Chronic nitrogen fertilization and carbon sequestration in grassland soils: evidence of a microbial enzyme link

Chronic nitrogen (N) fertilization can greatly affect soil carbon (C) sequestration by altering biochemical interactions between plant detritus and soil microbes. In lignin-rich forest soils, chronic N additions tend to increase soil C content partly by decreasing the activity of lignin-degrading enzymes. In cellulose-rich grassland soils it is not clear whether cellulose-degrading enzymes are also inhibited by N additions and what consequences this might have on changes in soil C content. Here we address whether chronic N fertilization has affected (1) the C content of light versus heavier soil fractions, and (2) the activity of four extracellular enzymes including the C-acquiring enzyme β-1,4-glucosidase (BG; necessary for cellulose hydrolysis). We found that 19 years of chronic N-only addition to permanent grassland have significantly increased soil C sequestration in heavy but not in light soil density fractions, and this C accrual was associated with a significant increase (and not decrease) of BG activity. Chronic N fertilization may increase BG activity because greater N availability reduces root C:N ratios thus increasing microbial demand for C, which is met by C inputs from enhanced root C pools in N-only fertilized soils. However, BG activity and total root mass strongly decreased in high pH soils under the application of lime (i.e. CaCO3), which reduced the ability of these organo-mineral soils to gain more C per units of N added. Our study is the first to show a potential ‘enzyme link’ between (1) long-term additions of inorganic N to grassland soils, and (2) the greater C content of organo-mineral soil fractions. Our new hypothesis is that the ‘enzyme link’ occurs because (a) BG activity is stimulated by increased microbial C demand relative to N under chronic fertilization, and (b) increased BG activity causes more C from roots and from microbial metabolites to accumulate and stabilize into organo-mineral C fractions. We suggest that any combination of management practices that can influence the BG ‘enzyme link’ will have far reaching implications for long-term C sequestration in grassland soils

Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP.

Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction, involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement, nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S, are known. To screen for functionally important regions within protein S LG1 we generated seven variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T, displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed four protein S variants in which 4-6 surface exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high affinity C4BP-binding. The C4BP β-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP β-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423 and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP β-chain blocks this function

Protective Effects of Non-Anticoagulant Activated Protein C Variant (D36A/L38D/A39V) in a Murine Model of Ischaemic Stroke

Ischaemic stroke is caused by occlusive thrombi in the cerebral vasculature. Although tissue-plasminogen activator (tPA) can be administered as thrombolytic therapy, it has major limitations, which include disruption of the blood-brain barrier and an increased risk of bleeding. Treatments that prevent or limit such deleterious effects could be of major clinical importance. Activated protein C (APC) is a natural anticoagulant that regulates thrombin generation, but also confers endothelial cytoprotective effects and improved endothelial barrier function mediated through its cell signalling properties. In murine models of stroke, although APC can limit the deleterious effects of tPA due to its cell signalling function, its anticoagulant actions can further elevate the risk of bleeding. Thus, APC variants such as APC(5A), APC(Ca-ins) and APC(36-39) with reduced anticoagulant, but normal signalling function may have therapeutic benefit. Human and murine protein C (5A), (Ca-ins) and (36-39) variants were expressed and characterised. All protein C variants were secreted normally, but 5-20% of the protein C (Ca-ins) variants were secreted as disulphide-linked dimers. Thrombin generation assays suggested reductions in anticoagulant function of 50- to 57-fold for APC(36-39), 22- to 27-fold for APC(Ca-ins) and 14- to 17-fold for APC(5A). Interestingly, whereas human wt APC, APC(36-39) and APC(Ca-ins) were inhibited similarly by protein C inhibitor (t½ - 33 to 39 mins), APC(5A) was inactivated ~9-fold faster (t½ - 4 mins). Using the murine middle cerebral artery occlusion ischaemia/repurfusion injury model, in combination with tPA, APC(36-39), which cannot be enhanced by its cofactor protein S, significantly improved neurological scores, reduced cerebral infarct area by ~50% and reduced oedema ratio. APC(36-39) also significantly reduced bleeding in the brain induced by administration of tPA, whereas wt APC did not. If our data can be extrapolated to clinical settings, then APC(36-39) could represent a feasible adjunctive therapy for ischaemic stroke

Conformational activation of ADAMTS13

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a “closed” conformation of WT ADAMTS13 and suggested a more “open” conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB–spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition

