Search

Abstract Embedding of cell-surface receptors into a membrane defines their dynamics but also complicates experimental characterization of their signaling complexes. The hepatocyte growth factor receptor MET is a receptor tyrosine kinase involved in cellular processes such as proliferation, migration, and survival. It is also targeted by the pathogen Listeria monocytogenes, whose invasion protein, internalin B (InlB), binds to MET, forming a signaling dimer that triggers pathogen internalization. Here we use an integrative structural biology approach, combining molecular dynamics simulations and single-molecule Förster resonance energy transfer (smFRET) in cells, to investigate the early stages of MET activation. Our simulations show that InlB binding stabilizes MET in a conformation that promotes dimer formation. smFRET reveals that the in situ dimer structure closely resembles one of two previously published crystal structures, though with key differences. This study refines our understanding of MET activation and provides a methodological framework for studying other plasma membrane receptors.

The Wadden Sea is a coastal system along the fringe of the land–sea borders of Denmark, Germany and the Netherlands. The Wadden Sea is extremely productive and influenced by strong variations in physical and biological forcing factors that act on timescales of hours to seasons. Productive coastal seas are known to dominate the ocean's methane emission to the atmosphere, but knowledge of controls and temporal variations in methane dynamics in these vastly dynamic systems is scarce. Here we address this knowledge gap by measuring methane inventories and methanotrophic activity at a temporal resolution of 1 h over a period of 2 d, repeatedly during four successive seasons in the central Dutch Wadden Sea. We found that methane dynamics varied between colder and warmer seasons, with generally higher water column methane concentrations and methanotrophic activity in the warmer seasons. The efflux of methane to the atmosphere was, on the other hand, lower in the warmer seasons because of lower wind speeds. On a diel scale, tides controlled methanotrophic activity, which increased ∼40 % at low tide compared to high tide. We estimate that methane oxidizing bacteria reduce the methane budget of the Dutch Wadden Sea by only 2 %, while ∼1/3 escapes to the atmosphere and ∼2/3 are flushed out into the open North Sea at ebb tide. Our findings indicate that tides play a key role in controlling methane dynamics and methanotrophic activity and highlight the importance of high-resolution and repeated sampling strategies to resolve methane dynamics in fast-changing coastal systems.

Recruitment of substrates to virulent bacterial type III secretion systems remains enigmatic. Here, a crystal structure reveals two binding sites between a secretion substrate in complex with its chaperone and the cytosolic domain of the export gate.

Hydrothermal vents modify and displace subsurface dissolved organic matter (DOM) into the ocean. Once in the ocean, this DOM is transported together with elements, particles, dissolved gases and biomass along with the neutrally buoyant plume layer. Considering the number and extent of actively venting hydrothermal sites in the oceans, their contribution to the oceanic DOM pool may be substantial. Here, we investigate the dynamics of DOM in relation to hydrothermal venting and related processes at the as yet unexplored Aurora hydrothermal vent field within the ultraslow-spreading Gakkel Ridge in the Arctic Ocean at 82.9∘ N. We examined the vertical distribution of DOM composition from sea ice to deep waters at six hydrocast stations distal to the active vent and its neutrally buoyant plume layer. In comparison to background seawater, we found that the DOM in waters directly affected by the hydrothermal plume was molecularly less diverse and 5 %–10 % lower in number of molecular formulas associated with the molecular categories related to lipid and protein-like compounds. On the other hand, samples that were not directly affected by the plume were chemically more diverse and had a higher percentage of chemical formulas associated with the carbohydrate-like category. Our results suggest that hydrothermal processes at Aurora may influence the DOM distribution in the bathypelagic ocean by spreading more thermally and/or chemically induced compositions, while DOM compositions in epipelagic and mesopelagic layers are mainly governed by the microbial carbon pump dynamics and surface-ocean–sea-ice interactions.

