Geochemistry and Microbial Populations in Sediments of the Northern Baffin Bay,Arctic

The Northern Baffin Bay between Greenland and Canada is a remote Arctic area restricted in primary production by seasonal ice cover, with presumably low sedimentation rates, carbon content and microbial activities in its sediments. Our aim was to study the so far unknown subseafloor geochemistry and microbial populations driving seafloor ecosystems. Shelf sediments had the highest organic carbon content, numbers of Bacteria and Archaea, and microcosms inoculated from Shelf sediments showed highest sulfate reduction and methane production rates. Sediments in the central deep area and on the southern slope contained less organic carbon and overall lower microbial numbers. Similar 16S rRNA gene copy numbers of Archaea and Bacteria were found for the majority of the sites investigated. Sulfate in pore water correlated with dsrA copy numbers of sulfate-reducing prokaryotes and differed between sites. No methane was found as free gas in the sediments, and mcrA copy numbers of methanogenic Archaea were low. Methanogenic and sulfate-reducing cultures were enriched on a variety of substrates including hydrocarbons. In summary, the Greenlandic shelf sediments contain vital microbial communities adapted to their specific environmental conditions.     

Human transformations of the Wadden Sea ecosystem through time: a synthesis

Todays Wadden Sea is a heavily human-altered ecosystem. Shaped by natural forces since its origin 7,500 years ago, humans gradually gained dominance in influencing ecosystem structure and functioning. Here, we reconstruct the timeline of human impacts and the history of ecological changes in the Wadden Sea. We then discuss the ecosystem and societal consequences of observed changes, and conclude with management implications. Human influences have intensified and multiplied over time. Large-scale habitat transformation over the last 1,000 years has eliminated diverse terrestrial, freshwater, brackish and marine habitats. Intensive exploitation of everything from oysters to whales has depleted most large predators and habitat-building species since medieval times. In the twentieth century, pollution, eutrophication, species invasions and, presumably, climate change have had marked impacts on the Wadden Sea flora and fauna. Yet habitat loss and overexploitation were the two main causes for the extinction or severe depletion of 144 species (~20% of total macrobiota). The loss of biodiversity, large predators, special habitats, filter and storage capacity, and degradation in water quality have led to a simplification and homogenisation of the food web structure and ecosystem functioning that has affected the Wadden Sea ecosystem and coastal societies alike. Recent conservation efforts have reversed some negative trends by enabling some birds and mammals to recover and by creating new economic options for society. The Wadden Sea history provides a unique long-term perspective on ecological change, new objectives for conservation, restoration and management, and an ecological baseline that allows us to envision a rich, productive and diverse Wadden Sea ecosystem and coastal society.     

Crustacean cardioactive peptide-immunoreactive neurons innervating brain neuropils,retrocerebral complex and stomatogastric nervous system of the locust,Locusta migratoria

The distribution and morphology of crustacean cardioactive peptide-immunoreactive neurons in the brain of the locust Locusta migratoria has been determined. Of more than 500 immunoreactive neurons in total, about 380 are interneurons in the optic lobes. These neurons invade several layers of the medulla and distal parts of the lobula. In addition, a small group of neurons projects into the accessory medulla, the lamina, and to several areas in the median protocerebrum. In the midbrain, 12 groups or individual neurons have been reconstructed. Four groups innervate areas of the superior lateral and ventral lateral protocerebrum and the lateral horn. Two cell groups have bilateral arborizations anterior and posterior to the central body or in the superior median protocerebrum. Ramifications in subunits of the central body and in the lateral and the median accessory lobes arise from four additional cell groups. Two local interneurons innervate the antennal lobe. A tritocerebral cell projects contralaterally into the frontal ganglion and appears to give rise to fibers in the recurrent nerve, and in the hypocerebral and ingluvial ganglia. Varicose fibers in the nervi corporis cardiaci III and the corpora cardiaca, and terminals on pharyngeal dilator muscles arise from two subesophageal neurons. Some of the locust neurons closely resemble immunopositive neurons in a beetle and a moth. Our results suggest that the peptide may be (1) a modulatory substance produced by many brain interneurons, and (2) a neurohormone released from subesophageal neurosecretory cells.     

Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P₂) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P₂ were determined to be K_d∼12 µM and 0.4 µM, respectively, and K_d∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P₂. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P₂ and an increased apparent affinity to PI(3,4,5)P₃, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P₂ and no synergy in its binding with PS and PI(4,5)P₂. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.     