Defective fibrin deposition and thrombus stability in Bambi ‐/‐ mice is mediated by elevated anticoagulant function

BACKGROUND: Bone morphogenetic and activin membrane-bound inhibitor (BAMBI) is a transmembrane protein related to the type I transforming growth factor- β (TGF-β) receptor family that is present on both platelets and endothelial cells (ECs). Bambi-deficient mice exhibit reduced hemostatic function and thrombus stability characterized by an increased embolization. OBJECTIVE: We aimed to delineate how BAMBI influences endothelial function and thrombus stability. METHODS: Bambi-deficient mice were subjected to the laser-induced thrombosis model where platelet and fibrin accumulation was evaluated. Expression of thrombomodulin and tissue factor pathway inhibitor (TFPI) was also assessed in these mice. RESULTS: Thrombus instability in Bambi-/- mice was associated with a profound defect in fibrin deposition. Injection of hirudin into Bambi+/+ mice prior to thrombus formation recapitulated the Bambi-/- thrombus instability phenotype. In contrast, hirudin had no additional effect upon thrombus formation in Bambi-/- mice. Deletion of Bambi in ECs resulted in mice with defective thrombus stability caused by decreased fibrin accumulation. Increased levels of the anticoagulant proteins TFPI and thrombomodulin were detected in Bambi-/- mouse lung homogenates. Endothelial cells isolated from Bambi-/- mouse lungs exhibited enhanced ability to activate protein C due to elevated thrombomodulin levels. Blocking thrombomodulin and TFPI in vivo fully restored fibrin accumulation and thrombus stability in Bambi-/- mice. CONCLUSIONS: We demonstrate that endothelial BAMBI influences fibrin generation and thrombus stability by modulating thrombomodulin and TFPI anticoagulant function of the endothelium; we also highlight the importance of these anticoagulant proteins in the laser-induced thrombosis model

Enhanced activity of an ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) against platelet agglutination in vitro and in a murine model of acute ischemic stroke.

Essentials ADAMTS13 requires a substrate-induced conformational change to attain full activity in vitro. The efficacy of wild type ADAMTS13 in models of thrombosis/stroke may be enhanced by pre-activation. A pre-activated ADAMTS13 variant exhibits enhanced proteolysis of platelet agglutinates. This ADAMTS13 variant is protective in a murine model of stroke at a lower dose than WT ADAMTS13. SUMMARY: Background ADAMTS-13 circulates in a closed conformation, only achieving full proteolytic activity against von Willebrand factor (VWF) following a substrate-induced conformational change. A gain-of-function (GoF) ADAMTS-13 variant (R568K/F592Y/R660K/Y661F/Y665F) is conformationally preactivated. Objectives To establish how the hyperactivity of GoF ADAMTS-13 is manifested in experimental models mimicking the occlusive arterial thrombi present in acute ischemic stroke. Methods The ability of GoF ADAMTS-13 to dissolve VWF-platelet agglutinates was examined with an assay of ristocetin-induced platelet agglutination and in parallel-flow models of arterial thrombosis. A murine model of focal ischemia was used to assess the thrombolytic potential of GoF ADAMTS-13. Results Wild-type (WT) ADAMTS-13 required conformational activation to attain full activity against VWF-mediated platelet capture under flow. In this assay, GoF ADAMTS-13 had an EC50 value more than five-fold lower than that of WT ADAMTS-13 (0.73 ± 0.21 nm and 3.81 ± 0.97 nm, respectively). The proteolytic activity of GoF ADAMTS-13 against preformed platelet agglutinates under flow was enhanced more than four-fold as compared with WT ADAMTS-13 (EC50 values of 2.5 ± 1.1 nm and 10.2 ± 5.6 nm, respectively). In a murine stroke model, GoF ADAMTS-13 restored cerebral blood flow at a lower dose than WT ADAMTS-13, and partially retained the ability to recanalize vessels when administration was delayed by 1 h. Conclusions The limited proteolytic activity of WT ADAMTS-13 in in vitro models of arterial thrombosis suggests an in vivo requirement for conformational activation. The enhanced activity of the GoF ADAMTS-13 variant translates to a more pronounced protective effect in experimental stroke

Ambulatory dispersal in Tetranychus urticae: an artificial selection experiment on propensity to disperse yields no response