Northern latitude peatlands act as important carbon sources and sinks, but little is known about the greenhouse gas (GHG) budgets of peatlands that were submerged beneath the North Sea during the last glacial–interglacial transition. We found that whilst peat formation was diachronous, commencing between 13 680 and 8360 calibrated years before the present, stratigraphic layering and local vegetation succession were consistent across a large study area. Large carbon stores were measured. In situ methane (CH4) concentrations of sediment pore waters were widespread but low at most sites, with the exception of two locations. Incubation experiments in the laboratory revealed molecular signatures of methanogenic archaea, with strong increases in rates of activity upon methylated substrate amendment. Remarkably, methanotrophic activity and the respective diagnostic molecular signatures could not be detected. Heterotrophic Bathyarchaeota dominated the archaeal communities, and bacterial populations were dominated by candidate phylum JS1 bacteria. In the absence of active methanogenic microorganisms, we conclude that these sediment harbour low concentrations of widespread millennia-old CH4. The presence of large widespread stores of carbon and in situ methanogenic microorganisms, in the absence of methanotrophic microorganisms, holds the potential for microbial CH4 production if catalysed by a change in environmental conditions.

Graphical Abstract

BackgroundThe clinical high-risk for psychosis (CHR-P) paradigm was introduced to detect individuals at risk of developing psychosis and to establish preventive strategies. While current prediction of outcomes in the CHR-P state is based mostly on the clinical assessment of presenting features, several emerging biomarkers have been investigated in an attempt to stratify CHR-P individuals according to their individual trajectories and refine the diagnostic process. However, heterogeneity across subgroups is a key challenge that has limited the impact of the CHR-P prediction strategies, as the clinical validity of the current research is limited by a lack of external validation across sites and modalities. Despite these challenges, electroencephalography (EEG) biomarkers have been studied in this field and evidence suggests that EEG used in combination with clinical assessments may be a key measure for improving diagnostic and prognostic accuracy in the CHR-P state. The PSYSCAN EEG study is an international, multi-site, multimodal longitudinal project that aims to advance knowledge in this field.MethodsParticipants at 6 international sites take part in an EEG protocol including EEG recording, cognitive and clinical assessments. CHR-P participants will be followed up after 2 years and subcategorised depending on their illness progression regarding transition to psychosis. Differences will be sought between CHR-P individuals and healthy controls and between CHR-P individuals who transition and those who do not transition to psychosis using data driven computational analyses.DiscussionThis protocol addresses the challenges faced by previous studies of this kind to enable valid identification of predictive EEG biomarkers which will be combined with other biomarkers across sites to develop a prognostic tool in CHR-P. The PSYSCAN EEG study aims to pave the way for incorporating EEG biomarkers in the assessment of CHR-P individuals, to refine the diagnostic process and help to stratify CHR-P subjects according to risk of transition. This may improve our understanding of the CHR-P state and therefore aid the development of more personalized treatment strategies.

During its second operational campaign (OP 1.2a) W7-X operated with an edge island divertor. Much higher densities (1020m−3) than in to the previous, limiter campaign were achieved. In order to asses this improved performance and the nature of transport with regard to the expected higher densities and other parameters such as the electron and ion temperature, the electric field and plasma flow, measurements at the plasma edge were conducted with reciprocating probe, mounted on a Manipulator. The probe contains a set of Langmuir pins, Mach probes, compensation coils and a set of 3D pick up coils for the measurement of the local magnetic field and fluctuations, and an ion sensitive probe. It has been observed, in addition to differences due to the magnetic configurations, that the magnetic topology is sensitive to the plasma conditions, especially to changes due to a evolving toroidal plasma current. Measurements from divertor cameras showed that an increasing toroidal current in the standard configuration broadened the heat flux and shifted the strike line considerably. These down-stream observation can be complemented with the measurements at the mid-plane with the manipulator mounted probes. During the experiments the edge island remnant was identified with the measured edge plasma profiles and compared with the predictions from the field line tracing code. Those electron densities and temperatures have been used as an input for understanding edge transport with the EMC3-EIRENE modeling. The results from EMC3-EIRENE also help improve the caculated density profiles, since the modelling provides a profile of the effective charge Zeff. Keywords: W7-X, Langmuir probes, Magnetic islands, Plasma edge transport

