The global nitrogen cycle in the twenty-first century

Global nitrogen fixation contributes 413 Tg of reactive nitrogen (N_r) to terrestrial and marine ecosystems annually of which anthropogenic activities are responsible for half, 210 Tg N. The majority of the transformations of anthropogenic N_r are on land (240 Tg N yr⁻¹) within soils and vegetation where reduced N_r contributes most of the input through the use of fertilizer nitrogen in agriculture. Leakages from the use of fertilizer N_r contribute to nitrate (NO₃⁻) in drainage waters from agricultural land and emissions of trace N_r compounds to the atmosphere. Emissions, mainly of ammonia (NH₃) from land together with combustion related emissions of nitrogen oxides (NO_x), contribute 100 Tg N yr⁻¹ to the atmosphere, which are transported between countries and processed within the atmosphere, generating secondary pollutants, including ozone and other photochemical oxidants and aerosols, especially ammonium nitrate (NH₄NO₃) and ammonium sulfate (NH₄)₂SO₄. Leaching and riverine transport of NO₃ contribute 40–70 Tg N yr⁻¹ to coastal waters and the open ocean, which together with the 30 Tg input to oceans from atmospheric deposition combine with marine biological nitrogen fixation (140 Tg N yr⁻¹) to double the ocean processing of N_r. Some of the marine N_r is buried in sediments, the remainder being denitrified back to the atmosphere as N₂ or N₂O. The marine processing is of a similar magnitude to that in terrestrial soils and vegetation, but has a larger fraction of natural origin. The lifetime of N_r in the atmosphere, with the exception of N₂O, is only a few weeks, while in terrestrial ecosystems, with the exception of peatlands (where it can be 10²–10³ years), the lifetime is a few decades. In the ocean, the lifetime of N_r is less well known but seems to be longer than in terrestrial ecosystems and may represent an important long-term source of N₂O that will respond very slowly to control measures on the sources of N_r from which it is produced.     

Inhibition of melanoma development in the Nras(Q61K)::Ink4a−/− mouse model by the small molecule BI‐69A11

To date, there are no effective therapies for tumors bearing NRAS mutations, which are present in 15–20% of human melanomas. Here we extend our earlier studies where we demonstrated that the small molecule BI‐69A11 inhibits the growth of melanoma cell lines. Gene expression analysis revealed the induction of interferon‐ and cell death‐related genes that were associated with responsiveness of melanoma cell lines to BI‐69A11. Strikingly, the administration of BI‐69A11 inhibited melanoma development in genetically modified mice bearing an inducible form of activated Nras and a deletion of the Ink4a gene (Nras^(Q61K)::Ink4a^?/?). Biweekly administration of BI‐69A11 starting at 10 weeks or as late as 24 weeks after the induction of mutant Nras expression inhibited melanoma development (100 and 36%, respectively). BI‐69A11 treatment did not inhibit the development of histiocytic sarcomas, which constitute about 50% of the tumors in this model. BI‐69A11‐resistant Nras^(Q61K)::Ink4a^?/? tumors exhibited increased CD45 expression, reflective of immune cell infiltration and upregulation of gene networks associated with the cytoskeleton, DNA damage response, and small molecule transport. The ability to attenuate the development of NRAS mutant melanomas supports further development of BI‐69A11 for clinical assessment.     

Murine Cell Glycolipids Customization by Modular Expression of Glycosyltransferases

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.     

Roles for Ordered and Bulk Solvent in Ligand Recognition and Docking in Two Related Cavities

A key challenge in structure-based discovery is accounting for modulation of protein-ligand interactions by ordered and bulk solvent. To investigate this, we compared ligand binding to a buried cavity in Cytochrome c Peroxidase (CcP), where affinity is dominated by a single ionic interaction, versus a cavity variant partly opened to solvent by loop deletion. This opening had unexpected effects on ligand orientation, affinity, and ordered water structure. Some ligands lost over ten-fold in affinity and reoriented in the cavity, while others retained their geometries, formed new interactions with water networks, and improved affinity. To test our ability to discover new ligands against this opened site prospectively, a 534,000 fragment library was docked against the open cavity using two models of ligand solvation. Using an older solvation model that prioritized many neutral molecules, three such uncharged docking hits were tested, none of which was observed to bind; these molecules were not highly ranked by the new, context-dependent solvation score. Using this new method, another 15 highly-ranked molecules were tested for binding. In contrast to the previous result, 14 of these bound detectably, with affinities ranging from 8 µM to 2 mM. In crystal structures, four of these new ligands superposed well with the docking predictions but two did not, reflecting unanticipated interactions with newly ordered waters molecules. Comparing recognition between this open cavity and its buried analog begins to isolate the roles of ordered solvent in a system that lends itself readily to prospective testing and that may be broadly useful to the community.     

