteriochlorophyll c, d or e were examined using a high-resolution high-performance liquid chromatography (HPLC) method and APCI-LC/MS/MS employing post-column addition of formic acid. The results reveal complex distributions of bacteriochlorophyll homologues, with some closely eluting species giving isobaric protonated molecules. On-line LC/MS/MS studies reveal characteristic fragment ions for bacteriochlorophylls c, d, and e. Fragmentations involving loss of the extended alkyl substituents that are unique to bacteriochlorophylls c, d and e and their derivatives have been rationalised by studying the phaeophorbides and the results applied to the direct study of the bacteriochlorophylls.

Identification of the bacteriochlorophyll homologues of Chlorobium phaeobacteroides strain UdG6053 grown at low light intensity Identification of the bacteriochlorophyll homologues of Chlorobium phaeobacteroides strain UdG6053 grown at low light intensity

Detailed APCI LC-MS/MS analysis using an improved HPLC separation reveals the green sulphur bacterium Chlorobium phaeobacteroides strain UdG6053 to contain a wider range of distinct bacteriochlorophyll homologues than has been previously recognised in Chlorobiaceae. The diversity in the homologue distribution is confirmed as arising from differences in the extent of alkylation of the macrocycle and variation in the nature of the esterifying alcohol and a novel series of bacteriochlorophyll structures has been recognised. Homologues containing esterifying alcohols other than farnesol, a number of which have not previously been reported in Chlorobiaceae, are present in high relative abundance. Confirmation of the structures of the esterifying alcohols has been obtained by hydrolysis and analysis by GC-MS.

A method for semi-automated objective quantification of linear bedforms from multi-scale Digital Elevation Models

The increasing availability of large, detailed digital representations of the Earth’s surface demands the application of objective and quantitative analyses. Given recent advances in the understanding of the mechanisms of formation of linear bedform features from a range of environments, objective measurement of their wavelength, orientation, crest and trough positions, height and asymmetry is highly desirable. These parameters are also of use when determining observation-based parameters for use in many applications such as numerical modelling, surface classification and sediment transport pathway analysis. Here, we (i) adapt and extend extant techniques to provide a suite of semi-automatic tools which calculate crest orientation, wavelength, height, asymmetry direction and asymmetry ratios of bedforms, and then (ii) undertake sensitivity tests on synthetic data, increasingly complex seabeds and a very large-scale (39 000km2) aeolian dune system. The automated results are compared with traditional, manually derived,measurements at each stage. This new approach successfully analyses different types of topographic data (from aeolian and marine environments) from a range of sources, with tens of millions of data points being processed in a semi-automated and objective manner within minutes rather than hours or days. The results from these analyses show there is significant variability in all measurable parameters in what might otherwise be considered uniform bedform fields. For example, the dunes of the Rub’ al Khali on the Arabian peninsula are shown to exhibit deviations in dimensions from global trends. Morphological and dune asymmetry analysis of the Rub’ al Khali suggests parts of the sand sea may be adjusting to a changed wind regime from that during their formation 100 to 10 ka BP.

Identidad territorial y paisaje. Evolución morfológica de los núcleos en Castilla y León.

Este estudio intenta esclarecer las transformaciones físicas y socioeconómicas de los asentamientos rurales de la región española de Castilla y León, durante la segunda mitad del siglo XX. Se analiza la evolución temporal de la forma urbana a través de un Sistema de Información Geográfico (SIG), calculando unos índices métricos y comparándolos con la información demográfica histórica. Los resultados pretenden mostrar los efectos de la especialización funcional económica, causada por la integración en las jerarquías productivas globales, sobre la estructura urbana. La pérdida gradual de las características tradicionales de los pueblos castellanos, como la compacidad y la integración en el entorno, debido a la pérdida o degradación de la arquitectura popular y la construcción de nuevas edificaciones industriales, supone un riesgo para las futuras políticas de desarrollo local. Se considera necesario preservar la identidad paisajística y evitar la destrucción del patrimonio cultural para poder revitalizar estos territorios.

Variaciones termométricas en la planta del pie y piernas valorada en corredores antes y después de correr 30 km

La termometría es una técnica no invasiva que permite cuantificar los cambios en la temperatura cutánea y evaluarla de forma cuantitativa. El aumento significativo de la temperatura puede indicar la existencia de patología. Se ha demostrado que la actividad muscular induce procesos de transferencia de calor entre los músculos y las capas superficiales de tejido. En este estudio queremos cuantificar los cambios de temperatura que se producen en los músculos del pie y miembro inferior tras una carrera de 30 km, para ello hemos utilizado una cámara termográfica de alta resolución. Contamos con la colaboración voluntaria de 32 sujetos sanos a los que procedimos a tomar fotografías de la planta del pie, parte anterior de la pierna, parte posterior de la pierna, parte anterior del muslo y parte posterior del muslo en dos etapas, primero antes de la carrera y segunda toma después de la carrera de 30 km, de esta manera pudimos valorar si había o no variación de temperatura en las zonas seleccionadas. Tras el análisis de los datos obtenidos encontramos significativas variaciones térmicas en Talón, cabeza primer metatarsiano, cabeza segundo metatarsiano, cabeza tercer metatarsiano, cabeza cuarto metatarsiano, cabeza quinto metatarsiano, apófisis estiloides quinto metatarsiano, arco longitudinal interno, maléolo interno, maléolo externo, peroneo lateral largo, vasto interno, vasto externo, recto femoral, tensor de la fascia lata, inserción cuádriceps, gemelo interno, tendón de Aquiles y Biceps femoral.

