Dissolved gases in the deep North Atlantic track ocean ventilation processes.

Gas exchange between the atmosphere and ocean interior profoundly impacts global climate and biogeochemistry. However, our understanding of the relevant physical processes remains limited by a scarcity of direct observations. Dissolved noble gases in the deep ocean are powerful tracers of physical air-sea interaction due to their chemical and biological inertness, yet their isotope ratios have remained underexplored. Here, we present high-precision noble gas isotope and elemental ratios from the deep North Atlantic (~32°N, 64°W) to evaluate gas exchange parameterizations using an ocean circulation model. The unprecedented precision of these data reveal deep-ocean undersaturation of heavy noble gases and isotopes resulting from cooling-driven air-to-sea gas transport associated with deep convection in the northern high latitudes. Our data also imply an underappreciated and large role for bubble-mediated gas exchange in the global air-sea transfer of sparingly soluble gases, including O2, N2, and SF6. Using noble gases to validate the physical representation of air-sea gas exchange in a model also provides a unique opportunity to distinguish physical from biogeochemical signals. As a case study, we compare dissolved N2/Ar measurements in the deep North Atlantic to physics-only model predictions, revealing excess N2 from benthic denitrification in older deep waters (below 2.9 km). These data indicate that the rate of fixed N removal in the deep Northeastern Atlantic is at least three times higher than the global deep-ocean mean, suggesting tight coupling with organic carbon export and raising potential future implications for the marine N cycle.

Influence of sample volume on nitrate N and O isotope ratio analyses with the denitrifier method.

RATIONALE: Analyses of the isotope ratios of nitrogen (15 N/14 N) and oxygen (18 O/16 O) in nitrate (NO3 - ) with the denitrifier method require relatively high sample volumes at low concentrations (≤1 µM) to afford sufficient analyte for mass spectrometry, resulting in isotopic offsets compared to more concentrated samples of the same isotopic composition. METHODS: To uncover the origins of isotopic offsets, we analyzed the N and O isotope ratios of NO3 - reference materials spanning concentrations of 0.5-20 µM. We substantiated the incidence of volume-dependent isotopic offsets, then investigated whether they resulted from (a) incomplete sample recovery during N2 O sparging, (b) blanks - bacterial, atmospheric, or in reference material solutions - and (c) oxygen atom exchange with water during the bacterial conversion of NO3 - to N2 O. RESULTS: Larger sample volumes resulted in modest offsets in δ15 N, but substantial offsets in δ18 O. N2 O recovery from sparging was less complete at higher volumes, resulting in decreases in δ15 N and δ18 O due to associated isotope fractionation. Blanks increased detectably with volume, whereas oxygen atom exchange with water remained constant within batch analyses, being sensitive to neither sample volume nor salinity. The sizeable offsets in δ18 O with volume are only partially explained by the factors considered in our analysis. CONCLUSIONS: Our observations argue for bracketing of NO3 - samples with reference materials that emulate sample volumes (concentrations) to achieve improved measurement accuracy and foster inter-comparability.

Global reconstruction reduces the uncertainty of oceanic nitrous oxide emissions and reveals a vigorous seasonal cycle.

Assessment of the global budget of the greenhouse gas nitrous oxide ([Formula: see text]O) is limited by poor knowledge of the oceanic [Formula: see text]O flux to the atmosphere, of which the magnitude, spatial distribution, and temporal variability remain highly uncertain. Here, we reconstruct climatological [Formula: see text]O emissions from the ocean by training a supervised learning algorithm with over 158,000 [Formula: see text]O measurements from the surface ocean-the largest synthesis to date. The reconstruction captures observed latitudinal gradients and coastal hot spots of [Formula: see text]O flux and reveals a vigorous global seasonal cycle. We estimate an annual mean [Formula: see text]O flux of 4.2 ± 1.0 Tg N[Formula: see text], 64% of which occurs in the tropics, and 20% in coastal upwelling systems that occupy less than 3% of the ocean area. This [Formula: see text]O flux ranges from a low of 3.3 ± 1.3 Tg N[Formula: see text] in the boreal spring to a high of 5.5 ± 2.0 Tg N[Formula: see text] in the boreal summer. Much of the seasonal variations in global [Formula: see text]O emissions can be traced to seasonal upwelling in the tropical ocean and winter mixing in the Southern Ocean. The dominant contribution to seasonality by productive, low-oxygen tropical upwelling systems (>75%) suggests a sensitivity of the global [Formula: see text]O flux to El Niño-Southern Oscillation and anthropogenic stratification of the low latitude ocean. This ocean flux estimate is consistent with the range adopted by the Intergovernmental Panel on Climate Change, but reduces its uncertainty by more than fivefold, enabling more precise determination of other terms in the atmospheric [Formula: see text]O budget.