We report a rare observation of a mini-fracture in near-surface sediments (30 cm below the seafloor) visualized using a rotational scanning X-ray of a core recovered from the Lomvi pockmark, Vestnesa Ridge, west of Svalbard (1200 m water depth). Porewater geochemistry and lipid biomarker signatures revealed clear differences in the geochemical and biogeochemical regimes of this core compared with two additional unfractured cores recovered from pockmark sites at Vestnesa Ridge, which we attribute to differential methane transport mechanisms. In the sediment core featuring the shallow mini-fracture at pockmark Lomvi, we observed high concentrations of both methane and sulfate throughout the core in tandem with moderately elevated values for total alkalinity, 13C-depleted dissolved inorganic carbon (DIC), and 13C-depleted lipid biomarkers (diagnostic for the slow-growing microbial communities mediating the anaerobic oxidation of methane with sulfate – AOM). In a separate unfractured core, recovered from the same pockmark about 80 m away from the fractured core, we observed complete sulfate depletion in the top centimeters of the sediment and much more pronounced signatures of AOM than in the fractured core. Our data indicate a gas advection-dominated transport mode in both cores, facilitating methane migration into sulfate-rich surface sediments. However, the moderate expression of AOM signals suggest a rather recent onset of gas migration at the site of the fractured core, while the geochemical evidence for a well-established AOM community at the second coring site suggest that gas migration has been going on for a longer period of time. A third core recovered from another pockmark along the Vestnesa Ridge Lunde pockmark was dominated by diffusive transport with only weak geochemical and biogeochemical evidence for AOM. Our study highlights that advective fluid and gas transport supported by mini-fractures can be important in modulating methane dynamics in surface sediments.

One of the main aims of Wendelstein 7-X (W7-X), an advanced stellarator, is to investigate the quasi-steady state operation of magnetic confinement devices for nuclear fusion, for which power exhaust is an important issue. A dominant fraction of the energy leaving from the confined plasma region will be removed by 10 so-called island divertor units, which are designed to sustain a maximum heat flux of up to 10 MWm−2. An essential prerequisite for the safe operation of a steady-state device is automatic detection of hot spots and other abnormal events. Simple temperature limits in infrared (IR) thermographic images will not be enough on their own, because of plasma-generated surface coatings and other effects summarized in the following. To protect divertor elements from overheating, and to monitor power deposition onto the divertor elements, near real-time hotspot detection algorithms for the analysis of carbon plasma facing components (PFCs) were implemented and tested in the GLADIS facility.One of the difficulties in hotspot detection in a carbon-based machine is the deposition of plasma impurities as layers with a reduced thermal connection to the underlying bulk material. We have developed and successfully tested a method to classify surface layers and benchmarked the performance of the method with the Tore Supra IR data operating with actively cooled carbon PFCs. The surface layers can be detected in a steady plasma discharge during the initial rise and decay in temperature when a strike line touches parts of the divertor or wall. It can also be detected by modulating electron cyclotron resonance heating (ECRH) input power. This feature allows detection of overheated areas while reducing false positives. For the recent operational campaign, inertially cooled test divertor units (TDU) were installed to prepare for steady-state operation with water-cooled divertor units. Automatic, near real-time detection of hot spots and identification of surface layers in the W7-X divertor are presented. Results are compared with a best fit estimate of the heat transmission coefficient α which is used to calculate heat flux onto the divertor in the presence of surface layers. Keywords: W7-X, Divertor, Plasma facing components, Heat load, Infrared, Surface layers, Tore supra