Fluxomics with Ratiometric Metabolite Dyes

Today''s major excitement in biology centers on signaling: How can a cell or organism measure the myriad of environmental cues, integrate it, and acclimate to the new conditions? Hormonal signals and second messengers are in the focus of most of these studies, e.g., regulation of glucose transporter GLUT4 cycling by insulin, or regulation of plant growth by auxin or brassinosteroids.^1–3 In comparison, we generally assume that we know almost everything about basic metabolism since it has been studied for many decades; for example we know since the early 80s that allosteric regulation by fructose-2,6-bisphophate plays an important role in regulating glycolysis in plants and animals.⁴ This may be the reason why studies of metabolism appear to be a bit out of fashion. But if we look to other organisms such as E. coli or yeast, we rapidly realize that metabolism is controlled by complex interconnected signaling networks, and that we understand little of these signaling networks in humans and plants.^5,6 As it turns out, the cell registers many metabolites, and flux through the pathways is regulated using complex signaling networks that involve calcium as well as hormones.Key Words: flux, fluxome, glucose, glutamate, phosphate, sucrose, fluorescence resonance energy transfer, biosensorOne of the reasons for the fable for hormones lies in the simple fact that it is easier to observe macroscopic changes, such as changes in the architecture of a plant than to determine metabolite levels, but also here new tools are urgently needed that allow quantification of these small molecules. Visualization of starch levels provided a significant advance, and in combination with mutant screens allowed to identify fundamental components of starch metabolism.^7–9 The biggest advance for the signaling field was the development of advanced chemical and genetically encoded calcium dyes.^10–12 No such dyes are available for hormones or metabolites, as soon as we try to determine levels of metabolites (or signaling molecules), we run into the issues of compartmentation and cellular differences in tissues. Today, the same enzymatic assays used decades ago are still widely used to determine metabolite levels. Although significant advances in chromatography and mass spectrometry based metabolite analysis have moved the study of metabolism to ‘omics’ era, compartmentalization of metabolism still presents a major challenge. Especially the large vacuoles of plant cells are a major obstacle, since even fractionation studies suffer from contamination. Moreover, with the current set of tools it is not possible to determine the dynamic changes in metabolite levels in different subcellular compartments in real time in vivo. Radiotracers have helped a lot to identify and quantify intermediates and to assemble pathways, originally using pulse labeling followed by paper chromatography. Today ¹³C-labeling is used together with mass spectrometry to obtain insights into metabolic flux control.¹³ This tool set for the first time enabled the comparison of mutants and study regulatory networks involved in sugar signaling. While significant, advances in radiotracer experiments do not provide cellular or subcellular information and only limited temporal resolution, they do provide efficient means for studying metabolite fluxes through complex and/or not well-defined pathways. Thus there is a clear need for metabolite specific dyes that can be targeted to subcellular compartments and that would enable flux measurements in response to environmental cues helping to push metabolic research back into the focus of signaling-related biology.In 2002, we developed the first prototype “metabolic dye” FRET sensor for maltose.^14,15 A similar glucose sensor was recently employed for measuring tracer-independent transport of glucose across the ER membrane of liver cells.¹⁶ After resolving some issues such as low signal-to-noise and gene silencing in plants, we are now able to compare glucose levels between cells in an intact root in real time.¹⁷ The parallel development of sucrose and phosphate sensors complements the set of tools, in future experiments providing a comparison of sucrose, phosphate and glucose fluxes in intact tissues with both temporal (below seconds) and spatial resolution (cellular and subcellular).^18,19The first experiments already led to a big surprise: glucose supplied to the root is rapidly taken up and is rapidly metabolized.¹⁷ Roots expressing the highest affinity sensor FLIPglu170n responded to glucose perfusion suggesting that the steady state glucose level in the root is less than 100 nM, the estimated detection limit for this sensor in these first experiments. The first experiments were limited by the mixing kinetics in the bath used for perfusion, while improvement of the chamber now allow for faster for glucose exchange. We estimate that glucose levels fall from a steady state level of approximately 5 mM in the cytosol when perfused with 5 mM glucose to below 100 nM in about three minutes. For the sensor with an affinity of 600 µM the rate of glucose accumulation, which is composed of the various rates that affect the steady state in the cytosol such as metabolism, compartmentation and transport across the plasma membrane, is in the range of 527 ± 77 µM glucose/min and that for glucose removal is 317 ± 37 (Fig. 1; Chaudhuri B, Frommer WB, unpublished). Questions that arise are: Which transport systems drive uptake? How much does the vacuole contribute to the observed flux and steady state levels? Is the capacity of hexokinase at levels below its K_m still sufficient to phosphorylate glucose efficient enough to pull glucose below 100 nM or does hexokinase have different properties in vivo compared to what we know from the purified enzyme? Are there different transporters and enzymes contributing to flux in the low (1–10 mM) and the ultrahigh affinity (low µM) phases? Are there spatial differences in the root? Why do roots take up glucose so efficiently in the first place? The combination of the sensors with information from the expression-LEDs from Birnbaum and Benfey²⁰ and specific knock-out mutants should help answering some of these questions.Open in a separate windowFigure 1Quantitative analysis of glucose flux from an Arabidopsis root expressing FLIPglu-600µΔ13, a FRET sensor for glucose with an affinity of 600 µM. The root of a 10 day-old seedling was placed into a perfusion chamber and perfused with hydroponic medium with or without 5 mM glucose. eCFP was excited and emission was recorded for eCFP and eYFP every 10 seconds (essentially as decsribed in ref. 17). The emission intensities for a region-of-interest were averaged and the emission ratio was determined at the two wavelengths for each image of a time series and plotted on the Y-axis against time on the X-axis. Addition of glucose is indicated.Another big surprise is the dramatic gradient of glucose across the plasma membrane, which has important implications for our understanding of transport processes across the plasma membrane as well as the intracellular membranes.¹⁷ Information about the gradients is relevant in the context of apo- and symplasmic unloading routes in roots²¹ and the contribution of proton-coupled transporters in cellular export.²² It will thus be interesting to follow the extracellular levels using surface-anchored sensors. Now that besides high sensitivity glucose FLIPs¹⁷ we also generated nanosensors for sucrose¹⁹ and phosphate,¹⁸ complementing the similar tool sets for calcium²³ and pH,²⁴ it is possible to compare multiple parameters and to follow flux at different levels and to calibrate against other influences.The improvements of the signal-to-noise ratio of the FRET-based metabolite sensors²⁵ makes the FLIPs a standard tool for every lab interested in measuring ion-, sugar- or amino acid flux in living cells. Since the nanosensors are genetically encoded, they can be used to characterize intracellular fluxes^16,26 in any organism for which transformation protocols have been established. The existing sets of sensors are simple to use, constructs are available through Addgene and Arabidopsis lines from the Arabidopsis Stock Center. Detailed instructions for imaging can be found at: http://carnegiedpb.stanford.edu/research/frommer/research_frommer_protocols.php. These tools will hopefully become a standard system not only for physiological analyses, but in addition provide a new way for high throughput fluxomics studies.     