Microbial ecosystem dynamics drive fluctuating nitrogen loss in marine anoxic zones.

The dynamics of nitrogen (N) loss in the ocean's oxygen-deficient zones (ODZs) are thought to be driven by climate impacts on ocean circulation and biological productivity. Here we analyze a data-constrained model of the microbial ecosystem in an ODZ and find that species interactions drive fluctuations in local- and regional-scale rates of N loss, even in the absence of climate variability. By consuming O2 to nanomolar levels, aerobic nitrifying microbes cede their competitive advantage for scarce forms of N to anaerobic denitrifying bacteria. Because anaerobes cannot sustain their own low-O2 niche, the physical O2 supply restores competitive advantage to aerobic populations, resetting the cycle. The resulting ecosystem oscillations induce a unique geochemical signature within the ODZ-short-lived spikes of ammonium that are found in measured profiles. The microbial ecosystem dynamics also give rise to variable ratios of anammox to heterotrophic denitrification, providing a mechanism for the unexplained variability of these pathways observed in the ocean.

Utilization of urea and cyanate in waters overlying and within the eastern tropical north Pacific oxygen deficient zone.

In marine oxygen deficient zones (ODZs), which contribute up to half of marine N loss, microbes use nitrogen (N) for assimilatory and dissimilatory processes. Here, we examine N utilization above and within the ODZ of the Eastern Tropical North Pacific Ocean, focusing on distribution, uptake and genes for the utilization of two simple organic N compounds, urea and cyanate. Ammonium, urea and cyanate concentrations generally peaked in the oxycline while uptake rates were highest in the surface. Within the ODZ, concentrations were lower, but urea N and C and cyanate C were taken up. All identified autotrophs had an N assimilation pathway that did not require external ammonium: ODZ Prochlorococcus possessed genes to assimilate nitrate, nitrite and urea; nitrite oxidizers (Nitrospina) possessed genes to assimilate nitrite, urea and cyanate; anammox bacteria (Scalindua) possessed genes to utilize cyanate; and ammonia-oxidizing Thaumarchaeota possessed genes to utilize urea. Urease genes were present in 20% of microbes, including SAR11, suggesting the urea utilization capacity was widespread. In the ODZ core, cyanate genes were largely (∼95%) associated with Scalindua, suggesting that, within this ODZ, cyanate N is primarily used for N loss via anammox (cyanammox), and that anammox does not require ammonium for N loss.

Biological nitrogen fixation in the oxygen-minimum region of the eastern tropical North Pacific ocean.

Biological nitrogen fixation (BNF) was investigated above and within the oxygen-depleted waters of the oxygen-minimum zone of the Eastern Tropical North Pacific Ocean. BNF rates were estimated using an isotope tracer method that overcame the uncertainty of the conventional bubble method by directly measuring the tracer enrichment during the incubations. Highest rates of BNF (~4 nM day-1) occurred in coastal surface waters and lowest detectable rates (~0.2 nM day-1) were found in the anoxic region of offshore stations. BNF was not detectable in most samples from oxygen-depleted waters. The composition of the N2-fixing assemblage was investigated by sequencing of nifH genes. The diazotrophic assemblage in surface waters contained mainly Proteobacterial sequences (Cluster I nifH), while both Proteobacterial sequences and sequences with high identities to those of anaerobic microbes characterized as Clusters III and IV type nifH sequences were found in the anoxic waters. Our results indicate modest input of N through BNF in oxygen-depleted zones mainly due to the activity of proteobacterial diazotrophs.

Cultivation of a chemoautotroph from the SUP05 clade of marine bacteria that produces nitrite and consumes ammonium.

Marine oxygen minimum zones (OMZs) are expanding regions of intense nitrogen cycling. Up to half of the nitrogen available for marine organisms is removed from the ocean in these regions. Metagenomic studies have identified an abundant group of sulfur-oxidizing bacteria (SUP05) with the genetic potential for nitrogen cycling and loss in OMZs. However, SUP05 have defied cultivation and their physiology remains untested. We cultured, sequenced and tested the physiology of an isolate from the SUP05 clade. We describe a facultatively anaerobic sulfur-oxidizing chemolithoautotroph that produces nitrite and consumes ammonium under anaerobic conditions. Genetic evidence that closely related strains are abundant at nitrite maxima in OMZs suggests that sulfur-oxidizing chemoautotrophs from the SUP05 clade are a potential source of nitrite, fueling competing nitrogen removal processes in the ocean.