The 3D effects on divertor heat loads have been investigated for performance-optimized island divertor configurations at Wendelstein 7-X with 3D modeling and IR camera measurements. A new high mirror configuration optimized for more stable island divertor operation due to reduced bootstrap currents and a more even heat load distribution between the main divertor targets has been investigated for the first time numerically and experimentally.Transport calculations with EMC3-EIRENE show a strong dependence of the heat flux distributions on the configurations and the details of the 3D island geometry. IR camera measurements confirm the predictions concerning the global heat load distributions for the standard configuration but show more even distributions between the main wetted divertor plates in the high mirror configurations. The local heat load profiles show offsets in their position of up to 5 cm to each other and averaged peak heat fluxes varying between 0.5 MWm−2 and 2.2 MWm−2 depending on the divertor module considered. These heat flux asymmetries complicate the local matching of profiles between experiment and 3D modeling.The 3D equilibrium of a high mirror high-performance scenario predicted by the HINT code has been investigated with EMC3-EIRENE for the first time to anticipate plasma response in higher performance scenarios. The island divertor is preserved for β=3% despite enhanced stochasticity. However, the islands are increased in size while the path lengths for parallel transport are reduced causing a substantial change in the divertor heat flux patterns. Keywords: Stellarator, Island divertor, Scrape-off layer, Heat fluxes, Equilibrium

In this study, we report lipid biomarker patterns and phylogenetic identities of key microbial communities mediating anaerobic oxidation of methane (AOM) in active mud volcanoes (MVs) on the continental slope of the Canadian Beaufort Sea. The carbon isotopic compositions (δ13C) of sn-2- and sn-3-hydroxyarchaeol showed the highly 13C-depleted values (−114 ‰ to −82 ‰) associated with a steep depletion in sulfate concentrations within 0.7 m of sediment depths. This suggested the presence of methanotrophic archaea involved in sulfate-dependent AOM, albeit in a small amount. The ratio of sn-2-hydroxyarchaeol to archaeol (> 1) and operational taxonomic units (OTUs) indicated that the anaerobic methanotrophic archaea (ANME) clades ANME-2c and ANME-3 were involved in AOM. Higher δ13C values of archaeol and biphytanes (BPs; -55.2±10.0 ‰ and -39.3±13.0 ‰, respectively) suggested that archaeal communities were also assimilating AOM-derived inorganic carbon. Furthermore, the distinct distribution patterns of methanotrophs in the three MVs appears to be associated with varying intensities of ascending gas fluids. Consequently, our results suggest that the niche diversification of active mud volcanoes has shaped distinct archaeal communities that play important roles in AOM in the Beaufort Sea.

It remains unclear how oligotrophic habitats in subterranean estuaries sustain complex ecosystems. Here, using stable isotopic evidence from organic matter and pelagic shrimp, the authors show that a microbial loop fuelled by methane and dissolved organic carbon sustains the anchialine food web.

The human MET receptor tyrosine kinase contributes to vertebrate development and cell proliferation. As a proto‐oncogene, it is a target in cancer therapies. MET is also relevant for bacterial infection by Listeria monocytogenes and is activated by the bacterial protein internalin B. The processes of ligand binding, receptor activation, and the diffusion behavior of MET within the plasma membrane as well as its interconnections with various cell components are not fully understood. We investigated the receptor diffusion dynamics using single‐particle tracking and imaging fluorescence correlation spectroscopy and elucidated mobility states of resting and internalin B‐bound MET. We show that internalin B‐bound MET exhibits lower diffusion coefficients and diffuses in a more confined area in the membrane. We report that the fraction of immobile receptors is larger for internalin B‐bound receptors than for resting MET. Results of single‐particle tracking in cells treated with various cytotoxins depleting cholesterol from the membrane and disrupting the actin cytoskeleton and microtubules suggest that cholesterol and actin influence MET diffusion dynamics, while microtubules do not have any effect.