English Trade Mark Law in the Eighteenth Century: Blanchard v Hill Revisited - Another 'Case of Monopolies'?

The decision of Lord Hardwicke LC in Blanchard v Hill in 1742 is the earliest reported case on the equitable jurisdiction to grant injunctive relief against trade mark piracy. The ambiguous manner in which the case was reported led to the decision being interpreted as either the basis of equitable jurisdiction or a denial of jurisdiction. This article seeks to establish the background to the case, what actually happened, and the immediate impact of the decision. The scene is set, however, in a parallel symbolic universe – heraldry – because in 1740, the officers of arms were confronted with a trade mark case.

Identification of an unknown b-agonist in feed by liquid chromatography/bioassay/quadrupole time-of-flight tandem mass spectrometry with accurate mass measurement.

A new approach to the search for residues of unknown growth promoting agents such as anabolic steroids and -agonists in feed is presented. Following primary extraction and clean-up, samples are separated using gradient liquid chromatography (LC). The effluent is split towards two identical 96-well fraction collectors and an optional electrospray quadrupole time-of-flight mass spectrometry (QTOFMS) system for accurate mass measurement. One 96-well plate is used for a bioassay (enzyme-immuno assay, receptor assay) and will detect the bioactivity and position of the relevant peak in the chromatogram. The positive well in the second 96-well plate is used for identification by LC/QTOFMS/MS. The value of this LC/bioassay/QTOFMS/MS methodology is highlighted by the finding and structure elucidation of a new -agonist in a feed extract.

Dynamics and Function of Langerhans Cells In Vivo: Dermal Dendritic Cells Colonize Lymph Node Areas Distinct from Slower Migrating Langerhans Cells

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.

Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.

Identification of mouse langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues

Human (h)Langerin/CD207 is a C-type lectin of Langerhans cells (LC) that induces the formation of Birbeck granules (BG). In this study, we have cloned a cDNA-encoding mouse (m)Langerin. The predicted protein is 66% homologous to hLangerin with conservation of its particular features. The organization of human and mouse Langerin genes are similar, consisting of six exons, three of which encode the carbohydrate recognition domain. The mLangerin gene maps to chromosome 6D, syntenic to the human gene on chromosome 2p13. mLangerin protein, detected by a mAb as a 48-kDa species, is abundant in epidermal LC in situ and is down-regulated upon culture. A subset of cells also expresses mLangerin in bone marrow cultures supplemented with TGF-beta. Notably, dendritic cells in thymic medulla are mLangerin-positive. By contrast, only scattered cells express mLangerin in lymph nodes and spleen. mLangerin mRNA is also detected in some nonlymphoid tissues (e.g., lung, liver, and heart). Similarly to hLangerin, a network of BG form upon transfection of mLangerin cDNA into fibroblasts. Interestingly, substitution of a conserved residue (Phe(244) to Leu) within the carbohydrate recognition domain transforms the BG in transfectant cells into structures resembling cored tubules, previously described in mouse LC. Our findings should facilitate further characterization of mouse LC, and provide insight into a plasticity of dendritic cell organelles which may have important functional consequences.

Dermatoxin and phylloxin from the waxy monkey frog, Phyllomedusa sauvagei: cloning of precursor cDNAs and structural characterization from lyophilized skin secretion

Amphibian skin is a morphologically, biochemically and physiologically complex organ that performs the wide range of functions necessary for amphibian survival. Here we describe the primary structures of representatives of two novel classes of amphibian skin antimicrobials, dermatoxin and phylloxin, from the skin secretion of Phyllomedusa sauvagei, deduced from their respective precursor encoding cDNAs cloned from a lyophilized skin secretion library. A degenerate primer, designed to a highly conserved domain in the 5'-untranslated region of analogous peptide precursor cDNAs from Phyllomedusa bicolor, was employed in a 3'-RACE reaction. Peptides with molecular masses coincident with precursor-deduced mature toxin peptides were identified in LC/MS fractions of skin secretion and primary structures were confirmed by MS/MS fragmentation. This integrated experimental approach can thus rapidly expedite the primary structural characterization of amphibian skin peptides in a manner that circumvents specimen sacrifice whilst preserving robustness of scientific data.