Biomarker discovery and transcriptomic responses in Daphnia magna exposed to munitions constituents.

Ecotoxicogenomic approaches are emerging as alternative methods in environmental monitoring because they allow insight into pollutant modes of action and help assess the causal agents and potential toxicity beyond the traditional end points of death, growth, and reproduction. Gene expression analysis has shown particular promise for identifying gene expression biomarkers of chemical exposure that can be further used to monitor specific chemical exposures in the environment. We focused on the development of gene expression markers to detect and discriminate between chemical exposures. Using a custom cDNA microarray for Daphnia magna, we identified distinct expression fingerprints in response to exposure at sublethal concentrations of Cu, Zn, Pb, and munitions constituents. Using the results obtained from microarray analysis, we chose a suite of potential biomarkers for each of the specific exposures. The selected potential biomarkers were tested in independent chemical exposures for specificity using quantitative reverse transcription polymerase chain reaction. Six genes were confirmed as differentially regulated bythe selected chemical exposures. Furthermore, each exposure was identified by response of a unique combination (suite) of individual gene expression biomarkers. These results demonstrate the potential for discovery and validation of novel biomarkers of chemical exposures using gene expression analysis, which could have broad applicability in environmental monitoring.

Daphnia magna ecotoxicogenomics provides mechanistic insights into metal toxicity.

Toxicogenomics has provided innovative approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to understand the modes of toxicity in environmentally relevant organisms. Daphnia magna, a small aquatic crustacean, is considered a "keystone" species in ecological food webs and is an indicator species for toxicant exposure. Our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology by identifying novel biomarkers and uncovering potential modes of action in D. magna. Using a custom D. magna cDNA microarray, we identified distinct expression profiles in response to sublethal copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. By integrating gene expression profiling into an environmentally important organism, this study provides experimental support for the utility of ecotoxicogenomics.

Immunostimulatory oligonucleotides block allergic airway inflammation by inhibiting Th2 cell activation and IgE-mediated cytokine induction.

A single treatment with a CpG-containing immunostimulatory DNA sequence (ISS) given before allergen challenge can inhibit T helper type 2 cell (Th2)-mediated airway responses in animal models of allergic asthma; however, the mechanism of this inhibition remains largely undefined. Here, we demonstrate that airway delivery of ISS before allergen challenge in Th2-primed mice acts in two distinct ways to prevent the allergic responses to this challenge. The first is to prevent induction of cytokines from allergen-specific Th2 cells, as demonstrated by the nearly complete inhibition of Th2 cytokine production, Th2-dependent functional responses, and gene induction patterns. ISS inhibits the Th2 response by rendering lung antigen-presenting cells (APCs) unable to effectively present antigen to Th2 cells, but not to Th1 cells. This loss of APC function correlates with a reduced expression of costimulatory molecules, including programmed cell death ligand (PD-L)1, PD-L2, CD40, CD80, CD86, and inducible T cell costimulator, and of major histocompatibility complex class II on CD11c(+ )APCs from the airways of ISS-treated mice. The second important action of ISS is inhibition of immunoglobulin E-dependent release of Th2 cytokines, especially interleukin 4, from basophils and/or mast cells in the airways of Th2-primed mice. Thus, inhibition by ISS of allergic responses can be explained by two novel mechanisms that culminate in the inhibition of the principal sources of type 2 cytokines in the airways.

Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus.

Raised serum levels of interferon (IFN)-alpha have been observed in systemic lupus erythematosus (SLE) patients, and these levels are correlated with both disease activity and severity. The origin of this IFN-alpha is still unclear, but increasing evidence suggests the critical involvement of activated plasmacytoid predendritic cells (PDCs). In SLE patients, DNA and RNA viruses, as well as immune complexes (ICs), that consist of autoantibodies specific to self-DNA and RNA protein particles can stimulate production of IFN-alpha. We have developed three series of oligonucleotide (ODN)-based inhibitors of Toll-like receptor (TLR) signaling. These ODNs include inhibitors of TLR9, inhibitors of TLR7 but not TLR9, and sequences that inhibit both TLR7 and TLR9. Specificity of these inhibitors is confirmed by inhibition of IFN-alpha production by PDCs in response to DNA or RNA viruses. We show that mammalian DNA and RNA, in the form of ICs, are potent self-antigens for TLR9 and TLR7, respectively, and induce IFN-alpha production by PDCs. This work suggests that TLRs may have a critical role in the promotion of lupus through the induction of IFN-alpha by PDCs. These inhibitors of TLR signaling thus represent novel therapeutic agents with potential for the treatment of lupus.

Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation.

In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-alpha and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.