Coastal seas may account for more than 75 % of global oceanic methane emissions. There, methane is mainly produced microbially in anoxic sediments from which it can escape to the overlying water column. Aerobic methane oxidation (MOx) in the water column acts as a biological filter, reducing the amount of methane that eventually evades to the atmosphere. The efficiency of the MOx filter is potentially controlled by the availability of dissolved methane and oxygen, as well as temperature, salinity, and hydrographic dynamics, and all of these factors undergo strong temporal fluctuations in coastal ecosystems. In order to elucidate the key environmental controls, specifically the effect of oxygen availability, on MOx in a seasonally stratified and hypoxic coastal marine setting, we conducted a 2-year time-series study with measurements of MOx and physico-chemical water column parameters in a coastal inlet in the south-western Baltic Sea (Eckernförde Bay). We found that MOx rates generally increased toward the seafloor, but were not directly linked to methane concentrations. MOx exhibited a strong seasonal variability, with maximum rates (up to 11.6 nmol L−1 d−1) during summer stratification when oxygen concentrations were lowest and bottom-water temperatures were highest. Under these conditions, 2.4–19.0 times more methane was oxidized than emitted to the atmosphere, whereas about the same amount was consumed and emitted during the mixed and oxygenated periods. Laboratory experiments with manipulated oxygen concentrations in the range of 0.2–220 µmol L−1 revealed a submicromolar oxygen optimum for MOx at the study site. In contrast, the fraction of methane–carbon incorporation into the bacterial biomass (compared to the total amount of oxidized methane) was up to 38-fold higher at saturated oxygen concentrations, suggesting a different partitioning of catabolic and anabolic processes under oxygen-replete and oxygen-starved conditions, respectively. Our results underscore the importance of MOx in mitigating methane emission from coastal waters and indicate an organism-level adaptation of the water column methanotrophs to hypoxic conditions.

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

Flavin-dependent halogenases catalyse halogenation of aromatic compounds. In most cases, this reaction proceeds with high regioselectivity and requires only the presence of FADH2, oxygen, and halide salts. Since marine habitats contain high concentrations of halides, organisms populating the oceans might be valuable sources of yet undiscovered halogenases. A new Hidden-Markov-Model (HMM) based on the PFAM tryptophan halogenase model was used for the analysis of marine metagenomes. Eleven metagenomes were screened leading to the identification of 254 complete or partial putative flavin-dependent halogenase genes. One predicted halogenase gene (brvH) was selected, codon optimised for E. coli, and overexpressed. Substrate screening revealed that this enzyme represents an active flavin-dependent halogenase able to convert indole to 3-bromoindole. Remarkably, bromination prevails also in a large excess of chloride. The BrvH crystal structure is very similar to that of tryptophan halogenases but reveals a substrate binding site that is open to the solvent instead of being covered by a loop.

Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion.