In vitro and in vivo activity of ribavirin against Andes virus infection

Pathogenic hantaviruses are a closely related group of rodent-borne viruses which are responsible for two distinct diseases in humans, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome (HPS, otherwise known as hantavirus cardiopulmonary syndrome, HCPS). The antiviral effect of ribavirin against Old World hantaviruses, most notably Hantaan virus, is well documented; however, only a few studies have addressed its inhibitory effect on New World hantaviruses. In the present study, we demonstrate that ribavirin is highly active against Andes virus (ANDV), an important etiological agent of HPS, both in vitro and in vivo using a lethal hamster model of HPS. Treatment of ANDV infected Vero E6 cells with ribavirin resulted in dose-dependent reductions in viral RNA and protein as well as virus yields with a half maximal inhibitory concentration between 5 and 12.5 μg ml(-1). In hamsters, treatment with as little as 5 mg kg(-1) day(-1) was 100% effective at preventing lethal HPS disease when therapy was administered by intraperitoneal injection from day 1 through day 10 post-infection. Significant reductions were observed in ANDV RNA and antigen positive cells in lung and liver tissues. Ribavirin remained completely protective when administered by intraperitoneal injections up to three days post-infection. In addition, we show that daily oral ribavirin therapy initiated 1 day post-infection and continuing for ten days is also protective against lethal ANDV disease, even at doses of 5 mg kg(-1) day(-1). Our results suggest ribavirin treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses and should be considered in scenarios where exposure to the virus is probable. The similarities between the results obtained in this study and those from previous clinical evaluations of ribavirin against HPS, further validate the hamster model of lethal HPS and demonstrate its usefulness in screening antiviral agents against this disease.