Dexamethasone Attenuates VEGF Expression and Inflammation but Not Barrier Dysfunction in a Murine Model of Ventilator–Induced Lung Injury

Background

Ventilator–induced lung injury (VILI) is characterized by vascular leakage and inflammatory responses eventually leading to pulmonary dysfunction. Vascular endothelial growth factor (VEGF) has been proposed to be involved in the pathogenesis of VILI. This study examines the inhibitory effect of dexamethasone on VEGF expression, inflammation and alveolar–capillary barrier dysfunction in an established murine model of VILI.

Methods

Healthy male C57Bl/6 mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with an inspiratory pressure of 10 cmH₂O (“lower” tidal volumes of ∼7.5 ml/kg; LV_T) or 18 cmH₂O (“higher” tidal volumes of ∼15 ml/kg; HV_T). Dexamethasone was intravenously administered at the initiation of HV_T–ventilation. Non–ventilated mice served as controls. Study endpoints included VEGF and inflammatory mediator expression in lung tissue, neutrophil and protein levels in bronchoalveolar lavage fluid, PaO₂ to FiO₂ ratios and lung wet to dry ratios.

Results

Particularly HV_T–ventilation led to alveolar–capillary barrier dysfunction as reflected by reduced PaO₂ to FiO₂ ratios, elevated alveolar protein levels and increased lung wet to dry ratios. Moreover, VILI was associated with enhanced VEGF production, inflammatory mediator expression and neutrophil infiltration. Dexamethasone treatment inhibited VEGF and pro–inflammatory response in lungs of HV_T–ventilated mice, without improving alveolar–capillary permeability, gas exchange and pulmonary edema formation.

Conclusions

Dexamethasone treatment completely abolishes ventilator–induced VEGF expression and inflammation. However, dexamethasone does not protect against alveolar–capillary barrier dysfunction in an established murine model of VILI.     

Enhanced Human Tissue Microdialysis Using Hydroxypropyl-?-Cyclodextrin as Molecular Carrier

Microdialysis sampling of lipophilic molecules in human tissues is challenging because protein binding and adhesion to the membrane limit recovery. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) forms complexes with hydrophobic molecules thereby improving microdialysis recovery of lipophilic molecules in vitro and in rodents. We tested the approach in human subjects. First, we determined HP-ß-CD influences on metabolite stability, delivery, and recovery in vitro. Then, we evaluated HP-ß-CD as microdialysis perfusion fluid supplement in 20 healthy volunteers. We placed 20 kDa microdialysis catheters in subcutaneous abdominal adipose tissue and in the vastus lateralis muscle. We perfused catheters with lactate free Ringer solution with or without 10% HP-ß-CD at flow rates of 0.3–2.0 µl/min. We assessed tissue metabolites, ultrafiltration effects, and blood flow. In both tissues, metabolite concentrations with Ringer+HP-ß-CD perfusate were equal or higher compared to Ringer alone. Addition of HP-ß-CD increased dialysate volume by 10%. Adverse local or systemic reactions to HP-ß-CD did not occur and analytical methods were not disturbed. HP-ß-CD addition allowed to measure interstitial anandamide concentrations, a highly lipophilic endogenous molecule. Our findings suggest that HP-ß-CD is a suitable supplement in clinical microdialysis to enhance recovery of lipophilic molecules from human interstitial fluid.     

Hereditary Angioedema Attacks Resolve Faster and Are Shorter after Early Icatibant Treatment

Background

Attacks of hereditary angioedema (HAE) are unpredictable and, if affecting the upper airway, can be lethal. Icatibant is used for physician- or patient self-administered symptomatic treatment of HAE attacks in adults. Its mode of action includes disruption of the bradykinin pathway via blockade of the bradykinin B₂ receptor. Early treatment is believed to shorten attack duration and prevent severe outcomes; however, evidence to support these benefits is lacking.

Objective

To examine the impact of timing of icatibant administration on the duration and resolution of HAE type I and II attacks.

Methods

The Icatibant Outcome Survey is an international, prospective, observational study for patients treated with icatibant. Data on timings and outcomes of icatibant treatment for HAE attacks were collected between July 2009–February 2012. A mixed-model of repeated measures was performed for 426 attacks in 136 HAE type I and II patients.

Results

Attack duration was significantly shorter in patients treated <1 hour of attack onset compared with those treated ≥1 hour (6.1 hours versus 16.8 hours [p<0.001]). Similar significant effects were observed for <2 hours versus ≥2 hours (7.2 hours versus 20.2 hours [p<0.001]) and <5 hours versus ≥5 hours (8.0 hours versus 23.5 hours [p<0.001]). Treatment within 1 hour of attack onset also significantly reduced time to attack resolution (5.8 hours versus 8.8 hours [p<0.05]). Self-administrators were more likely to treat early and experience shorter attacks than those treated by a healthcare professional.

Conclusion

Early blockade of the bradykinin B₂ receptor with icatibant, particularly within the first hour of attack onset, significantly reduced attack duration and time to attack resolution.