The bacterially mediated aerobic methane oxidation (MOx) is a key mechanism in controlling methane (CH4) emissions from the world's oceans to the atmosphere. In this study, we investigated MOx in the Arctic fjord Storfjorden (Svalbard) by applying a combination of radio-tracer-based incubation assays (3H-CH4 and 14C-CH4), stable C-CH4 isotope measurements, and molecular tools (16S rRNA gene Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, pmoA- and mxaF gene analyses). Storfjorden is stratified in the summertime with melt water (MW) in the upper 60 m of the water column, Arctic water (ArW) between 60 and 100 m, and brine-enriched shelf water (BSW) down to 140 m. CH4 concentrations were supersaturated with respect to the atmospheric equilibrium (about 3–4 nM) throughout the water column, increasing from ∼20 nM at the surface to a maximum of 72 nM at 60 m and decreasing below. MOx rate measurements at near in situ CH4 concentrations (here measured with 3H-CH4 raising the ambient CH4 pool by −1 at 60 m, followed by a decrease in the deeper ArW/BSW. In contrast, rate measurements with 14C-CH4 (incubations were spiked with ∼450 nM of 14C-CH4, providing an estimate of the CH4 oxidation at elevated concentration) showed comparably low turnover rates (−1) at 60 m, and peak rates were found in ArW/BSW at ∼100 m water depth, concomitant with increasing 13C values in the residual CH4 pool. Our results indicate that the MOx community in the surface MW is adapted to relatively low CH4 concentrations. In contrast, the activity of the deep-water MOx community is relatively low at the ambient, summertime CH4 concentrations but has the potential to increase rapidly in response to CH4 availability. A similar distinction between surface and deep-water MOx is also suggested by our molecular analyses. The DGGE banding patterns of 16S rRNA gene fragments of the surface MW and deep water were clearly different. A DGGE band related to the known type I MOx bacterium Methylosphaera was observed in deep BWS, but absent in surface MW. Furthermore, the Polymerase Chain Reaction (PCR) amplicons of the deep water with the two functional primers sets pmoA and mxaF showed, in contrast to those of the surface MW, additional products besides the expected one of 530 base pairs (bp). Apparently, different MOx communities have developed in the stratified water masses in Storfjorden, which is possibly related to the spatiotemporal variability in CH4 supply to the distinct water masses.

A novel proxy for continental mean annual air temperature (MAAT) and soil pH, the MBT/CBT-paleothermometer, is based on the temperature (T) and pH-dependent distribution of specific bacterial membrane lipids (branched glycerol dialkyl glycerol tetraethers – GDGTs) in soil organic matter. Here, we tested the applicability of the MBT/CBT-paleothermometer to sediments from Lake Cadagno, a high Alpine lake in southern Switzerland with a small catchment of 2.4 km2. We analysed the distribution of bacterial GDGTs in catchment soils and in a radiocarbon-dated sediment core from the centre of the lake, covering the past 11 000 yr. The distribution of bacterial GDGTs in the catchment soils is very similar to that in the lake's surface sediments, indicating a common origin of the lipids. Consequently, their transfer from the soils into the sediment record seems undisturbed, probably without any significant alteration of their distribution through in situ production in the lake itself or early diagenesis of branched GDGTs. The MBT/CBT-inferred MAAT estimates from soils and surface sediments are in good agreement with instrumental values for the Lake Cadagno region (~0.5 °C). Moreover, downcore MBT/CBT-derived MAAT estimates match in timing and magnitude other proxy-based T reconstructions from nearby locations for the last two millennia. Major climate anomalies recorded by the MBT/CBT-paleothermometer are, for instance, the Little Ice Age (~14th to 19th century) and the Medieval Warm Period (MWP, ~9th to 14th century). Together, our observations indicate the quantitative applicability of the MBT/CBT-paleothermometer to Lake Cadagno sediments. In addition to the MWP, our lacustrine paleo T record indicates Holocene warm phases at about 3, 5, 7 and 11 kyr before present, which agrees in timing with other records from both the Alps and the sub-polar North-East Atlantic Ocean. The good temporal match of the warm periods determined for the central Alpine region with north-west European winter precipitation strength implies a strong and far-reaching influence of the North Atlantic Oscillation on continental European T variations during the Holocene.