Dispersal to new hosts is an important process for an invasive herbivore, such as the two-spotted spider mite. A recent study, using artificial selection experiments, has suggested that genetic variation and genetic trade-offs are present for propensity to disperse in this species. However, due to the experimental setup alternative explanations for the response to selection could not be ruled out. Using an altered setup, we investigated whether the propensity for ambulatory dispersal differs genetically between individuals and whether genetic correlations with life-history traits exist. Upward and downward selection on propensity to leave the colony was performed for seven generations in four replicate artificial selection experiments and the results were compared to control lines. No consistent responses to selection were found and no significant effect on life-history traits (oviposition rate, juvenile survival, development rate and number of adult offspring) or sex ratio was present across the replicates. The data suggest that our base population of spider mites harbours at best a low amount of additive genetic variation for this behaviour

Effects of esomeprazole treatment for gastroesophageal reflux disease on quality of life in 12- to 17-year-old adolescents: an international health outcomes study

<p>Abstract</p> <p>Background</p> <p>Although gastroesophageal reflux disease (GERD) is common in adolescents, the burden of GERD on health-related quality of life (HRQOL) in adolescents has not been previously evaluated. Therefore, the objective of the study was to examine the effect of GERD on HRQOL in adolescents.</p> <p>Methods</p> <p>This international, 31-site, 8-week safety study randomized adolescents, aged 12 to 17 years inclusive, with GERD to receive esomeprazole 20 or 40 mg once daily. The Quality of Life in Reflux and Dyspepsia questionnaire (QOLRAD), previously validated in adults, consists of 25 questions grouped into 5 domains: emotional distress, sleep disturbance, food/drink problems, physical/social functioning, and vitality. The QOLRAD was administered at the baseline and week-8 (final) visits.</p> <p>Results</p> <p>Of the 149 patients randomized, 134 completed the QOLRAD at baseline and final visits and were eligible for analysis of their HRQOL data. Baseline QOLRAD scores indicated GERD had a negative effect on the HRQOL of these adolescents, especially in the domains of vitality and emotional distress, and problems with food/drink. At the final visit, mean scores for all 5 QOLRAD domains improved significantly (<it>P </it>< .0001); change of scores (ie, delta) for all domains met or exceeded the adult QOLRAD minimal clinically significant difference standard of 0.5 units.</p> <p>Conclusion</p> <p>GERD had a negative effect on QOL in adolescents. After esomeprazole treatment, statistically and clinically significant improvements occurred in all domains of the QOLRAD for these adolescents.</p> <p>Trial Registration</p> <p>D9614C00098; ClinicalTrials.gov Identifier NCT00241501</p

Effective Long-Distance Pollen Dispersal in Centaurea jacea

BACKGROUND: Agri-environment schemes play an increasingly important role for the conservation of rare plants in intensively managed agricultural landscapes. However, little is known about their effects on gene flow via pollen dispersal between populations of these species. METHODOLOGY/PRINCIPAL FINDINGS: In a 2-year experiment, we observed effective pollen dispersal from source populations of Centaurea jacea in restored meadows, the most widespread Swiss agri-environment scheme, to potted plants in adjacent intensively managed meadows without other individuals of this species. Potted plants were put in replicated source populations at 25, 50, 100 m and where possible 200 m distance from these source populations. Pollen transfer among isolated plants was prevented by temporary bagging, such that only one isolated plant was accessible for flower visitors at any one time. Because C. jacea is self-incompatible, seed set in single-plant isolates indicated insect mediated effective pollen dispersal from the source population. Seed set was higher in source populations (35.7+/-4.4) than in isolates (4.8+/-1.0). Seed set declined from 18.9% of that in source populations at a distance of 25 m to 7.4% at 200 m. At a distance of 200 m seed set was still significantly higher in selfed plants, indicating long-distance effective pollen dispersal up to 200 m. Analyses of covariance suggested that bees contributed more than flies to this long-distance pollen dispersal. We found evidence that pollen dispersal to single-plant isolates was positively affected by the diversity and flower abundance of neighboring plant species in the intensively managed meadow. Furthermore, the decline of the dispersal was less steep when the source population of C. jacea was large. CONCLUSIONS: We conclude that insect pollinators can effectively transfer pollen from source populations of C. jacea over at least 200 m, even when "recipient populations" consisted of single-plant isolates, suggesting that gene flow by pollen over this distance is very likely. Source population size and flowering environment surrounding recipient plants appear to be important factors affecting pollen dispersal in C. jacea. It is conceivable that most insect-pollinated plants in a network of restored sites within intensively managed grassland can form metapopulations, if distances between sites are of similar magnitude as tested here