First videographic indication of an Antarctic cold seep ecosystem was recently obtained from the collapsed Larsen B ice shelf, western Weddell Sea (Domack et al., 2005). Within the framework of the R/V Polarstern expedition ANTXXIII-8, we revisited this area for geochemical, microbiological and further videographical examinations. During two dives with ROV Cherokee (MARUM, Bremen), several bivalve shell agglomerations of the seep-associated, chemosynthetic clam Calyptogena sp. were found in the trough of the Crane and Evans glacier. The absence of living clam specimens indicates that the flux of sulphide and hence the seepage activity is diminished at present. This impression was further substantiated by our geochemical observations. Concentrations of thermogenic methane were moderately elevated with 2 μM in surface sediments of a clam patch, increasing up to 9 μM at a sediment depth of about 1 m in the bottom sections of the sediment cores. This correlated with a moderate decrease in sulphate from about 28 mM at the surface down to 23.4 mM, an increase in sulphide to up to 1.43 mM and elevated rates of the anaerobic oxidation of methane (AOM) of up to 600 pmol cm−3 d−1 at about 1 m below the seafloor. Molecular analyses indicate that methanotrophic archaea related to ANME-3 are the most likely candidates mediating AOM in sediments of the Larsen B seep.

Fluid flow related seafloor structures and gas seeps were detected in the North Sea in the 1970s and 1980s by acoustic sub-bottom profiling and oil rig surveys. A variety of features like pockmarks, gas vents and authigenic carbonate cements were found to be associated with sites of oil and gas exploration, indicating a link between these surface structures and the underlying, deep hydrocarbon reservoirs. In this study we performed acoustic surveys and videographic observation at Gullfaks, Holene Trench, Tommeliten, Witch's Hole and the giant pockmarks of the UK Block 15/25, to investigate the occurrence and distribution of cold seep ecosystems in the Northern North Sea. The most active gas seep sites, i.e. Gullfaks and Tommeliten, were investigated in detail. At both sites, gas bubbles escaped continuously from small holes in the seabed to the water column, reaching the upper mixed surface layer. At Gullfaks a gas emitting, flat area of 0.1 km2 of sandy seabed covered by filamentous sulfur-oxidizing bacteria was detected. At Tommeliten, we found a patchy distribution of small bacterial mats indicating sites of gas seepage. Below the patches the seafloor consisted of sand from which gas emissions were observed. At both sites, the anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) was the major source of sulfide. Molecular analyses targeting specific lipid biomarkers and 16S rRNA gene sequences identified an active microbial community dominated by sulfur-oxidizing and sulfate-reducing bacteria (SRB) as well as methanotrophic bacteria and archaea. Stable carbon isotope values of specific, microbial fatty acids and alcohols from both sites were highly depleted in the heavy isotope 13C, indicating that the microbial community incorporates methane or its metabolites. The microbial community composition of both shallow seeps shows high similarities to the deep water seeps associated with gas hydrates such as Hydrate Ridge or the Eel River basin.

The Tommeliten seepage area is part of the Greater Ekofisk area, which is situated above the Tommeliten Delta salt diapir in the central North Sea (56°29.90' N, 2°59.80' E, Norwegian Block 1/9, 75 m water depth). Here, cracks in a buried marl horizon allow methane to migrate into overlying clay-silt and sandy sediments. Hydroacoustic sediment echosounding showed several venting spots coinciding with the apex of marl domes where methane is released into the water column and potentially to the atmosphere. In the vicinity of the gas seeps, sea floor observations showed small mats of giant sulphide-oxidizing bacteria above patches of black sediments as well as carbonate crusts, which are exposed 10 to 50 cm above seafloor forming small reefs. These Methane-Derived Authigenic Carbonates (MDACs) contain 13C-depleted, archaeal lipids indicating previous gas seepage and AOM activity. High amounts of sn2-hydroxyarchaeol relative to archaeol and low abundances of biphytanes in the crusts give evidence that ANaerobic MEthane-oxidising archaea (ANME) of the phylogenetic cluster ANME-2 were the potential mediators of Anaerobic Oxidation of Methane (AOM) at the time of carbonate formation. Small pieces of MDACs were also found subsurface at about 1.7 m sediment depth, associated with the AOM zone. This zone is characterized by elevated AOM and Sulphate Reduction (SR) rates, increased concentrations of 13C-depleted tetraether derived biphytanes, and specific bacterial Fatty Acids (FA). Further biomarker and 16S rDNA based analyses of this horizon give evidence that AOM is mediated by archaea belonging to the ANME-1b group and Sulphate Reducing Bacteria (SRB) most likely belonging to the Seep-SRB1 cluster. The zone of active methane consumption was restricted to a distinct horizon of about 20 cm. Concentrations of 13C-depleted lipid biomarkers (e.g. 500 ng g-dw−1 biphythanes, 140 ng g-dw−1 fatty acid ai-C15:0), cell numbers (1.5×108 cells cm−3), AOM and SR rates (3 nmol cm−3 d−1) in the Tommeliten AOM zone are 2–3 orders of magnitude lower compared to AOM zones of highly active deep water cold seeps such as Hydrate Ridge or the Gulf of Mexico.

Array technology is a widely used tool for gene expression profiling in various biological systems. However, the application of this method to mammalian preimplantation embryos is limited by the small amount of mRNA that can be extracted from a single embryo, which is not sufficient for array analysis. Here we report a protocol for the rapid global amplification of embryonic mRNA that permits the generation of expression profiles from single murine blastocysts. The approach combines global PCR and T7 RNA polymerase amplification and allows the preparation of labeled, amplified RNA for array hybridization from single murine blastocysts containing approximately 1.5 pg mRNA in less than 12 h. We demonstrate that this amplification procedure is highly reproducible and does not bias original relative mRNA levels. Signal patterns from various embryonic stages of murine development revealed marked differences in mRNA expression that were in accordance with previously published data. We found genes known to be involved in embryonic apoptosis expressed at different levels in individual murine day 3.5 blastocysts. This technique can thus be used to assess embryonic viability and investigate molecular mechanisms of embryonic development.

We have developed a protocol for fast, nonradioactive, mRNA differential display reverse transcription PCR (DDRT-PCR) based on a commerical automated sequencer with RNA isolated from pig granulosa cells. We sought to discover conditions that would minimize the problem of using relatively small primers labeled with large infrared dye molecule, IR41, required for the sequencer. Extended IR41-labeled primers IR41-AAGC-T11-A, IR41-AAGC-T11-C and IR41-AAGC-T11-G gave more consistent differential display patterns than shorter anchored primers (IR41-T11A, IR41-T11C and IR41-T11G) without the additional (AAGC) cloning site. The optimal concentration of the extended labeled (downstream) primers was 20 pmol when 13-mer arbitrary (upstream) primers were used at a concentration of 4 pmol. Background smear and the intensity of amplified bands was significantly improved by changing from conventional Taq DNA polymerase to AmpliTaq® Gold™ polymerase, which permits an improved “hot start” for the reaction. Running time (during which a digitized gel image is recorded) for a 26-cm polyacrylamide gel was 4 h, enabling us to analyze 90 reactions in an 8-h day. This protocol offers a rapid and reliable nonradioactive method for comparing gene expression patterns for various research or diagnostic purposes.

We have demonstrated efficient injection and trapping of a cold positron beam in a dipole magnetic field configuration. The intense 5 eV positron beam was provided by the NEutron induced POsitron source MUniCh facility at the Heinz Maier-Leibnitz Zentrum, and transported into the confinement region of the dipole field trap generated by a supported, permanent magnet with 0.6 T strength at the pole faces. We achieved transport into the region of field lines that do not intersect the outer wall using the ${\bf{E}}\times {\bf{B}}$ drift of the positron beam between a pair of tailored plates that created the electric field. We present evidence that up to 38% of the beam particles are able to reach the intended confinement region and make at least a 180° rotation around the magnet where they annihilate on an insertable target. When the target is removed and the ${\bf{E}}\times {\bf{B}}$ plate voltages are switched off, confinement of a small population persists for on the order of 1 ms. These results lend optimism to our larger aims to apply a magnetic dipole field configuration for trapping of both positrons and electrons in order to test predictions of the unique properties of a pair plasma.